JP6921797B2 - Modified polynucleotides for the production of biologics and proteins associated with human disease - Google Patents

Description

Sequence Listing Reference This application has been filed in electronic form with the sequence listing. M300_PCTSQLST. The sequence listing file, entitled txt, was created on March 9, 2013 and is 49,417,315 bytes in size. The information in electronic form of this sequence listing is incorporated herein by reference in its entirety.

Mutual reference of related applications This application is a US provisional patent application No. 61 / 681,742 filed on August 10, 2012, entitled "Modified Polynucleotides for the Production of Oncology-Related Protocols and Pedides, 2012", 2012. US Provisional Patent Application No. 61 / 737,224 filed in Japan, title "Terminally Adaptive Modified RNAs", International Application No. PCT / US2012 / 069610 filed on December 14, 2012, title "Modified Nucleoside, Nucleotide". "Acid Compositions", US provisional patent application No. 61 / 618,862 filed on April 2, 2012, title "Modified Polynucleotides for the Productions of Biologics", US provisional patent application filed on August 10, 2012. No. 681,645, title "Modified Polynucleotides for the Production of"
Biologics ”, US Provisional Patent Application No. 61 / 737,130 filed December 14, 2012, Title“ Modified Polynucleotides for the Production of Biologics ”, US Provisional Patent Application No. 61/618 filed April 2, 2012 , 866, Title "Modified Polynucleotides for the Productions of Antibodies", US Provisional Patent Application No. 61 / 681,647 filed on August 10, 2012, Title "Modified Plastics
"of Antibodies", US provisional patent application No. 61 / 737,134 filed on December 14, 2012, title "Modified Polynucleotides for the Productions of Antibodies", US provisional patent application filed on April 2, 2012. No. 618,868, title "Modified Polynucleotides for the Production of Vaccines", US provisional patent application No. 61 / 681,648 filed on August 10, 2012, title "Modified Polynuclecitudes 2012 Vaccines" US provisional patent application No. 61 / 737,135 filed on March 14, 2012, title "Modified Polynucreotides for the Production of Vaccines", US provisional patent application No. 61 / 618,870 filed on April 2, 2012, title " Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides ", US provisional patent application, filed Aug. 10, 2012 No. 61 / 681,649, entitled" Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides ", December 2012 U.S. provisional patent application No. 61 / 737,139 filed on 14th, title "Modified Polynucreotides for the Productions of Therapeutic Proteins and Peptides", filed on April 2, 2012, U.S. provisional patent application No. 87/3, 2012. Title "Modified Polynucleotides for the Production of Selected Products", US Provisional Patent Application No. 61 / 681,650 filed on August 10, 2012, Title "Modified PolynucletionProtesys12 US Provisional Patent Application No. 61 / 737,147, entitled "Modified Polynuc" secrets for the Production of Secreted
"Proteins", US provisional patent application No. 61 / 618,878 filed April 2, 2012, title "Modified Polynucleotides for the Production of Plasma Membrane Proteins", filed August 10, 2012, US provisional patent application. / 681,654, title "Modified Polynucleotides for the Production of Plasma Membrane Proteins", US provisional patent application No. 61 / 737,152 filed on December 14, 2012, title "Modified Cytoskeleton , U.S. Provisional Patent Application No. 61 / 618,885 filed on April 2, 2012, title "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletonal Proteins", filing August 10, 2012, U.S.A. / 681,658, title "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletonal Proteins", US provisional patent application No. 61 / 737,155 Cytoplasm
Cytoskeletonal Proteins ”, US provisional patent application No. 61 / 618,896 filed April 2, 2012, title“ Modified Polynucleotides for the Productions of Intracellular Membrane 7th year provisional patent application 20th year of the United States No. 61 / 668,157, entitled "Modified Protein patents for the Production of Intracellular"
"Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 681,661 filed on August 10, 2012, title "Modified Polynucleotides for the Productions for The Production of Intracellular Protein 12 May, 2012 U.S.A." Application No. 61 / 737,160, Title "Modified Polynucleotides for the Production of International Protein Bound Proteins", US Provisional Patent Application No. 61/618 of Nuclear
"Proteins", US provisional patent application No. 61 / 681,667 filed on August 10, 2012, title "Modified Polynucleotides for the Production of Nuclear Products", US provisional patent application No. 61 / filed on December 14, 2012. 737,168, title "Modified Polynucleotides for the Production of Nuclear Products", US provisional patent application No. 61 / 618,922 filed April 2, 2012, title "Modified Polythe U.S. provisional patent application No. 61 / 681,675 filed on August 10, 2012, title "Modified Polynucleotides for the Production of Products", U.S. provisional patent application No. 61 / 737,174 filed on December 14, 2012, title. "Modified Polynucleotides for the Productions of Proteins", US Provisional Patent Application No. 61 / 618,935 filed April 2, 2012, entitled "Modified Polynucleotides for the"
Production of Products Assisted with Human Disease ”, US provisional patent application No. 61 / 681,687 filed on August 10, 2012, title“ Modified Polynetides for the Production U.S. Provisional Patent Application No. 61 / 737,184 of the application, title "Modified Polynucleotides for the Production of Products Associates Assisted with Human Disease", April 2, 2012, U.S. Provisional Patent Application No. 61, "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", US provisional patent application, filed Aug. 10, 2012 No. 61 / 681,696, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", 2012 US provisional patent application No. 61 / 737,191 filed on December 14, 2012, title "Modified Polynucleotides for the Productions Assisted with Human Disease", filed on April 2, 2012, US provisional patent. , 953, Title "Modified Polynucleotides for the Production of Products Associates Assisted with Human Disease", US Provisional Patent Application No. 61 / 681, 704 "Human Disease", US Provisional Patent Application No. 61 / 737,203 filed December 14, 2012, entitled "Modified Polynucleotides for the Production of P" Rotains Associated with Human Disease ”, US Provisional Patent Application No. 61 / 618,961 filed April 2, 2012, Title“ Dosing Methods for Modified mRNA ”, US Provisional Patent Application No. 61 filed May 17, 2012 / 648,286, which claims priority over the title "Dosing Methods for Modified mRNA", the contents of each of which are incorporated herein by reference in their entirety.

This application is published in International Publication No. PCT / US2012 / 58519 filed on October 3, 2012, entitled "Modified Nucleosides, Nucleosides, and Nucleic Acids, and Uses Thereof", and published on December 14, 2012. It is also related to PCT / US2012 / 96610, entitled "Modified Nucleoside, Nucleoside, and Nucleic Acid Compositions".

This application is also related to a co-pending application, which was filed simultaneously with this application on March 9, 2013, respectively, and has an agent reference number M301.20 (PCT / US13 / XXXXXX) under the title "Modified Polynucleotides", title. "Modified Polynucleotides for the Production of the"
Proxy reference number M304.20 (PCT / US13 / XXXXX) of "Secreted Proteins", proxy reference number M305.20 (PCT / US13 / XXXXX), title "Modified Polynucleotides for the Production of Membrane Proteins" for the Production of
Agent reference number M306.20 (PCT / US13 / XXXXXX) of "Cytoplasmic and Cytoskeleton Proteins", title "Modified Polynucleotides for the Production of"
Agent reference number M308.20 (PCT / US13 / XXXXX) of "Nuclear Proteins", agent reference number M309.20 (PCT / US13 / XXXX) of the title "Modified Polynucleotides for the Production of Proteins" The Production of Proteins Assisted with Human Disease "agent reference number M310.20 (PCT / US13 / XXXXXX), title" Modified Polynucleotides for the Product ".
The agent reference number MNC1.20 (PCT / US13 / XXXXX) of "Proteins and Peptides" and the title "Modified Polynucleotides for the Products for the Production of Oncology-Related X ), And the contents of each of these are incorporated herein by reference in their entirety.

Field of invention The present invention relates to the composition of polynucleotides, primary constructs, and modified mRNA molecules (m mRNAs), the methods, processes, kits, and devices of their design, preparation, manufacture, and / or formulation.

There are many problems with conventional methods of producing protein expression. For example, the introduced DNA can frequently integrate into the host cell genomic DNA, resulting in degeneration and / or damage to the host cell genomic DNA. Alternatively, the heterologous deoxyribonucleic acid (DNA) introduced into the cell can be inherited by daughter cells (whether or not the heterologous DNA is integrated into the chromosome) or by progeny.
In addition, there are many steps that must occur before the encoded protein is made, assuming that it is properly delivered and that there is no damage or integration of the host genome. Once inside the cell, the DNA must be transported into the nucleus where it is RNA transcribed. The RNA transcribed from the DNA must then enter the cytoplasm where it is translated into protein. Not only do multiple processing steps from administered DNA to protein create a time lag before the production of functional proteins, but each step also provides an opportunity for cell error and damage. In addition, it is known that it is difficult to achieve DNA expression in cells because DNA frequently enters cells but is not expressed or is not expressed at a reasonable rate or concentration. This can be especially problematic when DNA is introduced into primary cells or modified cell lines.

In the early 1990s, Bloom and colleagues successfully rescued vasopressin-deficient rats by injecting in vitro transcribed vasopressin mRNA into the hypothalamus (Science 255: 996-998; 1992). However, low levels of translation and molecular immunogenicity impede the development of mRNA as a therapeutic agent, and since then, rather, an alternative application that could take advantage of these pitfalls, namely immunization with mRNA encoding a cancer antigen. The effort is concentrated on.

Others are studying the use of mRNA to deliver the polypeptide of interest, and certain chemical modifications of the mRNA molecule, specifically pseudouridine and 5-methylcytosine, have immunostimulatory effects. It shows that it has been lowered.

These studies were, for example, published as US Patent Application No. 0316089.2, International Publication No. WO2005005622, which was filed on July 9, 2003 by Ribotem Limited and is now abandoned, July 9, 2004. US Patent Application National Stage Registration No. 10 / 563,897, published as PCT Application No. PCT / GB2004 / 002981, US Pat. No. US20060247195, and now abandoned, filed June 8, 2006. And European Patent Application National Stage Registration EP2004743322, published as European Patent EP1646714 and currently withdrawn, filed July 9, 2004; Novozimes, Inc. PCT application No. PCT / US2007 / 88060 published as International Publication No. WO2008140615 on December 19, 2007, US patent application filed on July 2, 2009 published as US Patent No. US20100028943 Domestic stage Registration 12 / 520,072, and European Patent Application National Stage Registration EP2007874376 published on July 7, 2009, published as European Patent EP2104739; 2006 published as International Publication WO2007064952 of the University of Rochester. PCT application No. PCT / US2006 / 46120 filed on December 4, 2006, and US patent application No. 11 / 606,995, filed on December 1, 2006, published as US patent US200701141030; BioNTech AG 2007. European Patent Application No. EP2007024312, filed on December 14, 2008 and now abandoned, PCT Application No. PCT / EP2008 / 01509, filed on December 12, 2008, published as International Publication No. WO200907134, Europe European Patent Application National Stage Registration EP2008861423 published as Patent EP2240572 June 2, 2010, US Patent Application National Stage 12 published as US Patent US20110065103 on November 24, 2010 /, 735,060, German patent application DE 10 2005 046 490 filed September 28, 2005, PCT application PCT / EP 2006 filed September 28, 2006 published as International Publication No. WO200703366 / 0448, National Stage European Patent No. 1934345, filed March 21, 2012, and National Stage US Patent Application No. 11 / 992,638, published as August 14, 2009, published as 201209877; Immune. Disease Institute Inc. U.S. Patent Application No. 13 / 088,009, filed April 15, 2011, published as U.S. Pat. No. US 20120046346, and PCT Application No. PCT, filed April 15, 2011, published as International Publication No. WO20111030624. / US2011 / 32679; U.S. Patent Application No. 12 / 957,340, filed November 20, 2010, published as US Pat. PCT application No. PCT / US 1998/09492 filed on September 18, 1998, published as International Publication No. WO1999014346; US Patent Application National Stage Registration Nos. 13 / 203,229, published as PCT Application No. PCT / US2010 / 00377, and US Patent No. US20120053333, filed November 3, 2011; International Publication No. 13/203,229 of Ludwig Maximilian University PCT application No. PCT / EP2010 / 004681, filed July 30, 2010, published as WO20110112316; Cellscript Inc. US Pat. No. 8,039,214, filed on June 30, 2008 and granted on October 18, 2011, and US Pat. Application No. 12 / 962,498, published as US Patent No. US201101143397 December 7, 2010 Application No. 12 / 962,468, published as US Patent No. US201209649 September 20, 2011 Application Nos. 13 / 237,451 and PCT application PCT / US2010 / 59305 filed on December 7, 2010, published as WO2011071931, and 201012 published as WO2011071936. PCT / US2010 / 59317 filed on 7th May; PCT application PCT / US2006 / 32372 filed on 21st August 2006 published as International Publication No. WO2007024708 of the Board of Directors of the University of Pennsylvania, and US Patent No. US Patent Application No. 11 / 990,646 published as US 20090286852, filed March 27, 2009; German Patent Application No. DE10 2001 027 283.9 filed June 5, 2001 by Curevac GMBH. , DE10 2001 062 480.8 filed December 19, 2001, and DE 20 2006 051 516 filed October 31, 2006 (all of which have been abandoned), March 30, 2005. PCT application No. PCT / of June 5, 2002, published as European Patent EP1392341 granted on the date, European Patent EP1458410 granted on January 2, 2008, and WO2002098443. EP2002 / 06180, International Publication No. WO2003051401 published on December 19, 2002, PCT / EP2002 / 14577, International Publication No. WO2008052770, published on December 31, 2007. PCT / EP2007 / 09469, published as International Publication No. WO20092127230, filed on April 16, 2008, PCT / EP2008 / 03033, published as International Publication No. WO2006122828, filed on May 19, 2005. Same as PCT / EP2006 / US Pat. 10 / 729,830, US Pat. No. US20050059624, filed on June 18, 2004, filed on 10 / 870,110, US Pat. No. US2008,0267,873, filed on July 7, 2008. Published as US Pat. No. 11 / 914,945, US Pat. No. US20100047261, and now abandoned, filed on October 27, 2009, No. 12 / 446,912, US Pat. As No. 12 / 522,214 of the same application filed on January 4, 2010, No. 12 / 787,566 of the same application filed on May 26, 2010 published as US Pat. No. US20110077287, and US Pat. No. US201000239608. Published May 26, 2010, No. 12 / 787,755, U.S. Patent No. US201110269950, No. 13 / 185,119, published on July 18, 2011, and U.S. Patent No. It is described in No. 13 / 106,548 of the May 12, 2011 application published as US20110311472, all of which are incorporated herein by reference in their entirety.

Despite these reports being limited to the selection of chemical modifications including pseudouridine and 5-methylcytosine, it surrounds the effective regulation of intracellular translation and the processing of nucleic acids encoding the polypeptide or fragments thereof. There is still a need in the art for treatments that address myriad barriers.

To this end, we have the potential as a therapeutic agent in which certain modified mRNA sequences have advantages that do not merely evade, avoid, or reduce the immune response. I showed that. Such studies include published co-pending applications, International Application No. PCT / US2011 / 046861, filed August 5, 2011, and PCT / US2011 / 054636, filed October 3, 2011. It is detailed in International Application No. PCT / US2011 / 054617 filed October 3, 2011, the contents of which are incorporated herein by reference in their entirety.

The present invention encodes a polypeptide of interest (eg, modified mRNA or mRNA) and has structural and / or chemical features such as structural and function that avoid one or more of the challenges in the art. Maintaining completeness, overcoming expression thresholds, improving expression rate, half-life, and / or protein concentration, optimizing protein localization, and detrimental biological responses such as immune responses and / or degradation This need is addressed by providing nucleic acid compounds or polynucleotides with features that are useful in optimizing the formulation and delivery of nucleic acid therapeutics while bypassing the pathway.

The composition of the modified mRNA (m mRNA) molecule, the design, preparation, production, and / or formulation method, process, kit, and device of the modified mRNA (m mRNA) molecule are described herein.

Details of various embodiments of the present invention will be described in the following embodiments for carrying out the invention. Other features, objectives, and advantages of the invention will become apparent from the embodiments and drawings for carrying out the invention, as well as the claims.

The aforementioned and other objectives, features, and advantages become apparent from the following description of a particular embodiment of the invention, as illustrated in the accompanying drawings, in which similar reference characters are different drawings. Refers to the same part through. The drawings are not necessarily proportional to the actual size, but rather the emphasis is on exemplifying the principles of various embodiments of the invention.

It is a schematic diagram of the primary structure of this invention. The lipid structure in the prior art useful in the present invention is illustrated. 98N12-5 (TETA5-LAP), DLin-DMA, DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane), DLin-KC2-DMA, DLin-MC3- The structures of DMA and C12-200 are shown. Same as above. It is a representative plasmid useful in the IVT reaction taught herein. This plasmid contains inserts 64818 designed by us. A gel profile of modified mRNA encapsulated in PLGA microspheres. It is a histogram of the factor IX protein production PLGA preparation factor IX modified mRNA. Histogram showing VEGF protein production in human keratinocyte cells after transfection of modified mRNA at various doses. FIG. 6A shows protein production after transfection of modified mRNA containing native nucleoside triphosphate (NTP). Histogram showing VEGF protein production in human keratinocyte cells after transfection of modified mRNA at various doses. FIG. 6B shows post-transfection protein production of modified mRNA fully modified with pseudouridine (pseudouridine) and 5-methylcytosine (5 mC). Histogram showing VEGF protein production in human keratinocyte cells after transfection of modified mRNA at various doses. FIG. 6C shows post-transfection protein production of modified mRNA fully modified with N1-methyl-pseudouridine (N1-methyl-pseudouridine) and 5-methylcytosine (5 mC). It is a histogram of VEGF protein production in HEK293 cells. Histogram of VEGF expression and IFN-α induction after transfection of VEGF-modified mRNA in peripheral blood mononuclear cells (PBMC). FIG. 8A shows VEGF expression. Histogram of VEGF expression and IFN-α induction after transfection of VEGF-modified mRNA in peripheral blood mononuclear cells (PBMC). FIG. 8B shows the IFN-α lead. FIG. 6 is a histogram of VEGF protein production in HeLa cells from VEGF-modified mRNA. Histogram of VEGF protein production from lipoplexed VEGF-modified mRNA in mice. 9 is a histogram of G-CSF protein production in HeLa cells from G-CSF modified mRNA. 9 is a histogram showing G-CSF protein production from lipoplexed G-CSF modified mRNA in mice. 9 is a histogram showing factor IX protein production from factor IX modified mRNA in HeLa cell supernatant. FIG. 6 is a histogram showing APOA1 protein production from APOA1 wild-type modified mRNA, APOA1 Milano-modified mRNA or APOA1 Paris-modified mRNA in HeLa cells. It is a gel profile of APOA1 protein derived from APOA1 wild-type modified mRNA. It is a gel profile of APOA1 protein derived from APOA1 Paris modified mRNA. It is a gel profile of APOA1 protein derived from APOA1 Milano-modified mRNA. It is a gel profile of FGA protein derived from fibrinogen α (FGA) modified mRNA. 9 is a histogram showing plasminogen protein production from plasminogen-modified mRNA in HeLa cell supernatant. A gel profile of a plasminogen protein derived from plasminogen-modified mRNA. It is a gel profile of GALT protein derived from galactose-1-phosphate uridyl transferase (GALT) -modified mRNA. A gel profile of ASL protein derived from argininosuccinate lyase (ASL) -modified mRNA. A gel profile of a TAT protein derived from tyrosine aminotransferase (TAT) -modified mRNA. It is a gel profile of GBE1 protein derived from glucan (1,4-α-), branching enzyme 1 (GBE1) -modified mRNA. 9 is a histogram showing prothrombin protein production from prothrombin-modified mRNA in HeLa cell supernatant. 9 is a histogram showing prothrombin protein production from prothrombin-modified mRNA in HeLa cell supernatant. A gel profile of a CP protein derived from ceruloplasmin (CP or CLP) modified mRNA. 9 is a histogram showing the production of TGF-β1 protein from transforming growth factor β1 (TGF-β1) -modified mRNA in HeLa cell supernatant. A gel profile of an OTC protein derived from ornithine carbamoyl transferase (OTC) -modified mRNA. It is a flow cytometric plot of low density lipoprotein receptor (LDLR) modified mRNA. A gel profile of the UGT1A1 protein derived from the UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) modified mRNA. 6 is a histogram showing factor XI protein production in HEK293 cells. It is a gel profile of aquaporin-5 protein derived from aquaporin-5 modified mRNA. 9 is a histogram showing the production of Factor VII protein from Factor VII modified RNA in HeLa cells. 9 is a histogram showing insulin glargine protein production from insulin glargine-modified mRNA in HeLa cells. 9 is a histogram showing tissue factor protein production from tissue factor modified RNA in HeLa cells. FIG. 5 is a histogram showing factor XI protein production from factor XI modified mRNA in HeLa cells. 9 is a histogram showing factor XI protein production from factor XI modified mRNA in HeLa cell supernatant. 9 is a histogram showing insulin aspart protein production from insulin aspart-modified mRNA. 9 is a histogram showing insulin lisproprotein production from insulin lispro-modified mRNA in HeLa cells. 9 is a histogram showing insulin glulisine protein production from insulin glulisine-modified mRNA in HeLa cells. 9 is a histogram showing human growth hormone protein production from human growth hormone modified mRNA in HeLa cells. It is a gel profile of p53 protein derived from tumor protein 53 (p53) modified mRNA. FIG. 43A shows the predicted size of p53. FIG. 43B shows the predicted size of p53. It is a gel profile of TUFT1 protein derived from TUFT1 modified mRNA. It is a gel profile of GALK1 protein derived from galactokinase 1 (GALK1) modified mRNA. FIG. 45A shows the predicted size of GALK1. FIG. 45B shows the predicted size of GALK1. A gel profile of the DEFB103A protein derived from defensin, β103A (DEFB103A) modified mRNA. It is a flow cytometric plot of LDLR-modified mRNA. 6 is a histogram showing the expression of vascular endothelial growth factor in HeLa. 6 is a histogram showing the cell viability of HeLa cells transfected with vascular endothelial growth factor mRNA. It is a histogram showing the expression of insulin aspart protein. It is a histogram showing the expression of insulin glargine protein. It is a histogram showing the expression of insulin glulisine protein. 6 is a histogram showing the expression of interleukin 7 (IL-7) protein. 6 is a histogram showing the expression of erythropoietin (EPO) protein. It is a gel profile of a lysosomal acid lipase protein derived from a lysosomal acid lipase-modified mRNA. A gel profile of a glucocerebrosidase protein derived from glucocerebrosidase-modified mRNA. It is a gel profile of the isulonic acid-2-sulfatase protein derived from the isulonic acid-2-sulfatase-modified mRNA. A gel profile of a luciferase protein derived from luciferase-modified mRNA. It is a histogram which shows the IgG concentration after the administration of the formulated Herceptin modified mRNA in a mammal. 6 is a histogram showing the IgG concentration (ng / ml) after transfection of Herceptin-modified mRNA. It is a gel profile of Herceptin protein derived from Herceptin-modified mRNA. It is a histogram of the glucocerebrosidase enzyme activity. It is a histogram of the lysosomal acid lipase enzyme activity. 6 is a histogram showing factor VIII protein expression. It is a histogram which shows the chromogenic activity of the factor VIII. It is a graph which shows the LDLR expression. FIG. 66A shows cellular LDL receptor expression for the addition of LDLR mRNA. It is a graph which shows the LDLR expression. FIG. 66B shows LDL receptor expression in cells after transfection. It is a graph which shows the LDLR expression. FIG. 66C shows the saturation of BODIPY® labeled LRL. It is a graph which shows the LDLR expression. FIG. 66D shows the binding affinity of BODIPY-LDL for cells. It is a graph which shows the percentage of cells which are positive for UGT1A1 expression. It is a graph which shows the accumulation of UGT1A1 protein. It is a gel profile of UGT1A1 protein and OTC derived from UGT1A1 modified mRNA or OTC modified mRNA. It is a flow cytometric plot of HEK293 cells transfected with PAh or UGT1A1. It is a gel profile of UGT1A1 protein derived from UGT1A1 modified mRNA. A gel profile of a mouse microsome extract treated with LNP containing UGT1A1.

In the field of therapeutics, diagnostics, reagents, and biological assays, whether in vitro, in vivo, insights, or ex vivo, for example, to cause intracellular translation of nucleic acids and production of the encoded polypeptide of interest. It is very interesting to be able to deliver nucleic acids, such as ribonucleic acids (RNAs), into cells. Delivery and function of non-incorporated polynucleotides is of particular importance.

Compositions (including pharmaceutical compositions) of polynucleotides encoding one or more polypeptides of interest, as well as methods of designing, preparing, producing, and / or formulating them are described herein. Systems, processes, devices, and kits for selecting, designing, and / or utilizing polynucleotides encoding the polypeptides of interest described herein are also provided.

According to the present invention, these polynucleotides are preferably modified to avoid defects in molecules encoding other polypeptides in the art. Therefore, these polynucleotides are referred to as modified mRNAs or mRNAs.

The use of modified polynucleotides in antibodies, viruses, fields of application in veterinary medicine, and in various in vivo environments has been studied by us, and these studies are, for example, co-pending and co-owned in vivo of mmRNA. US Provisional Patent Application No. 61 / 470,451 filed March 31, 2011 to teach application; the same No. 61 filed April 26, 2011 to teach nucleic acids engineered for the production of antibody polypeptides. / 517,784; No. 61 / 519,158, filed May 17, 2011, which teaches the application of mmRNA technology in veterinary medicine; filed September 12, 2011, which teaches the application of mmRNA technology in antibacterial agents. No. 61 / 533,537; No. 61 / 533,554; No. 61 / 542,533 filed on 3rd; No. 61 / 570,690 filed on December 14, 2011, which teaches mobile devices to be used in the fabrication and use of mmRNA technology; mmRNA under emergency treatment conditions. No. 61 / 570,708, filed December 14, 2011; using Lipidoid, No. 61 / 576,651, filed December 16, 2011, which teaches the end-modified structure of mmRNA. No. 61 / 576,705, filed December 16, 2011, which teaches how to deliver the mmRNA that was present; December 21, 2011, which teaches how to increase the viability of an organ or tissue using mmRNA. No. 61 / 578,271 of the application; teaching mmRNA encoding a cell-permeable peptide; No. 61 / 581,322 of the same application of December 29, 2011; teaching the incorporation of cytotoxic nucleoside into the mmRNA. No. 61 / 581,352, filed December 29, 2011; and No. 61 / 631,729, filed January 10, 2012, which teaches how to use mmRNA to cross the blood-brain barrier. All of these are disclosed and are incorporated herein by reference in their entirety.

Stability and / or elimination in tissues, receptor uptake and / or kinetics, cell arrival by composition, involvement with translational mechanisms, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretory efficiency (application) Designed to improve reachability to blood circulation, protein half-life, and / or regulation of cell status, function, and / or activity, if done). Polynucleotides, primary constructs, and / or mRNAs encoding polypeptides are provided in parts of this specification.

I. Compositions of the Invention (mmRNA) The present invention provides nucleic acid molecules encoding one or more polypeptides of interest, specifically polynucleotides, primary constructs, and / or mmRNAs. The term "nucleic acid", in its broadest sense, includes any compound and / or substance, including polymers of nucleotides. These polymers are often referred to as polynucleotides. Examples of nucleic acids or polynucleotides of the present invention include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, β-D). LNA with -ribo configuration, α-LNA with α-L-ribo configuration (diastereomer of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino functionality Includes, but is not limited to, 2'-amino-α-LNA) or hybrids thereof.

In a preferred embodiment, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA (mRNA)" encodes a polypeptide of interest and is translated and encoded in vitro, in vivo, in vivo, or ex vivo. Refers to any polynucleotide capable of producing.

Traditionally, the major components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5'cap, and a poly A tail. Based on this wild-type modular structure, the invention maintains the modular tissue, but in some embodiments comprises a lack of substantial induction of the cell's spontaneous immune response to the moiety into which the polynucleotide is introduced. Extending the scope of function of conventional mRNA molecules by providing polynucleotides or primary RNA constructs that include one or more structures and / or chemical modifications or modifications that impart useful properties to the polynucleotide. Therefore, the modified mRNA molecule of the present invention is referred to as "m mRNA". As used herein, a "structural" feature or modification is that two or more binding nucleotides are inserted, deleted, replicated in a polynucleotide, primary construct, or mmRNA without significant chemical modification to the nucleotide itself. A feature or modification that is inverted or randomized. Structural modifications are of chemical nature and are therefore chemical modifications, as chemical bonds are inevitably broken and reformed to result in structural modifications. However, structural modifications result in different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" is inserted, resulting in a structural modification to the polynucleotide.

mmRNA Structure The mmRNAs of the present invention are in their functional and / or structural design features that help overcome the existing problems of effective polypeptide production with nucleic acid therapeutics as demonstrated herein. Distinguished from wild-type mRNA.

FIG. 1 shows a representative polynucleotide primary construct 100 of the present invention. As used herein, the term "primary construct" or "primary mRNA construct" encodes one or more polypeptides of interest, wherein the polypeptide of interest encoded therein is translated. Refers to a polynucleotide transcript that retains sufficient structural and / or chemical characteristics to make it possible. The primary construct can be the polynucleotide of the invention. When structurally or chemically modified, the primary construct can be referred to as mmRNA.

With reference to FIG. 1, here the primary construct 100 contains a first region of binding nucleotide 102 flanking the first flanking region 104 and the second flanking region 106. As used herein, the "first region" may be referred to as the "coding region" or "coding region", or simply the "first region". This first region may include, but is not limited to, the encoded polypeptide of interest. The polypeptide of interest may contain one or more signal sequences encoded by the signal sequence region 103 at its 5'end. Adjacent region 104 may include a region of binding nucleotide that contains one or more complete or incomplete 5'UTR sequences. Adjacent region 104 may also include a 5'end cap 108. The second flanking region 106 may include a region of the binding nucleotide containing one or more complete or incomplete 3'UTRs. Adjacent region 106 may also include a 3'tail sequence 110.

The crosslink at the 5'end of the first region 102 and the first adjacent region 104 is the first operating region 105. Traditionally, this manipulation area contains the start codon. Alternatively, this working region may include any translation initiation sequence or signal that includes the initiation codon.

The cross-linking at the 3'end of the first region 102 and the second adjacent region 106 is the second operating region 107. Traditionally, this manipulation area contains a stop codon. Alternatively, this manipulation region may include any translation initiation sequence or signal that includes a stop codon. According to the present invention, multiple consecutive stop codons may also be used.

In general, the shortest length of the first region of the primary construct of the invention is sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. Can be the length of a peptide sequence. In another embodiment, this length comprises peptides of 2 to 30 amino acids, such as 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. Can be enough to code. This length is at least 11, 12, 13, 14, 15, 17, 20, 25, or a peptide of 30 amino acids, or a peptide that is not longer than 40 amino acids, eg, 35, 30, 25, 20, It may be sufficient to encode a peptide that is no longer than 17, 15, 14, 13, 12, 11, or 10 amino acids. Examples of dipeptides that the polynucleotide sequence can encode include, but are not limited to, carnosine and anserine.

In general, the length of the first region encoding the polypeptide of interest of the present invention exceeds about 30 nucleotides in length (eg, at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7 000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100, Up to 000 nucleotides in length (including 100,000) or more. As used herein, the "first region" may be referred to as the "coding region" or "coding region", or simply the "first region".

In some embodiments, the polynucleotide, primary construct, or mmRNA is about 30 to about 100,000 nucleotides (eg, 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1, 000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1, 000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7, 000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000).

According to the present invention, the first and second adjacent regions independently range from 15 to 1,000 nucleotides in length (eg, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and more than 900 nucleotide lengths, or at least 30, 40, 45, 50, 55, 60, 70 , 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides in length).

According to the present invention, tail sequences range from 0 to 500 nucleotides in length (eg, at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides. Can be long). If the tail region is a poly A tail, the length can be determined in units of poly A binding protein binding or as a function thereof. In this embodiment, the poly A tail is long enough to bind to at least 4 monomers of the poly A binding protein. The monomer of the poly A binding protein binds to a series of about 38 nucleotides. Therefore, it has been observed that the polyA tail of about 80 nucleotides and 160 nucleotides is functional.

According to the present invention, the capping region may contain a single cap or a set of nucleotides forming the cap. In this embodiment, the capping region can be 1-10, eg, 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2 or 10 or less nucleotides in length. In some embodiments, the cap is absent.

According to the present invention, the first and second operating regions can be in the range of 3-40, eg, 5-30, 10-20, 15, or at least 4, or 30 nucleotide lengths, starting codons and /. Or it may contain one or more signals and / or restriction sequences in addition to the stop codon.

Circular mmRNA
According to the present invention, the primary construct or mmRNA can be cyclized or concategorized to produce a translation competent molecule and assist in the interaction between the poly A binding protein and the 5'end binding protein. The mechanism of cyclization or concateration can occur through at least three different pathways: 1) chemical pathways, 2) enzymatic pathways, and 3) ribozyme-catalyzed pathways. The newly formed 5'/3'linkage can be intramolecular or intermolecular.

In the first pathway, the 5'and 3'ends of nucleic acids contain chemical reactive groups that form new covalent bonds between the 5'and 3'ends of the molecule when in close proximity. In an organic solvent, the 3'-amino-terminal nucleotide on the 3'-terminal of the synthetic mRNA molecule undergoes a nucleophilic attack on the 5'-NHS-ester moiety to form a new 5'/3' amide bond. The 5'terminal may contain an NHS-ester reactive group and the 3'terminal may contain a 3'-amino-terminal nucleotide.

In the second pathway, T4 RNA ligase can be used to enzymatically bind a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of the nucleic acid to form a new phosphorylester bond. In an example of the reaction, 1 μg of nucleic acid molecule is incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) for 1 hour at 37 ° C. according to the manufacturer's protocol. The ligation reaction can occur in the presence of split oligonucleotides that can base pair alongside both the 5'and 3'regions to assist in the enzymatic ligation reaction.

In the third pathway, either the 5'end or the 3'end of the cDNA template allows the resulting nucleic acid molecule to link the 5'end of the nucleic acid molecule to the 3'end of the nucleic acid molecule during in vitro transcription. The ligase ribozyme sequence is encoded so that it can contain a capable active ribozyme sequence. Rigase ribozymes can be derived from group I introns, group I introns, delta hepatitis virus, hairpin ribozymes, or can be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction can take 1 to 24 hours at a temperature of 0 to 37 ° C.

mmRNA Multimeric According to the present invention, a plurality of distinctly different polynucleotides, primary constructs, or mmRNAs can be linked via the 3'end using nucleotides modified at the 3'end. Chemical complex formation can be used to control stoichiometry of cell delivery. For example, glyoxylate cycle enzyme, isocitrate lyase, and malate synthase can be fed to HepG2 cells in a 1: 1 ratio to alter the metabolism of cellular fatty acids. This ratio is chemically determined using 3'-azido-terminated nucleotides for one polynucleotide, primary construct, or mMV species and C5-ethynyl or alkynyl-containing nucleotides for the opposite polynucleotide, primary construct, or mMV species. It can be controlled by chemically binding a binding polynucleotide, primary construct, or mmRNA. This modified nucleotide is added post-transcriptionally using terminal transferases (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. After the addition of the 3'end-modified nucleotide, these two polynucleotides, the primary construct, or the mMr species are combined in aqueous solution in the presence or absence of copper and via the click chemistry described in the literature. New covalent bonds can be formed.

In another example, three or more polynucleotides can be attached using a functionalized linker molecule. For example, functionalized saccharide molecule, a plurality of chemically reactive group _(SH-, NH 2 -, _{N 3,} etc.) cognate moiety on containing 3'functionalized mRNA molecule (i.e., 3'-maleimide ester, 3 It can be chemically modified to react with'-NHS-ester, alkynyl). The number of reactive groups on this modified saccharide can be controlled in a stoichiometric manner to directly control the stoichiometric ratio of the complexed polynucleotide, primary construct, or mmRNA.

To further enhance mmRNA complex and combination protein production, the primary constructs or mmRNAs of the invention are other polypeptides, dyes, inserts (eg, acridin), cross-linking agents (eg, psolarene, mitomycin C), porphyrin. (TPPC4, texaphyllin, sapphirine), polycyclic aromatic hydrocarbons (eg, phenazine, dihydrophenazine), artificial endonucleases (eg, EDTA), alkylating agents, phosphates, amino, mercapto, PEG (eg, PEG- 40K), MPEG, [MPEG] ₂ , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (eg biotin), transport / absorption enhancers (eg aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins , For example, glycoproteins, or peptides, for example, molecules or antibodies that have specific affinity for coligants, for example, antibodies, hormones and hormone receptors that bind to specific cell types such as cancer cells, endothelial cells, or bone cells. It can be designed to be complexed with the body, non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or drugs.

Complexization can result in increased stability and / or half-life and can be particularly useful when targeting polynucleotides, primary constructs, or mmRNAs to specific sites in cells, tissues, or organisms. ..

According to the present invention, the mmRNA or primary construct is among RNAi agents, siRNA, shRNA, miRNA, miRNA binding sites, antisense RNA, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors and the like. It can be administered with one or more, or these can be further encoded.

Bifunctional mmRNA
One embodiment of the invention is a bifunctional polynucleotide (eg, a bifunctional primary construct or a bifunctional mmRNA). As the name implies, a bifunctional polynucleotide is a polynucleotide that has at least two functions or is capable of at least two functions. By convention, these molecules can also be referred to as multifunctional.

Multiple functions of a bifunctional polynucleotide can be encoded by RNA (this function cannot appear until the encoded product is translated) or can be a property of the polynucleotide itself. It can be structural or chemical. Bifunctional modified polynucleotides may include functions that are covalently or electrostatically associated with the polynucleotide. In addition, these two functions can be provided in the context of mmRNA and complex of another molecule.

The bifunctional polynucleotide may encode an antiproliferative peptide. These peptides can be linear, cyclic, constrained, or random coils. They can function as aptamers, signaling molecules, ligands, or mimics or mimetics thereof. Antiproliferative peptides can be 3 to 50 amino acids long when translated. They can be 5-40, 10-30, or about 15 amino acids long. They can be single-stranded, multi-stranded, or branched and, when translated, can form complexes, aggregates, or any multi-unit structure.

Non-coding polynucleotides and primary constructs As described herein, polynucleotides and primary constructs with partially or substantially non-translatable sequences, such as non-coding regions, are provided. Such a non-coding region can be the "first region" of the primary construct. Alternatively, the non-coding region can be an region other than the first region. Such molecules are usually untranslated, but affect protein production by one or more of binding and sequestration to one or more translational machinery components such as ribosomal proteins or transfer RNAs (tRNAs), thereby thereby affecting protein production. It can effectively reduce protein expression in cells or regulate one or more pathways or cascades in cells, which in turn alter protein levels. Polynucleotides or primary constructs can be one or more long non-coding RNAs (lncRNA or lincRNA) or portions thereof, small interfering RNAs (sno-RNAs), microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi. Interfering RNAs (piRNAs) can be contained or encoded.

Polypeptides of Interest According to the present invention, primary constructs are designed to encode one or more polypeptides of interest or fragments thereof. The polypeptide of interest may include, but is not limited to, the entire polypeptide, multiple polypeptides, or fragments of the polypeptide, which may independently include one or more nucleic acids, multiple nucleic acids, nucleic acids. Can be encoded by a fragment of, or a variant of any of the above. As used herein, the term "polypeptide of interest" refers to any polypeptide selected and encoded in the primary construct of the invention. As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) that are most often attached by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of arbitrary size, structure, or function. In some examples, if the encoded polypeptide is less than about 50 amino acids, then the polypeptide is referred to as a peptide. If the polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residue lengths. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologs, paralogs, the aforementioned fragments and other equivalents, variants, and analogs. The polypeptide can be a single molecule or a multimolecular complex such as a dimer, trimer, or tetramer. They can also include single-stranded or multi-stranded polypeptides such as antibodies or insulin and can be associated or bound. In most cases, disulfide bonds are found in multi-stranded polypeptides. The term "polypeptide" can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from its native or reference sequence. Amino acid sequence variants can have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence as compared to the native or reference sequences. Usually, the variants have at least about 50% identity (homology) with the native or reference sequence, preferably they are at least about 80%, more preferably at least about about, with the native or reference sequence. It is 90% identical (homologous).

In some embodiments, "mutant mimics" are provided. As used herein, the term "mutant mimetic" is one that contains one or more amino acids that mimic the activation sequence. For example, glutamate can act as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, the variant mimetic can result in the inactivation or inactivation of the product containing the mimic, for example, phenylalanine can act as an inactivating substitution for tyrosine, or alanine can be serine. Can serve as an inactivating substitution for.

"Homogeneity", when applied to an amino acid sequence, is with the residues in the amino acid sequence of the second sequence after aligning the sequences and introducing gaps as needed to obtain the maximum percentage of homology. It is defined as the percentage of residues within the same candidate amino acid sequence. Methods and computer programs for alignment are well known in the art. It is understood that homology depends on the calculation of percent identity, but the values can vary due to the gaps and penalties introduced in the calculation.

By "homology", when applied to a polypeptide sequence, it means the corresponding sequence of another species that has a significant proportion of identity to the second sequence of the second species.

An "similar" is a polypeptide variant that differs by one or more amino acid modifications, eg, substitutions, additions, or deletions of amino acid residues that still retain one or more of the properties of the parent or starting polypeptide. Is intended to include.

The present invention contemplates several types of compositions of polypeptide systems, including variants and derivatives. These include substitutions, insertions, deletions, and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant" but generally refers to a molecule that has been modified and / or altered with respect to a reference or starting molecule in any way.

Thus, an mRNA encoding a polypeptide containing a reference sequence, specifically a polypeptide containing substitutions, insertions and / or additions, deletions, and covalent modifications to the polypeptide sequences disclosed herein, is described in the present invention. Is included in the range of. For example, a sequence tag or amino acid, such as one or more lysines, can be added to the peptide sequence of the invention (eg, at the N-terminus or C-terminus). Sequence tags can be used for peptide purification or localization. Lysine is used to increase peptide solubility or allow biotinylation. Alternatively, the carboxy and amino acid residues located in the amino-terminal region of the amino acid sequence of the peptide or protein may be optionally deleted to provide a cleavage sequence. Alternatively, certain amino acids (eg, C-terminal or N-terminal residues) can be deleted upon expression of the sequence, eg, as part of a larger soluble sequence, depending on the use of the sequence, or solid support. Can be bound to the body.

When referring to a polypeptide, a "substitution variant" is one in which at least one amino acid residue in the native or starting sequence has been removed and different amino acids have been inserted at the same location. These substitutions are single substitutions and can be one in which only one amino acid in the molecule is substituted, or these substitutions are multiple substitutions and two or more amino acids in the same molecule are substituted. It can be a replacement.

As used herein, the term "conserved amino acid substitution" refers to replacing an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include substitution of non-polar (hydrophobic) residues such as isoleucine, valine, and leucine with other non-polar residues. Similarly, examples of conservative substitutions include substitution of polar (hydrophilic) residues such as arginine with lysine, glutamine with asparagine, and glycine with serine. In addition, replacement of a basic residue such as lysine, arginine, or histidine with another basic residue, or replacement of one acidic residue such as aspartic acid or glutamic acid with another acidic residue is a conservative substitution. A further example. Examples of non-conservative substitutions include substitution of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, and methionine with polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, or lysine. , And / or substitution of polar residues with non-polar residues.

An "insertion variant" is one in which one or more amino acids are inserted immediately adjacent to an amino acid at a particular position in the native or starting sequence when referring to a polypeptide. By "immediately adjacent" to an amino acid is meant linked to either the α-carboxy or α-amino functional group of the amino acid.

A "deletion variant" is one in which one or more amino acids in the natural or starting amino acid sequence have been removed when referring to a polypeptide. Deletion variants usually delete one or more amino acids in a particular region of the molecule.

"Covalent derivatives" include, when referring to polypeptides, modifications of natural or starting proteins with organic proteinaceous or non-proteinaceous derivatizing agents, and / or post-translational modifications. Covalent modification has traditionally worked by reacting a protein's target amino acid residue with an organic derivatizing agent capable of reacting with selected side chains or terminal residues, or in selected recombinant host cells. It is introduced by utilizing the mechanism of post-translational modification. The resulting covalent derivatives are useful in antiprotein antibody biological activity for immunoaffinity purification of recombinant glycoproteins, immunological assays, or programs aimed at identifying residues important for preparation. Is. Such modifications are within the skill of the art and are carried out without undue experimentation.

Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues after translation. Alternatively, these residues are deamidated under weakly acidic conditions. Any form of these residues may also be present in the polypeptide produced according to the present invention.

Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of ceryl or threonyl residues, and methylation of the α-amino group of the lysine, arginine, and histidine side chains (TE Protein). , Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)).

A "characteristic" is defined as a component based on a distinctly different amino acid sequence of a molecule when referring to a polypeptide. Features of the polypeptides encoded by the mmRNAs of the invention include surface appearance, local conformational shape, folding, loops, half-loops, domains, half-domains, sites, ends, or any combination thereof.

As used herein, when referring to polypeptides, the term "surface appearance" refers to the appearance of the polypeptide-based components of a protein on the outermost surface.

As used herein, when referring to a polypeptide, the term "local conformational shape" means the emergence of a polypeptide-based structure of a protein located within a definable space of the protein.

As used herein, when referring to a polypeptide, the term "folding" refers to the conformation that results from the amino acid sequence during energy minimization. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary level folding include β-sheets and α-helices. Examples of tertiary level folding include domains and regions formed due to the aggregation or separation of energy forces. The region thus formed includes hydrophobic and hydrophilic pockets and the like.

As used herein, the term "rotation" refers to a bend that alters the skeleton of a peptide or polypeptide when it comes to protein structure, and may involve one, two, or three or more amino acid residues. ..

As used herein, when referring to a polypeptide, the term "loop" refers to a structural feature of a polypeptide that can serve to reverse the orientation of the peptide or the skeleton of the polypeptide. If the loop is found in the polypeptide and changes only the orientation of the skeleton, it may contain 4 or more amino acid residues. Oliva et al. Have identified at least five classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997). The loop can be an open loop or a closed loop. A closed or "cyclic" loop may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids between the crosslinked moieties. Such cross-linked moieties may include cysteine-cysteine cross-links (Cys-Cys) typical of polypeptides having disulfide bridges, or the cross-linked moieties may be dibromozylyl agents as used herein. ) Etc. can be non-protein type.

As used herein, when referring to a polypeptide, the term "half-loop" refers to the portion of the loop that has at least half of the amino acid residues identified as the loop from which it is derived. It is understood that the loop does not necessarily contain an even number of amino acid residues. Therefore, where a loop contains an odd number of amino acids or is identified as containing an odd number of amino acids, the half-loop of the odd loop is the integer part or the next integer part of the loop (number of amino acids in the loop / 2+). / -0.5 amino acids). For example, a loop identified as a 7 amino acid loop can result in a half loop of 3 amino acids or 4 amino acids (7/2 = 3.5 +/- 0.5 becomes 3 or 4).

As used herein, when referring to a polypeptide, the term "domain" refers to one or more identifiable structural or functional features or properties (eg, binding ability, role as a site of protein-protein interaction). Refers to the motif of a polypeptide having).

As used herein, when referring to a polypeptide, the term "half domain" means the portion of the domain that has at least half of the amino acid residues identified as the domain from which it is derived. It is understood that the domain does not necessarily contain an even number of amino acid residues. Therefore, where a domain contains an odd number of amino acids or is identified as containing an odd number of amino acids, the half domain of the odd domain is the integer part or the next integer part of the domain (number of amino acids in the domain / 2+). / -0.5 amino acids). For example, a domain identified as a 7 amino acid domain can result in a half domain of 3 amino acids or 4 amino acids (7/2 = 3.5 +/- 0.5 becomes 3 or 4). Subdomains can be identified within a domain or semi-domain, and these subdomains may have structural or functional characteristics that are less than all of the structural or functional characteristics identified in the domain or semi-domain from which they originate. Understood. Amino acids, including any of the domain types herein, need not be adjacent along the backbone of the polypeptide (ie, non-adjacent amino acids are structurally folded into domains, semi-domains, or subdomains. It is also understood that it can bring about).

As used herein, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when referring to embodiments of an amino acid system. The site represents a position within the peptide or polypeptide that can be modified, manipulated, modified, derivatized, or altered within the polypeptide-based molecule of the invention.

As used herein, when referring to a polypeptide, the term "termini" or "terminus" refers to the end of a peptide or polypeptide. Such ends are not only limited to the first or final site of the peptide or polypeptide, but may also contain additional amino acids in the terminal region. The polypeptide-based molecule of the present invention may be characterized by having both an N-terminal (terminated by an amino acid at a free amino group (NH2)) and a C-terminal (terminated by an amino acid at a free carboxyl group (COOH)). In some cases, the proteins of the invention can consist of multiple polypeptide chains linked by disulfide bonds or non-covalent bonds (multimers, oligomers). These types of proteins have multiple N-terminus and C-terminus. Alternatively, the ends of the polypeptides can be modified such that they optionally begin or end with a non-polypeptide moiety such as an organic complex.

Of some manipulations and / or modifications of these features, if any of these features is identified or defined as the desired component of the primary construct of the invention or the polypeptide encoded by the mmRNA. Any of these can be done by transfer, exchange, inversion, deletion, randomization, or replication. Furthermore, it is understood that manipulation of features yields the same results as modifications to the molecules of the invention. For example, an operation involving a domain deletion results in a modification of the length of the molecule, such as a modification of a nucleic acid encoding a less than full-length molecule.

Modifications and manipulations can be accomplished by methods known in the art, such as, but not limited to, site-directed mutagenesis. The resulting modified molecule can then be tested for activity using an in vitro or in vivo assay such as the assay described herein, or any other suitable screening assay known in the art.

According to the present invention, the polypeptide may contain a consensus sequence found by a series of experiments. As used herein, a "consensus" sequence is a single sequence that represents a set of sequences that allow variability at one or more sites.

As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the polypeptide of interest of the present invention. For example, any protein fragment of a reference protein having an amino acid length greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 100 (at least one amino acid residue is from the reference polypeptide sequence). Also short, but otherwise identical (meaning a polypeptide sequence) is provided herein. In another example, it is about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein. Any protein containing a series of about 20, about 30, about 40, about 50, or about 100 amino acids can be utilized in accordance with the present invention. In certain embodiments, the polypeptides utilized in accordance with the present invention are 2, 3, 4, 5, 6, 7, 8, 9 shown in any of the sequences provided or referenced herein. Includes 10 or more mutations.

Encoded Polypeptides The primary constructs or mmss of the invention are biologics, antibodies, vaccines, therapeutic proteins or peptides, cell-penetrating peptides, secretory proteins, progenitor membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane binding. These include proteins, nuclear proteins, proteins associated with human disease, target moieties, or proteins encoded by the human genome that have utility in the field of research and discovery, even though no therapeutic indicators have been identified. It can be designed to encode the polypeptide of interest selected from any of several target categories, not limited to.

In one embodiment, the primary construct or mmRNA may encode a variant polypeptide having certain identity with the reference polypeptide sequence. As used herein, "reference polypeptide sequence" refers to the starting polypeptide sequence. The reference sequence can be a wild-type sequence or any sequence referenced in the design of another sequence. The "reference polypeptide sequence" is, for example, any one of SEQ ID NOs: 769 to 1392 disclosed herein, eg, SEQ ID NOs: 769, 770, 771, 772, 773, 774, 775, 767, 777. , 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801 and 802. , 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 827, 827 , 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852. , 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877. , 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902 , 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927. , 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952. , 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977. , 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002 , 1003 , 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1101, 1101, 1103 , 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128. , 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153. , 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178. , 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203. , 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228 , 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253. , 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278. , 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303 , 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328 , 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353. , 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1376, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, It can be any of 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392.

The term "identity" known in the art refers to the relationship between the sequences of two or more peptides determined by comparing the sequences. In the art, identity also means the degree of sequence association between peptides as determined by the number of matches between strings of two or more amino acid residues. Identity is the percentage of identical matches between smaller sequences of two or more sequences that have gap alignments (if any) that are processed by a particular mathematical model or computer program (ie, an "algorithm"). %) Is measured. The identity of the relevant peptides can be easily calculated by known methods. Such methods include Computational Molecular Biology, Lesk, A. et al. M. , Ed. , Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, D.M. W. , Ed. , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part 1, Griffin, A. et al. M. , And Griffin, H. et al. G. , Eds. , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von
Heinje, G.M. , Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. et al. and Deverex, J. et al. , Eds. , M. Stockton Press, New York, 1991, and Carillo et al. , SIAM J. Applied Math. 48, 1073 (1988), but is not limited to.

In some embodiments, the polypeptide variant may have the same or similar activity as the reference polypeptide. Alternatively, the variant may have altered (eg, increased or decreased) activity compared to the reference polypeptide. In general, variants of a particular polynucleotide or polypeptide of the invention will be determined by a sequence alignment program and parameters described herein and known to those of skill in the art. And at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%. , 95%, 96%, 97%, 98%, 99%, but with less than 100% sequence identity. Such alignment tools include a complete BLAST program (Stephen).
F. Altschul, Thomas L. et al. Madden, Alejandro A. Sch 艪 ・ ・ ・ Chi CJinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database programs", Nucleic Acids Res. 25: 3389-3402). Other tools are described herein, specifically in the definition of "identity".

The initialization parameters of the BLAST algorithm include, for example, a prediction threshold of 10, a word size of 28, a match / mismatch score of 1, -2, and a linear gap cost. Any filter, as well as selection of species-specific repeat sequences, such as human-specific repeat sequences, may be applied.

Bioforms The polynucleotides, primary constructs, or mmRNAs disclosed herein may encode one or more bioforms. As used herein, a "biologic" is used to treat, cure, alleviate, prevent, or diagnose a serious or life-threatening disease or condition produced by the methods provided herein. It is a possible polypeptide-based molecule. Biologics include allergen extracts (eg, for allergic injections and tests), blood components, genetic therapeutic products, human tissue or cell products used for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, according to the invention. , Thrombolytic agents, immunomodulators, and the like, but are not limited to these.

According to the present invention, one or more bioforms currently on the market or under development may be encoded by the polynucleotides, primary constructs, or mmRNAs of the present invention. We do not want to be bound by theory, but at least in part, due to specificity, purity, and / or construct design selectivity, to the primary construct or mMV of the invention of a polynucleotide encoding a known bioform. It is considered that the incorporation of Nucleotides brings about an improvement in the therapeutic effect.

Antibodies The primary constructs or mmRNAs disclosed herein may encode one or more antibodies or fragments thereof. The term "antibody" refers to monoclonal antibodies (including full-length antibodies having immunoglobulin Fc regions), antibody compositions with polyepitogenic specificity, multispecific antibodies (eg, bispecific antibodies, bispecific antibodies (eg, bispecific antibodies, bispecific antibodies). Diabody), and single-stranded molecules), as well as antibody fragments. The term "immunoglobulin (Ig)" is used synonymously herein with "antibody." As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., naturally occurring mutations and / or post-translational modifications that may be present in small amounts. Refers to individual antibodies containing that population that are identical except (eg, isomerization, amidation). Monoclonal antibodies are highly specific and are directed against a single antigenic site.

Monoclonal antibodies herein are antibodies in which the heavy chain and / or part of the light chain is derived from a particular species or belongs to a particular antibody class or subclass, as long as they exhibit the desired biological activity. The rest of the chain (s) are identical or homologous to the corresponding sequence of an antibody that is derived from another species or belongs to another antibody class or subclass, while being identical or homologous to the corresponding sequence of. Includes "chimeric" antibodies (immunoglobulins) that are, as well as fragments of such antibodies. Chimeric antibodies of interest herein include "primate" antibodies that include variable domain antigen binding sequences and human constant region sequences from non-human primates (eg, Old World monkeys, apes, etc.). , Not limited to this.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F (ab') ₂ , and Fv fragments; bispecific antibodies; linear antibodies; Nanobodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Can be mentioned.

One of five classes of immunoglobulin, IgA, IgD, IgE, IgG, and IgM, each containing heavy chains designated α, δ, ε, γ, and μ, is encoded by the mMV of the present invention. Can be done. Also included are polynucleotide sequences encoding subclasses, γ and μ. Thus, any of the following subclasses: Subclasses of antibodies, including IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, may be partially or wholly encoded.

One or more antibodies or fragments currently on the market or under development in accordance with the present invention may be encoded by the polynucleotides, primary constructs, or mmRNAs of the present invention. Although not bound by theory, it is believed that incorporation into the primary construct of the invention will result in improved therapeutic efficacy, at least in part, due to specificity, purity, and selectivity of mmRNA design.

Utilizing the antibodies encoded by the polynucleotides, primary constructs, or mMVs of the invention, blood, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, tumors. It can treat conditions or diseases in many therapeutic areas, such as, but not limited to, science, immunology, respiratory, sensory, and anti-infection.

In one embodiment, the primary construct or mMV disclosed herein can encode a monoclonal antibody and / or a variant thereof. Antibody variants can also include, but are not limited to, substitution variants, conserved amino acid substitutions, insertion variants, deletion variants, and / or covalent derivatives. In one embodiment, the primary construct and / or mmRNA disclosed herein can encode an immunoglobulin Fc region. In another embodiment, the primary construct and / or mRNA may encode a mutant immunoglobulin Fc region. As a non-limiting example, the primary construct and / or mRNA encodes an antibody having the mutant immunoglobulin Fc region described in US Pat. No. 8,217,147, which is incorporated herein by reference in its entirety. obtain.

Vaccines The primary constructs or mmRNAs disclosed herein can encode one or more vaccines. As used herein, a "vaccine" is a biological preparation that enhances immunity to a particular disease or infectious agent. One or more vaccines currently on the market or under development in accordance with the present invention may be encoded by the polynucleotides, primary constructs, or mMVs of the present invention. Although not bound by theory, it is believed that integration into the primary construct or mmRNA of the invention results in improved therapeutic efficacy, at least in part, due to specificity, purity, and construct design selectivity.

Utilizing the vaccines encoded by the polynucleotides, primary constructs, or mmRNAs of the invention, such as cardiovascular, CNS, dermatology, endocrinology, oncology, immunology, respiratory, and anti-infection. It can treat conditions or diseases in many therapeutic areas, not limited to.

Therapeutic Proteins or Peptides The primary constructs or mMVs disclosed herein may encode one or more effective or "test" Therapeutic proteins or peptides.

According to the invention, one or more therapeutic proteins or peptides currently on the market or under development may be encoded by the polynucleotides, primary constructs, or mmRNAs of the invention. Although not bound by theory, it is believed that integration into the primary construct or mmRNA of the invention results in improved therapeutic efficacy, at least in part, due to specificity, purity, and construct design selectivity.

Utilizing the therapeutic proteins and peptides encoded by the polynucleotides, primary constructs, or mmRNAs of the invention, blood, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, genetics, urogenital organs , Gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory, and anti-infection, and can treat conditions or diseases in many therapeutic areas, including but not limited to these.

Cell-Permeable Polypeptides The primary constructs or mmss disclosed herein can encode one or more cell-permeable polypeptides. As used herein, "cell-penetrating polypeptide" or CPP refers to a polypeptide that can promote cellular uptake of a molecule. The cell permeable polypeptides of the invention may contain one or more detectable labels. The polypeptide can be partially labeled or fully labeled throughout. The polynucleotide, primary construct, or mmRNA can fully, partially, or not encode the detectable label. The cell-permeable peptide may also include a signal sequence. As used herein, "signal sequence" refers to the sequence of amino acid residues that are attached to the amino terminus of a nascent protein during protein translation. The signal sequence can be used to signal the secretion of a cell-permeable polypeptide.

In one embodiment, the polynucleotide, primary construct, or mmRNA may also encode a fusion protein. Fusion proteins can be created by operably binding charged proteins to therapeutic proteins. As used herein, "operably binds" means that the therapeutic and charged proteins are bound in such a way as to allow expression of the complex when introduced into the cell. Point to. As used herein, "charged protein" refers to a protein that has a positive charge, a negative charge, or an overall neutral charge. Preferably, the therapeutic protein can be covalently attached to the charged protein during the formation of the fusion protein. The ratio of surface charge to total or surface amino acids is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9. could be.

Cell-permeable polypeptides encoded by polynucleotides, primary constructs, or mmRNAs can form complexes after translation. This complex may comprise a charged protein that is bound, eg, covalently linked, to a cell-permeable polypeptide. "Therapeutic protein" refers to a protein that, when administered to cells, has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect.

In one embodiment, the cell permeable polypeptide may include a first domain and a second domain. The first domain may include a hypercharged polypeptide. The second domain may include protein binding partners. As used herein, "protein binding partners" include, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. Cell-permeable polypeptides may further include intracellular binding partners of protein binding partners. Cell-permeable polypeptides can be secreted from cells into which polynucleotides, primary constructs, or mmRNA can be introduced. The cell-permeable polypeptide can also penetrate the first cell.

In a further embodiment, the cell permeable polypeptide is capable of penetrating a second cell. The second cell can be derived from the same region as the first cell, or can be derived from a different region. This area may include, but is not limited to, tissues and organs. The second cell can be proximal to or distal to the first cell.

In one embodiment, the polynucleotide, primary construct, or mmRNA may encode a cell-permeable polypeptide that may include a protein binding partner. Protein binding partners can include, but are not limited to, antibodies, hypercharged antibodies, or functional fragments. A polynucleotide, primary construct, or mmRNA can be introduced into a cell into which a cell-permeable polypeptide containing a protein binding partner is introduced.

Secretory proteins Humans and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane-bound compartment, or organelle, contains a different protein that is essential for organelle function. The cells use "selection signals," which are amino acid motifs located within the protein, to target the protein to specific organelles.

A type of sorting signal, called a signal sequence, signal peptide, or leader sequence, directs a class of proteins to organelles called the endoplasmic reticulum (ER).

Proteins targeted to the ER by the signal sequence can be released into the extracellular space as secretory proteins. Similarly, proteins present on the cell membrane can be secreted into the extracellular space by proteolytic cleavage of the "linker" that holds the protein on the membrane. Although not bound by theory, the molecules of the invention can be used to take advantage of the cell transport described above. Therefore, in some embodiments of the invention, polynucleotides, primary constructs, or mmRNAs that express secretory proteins are provided. Secreted proteins may be selected from those described herein or in US Patent Publication No. 2010255574, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, they can be used in the production of large amounts of beneficial human gene products.

Protoplasmic Membrane Protein In some embodiments of the invention, polynucleotides, primary constructs, or mmRNAs that express the plasma membrane protein are provided.

Cytoplasmic or Cytoskeletal Protein In some embodiments of the invention, a polynucleotide, primary construct, or mMr expressing the cytoplasmic or cytoskeletal protein is provided.

Intramembrane Membrane Binding Proteins In some embodiments of the invention, polynucleotides, primary constructs, or mMVs that express an intracellular membrane binding protein are provided.

Nucleoproteins In some embodiments of the invention, polynucleotides, primary constructs, or mmRNAs that express nucleoproteins are provided.

Proteins Related to Human Diseases In some embodiments of the invention, polynucleotides, primary constructs, or mRNAs that express proteins associated with human disease are provided.

Various Proteins In some embodiments of the invention, polynucleotides, primary constructs, or mmRNAs that express proteins with currently unknown therapeutic functions are provided.

Target Partitions In some embodiments of the invention, polynucleotides, primary constructs, or mmRNAs that express the target moiety are provided. These include protein binding partners or receptors on the cell's surface that act to target or interact with specific tissue spaces, either in vivo or in vitro. Suitable protein binding partners include, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. In addition, polynucleotides, primary constructs, or mmRNAs can be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological parts or biomolecules.

Polypeptide Library In one embodiment, polynucleotides, primary constructs, or mmRNAs can be used to generate polypeptide libraries. Each of these libraries can result from the production of populations of polynucleotides, primary constructs, or mmRNAs with different structural or chemically modified designs. In this embodiment, a population of polynucleotides, primary constructs, or mMVs is an antibody or antibody fragment, protein binding partner, scaffold protein, and other polypeptide as taught herein or known in the art. Can include a plurality of encoded polypeptides including, but not limited to. In a preferred embodiment, the polynucleotide is the primary construct of the invention comprising mmRNA that may be suitable for direct introduction into a target cell or culture capable of subsequently synthesizing the encoded polypeptide.

In certain embodiments, multiple variants of a protein, each with a different amino acid modification (s), are biophysical such as pharmacokinetics, stability, biocompatibility, and / or biological activity, or expression level. It can be produced and tested to determine the best variant in terms of biophysical properties. Such ^libraries 10, 10 ^{2, 10 3,} 10 ^4, 10 ^5, 10 6, 10 ^7, 10 8, 10 ^{9-substituted} or 10 ⁹ capable than such variants (1 or more residues, , Deletions, and insertions of one or more residues, but not limited to).

Antibacterial and Antiviral Polypeptides The polynucleotides, primary constructs, and mmRNAs of the invention can be designed to encode one or more antibacterial peptides (AMPs) or antiviral peptides (AVPs). AMP and AVP have been isolated and described from a variety of animals including, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals (Wang et al., Nucleic). Acids Res. 2009; 37 (published in the database): D933-7). For example, an antibacterial polypeptide is an antibacterial peptide database (http://apps.unmc.edu/AP/main.php; Wang et al., Nucleic Acids Res. 2009; 37 (database publication): D933-7). , CAMP: Collection of Anti-Microbial Peptides (http://www.vicnirrh.res.in/antimicrobial/); Thomas et al. , Nucleic Acids Res. 2010; 38 (database publication): D774-80), US Pat. Nos. US5221732, US54479114, US5519115, US5607914, US5714577, US5734015, US5798336, US Pat. US582124, US58249490, US585612, US5905187, US5994308, US5993774, US6107460, US6191254, US621148, US6300489, US6300489. US 6329504, US 6399370, US 6476189, US 6478825, US 6492328, US 6514701, US 6573361, US 6573361, US 6576755, US 6605698, US 6605698. US662140, US6668531, US664203, US6653280, US666238, US6727066, US6730659, US67435898, US6743769, US674007, and US674007. US67990833, US6794490, US6818407, US6835536, US6835713, US6838435, US678275, US6875907, US6884776, US68878747, US6906035, US6911524, US6936432, US700124, US7071293, US707380, US7091185, US7094759, US7166769, US7244710, US7314858 and US7582301 have the same, the contents of which are incorporated by reference in their entirety.

The antibacterial polypeptides described herein can block cell fusion and / or viral entry by one or more enveloped viruses (eg, HIV, HCV). For example, the antimicrobial polypeptide is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 of a transmembrane subunit of a region, eg, a viral envelope protein, eg, HIV-1 gp120 or gp41. , 50, 55, or 60 amino acids can contain or consist of synthetic peptides corresponding to the sequence. The amino acid and nucleotide sequences of HIV-1 gp120 or gp41 are described, for example, in Kuiken et al. , (2008) "HIV Sequence Compendium," Los Alamos National Laboratory.

In some embodiments, the antibacterial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In some embodiments, the antibacterial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein sequence.

In other embodiments, the antimicrobial polypeptide comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 of a region, eg, the binding domain of a capsid-binding protein. It can contain or consist of synthetic peptides that correspond to a contiguous sequence of individual amino acids. In some embodiments, the antibacterial polypeptide has at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding sequence of the capsid binding protein. obtain.

The antibacterial polypeptides described herein block protease dimerization and inhibit the cleavage of viral proproteins (eg, HIV Envelope treatment) into functional proteins, thereby one or more. Can block the release of enveloped viruses (eg, HIV, HCV). In some embodiments, the antibacterial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence.

In other embodiments, the antimicrobial polypeptide comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 of a region, eg, the binding domain of a protease binding protein. It can contain or consist of synthetic peptides that correspond to a contiguous sequence of individual amino acids. In some embodiments, the antibacterial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of the protease binding protein. ..

The antibacterial polypeptides described herein can include in vitro evolved polypeptides directed against viral pathogens.

Antibacterial Polypeptides Antibacterial polypeptides (AMPs) are variable with broad-spectrum activity against a variety of microorganisms, including but not limited to bacteria, viruses, fungi, protozoans, parasites, prions, and tumor / cancer cells. Small peptides of length, variable sequence, and variable structure (see, eg, Zaio, J Mol Med, 2007; 85: 317, which are incorporated herein by reference in their entirety). It has been shown that AMP has a wide range of rapidly developing killing activity and may have low levels of induced resistance and a wide range of concomitant anti-inflammatory effects.

In some embodiments, the antibacterial polypeptide (eg, antibacterial polypeptide) can be less than 10 kDa, such as less than 8 kDa, 6 kDa, 4 kDa, 2 kDa, or less than 1 kDa. In some embodiments, an antibacterial polypeptide (eg, an antibacterial polypeptide) is about 6 to about 100 amino acids, such as about 6 to about 75 amino acids, about 6 to about 50 amino acids. Consists of about 6 to about 25 amino acids, about 25 to about 100 amino acids, about 50 to about 100 amino acids, or about 75 to about 100 amino acids. In certain embodiments, an antibacterial polypeptide (eg, an antibacterial polypeptide) can consist of about 15 to about 45 amino acids. In some embodiments, the antibacterial polypeptide (eg, antibacterial polypeptide) is substantially cationic.

In some embodiments, the antibacterial polypeptide (eg, an antibacterial polypeptide) can be substantially amphipathic. In certain embodiments, the antibacterial polypeptide (eg, an antibacterial polypeptide) can be substantially cationic and amphipathic. In some embodiments, the antibacterial polypeptide (eg, an antibacterial polypeptide) can be cell proliferation inhibitory against Gram-positive bacteria. In some embodiments, the antibacterial polypeptide (eg, antibacterial polypeptide) can be cytotoxic to Gram-positive bacteria. In some embodiments, the antibacterial polypeptide (eg, an antibacterial polypeptide) can be cell proliferation inhibitory and cytotoxic to Gram-positive bacteria. In some embodiments, the antibacterial polypeptide (eg, antibacterial polypeptide) can be cell proliferation inhibitory against Gram-negative bacteria. In some embodiments, the antibacterial polypeptide (eg, antibacterial polypeptide) can be cytotoxic to Gram-negative bacteria. In some embodiments, the antibacterial polypeptide (eg, an antibacterial polypeptide) can be cell proliferation inhibitory and cytotoxic to Gram-positive bacteria. In some embodiments, the antimicrobial polypeptide can be cell proliferation inhibitory against viruses, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antibacterial polypeptide can be cytotoxic to viruses, fungi, protozoa, parasites, prions, or combinations thereof. In certain embodiments, the antibacterial polypeptide can be cell proliferation inhibitory and cytotoxic to viruses, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antibacterial polypeptide can be cytotoxic to a tumor or cancer cell (eg, human tumor and / or cancer cell). In some embodiments, the antibacterial polypeptide can be cell proliferation inhibitory to tumors or cancer cells (eg, human tumors and / or cancer cells). In certain embodiments, the antibacterial polypeptide can be cytotoxic and antiproliferative to tumor or cancer cells (eg, human tumor or cancer cells). In some embodiments, the antibacterial polypeptide (eg, an antibacterial polypeptide) can be a secretory polypeptide.

In some embodiments, the antibacterial polypeptide comprises or consists of a defensin. Examples of defensins include α-defensins (eg, neutrophil defensins 1, defensins α1, neutrophil defensins 3, neutrophil defensins 4, defensins 5, defensins 6), β-defensins (eg, β-defensins 1). , Β-Defensin 2, β-Defensin 103, β-Defensin 107, β-Defensin 110, β-Defensin 136), and θ-Defensin, but is not limited thereto. In other embodiments, the antibacterial polypeptide comprises or consists of cathelicidin (eg, hCAP18).

Antiviral Polypeptides Antiviral polypeptides (AVPs) are small peptides of variable length, variable sequence, and variable structure that have broad-spectrum activity against a variety of viruses. For example, Zaio, J Mol
See Med, 2007; 85: 317. It has been shown that AVP has a wide range of rapidly occurring killing activity and may have low levels of induced resistance and a wide range of concomitant anti-inflammatory effects. In some embodiments, the antiviral polypeptide is less than 10 kDa, eg, less than 8 kDa, 6 kDa, 4 kDa, 2 kDa, or less than 1 kDa. In some embodiments, the antiviral polypeptide comprises about 6 to about 100 amino acids, such as about 6 to about 75 amino acids, about 6 to about 50 amino acids, and about 6 to about 25 amino acids. Contains or consists of amino acids, about 25 to about 100 amino acids, about 50 to about 100 amino acids, or about 75 to about 100 amino acids. In certain embodiments, the antiviral polypeptide comprises or comprises from about 15 to about 45 amino acids. In some embodiments, the antiviral polypeptide is substantially cationic. In some embodiments, the antiviral polypeptide is substantially amphipathic. In certain embodiments, the antiviral polypeptide is substantially cationic and amphipathic. In some embodiments, the antiviral polypeptide is cell proliferation inhibitory to the virus. In some embodiments, the antiviral polypeptide is cytotoxic to the virus. In some embodiments, the antiviral polypeptide is cell proliferation inhibitory and cytotoxic to the virus. In some embodiments, the antiviral polypeptide is cell proliferation inhibitory against bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic to bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In certain embodiments, the antiviral polypeptide is cell proliferation inhibitory and cytotoxic to bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic to a tumor or cancer cell (eg, human cancer cell). In some embodiments, the antiviral polypeptide is cell proliferation inhibitory to tumor or cancer cells (eg, human cancer cells). In certain embodiments, the antiviral polypeptide is cytotoxic to a tumor or cancer cell (eg, human cancer cell) and antiproliferative against. In some embodiments, the antiviral polypeptide is a secretory polypeptide.

Cytotoxic Nucleoside In one embodiment, the polynucleotide, primary construct, or mmRNA of the invention may incorporate one or more cytotoxic nucleosides. For example, a cytotoxic nucleoside can be integrated into a polynucleotide, primary construct, or mRNA, such as a bifunctional modified RNA or mRNA. Cytotoxic nucleoside anticancer agents include adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxiuridine, FTORAFUR® (combination of tegafur and uracil), tegafur ((RS) -5-fluoro). -1, but not limited to -1- (tetra-2-yl) pyrimidine-2,4 (1H, 3H) -dione), and 6-mercaptopurine.

Some cytotoxic nucleoside analogs have been used clinically or have been the subject of clinical trials as anticancer agents. Examples of such analogs include cytorabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methyridenecytodin (DMDC), cladribine, clofarabine, 5-azacitidine, 4'-thio-aracytidine, Cyclopentenylcytosine and 1- (2-C-cyano-2-deoxy-β-D-arabino-pentoflanosyl) -cytosine are included, but not limited to. Another example of such a compound is fludarabine phosphate. These compounds can be administered systemically and may have side effects typical of cytotoxic agents, such as, but not limited to, side effects such as little or no specificity for tumor cells during normal cell proliferation. ..

Several prodrugs of cytotoxic nucleoside analogs have also been reported in the art. Examples include N4-behenoyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl-1-β-D-arabinofuranosylcytosine, N4-palmitoyl-1- (2-C-cyano-2). Includes, but is not limited to, -deoxy-β-D-arabino-pentoflanosyl) cytosine, and P-4055 (cytarabine 5'-elaidate). In general, these prodrugs are converted to active drugs primarily in the liver and systemic circulation and exhibit little or no selective release of active drugs in tumor tissue. For example, capecitabine, a prodrug of 5'-deoxy-5-fluorocytidine (eventually 5-fluorouracil), is metabolized in liver and tumor tissue. A series of capecitabine analogs containing "radicals that can be easily hydrolyzed under physiological conditions" are claimed by Fujiu et al. (US Pat. No. 4,966,891) and are hereby referred to herein. Be incorporated. This series of capecitabine analogs described by Fujiu contains the N4 alkyl and aralkylcarbamates of 5'-deoxy-5-fluorocytidine, and these compounds are activated by hydrolysis under normal physiological conditions. Implies that it results in 5'-deoxy-5-fluorocytidine.

A series of cytarabine N4-carbamates has been reported by Fadl et al. (Pharmazie. 1995, 50, 382-7, incorporated herein by reference), where the compound is converted to cytarabine in liver and plasma. Designed to do. WO 2004/041203, incorporated herein by reference, discloses a prodrug of gemcitabine, wherein some of the prodrugs are N4-carbamates. These compounds were designed to overcome the gastrointestinal toxicity of gemcitabine and were intended to provide gemcitabine by hydrolytic release in the liver and plasma after absorption of the intact prodrug from the gastrointestinal tract. Nomura et al. (Bioorg Med. Chem. 2003, 11,453-61, incorporated herein by reference) produced intermediates that required further hydrolysis under acidic conditions during in vivo reduction to produce cells. Describes an acetal derivative of 1- (3-C-ethynyl-β-D-ribo-pentopharanosyl) cytosine that produced a damaging nucleoside compound.

Cytotoxic nucleotides that can be chemotherapeutic agents include pyrazolo [3,4-D] -pyrimidine, alloprinol, azathiopurine, capecitabin, citocin arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, Liverabilin, 7-deaza-adenosine, 7-deraza-guanosine, 6-aza-uracil, 6-aza-citidine, thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, and inosine, or combinations thereof. Included, but not limited to.

Adjacent region: Untranslated region (UTR)
The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR begins at the transcription start site and follows the start codon, but does not contain the start codon, while the 3'UTR begins immediately after the stop codon and continues to the transcription termination signal. There have been a series of reports on the regulatory role that UTRs play in the stability of nucleic acid molecules and translations. The regulatory features of the UTR can be incorporated into the polynucleotides, primary constructs, and / or mmRNAs of the invention to enhance molecular stability. Certain features are also incorporated to ensure controlled downregulation of transcripts in the unlikely event that they are misdirected to unwanted organ sites.

5'UTR and translation initiation Natural 5'UTR has characteristics involved in translation initiation. They have characteristics similar to the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has a consensus CCR (A / G) CCAUGG, where R is a purine (adenine or guanine) 3 bases upstream of the start codon (AUG), followed by another "G". .. The 5'UTR is also known to form secondary structures involved in elongation factor binding.

By manipulating the features typically found in genes that are highly expressed in a particular target organ, the stability and protein production of the polynucleotides, primary constructs, or mmRNAs of the invention can be enhanced. For example, in a hepatocyte line or liver using 5'UTR introduction of liver-expressed mRNA such as albumin, serum amyloid A, apolypoprotein A / B / E, transferrin, α-fetoprotein, erythropoetin, or factor VIII. It is possible to enhance the expression of nucleic acid molecules such as mmRNA. Similarly, enhancing expression in that tissue with 5'UTR from other tissue-specific mRNAs can be used in muscle (MyoD, myosin, myoglobin, myogenin, Herculin), endothelial cells (Tie-1, Tie-1, CD36), bone marrow cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), white blood cells (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin) ), And in lung epithelial cells (SP-A / B / C / D).

Other non-UTR sequences may be incorporated into the 5'UTR (or 3'UTR). For example, an intron or part of an intron sequence can be integrated into an adjacent region of the polynucleotide, primary construct, or mmRNA of the invention. Intron sequence integration can increase protein production, as well as mRNA levels.

3'UTRs and AU-rich elements 3'UTRs are known to have a series of adenosine and uridine encapsulated in them. These AU-rich features are especially common in genes with high turnover rates. Based on their sequence and functional characteristics, AU-rich elements (ARE) can be classified into three classes (Chen et al, 1995): Class I ARE is a UUUA motif within a U-rich region. Contains several dispersed copies. C-Myc and MyoD contain Class I ARE. Class II ARE has two or more overlapping UUAUUUA (U / A) (U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III ARE is less well defined. These U-rich regions do not contain the AUUUA motif. C-Jun and myogenin are two well-studied examples of this class. Most proteins that bind to ARE are known to destabilize messengers, but members of the ELAV family, notably HuR, have been demonstrated to improve mRNA stability. HuR binds to these three classes of ARE. Manipulating the HuR-specific binding site into the 3'UTR of the nucleic acid molecule leads to HuR binding and thus stabilization of the message in vivo.

The introduction, removal, or modification of the AU-rich element (ARE) of the 3'UTR can be used to regulate the stability of the polynucleotides, primary constructs, or mmRNAs of the invention. When manipulating a particular polynucleotide, primary construct, or mmRNA, one or more copies of the ARE are introduced, reducing the stability of the polynucleotide, primary construct, or mmRNA of the invention, thereby resulting. It can suppress the translation of the resulting protein and reduce its production. Similarly, ARE can be identified and eliminated or mutated to improve intracellular stability and thus increase the translation and production of the resulting protein. Transfection experiments using the polynucleotides, primary constructs, or mmRNAs of the invention can be performed on related cell lines and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-manipulated molecules and produced for related proteins 6 hours, 12 hours, 24 hours, 48 hours, and 7 days after transfection using an ELISA kit. Proteins can be assayed.

Incorporation of MicroRNA Binding Sites MicroRNAs (or miRNAs) bind to 3'UTRs of nucleic acid molecules and down-regulate gene expression by either reducing nucleic acid molecule stability or inhibiting translation. It is a non-coding RNA 19-25 nucleotides in length. The polynucleotides, primary constructs, or mRNAs of the invention may comprise one or more microRNA target sequences, microRNA sequences, or microRNA species. Such sequences may correspond to any known microRNA, such as those taught in US Patent Publication No. US2005 / 0261218 and US Patent Publication No. US2005 / 0059005, the contents of which are these by reference. The whole is incorporated herein.

The microRNA sequence comprises a sequence in the "seed" region, i.e., the region at positions 2-8 of the mature microRNA, which sequence has full Watson-Crick complementarity to the miRNA target sequence. MicroRNA species can include positions 2-8 or 2-7 of mature microRNAs. In some embodiments, the microRNA species may contain 7 nucleotides (eg, nucleotides 2-8 of the mature microRNA), and this species-complementary site in the corresponding miRNA target is opposite to the microRNA 1 position. Adjacent to Adenine (A). In some embodiments, the microRNA species may contain 6 nucleotides (eg, nucleotides 2-7 of the mature microRNA) and the species-complementary site in the corresponding miRNA target is opposite to the microRNA 1 position. Adjacent to adenine (A). For example, Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6; 27 (1): 91-105, each of which is incorporated herein by reference in its entirety. The bases of microRNA species have perfect complementarity with the target sequence. The molecule can be targeted for degradation or translational reduction by manipulating the microRNA target sequence into the 3'UTR of the polynucleotides, primary constructs, or mRNAs of the invention, provided that the problem. Subject to the availability of microRNAs. This process reduces the risk of off-target effects during nucleic acid molecule delivery. Identification of microRNAs, microRNA target regions, and their expression patterns, as well as their role in biology, have been reported (both Auer et al., Curr Drag Targets, respectively, which are incorporated herein by reference in their entirety. 2010 11: 943-949, Andand Cheresh Curr Opin Hematol 2011 18: 171-176, Controlras and Lao Leukemia 2012 26: 404-413 (2011 Dec 20. Doi: 10.1038 / leu.2011. 2009 136: 215-233, Landgraf et al, Cell, 2007
129: 1401-1414).

For example, if the nucleic acid molecule is mRNA and is not intended to be delivered to the liver, but settles there, the miR-122, which is a microRNA abundant in the liver, can be targeted at one or more target sites of miR-122. When engineered into the 3'UTR of a polynucleotide, primary construct, or mRNA, it can inhibit the expression of the gene of interest. The introduction of one or more binding sites of different microRNAs can be engineered to further reduce longevity, stability, and protein translation of polynucleotides, primary constructs, or mRNAs.

As used herein, the term "microRNA site" refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It is understood that "binding" can follow conventional Watson-click hybridization rules or can exhibit any stable association of microRNAs with target sequences at or adjacent to the microRNA site. sea bream.

Conversely, for the purposes of the polynucleotides, primary constructs, or mRNAs of the invention, naturally occurring microRNA binding sites to increase protein expression in specific tissues are manipulated out of the sequence (ie, sequence). Can be removed from). For example, the miR-122 binding site can be removed to improve protein expression in the liver. Control of expression in multiple tissues can be achieved by the introduction or removal of one or several microRNA binding sites.

Examples of tissues in which microRNAs are known to regulate mRNA and thus protein expression include liver (miR-122), muscle (miR-133, miR-206, miR-208), and endothelium. Cells (miR-17-92, miR-126), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), fat Tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, MiR-126), but is not limited to these. MicroRNAs can also control complex biological processes such as angiogenesis (miR-132) (And and Cheresh Curr Opin Hematol 2011 18: 171-176, which is incorporated herein by reference in its entirety). In the polynucleotides, primary constructs, or mmRNAs of the invention, the binding sites of microRNAs involved in such processes are poly-based on or in connection with biologically related cell types. It can be removed or introduced to regulate the expression of nucleotides, primary constructs, or mRNA. A list of microRNAs, miR sequences, and miR binding sites can be found in Table 9, US Patent Provisional Application No. 61 / 753,661 filed January 17, 2013, US Patent Provisional Application No. 18 January 2013. They are listed in Table 9 of 61 / 754,159 and Table 7 of US Patent Provisional Application No. 61 / 758,921 filed January 31, 2013, respectively, all of which are hereby incorporated by reference. Incorporated in.

Finally, through an understanding of microRNA expression patterns in different cell types, polynucleotides, primary constructs, or mRNAs are more targeted expression in specific cell types or more targeted only under specific biological conditions. Can be engineered for personalized expression. By introducing tissue-specific microRNA binding sites, polynucleotides, primary constructs, or mRNAs that are optimal for protein expression in tissues or protein expression related to biological conditions can be designed. Examples of the use of microRNAs to drive tissue or disease-specific gene expression are listed (Getner, which is incorporated herein by reference in its entirety).
and Naldini, Tissue Antigens. 2012, 80: 393-403). In addition, microRNA species sites can reduce expression in certain cells that are integrated into mRNA, which results in biological improvement. An example of this is the incorporation of the miR-142 site into a UGT1A1-expressing lentiviral vector. The presence of the miR-142 site reduced expression in hematopoietic cells, resulting in reduced expression in antigen-presenting cells, resulting in the absence of an immune response to virally expressed UGT1A1. Schmitt et al., Gastroenterology 2010; 139: 999-1007, Gonzarez-Asecuinolaza et al. Gastroenterology 2010, 139: 726-729), which are incorporated herein. Incorporation of the miR-142 site into the modified mRNA could not only reduce the expression of the encoded protein in hematopoietic cells, but also reduce or abolish the immune response to the mRNA-encoded protein. did it. Integration of miR-142 sites (s) into mRNA is important during the treatment of patients with complete protein deficiency (Type I UGT1A1, LDLR deficient patients, CRIM-negative Poppe patients, etc.).

Transfection experiments with engineered polynucleotides, primary constructs, or mmRNAs can be performed on related cell lines and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different microRNA binding site manipulation polynucleotides, primary constructs, or mmRNAs and related proteins 6 hours, 12 hours, 24 hours, 48 hours after transfection with an ELISA kit. Proteins produced at time, 72 hours, and 7 days can be assayed. In vivo experiments with microRNA binding site manipulation molecules can also be performed to test changes in tissue-specific expression of formulated polynucleotides, primary constructs, or mRNAs.

The 5'cap structure of 5'capping mRNA is involved in nuclear export, improves the stability of mRNA and binds to the mRNA cap binding protein (CBP), which is associated with the poly (A) binding protein of CBP. Through association, it is involved in mRNA stability and translational eligibility in cells to form mature cyclic mRNA species. This cap also assists in the removal of 5'proximal introns during mRNA splicing.

The endogenous mRNA molecule can be capped at the 5'end to result in a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the 5'end transcription sense nucleotide of the mRNA molecule. The 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylic acid residue. Ribose sugars at the 5'end and / or anterior end transcription nucleotides of mRNA can also be optionally 2'-O-methylated. 5'decapping via hydrolysis and cleavage of the guanylic cap structure can target nucleic acid molecules such as mRNA molecules for degradation.

Modifications of the polynucleotides, primary constructs, and mRNAs of the invention can produce non-hydrolyzable cap structures that block decapping, resulting in an increase in the half-life of mRNAs. Modified nucleotides can be used during the capping reaction because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5'phosphologiester bond. For example, the New England Biolabs (Ipswich, MA) vaccinia capping enzyme can be used with α-thio-guanosine nucleotides as directed by the manufacturer to trigger phosphorothioate binding in the 5'-ppp-5'cap. Further modified guanosine nucleotides such as α-methyl-phosphonate and seleno-phosphate nucleotides can be used.

Further modifications include 2'-O-methylation of the ribose sugar of the 5'end and / or 5'pre-terminal nucleotides of the mRNA at the 2'-hydroxyl group of the sugar ring (as described above). Not limited. Multiple distinctly different 5'-cap structures can be used to generate 5'caps for nucleic acid molecules, such as mRNA molecules.

Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, are natural (ie, endogenous, wild) in terms of their chemical structure. , Or physiological) Different from the 5'cap, but retains the cap function. Cap analogs can be chemically (ie, non-enzymatically) or enzymatically synthesized and / or bound to nucleic acid molecules.

For example, a rebellious cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, one of which is an N7 methyl group and a 3'-O-methyl. The group (ie, N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m ⁷ G-3'mppp-G, which is equivalently 3'O-Me-m7G (5'). ) Ppp (5') G) is contained. The 3'-O atom of the other unmodified guanine is attached to the 5'terminal nucleotide of the capped nucleic acid molecule (eg, mRNA or mRNA). . N7- and 3'-O-methylated guanines provide a terminal portion of a capped nucleic acid molecule (eg, mRNA or mRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (ie, N7,2'-O-dimethyl-guanosine-5'-tri". ^{phosphate-5'-guanosine,} having m 7 Gm-ppp-G) .

While cap analogs allow simultaneous capping of nucleic acid molecules in in vitro transcription reactions, up to 20% of transcripts can remain uncapped. This, as well as structural differences in cap analogs with the endogenous 5'-cap structure of nucleic acids produced by the endogenous cell transcription mechanism, can result in reduced translational eligibility and reduced cell stability.

The polynucleotides, primary constructs, and mmRNAs of the invention can also be capped after transcription using enzymes to produce more authentic 5'-cap structures. As used herein, the expression "more authentic" refers to a feature that closely mimics or mimics an intrinsic or wild-type feature, either structurally or functionally. That is, "more authentic" features better represent endogenous, wild-type, natural, or physiological cellular function and / or structure compared to prior art synthetic features or analogs, etc. It surpasses the corresponding endogenous, wild-type, natural or physiological features in one or more respects. Non-limiting examples of more authentic 5'cap structures of the invention are compared to synthetic 5'cap structures known in the art (or wild-type, natural, or physiological 5'cap structures). Among others, those with enhanced cap-binding protein binding, increased half-life, reduced sensitivity to 5'endonucleases, and / or reduced 5'decapping. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme may generate a reference 5'-5'-triphosphate bond between the 5'end nucleotide of mRNA and a guanine cap nucleotide. , Capguanine contains N7 methylation, and the 5'terminal nucleotide of mRNA contains 2'-O-methyl. Such a structure is referred to as a cap 1 structure. This cap results in higher translational eligibility and cell stability, as well as reduced activation of cell inflammation-inducing cytokines, as compared to, for example, other 5'cap analog structures known in the art. Cap structures include 7mG (5') ppp (5') N, pN2p (cap 0), 7mG (5') ppp (5') NlpNp (cap 1), and 7mG (5') -ppp (5'). ) NlpN2mp (cap 2) is included, but not limited to these.

Almost 100% of polynucleotides, primary constructs, or mmRNAs can be capped because polynucleotides, primary constructs, or mmRNAs can be capped after transcription, and because this process is more efficient. This is very different from about 80% when cap analogs are bound to mRNA during the in vitro transcription reaction.

According to the present invention, the 5'end cap may include an endogenous cap or a cap analog. According to the present invention, the 5'end cap may contain a guanine analog. Useful guanosine analogs include inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deraza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. Includes, but is not limited to.

Viral Sequences Translated enhancer sequences of barley stripe dwarf virus (BYDV-PAV), Jaagsiekte sheep retrovirus (JSRV), and / or local pathogenic nasal oncoviruses (eg, incorporated herein by reference in their entirety, international publication). Further viral sequences, such as, but not limited to, WO 20121229648) can be engineered and inserted into the polynucleotides, primary constructs, or 3'UTRs of the mRNA of the invention and constructs in vitro and in vivo. Can stimulate translation. Transfection experiments can be performed on the relevant cell line and assay protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and 7 days after transfection.

IRES Sequence In addition, a polynucleotide, primary construct, or mmRNA that can contain an internal ribosome entry site (IRES) is provided. The IRES, initially identified as a characteristic picornavirus RNA, plays an important role in initiating protein synthesis in the absence of a 5'cap structure. The IRES can serve as the sole ribosome binding site for mRNA or can serve as one of multiple ribosome binding sites. A polynucleotide, primary construct, or mMV containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by the ribosome (“polycistron nucleic acid molecule”). .. When an IRES is provided for a polynucleotide, primary construct, or mmRNA, a second translatable region is further optionally provided. Examples of IRES sequences that can be used in accordance with the present invention include, without limitation, picornavirus (eg, FMDV), pestvirus (CFFV), poliovirus (PV), encephalomyelitis virus (ECMV), foot-and-mouth disease virus (FMDV). , Hepatitis C virus (HCV), porcine cholera virus (CSFV), murine leukemia virus (MLV), monkey immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

Poly A tail During RNA treatment, a long adenine nucleotide chain (poly A tail) can be added to polynucleotides such as mRNA molecules to improve stability. Immediately after transfer, the 3'end of the transcript can be cleaved to release the 3'hydroxyl. The poly A polymerase then adds an adenine nucleotide strand to the RNA. This process, called polyadenylation, adds a poly A tail, which can be, for example, about 100-250 residue length.

It has been found that the unique poly A tail length provides certain advantages to the polynucleotides, primary constructs, or mmRNAs of the invention.

Generally, the length of the poly A tail of the present invention exceeds 30 nucleotides in length. In another embodiment, the poly A tail exceeds 35 nucleotides in length (eg, at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200). , 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1 , 700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides in length, or more). In some embodiments, the polynucleotide, primary construct, or mmRNA is about 30 to about 3,000 nucleotides (eg, 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1, 500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2, 500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2, 000-3,000, 2,000-2,500, and 2,500-3,000).

In one embodiment, the poly A tail is designed for the length of the entire polynucleotide, primary construct, or mmRNA. This design can be based on the length of the coding region, the length of a particular feature or region (such as the first or adjacent region), or the length of the final product expressed from a polynucleotide, primary construct, or mmRNA. obtain.

In this context, polyA tail is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, more than a polynucleotide, primary construct, or mmRNA, or its characteristics. Or it can be 100% longer. The poly A tail can also be designed as a fraction of the polynucleotide, primary construct, or mm mRNA to which it belongs. In this context, the poly A tail is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the total length of the structure or the total length of the structure minus the poly A tail. Or it can be 90% or more. In addition, manipulation of the binding site and complexation of polynucleotides, primary constructs, or mmRNAs to poly A-binding proteins can enhance expression.

In addition, multiple distinctly different polynucleotides, primary constructs, or mMVs can be attached to PABPs (polyA-binding proteins) via the 3'end using nucleotides modified at the 3'end of the polyA tail. Transfection experiments can be performed on the relevant cell line and assay protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and 7 days after transfection.

In one embodiment, the polynucleotide primary constructs of the invention are designed to include polyA through G quadruple terms. The G quadruple term is a cyclic hydrogen bond array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G quadruple is incorporated at the end of the poly A tail. The resulting mmRNA construct is assayed for stability, protein production, and half-life and other parameters at various time points. It has been found that the poly A-G quadruple term results in protein production corresponding to at least 75% of the protein production produced by using the poly A tail of 120 nucleotides alone.

Quantification In one embodiment, the polynucleotides, primary constructs, or mmRNAs of the invention can be quantified in exosomes derived from one or more body fluids. As used herein, "body fluid" includes peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, lymph, tufts, sheep's water, chyle, breast milk, Bronchial alveolar lavage fluid, semen, prostatic fluid, Cooper's gland fluid or pre-ejaculate fluid, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, heart sac fluid, lymph fluid, plasma fluid, chyle, bile, interstitial fluid Includes chyle, pus, sebum, vomitus, vaginal secretions, mucosal secretions, water stools, pancreatic fluid, nasal lavage fluid, bronchopulmonary aspirates, scutellum cyst fluid, and umbilical cord blood. Alternatively, exosomes are recovered from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testicle, skin, colon, breast, prostate, brain, esophagus, liver, and placenta. obtain.

In the quantification method, less than 2 mL of sample was obtained from the subject and the exosomes were size exclusion chromatography, density gradient centrifugation, fractional centrifugation, nanomembrane filtration, immunoabsorption capture, affinity purification, microfluidic separation. , Or a combination of these. In the analysis, the level or concentration of polynucleotide, primary construct, or mmRNA can be the expression level, presence, absence, cleavage, or modification of the administered construct. It is advantageous to correlate this level with assays for one or more clinical phenotypes or human disease biomarkers. While this assay can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass analysis, or a combination thereof, exosomes are enzymatically bound. It can be isolated using immunohistochemical methods such as immunosorbent assay (ELISA). Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, fractional centrifugation, nanomembrane ultrafiltration, immunoabsorption capture, affinity purification, microfluidic separation, or a combination thereof.

These methods provide researchers with the ability to monitor levels of residual or delivered polynucleotides, primary constructs, or mmRNAs in real time. This is possible because the polynucleotides, primary constructs, or mmRNAs of the invention differ from their intrinsic morphology due to structural or chemical modifications.

II. Design and Synthesis of mMRNA The polynucleotides, primary constructs, or mmRNAs used in accordance with the present invention include chemical synthesis, enzymatic synthesis commonly referred to as in vitro transcription (IVT), or enzymatic or chemical cleavage of longer precursors. Can be prepared according to any available technique, but not limited to these. Methods of synthesizing RNA are known in the art (eg, Gait, MJ (ed.) Oligonoculotide synthesis: a practical application, Oxford [Oxfordshire], both of which are incorporated herein by reference. Washington, DC: IRL Press, 1984, and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular BioN. , 2005).

The process of designing and synthesizing the primary construct of the present invention generally includes a gene construction step, an mRNA production step (with or without modification), and a purification step. In enzyme synthesis methods, the target polynucleotide sequence encoding the polypeptide of interest is first selected for integration into a vector that is amplified to produce a cDNA template. Optionally, the target polynucleotide sequence and / or any flanking sequence can be codon-optimized. The cDNA template is then used to produce mRNA by in vitro transcription (IVT). After production, mRNA can undergo purification and purification processes. These steps are provided in more detail below.

Gene Construction Gene construction steps can include, but are not limited to, gene synthesis, vector amplification, plasmid purification, plasmid linearization and purification, and cDNA template synthesis and purification.

Gene Synthesis The primary construct is designed when the polypeptide or target of interest is selected for production. Within the primary construct, the first region of the binding nucleoside encoding the polypeptide of interest can be constructed using an open reading frame (ORF) of the selected nucleic acid (DNA or RNA) transcript. The ORF may include a wild-type ORF, an isoform, a variant, or a fragment thereof. As used herein, the "open reading frame" or "ORF" is intended to refer to a nucleic acid sequence (DNA or RNA) that can encode the polypeptide of interest. The ORF often begins with the start codon ATG and ends with a nonsense or stop codon or signal.

In addition, the nucleotide sequence of the first region can be codon-optimized. Codon optimization methods are known in the art and can be useful in efforts to achieve one or more of several goals. These goals include matching codon frequencies in target and host organisms to ensure proper folding, entraining GC content to increase mRNA stability, or reduce secondary structures. Minimize tandem repetitive codons or basation that can impair gene construction or expression, customize transcription and translational control regions, insert or remove protein transport sequences, post-translational modifications in encoded proteins Removing / adding sites (eg, glycosylation sites), adding, removing, or rearranging protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites. Includes adjusting the translation rate to allow proper folding of various domains of the protein, or reducing or eliminating the secondary structure of the problem within the mRNA. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include GeneArt's services (Life Technologies), DNA 2.0 (Menlo Park CA), and / or ownership. The method to have is mentioned. In one embodiment, the ORF sequence is optimized using an optimization algorithm. The codon choices for each amino acid are provided in Table 1.
Table 1. Codon choice

Features that may be considered beneficial in some embodiments of the invention may be encoded by the primary construct and may be adjacent to the ORF as a first or second adjacent region. This adjacent region can be incorporated into the primary construct before and / or after the optimization of the ORF. The primary construct need not contain both 5'adjacent regions and 3'adjacent regions. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, oligo (dT) sequences, and detectable tags, but multiple clonings that may have XbaI recognition. Sites can also be mentioned.

In some embodiments, 5'UTRs and / or 3'UTRs may be provided as adjacent regions. Multiple 5'or 3'UTRs can be contained in adjacent regions and can be the same or different sequences. Any part of the adjacent region (including the absence of such a part) can be codon-optimized, and any of them can independently have one or more different structures or one or more before and / or after codon-optimization. May contain chemical modifications. The combination of features may be included in the first and second adjacent regions and may be contained within other features. For example, the ORF may be flanked by a 5'UTR that may contain a strong Kozak translation initiation signal and / or a 3'UTR that may contain an oligo (dT) sequence for template addition of the poly A tail. 5'UTRs, such as the 5'UTRs described in U.S. Patent Application Publication No. 201029263625, which are incorporated herein by reference in their entirety, are a first polynucleotide fragment and a second poly from the same and / or different genes. It may contain nucleotide fragments.

Tables 2 and 3 provide a list of exemplary UTRs that can be used in the primary construct of the invention as adjacent regions. A list of 5'untranslated regions of the present invention is shown in Table 2. Variants of the 5'UTR can be utilized in which one or more nucleotides, including A, T, C, or G, are added to the end or removed from the end.
Table 2.5'Untranslated Region

A representative list of the 3'untranslated regions of the present invention is shown in Table 3. Variants of the 3'UTR can be utilized in which one or more nucleotides, including A, T, C, or G, are added to the end or removed from the end.
Table 3.3'Untranslated region

It should be understood that the ones listed in the above table are examples and that any UTR from any gene can be integrated into the respective first or second adjacent region of the primary construct. In addition, multiple wild-type UTRs of any known gene can be utilized. It is also within the scope of the present invention to provide an artificial UTR that is not a variant of a wild-type gene. These UTRs or parts thereof may be positioned in the same orientation as the UTRs or parts of the transcript from which they may be selected, or may be oriented or repositioned. Thus, the 5'or 3'UTR can be inverted, shortened, extended and chimerized with one or more other 5'UTRs or 3'UTRs. As used herein, the term "altered" means that when referring to a UTR sequence, the UTR has changed in that it is associated with a reference sequence. For example, the 3'or 5'UTR can be altered relative to the wild-type or native UTR by the orientation or positional changes taught above, or additional nucleotide inclusions, nucleotide deletions, nucleotide exchanges or transposes. Can be changed by Any of these changes that result in a "modified" UTR (whether 3'or 5') include a mutant UTR.

In one embodiment, double, triple, or quadruple UTRs such as 5'or 3'UTRs may be used. As used herein, a "double" UTR is one in which two copies of the same UTR are encoded either contiguously or substantially contiguously. For example, the double β-globin 3'UTR described in US Patent Publication No. 2010129877, the contents of which are incorporated herein by reference in its entirety, may be used.

Having a patterned UTR is also within the scope of the present invention. As used herein, a "patterned UTR" is a UTR that indicates a repeating or alternating pattern that is repeated once, twice, or three or more times, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof. Is. In these patterns, each letter, A, B, or C represents a different UTR at the nucleotide level.

In one embodiment, adjacent regions are selected from a family of transcripts in which the proteins share common functional, structural and characteristic characteristics. For example, a polypeptide of interest may belong to a family of proteins that are expressed in a particular cell, tissue, or at some point during development. Any UTR of these genes can be exchanged for any other UTR of the same or different protein family to create a new chimeric primary transcript. As used herein, a "protein family" is, in the broadest sense, two or more intended polypeptides that share at least one function, structure, characteristic, localization, origin, or expression pattern. It is used to refer to a group of.

After optimization (if desired), the primary construct components can be reconstituted and converted into vectors such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, optimized constructs can be reconstituted and converted to chemically competent Escherichia coli, yeast, Neurospora crassa, maize, Drosophila, etc., and high copy count plasmid-like or chromosomal structures are described herein. Caused by the method.

The untranslated region may also include a translation enhancer element (TEE). As a non-limiting example, the TEE may include the TEE described in U.S. Patent Application No. 20090226470, which is incorporated herein by reference in its entirety, and the TEE known in the art.

Stop Codon In one embodiment, the primary construct of the invention may include at least two stop codons before the 3'untranslated region (UTR). The stop codon can be selected from TGA, TAA, and TAG. In one embodiment, the primary construct of the invention comprises a stop codon TGA and another stop codon. In a further embodiment, another stop codon can be TAA. In another embodiment, the primary construct of the invention comprises three stop codons.

Vector Amplification Vectors containing primary constructs are then amplified, such as maxiprep using the Invitrogen PURELINK ™ HiPure Maximprep kit (Carlsbad, CA), but not limited to methods known in the art. The plasmid is isolated and purified using.

Plasmid linearization The plasmid can then be linearized using methods known in the art, such as, but not limited to, the use of restriction enzymes and buffers. The linearized reactants include, for example, Invitrogen's PURELINK ™ PCR Micro Kit (Carlsbad, CA), as well as strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC ( HIC-HPLC) and the like, but not limited to HPLC-based purification methods, as well as methods including Invitrogen's standard PURELINK ™ PCR kit (Carlsbad, CA). The purification method can be modified depending on the size of the resulting linearization reactant. The linearized plasmid is then used to generate cDNA for the in vitro transcription (IVT) reaction.

＊ＵＦＰはユニバーサル順方向プライマーであり、ＵＲＰはユニバーサル逆方向プライマーである。 Synthesis of cDNA template A cDNA template can be synthesized by subjecting a linearized plasmid to a polymerase chain reaction (PCR). Table 4 is a list of primers and probes that may be useful in the PCR reaction of the present invention. It should be understood that this list is not exhaustive and that primer-probe designs for any amplification are within the technical scope of the art. The probe may also contain bases that have been chemically modified to increase base pairing fidelity and base pairing strength to the target molecule. Such modifications may include 5-methyl-cytidine, 2,6-di-amino-purine, 2'-fluoro, phosphoro-thioate, or locked nucleic acid.
Table 4. Primers and probes

* UFP is a universal forward primer and URP is a universal reverse primer.

In one embodiment, the cDNA can be submitted for sequencing analysis prior to undergoing transcription.

mRNA Production The process of mRNA or mRNA production can include, but is not limited to, in vitro transcription, cDNA template removal and RNA purification, and mRNA capping and / or tailing reactions.

In vitro Transcription The cDNA produced in the previous step can be transcribed using an in vitro transcription (IVT) system. This system typically comprises transcription buffers, nucleotide triphosphates (NTPs), RNase inhibitors, and polymerases. NTPs can be manufactured in-house, selected from suppliers, or synthesized as described herein. NTPs can be selected from, but not limited to, the NTPs described herein, including natural and non-natural (modified) NTPs. Polymerases can be selected from, but are not limited to, variant polymerases such as, but not limited to, T7 RNA polymerase, T3 RNA polymerase, and polymerases capable of incorporating modified nucleic acids.

RNA polymerase Any number of RNA polymerases or variants can be used in the design of the primary constructs of the invention.

RNA polymerase can be modified by inserting or deleting amino acids in the RNA polymerase sequence. As a non-limiting example, RNA polymerases can be modified to show an increased ability to incorporate 2'-modified nucleotide triphosphates as compared to unmodified RNA polymerases (see herein by reference in their entirety. See International Publication No. WO20080878180 and US Pat. No. 8,101,385, which are incorporated).

Variants can be obtained by evolving RNA polymerase, optimizing RNA polymerase amino acid and / or nucleic acid sequences, and / or using other methods known in the art. As a non-limiting example, the T7 RNA polymerase variant incorporates a continuous directed evolutionary system devised by Esvelt et al. (Nature (2011) 472 (7344): 499-503, which is incorporated herein by reference in its entirety). T7 RNA polymerase clones can be evolved using T7 RNA polymerase mutations (K93T) in which the lysine at position 93 is replaced with treonine, I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S. , G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P , K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, N748D, Q754R, E775K, A827V, D851N, etc. It can encode at least one mutation. As another non-limiting example, the T7 RNA polymerase variant may encode at least one mutation described in US Patent Publication No. 2011001224 and No. 20070117112, which is incorporated herein by reference in its entirety. Variants of RNA polymerase can also include, but are not limited to, substitution variants, conserved amino acid substitutions, insertion variants, deletion variants, and / or covalent derivatives.

In one embodiment, the primary construct can be designed to be recognized by wild-type or mutant RNA polymerase. In doing so, the primary construct can be modified to contain a sequence change site or region from the wild-type or parent primary construct.

In one embodiment, the primary construct has at least one substitution and / or insertion within the 5'UTR, before the 5'UTR, and / or after the 5'UTR, upstream of the RNA polymerase binding or recognition site of the primary construct. , Downstream of RNA polymerase binding or recognition sites, upstream of TATA box sequences, downstream of TATA box sequences, but may be designed to include upstream of the coding region of the primary construct.

In one embodiment, the 5'UTR of the primary construct can be replaced by inserting at least one region and / or string of nucleotides of the same base. Regions and / or strings of nucleotides can include, but are not limited to, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides, which nucleotides are native and / or non-natural. Can be natural. As a non-limiting example, this nucleotide group may comprise 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein, and / or a combination thereof.

In one embodiment, the 5'UTR of the primary construct is, but is not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein, and / or a combination thereof, etc. 2 It can be replaced by inserting at least two regions and / or strings of nucleotides of two different bases. For example, the 5'UTR can be replaced by inserting 5-8 adenine bases followed by 5-8 cytosine bases. In another example, the 5'UTR can be replaced by inserting 5-8 cytosine bases followed by 5-8 adenine bases.

In one embodiment, the primary construct may include at least one substitution and / or insertion downstream of a transcription initiation site that can be recognized by RNA polymerase. As a non-limiting example, at least one substitution and / or insertion replaces at least one nucleic acid in a region immediately downstream of the transcription initiation site (such as, but not limited to, +1 to +6). Can occur downstream of the transcription initiation site. Transcription complex by altering the nucleotide region immediately downstream of the transcription initiation site, affecting the initiation rate, increasing the apparent constant nucleotide triphosphate (NTP) reaction, and curing the initial transcript. It can enhance the dissociation of short transcripts from (Brieba et al, Biochemistry (2002) 41: 5144-5149, which is incorporated herein by reference in its entirety). Modifications, substitutions, and / or insertions of at least one nucleic acid can cause silent mutations in the nucleic acid sequence or can cause mutations in the amino acid sequence.

In one embodiment, the primary construct is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least downstream of the transcription initiation site. It may contain substitutions of 12, or at least 13 guanine bases.

In one embodiment, the primary construct may comprise at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 guanine base substitutions in the region immediately downstream of the transcription initiation site. As a non-limiting example, when the nucleotide in the region is GGGAGA, the guanine base can be replaced by at least 1, at least 2, at least 3, or at least 4 adenine nucleotides. In another non-limiting example, when the nucleotide in the region is GGGAGA, the guanine base can be replaced by at least 1, at least 2, at least 3, or at least 4 cytosine bases. In another non-limiting example, when the nucleotide in the region is GGGAGA, the guanine base is at least 1, at least 2, at least 3, or at least 4 thymines, and / or the nucleotides described herein. Can be replaced by any of.

In one embodiment, the primary construct may include at least one substitution and / or insertion upstream of the start codon. For clarity, one of ordinary skill in the art will understand that the initiation codon is the first codon in the protein coding region, while the transcription initiation site is the site where transcription begins. The primary construct may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight substitutions and / or insertions of nucleotide bases. Not limited to. Nucleotide bases can be inserted or substituted at one, at least one, at least two, at least three, at least four, or at least five positions upstream of the start codon. The inserted and / or substituted nucleotides are the same base (eg, all A or all C or all T or all G) and two different bases (eg, A and C, A and T, or C and T). It can be three different bases (eg, A, C, and T, or A, C, and T), or at least four different bases. As a non-limiting example, the guanine base upstream of the coding region in the primary construct can be replaced with either adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example, the substitution of a guanine base in the primary construct can be designed to leave one guanine base downstream of the transcription initiation site and in the region prior to the initiation codon (whole by reference). Is incorporated herein by Esvelt et al. Nature (2011) 472 (7344): 499-503). As a non-limiting example, at least 5 nucleotides can be inserted at one position downstream of the transcription initiation site and upstream of the initiation codon, and at least 5 nucleotides can be of the same base type.

CDNA Template Removal and Purification The cDNA template can be removed using methods known in the art, such as, but not limited to, treatment with deoxyribonuclease I (DNase I). RNA purification is performed by Beckman Coulter (Dambers, MA) AGENCOURT® CLEANSEQ® system; strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC ( HIC-HPLC) and the like, but the purification method based on HPLC is not limited thereto, but the purification method is not limited thereto.

Capping and / or tailing reaction The primary construct or mRNA may also undergo a capping and / or tailing reaction. The capping reaction can be performed using a method known in the art in which a 5'cap is added to the 5'end of the primary construct. Methods for capping include, but are not limited to, the use of vaccinia capping enzymes (New England Biolabs, Ipswich, MA).

The polyA tailing reaction can be carried out using methods known in the art, such as, but not limited to, 2'O-methyltransferase and the methods described herein. If the primary construct produced from the cDNA does not contain poly T, it may be beneficial to perform a poly A tailing reaction before the primary construct is purified.

mRNA Purification Primary constructs or mRNA purification may include, but are not limited to, mRNA or mRNA purification, quality assurance, and quality control. For mRNA or mRNA purification, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA); poly T beads; LNA® oligo T capture probes (EXIQON® Inc, Vedbaek, Denmark); or strong anions. Exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), hydrophobic interaction HPLC (HIC-HPLC), etc., but are not limited to HPLC-based purification methods, but are limited thereto. It can be done using methods known in the art that are not. The term "purified", such as "purified mRNA or mRNA", refers to one that is separated from at least one contaminant when used in the context of a polynucleotide. As used herein, a "contamination substance" is any substance that makes another substance inappropriate, impure, or recessive. Thus, purified polynucleotides (eg, DNA and RNA) are in a form or environment that differs from the naturally occurring form or environment, or a form or environment that is different from the form or environment that existed prior to the treatment or purification method. Or it exists in the environment.

Quality assurance and / or quality control inspections can be performed using methods such as, but not limited to, gel electrophoresis, UV absorption, or analytical HPLC.

In another embodiment, mRNA or mRNA can be sequenced using methods that include, but are not limited to, reverse transcription PCR.

In one embodiment, mRNA or mRNA can be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is the NANODROP® spectrometer (Thermo Fisher, Waltham, MA). The quantified mRNA or mRNA can be analyzed to determine if the mRNA or mRNA can be of suitable size and to confirm that no degradation of the mRNA or mRNA has occurred. Degradation of mRNA and / or m mRNA includes agarose gel electrophoresis; strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). Purification methods based on HPLC, such as, but not limited to, liquid chromatography mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE), but can be confirmed using methods not limited thereto.

The signal sequence primary construct or mmRNA may also encode an additional feature that facilitates transport of the polypeptide to a therapy-related site. One such feature that supports protein transport is the signal sequence. As used herein, a "signal sequence" or "signal peptide" is each incorporated into the coding region or the 5'(or N-terminus) of the encoded polypeptide, each approximately 9-200 nucleotides in length. A polynucleotide or polypeptide (3-60 amino acid length). Addition of these sequences results in the transport of the encoded polypeptide through one or more secretory pathways into the endoplasmic reticulum. Some signal peptides are cleaved from the protein by signal peptidase after the protein has been transported.

Table 5 is a representative list of protein signal sequences that can be incorporated to encode the polynucleotides, primary constructs, or mMVs of the invention.
Table 5. Signal sequence

In this table, SS is a secretory signal and MLS is a mitochondrial leader signal. The primary construct or mmRNA of the present invention may be designed to encode any of the signal sequences of SEQ ID NOs: 94-155, or fragments or variants thereof. These sequences may be included at the beginning, middle, or end of the polypeptide coding region, or in adjacent regions. In addition, any of the polynucleotide primary constructs of the invention may also include one or more of the sequences defined by SEQ ID NOs: 32-93. These may be in the first region or any adjacent region.

Additional signal sequences that can be utilized in the present invention are described, for example, at http: // www. signal peptide. de / or http: // proline. bic. nus. edu. Contains a signal sequence taught in a database such as the database found at sg / spdb /. U.S. Pat. Nos. 8,124,379, 7,413,875, and 7,385,034 are also within the scope of the present invention, the contents of each of which are described by reference. Is incorporated herein in its entirety.

Target Selection According to the present invention, the primary construct comprises at least one first region of a binding nucleoside encoding the polypeptide of interest. The polypeptides or "targets" of interest in the present invention are listed in Table 6. In addition to the name and description of the gene encoding the polypeptide of interest, the ENSEMBL transcript SEQ ID NO: (ENST), ENSEMBL protein SEQ ID NO: (ENSP), and, if available, the optimized transcription sequence number (ENSP). The Optim Trans sequence number) or the optimized open reading frame sequence number (Optim ORF sequence number) is also shown in Table 6. One or more variants or isoforms may be present for any particular gene. If they are present, they are also shown in the table. Those skilled in the art will understand that what is disclosed in the table is a potential adjacent area. These are encoded as either 5'(upstream) or 3'(downstream) of the ORF or coding region in each ENST transcript. Coding regions are categorically and specifically disclosed by teaching ENSP sequences. As a result, adjacent teaching sequences encoding proteins are considered adjacent regions. It is also possible to further characterize the 5'and 3'adjacent regions by utilizing one or more available databases or algorithms. The database annotates the features contained in the adjacent regions of the ENST transcript, which are available in the art.
Table 6. target

Protein Cleavage Signals and Sites In one embodiment, the polypeptides of the invention may comprise at least one protein cleavage signal containing at least one protein cleavage site. Protein cleavage sites include, but are not limited to, the middle of the N-terminus and the C-terminus, between the N-terminus and the midpoint, between the midpoint and the C-terminus, and combinations thereof. It can be located at the N-terminus or C-terminus in any space between.

Polypeptides of the invention may include, but are not limited to, proprotein convertase (or prohormone convertase), thrombin, or factor Xa protein cleavage signals. Proprotein convertases are associated with prohormone convertase 1/3 (PC1 / 3), PC2, furin, PC4, PC5 / 6, subtilisin amino acid cleaving enzyme 4 (PACE4), and yeast kexin known as PC7. Two basic amino acid-specific subtilisin-like serine proteinases and two other non-basic residues called subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9). It is a family of 9 proteinases, including 9 subtilises. Non-limiting examples of protein cleavage signal amino acid sequences are listed in Table 7. In Table 7, "X" refers to any amino acid, "n" can be 0, 2, 4, or 6 amino acids, and " ^* " refers to a protein cleavage site. In Table 7, SEQ ID NO: 21426 refers to when n is 4, and SEQ ID NO: 21427 refers to when n is 6.
Table 7. Protein cleavage site sequence

In one embodiment, the primary construct and mmRNA of the invention can be engineered such that the primary construct or mmRNA contains at least one encoded protein cleavage signal. The encoded protein cleavage signal can be located before the start codon, after the start codon, before the coding region, within the coding region, eg, in the middle of the coding region, between the start codon and the midpoint, with the midpoint. Between, but not limited to, the stop codon, after the coding region, after the stop codon, between the two stop codons, after the stop codon, and combinations thereof.

In one embodiment, the primary construct or mMV of the invention may comprise at least one encoded protein cleavage signal containing at least one protein cleavage site. The encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin, and / or a factor Xa protein cleavage signal. One of ordinary skill in the art can use Table 1 above or other known methods to determine the appropriate encoded protein cleavage signal to include in the primary construct or mmRNA of the invention. For example, starting with the signals in Table 7 and considering the codons in Table 1, the signals of the primary constructs capable of producing protein signals in the resulting polypeptide can be designed.

In one embodiment, the polypeptide of the invention comprises at least one protein cleavage signal and / or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Patent Publication No. 20090227660, which are incorporated herein by reference in their entirety, use a furin cleavage site to GLP-in the expression product. The N-terminal methionine of 1 is cleaved from the Golgi apparatus of the cell. In one embodiment, the polypeptide of the invention comprises at least one protein cleavage signal and / or site, provided that the polypeptide is not GLP-1.

In one embodiment, the primary construct or mmRNA of the invention comprises at least one encoded protein cleavage signal and / or site.

In one embodiment, the primary construct or mmRNA of the invention comprises at least one encoded protein cleavage signal and / or site, provided that the primary construct or mmRNA does not encode GLP-1. ..

In one embodiment, the primary construct or mmRNA of the invention may comprise more than one coding region. If multiple coding regions are present in the primary construct or mmRNA of the invention, the multiple coding regions can be separated by the encoded protein cleavage site. As a non-limiting example, the primary construct or mmRNA can be written in an ordered pattern. Such a pattern follows the AXBY form, where A and B are coding regions that can be the same or different coding regions and / or can encode the same or different polypeptides, and X and Y are the same. Alternatively, it is a encoded protein cleavage signal that can encode a different protein cleavage signal. The second such pattern follows the AXYBZ form, in which A and B are coding regions that can be the same or different coding regions and / or can encode the same or different polypeptides, X, Y. , And Z are encoded protein cleavage signals that can encode the same or different protein cleavage signals. The third pattern follows the ABXCY form, where A, B, and C are coding regions that can be the same or different coding regions and / or can encode the same or different polypeptides, and X and Y are the same. Alternatively, it is a encoded protein cleavage signal that can encode a different protein cleavage signal.

In one embodiment, the polypeptide, primary construct and mmRNA are described above so that the polypeptide, primary construct and mmRNA can be released from the carrier region or fusion partner by treatment with a protease specific for the protein cleavage site. It may also contain a sequence encoding a protein cleavage site.

In one embodiment, the polypeptides, primary constructs, and mmRNAs of the invention may comprise a sequence encoding a 2A peptide. In one embodiment, this sequence can be used to separate the coding regions of two or more polypeptides of interest. As a non-limiting example, the sequence encoding the 2A peptide can be located between the coding region A and the coding region B (A-2Apep-B). The presence of the 2A peptide results in cleavage of one long protein into protein A, protein B, and 2A peptides. Protein A and protein B can be polypeptides of the same or different purpose. In another embodiment, the 2A peptide is used in the polynucleotides, primary constructs, and / or mRNAs of the invention to produce 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins. be able to.

Incorporation of Post-transcriptional Regulators In one embodiment, the polynucleotides, primary constructs, and / or mRNAs of the invention may comprise at least one post-transcriptional regulator. These post-transcriptional regulators can be, but are not limited to, small molecules, compounds, and regulatory sequences. As a non-limiting example, post-transcriptional regulation is performed using PTC Therapeutics Inc. using the GEMS ™ (Gene Expression Modulation by Small-Moleculars) screening technique. It can be achieved using the small molecule identified by (South Plainfield, NJ).

Post-transcriptional regulators can be gene expression regulators screened by the methods detailed in WO 2006022712, which are incorporated herein by reference in their entirety, or the gene expression regulators described herein. .. Methods for identifying RNA regulatory sequences involved in translational regulation are described in WO20040677728, which is incorporated herein by reference in its entirety, and identifies compounds that regulate untranslated region-dependent expression of genes. The method of doing so is described in WO20040655561, which is incorporated herein by reference in its entirety.

In one embodiment, the polynucleotide, primary construct, and / or mmRNA of the invention is at least one located in the 5'and / or 3'untranslated region of the polynucleotide, primary construct, and / or mmRNA of the invention. May include post-transcriptional regulatory regulators.

In another embodiment, the polynucleotides, primary constructs, and / or mmRNAs of the invention may contain at least one post-transcriptional regulatory regulator to regulate immature translation termination. Post-transcriptional regulators are compounds described in WO2004010106, WO200604456, WO200604682, WO200604453, and WO200604405, respectively, which are incorporated herein by reference in their entirety. , Or compounds found in these by the methods outlined above. As a non-limiting example, this compound may bind to a region of 28S ribosomal RNA to regulate immature translation termination (eg, WO2004010106, which is incorporated herein by reference in its entirety). See).

In one embodiment, the polynucleotides, primary constructs, and / or mMVs of the invention may contain at least one post-transcriptional regulator to alter protein expression. As a non-limiting example, the expression of VEGF is the compounds described in WO20051518857, WO200665480, WO2006605479, and WO2006058088, respectively, which are incorporated herein by reference in their entirety. , Or can be controlled using compounds that can be found by the methods described herein.

The polynucleotides, primary constructs, and / or mRNAs of the present invention may contain at least one post-transcriptional regulatory regulator to control translation. In one embodiment, the post-transcriptional regulatory regulator can be an RNA regulatory sequence. As a non-limiting example, RNA control sequences can be identified by the method described in WO 20060171903, which is incorporated herein by reference in its entirety.

III. Modifications In polynucleotides (primary constructs or mRNA molecules, etc.) herein, the term "modified" or optionally "modified" refers to modifications to A, G, U, or C ribonucleotides. In general, these terms are not intended herein to refer to ribonucleotide modifications at the naturally occurring 5'end mRNA cap moiety. In a polypeptide, the term "modification" refers to a modification compared to the 20 amino acids (parts) of the reference set.

This modification can be a variety of distinctly different modifications. In some embodiments, the coding region, adjacent region, and / or terminal region may contain one, two, or two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide, primary construct, or mmRNA introduced into the cell may exhibit reduced degradation in the cell as compared to the unmodified polynucleotide, primary construct, or mmRNA.

Polynucleotides, primary constructs, and mmRNAs may contain any useful modifications to sugars, nucleobases, or nucleoside bonds (eg, to bound phosphate / phosphodiester bonds / phosphodiester skeletons). One or more atoms of the pyrimidine nucleobase are replaced with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (eg, methyl or ethyl), or halo (eg, chloro or fluoro). It can be obtained or replaced by these. In certain embodiments, modifications (eg, one or more modifications) are present at each of the sugar and nucleoside bonds. Modifications according to the present invention are modifications of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof). could be. Further modifications are described herein.

As described herein, the polynucleotides, primary constructs, and mmRNAs of the invention do not substantially induce the innate immune response of the cells into which the mRNA is introduced. The characteristics of the induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc., and / or 3) termination or decrease of protein translation. included.

In certain embodiments, it may be desirable to intracellularly degrade the modified nucleic acid molecule introduced into the cell. Degradation of modified nucleic acid molecules, for example, may be preferred when precise timing of protein production is desired. Thus, in some embodiments, the invention provides a modified nucleic acid molecule containing a degradation domain that can act intracellularly in a directed manner. In another aspect, the disclosure provides a polynucleotide comprising a nucleoside or nucleotide that can disrupt the binding of a main groove interaction (eg, binding) partner with a polynucleotide (eg, a modified nucleotide with an unmodified nucleotide). By comparison, if it has a reduced binding affinity for the main groove interaction partner).

Polynucleotides, primary constructs, and mRNAs are other agents (eg, RNAi inducers, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple helix formation, aptamers. , Vector, etc.) can be optionally included. In some embodiments, the polynucleotide, primary construct, or mRNA may comprise one or more messenger RNAs (mRNAs) and one or more modified nucleosides or nucleotides (eg, mRNA molecules). Details about these polynucleotides, primary constructs, and mmRNA follow below.

Polynucleotides and Primary Constructs The polynucleotides, primary constructs, and mmRNAs of the invention are the first region of the binding nucleoside encoding the polypeptide of interest, the first adjacent region located at the 5'end of the first region. , And a second adjacent region located at the 3'end of the first region.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is of formula (Ia) or formula (Ia-1) :.

Containing n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof,

During the ceremony

U is, O, a ^{S, N} (R _{U) nu} or ^C (R _{U) nu,,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo , Or an optionally substituted alkyl,

--- is a single bond or absent,

H, halo, hydroxy, thiol if each of R ^1' , R ^2' , R ^{1 "} , R ^2" , R ¹ , R ² , R ³ , R ⁴ , and R ^{5 are present independently.} , Arbitrarily substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted Hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent And a combination of R3 and one or more of R1', R1 ", R2', R2", or R5 (eg, a combination of R1'and R3, a combination of R1 "and R3, R2' and R3. , A combination of R2 "and R3, or a combination of R5 and R3) can be combined to form an arbitrarily substituted alkylene or an arbitrarily substituted heteroalkylene, and with the carbon to which they are attached. Together, optionally substituted heterocyclyls (eg, bicyclic, tricyclic, or tetracyclic heterocyclyls) may be provided, of R5 and R1', R1 ", R2', or R2". Combinations with one or more of (eg, combinations of R1'and R5, combinations of R1'and R5, combinations of R2' and R5, or combinations of R2' and R5) have been arbitrarily substituted. Arbitrarily substituted heterocyclyls (eg, bicyclic, tricyclic, or tetracyclic heterocyclyls) that can form alkylene or optionally substituted heteroalkylenes and, together with the carbon to which they are attached, Can be provided, and combinations of R ⁴ with ^{one or more of R 1'} , R ^{1 "} , R ^2' , R ^2" , R ³ , or R ⁵ are optionally substituted together. Or alkylene or optionally substituted heteroalkylenes can be formed, and together with the carbon to which they are attached, optionally substituted heterocyclyls (eg, bicyclic, tricyclic, or tetracyclic heterocyclyls). ), Each of m'and m'is independently an integer of 0-3 (eg 0-2, 0-1, 1-3, or 1-2).

Each of Y ¹ , Y ² , and Y ³ is independently O, S, Se, -NR ^N1- , an optionally substituted alkylene, or an optionally substituted heteroalkylene, R in the formula. ^N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent.

Each Y ⁴ are, independently, H, hydroxy, thiol, substituted boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally Alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino,

Each Y ⁵ is independently O, S, Se, optionally substituted alkylene (eg, methylene), or optionally substituted heteroalkylene.

n is an integer of 1-100,000

B is a nucleic acid base (eg, purine, pyrimidine, or a derivative thereof), ^{a combination of B and R 1',} a combination of B and R ^2', a combination of B and R ^{1 "} , or a combination of B and R ^2". The combination of can optionally form a bicyclic group (eg, a bicyclic heterocyclyl) together with the carbon to which they are attached, or a combination of ^{B, R 1 "} , and R ^{3, or B.} , R ^{2 ”} , and R ³ optionally form tricyclic or tetracyclic groups (eg, tricyclic or tetracyclic heterocyclyls such as formulas (IIo)-(IIp) herein). Can be. In some embodiments, the polynucleotide, primary construct, or mmRNA comprises modified ribose. In some embodiments, the polynucleotide, primary construct, or mmRNA (eg, first region, first adjacent region, or second adjacent region) is represented by formulas (Ia-2)-(Ia-5). Includes n bound nucleosides with, or pharmaceutically acceptable salts or stereoisomers thereof.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is of formula (Ib) or formula (Ib-1) :.

Containing n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof,

During the ceremony

U is, O, a ^{S, N} (R _{U) nu} or ^C (R _{U) nu,,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo , Or an optionally substituted alkyl,

--- is a single bond or absent,

Each of R ¹ , R ^3' , R ^3' , and R ⁴ independently H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, Arbitrarily substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl, optionally substituted amino alkyl, amino alkenyl, optionally substituted, or an amino alkynyl optionally substituted, or is absent, together combination of R ¹ and R ³ combined, or R ¹ and R ³ of the ^'' Can form optionally substituted alkylenes or optionally substituted heteroalkylenes (eg, to produce locked nucleic acids).

Each R ⁵ was independently substituted with H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted. Aminoalkoxy, optionally substituted alkoxyalkoxy, or absent,

Each of Y ¹ , Y ² , and Y ³ is independently O, S, Se, -NR ^N1- , an optionally substituted alkylene, or an optionally substituted heteroalkylene, R in the formula. ^N1 is H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl.

Each Y ⁴ are, independently, H, hydroxy, thiol, substituted boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally Alkenyloxy, optionally substituted alkynyloxy, optionally substituted alkoxyalkoxy, or optionally substituted amino,

n is an integer of 1-100,000

B is a nucleobase.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first flanking region, or second flanking region) is of formula (Ic) :.

Containing n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof,

During the ceremony

U is, O, a ^{S, N} (R _{U) nu} or ^C (R _{U) nu,,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo , Or an optionally substituted alkyl,

--- is a single bond or absent,

Each of B ¹ , B ² , and B ³ was independently substituted with a nucleic acid base (eg, purine, pyrimidine, or derivative thereof described herein), H, halo, hydroxy, thiol, optionally. Alkoxy, optionally substituted alkoxy, optionally substituted alkoxyoxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted Amino, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkoxy, or optionally substituted aminoalkoxyl, B ¹ , B ² , and B. ^{Only one of the three} is a nucleic acid base,

Each of R ^b1 , R ^b2 , R ^b3 , R ³ , and R ⁵ was independently substituted with H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted. Alkoxyoxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl , Arbitrarily substituted aminoalkyl, optionally substituted aminoalkoxy, or optionally substituted aminoalkoxyl,

Each of Y ¹ , Y ² , and Y ³ is independently O, S, Se, -NR ^N1- , an optionally substituted alkylene, or an optionally substituted heteroalkylene, R in the formula. ^N1 is H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl.

Each Y ⁴ are, independently, H, hydroxy, thiol, substituted boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally Alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino,

Each Y ⁵ is independently O, S, Se, optionally substituted alkylene (eg, methylene), or optionally substituted heteroalkylene.

n is an integer of 1-100,000

The ring containing U may contain one or more double bonds.

In certain embodiments, the ring containing the U has no double bond between ^{the U-CB 3} R ^b3 or between the CB ³ R ^b3- C ^B2 R ^b2.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is of formula (Id):

Containing n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof,

During the ceremony

U is, O, a ^{S, N} (R _{U) nu} or ^C (R _{U) nu,,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo , Or an optionally substituted alkyl,

Each R ³ is independently H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkoxyoxy, optionally substituted alkynyloxy, optionally substituted. Aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted Aminoalkoxy, or optionally substituted aminoalkoxyl,

Each of Y ¹ , Y ² , and Y ³ is independently O, S, Se, -NR ^N1- , an optionally substituted alkylene, or an optionally substituted heteroalkylene, R in the formula. ^N1 is H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl.

Each Y ⁴ are, independently, H, hydroxy, thiol, substituted boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally Alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino,

Each Y ⁵ is independently O, S, optionally substituted alkylene (eg, methylene), or optionally substituted heteroalkylene.

n is an integer of 1-100,000

B is a nucleobase (eg, purine, pyrimidine, or derivative thereof).

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first flanking region, or second flanking region) is of formula (Ie) :.

Containing n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof,

During the ceremony

Each of U 'and U "are independently, O, a ^{S, N} (R _{U) nu} or ^C (R _{U) nu,,} wherein, nu is an integer of 0 to 2, each R ^U is independently an H, a halo, or an optionally substituted alkyl,

Each R ⁶ is independently H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkoxyoxy, optionally substituted alkynyloxy, optionally substituted. Aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted Aminoalkoxy, or optionally substituted aminoalkoxyl,

Each Y ^{5 'is,} independently, O, S, optionally substituted alkylene (e.g., methylene or ethylene), or an optionally substituted heteroalkylene,

n is an integer of 1-100,000

B is a nucleobase (eg, purine, pyrimidine, or derivative thereof).

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is of formula (If) or (If-1) :.

Containing n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof,

During the ceremony

Each of U 'and U "are independently, O, a _{^{S, N, N (R U}} ) nu or ^C (R _{U) nu,,} wherein, nu is an integer of 0 to 2, Each ^RU is an independently H, halo, or optionally substituted alkyl (eg, U'is O and U'is N).

--- is a single bond or absent,

Each of R ^1' , R ^2' , R ^{1 "} , R ^2" , R ³ and R ⁴ was independently substituted with H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted. Alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide , optionally substituted aryl, amino optionally substituted alkyl, amino alkenyl, optionally substituted, or an amino alkynyl optionally substituted, or is absent, the combination of R 1 ^'and R ^3, R ^{The combination of 1 "} and R ^3, the combination of R ^2'and R ³ , or the combination of R ^2" and R ³ can be combined to form an arbitrarily substituted alkylene or an arbitrarily substituted heteroalkylene. Each of m'and m'(eg, to produce locked nucleic acids) is independently an integer of 0-3 (eg, 0-2, 0-1, 1-3, or 1-2). can be,

Each of Y ¹ , Y ² , and Y ³ is independently O, S, Se, -NR ^N1- , an optionally substituted alkylene, or an optionally substituted heteroalkylene, R in the formula. ^N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent.

Each Y ⁴ are, independently, H, hydroxy, thiol, substituted boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally Alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino,

Each Y ⁵ is independently O, S, Se, optionally substituted alkylene (eg, methylene), or optionally substituted heteroalkylene.

n is an integer of 1-100,000

B is a nucleobase (eg, purine, pyrimidine, or derivative thereof).

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia), (Ia-1) to (Ia-3), (Ib) to (If), and (IIa) to (IIp). )), The ring containing U has one or two double bonds.

In some embodiments of polynucleotides, primary constructs, or mRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , ^R 1, R ^{1 ',} and ^{R 1 "each,} if present, in a. further embodiment a ^{H, R 2, R 2'} , and ^{R 2"} each, if present, independently H, halo (eg, fluoro), hydroxy, optionally substituted alkoxy (eg, methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, the alkoxyalkoxy is − (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is 1-10 (eg, 1-6 or 1). ~ 4), and each of s2 and s3 is an independent integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). _{, R'are} H or C 1-20 alkyl. In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R'is C _1-6 alkyl.

In some embodiments of polynucleotides, primary constructs, or mRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , ^R 2, R ^{2 ',} and ^{R 2 "each,} when present, is H. in a further ^{embodiment, R} 1, R ^1', and ^{R 1"} each, if present, independently H, halo (eg, fluoro), hydroxy, optionally substituted alkoxy (eg, methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, the alkoxyalkoxy is − (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is 1-10 (eg, 1-6 or 1). ~ 4), and each of s2 and s3 is an independent integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). _{, R'are} H or C 1-20 alkyl. In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R'is C _1-6 alkyl.

In some embodiments of polynucleotides, primary constructs, or mRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , ^R 3, ^{R 4,} and each ^{R 5} is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), Alternatively, it is an optionally substituted alkoxyalkoxy. In certain embodiments, R ³ is H, R ⁴ is H, R ⁵ is H, or all of R ³ , R ⁴ , and R ⁵ are H. In certain embodiments, R ³ is C _1-6 alkyl, R ⁴ is C _1-6 alkyl, R ⁵ is C _1-6 alkyl, or R ³ , R ⁴ , and R ⁵ All are C _1-6 alkyl. In certain embodiments, both R ³ and R ⁴ are H and R ⁵ is C _1-6 alkyl.

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , R ³ and R ⁵ together to form an arbitrarily substituted alkylene or an arbitrarily substituted heteroalkylene, and together with the carbon to which they are attached, trans-3', 4'similar. optionally substituted heterocyclyl of the body such as (e.g., bicyclic, tricyclic, or tetracyclic heterocyclyl) provides, R ³ and R ⁵ are taken together to form a heteroalkylene (e.g., - (CH ₂ ) _b1 O (CH ₂ ) _b2 O (CH ₂ ) _b3 −, and each of b1, b2, and b3 in the equation is independently an integer of 0 to 3).

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , R ³ and ^{one or more of R 1'} , R ^{1 "} , R ^2' , R ^2" , or R ⁵ together, optionally substituted alkylene or optionally substituted heteroalkylene And together with the carbons to which they bind, provide optionally substituted heterocyclyls (eg, bicyclic, tricyclic, or tetracyclic heterocyclyls) with R ³ and R ^1' , One or more of R ^{1 "} , R ^2' , R ^2" , or R ⁵ together form a heteroalkylene (eg-(CH ₂ ) _b1 O (CH _{2 ) b2} O (CH). ₂ ) _b3 −, where each of b1, b2, and b3 in the equation is an independent integer from 0 to 3).

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , R ⁵ and ^{one or more of R 1'} , R ^{1 "} , R ^2' , or R ^2" together to form an arbitrarily substituted alkylene or an arbitrarily substituted heteroalkylene. , Along with the carbon to which they are attached, provide optionally substituted heterocyclyls (eg, bicyclic, tricyclic, or tetracyclic heterocyclyls) with R ⁵ and R ^1' , R ^{1 ".} , R ^{2 ',} or one or more of ^{R 2 "together} form a heteroalkylene _{(e.g., - (CH 2) b1 O} (CH 2) b2 O (CH 2) b3 - met In the equation, each of b1, b2, and b3 is independently an integer of 0 to 3).

In some embodiments of polynucleotides, primary constructs, or mRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , Each Y ² is independently O, S, or -NR ^N1- , in which ^RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted. Alkynyl, or optionally substituted aryl. In certain embodiments, Y ² is NR ^N1- , in which ^{RN 1} is H or an optionally substituted alkyl (eg, C _1-6 alkyl, eg, methyl, ethyl, isopropyl, or n). -Propyl).

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , Each Y ³ is independently O or S.

In some embodiments of a polynucleotide, primary construct, or mRNA (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , R ¹ is H, and each R ² is independently H, halo (eg, fluoro), hydroxy, optionally substituted alkoxy (eg, methoxy or ethoxy), or optionally substituted alkoxy. It is an alkoxy (for example, − (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', and in the formula, s1 is 1 to 10 (for example, 1 to 6 or 1 to 4). It is an integer, and each of s2 and s3 is independently an integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R'is , H or C _{1 to 20} alkyl, for example, in the formula, s2 is 0, s1 is 1 or 2, and s3 is 0 or 1 (eg, _{R'is C 1 to 6} alkyl. Yes), each Y ² is independently O or -NR ^N1- , in which ^RN1 is H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkynyl. , Or optionally substituted aryl (eg, in the formula, ^RN1 is H or optionally substituted alkyl (eg, C _1-6 alkyl, eg, methyl, ethyl, isopropyl, or n-propyl). Each Y ³ is independently O or S (eg, S). In a further embodiment, R ³ is H, halo (eg, fluoro), hydroxy, optionally substituted alkyl. , Arbitrarily substituted alkoxy (eg, methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In a further embodiment, each Y ¹ is independently O or −NR ^N1 −. In the formula, ^RN1 is H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkynyl, or optionally substituted aryl (eg, in the formula, ^RN1 is H. Or optionally substituted alkyl (eg, C _1-6 alkyl, eg, methyl, ethyl, isopropyl, or n-propyl), each Y ⁴ is independently H, hydroxy, Thiols, optionally substituted alkyls, optionally substituted alkoxys, optionally substituted thioalkoxys, optionally substituted alkoxyalkoxys, or optionally substituted aminos.

In some embodiments of a polynucleotide, primary construct, or mRNA (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , Each R ¹ is independently H, halo (eg, fluoro), hydroxy, optionally substituted alkoxy (eg, methoxy or ethoxy), or optionally substituted alkoxyalkoxy (eg,-(eg,-(eg,). CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer of 1-10 (eg, 1-6 or 1-4) and of s2 and s3. Each is independently an integer from 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), where R'is H or C _{1 to 20} alkyl. (For example, in the formula, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, R'is C _1-6 alkyl), R ² is H, and each. Y ² is independently O or −NR ^N1- , in which ^RN1 is H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkynyl, or optionally. It is a substituted aryl (eg, in the formula, ^RN1 is H or an optionally substituted alkyl (eg, C _1-6 alkyl, eg, methyl, ethyl, isopropyl, or n-propyl)). Each Y ³ is independently O or S (eg, S). In a further embodiment, R ³ is H, halo (eg, fluoro), hydroxy, optionally substituted alkyl, optionally substituted. Alkoxy (eg, methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In a further embodiment, each Y ¹ is independently O or −NR ^N1 − and is R in the formula. ^N1 is H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkynyl, or optionally substituted aryl (eg, in the formula, ^RN1 is H or optionally substituted. _Alkoxy (eg, C 1-6 alkyl, eg, methyl, ethyl, isopropyl, or n-propyl), each Y ⁴ is independently H, hydroxy, thiol, Any substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)) The ring containing, U is in a β-D (eg, β-D-ribo) configuration.

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) The ring containing, U is in the α-L (eg, α-L-ribo) configuration.

In some embodiments of polynucleotides, primary constructs, or mmRNAs (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , one or more B, pseudouridine ([psi) or 5-methyl - never cytidine ^(m 5 C). In some embodiments, from about 10% to about 100% of n B nucleobases neither ^{m 5} C even [psi (e.g., 10% to 20% of n B, 10% to 35%, 10% -50%, 10% -60%, 10% -75%, 10% -90%, 10% -95%, 10% -98%, 10% -99%, 20% -35%, 20% ~ 50%, 20% -60%, 20% -75%, 20% -90%, 20% -95%, 20% -98%, 20% -99%, 20% -100%, 50% -60 %, 50% -75%, 50% -90%, 50% -95%, 50% -98%, 50% -99%, 50% -100%, 75% -90%, 75% -95%, 75% to 98%, 75% to 99%, and 75% to 100% is, ^m nor ⁵ C even [psi). In some embodiments, B is neither ^{ψ nor m 5 C.}

In some embodiments of polynucleotides, primary constructs, or nucleic acids (eg, formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-). 1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr)) , B is cytosine, guanine, uracil, and when it is unmodified nucleobase selected from adenine, at least one of Y ^{1, Y} 2 or Y ^3, is not a O.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含む。特定の実施形態において、Ｕは、ＯまたはＣ（Ｒ^Ｕ）_ｎｕであり、式中、ｎｕが、０〜２の整数であり、各Ｒ^Ｕが、独立して、Ｈ、ハロ、または任意に置換されたアルキルである（例えば、Ｕが、−ＣＨ_２−または−ＣＨ−である）。他の実施形態では、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５の各々は、独立して、Ｈ、ハロ、ヒドロキシ、チオール、任意に置換されたアルキル、任意に置換されたアルコキシ、任意に置換されたアルケニルオキシ、任意に置換されたアルキニルオキシ、任意に置換されたアミノアルコキシ、任意に置換されたアルコキシアルコキシ、任意に置換されたヒドロキシアルコキシ、任意に置換されたアミノ、アジド、任意に置換されたアリール、任意に置換されたアミノアルキル、任意に置換されたアミノアルケニル、任意に置換されたアミノアルキニルであるか、または不在であり（例えば、各Ｒ^１およびＲ^２は、独立して、Ｈ、ハロ、ヒドロキシ、任意に置換されたアルキル、または任意に置換されたアルコキシであり、各Ｒ^３およびＲ^４は、独立して、Ｈまたは任意に置換されたアルキルであり、Ｒ^５は、Ｈまたはヒドロキシであり）、−−−は、一重結合または二重結合である。 In some embodiments, the polynucleotide, primary construct, or mmRNA comprises modified ribose. In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is of formulas (IIa)-(IIc):

Includes n bound nucleosides with, or pharmaceutically acceptable salts or stereoisomers thereof. In certain embodiments, U is O or ^C (R _{U) nu,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo, or optionally, It is a substituted alkyl (eg, U is -CH _2- or -CH-). In other embodiments, each of R ¹ , R ² , R ³ , R ⁴ , and R ⁵ is independently H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy. , Arbitrarily substituted alkoxyoxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, Arbitrarily substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkoxy, optionally substituted aminoalkoxyl, or absent (eg, each R ¹ and R ² are independent). H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, and each R ³ and R ⁴ are independently H or optionally substituted alkyl, R. ⁵ is H or hydroxy), --- is a single bond or a double bond.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含む。いくつかの実施形態において、Ｕは、ＯまたはＣ（Ｒ^Ｕ）_ｎｕであり、式中、ｎｕが、０〜２の整数であり、各Ｒ^Ｕが、独立して、Ｈ、ハロ、または任意に置換されたアルキルである（例えば、Ｕは、−ＣＨ_２−または−ＣＨ−である）。他の実施形態では、Ｒ^１およびＲ^２ の各々は、独立して、Ｈ、ハロ、ヒドロキシ、チオール、任意に置換されたアルキル、任意に置換されたアルコキシ、任意に置換されたアルケニルオキシ、任意に置換されたアルキニルオキシ、任意に置換されたアミノアルコキシ、任意に置換されたアルコキシアルコキシ、任意に置換されたヒドロキシアルコキシ、任意に置換されたアミノ、アジド、任意に置換されたアリール、任意に置換されたアミノアルキル、任意に置換されたアミノアルケニル、任意に置換されたアミノアルキニルであるか、または不在である（例えば、各Ｒ^１およびＲ^２は、独立して、Ｈ、ハロ、ヒドロキシ、任意に置換されたアルキル、または任意に置換されたアルコキシ、例えば、Ｈ、ハロ、ヒドロキシ、アルキル、またはアルコキシである）。特定の実施形態において、Ｒ^２は、ヒドロキシまたは任意に置換されたアルコキシ（例えば、メトキシ、エトキシ、または本明細書に記載のいずれか）である。 In certain embodiments, the polynucleotide or mmRNA is expressed in formulas (IIb-1)-(IIb-2):

Includes n bound nucleosides with, or pharmaceutically acceptable salts or stereoisomers thereof. In some embodiments, U is O or ^C (R _{U) nu,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo, or optionally, It is an alkyl substituted with (eg, U is -CH _2- or -CH-). In other embodiments, ^{each of R 1} and R ² is independently H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally. Alkinyloxy substituted with, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl, optionally substituted Aminoalkyl, optionally substituted aminoalkoxy, optionally substituted aminoalkoxyl, or absent (eg, each R ¹ and R ² are independently H, halo, hydroxy, optional. Alkoxy substituted with, or alkoxy optionally substituted (eg, H, halo, hydroxy, alkyl, or alkoxy). In certain embodiments, R ² is hydroxy or optionally substituted alkoxy (eg, either methoxy, ethoxy, or any of those described herein).

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含む。いくつかの実施形態において、Ｕは、ＯまたはＣ（Ｒ^Ｕ）_ｎｕであり、式中、ｎｕが、０〜２の整数であり、各Ｒ^Ｕが、独立して、Ｈ、ハロ、または任意に置換されたアルキルである（例えば、Ｕが、−ＣＨ_２−または−ＣＨ−である）。いくつかの実施形態において、Ｒ^１、Ｒ^２、およびＲ^３の各々は、独立して、Ｈ、ハロ、ヒドロキシ、チオール、任意に置換されたアルキル、任意に置換されたアルコキシ、任意に置換されたアルケニルオキシ、任意に置換されたアルキニルオキシ、任意に置換されたアミノアルコキシ、任意に置換されたアルコキシアルコキシ、任意に置換されたヒドロキシアルコキシ、任意に置換されたアミノ、アジド、任意に置換されたアリール、任意に置換されたアミノアルキル、任意に置換されたアミノアルケニル、任意に置換されたアミノアルキニルであるか、または不在である（例えば、各Ｒ^１およびＲ^２は、独立して、Ｈ、ハロ、ヒドロキシ、任意に置換されたアルキル、または任意に置換されたアルコキシ、例えば、Ｈ、ハロ、ヒドロキシ、アルキル、またはアルコキシであり、各Ｒ^３は、独立して、Ｈまたは任意に置換されたアルキル）である）。特定の実施形態において、Ｒ^２は、任意に置換されたアルコキシ（例えば、メトキシもしくはエトキシ、または本明細書に記載のいずれか）である。特定の実施形態において、Ｒ^１は、任意に置換されたアルキルであり、Ｒ^２は、ヒドロキシである。他の実施形態では、Ｒ^１は、ヒドロキシであり、Ｒ^２は、任意に置換されたアルキルである。さらなる実施形態において、Ｒ^３は、任意に置換されたアルキルである。 In certain embodiments, the polynucleotide, primary construct, or mmRNA is expressed in formulas (IIc-1) through (IIc-4):

Includes n bound nucleosides with, or pharmaceutically acceptable salts or stereoisomers thereof. In some embodiments, U is O or ^C (R _{U) nu,} wherein, nu is an integer of 0 to 2, each ^{R U,,} independently, H, halo, or optionally, It is an alkyl substituted with (eg, U is -CH _2- or -CH-). In some embodiments, each of R ¹ , R ² , and R ³ is independently substituted with H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted. Alkoxyoxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted Aryl, optionally substituted aminoalkyl, optionally substituted aminoalkoxy, optionally substituted aminoalkoxyl or absent (eg, each R ¹ and R ² are independently H, Halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, such as H, halo, hydroxy, alkyl, or alkoxy, each R ³ being independently H or optionally substituted. Alkoxy)). In certain embodiments, R ² is an optionally substituted alkoxy (eg, either methoxy or ethoxy, or either described herein). In certain embodiments, R ¹ is an optionally substituted alkyl and R ² is a hydroxy. In other embodiments, R ¹ is hydroxy and R ² is an optionally substituted alkyl. In a further embodiment, R ³ is optionally substituted alkyl.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含む。 In some embodiments, the polynucleotide, primary construct, or mmRNA comprises acyclic modified ribose. In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is represented by formulas (IId)-(IIf):

Includes n bound nucleosides with, or pharmaceutically acceptable salts or stereoisomers thereof.

のｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含む。 In some embodiments, the polynucleotide, primary construct, or mmRNA comprises an acyclic modified hexitol. In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first flanking region, or second flanking region) is of formulas (IIg)-(IIj) :.

Includes n bound nucleosides, or pharmaceutically acceptable salts or stereoisomers thereof.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、式中、Ｒ^１’、Ｒ^１”、Ｒ^２’、およびＲ^２”の各々が、独立して、Ｈ、ハロ、ヒドロキシ、任意に置換されたアルキル、任意に置換されたアルコキシ、任意に置換されたアルケニルオキシ、任意に置換されたアルキニルオキシ、任意に置換されたアミノアルコキシ、任意に置換されたアルコキシアルコキシであるか、または不在であり、Ｒ^２’とＲ^３の組み合わせまたはＲ^２”とＲ^３の組み合わせが一緒になって、任意に置換されたアルキレンまたは任意に置換されたヘテロアルキレンを形成し得る。 In some embodiments, the polynucleotide, primary construct, or mmRNA comprises a sugar moiety having a shortened or extended ribose ring. In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first adjacent region, or second adjacent region) is of formulas (IIk)-(IIm) :.

^{Each of R 1'} , R ^{1 "} , R ^2' , and R ^{2" in} the formula contains n bound nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, independently of each other. , H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy or where absent, becomes R 2 ^'combination of R ³ or R ^{2 "and} with the combination of R ³ are forming a heteroalkylene substituted with alkylene or optionally substituted optionally Can be done.

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、式中、Ｒ^３’が、Ｏ、Ｓ、または−ＮＲ^Ｎ１−であり、式中、Ｒ^Ｎ１が、Ｈ、任意に置換されたアルキル、任意に置換されたアルケニル、任意に置換されたアルキニル、または任意に置換されたアリールであり、Ｒ^３”が、任意に置換されたアルキレン（例えば、−ＣＨ_２−、−ＣＨ_２ＣＨ_２−、もしくは−ＣＨ_２ＣＨ_２ＣＨ_２−）または任意に置換されたヘテロアルキレン（例えば、−ＣＨ_２ＮＨ−、−ＣＨ_２ＣＨ_２ＮＨ−、−ＣＨ_２ＯＣＨ_２−、もしくは−ＣＨ_２ＣＨ_２ＯＣＨ_２−）である（例えば、Ｒ^３’がＯであり、Ｒ^３”が任意に置換されたアルキレン（例えば、−ＣＨ_２−、−ＣＨ_２ＣＨ_２−、もしくは−ＣＨ_２ＣＨ_２ＣＨ_２−）である）。 In some embodiments, the polynucleotide, primary construct, or mmRNA comprises locked modified ribose. In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first flanking region, or second flanking region) is of formula (IIn):

It includes n bound nucleoside or a pharmaceutically acceptable salt or stereoisomer thereof, having the formula, R ^{3 'is,} O, S, or ^{-NR N1} - a and, ^{wherein, R N1} is , H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl, where R3 ^" is optionally substituted alkylene (eg, -CH). _{2 -, - CH 2 CH 2} -, or _-CH 2 _CH 2 CH ₂ -), or an optionally substituted heteroalkylene _{(e.g., -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 OCH 2 -, or _{-CH 2 CH} 2 OCH 2 - is a) (e.g., R ^{3 'is} O, ^{alkylene R 3 "is} optionally substituted _{(e.g., -CH 2 -, - CH 2} CH 2 -, Or −CH ₂ CH ₂ CH ₂ −)).

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、式中、Ｒ^３’が、Ｏ、Ｓ、または−ＮＲ^Ｎ１−であり、式中、Ｒ^Ｎ１が、Ｈ、任意に置換されたアルキル、任意に置換されたアルケニル、任意に置換されたアルキニル、または任意に置換されたアリールであり、Ｒ^３”が、任意に置換されたアルキレン（例えば、−ＣＨ_２−、−ＣＨ_２ＣＨ_２−、もしくは−ＣＨ_２ＣＨ_２ＣＨ_２−）または任意に置換されたヘテロアルキレン（例えば、−ＣＨ_２ＮＨ−、−ＣＨ_２ＣＨ_２ＮＨ−、−ＣＨ_２ＯＣＨ_２−、もしくは−ＣＨ_２ＣＨ_２ＯＣＨ_２−）である（例えば、Ｒ^３’がＯであり、Ｒ^３”が任意に置換されたアルキレン（例えば、−ＣＨ_２−、−ＣＨ_２ＣＨ_２−、もしくは−ＣＨ_２ＣＨ_２ＣＨ_２−）である）。 In some embodiments, the polynucleotide, primary construct, or mMV is of formulas (IIn-1) to (II-n2):

It includes n bound nucleoside or a pharmaceutically acceptable salt or stereoisomer thereof, having the formula, R ^{3 'is,} O, S, or ^{-NR N1} - a and, ^{wherein, R N1} is , H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl, where R3 ^" is optionally substituted alkylene (eg, -CH). _{2 -, - CH 2 CH 2} -, or _-CH 2 _CH 2 CH ₂ -), or an optionally substituted heteroalkylene _{(e.g., -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 OCH 2 -, or _{-CH 2 CH} 2 OCH 2 - is a) (e.g., R ^{3 'is} O, ^{alkylene R 3 "is} optionally substituted _{(e.g., -CH 2 -, - CH 2} CH 2 -, Or −CH ₂ CH ₂ CH ₂ −)).

を有するｎ個の結合ヌクレオシド、またはその薬学的に許容される塩もしくは立体異性体を含み、式中、Ｒ^１２ａ、Ｒ^１２ｃ、Ｔ^１’、Ｔ^１”、Ｔ^２’、Ｔ^２”、Ｖ^１、およびＶ^３は、本明細書に記載の通りである。 In some embodiments, the polynucleotide, primary construct, or mMV comprises locked modified ribose that forms tetracyclic heterocyclyl. In some embodiments, the polynucleotide, primary construct, or mMV (eg, first region, first flanking region, or second flanking region) is of formula (IIo):

Containing n bound nucleosides with, or pharmaceutically acceptable salts or stereoisomers thereof, in the formulas R ^12a , R ^12c , T ^1' , T ^{1 "} , T ^2' , T ^2" , V. ^1, and V ³ are as described herein.

Any of the formulas for polynucleotides, primary constructs, or mmRNAs may comprise one or more nucleobases described herein (eg, formulas (b1)-(b43)).

を有するｎ個のヌクレオシドを含み、この方法は、本明細書で定義される式（ＩＩＩａ）：

の化合物を、ＲＮＡポリメラーゼおよびｃＤＮＡ鋳型と反応させることを含む。 In one embodiment, the invention provides a method of preparing a polynucleotide, primary construct, or mmRNA, which polynucleotide is defined herein in formula (Ia):

The method comprises n nucleosides having a formula (IIIa) as defined herein:

Includes reacting a compound of the above with an RNA polymerase and a cDNA template.

In a further embodiment, the invention provides a method of amplifying a polynucleotide, primary construct, or mRNA containing at least one nucleotide (eg, an mRNA molecule), the method of which is defined herein. It comprises reacting the compound of (IIIa) with a primer, a cDNA template, and an RNA polymerase.

を有するｎ個のヌクレオシドを含み、この方法は、本明細書で定義される式（ＩＩＩａ−１）：

の化合物を、ＲＮＡポリメラーゼおよびｃＤＮＡ鋳型と反応させることを含む。 In one embodiment, the invention provides a method of preparing a polynucleotide, a primary construct, or mmRNA containing at least one nucleotide (eg, an mm mRNA molecule), the polynucleotide being defined herein. Equation (Ia):

The method comprises n nucleosides having the formula (IIIa-1) as defined herein:

Includes reacting a compound of the above with an RNA polymerase and a cDNA template.

In a further embodiment, the invention provides a method of amplifying a polynucleotide, primary construct, or mmRNA containing at least one nucleotide (eg, an mm mRNA molecule).

It comprises reacting a compound of formula (IIIa-1) as defined herein with a primer, a cDNA template, and an RNA polymerase.

を有するｎ個のヌクレオシドを含み、この方法は、本明細書で定義される式（ＩＩＩａ−２）：

の化合物を、ＲＮＡポリメラーゼおよびｃＤＮＡ鋳型と反応させることを含む。 In one embodiment, the invention provides a method of preparing a modified mRNA containing at least one nucleotide (eg, an mRNA molecule), wherein the polynucleotide is of formula (Ia-2) as defined herein. :

The method comprises n nucleosides having the formula (IIIa-2) as defined herein:

Includes reacting a compound of the above with an RNA polymerase and a cDNA template.

In a further embodiment, the invention provides a method of amplifying a modified mRNA containing at least one nucleotide (eg, an mRNA molecule).

It comprises reacting a compound of formula (IIIa-2) as defined herein with a primer, a cDNA template, and an RNA polymerase.

In some embodiments, the reaction can be repeated 1 to about 7,000 times. In any of the embodiments herein, B can be a nucleobase of formulas (b1)-(b43).

Polynucleotides, primary constructs, and mmRNAs can optionally contain the 5'and / or 3'adjacent regions described herein.

、またはその薬学的に許容される塩もしくは立体異性体を有し、式中、置換基は、本明細書に記載のもの（例えば、式（Ｉａ）および（Ｉａ−１））であり、Ｂが、シトシン、グアニン、ウラシル、およびアデニンから選択される未修飾核酸塩基であるとき、Ｙ^１、Ｙ^２、またはＹ^３のうちの少なくとも１つは、Ｏではない。 Modified RNA (mmRNA) Molecules The present invention also includes building blocks of modified RNA (mmRNA) molecules, such as modified ribonucleosides, modified ribonucleotides. For example, these building blocks can be useful in the preparation of polynucleotides, primary constructs, or mmRNAs of the invention. In some embodiments, the building block molecule is of formula (IIIa) or (IIIa-1) :.

, Or a pharmaceutically acceptable salt or stereoisomer thereof, wherein in the formula, the substituents are those described herein (eg, formulas (Ia) and (Ia-1)), and B. but cytosine, guanine, uracil, and when it is unmodified nucleobase selected from adenine, at least one of Y ^{1, Y} 2 or Y ^3, is not a O.

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。特定の実施形態において、式（ＩＶａ）または（ＩＶｂ）は、修飾ウラシル（例えば、式（ｂ１）〜（ｂ９）、（ｂ２１）〜（ｂ２３）、および（ｂ２８）〜（ｂ３１）のうちのいずれか１つ、例えば、式（ｂ１）、（ｂ８）、（ｂ２８）、（ｂ２９）、または（ｂ３０））と組み合わせられる。特定の実施形態において、式（ＩＶａ）または（ＩＶｂ）は、修飾シトシン（例えば、式（ｂ１０）〜（ｂ１４）、（ｂ２４）、（ｂ２５）、および（ｂ３２）〜（ｂ３６）のうちのいずれか１つ、例えば、式（ｂ１０）または（ｂ３２））と組み合わせられる。特定の実施形態において、式（ＩＶａ）または（ＩＶｂ）は、修飾グアニン（例えば、式（ｂ１５）〜（ｂ１７）および（ｂ３７）〜（ｂ４０）のうちのいずれか１つ）と組み合わせられる。特定の実施形態において、式（ＩＶａ）または（ＩＶｂ）は、修飾アデニン（例えば、式（ｂ１８）〜（ｂ２０）および（ｂ４１）〜（ｂ４３）のうちのいずれか１つ）と組み合わせられる。 In some embodiments, the building block molecules that can be integrated into the polynucleotide, primary construct, or mmRNA are of formulas (IVa)-(IVb):

Alternatively, it has a pharmaceutically acceptable salt or stereoisomer thereof, and in the formula, B is one described herein (for example, any one of (b1) to (b43)). .. In certain embodiments, the formula (IVa) or (IVb) is any of the modified uracils (eg, formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31). In combination with one, eg, equations (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, the formula (IVa) or (IVb) is any of the modified cytosines (eg, formulas (b10)-(b14), (b24), (b25), and (b32)-(b36). In combination with one, eg, equation (b10) or (b32)). In certain embodiments, formula (IVa) or (IVb) is combined with modified guanine (eg, any one of formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, formula (IVa) or (IVb) is combined with modified adenine (eg, any one of formulas (b18)-(b20) and (b41)-(b43)).

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。特定の実施形態において、式（ＩＶｃ）〜（ＩＶｋ）のうちの１つは、修飾ウラシル（例えば、式（ｂ１）〜（ｂ９）、（ｂ２１）〜（ｂ２３）、および（ｂ２８）〜（ｂ３１）のうちのいずれか１つ、例えば、式（ｂ１）、（ｂ８）、（ｂ２８）、（ｂ２９）、または（ｂ３０））と組み合わせられる。特定の実施形態において、式（ＩＶｃ）〜（ＩＶｋ）のうちの１つは、修飾シトシン（例えば、式（ｂ１０）〜（ｂ１４）、（ｂ２４）、（ｂ２５）、および（ｂ３２）〜（ｂ３６）のうちのいずれか１つ、例えば、式（ｂ１０）または（ｂ３２））と組み合わせられる。特定の実施形態において、式（ＩＶｃ）〜（ＩＶｋ）のうちの１つは、修飾グアニン（例えば、式（ｂ１５）〜（ｂ１７）および（ｂ３７）〜（ｂ４０）のうちのいずれか１つ）と組み合わせられる。特定の実施形態において、式（ＩＶｃ）〜（ＩＶｋ）のうちの１つは、修飾アデニン（例えば、式（ｂ１８）〜（ｂ２０）および（ｂ４１）〜（ｂ４３）のうちのいずれか１つ）と組み合わせられる。 In some embodiments, the building block molecules that can be integrated into the polynucleotide, primary construct, or mmRNA are of formulas (IVc)-(IVk):

Alternatively, it has a pharmaceutically acceptable salt or stereoisomer thereof, and in the formula, B is one described herein (for example, any one of (b1) to (b43)). .. In certain embodiments, one of the formulas (IVc)-(IVk) is a modified uracil (eg, formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31). ), For example, in combination with the formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of formulas (IVc)-(IVk) is a modified cytosine (eg, formulas (b10)-(b14), (b24), (b25), and (b32)-(b36). ), For example, in combination with the formula (b10) or (b32)). In certain embodiments, one of formulas (IVc)-(IVk) is a modified guanine (eg, any one of formulas (b15)-(b17) and (b37)-(b40)). Combined with. In certain embodiments, one of formulas (IVc)-(IVk) is a modified adenine (eg, any one of formulas (b18)-(b20) and (b41)-(b43)). Combined with.

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。 In other embodiments, the building block molecule that can be integrated into a polynucleotide, primary construct, or mmRNA is of formula (Va) or (Vb) :.

Alternatively, it has a pharmaceutically acceptable salt or stereoisomer thereof, and in the formula, B is one described herein (for example, any one of (b1) to (b43)). ..

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。特定の実施形態において、式（ＩＸａ）〜（ＩＸｄ）のうちの１つは、修飾ウラシル（例えば、式（ｂ１）〜（ｂ９）、（ｂ２１）〜（ｂ２３）、および（ｂ２８）〜（ｂ３１）のうちのいずれか１つ、例えば、式（ｂ１）、（ｂ８）、（ｂ２８）、（ｂ２９）、または（ｂ３０））と組み合わせられる。特定の実施形態において、式（ＩＸａ）〜（ＩＸｄ）のうちの１つは、修飾シトシン（例えば、式（ｂ１０）〜（ｂ１４）、（ｂ２４）、（ｂ２５）、および（ｂ３２）〜（ｂ３６）のうちのいずれか１つ、例えば、式（ｂ１０）または（ｂ３２））と組み合わせられる。特定の実施形態において、式（ＩＸａ）〜（ＩＸｄ）のうちの１つは、修飾グアニン（例えば、式（ｂ１５）〜（ｂ１７）および（ｂ３７）〜（ｂ４０）のうちのいずれか１つ）と組み合わせられる。特定の実施形態において、式（ＩＸａ）〜（ＩＸｄ）のうちの１つは、修飾アデニン（例えば、式（ｂ１８）〜（ｂ２０）および（ｂ４１）〜（ｂ４３）のうちのいずれか１つ）と組み合わせられる。 In other embodiments, the building block molecules that can be integrated into a polynucleotide, primary construct, or mmRNA are of formulas (IXa)-(IXd):

Alternatively, it has a pharmaceutically acceptable salt or stereoisomer thereof, and in the formula, B is one described herein (for example, any one of (b1) to (b43)). .. In certain embodiments, one of the formulas (IXa)-(IXd) is a modified uracil (eg, formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31). ), For example, in combination with the formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of the formulas (IXa)-(IXd) is a modified cytosine (eg, formulas (b10)-(b14), (b24), (b25), and (b32)-(b36). ), For example, in combination with the formula (b10) or (b32)). In certain embodiments, one of the formulas (IXa)-(IXd) is a modified guanine (eg, any one of the formulas (b15)-(b17) and (b37)-(b40)). Combined with. In certain embodiments, one of the formulas (IXa)-(IXd) is a modified adenine (eg, any one of the formulas (b18)-(b20) and (b41)-(b43)). Combined with.

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。特定の実施形態において、式（ＩＸｅ）〜（ＩＸｇ）のうちの１つは、修飾ウラシル（例えば、式（ｂ１）〜（ｂ９）、（ｂ２１）〜（ｂ２３）、および（ｂ２８）〜（ｂ３１）のうちのいずれか１つ、例えば、式（ｂ１）、（ｂ８）、（ｂ２８）、（ｂ２９）、または（ｂ３０））と組み合わせられる。特定の実施形態において、式（ＩＸｅ）〜（ＩＸｇ）のうちの１つは、修飾シトシン（例えば、式（ｂ１０）〜（ｂ１４）、（ｂ２４）、（ｂ２５）、および（ｂ３２）〜（ｂ３６）のうちのいずれか１つ、例えば、式（ｂ１０）または（ｂ３２））と組み合わせられる。特定の実施形態において、式（ＩＸｅ）〜（ＩＸｇ）のうちの１つは、修飾グアニン（例えば、式（ｂ１５）〜（ｂ１７）および（ｂ３７）〜（ｂ４０）のうちのいずれか１つ）と組み合わせられる。特定の実施形態において、式（ＩＸｅ）〜（ＩＸｇ）のうちの１つは、修飾アデニン（例えば、式（ｂ１８）〜（ｂ２０）および（ｂ４１）〜（ｂ４３）のうちのいずれか１つ）と組み合わせられる。 In other embodiments, the building block molecules that can be integrated into a polynucleotide, primary construct, or mmRNA are of formulas (IXe)-(IXg):

Alternatively, it has a pharmaceutically acceptable salt or stereoisomer thereof, and in the formula, B is one described herein (for example, any one of (b1) to (b43)). .. In certain embodiments, one of the formulas (IXe)-(IXg) is a modified uracil (eg, formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31). ), For example, in combination with the formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of the formulas (IXe)-(IXg) is a modified cytosine (eg, formulas (b10)-(b14), (b24), (b25), and (b32)-(b36). ), For example, in combination with the formula (b10) or (b32)). In certain embodiments, one of the formulas (IXe)-(IXg) is a modified guanine (eg, any one of the formulas (b15)-(b17) and (b37)-(b40)). Combined with. In certain embodiments, one of the formulas (IXe)-(IXg) is a modified adenine (eg, any one of the formulas (b18)-(b20) and (b41)-(b43)). Combined with.

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。特定の実施形態において、式（ＩＸｈ）〜（ＩＸｋ）のうちの１つは、修飾ウラシル（例えば、式（ｂ１）〜（ｂ９）、（ｂ２１）〜（ｂ２３）、および（ｂ２８）〜（ｂ３１）のうちのいずれか１つ、例えば、式（ｂ１）、（ｂ８）、（ｂ２８）、（ｂ２９）、または（ｂ３０））と組み合わせられる。特定の実施形態において、式（ＩＸｈ）〜（ＩＸｋ）のうちの１つは、修飾シトシン（例えば、式（ｂ１０）〜（ｂ１４）、（ｂ２４）、（ｂ２５）、および（ｂ３２）〜（ｂ３６）のうちのいずれか１つ、例えば、式（ｂ１０）または（ｂ３２））と組み合わせられる。特定の実施形態において、式（ＩＸｈ）〜（ＩＸｋ）のうちの１つは、修飾グアニン（例えば、式（ｂ１５）〜（ｂ１７）および（ｂ３７）〜（ｂ４０）のうちのいずれか１つ）と組み合わせられる。特定の実施形態において、式（ＩＸｈ）〜（ＩＸｋ）のうちの１つは、修飾アデニン（例えば、式（ｂ１８）〜（ｂ２０）および（ｂ４１）〜（ｂ４３）のうちのいずれか１つ）と組み合わせられる。 In other embodiments, the building block molecules that can be integrated into the polynucleotide, primary construct, or mmRNA are of formulas (IXh) to (IXk):

Alternatively, it has a pharmaceutically acceptable salt or stereoisomer thereof, and in the formula, B is one described herein (for example, any one of (b1) to (b43)). .. In certain embodiments, one of the formulas (IXh) to (IXk) contains modified uracils (eg, formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31). ), For example, in combination with the formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of the formulas (IXh)-(IXk) is a modified cytosine (eg, formulas (b10)-(b14), (b24), (b25), and (b32)-(b36). ), For example, in combination with the formula (b10) or (b32)). In certain embodiments, one of the formulas (IXh) to (IXk) is a modified guanine (eg, any one of the formulas (b15) to (b17) and (b37) to (b40)). Combined with. In certain embodiments, one of the formulas (IXh) to (IXk) is a modified adenine (eg, any one of the formulas (b18) to (b20) and (b41) to (b43)). Combined with.

またはその薬学的に許容される塩もしくは立体異性体を有し、式中、各ｒ１およびｒ２は、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数であり、Ｂは、本明細書に記載のもの（例えば、（ｂ１）〜（ｂ４３）のうちのいずれか１つ）である。特定の実施形態において、式（ＩＸｌ）〜（ＩＸｒ）のうちの１つは、修飾ウラシル（例えば、式（ｂ１）〜（ｂ９）、（ｂ２１）〜（ｂ２３）、および（ｂ２８）〜（ｂ３１）のうちのいずれか１つ、例えば、式（ｂ１）、（ｂ８）、（ｂ２８）、（ｂ２９）、または（ｂ３０））と組み合わせられる。特定の実施形態において、式（ＩＸｌ）〜（ＩＸｒ）のうちの１つは、修飾シトシン（例えば、式（ｂ１０）〜（ｂ１４）、（ｂ２４）、（ｂ２５）、および（ｂ３２）〜（ｂ３６）のうちのいずれか１つ、例えば、式（ｂ１０）または（ｂ３２））と組み合わせられる。特定の実施形態において、式（ＩＸｌ）〜（ＩＸｒ）のうちの１つは、修飾グアニン（例えば、式（ｂ１５）〜（ｂ１７）および（ｂ３７）〜（ｂ４０）のうちのいずれか１つ）と組み合わせられる。特定の実施形態において、式（ＩＸｌ）〜（ＩＸｒ）のうちの１つは、修飾アデニン（例えば、式（ｂ１８）〜（ｂ２０）および（ｂ４１）〜（ｂ４３）のうちのいずれか１つ）と組み合わせられる。 In other embodiments, the building block molecules that can be integrated into a polynucleotide, primary construct, or mmRNA are of formulas (IXl)-(IXr) :.

Or have a pharmaceutically acceptable salt or stereoisomer thereof, in which each r1 and r2 are independently 0-5 (eg 0-3, 1-3, or 1-5). It is an integer, and B is that described herein (eg, any one of (b1) to (b43)). In certain embodiments, one of the formulas (IXl)-(IXr) is a modified uracil (eg, formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31). ), For example, in combination with the formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of the formulas (IXl)-(IXr) is a modified cytosine (eg, formulas (b10)-(b14), (b24), (b25), and (b32)-(b36). ), For example, in combination with the formula (b10) or (b32)). In certain embodiments, one of the formulas (IXl) to (IXr) is a modified guanine (eg, any one of the formulas (b15) to (b17) and (b37) to (b40)). Combined with. In certain embodiments, one of the formulas (IXl) to (IXr) is a modified adenine (eg, any one of the formulas (b18) to (b20) and (b41) to (b43)). Combined with.

またはその薬学的に許容される塩もしくは立体異性体からなる群から選択され得、式中、各ｒは、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数である。 In some embodiments, the building block molecule that can be integrated into a polynucleotide, primary construct, or mmRNA is

Alternatively, it may be selected from the group consisting of pharmaceutically acceptable salts or stereoisomers thereof, in which each r is independently 0-5 (eg 0-3, 1-3, or 1-5). ) Is an integer.

、またはその薬学的に許容される塩もしくは立体異性体からなる群から選択され得、式中、各ｒは、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数であり、ｓ１は、本明細書に記載の通りである。 In some embodiments, the building block molecule that can be integrated into a polynucleotide, primary construct, or mmRNA is

, Or a group consisting of pharmaceutically acceptable salts or stereoisomers thereof, wherein each r is independently 0-5 (eg, 0-3, 1-3, or 1-) in the formula. It is an integer of 5), and s1 is as described in the present specification.

In some embodiments, the building block molecule that can be integrated into a nucleic acid (eg, RNA, mRNA, polynucleotide, primary construct, or mRNA) is a modified uridine (eg, one selected from the group consisting of: Or a pharmaceutically acceptable salt or steric isomer thereof, wherein Y ¹ , Y ³ , Y ⁴ , Y ⁶ , and r are as described herein (eg, each r is). , Independently, 0-5, eg, an integer of 0-3, 1-3, or 1-5):

例えば、ポリヌクレオチド、一次構築物、またはｍｍＲＮＡに組み込まれ得るビルディングブロック分子は、

、またはその薬学的に許容される塩もしくは立体異性体であり得、式中、各ｒは、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数である。 In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified cytidine (eg, one selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof). Alternatively, they are stereoisomers, in which Y ¹ , Y ³ , Y ⁴ , Y ⁶ and r are as described herein (eg, each r is independently 0-5, For example, an integer of 0-3, 1-3, or 1-5):

For example, a polynucleotide, a primary construct, or a building block molecule that can be integrated into an mRNA

, Or a pharmaceutically acceptable salt or stereoisomer thereof, in which each r is independently an integer of 0-5 (eg, 0-3, 1-3, or 1-5). Is.

In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified adenosine (eg, one selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof). Alternatively, they are stereoisomers, in which Y ¹ , Y ³ , Y ⁴ , Y ⁶ and r are as described herein (eg, each r is independently 0-5, For example, an integer of 0-3, 1-3, or 1-5):

In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified guanosine (eg, one selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof). Alternatively, they are stereoisomers, in which Y ¹ , Y ³ , Y ⁴ , Y ⁶ and r are as described herein (eg, each r is independently 0-5, For example, an integer of 0-3, 1-3, or 1-5):

、またはその薬学的に許容される塩もしくは立体異性体であり得、式中、各ｒは、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数である。 In some embodiments, the chemical modification is the substitution of the C-5 C group N of the ring (eg, pyrimidine nucleoside, eg cytosine or uracil) with the N of the ring (eg C-5> CH group> NR). Substitution with an ^{N1 group (wherein RN} 1 is H or an optionally substituted alkyl)) can be included. For example, a polynucleotide, a primary construct, or a building block molecule that can be integrated into an mRNA

, Or a pharmaceutically acceptable salt or stereoisomer thereof, in which each r is independently an integer of 0-5 (eg, 0-3, 1-3, or 1-5). Is.

、またはその薬学的に許容される塩もしくは立体異性体であり得、式中、各ｒは、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数である。 In another embodiment, the chemical modification may include substitution of the cytosine C-5 with a hydrogen halo (eg, Br, Cl, F, or I) or an optionally substituted alkyl (eg, methyl). For example, a polynucleotide, a primary construct, or a building block molecule that can be integrated into an mRNA

, Or a pharmaceutically acceptable salt or stereoisomer thereof, in which each r is independently an integer of 0-5 (eg, 0-3, 1-3, or 1-5). Is.

、またはその薬学的に許容される塩もしくは立体異性体であり得、式中、各ｒは、独立して、０〜５（例えば、０〜３、１〜３、または１〜５）の整数である。 In a further embodiment, the chemical modification _{may include a fused ring formed by NH 2} at the C-4 position and by a carbon atom at the C-5 position. For example, a polynucleotide, a primary construct, or a building block molecule that can be integrated into an mRNA

, Or a pharmaceutically acceptable salt or stereoisomer thereof, in which each r is independently an integer of 0-5 (eg, 0-3, 1-3, or 1-5). Is.

Modifications in Sugars Modified polynucleotides, primary constructs, or modified nucleosides and nucleotides (eg, building block molecules) that can be incorporated into mRNAs (eg, RNA or mRNA described herein) can be modified in the sugar of ribonucleic acid. For example, the 2'hydroxyl group (OH) can be modified or substituted with several different substituents. Illustrative substitutions at the 2'position include H, halo, optionally substituted C _1-6 alkyl, optionally substituted C _{1 to 6} alkoxy, optionally substituted C _{6 to 10} aryloxy, optionally. C _3-8 cycloalkyl substituted with, optionally substituted C _3-8 cycloalkoxy, optionally substituted C _6-10 aryloxy, optionally substituted C _6-10 aryl-C _1-6 Alkoxy, optionally substituted C _1-12 (heterocyclyl) oxy, sugars (eg, ribose, pentose, or any of those described herein), polyethylene glycol (PEG), -O _{(CH 2 CH 2} O). _n CH ₂ CH ₂ OR (in the formula, R is H or an optionally substituted alkyl, n is 0 to 20 (eg 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4). , 1-8, 1-10, 1-16, 1-20, 2-4, 2-8, 2-10, 2-16, 2-20, 4-8, 4-10, 4-16, and A "locked" nucleic acid (LNA) (LNA) _{in which the 2'-hydroxyl is attached to the 4'-carbon of the same ribose sugar by C 1-6} alkylene or C _1-6 heteroalkylene bridges (which is an integer of 4-20). Illustrated cross-linking includes methylene, propylene, ether, or amino cross-linking), aminoalkyl as defined herein, aminoalkoxy as defined herein, amino as defined herein, and. Includes, but is not limited to, amino acids as defined herein. Generally, RNA contains a 5-membered ring sugar ribose with oxygen. Non-limiting exemplary modified nucleotides include the substitution of oxygen for ribose (eg, with S, Se, or alkylene, eg, methylene or ethylene), the addition of double bonds (eg, ribose to cyclopentenyl or cyclohexenyl). Ribose ring reduction (eg, to form a 4-membered ring of cyclobutane or oxetane), ribose ring expansion (eg, hexitol anhydride, altritor, mannitol, cyclohexanyl, cyclohexenyl, and phospho). To form a 6- or 7-membered ring with additional carbon or heteroatoms such as morpholino that also has a lamidate skeleton), polycyclic form (eg, tricyclo, and "unlocked" form, such as glycol nucleic acid (GNA) (eg, eg, , R-GNA or S-GNA, the moiety where ribose is replaced by glycol units that bind to phosphodiester bonds), and treose nucleic acids (TNA, ribose is replaced by α-L-treoflanosyl- (3'→ 2'). Part), as well as peptide nucleic acids (PNA, the part where the 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone). It may also contain one or more carbons with a chemical configuration. Thus, a polynucleotide, primary construct, or mmRNA molecule may contain a nucleotide containing, for example, arabinose as a sugar.

Modifications in Nucleobases The present disclosure provides modified nucleosides and nucleotides. The "nucleoside" described herein is a sugar molecule (eg, pentose or ribose) or a sugar molecule (eg, pentose or ribose) in combination with an organic base (eg, purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). It is defined as a compound containing the derivative. As used herein, a "nucleotide" is defined as a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method described herein (eg, chemically, enzymatically, or recombinantly to include one or more modified or unnatural nucleosides).

Modified nucleotide base pairs include not only standard adenosine-timine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides. Including, the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard bases and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil.

Modified nucleosides and nucleotides may contain modified nucleobases. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases may be modified or completely substituted to provide polynucleotides, primary constructs, or mmRNA molecules that have enhanced properties, eg, resistance to nucleases by disrupting the binding of the main groove binding partner. Table 8 below identifies the chemical appearance of each reference nucleotide. The circle identifies the atom containing each chemical region.
Table 8

を有するもの、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, B is a modified uracil. Illustrative modified uracils are in formulas (b1)-(b5):

Or containing a pharmaceutically acceptable salt or stereoisomer thereof

During the ceremony

が、一重または二重結合であり、

Is a single or double bond,

Each of T ^1' , T ^{1 "} , T ^2' , and T ^2" is independently H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy. or a combination of T ^{1 'and T 1 "combination} or T ² of ^the' and ^{T 2"} together (e.g., as in ^{T 2),} O (oxo), S (thio), or Se Form (Seleno),

Each of V ¹ and V ² is independently O, S, N (R ^Vb ) _nv , or C (R ^Vb ) _nv , where nv is an integer of 0 to 2 and each R. ^Vb is independently H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted. Alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl (For example, an N-protecting group, eg, any of those described herein, eg, substituted with trifluoroacetyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted. Acylaminoalkyl (eg, N-protecting group, eg, any of those described herein, eg, substituted with trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted. Alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, or optionally substituted alkynyloxy (eg, any of the substituents described herein, eg, selected from alkyl (1)-(21). (Arbitrarily substituted with a substituent)

R ¹⁰ is H, halo, optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted. Hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl Alkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted Carbamoylalkyl,

R ¹¹ is H or an optionally substituted alkyl,

R ^12a is H, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted. Aminoalkenyl, or optionally substituted aminoalkynyl, optionally substituted carboxyalkyl (eg, optionally substituted with hydroxy), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or Arbitrarily substituted carbamoylalkyl,

R ^12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted. Hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.

を有するもの、またはその薬学的に許容される塩もしくは立体異性体を含み、 Other exemplary modified uracils have the formulas (b6)-(b9) :.

Or containing a pharmaceutically acceptable salt or stereoisomer thereof

During the ceremony

が、一重または二重結合であり、

Is a single or double bond,

Each of T ^1' , T ^{1 "} , T ^2' , and T ^2" is independently H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy. or T ^{1 'and'} become combination together (e.g., as in ^{T 1),} or T ^{2 T 1 'and T 2 "combinations} together (e.g., in ^{T 2} Like), O (oxo), S (thio), or Se (seleno), or each T ¹ and T ² independently O (oxo), S (thio), or Se ( Sereno)

Each of W ¹ and W ² is independently N (R ^Wa ) _nw or C (R ^Wa ) _nw , where nw is an integer of 0 to 2 and each R ^Wa is independent. H, optionally substituted alkyl, or optionally substituted alkoxy,

Each V ³ is independently O, S, N (R ^Va ) _nv , or C (R ^Va ) _nv , where nv is an integer of 0 to 2 and each R ^Va is independent. H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkoxy, optionally substituted hydroxyalkoxyyl, optionally substituted Alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted alkyl heterocyclyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy, optionally Substituted aminoalkyl (eg, N-protecting group, eg, any of those described herein, eg, substituted with trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkoxy, optionally. Substituted aminoalkoxyl, optionally substituted acylaminoalkyl (eg, N-protecting group, eg, any of those described herein, eg, substituted with trifluoroacetyl), optionally substituted. Alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (eg, optionally substituted carboxyalkyl) Arbitrarily substituted with hydroxy and / or O-protective groups), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (eg, described herein). Arbitrary substituents of, for example, optionally substituted with a substituent selected from (1)-(21) of alkyl), with R ^Va and R ^12c together with the carbon atom to which they are attached. Can form optionally substituted cycloalkyls, optionally substituted aryls, or optionally substituted heterocyclyls (eg, 5- or 6-membered rings).

R ^12a is H, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted. Aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted carboxyalkyl (eg, optionally substituted with hydroxy and / or O-protecting group), optionally substituted carboxyalkoxy, optionally substituted Carboxaminoalkyl, optionally substituted carbamoylalkyl, or absent,

R ^12b is H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkoxyyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkoxy, optionally substituted hydroxyalkoxyl. , Arbitrarily substituted aminoalkyl, optionally substituted aminoalkoxy, optionally substituted aminoalkoxyyl, optionally substituted alkylaryl, optionally substituted heterocyclyl, optionally substituted alkyl heterocyclyl, optionally Substituted amino acids, optionally substituted alkoxycarbonylacyls, optionally substituted alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, Arbitrarily substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (eg, optionally substituted with hydroxy and / or O-protective groups), optionally substituted carboxyalkoxy, optionally substituted carboxy Aminoalkyl, or optionally substituted carbamoylalkyl,

Combinations combinations or ^{R 12b} and ^{R 12c} of R ^12b and T ^{1 'are} taken together to form a optionally substituted heterocyclyl,

R ^12c was H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted. Aminoalkenyl, or optionally substituted aminoalkynyl.

を有するもの、またはその薬学的に許容される塩もしくは立体異性体を含み、 Further exemplary modified uracils are described in Formulas (b28)-(b31):

Or containing a pharmaceutically acceptable salt or stereoisomer thereof

During the ceremony

Each of T ¹ and T ² is independently O (oxo), S (thio), or Se (seleno).

Each R ^Vb'and R ^Vb'is independently H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted hydroxyalkyl, optionally substituted. Hydroxylalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, Arbitrarily substituted aminoalkyl (eg, N-protecting group, eg, any of those described herein, eg, substituted with trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, Arbitrarily substituted aminoalkynyl, optionally substituted acylaminoalkyl (eg, N-protecting group, eg, any of those described herein, eg, substituted with trifluoroacetyl), optionally substituted. Alkoxycarbonylalkyl optionally substituted, alkoxycarbonylalkenyl optionally substituted, alkoxycarbonylalkynyl optionally substituted, alkoxycarbonylacyl optionally substituted, alkoxycarbonylalkoxy optionally substituted, carboxyalkyl optionally substituted ( For example, optionally substituted with hydroxy and / or O-protecting groups), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (eg, herein). ^{(Eg, an alkyl in which R Vb'is} optionally substituted), for example, any substituent selected from (1) to (21) of the alkyl (1) to (21). Any substituted alkenyl, or optionally substituted aminoalkyl, eg, N-protecting group, eg, any of those described herein, eg, trifluoroacetyl, or sulfoalkyl substituted. ),

R ^12a is H, an optionally substituted alkyl, an optionally substituted carboxyaminoalkyl, an optionally substituted aminoalkyl (eg, for example, an N-protecting group, eg, any of those described herein. For example, trifluoroacetyl or sulfoalkyl substituted), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.

R ^12b is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl. , Arbitrarily substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl (eg, N-protecting group, eg, any of those described herein, eg, trifluoroacetyl). , Or substituted with sulfoalkyl),

Arbitrarily substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted Alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl.

In certain embodiments, T ¹ is O (oxo) and T ² is S (thio) or Se (seleno). In other embodiments, T ¹ is S (thio) and T ² is O (oxo) or Se (seleno). In some embodiments, R ^Vb'is H, an optionally substituted alkyl, or an optionally substituted alkoxy.

In other embodiments, each R ^12a and R ^12b is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted hydroxyalkyl. be. In certain embodiments, R ^12a is H. In other embodiments, ^{both R 12a} and R ^12b are H.

In some embodiments, each R ^{Vb'of R 12b} is an independently optionally substituted aminoalkyl (eg, an N-protecting group, eg, any of those described herein, eg, trifluoro). Acetyl or sulfoalkyl substituted), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or optionally substituted acylaminoalkyl (eg, N-protecting group, eg, herein). , For example, substituted with trifluoroacetyl). In some embodiments, the amino and / or alkyl of the optionally substituted aminoalkyl is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted sulfoalkyl, optionally substituted carboxy. (For example, substituted with an O-protecting group), optionally substituted hydroxy (eg, substituted with an O-protecting group), optionally substituted carboxyalkyl (eg, substituted with an O-protecting group). Is substituted), optionally substituted alkoxycarbonylalkyl (eg, substituted with an O-protecting group), or substituted with one or more of the N-protecting groups. In some embodiments, the optionally substituted aminoalkyl is substituted with an optionally substituted sulfoalkyl or an optionally substituted alkenyl. In certain embodiments, both R ^12a and R ^{Vb "} are H. In certain embodiments, T ¹ is O (oxo) and T ² is S (thio) or Se (seleno). be.

In some embodiments, R ^Vb'is an optionally substituted alkoxycarbonylalkyl or an optionally substituted carbamoylalkyl.

In certain embodiments, ^{any substituent of R 12a} , R ^12b , R ^12c , or R ^Va is a polyethylene glycol group (eg, − (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR. ', In the formula, s1 is an integer of 1 to 10 (eg, 1 to 6 or 1 to 4), and each of s2 and s3 is independently 0 to 10 (eg, 0 to 4, An integer of 0-6, 1-4, 1-6, or 1-10), where R'is H or C _1-20 alkyl), or an amino-polyethylene glycol group (eg, -NR ^N1 (eg, -NR N1). CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 , in which s1 is an integer of 1-10 (eg 1-6 or 1-4), s2 and s3. each, independently, 0 (e.g., 0～4,0～6,1～4,1～6, or 10) is an integer, and each ^{R N1} is independently a hydrogen Alternatively, it is an arbitrarily substituted C _1-6 alkyl).

の化合物、またはその薬学的に許容される塩もしくは立体異性体を含み、
式中、 In some embodiments, B is a modified cytosine. Illustrative modified cytosines are given in formulas (b10)-(b14):

Containing a compound of, or a pharmaceutically acceptable salt or stereoisomer thereof,
During the ceremony

T 3 ^'are each and T ^{3 ",} independently, H, optionally substituted alkyl, an optionally substituted alkoxy or optionally substituted thioalkoxy, or T ^3' and T ³ the combination of ^"together (e.g., as in ^{T 3),} O (oxo), to form a S (thio), or Se (seleno)

Each V ⁴ is independently O, S, N (R ^Vc ) _nv , or C (R ^Vc ) _nv , where nv is an integer of 0 to 2 and each R ^Vc is independent. H, halo, optionally substituted amino acids, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, Select from any substituted heterocyclyl, optionally substituted alkyl heterocyclyl, or optionally substituted alkynyloxy (eg, any substituents described herein, eg, alkyl (1)-(21)). The combination of R ^13b and R ^Vc can be combined to form an arbitrarily substituted heterocyclyl.

Each V ⁵ is independently N (R ^Vd ) _nv , or C (R ^Vd ) _nv , where nv is an integer from 0 to 2, and each R ^Vd is independently H. , Halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted Heterocyclyl, optionally substituted alkyl heterocyclyl, or optionally substituted alkynyloxy (eg, any substituent described herein, eg, a substituent selected from alkyl (1)-(21). is in optionally those substituted) (e.g., ^{V 5} is be -CH or N),

Each of R ^13a and R ^13b is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, and R ^13b . the combination of R ¹⁴ are taken together to form a optionally substituted heterocyclyl,

Each R ¹⁴ was independently substituted with H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted. Alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (eg, substituted with an O-protecting group), optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted Alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino ( For example, -NHR (where R is H, alkyl, aryl, or phosphoryl in the formula), azide, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkyl heterocyclyl, optionally. Substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkyl,

Each of R ¹⁵ and R ¹⁶ is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

を有するもの、またはその薬学的に許容される塩もしくは立体異性体を含み、 Further exemplary modified cytosines are described in Formulas (b32)-(b35):

Or containing a pharmaceutically acceptable salt or stereoisomer thereof

During the ceremony

Each of T ¹ and T ³ is independently O (oxo), S (thio), or Se (seleno).

Each of R ^13a and R ^13b is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, and R ^13b . the combination of R ¹⁴ are taken together to form a optionally substituted heterocyclyl,

Each R ¹⁴ was independently substituted with H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted. Alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (eg, substituted with an O-protecting group), optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted Alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino ( For example, -NHR (where R is H, alkyl, aryl, or phosphoryl in the formula), azide, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkyl heterocyclyl, optionally substituted. Aminoalkyl (eg, hydroxyalkyl, alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.

Each of R ¹⁵ and R ¹⁶ is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (eg, R ¹⁵ is H and R). ¹⁶ is H or an optionally substituted alkyl).

In some embodiments, R ¹⁵ is H and R ¹⁶ is H or an optionally substituted alkyl. In certain embodiments, R ¹⁴ is an H, acyl, or hydroxyalkyl. In some ^{embodiments, R 14} is halo. In some embodiments, both R ¹⁴ and R ¹⁵ are H. In some embodiments, both R ¹⁵ and R ¹⁶ are H. In some embodiments, each of ^{R 14} and R ¹⁵ and R ^{16 is H.} In a further embodiment, ^{each of R 13a} and R ^13b is an independently H or optionally substituted alkyl.

の化合物、またはその薬学的に許容される塩もしくは立体異性体が挙げられ、 Further non-limiting examples of modified cytosines include formula (b36) :.

Compounds, or pharmaceutically acceptable salts or stereoisomers thereof.

During the ceremony

Each R ^13b is independently H, an optionally substituted acyl, an optionally substituted acyloxyalkyl, an optionally substituted alkyl, or an optionally substituted alkoxy, a combination of ^{R 13b} and R ^14b. Can together form an arbitrarily substituted heterocyclyl,

Each R ^14a and R ^14b can independently be H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally. Substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (eg, substituted with an O-protecting group), optionally substituted hydroxyalkenyl, optionally substituted alkoxy, optionally Substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (eg-NHR (eg, -NHR) In the formula, R is H, alkyl, aryl, phosphoryl, optionally substituted aminoalkyl, or optionally substituted carboxyaminoalkyl))), azide, optionally substituted aryl, optionally substituted. Heterocyclyl, optionally substituted alkyl heterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkoxy, or optionally substituted aminoalkoxyl,

Each of R ¹⁵ is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In certain embodiments, R ^14b is an optionally substituted amino acid (eg, an optionally substituted lysine). In some embodiments, R ^14a is H.

の化合物、またはその薬学的に許容される塩もしくは立体異性体を含み、 In some embodiments, B is a modified guanine. Illustrative modified guanines are represented by formulas (b15)-(b17):

Containing a compound of, or a pharmaceutically acceptable salt or stereoisomer thereof,

During the ceremony

Each of T ^4' , T ^{4 "} , T ^5' , T ^5" , T ^6' , and T ^{6 "} is independently H, optionally substituted alkyl, or optionally substituted alkoxy. , T ^4'and T ^{4 "} combination (eg, as in T ⁴ ) or T ^5'and T ^5" combination (eg, as in T ⁵ ) or T ^6'and T ^{6 "} combination (e.g., as in ^{T 6)} together form a O (oxo), S (thio), or Se (seleno)

Each of V ⁵ and V ⁶ is independently O, S, N (R ^Vd ) _nv , or C (R ^Vd ) _nv , where nv is an integer of 0 to 2 and each R. ^Vd is independently H, halo, thiol, optionally substituted amino acid, cyano, amidine, optionally substituted aminoalkyl, optionally substituted aminoalkoxy, optionally substituted aminoalkynyl, optionally Substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (eg, described herein). Any substituents of (eg, optionally substituted with a substituent selected from (1)-(21) of alkyl), optionally substituted thioalkoxy, or optionally substituted amino.

Each of R ¹⁷ , R ¹⁸ , R ^19a , R ^19b , R ²¹ , R ²² , R ²³ , and R ²⁴ was independently substituted with H, halo, thiol, optionally substituted alkyl, optionally substituted. Alkoxy, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.

の化合物、またはその薬学的に許容される塩もしくは立体異性体を含み、 Illustrative modified guanosines are in formulas (b37)-(b40):

Containing a compound of, or a pharmaceutically acceptable salt or stereoisomer thereof,

During the ceremony

Each of T 4 ^'are, independently, H, an optionally substituted alkyl or optionally substituted alkoxy, each T ⁴ is independently, O (oxo), S (thio), or Se (Sereno),

Each of R ¹⁸ , R ^19a , R ^19b , and R ²¹ independently substitutes H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted. Thiolalkic, optionally substituted amino, or optionally substituted amino acid.

In some embodiments, R ¹⁸ is alkyl H or optionally substituted. In a further embodiment, T ⁴ is oxo. In some embodiments, ^{each of R 19a} and R ^19b is an independently H or optionally substituted alkyl.

の化合物、またはその薬学的に許容される塩もしくは立体異性体を含み、式中、各Ｖ^７が、独立して、Ｏ、Ｓ、Ｎ（Ｒ^Ｖｅ）_ｎｖ、またはＣ（Ｒ^Ｖｅ）_ｎｖであり、式中、ｎｖが、０〜２の整数であり、各Ｒ^Ｖｅが、独立して、Ｈ、ハロ、任意に置換されたアミノ酸、任意に置換されたアルキル、任意に置換されたアルケニル、任意に置換されたアルキニル、任意に置換されたアルコキシ、任意に置換されたアルケニルオキシ、または任意に置換されたアルキニルオキシ（例えば、本明細書に記載の任意の置換基、例えば、アルキルの（１）〜（２１）から選択される置換基で任意に置換されたもの）であり、 In some embodiments, B is a modified adenine. Illustrative modified adenine is described in formulas (b18)-(b20):

In the formula, each V ⁷ is independently O, S, N (R ^Ve ) _nv , or C (R ^Ve ) _nv . Yes, in the formula, nv is an integer of 0 to 2, and each R ^Ve is independently H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkoxy, Arbitrarily substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (eg, any substituent described herein, eg, alkyl (1). ) To (21) arbitrarily substituted with a substituent selected from).

Each R ²⁵ was independently substituted with H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted. Amino and

Each of R ^26a and R ^26b is independently H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoyl alkyl, optionally substituted alkyl, optionally substituted alkenyl. , Arbitrarily substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group (eg,-(CH _2). ) _S2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR'(In the formula, s1 is an integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and each of s2 and s3 is independent. Then, it is an integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R'is H or C _{1 to 20} alkyl). ), Or aminopolyethylene glycol group (eg, -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 (in the formula, s1 is 1-10 (eg 1-6 or It is an integer of 1 to 4), and each of s2 and s3 is independently an integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). Yes, each ^RN1 is independently a hydrogen or an optionally substituted C _1-6 alkyl)).

Each R ²⁷ is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally Substituted amino,

Each R ²⁸ is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

Each R ²⁹ is independently H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoyl alkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted. Alkinyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted alkoxy, or optionally substituted amino.

Illustrative modified adenine is from formulas (b41) to (b43):

の化合物、またはその薬学的に許容される塩もしくは立体異性体を含み、

Containing a compound of, or a pharmaceutically acceptable salt or stereoisomer thereof,

During the ceremony

Each R ²⁵ was independently substituted with H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted. Amino and

Each of R ^26a and R ^26b is independently H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoyl alkyl, optionally substituted alkyl, optionally substituted alkenyl. , Arbitrarily substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group (eg,-(CH _2). ) _S2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR'(In the formula, s1 is an integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and each of s2 and s3 is independent. Then, it is an integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R'is H or C _{1 to 20} alkyl.) , Or aminopolyethylene glycol group (eg, -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 (in the formula, s1 is 1-10 (eg 1-6 or 1). ~ 4), and each of s2 and s3 is an independent integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). , Each ^RN1 is independently hydrogen or optionally substituted C _1-6 alkyl)).

Each R ²⁷ is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally It is a substituted amino.

In some embodiments, R ^26a is H and R ^26b is an optionally substituted alkyl. In some embodiments, ^{each of R 26a} and R ^26b is an independently, optionally substituted alkyl. In certain embodiments, R ²⁷ is optionally substituted alkyl, optionally substituted alkoxy or optionally substituted thioalkoxy,. In another embodiment, R ²⁵ is optionally substituted alkyl, optionally substituted alkoxy or optionally substituted thioalkoxy,.

In certain embodiments, ^{any substituent of R 26a} , R ^26b , or R ²⁹ is a polyethylene glycol group (eg, − (CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH _{2 ) s3} OR'(formula). Among them, s1 is an integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and each of s2 and s3 is independently 0 to 10 (for example, 0 to 4, 0 to 6, 1). ~ 4, 1-6, or 1-10), where R'is H or C _1-20 alkyl)), or an aminopolyethylene glycol group (eg, -NR ^N1 (CH _{2 ) s2} (eg, -NR N1 (CH 2)) CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 (In the formula, s1 is an integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and each of s2 and s3 is independent. 0-10 (e.g., 0～4,0～6,1～4,1～6 or 10) is an integer of, C each ^{R N1} is independently substituted with hydrogen or optionally _{It is 1 to 6} alkyl)).

を有し得、式中、Ｘ^１２は、独立して、Ｏ、Ｓ、任意に置換されたアルキレン（例えば、メチレン）、または任意に置換されたヘテロアルキレンであり、ｘａは、０〜３の整数であり、Ｒ^１２ａおよびＴ^２は、本明細書に記載の通りである。 In some embodiments, B is the formula (b21) :.

In the formula, X ¹² is independently O, S, optionally substituted alkylene (eg, methylene), or optionally substituted heteroalkylene, and xa is 0 to 3. It is an integer and R ^12a and T ² are as described herein.

を有し得、式中、Ｒ^１０’は、独立して、任意に置換されたアルキル、任意に置換されたアルケニル、任意に置換されたアルキニル、任意に置換されたアリール、任意に置換されたヘテロシクリル、任意に置換されたアミノアルキル、任意に置換されたアミノアルケニル、任意に置換されたアミノアルキニル、任意に置換されたアルコキシ、任意に置換されたアルコキシカルボニルアルキル、任意に置換されたアルコキシカルボニルアルケニル、任意に置換されたアルコキシカルボニルアルキニル、任意に置換されたアルコキシカルボニルアルコキシ、任意に置換されたカルボキシアルコキシ、任意に置換されたカルボキシアルキル、または任意に置換されたカルバモイルアルキルであり、Ｒ^１１、Ｒ^１２ａ、Ｔ^１、およびＴ^２は、本明細書に記載の通りである。 In some embodiments, B is the formula (b22) :.

In a resulting formula, R ^{10 'are} independently substituted alkyl, optionally substituted, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally Heterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl , Arbitrarily substituted alkoxycarbonylalkoxyyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl, R ¹¹ , R. ^12a , T ¹ , and T ² are as described herein.

を有し得、式中、Ｒ^１０は、任意に置換されたヘテロシクリル（例えば、任意に置換されたフリル、任意に置換されたチエニル、もしくは任意に置換されたピロリル）、任意に置換されたアリール（例えば、任意に置換されたフェニルもしくは任意に置換されたナフチル）、または本明細書に記載の任意の置換基（例えば、Ｒ^１０のもの）であり、Ｒ^１１（例えば、Ｈまたは本明細書に記載の任意の置換基）、Ｒ^１２ａ（例えば、Ｈまたは本明細書に記載の任意の置換基）、Ｔ^１（例えば、オキソまたは本明細書に記載の任意の置換基）、およびＴ^２（例えば、オキソまたは本明細書に記載の任意の置換基）は、本明細書に記載の通りである。
いくつかの実施形態において、Ｂは、式（ｂ２４）：

を有し得、式中、Ｒ^１４’は、独立して、任意に置換されたアルキル、任意に置換されたアルケニル、任意に置換されたアルキニル、任意に置換されたアリール、任意に置換されたヘテロシクリル、任意に置換されたアルキルアリール、任意に置換されたアルキルヘテロシクリル、任意に置換されたアミノアルキル、任意に置換されたアミノアルケニル、任意に置換されたアミノアルキニル、任意に置換されたアルコキシ、任意に置換されたアルコキシカルボニルアルケニル、任意に置換されたアルコキシカルボニルアルキニル、任意に置換されたアルコキシカルボニルアルキル、任意に置換されたアルコキシカルボニルアルコキシ、任意に置換されたカルボキシアルコキシ、任意に置換されたカルボキシアルキル、または任意に置換されたカルバモイルアルキルであり、Ｒ^１３ａ、Ｒ^１３ｂ、Ｒ^１５、およびＴ^３は、本明細書に記載の通りである。 In some embodiments, B is in formula (b23) :.

The resulting a, wherein, R ¹⁰ is an optionally substituted heterocyclyl (for example, optionally substituted furyl, optionally substituted thienyl or optionally substituted pyrrolyl,), an optionally substituted aryl (For example, optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (eg, of R ¹⁰ ) and R ¹¹ (eg, H or herein). R ^12a (eg, H or any substituent described herein), T ¹ (eg, oxo or any substituent described herein), and T ² (For example, oxo or any substituent described herein) are as described herein.
In some embodiments, B is the formula (b24) :.

In a resulting formula, R ^{14 'are} independently substituted alkyl, optionally substituted, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally Heterocyclyl, optionally substituted alkylaryl, optionally substituted alkyl heterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optional Alkoxycarbonylalkoxy substituted with, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl , Or optionally substituted carbamoylalkyl, R ^13a , R ^13b , R ¹⁵ and T ³ as described herein.

を有し得、式中、Ｒ^１４’は、任意に置換されたヘテロシクリル（例えば、任意に置換されたフリル、任意に置換されたチエニル、もしくは任意に置換されたピロリル）、任意に置換されたアリール（例えば、任意に置換されたフェニルもしくは任意に置換されたナフチル）、または本明細書に記載の任意の置換基（例えば、Ｒ^１４またはＲ^１４’のもの）であり、Ｒ^１３ａ（例えば、Ｈまたは本明細書に記載の任意の置換基）、Ｒ^１３ｂ（例えば、Ｈまたは本明細書に記載の任意の置換基）、Ｒ^１５（例えば、Ｈまたは本明細書に記載の任意の置換基）、およびＴ^３（例えば、オキソまたは本明細書に記載の任意の置換基）は、本明細書に記載の通りである。 In some embodiments, B is the formula (b25) :.

Have a wherein, R ^{14 'is} an optionally substituted heterocyclyl (for example furyl, optionally substituted thienyl, optionally substituted, or optionally substituted pyrrolyl), optionally substituted Aryl (eg, optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (eg, of R ¹⁴ or R ^14' ), and R ^13a (eg, R 13a). H or any substituent described herein), R ^13b (eg H or any substituent described herein), R ¹⁵ (eg H or any substituent described herein). ), And T ³ (eg, oxo or any substituent described herein) are as described herein.

であり得る。 In some embodiments, B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil. In some embodiments, B is

Can be.

In some embodiments, the modified nucleobase is modified uracil. Examples of nucleic acid bases and nucleosides with modified uracil include pseudouridine (ψ), pyridine-4-oneribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2 -Thio-pseudouridine (s ² U), 4-thio-pseudouridine (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-pseudouridine (ho ⁵ U), 5-aminoallyl- Pseudouridine, 5-halo-pseudouridine (eg, 5-iodo-pseudouridine or 5-bromo-pseudouridine), 3-methyl-pseudouridine (m ³ U), 5-methoxy-pseudouridine (mo ⁵ U), uridine 5-oxyacetic acid (Como ⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U) ), 5-Carboxyhydroxymethyl-pseudouridine (mchm ⁵ U), 5-methoxycarbonylmethyl-pseudouridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5 -Aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl- ^{uridine (nm m 5} U), 5-methylaminomethyl-2-thio-uridine (nm m ⁵ s ² U), 5 -Methylaminomethyl-2-seleno-uridine (nmm ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm ⁵ U), 5-carboxymethyl aminomethyl-uridine (cm nm ⁵ U), 5-carboxymethyl aminomethyl -2-thio-uridine (cmnm ⁵ s ² U), 5-propinyl-pseudouridine, 1-propinyl-pseudouridine, 5-taurinomethyl-pseudouridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2 -Thio-pseudouridine (τm ⁵ s ² U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-pseudouridine (m ⁵ U, that is, one having the nucleic acid base deoxytimine), 1-methyl pseudouridine ( m ¹ ψ), 5-methyl-2-thio-pseudouridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine , 3-Methyl-si Pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-desa-pseudouridine, dihydrouridine (D), dihydro pseudouridine, 5,6-dihydrouridine, 5-methyl - dihydro uridine ^(m 5 D) 2-thio - dihydro uridine, 2-thio - dihydro pseudouridine, 2-methoxy - uridine, 2-methoxy-4- Thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine ( ^{also known as 1} -methyl pseudouridine (m 1 ψ)), 3- (3) ^−Amino-3 -carboxypropyl) uridine (acp 3 U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp ³ ψ), 5- (isopentenylaminomethyl) uridine (inm) ⁵ U), 5- (isopentenylaminomethyl) -2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O -Dimethyl-uridine (m ⁵ Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2'- O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cm nm ⁵ Um) , 3,2'-O-dimethyl-uridine (m ³ Um), 5- (isopentenylaminomethyl) -2'-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2 '-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5- (2-carbomethoxyvinyl) uridine, and 5- [3- (1-E-propenylamino) uridine Can be mentioned.

In some embodiments, the modified nucleobase is a modified cytosine. Illustrative nucleic acid bases and nucleosides having modified cytosine, 5-aza - cytidine, 6-aza - cytidine, shoe Doi Société cytidine, 3-methyl - cytidine ^(m 3 C), N4- acetyl - cytidine ^(ac 4 C) , 5-formyl-cytidine (f ⁵ C), N4-methyl-cytidine (m ⁴ C), 5-methyl-cytidine (m ⁵ C), 5-halo-cytidine (eg 5-iodo-cytidine), 5 - hydroxymethyl - cytidine ^(hm 5 C), 1- methyl - shoe Doi Société cytidine, pyrrolo - cytidine, pyrrolo - shoe Doi Société cytidine, 2-thio - cytidine ^(s 2 C), 2-thio-5-methyl - cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebraline, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoiso cytidine, 4-methoxy-1-methyl - shoe Doi Société cytidine, Rishijin _(k 2 C), alpha-thio - cytidine, 2'-O-methyl - cytidine (Cm), 5,2'-O- dimethyl - cytidine ( m ⁵ Cm), N4-acetyl-2'-O-methyl-cytidine (ac ⁴ Cm), N4, 2'-O-dimethyl-cytidine (m ⁴ Cm), 5-formyl-2'-O-methyl- cytidine ^{(f 5 Cm), N4,} N4,2'-O-tri -methyl - cytidine _^(m 4 2 Cm), 1- thio - cytidine, 2'-F- Ara - cytidine, 2'-F- cytidine, and 2'-OH-ara-cytidine can be mentioned.

In some embodiments, the modified nucleobase is modified adenine. Examples of nucleic acid bases and nucleosides with modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (eg, 2-amino-6-chloro-purine), 6 -Halo-purine (eg, 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-daza-adenine, 7-daza-8-aza-adenine, 7-daza- 2-amino-purine, 7-daza-8-aza-2-amino-purine, 7-daza-2,6-diaminopurine, 7-daza-8-aza-2,6-diaminopurine, 1-methyl- Adenosine (m ¹ A), 2-methyl-adenine (m ² A), N6-methyl-adenosine (m ⁶ A), 2-methylthio-N6-methyl-adenosine (ms ² m ⁶ A), N6-isopentenyl -Adenosine (i ⁶ A), 2-methylthio-N6-isopentenyl-adenosine (ms ² i ⁶ A), N6- (cis-hydroxyisopentenyl) adenosine (io ⁶ A), 2-methylthio-N6- (cis) - hydroxy isopentenyl) adenosine ^{(ms 2 io 6 A),} N6- glycidyl senior carbamoyl - adenosine ^(g 6 A), N6- tray demon carbamoyl - adenosine ^(t 6 A), N6- methyl -N6- tray Oni carbamoyl - Adenosine (m ⁶ t ⁶ A), 2-methylthio-N6-threonyl carbamoyl-adenosine (ms ² g ⁶ A), N6, N6-dimethyl-adenosine (m ⁶ ₂ A), N6-hydroxynorvalyl carbamoyl-adenosine ( ^{Hn 6} A), 2-methylthio-N6-hydroxynorvalyl carbamoyl-adenosine (ms ² hn ⁶ A), N6-acetyl-adenosine (ac ⁶ A), 7-methyl-adenine, 2-methylthio-adenine, 2 -Methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m ⁶ Am), N6, N6,2'-O-trimethyl -Adenosine (m ⁶ ₂ Am), 1,2'-O-dimethyl-adenosine (m ¹ Am), 2'-O-ribosyl adenosine (phosphate) (Ar (p)), 2-amino-N6-methyl- Purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara- Included are adenosine and N6- (19-amino-pentaoxanonadesyl) -adenosine.

In some embodiments, the modified nucleobase is modified guanine. Illustrative nucleobases and nucleosides having modified guanine, inosine (I), 1-methyl - inosine ^(m 1 I), Waioshin (img), Mechiruwaioshin (mImg), 4-demethyl - Waioshin (img-14 ), Isowaioshin (imG2), wybutosine (yW), peroxy Wai but-Shin _(o 2 _yW), hydroxy Wai but-Shin (OHyW), unmodified hydroxy Wai but-Shin (OHyW *), 7- deaza - guanosine, Kueoshin (Q ), Epoxyqueosin (oQ), galactosyl-queosin (galQ), mannosyl-queosin (manQ), 7-cyano-7-diaza-guanosine (preQ ₀ ), 7-aminomethyl-7-diaza-guanosine (preQ _1). ), Alcaeosin (G ⁺ ), 7-Dather-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7 -Methyl-guanosine (m ⁷ G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m ¹ G), N2-methyl-guanosine (m) ² G), N2, N2- dimethyl - guanosine _^(m 2 2 G), N2,7- dimethyl - guanosine ^{(m 2,7 G), N2,} N2,7- dimethyl - guanosine ^{(m 2,2,7} G ), 8-Oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2, N2-dimethyl-6-thio-guanosine, α- thio - guanosine, 2'-O-methyl - guanosine (Gm), N2- methyl -2'-O-methyl - guanosine ^{(m 2 Gm), N2,} N2- dimethyl -2'-O-methyl - guanosine ( ^{M 2} ₂ Gm), 1-methyl-2'-O-methyl-guanosine (m ¹ Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m ^2,7 Gm), 2'- Included are O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m ¹ Im), and 2'-O-ribosylguanosine (phosphate) (Gr (p)).

Nucleobases of nucleotides can be selected independently of purines, pyrimidines, purines, or pyrimidine analogs. For example, each nucleobase can be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine, respectively. In another embodiment, the nucleic acid base includes, for example, pyrazolo [3,4-d] pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthin, hypoxanthin, 2-aminoadenine. , 6-Methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiotimine and 2-thiocytocin, 5-propynyluracil and citocin, 6-azouracil, Citocins and pyrimidines, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (eg 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and Guanine, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and pyrimidines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine , 3-Dazaguanine, Derazaadenine, 7-Derazaadenine, 3-Dazaadenine, Pyrazolo [3,4-d] pyrimidine, Imidazo [1,5-a] 1,3,5 Triazinone, 9-Dazapurine, Imidazo [4,5-a] d] Pyrazine, thiazolo [4,5-d] pyrimidines, pyrazine-2-one, 1,2,4-triazine, pyridazine, and naturally occurring synthetic derivatives of bases including 1,3,5 triazine may also be included. .. When nucleotides are indicated using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or a derivative thereof, eg, A is adenine, or, for example, 7-. Includes adenine analogs such as deazaadenine.

Modifications in Inter-Nucleoside Binding Polynucleotides, primary constructs, or modified nucleotides that can be incorporated into the mm mRNA molecule can be modified in inter-nucleoside binding (eg, phosphate backbone). As used herein, the terms "phosphate" and "phosphodiester" are used synonymously in the context of the polynucleotide backbone. Skeletal phosphate groups can be modified by substituting one or more of the oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include large-scale substitutions of unmodified phosphate moieties with other nucleoside interlinks described herein. Examples of modified phosphate groups include phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphorora midate, phosphorodiamidate, alkyl or arylphosphonate, and phosphotriester. , Not limited to these. Phosphorodithioates have both unbound oxygens substituted with sulfur. Phosphate linkers can also be modified by substitution of bound oxygen with nitrogen (crosslinked phosphoramidate), sulfur (crosslinked phosphorothioate), and carbon (crosslinked methylenephosphonate).

The α-thio-substituted phosphate moiety is provided to impart stability to RNA and DNA polymers via unnatural phosphorothioate backbone binding. Phosphorothioate DNA and RNA have increased nuclease resistance, followed by a longer half-life in the cellular environment. Phosphorothioate-binding polynucleotides, primary constructs, or mmRNA molecules are also expected to reduce the innate immune response by weaker binding / activation of the cell's innate immune molecules.

In certain embodiments, the modified nucleoside is an α-thio-nucleoside (eg, 5'-O- (1-thiophosphate) -adenosine, 5'-O- (1-thiophosphate) -cytidine (α-thio-). Cytidine), 5'-O- (1-thiophosphate) -guanosine, 5'-O- (1-thiophosphate) -uridine, or 5'-O- (1-thiophosphate) -pseudouridine).

Other nucleoside bonds that can be used in accordance with the present invention, including phosphorus atom-free nucleoside bonds, are described herein below.

Combination of Modified Sugar, Nucleobase, and Nucleoside Binding The polynucleotides, primary constructs, and mmRNAs of the present invention may contain a combination of modifications to sugar, nucleobase, and / or nucleoside binding. These combinations may include any one or more of the modifications described herein. For example, formulas (Ia), (Ia-1) to (Ia-3), (Ib) to (If), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-). 1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr) any of the nucleotides described herein. Can be combined with any of the nucleobases described herein (eg, formulas (b1)-(b43) or any other as described herein).

Synthesis of Polypeptides, Primary Constructs, and mm mRNA Molecules Polypeptides, primary constructs, and mm mRNA molecules used in accordance with the present invention can be prepared according to any useful technique described herein. The polynucleotides, primary constructs, and modified nucleosides and nucleotides used in the synthesis of the mmRNA molecule disclosed herein can be prepared from readily available starting materials using the following common methods and procedures. If typical or preferred process conditions (eg, reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are provided, those skilled in the art will be able to optimize and develop further process conditions. Will be able to. Optimal reaction conditions may vary depending on the particular reactant or solvent used, but such conditions may be determined by one of ordinary skill in the art using routine optimization procedures.

The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be achieved by nuclear magnetic resonance spectroscopy (eg, ¹ H or ¹³ C), infrared spectroscopy, spectroscopic spectroscopy (eg, ultraviolet visibility), spectroscopic means such as mass spectrometry, or high performance liquid chromatography. It can be monitored by chromatography such as (HPLC) or thin layer chromatography.

The preparation of the polypeptides, primary constructs, and mmRNA molecules of the invention can involve protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by one of ordinary skill in the art. The chemistry of protecting groups is, for example, incorporated herein by reference in its entirety, Greene, et al. , Protective Groups in Organic Synthesis, 2d. Ed. , Wiley & Sons, 1991.

The reactions of the processes described herein can be carried out in suitable solvents that can be readily selected by engineers in the field of organic synthesis. A suitable solvent is the temperature at which the reaction takes place, i.e., a temperature that can range from the freezing temperature of the solvent to the boiling point of the solvent, and is substantially non-reactive with the starting material (reactant), intermediate, or product. could be. A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the particular reaction step, a suitable solvent for the particular reaction step can be selected.

Degradation of racemic mixtures of modified nucleosides and nucleotides can be performed by any of a number of methods known in the art. Examples of methods include fractional recrystallization using an optically active salt-forming organic acid, a "chirally degrading acid". Degrading acids suitable for the fractional recrystallization method include, for example, tartaric acid, diacetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid, or optically active acids such as the D and L forms of various optically active camphorsulfonic acids. .. The decomposition of the racemic mixture can also be carried out by elution on a column packed with an optically active degrading agent (eg, dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one of ordinary skill in the art.
Modified nucleosides and nucleotides (eg, building block molecules), each of which is incorporated by reference in its entirety, Ogata et al. , J. Org. Chem. 74: 2585-2588 (2009), Pharmaceutical et al. , Nucl. Acids Res. 22 (1): 72-78, (1994), Fukuhara et al. , Biochemistry, 1 (4): 563-568 (1962), and Xu et al. , Tetrahedron, 48 (9): 1729-1740 (1992).

The polypeptides, primary constructs, and mmRNAs of the invention may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (eg, purines or pyrimidines, or any one or more or all of A, G, U, C) are the polynucleotides of the invention or given thereof. It may or may not be uniformly modified in a defined sequence region (eg, one or more of the sequence regions shown in FIG. 1). In some embodiments, all nucleotides X in the polynucleotide of the invention (or a given sequence region thereof) are modified where X is any one of nucleotides A, G, U, C. , Or a combination of A + G, A + U, A + C, G + U, G + C, U + C, A + G + U, A + G + C, G + U, or A + G + C.

Different sugar modifications, nucleotide modifications, and / or nucleoside interlinks (eg, skeletal structures) can be present at various positions in the polynucleotide, primary construct, or mmRNA. To those skilled in the art, any position of the polynucleotide, primary construct, or mmRNA such that the nucleotide analog or other modification (s) substantially reduces the function of the polynucleotide, primary construct, or mmRNA. Recognize that it can be located (s). The modification can also be a 5'or 3'end modification. Polynucleotides, primary constructs, or mmRNAs are about 1% to about 100% modified nucleotides (total nucleotide content or one or more nucleotides, ie, any one or more of A, G, U, or C. Any related) or any intermediate percentage (eg 1% -20%, 1% -25%, 1% -50%, 1% -60%, 1% -70%, 1% -80% 1, 1% -90%, 1% -95%, 10% -20%, 10% -25%, 10% -50%, 10% -60%, 10% -70%, 10% -80%, 10% % To 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100% , 70% -80%, 70% -90%, 70% -95%, 70% -100%, 80% -90%, 80% -95%, 80% -100%, 90% -95%, 90 % -100%, and 95% -100%).

を有するｎ個の結合ヌクレオシドを含むポリヌクレオチド、一次構築物、またはｍｍＲＮＡ（例えば、第１の領域、第１の隣接領域、または第２の隣接領域）を合成する方法を提供し、この方法は、
ａ）式（ＩＶ−１）：

のヌクレオチドを、式（Ｖ−１）：

のホスホラミダイト化合物と反応させて（式中、Ｙ^９が、Ｈ、ヒドロキシ、ホスホリル、ピロリン酸塩、硫酸塩、アミノ、チオール、任意に置換されたアミノ酸、またはペプチド（例えば、２〜１２個のアミノ酸を含む）であり、各Ｐ^１、Ｐ^２、およびＰ^３が、独立して、好適な保護基であり、

が、固体支持体を示す）、
式（ＶＩ−１）のポリヌクレオチド、一次構築物、またはｍｍＲＮＡを提供することと、

、および
ｂ）式（Ｖ）のポリヌクレオチド、一次構築物、またはｍｍＲＮＡを酸化または硫化して、式（ＶＩＩ−１）のポリヌクレオチド、一次構築物、またはｍｍＲＮＡを産出することと、

、および
ｃ）保護基を除去して、式（Ｉａ）のポリヌクレオチド、一次構築物、またはｍｍＲＮＡを産出することとを含む。 In some embodiments, the polynucleotide, primary construct, or mMV comprises a modified pyrimidine (eg, modified uracil / uridine / U or modified cytosine / cytidine / C). In some embodiments, the uracil or uridine (generally U) in the polynucleotide, primary construct, or mmRNA molecule is from about 1% to about 100% modified uracil or modified uridine (eg, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% ~ 25%, 10% -50%, 10% -60%, 10% -70%, 10% -80%, 10% -90%, 10% -95%, 10% -100%, 20% -25 %, 20% -50%, 20% -60%, 20% -70%, 20% -80%, 20% -90%, 20% -95%, 20% -100%, 50% -60%, 50% -70%, 50% -80%, 50% -90%, 50% -95%, 50% -100%, 70% -80%, 70% -90%, 70% -95%, 70% Substituted with ~ 100%, 80% ~ 90%, 80% ~ 95%, 80% ~ 100%, 90% ~ 95%, 90% ~ 100%, and 95% ~ 100% modified uracil or modified uridine) obtain. The modified uracil or uridine can be replaced with a compound having a single unique structure or multiple compounds having different structures (eg, a few unique structures described herein). In some embodiments, the cytosine or cytidine (generally C) in the polynucleotide, primary construct, or mmRNA molecule is from about 1% to about 100% modified cytosine or modified cytidine (eg, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% ~ 25%, 10% -50%, 10% -60%, 10% -70%, 10% -80%, 10% -90%, 10% -95%, 10% -100%, 20% -25 %, 20% -50%, 20% -60%, 20% -70%, 20% -80%, 20% -90%, 20% -95%, 20% -100%, 50% -60%, 50% -70%, 50% -80%, 50% -90%, 50% -95%, 50% -100%, 70% -80%, 70% -90%, 70% -95%, 70% Substituted with ~ 100%, 80% ~ 90%, 80% ~ 95%, 80% ~ 100%, 90% ~ 95%, 90% ~ 100%, and 95% ~ 100% modified cytosine or modified cytidine) obtain. The modified cytosine or cytidine can be replaced with a compound having a single unique structure or multiple compounds having different structures (eg, 2, 3, 4 or more unique structures described herein).
In some embodiments, the present disclosure comprises formula (Ia-1) :.

Provided is a method of synthesizing a polynucleotide containing n binding nucleosides, a primary construct, or an mRNA (eg, a first region, a first adjacent region, or a second adjacent region).
a) Equation (IV-1):

Nucleotides of the formula (V-1):

By the phosphoramidite compound reaction (wherein, Y ⁹ is, H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino, thiol, optionally substituted amino acid or peptide (e.g., 2-12 amino acids, , And each P ¹ , P ² , and P ³ are independently suitable protecting groups.

Shows a solid support),
To provide a polynucleotide of formula (VI-1), a primary construct, or mmRNA,

, And b) Oxidizing or sulfurizing the polynucleotide, primary construct, or mmRNA of formula (V) to produce the polynucleotide, primary construct, or mmRNA of formula (VII-1).

, And c) removing the protecting group to produce the polynucleotide, primary construct, or mmRNA of formula (Ia).

In some embodiments, steps a) and b) are repeated 1 to about 10,000 times. In some embodiments, the method further comprises a nucleotide (eg, an mRNA molecule) selected from the group consisting of A, C, G, and U (adenosine, cytosine, guanosine, and uracil). In some embodiments, the nucleobase can be a pyrimidine or a derivative thereof. In some embodiments, the polynucleotide, primary construct, or mmRNA may be translatable.

Polynucleotides, primary constructs, and other components of mmRNA are optional and beneficial in some embodiments. For example, 5'untranslated regions (UTRs) and / or 3'UTRs are provided, and either or both of these can independently contain one or more different nucleotide modifications. In such embodiments, nucleotide modifications may also be present in the translatable region. Polynucleotides, primary constructs, and mmRNAs containing the Kozak sequence are also provided.

Illustrative synthesis of modified nucleic acids or mRNAs, such as modified nucleotides that integrate into RNA or mRNA, is provided in Schemes 1 to 11 below. Scheme 1 provides a general method of phosphorylation of nucleosides, including modified nucleosides.
Scheme 1

Various protecting groups can be used to control the reaction. For example, Scheme 2 provides the use of multiple protection and deprotection steps to promote phosphorylation of the 5'position of sugars rather than the 2'and 3'hydroxyl groups.
Scheme 2

スキーム４

スキーム５

スキーム６

スキーム７

Modified nucleotides can be synthesized in any useful way. Schemes 3, 4, and 7 provide exemplary methods for synthesizing modified nucleotides with modified purine nucleobases, and Schemes 5 and 6 synthesize modified nucleotides with modified pseudouridine or pseudoisocytidine, respectively. An exemplary method for doing so is provided.
Scheme 3

Scheme 4

Scheme 5

Scheme 6

Scheme 7

スキーム９

スキーム１０

Schemes 8 and 9 provide exemplary synthesis of modified nucleotides. Scheme 10 provides a non-limiting biocatalytic method for producing nucleotides.
Scheme 8

Scheme 9

Scheme 10

Scheme 11 provides an exemplary synthesis of modified uracil, where the N1 position is ^{modified with R 12b} as provided elsewhere and the 5'position of ribose is phosphorylated. T ¹ , T ² , R ^12a , R ^12b , and r are as provided herein. This synthesis and its optimized version are used to modify other pyrimidine nucleobases and purine nucleobases (see, eg, formulas (b1)-(b43)) and / or one or more phosphorus. Acid groups can be introduced (eg, at the 5'position of the sugar). Using this alkylation reaction, any reactive group (eg, amino group) in any of the nucleobases described herein (eg, amino on the Watson-Crick base pairing surface of cytosine, uracil, adenine, and guanine). The group) can also contain one or more optionally substituted alkyl groups.
Scheme 11

Nucleotide Combinations in mmRNA Further examples of modified and modified nucleotide combinations are provided in Table 9 below. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mmRNAs of the invention. Unless otherwise stated, modified nucleotides can be completely replaced with the native nucleotides of the modified nucleic acids or mmRNAs of the invention. As a non-limiting example, the native nucleotide uridine can be replaced with the modified nucleosides described herein. In another non-limiting example, the native nucleotide uridine can be partially substituted with at least one of the modified nucleosides disclosed herein (eg, about 0.1%, 1%, 5%, etc.). 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, or 99.9%).
Table 9

Further examples of modified nucleotide combinations are provided in Table 10 below. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mmRNAs of the invention.
Table 10

In some embodiments, at least 25% of cytosine is replaced by compounds of formulas (b10)-(b14) (eg, at least about 30%, at least about 35%, at least about 40%, at least about 45%). , At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , Or about 100%).

In some embodiments, at least 25% of the uracil is replaced with a compound of formula (b1)-(b9) (eg, at least about 30%, at least about 35%, at least about 40%, at least about 45%. , At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , Or about 100%).

In some embodiments, at least 25% of cytosine is replaced with compounds of formulas (b10)-(b14) and at least 25% of uracil is replaced with compounds of formulas (b1)-(b9) ( For example, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75. %, At least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).

IV. Pharmaceutical Compositions Formulation, Administration, Delivery, and Dosing The present invention provides compositions and complexes of polynucleotides, primary constructs, and mmRNAs in combination with one or more pharmaceutically acceptable excipients. .. The pharmaceutical composition may optionally include one or more additional active substances, such as therapeutic and / or prophylactically active substances. General considerations in the formulation and / or manufacture of a medicament, for example, are incorporated herein by reference, Remington: The Science and Practice of Pharmacy 21 st ed. , Lippincott Williams & Wilkins, 2005.

In some embodiments, the composition is administered to a human, i.e. a human patient or subject. For the purposes of the present disclosure, the expression "active ingredient" generally refers to the polynucleotides, primary constructs, and mmRNAs to be delivered described herein.

The description of pharmaceutical compositions provided herein is, in principle, intended for pharmaceutical compositions suitable for administration to humans, but such compositions are generally any other animal. It will be appreciated by those skilled in the art that it is suitable for administration to, for example, non-human animals, eg, non-human mammals. Modifications of pharmaceutical compositions suitable for human administration to make the composition suitable for administration to various animals are well understood and are used by veterinary science experts, even if used. Such modifications can be designed and / or carried out using mere conventional design of experiments. Subjects for which administration of the pharmaceutical composition is intended are humans and / or other primates; commercially relevant such as cows, pigs, horses, ducks, cats, dogs, mice, and / or rats. Mammals, including mammals; and / or birds including, but not limited to, poultry, chickens, ducks, geese, and / or commercially suitable birds such as citrus butterflies.

The pharmaceutical compositions described herein can be prepared by any method known or developed in the art of pharmacology. In general, such preparation methods mix the active ingredient with excipients and / or one or more other sub-ingredients, and then, if necessary and / or desired, dose the product in the desired single or multiple doses. Includes steps to divide, mold, and / or package into units.

Pharmaceutical compositions according to the invention may be prepared, packaged and / or sold in bulk as a single dose and / or as multiple single doses. As used herein, a "unit dose" is an individual amount of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of active ingredient is generally the dosage of active ingredient that will be administered to the subject and / or of such dosage, for example, one-half or one-third of such dosage. Equal to a favorable fraction.

The relative amount of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredient in a pharmaceutical composition according to the invention is the identity, size, and / or condition of the subject to be treated. It will change depending on the route, and will change further depending on the route to which the composition should be administered. As an example, the composition is 0.1% to 100 w / w%, eg, 0.5 to 50 w / w%, 1 to 30 w / w%, 5 to 80 w / w%, at least 80 w / w% active ingredient. May include.

Formulations The polynucleotides, primary constructs, and in vivos of the invention: (1) increase stability, (2) increase cell transfection, and (3) (eg, polynucleotides, primary constructs, or in vivos). Allows sustained or delayed release (from depot formulations), (4) alters biodistribution (eg, targets polynucleotides, primary constructs, or mRNAs to specific tissues or cell types), and (5) codes. It can be formulated with one or more excipients to increase the translation of the protein in vivo and / or to alter the release profile of the encoded protein in vivo. In addition to all kinds of solvents, dispersion media, diluents, or other traditional excipients such as liquid vehicles, dispersion or suspension aids, surfactants, isotonics, thickeners or emulsifiers, preservatives, etc. Excipients of the invention are, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, polynucleotides, primary constructs, or mmRNAs (eg, for transplantation into a subject). Can include cells transfected with), hyaluronidases, nanoparticle mimics, and combinations thereof. Thus, the formulations of the invention can contain one or more excipients, each of which together increases the stability of the polynucleotide, primary construct, or mmRNA, cells with the polynucleotide, primary construct, or mmRNA. An amount that increases transfection, increases expression of a protein encoded by a polynucleotide, primary construct, or mmRNA, and / or alters the release profile of a protein encoded by a polynucleotide, primary construct, or mmRNA. .. In addition, the primary construct and mmRNA of the present invention may be formulated with self-assembling nucleic acid nanoparticles.

The pharmaceutical compositions described herein can be prepared by any method known or developed in the art of pharmacology. In general, such a preparation method comprises mixing the active ingredient with an excipient and / or one or more other sub-ingredients.

The pharmaceutical compositions according to the present disclosure may be prepared, packaged and / or sold in bulk as a single dose and / or as multiple single doses. As used herein, "unit dose" refers to an individual amount of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of active ingredient generally includes, but is not limited to, the dosage of active ingredient that will be administered to the subject and / or, for example, one-half or one-third of such dosage. Equal to a favorable fraction of such dosage.

The relative amount of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredient in the pharmaceutical composition according to the present disclosure is the identity, size, and / or condition of the subject being treated. It will change depending on the route, and will change further depending on the route to which the composition should be administered. For example, the composition may contain from 0.1 w / w% to 99% active ingredient.

In some embodiments, the formulations described herein may contain at least one m mRNA. As a non-limiting example, the formulation may contain 1, 2, 3, 4, or 5 mRNAs. In one embodiment, the formulations are human proteins, veterinary proteins, bacterial proteins, bioproteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secretory proteins, progenitor membrane proteins, cytoplasms and cells. Modifications encoding proteins selected from categories such as, but not limited to, skeletal proteins, intracellular membrane-binding proteins, nuclear proteins, proteins associated with human diseases and / or proteins associated with non-human diseases, etc. May contain mRNA. In one embodiment, the pharmaceutical product contains a protein encoded by at least three modified mRNAs. In one embodiment, the pharmaceutical product contains a protein encoded by at least 5 modified mRNAs.

The pharmaceutical formulation is suitable for the particular dosage form desired, in any solvent, dispersion medium, diluent, or other liquid vehicle, dispersion or suspension aid, surfactant when used herein. Additives, but not limited to, pharmaceutically acceptable excipients may be further included, including, but not limited to, agents, isotonic agents, thickeners or emulsifiers, preservatives and the like. Various excipients for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the art (remington: The Science, which is incorporated herein by reference in its entirety). ^{and Practice of Pharmacy, 21 st Edition} , A.R.Gennaro, Lippincott, Williams & Wilkins, Baltimore, see MD, 2006). A substance or substance in which any conventional excipient medium causes any unwanted biological effects or otherwise interacts with any other component (s) of the pharmaceutical composition in a detrimental manner. Its use may be contemplated within the scope of the present disclosure, except where it is incompatible with the derivative.

In some embodiments, the particle size of the lipid nanoparticles can increase and / or decrease. Changes in particle size can help counter biological reactions, such as, but not limited to, inflammation, or can increase the biological effect of modified mRNA delivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include inert diluents, surfactants and / or emulsifiers, preservatives, buffers, lubricants, and / or oils. Included, but not limited to. Such excipients may optionally be included in the pharmaceutical formulations of the present invention.

Lipidoids The synthesis of lipidoids is described in detail, and formulations containing these compounds are particularly suitable for delivery of polynucleotides, primary constructs, or mmRNAs (all of which are referred to herein in their entirety). Incorporated, Mahon et al., Bioconjug Chem. 2010 21: 1448-1454, Schroeder et al., J Intern Med. 2010 267: 9-21, Akinc et al., Nat Biotechnol. 2008 26: 561- et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869, Seegwart et al., Proc Natl Acad Sci USA. 2011108: 12996-3001).

These lipidoids have been used to effectively deliver double-stranded small interfering RNAs in rodents and non-human primates (all of which are incorporated herein by reference in Akinc et al. , Nat Biotechnol. 2008 26: 561-569, Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105: 11915-11920, Akinc et al., Mol Ther. 2009 17:872. , Proc Natl Acad Sci USA. 2010 107: 1864-1869, Leuschner et al., Nat Biotechnol. 2011 29: 1005-1010), the present disclosure contains single-stranded polynucleotides, primary constructs, or mmRNAs. Describe their formulation and use in delivery. Complexes, micelles, liposomes, or particles containing these lipidoids can be prepared and therefore poly when determined by the production of the encoded protein after injection of the lipidoid product via the topical and / or systemic route of administration. It can provide effective delivery of nucleotides, primary constructs, or mRNAs. Polynucleotides, primary constructs, or lipidoid complexes of mmRNA can be administered by a variety of means, including, but not limited to, the intravenous, intramuscular, or subcutaneous pathways.

In vivo delivery of nucleic acids includes, but is not limited to, formulation composition, particle PEGylation properties, degree of loading, oligonucleotide-to-lipid ratio, and many parameters, such as, but not limited to, biophysics. Can be influenced by physical parameters (Akinc et al., Mol Ther. 2009, which is incorporated herein by reference in its entirety).
17: 872-879). As an example, a slight change in anchor chain length of a poly (ethylene glycol) (PEG) lipid can have a significant effect on in vivo efficacy. Penta [3- (1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP (also known as 98N12-5)), which is incorporated herein by reference in its entirety, Murugaiah et al., Analytical Biochemistry. , 401: 61 (2010)), C12-200 (including derivatives and variants), and MD1, but not limited to, formulations with different lipidoids can be tested for in vivo activity.

The lipidoids referred to herein as "98N12-5" are incorporated by reference in their entirety, Akinc et al. , Mol Ther. It is disclosed by 2009 17: 872-879 (see FIG. 2) (see FIG. 2).

Lipidoids, both referred to herein as "C12-200," are incorporated herein by reference in their entirety, Love et al. , Proc Natl
Acad Sci USA. 2010 107: 1864-1869 (see FIG. 2) and Liu and Hung, Molecular Therapy. It is disclosed by 2010 669-670 (see FIG. 2). The lipidoid preparation can include particles containing any or more of three or four components in addition to the polynucleotide, primary construct, or mmRNA. As an example, a formulation having a particular lipidoid comprises, but is not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol, and 10% PEG (C14 alkyl chain length). As another example, formulations having a particular lipidoid include, but are not limited to, C12-200, 50% lipidoid, 10% disteroylpphosphatidyl choline, 38.5% cholesterol, and 1. It may contain 5% PEG-DMG.

In one embodiment, the polynucleotide, primary construct, or mmRNA formulated with lipidoid for systemic intravenous administration can target the liver. For example, using polynucleotides, primary constructs, or mmRNAs, containing a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipids, approximately 7.5-1 total lipid pairs. The final optimized intravenous formulation, which has a final weight ratio of polynucleotide, primary construct, or mm mRNA, and C14 alkyl chain length on PEG lipids and has an average particle size of approximately 50-60 nm, is the liver. Can result in a distribution of more than 90% of the formulation to (see Akinc et al., Mol Ther. 2009 17: 872-879, which is incorporated herein by reference in its entirety). In another example, C12-200 (see US Provisional Application No. 61 / 175,770 and Published International Application No. WO2010129709, each of which is incorporated herein by reference in its entirety) lipidoids. It can have a molar ratio of 50/10 / 38.5 / 1.5 C12-200 / disteroylphospatidyl choline / cholesterol / PEG-DMG, 7: 1 total lipid to polynucleotide, Intravenous formulations with a weight ratio of primary constructs, or mmRNAs, and an average particle size of 80 nm may be effective in delivering polynucleotides, primary constructs, or mmRNAs to hepatocytes (see throughout herein). See Love et al., Proc Nucle Acad Sci USA. 2010 107: 1864-1869). In another embodiment, the MD1 lipidoid-containing preparation can be used to effectively deliver a polynucleotide, primary construct, or mmRNA to hepatocytes in vivo. The properties of the lipidoid formulation optimized for the intramuscular or subcutaneous pathway can vary widely depending on the target cell type and the ability of the formulation to diffuse into the bloodstream via the extracellular matrix. Due to the size of the endothelial window, a particle size of less than 150 nm may be desired for effective hepatocyte delivery (Akinc et al., Mol Ther., Which is incorporated herein by reference in its entirety. (See 2009 17: 872-879), Lipidoid-formulated polynucleotides, primary constructs, for delivering the formulation to other cell types including, but not limited to, endothelial cells, bone marrow cells, and muscle cells. Alternatively, the use of mmRNA may not be size limited as well. The use of lipidoid preparations for delivering siRNA to bone marrow cells and other non-hepatocyte cells such as endothelium in vivo has been reported (each incorporated herein by reference in its entirety, Akinc et al., Nat. Biotechnol.2008 26:. 561-569, Leuschner et al, Nat Biotechnol.2011 29: 1005-1010, Cho et al.Adv.Funct.Mater.2009 19: 3112-3118,8 th International Judah Folkman Conference, Cambridge, MA October 8-9, 2010). Effective delivery to bone marrow cells such as monocytes, lipidoid formulations may have similar component molar ratios. Preparations of polynucleotides, primary constructs, or marrows with different proportions of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol, and PEG-DMG, in the liver. It can be optimized for delivery to different cell types, including but not limited to cells, bone marrow cells, muscle cells, etc. For example, component molar ratios may include, but are limited to, 50% C12-200, 10% disteroylpphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG. (See Leuschner et al., Nat Biotechnol 2011 29: 1005-1010, which is incorporated herein by reference in its entirety). The use of a lipidoid agent for local delivery of nucleic acids to cells (such as, but not limited to, adipocytes and muscle cells) via either subcutaneous or intramuscular delivery is desired for systemic delivery. It may not require all of the components of the product, and therefore may contain only lipidoids and polynucleotides, primary constructs, or nucleic acids.

Lipidoids can be used to increase cell transfection with polynucleotides, primary constructs, or mmRNAs and / or to increase translation of the encoded protein, so using a combination of different lipidoids, polynucleotides, The effectiveness of protein production as directed by the primary construct, or mmRNA, may be improved (see Whitehead et al., Mol. Ther. 2011, 19: 1688-1694, which is incorporated herein by reference in its entirety). ..

Liposomes, Lipoplexes, and Lipid Nanoparticles The polynucleotides, primary constructs, and mmRNAs of the invention can be formulated with one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, the polynucleotide, primary construct, or pharmaceutical composition of mmRNA comprises liposomes. Liposomes are artificially prepared vesicles that may consist primarily of a lipid bilayer and can be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments (MLVs), small unicellular vesicles that can be smaller than 50 nm in diameter. (SUV), and large monolayer vesicles (LUVs) that can be 50-500 nm in diameter, but are not limited to these and can be of different sizes. Liposomal designs include, but are not limited to, opsonins or ligands for improving the binding of liposomes to unhealthy tissues, or for activating events such as, but not limited to, endocytosis. Liposomes may contain low or high pH to improve delivery of pharmaceutical formulations.

Liposomal formation occurs during encapsulation of the pharmaceutical formulation and liposomal components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulating substance and its potential toxicity, the application and / or delivery of the vesicles. Any additional process involved, optimized vesicle size for intended application, polydispersity, and shelf life, as well as safe and efficient inter-batch reproducibility and large-scale production of liposome products. It may depend on physicochemical properties, such as sex, but not limited to these.

In one embodiment, the pharmaceutical compositions described herein are, without limitation, 1,2-dioreyloxy-N, N-dimethylaminopropane (DODA) liposomes, DiLa2 liposomes with Marina Biotech (Botell, WA). , 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3] -dioxolane (DLin- KC2-DMA), and liposomes such as those formed from MC3 (US Patent Publication No. 2010234120, which is incorporated herein by reference in its entirety), and Janssen Biotech, Inc. (Horsham, PA), such as DOXIL®, but not limited to these, may include liposomes capable of delivering small molecule drugs.

In one embodiment, the pharmaceutical compositions described herein have been previously described without limitation and have been shown to be suitable for oligonucleotide delivery in vitro and in vivo, stabilized plasmid-lipids. Liposomes, such as those formed from the synthesis of particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), may be included (all of which are incorporated herein by reference in their entirety, Wheeler et al. Gene Therapy. 1999 6: 271-281, Zhang et al. Gene Therapy. 1999 6: 1438-1447, Jeffs et al. Pharma Res. 2005 22: 362-372, Morrissey et al., Nat
Biotechnol. 2005 2: 1002-1007, Zimmermann et al. , Nature. 2006 441: 111-114, Heies et al. J Control Rel. 2005 107: 276-287, Simple et al. Nature Biotechnology. 2010 28: 172-176, Judge et al. J Clin Invest. 2009 119: 661-673, deFougelolesHum Gene Ther. 2008 19: 125-132). The original production method by Wheeler et al. Was a detergent dialysis method, which was later improved by Jeffs et al. And is referred to as a spontaneous vesicle formation method. Liposomal preparations are composed of 3-4 lipid constituents in addition to polynucleotides, primary constructs, or mmRNAs. As an example, liposomes are 55% cholesterol, 20% distearoylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2- Dioleyloxy-N, N-dimethylaminopropane (DODA) can be contained, but is not limited thereto. As another example, certain liposome formulations contain 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipids, as described by Heies et al. However, but not limited to these, the cationic lipid may be 1,2-distearoyloxy-N, N-dimethylaminopropane (DSDMA), DODA, DLin-DMA, or 1,2-. It can be dilinolenyloxy-3-dimethylaminopropane (DLenDMA).

In one embodiment, the pharmaceutical composition may comprise liposomes that can be formed to deliver mmRNA that can encode at least one immunogen. The m mRNA may be encapsulated by liposomes and / or it may be contained in an aqueous core, which may then be encapsulated by liposomes (each of which is incorporated herein by reference in its entirety. See International Publications WO2012031046, WO2012031043, WO2012030901, and WO2012006378, which are incorporated). In another embodiment, the mmRNA that can encode an immunogen may be formulated in a cationic oil-in-water emulsion, and the emulsion particles may interact with the mm mRNA to attach the molecule to the emulsion particles. Containing possible oil cores and cationic lipids (see WO 2012006380, which is incorporated herein by reference in its entirety). In yet another embodiment, the lipid formulation may comprise at least a cationic lipid, which is a lipid capable of improving transfection, and at least one lipid containing a hydrophilic parietal group linked to the lipid moiety (each containing). See International Publication No. WO2011076807 and US Publication No. 20110200582, which are incorporated herein by reference in their entirety). In another embodiment, the polynucleotide encoding the immunogen, the primary construct, and / or mmRNA may be formulated in lipid vesicles that may have crosslinks between the functionalized lipid bilayers (see by reference). See US Publication No. 201201777224, which is incorporated herein in its entirety).

In one embodiment, the polynucleotide, primary construct, and / or mmRNA may be formulated in lipid vesicles that may have crosslinks between the functionalized lipid bilayers.

In one embodiment, the polynucleotide, primary construct, and / or mRNA may be formulated in a liposome containing a cationic lipid. Liposomes are nitrogen atoms in cationic lipids of 1: 1 to 20: 1: phosphates in RNA (N:) as described in WO2013006825, which is incorporated herein by reference in its entirety. It may have a molar ratio of P ratio). In another embodiment, the liposomes may have an N: P ratio greater than 20: 1 or less than 1: 1.

In one embodiment, the polynucleotide, primary construct, and / or mRNA may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and / or as described in US Publication No. 2012178702, which is incorporated herein by reference in its entirety. As non-limiting examples, polycations include, but are not limited to, polylysine, polyornithin, and / or polyarginine, and internationally incorporated herein by reference in their entirety. The cationic peptide described in Publication No. WO2011133326 may be included. In another embodiment, the polynucleotide, primary construct, and / or mRNA may further include neutral lipids such as, but not limited to, cholesterol or dioreoil phosphatidylethanolamine (DOPE), lipid-polycations. It may be formulated in the complex.

Liposomal formulations can be influenced by, but limited to, biophysical parameters such as the choice of cationic lipid components, the degree of cationic lipid saturation, the nature of PEGylation, the proportions of all components, and size. Not done. In an example by Single et al. (Semple et al. Nature Biotech. 2010 28: 172-176, which is incorporated herein by reference in its entirety), the liposome formulation is 57.1% cationic lipid, 7.1%. It was composed of dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, by altering the composition of cationic lipids, siRNA could be effectively delivered by a variety of antigen presenting cells (Basha et al. Mol Ther, which is incorporated herein by reference in its entirety). .2011 19: 2186-2200).

In some embodiments, the proportion of PEG in the lipid nanoparticle (LNP) formulation can be increased or decreased and / or the carbon chain length of the PEG lipid is modified from C14 to C18 and the pharmacokinetics of the LNP formulation. And / or the biodistribution can be altered. As a non-limiting example, the LNP preparation may contain PEG-c-DOMG with a lipid molar ratio of 1-5% as compared to cationic lipids, DSPC, and cholesterol. In another embodiment, the PEG-c-DOMG is PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene). Glycol) and the like, but may be replaced with PEG lipids not limited to these. Cationic lipids may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA. ..

In one embodiment, the polynucleotide, primary construct, or mmRNA may be formulated into the lipid nanoparticles and the like described in WO2012170930, which is incorporated herein by reference in its entirety.

In one embodiment, the cationic lipids are incorporated herein by reference in their entirety, WO2011040184, WO2011153120, WO2011149733, WO2011109965, WO20110439313. , WO20110224660, WO2012061259, WO201004365, WO2012044638, WO20100724, WO201001865, and WO2008103276, US Pat. No. 7,893,302, No.7 , 404, 969, and 8,283,333, and US Patent Publication Nos. US20100036115 and US20120202871 may be selected from, but are not limited to. In another embodiment, the cationic lipids are incorporated herein by reference in their entirety, WO 201240184, WO 2011153120, WO 2011149733, WO 2011090965, WO 20110439313. No., WO2011224660, WO2011061259, WO2012054365, and WO2012044638 may be selected from, but are not limited to, the formula A. In yet another embodiment, the cationic lipids, each of which is incorporated herein by reference in its entirety, formula CLI-CLXXIX of WO 2008103276, formula CLI of US Pat. No. 7,893,302. -CLXXXIX, formula CLI-CLXXXXXII of US Pat. No. 7,404,969, and formula I-VI of US Pat. No. 7,20100036115, but are not limited thereto. As a non-limiting example, the cationic lipids are (20Z, 23Z) -N, N-dimethylnonacosa-20,23-diene-10-amine, (17Z, 20Z) -N, N-dimethylhexacosa ( dimemylhexacosa) -17,20-diene-9-amine, (1Z, 19Z) -N5N-dimethylpentacosa-16,19-diene-8-amine, (13Z, 16Z) -N, N-dimethyldocosa-13, 16-diene-5-amine, (12Z, 15Z) -N, N-dimethylhenicosa-12,15-dien-4-amine, (14Z, 17Z) -N, N-dimethyltricosa-14,17-diene- 6-amine, (15Z, 18Z) -N, N-dimethyltetracosa-15,18-diene-7-amine, (18Z, 21Z) -N, N-dimethylheptacosa-18,21-diene-10- Amine, (15Ζ, 18Ζ) -Ν, Ν-dimethyltetracosa-15,18-diene-5-amine, (14Z, 17Z) -N, N-dimethyltricosa-14,17-dien-4-amine, (19Z, 22Z) -N, N-dimethyloctacosa-19,22-diene-9-amine, (18Z, 21Z) -N, N-dimethylheptacosa-18,21-dien-8- Amine, (17Z, 20Z) -N, N-dimethylhexacosa-17,20-diene-7-amine, (16Z, 19Z) -N, N-dimethylpentacosa-16,19-dien-6-amine, (22Z, 25Z) -N, N-Dimethylhentoria Conta-22,25-diene-10-amine, (21Z, 24Z) -N, N-Dimethyltoria Conta-21,24-diene-9-amine, ( 18Z) -N, N-dimethylheptacos-18-en-10-amine, (17Z) -N, N-dimethylhexacosa-17-en-9-amine, (19Z, 22Z) -N, N-Dimethyloctacosa-19,22-diene-7-amine, N, N-dimethylheptacosan-10-amine, (20Z, 23Z) -N-ethyl-N-methylnonacosa-20,23-diene-10- Amine, 1-[(11Z, 14Z) -1-nonylikosa-11,14-dien-1-yl] pyrrolidine, (20Z) -N, N-dimethylheptacosa-20-ene-10-amine, (15Z) -N, N-dimethyl eptacosa-15-ene-10-amine , (14Z) -N, N-dimethylnonacosa-14-ene-10-amine, (17Z) -N, N-dimethylnonacosa-17-en-10-amine, (24Z) -N, N-dimethyl Tritoria Conta-24-en-10-amine, (20Z) -N, N-dimethylnonacosa-20-en-10-amine, (22Z) -N, N-dimethylhentoria Conta-22-en-10 -Amine, (16Z) -N, N-dimethylpentacosa-16-ene-8-amine, (12Z, 15Z) -N, N-dimethyl-2-nonylhenicosa-12,15-diene-1-amine, ( 13Z, 16Z) -N, N-dimethyl-3-nonyldocosa-13,16-diene-1-amine, N, N-dimethyl-1-[(lS, 2R) -2-octylcyclopropyl] eptadecan -8-amine, 1-[(1S, 2R) -2-hexylcyclopropyl] -N, N-dimethylnonadecan-10-amine, Ν, Ν-dimethyl-1-[(1S, 2R) -2- Octylcyclopropyl] nonadecan-10-amine, N, N-dimethyl-21-[(1S, 2R) -2-octylcyclopropyl] henicosan-10-amine, Ν, Ν-dimethyl-1-[(1S, 2S) ) -2-{[(1R, 2R) -2-pentylcyclopropyl] methyl} cyclopropyl] nonadecan-10-amine, Ν, Ν-dimethyl-1-[(1S, 2R) -2-octylcyclopropyl] Hexadecane-8-amine, Ν, Ν-dimethyl-[(1R, 2S) -2-undecylcyclopropyl] tetradecane-5-amine, N, N-dimethyl-3- {7-[(1S, 2R)- 2-octylcyclopropyl] heptyl} dodecane-1-amine, 1-[(1R, 2S) -2-heptylcyclopropyl] -Ν, Ν-dimethyloctadecane-9-amine, 1-[(1S, 2R)- 2-decylcyclopropyl] -N, N-dimethylpentadecane-6-amine, N, N-dimethyl-1-[(lS, 2R) -2-octylcyclopropyl] pentadecane-8-amine, RN, N -Dimethyl-1-[(9Z, 12Z) -Octadeca-9,12-diene-1-yloxy] -3- (octyloxy) propan-2-amine, S-N, N-dimethyl-1-[(9Z) , 12Z) -Octadeca-9,12-diene-1-yloxy] -3- (octyloxy) propan-2-amine, 1- {2-[(9Z, 12Z)- Octadeca-9,12-diene-1-yloxy] -1-[(octyloxy) methyl] ethyl} pyrrolidine, (2S) -N, N-dimethyl-1-[(9Z, 12Z) -octadeca-9,12 -Diene-1-yloxy] -3-[(5Z) -octa-5-ene-1-yloxy] propan-2-amine, 1- {2-[(9Z, 12Z) -octadeca-9,12-diene -1-Iloxy] -1-[(octyloxy) methyl] ethyl} azetidine, (2S) -1- (hexyloxy) -N, N-dimethyl-3-[(9Z, 12Z) -octadeca-9,12 -Diene-1-yloxy] Propan-2-amine, (2S) -1- (heptyloxy) -N, N-dimethyl-3-[(9Z, 12Z) -octadeca-9,12-diene-1-yloxy ] Propan-2-amine, Ν, Ν-dimethyl-1- (nonyloxy) -3-[(9Z, 12Z) -octadeca-9,12-diene-1-yloxy] Propan-2-amine, Ν, Ν- Dimethyl-1-[(9Z) -octadeca-9-ene-1-yloxy] -3- (octyloxy) propan-2-amine; (2S) -N, N-dimethyl-1-[(6Z, 9Z,) 12Z) -Octadeca-6,9,12-Triene-1-yloxy] -3- (octyloxy) propan-2-amine, (2S) -1-[(11Z, 14Z) -Icosa-11,14-diene -1-Iloxy] -N, N-dimethyl-3- (pentyloxy) propan-2-amine, (2S) -1- (hexyloxy) -3-[(11Z, 14Z) -Icosa-11, 14- Diene-1-yloxy] -N, N-dimethylpropan-2-amine, 1-[(11Z, 14Z) -Icosa-11,14-diene-1-yloxy] -Ν, Ν-dimethyl-3- (octyl) Oxy) Propan-2-amine, 1-[(13Z, 16Z) -docosa-l3,16-diene-1-yloxy] -N, N-dimethyl-3- (octyloxy) propan-2-amine, (2S) ) -1-[(13Z, 16Z) -docosa-13,16-dien-1-yloxy] -3- (hexyloxy) -N, N-dimethylpropan-2-amine, (2S) -1-[( 13Z) -docosa-13-en-1-yloxy] -3- (hexyloxy) -N, N-dimethylpropan-2-amine, 1-[(13Z) -docosa-13-en-1-yloxy]- N, N-dimethyl-3 -(Octyloxy) propan-2-amine, 1-[(9Z) -hexadeca-9-ene-1-yloxy] -N, N-dimethyl-3- (octyloxy) propan-2-amine, (2R) -N, N-dimethyl-H (1-methoyloctyl) oxy] -3-[(9Z, 12Z) -octadeca-9,12-diene-1-yloxy] propan-2-amine, (2R) -1 -[(3,7-Dimethyloctyl) oxy] -N, N-dimethyl-3-[(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] propan-2-amine, N, N- Dimethyl-1- (octyloxy) -3-({8-[(1S, 2S) -2-{[(1R, 2R) -2-pentylcyclopropyl] methyl} cyclopropyl] octyl} oxy) Propane-2 -Amine, N, N-dimethyl-1-{[8- (2-octylcyclopropyl) octyl] oxy} -3- (octyloxy) propan-2-amine, and (11E, 20Z, 23Z) -N, It may be selected from N-dimethylnonacosa-11,20,2-triene-10-amine, or pharmaceutically acceptable salts or stereoisomers thereof.

In one embodiment, the lipid may be a cleavable lipid, such as the lipid described in WO 2012170888, which is incorporated herein by reference in its entirety.

In one embodiment, the cationic lipids are incorporated herein by methods known in the art and / or by reference in their entirety, WO 201240184, WO 2011153120, the same. Synthesized as described in WO2011149733, WO2011109 May be done.

In one embodiment, the LNP preparation of polynucleotide, primary construct, and / or mmRNA may contain PEG-c-DOMG in a 3% lipid molar ratio. In another embodiment, the LNP-prepared polynucleotide, primary construct, and / or mmRNA may contain PEG-c-DOMG in a 1.5% lipid molar ratio.

In one embodiment, the pharmaceutical composition polynucleotide, primary construct, and / or mmRNA may comprise at least one of the PEGylated lipids described in WO 20120999755, which is incorporated herein by reference. good.

In one embodiment, the LNP preparation may contain PEG-DMG 2000 (1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000). In one embodiment, the LNP preparation may contain a cationic lipid known in the art, PEG-DMG 2000, and at least one other component. In another embodiment, the LNP preparation may contain the cationic lipids known in the art, PEG-DMG 2000, DSPC, and cholesterol. As a non-limiting example, the LNP preparation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, the LNP preparation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (eg, overall by reference). Is incorporated herein by Gall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID: 229082894). As another non-limiting example, the modified RNA described herein is a nanoparticle delivered by the parenteral route, as described in US Publication No. 20120207845, which is incorporated herein by reference in its entirety. It may be formulated in.
In one embodiment, the LNP formulations may be formulated by the methods described in WO2011127255 or WO2008103276, each of which is incorporated herein by reference in its entirety. As a non-limiting example, the modified RNAs described herein are described in WO2011127255 and / or WO2008103276, each of which is incorporated herein by reference in its entirety. It may be encapsulated in the LNP preparation.
In one embodiment, the LNP preparation described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be selected from Formulas 1-60 of US Patent Publication No. US20050222064, which is incorporated herein by reference in its entirety. In another embodiment, the LNP preparation containing the polycationic composition may be used for in vivo and / or in vitro delivery of the modified RNA described herein.

In one embodiment, the LNP preparation described herein may further comprise a permeability enhancer molecule. Non-limiting permeation enhancer molecules are described in US Patent Publication No. US20050222064, which is incorporated herein by reference in its entirety.

In one embodiment, the pharmaceutical composition is a DiLa2 liposome (Marina Biotech, Bothell, WA), SMARTICLES® (Marina).
Biotech, Bothell, WA), Neutral DOPC (1,2-dioleoil-sn-glycero-3-phosphocholine) liposomes (eg, siRNA delivery for ovarian cancer (incorporated herein by reference in its entirety, Landen et al). Cancer Biotechnology & Therapy 2006 5 (12) 1708-1713)), and hyaluronan-coated liposomes (Quiet Therapeutics, Isolael), but may be formulated in liposomes.

The nanoparticle preparation may be carbohydrate nanoparticles containing a carbohydrate carrier and a modified nucleic acid molecule (eg, mmRNA). As a non-limiting example, the carbohydrate carrier is an anhydride-modified plant glycogen or glycogen-type material, plant glycogen octenyl succinate, plant glycogen β-dextrin, anhydride-modified plant glycogen β-dextrin. May include, but are not limited to. (See, eg, WO 2012109121, which is incorporated herein by reference in its entirety).

The lipid nanoparticle preparation may be improved by replacing the cationic lipid with a biodegradable cationic lipid known as rapid eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time, potentially. Can be a source of toxicity. Rapid metabolism of rapidly excreted lipids can improve the resistance and therapeutic index of lipid nanoparticles by an order of magnitude from 1 mg / kg dose to 10 mg / kg dose in rats. Incorporation of enzymatically degraded ester bonds can improve the degradation and metabolic profile of cationic components while still maintaining the activity of the reLNP formulation. The ester bond can be located inside the lipid chain, or it may be located at the end of the lipid chain. The internal ester bond may replace any carbon in the lipid chain.

が挙げられる。 In one embodiment, the internal ester bond may be located on either side of the saturated carbon. A non-limiting example of reLNP is

Can be mentioned.

In one embodiment, an immune response can be elicited by delivering lipid nanoparticles that can include nanochemical species, polymers, and immunogens. (US Publication No. 2011089700 and International Publication No. WO 20120999805, each of which is incorporated herein by reference in its entirety). The polymer may be encapsulated with nano-species or partially encapsulated with nano-species. The immunogen may be a recombinant protein, modified RNA, and / or primary construct described herein. In one embodiment, the lipid nanoparticles may be formulated for use in vaccines such as, but not limited to, those against pathogens.

Lipid nanoparticles may be engineered to alter the surface properties of the particles so that the lipid nanoparticles penetrate the mucosal barrier. Mucus is the oral cavity (eg, peri-mouth and esophageal membranes and tonsillar tissue), eyes, gastrointestinal (eg, stomach, small intestine, large intestine, colon, rectum), nose, respiratory organs (eg, nasal, pharynx, trachea, and bronchi). Membrane), mucosal tissue located on the reproductive organs (eg, in the vagina, cervix, and esophageal membrane), but is not limited to these. Nanoparticles larger than 10-200 nm, preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wide range of drugs, are believed to be too large to diffuse rapidly through the mucosal barrier. Most of the captured particles can be removed from the mucosla tissue within seconds or hours, as mucus is continuously secreted, shed, discarded or digested and regenerated. Large polymer nanoparticles (200 nm to 500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) were only 4 to 6 times lower than the same particles diffused in water and diffused through the mucilage (each). Lai et al. PNAS 2007 104 (5): 1482-487, Lai et al. Adv Drug Driv Rev. 2009 61 (2): 158-171), all of which are incorporated herein by reference. Nanoparticle transport can be determined using transmission rate and / or fluorescence microscopy techniques, including, but not limited to, fluorescence post-fading recovery measurements (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions capable of penetrating the mucosal barrier may be made as described in US Pat. No. 8,241,670, which is incorporated herein by reference in its entirety.

Lipid nanoparticles engineered to permeate mucus may include polymeric materials (ie, polymeric cores) and / or polymeric-vitamin complexes and / or triblock copolymers. Polymer materials include polyamine, polyether, polyamide, polyester, polycarbamate, polyurea, polycarbonate, poly (styrene), polyimide, polysulfone, polyurethane, polyacetylene, polyethylene, polyethyleneimine, polyisocyanate, polyacrylate, polymethacrylate, poly. Acrylonitrile and polyarylate may be included, but are not limited thereto. The polymeric material may be biodegradable and / or biocompatible. The polymeric material may be further irradiated. As a non-limiting example, the polymeric material may be gamma-irradiated (see, eg, International Application WO 201282165, which is incorporated herein by reference in its entirety). Non-limiting examples of specific polymers include poly (caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly (lactic acid) (PLA), poly (L-lactic acid) (PLLA), poly (glycol). Acid) (PGA), Poly (lactic acid-co-glycolic acid) (PLGA), Poly (L-lactic acid-co-glycolic acid) (PLLGA), Poly (D, L-lactide) (PDLA), Poly (L-) Lactide) (PLLA), Poly (D, L-Lactide-co-Caprolactone), Poly (D, L-Lactide-co-Caprolactone-co-Glycolide), Poly (D, L-Lactide-co-PEO-co-) D, L-lactide), poly (D, L-lactide-co-PPO-co-D, L-lactide), polyalkylcyanoacrylate (acrylate), polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate Polyalkylenes such as (HPMA), polyethylene glycol, poly-L-glutamic acid, poly (hydroxy acid), polyan anhydride, polyorthoester, poly (esteramide), polyamide, poly (ester ether), polycarbonate, polyethylene and polypropylene. , Polyalkylene glycol such as poly (ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalate such as poly (ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl acetate, etc. Polylate halides such as esters and poly (vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxane, polystyrene (PS), polyurethane, alkylcellulose, hydroxyalkylcellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropylcellulose, carboxy Derivatized cellulose such as methyl cellulose, poly (methyl (meth) acrylate) (PMMA), poly (ethyl (meth) acrylate), poly (butyl (meth) acrylate), poly (isobutyl (meth) acrylate), poly (hexyl (hexyl) Meta) acrylate), poly (isodecyl (meth) acrylate), poly (lauryl (meth) acrylate), poly (phenyl (meth) acrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), Poly (octadecyl acrylate), etc. Acrylic acid polymers, and copolymers and mixtures thereof, polydioxanone and copolymers thereof, polyhydroxyalkanoate, polypropylene fumarate, polyoxymethylene, poloxamers, poly (ortho) esters, poly (butyric acid), poly (valvic acid), Poly (lactide-co-caprolactone), and trimethylene carbonate, polyvinylpyrrolidone can be mentioned. The lipid nanoparticles are block copolymers (such as the branched polyether-polyamide block copolymers described in WO2013012476, which is incorporated herein by reference in their entirety), and (poly (ethylene glycol)-(poly (propylene)). Oxide)-(Poly (ethylene glycol) triblock copolymers (eg, US Publication No. 20120121718 and US Publication No. 2001003337 and US Pat. No. 8,263,665, each of which is incorporated herein by reference in its entirety. The copolymer may be coated with or associated with a copolymer, such as, but not limited to, the copolymer may be a generally accepted (GRAS) polymer or a lipid nano. The formation of particles may be such that no new chemical entity is created. For example, lipid nanoparticles are still rapidly permeable to human mucus without forming new chemical entities. , Poroxamer-coated PLGA nanoparticles may be included (all incorporated herein by reference, Yang.
et al. Angew. Chem. Int. Ed. 2011 50: 2597-2600).

The vitamin in the polymer-vitamin complex may be vitamin E. The vitamin portion of this complex is the hydrophobic portion or hydrophobic constituents of vitamin A, vitamin E, other vitamins, cholesterol and other surfactants (eg, sterol chains, fatty acids, hydrocarbon chains, and alkylene oxide chains). ) Etc., but may be replaced with other suitable constituents not limited to these.

Lipid nanoparticles engineered to permeate the mucus include mmRNA, anionic proteins (eg, bovine serum albumin), surfactants (eg, dimethyldioctadecyl-ammonium butamide, etc.), eg, cationic surfactants. Agents), sugars or sugar derivatives (eg cyclodextrin), nucleic acids, polymers (eg heparin, polyethylene glycol, and poroxamer), mucolytic agents (eg N-acetylcysteine, rice cake grass, bromhexol, papain, clerodendron, acetyl) Various DNases including cysteine, bromhexine, carbocysteine, epradinone, mesna, ambroxol, sobrolol, domiodol, retostain, stepronin, thiopronin, gelzoline, thymosin β4 dronase α, nertenexin, erdosteine), and rhDNase, etc. Surface alteration agents not limited to these may be included. The surface alteration agent may be embedded or entangled on the surface of the particles or placed on the surface of the lipid nanoparticles (eg, by coating, adsorption, covalent bonding, or other process). (See, for example, US Publication No. 2014152580 and US Publication No. 20080166414, each of which is incorporated herein by reference in its entirety).

Mucus permeable lipid nanoparticles may contain at least one mmRNA described herein. The m mRNA may be encapsulated in lipid nanoparticles and / or placed on the surface of the particles. The m mRNA may be covalently attached to lipid nanoparticles. The preparation of mucus-permeable lipid nanoparticles may contain a plurality of nanoparticles. In addition, the formulation can interact with mucus to alter the structural and / or adherent properties of the surrounding mucus to reduce mucosal adhesion, thereby delivering mucus-permeable lipid nanoparticles to the mucosal tissue. It may contain particles that can be increased.

In one embodiment, polynucleotides, primary constructs, or mRNAs are, without limitation, ATUPLEX ™, DACC, DBTC, and other siRNA-lipoplex techniques, STEMGENT, by Silence Therapeutics (London, United Kingdom). Formulated as lipoplexes, such as STEMFECT ™ by Registered Trademarks) (Cambridge, MA), and targeted and untargeted nucleic acid delivery of polyethyleneimine (PEI) or protamine-based (all by reference). Alleku et al. Cancer Res. 2008 68: 9788-9798, Strumberg et al. Int J Clin Pharmacol Ther 2012 50: 76-78, Santel et al., Gene Ther6, which are incorporated herein in their entirety. -1234, Santel et al., Gene The 2006 13: 1360-1370, Gutbier et al., Palm Pharmacol. Ther. 2010 23: 334-344, Kaufmann et al. Microvasc Res 2010 80: 28
Immunother. 2009 32: 498-507, Weide et al. J Immunother. 2008 31: 180-188, Pascolo Expert Opin. Biol. The. 4: 1285-1294, Fotin-Mlekzek et al. , 2011 J.M. Immunother. 34: 1-15, Song et al. , Nature Biotechnology. 2005, 23: 709-717, Peer et al. , Proc Natl Acad Sci USA. 2007 6; 104: 4095-4100, deFougelolesHum Gene Ther. 2008 19: 125-132).

In one embodiment, such formulations passively or actively have different cell types, including, but not limited to, hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes. They can also be constructed to be oriented in vivo or have varying compositions (all incorporated herein by reference in their entirety, Akinc et al. Mol Ther. 2010 18: 1357-1364, Song et al. , Nat
Biotechnol. 2005 23: 709-717, Judge et al. , J Clin Invest. 2009 119: 661-673, Kaufmann
et al. , Microvasc Res 2010 80: 286-293, Santel et al. , Gene Ther 2006 13: 1222-1234, Santel et al. , Gene The 2006 13: 1360-1370, Gutbier et al. , Palm Pharmacol. The. 2010 23: 334-344, Basha et al. , Mol. The. 2011 19: 2186-2200, Fensuke and Cullis, Expert Opin Drag Deliv. 2008 5: 25-44, Peer et al. , Science. 2008 319: 627-630, Peer and Lieberman, Gene Ther. 2011 18: 1127-1133). Examples of passive targeting of the formulations to hepatocytes include DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA-based lipid nanoparticle formulations, which bind to apolipoprotein E and It has been shown to promote the binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18: 1357-1364, which is incorporated herein by reference in its entirety). The formulations are illustrated by an approach targeted by folate, transferrin, N-acetylgalactosamine (GalNAc), and antibodies, but selectively through the expression of different ligands on their surface, but not limited by these. Targets can also be defined (all incorporated herein by reference, Kolhatkar et al., Curr Drag Discov Technique. 2011 8: 197-206, Musaccio and Torchilin, Front Bioscii. 2011 16: 1388-14. , Yu et al., Mol Membr Biol. 2010 27: 286-298, Patil et al., Crit Rev The Drug Carrier System 2008 25: 1-61, Benoit et
al. , Biomacromolecules. 2011 12: 2708-2714, Zhao et al. , Expert Opin Drag Deliv. 2008
5: 309-319, Akinc et al. , Mol Ther. 2010 18: 1357-1364, Srinivasan et al. , Methods Mol
Biol. 2012 820: 105-116, Ben-Arie et al. , Methods Mol Biol. 2012 757: 497-507, Peer 2010 J Control Release. 20: 63-68, Peer et al. , Proc Natl Acad Sci USA. 2007 104: 4095-4100, Kim et al. , Methods Mol Biol. 2011 721: 339-353, Subramanya et al. , Mol Ther. 2010
18: 2028-2037, Song et al. , Nat Biotechnology. 2005 23: 709-717, Peer et al. , Science. 2008 319: 627-630, Peer and Lieberman, Gene Ther. 2011 18: 1127-1133).

In one embodiment, the polynucleotide, primary construct, or mmRNA is formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLN) can be spherical with an average diameter of 10 to 1000 nm. SLN has a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized by surfactants and / or emulsifiers. In a further embodiment, the lipid nanoparticles may be self-assembling lipid-polymer nanoparticles (Zhang et al., ACS Nano, 2008, 2 (8), which are incorporated herein by reference in their entirety). See pp 1696-1702).

Liposomes, lipoplexes, or lipid nanoparticles may allow these formulations to increase cell transfection with polynucleotides, primary constructs, or mmRNAs and / or increase translation of the encoded protein. Therefore, it may be used to improve the effectiveness of protein production as directed by polynucleotides, primary constructs, or mmRNAs. One such example involves the use of lipid encapsulation to allow effective systemic delivery of the polypeptide plasmid DNA (Heies et al., Mol Ther., Which is incorporated herein by reference in its entirety. 2007 15: 713-720). Liposomes, lipoplexes, or lipid nanoparticles can also be used to increase the stability of polynucleotides, primary constructs, or mmRNAs.

In one embodiment, the polynucleotides, primary constructs, and / or mRNAs of the invention can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that adapts to a particular release pattern and results in therapeutic outcomes. In one embodiment, the polynucleotide, primary construct, or mmRNA is encapsulated in a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. It may be enclosed. As used herein, the term "encapsulating" means encapsulating, enclosing, or enveloping. When it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial. The term "substantially encapsulated" refers to at least more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, 95% of the pharmaceutical compositions or compounds of the invention. More than 96%, more than 97%, more than 98%, more than 99%, more than 99.9%, more than 99.9%, or more than 99.999% are encapsulated or surrounded in the delivery agent. Or it means to be wrapped up. "Partial encapsulation" means that less than 10, 10, 20, 30, 40, 50 or less of the pharmaceutical compositions or compounds of the invention are encapsulated, enclosed, or encased in a delivery agent. means. Advantageously, encapsulation may be determined by measuring the escape or activity of the pharmaceutical composition or compound of the invention using fluorescence and / or electron micrographs. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9 of the pharmaceutical compositions or compounds of the invention. , 99.99, or more than 99.99% is encapsulated in the delivery agent.

In one embodiment, the controlled release formulation may include, but is not limited to, triblock copolymers. As a non-limiting example, the pharmaceutical product may contain two different types of triblock copolymers (International Publication Nos. WO2012131104 and WO2012131106, each of which is incorporated herein by reference in its entirety).

In another embodiment, the polynucleotide, primary construct, or mMV may be encapsulated in lipid nanoparticles or rapidly effluxing lipid nanoparticles, which are then described herein. They may be encapsulated in polymers, hydrogels, and / or surgical sealants described and / or known in the art. As a non-limiting example, polymers, hydrogels, or surgical sealants include PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (registered trademark). Halozyme Therapeutics, San Diego CA), Fibrinogen Polymer (Ethylene Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based Sealant, and COSE It may be a surgical sealant such as Inc. Deerfield, IL).

In another embodiment, the lipid nanoparticles may be encapsulated in any polymer known in the art that can form a gel when injected into the subject. As another non-limiting example, lipid nanoparticles may be encapsulated in a potentially biodegradable polymer matrix.

In one embodiment, the polynucleotide, primary construct, or mm mRNA formulation for controlled release and / or targeted delivery may also include at least one controlled release coating agent. Controlled release coatings include OPADRY®, polyvinylpyrrolidone / vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS®, and ethylcellulose. Includes, but is not limited to, cellulose derivatives such as aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the controlled release and / or targeted delivery formulation may comprise at least one degradable polyester which may contain a polycationic side chain. Degradable polyesters include poly (serine ester), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline ester), and combinations thereof. Not limited to. In another embodiment, the degradable polyester may include a PEG complex to form a PEGylated polymer.

In one embodiment, the polynucleotides, primary constructs, and / or mmRNAs of the invention may be encapsulated in therapeutic nanoparticles. Therapeutic nanoparticles are the methods described herein, and WO2010005740, WO20100030763, WO2010005721, WO2010005723, each of which is incorporated herein by reference in its entirety. , WO2011054923, US Publication No. US201101262491, US201001046445, US201000873337, US20100068285, US20110274759, US201000682886, and US Pat. No. 8,206. , 747, No. 8,293,276, No. 8,318,208, No. 8,318,211 and the like, but not limited to these, formulated by a method known in the art. May be done. In another embodiment, the therapeutic polymer nanoparticles may be identified by the method described in US Publication US 201102140790, which is incorporated herein by reference in its entirety.

In one embodiment, the therapeutic nanoparticles may be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that is compatible with a release rate over a particular period of time. The period may include, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained release nanoparticles may include, but are not limited to, polymers and therapeutic agents such as, but not limited to, the polynucleotides, primary constructs, and mmRNAs of the invention (each of which, by reference, in their entirety). See International Publication No. 20100075072, and US Publication Nos. US20100216804, US201110217377, and US20110201859), which are incorporated herein by the present invention).

In one embodiment, the therapeutic nanoparticles may be formulated to be target-specific. As a non-limiting example, therapeutic nanoparticles may include corticosteroids (see WO2011084518, which is incorporated herein by reference in its entirety). In one embodiment, the therapeutic nanoparticles may be formulated to be cancer-specific. As a non-limiting example, therapeutic nanoparticles are incorporated herein by reference in their entirety, WO 2008121949, WO 2010005726, WO 2010005725, WO 2011084521, and It may be formulated into the nanoparticles described in US Publication Nos. US201069426, US20110004293, and US20010014655.

In one embodiment, the nanoparticles of the invention may comprise a polymer matrix. As a non-limiting example, the nanoparticles are polyethylene, polycarbonate, polyanhydride, polyhydroxyic acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly (orthoester), poly. Cyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly (ethyleneimine), poly (serine ester), poly (L-lactide-co-) It may contain two or more polymers such as, but not limited to, L-lysine), poly (4-hydroxy-L-proline ester) or a combination thereof.

In one embodiment, the therapeutic nanoparticles include diblock copolymers. In one embodiment, the diblock copolymer includes polyethylene, polycarbonate, polyanhydrous, polyhydroxyic acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly (orthoester), poly. Cyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly (ethyleneimine), poly (serine ester), poly (L-lactide-co-) L-lysine), poly (4-hydroxy-L-proline ester) or a combination thereof, etc., but may include PEG in combination with a polymer which is not limited thereto.

As a non-limiting example, therapeutic nanoparticles include PLGA-PEG block copolymers, US Publication US 20110004293 and US Pat. No. 8,236,330, each of which is incorporated herein by reference in its entirety. See issue). In another non-limiting example, therapeutic nanoparticles are stealth nanoparticles comprising diblock copolymers of PEG and PLA or PEG and PLGA (each incorporated herein by reference in its entirety, USA). See Pat. No. 8,246,968 and WO2012166923).

In one embodiment, the therapeutic nanoparticles may comprise multi-block copolymers (eg, US Pat. Nos. 8,263,665 and 8,287, each of which is incorporated herein by reference in its entirety. , No. 910).

In one embodiment, the block copolymers described herein may be included in polyion complexes, including non-polymeric micelles and block copolymers. (See, eg, US Publication No. 20120076836, which is incorporated herein by reference in its entirety).

In one embodiment, the therapeutic nanoparticles may comprise at least one acrylic polymer. Acrylic polymers include acrylic acid, methacrylic acid, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), and polycyano. Acrylates, as well as combinations thereof, are included, but not limited to these.

In one embodiment, the therapeutic nanoparticles may comprise at least one cationic polymer described herein and / or known in the art.

In one embodiment, the therapeutic nanoparticles are polylysine, polyethyleneimine, poly (amide amine) dendrimer, poly (β-amino ester) (eg, incorporated herein by reference in their entirety, US Pat. No. 8,287, 849), and combinations thereof, etc., but may include at least one amine-containing polymer, but not limited to these.

In one embodiment, the therapeutic nanoparticles may comprise at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include poly (serine ester), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline ester), and combinations thereof. Not limited to. In another embodiment, the degradable polyester may include a PEG complex to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticles may include complexation of at least one targeting ligand. The targeting ligand may be any ligand known in the art, such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006 66: 6732-6740, which is incorporated herein by reference in its entirety).

In one embodiment, the therapeutic nanoparticles may be formulated in aqueous solution and may be used to target cancer (each of which is incorporated herein by reference in its entirety). See WO2011084513 and US Publication No. US201110294717).

In one embodiment, the polynucleotide, primary construct, or mMV may be encapsulated in a synthetic nanocarrier, linked to it, and / or associated with it. The synthetic nanocarriers, each of which is incorporated herein by reference in its entirety, are WO2010005740, WO20100030763, WO201213501, WO2012149252, WO2012149255, WO2012149259. No. WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454, and WO2011309669, and the United States. Includes, but is not limited to, the synthetic nanocarriers described in Publications US 201101262491, US 20101014645, US 201000873337, and US 20110244222. Synthetic nanocarriers can be formulated using methods known in the art and / or methods described herein. As a non-limiting example, synthetic nanocarriers are incorporated herein by reference in their entirety, WO2010005740, WO20100030763, and WO201213501, and US Publication US201101262491. , US20010014645, US201000873337, and US201204422. In another embodiment, the synthetic nanocarrier formulations are lyophilized by the methods described in WO2011072218 and US Pat. No. 8,211,473, each of which is incorporated herein by reference in its entirety. obtain.

In one embodiment, the synthetic nanocarriers may contain the polynucleotides, primary constructs, and / or reactive groups for releasing the mRNA described herein (each by reference in its entirety). See International Publication No. WO20110952552 and US Publication No. US20120171229, which are incorporated herein by reference).

In one embodiment, the synthetic nanocarrier may contain an immunostimulatory agent to enhance the immune response upon delivery of the synthetic nanocarrier. As a non-limiting example, synthetic nanocarriers may include Th1 immunostimulatory agents that may enhance the Th1-based response of the immune system (each of which is incorporated herein by reference in its entirety, WO 2010123569. (See US Publication No. US201102223201).

In one embodiment, the synthetic nanocarrier may be formulated for targeted release. In one embodiment, the synthetic nanocarrier is formulated to release the polynucleotide, primary construct, and / or mmRNA at the specified pH and / or after the desired time interval. As a non-limiting example, synthetic nanoparticles may be formulated to release polynucleotides, primary constructs, and / or mRNA after 24 hours and / or at a pH of 4.5 (each by reference). See International Publications WO2010138193 and WO2010138194, and US Publications US2011020038 and US20110027217, which are incorporated herein in their entirety).

In one embodiment, the synthetic nanocarrier may be formulated for controlled and / or sustained release of the polynucleotides, primary constructs, and / or mmRNAs described herein. As a non-limiting example, synthetic nanocarriers for sustained release are known in the art and are described herein and / or each of which is incorporated herein by reference in its entirety. It may be formulated by the method described in International Publication No. WO2010138192 and US Publication No. 2010030385.

In one embodiment, the synthetic nanocarrier may be formulated for use as a vaccine. In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide, primary construct, and / or mRNA encoding at least one antigen. As a non-limiting example, synthetic nanocarriers may contain excipients for at least one antigen and vaccine dosage form (each of which is incorporated herein by reference in its entirety, WO 2011150264). And US Publication No. US201102937723). As another non-limiting example, the vaccine dosage form may comprise at least two synthetic nanocarriers with the same or different antigens, and excipients, each of which is incorporated herein by reference in its entirety. , International Publication No. WO2011150249 and US Publication No. US201110293701). Vaccine dosage forms are described herein, are known in the art, and / or each of which is incorporated herein by reference in their entirety in WO201115258 and US Publication US20120027806. It may be selected by the method described.

In one embodiment, the synthetic nanocarrier may comprise at least one polynucleotide, a primary construct, and / or mmRNA that encodes at least one adjuvant. As a non-limiting example, adjuvants are dimethyldioctadecylammonium-bromid, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate, or dimethyldioctadecylammonium-acetate (DDA), and Mycobacterium. May include a non-polar fraction of the total lipid extract of or a portion of the non-polar fraction (see, eg, US Pat. No. 8,241,610, which is incorporated herein by reference in its entirety. ). In another embodiment, the synthetic nanocarrier may contain at least one polynucleotide, a primary construct, and / or mRNA, and an adjuvant. As a non-limiting example, synthetic nanocarriers containing an adjuvant may be formulated by the methods described in WO 2011150240 and US 201110293700, each of which is incorporated herein by reference in its entirety. ..

In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide, primary construct, and / or mmRNA that encodes a peptide, fragment, or region of viral origin. As a non-limiting example, synthetic nanocarriers are incorporated herein by reference in their entirety, WO 2012024621, WO 2012026229, WO 2012024632, and US Publication US 20120064110. , US 20120058153, and US 20120058154. The nanocarriers are included, but not limited to.

In one embodiment, the synthetic nanocarrier may be linked to a polynucleotide, primary construct, or mMr that may be capable of inspiring a humoral and / or cytotoxic T lymphocyte (CTL) response ( See, for example, WO20131969669, which is incorporated herein by reference in its entirety).

In one embodiment, the nanoparticles may be optimized for oral administration. The nanoparticles may include at least one cationic biopolymer, such as, but not limited to, chitosan or derivatives thereof. As a non-limiting example, nanoparticles may be formulated by the method described in US Publication No. 20120282343, which is incorporated herein by reference in its entirety.

Polymers, biodegradable nanoparticles, and core-shell nanoparticles The polynucleotides, primary constructs, and mmRNAs of the invention can be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that can be used for delivery include MIRUS® Bio (Madison, WI) and Roche.
DYNAMIC POLYCONJUGATE ™ (Registered Trademark) (Arrowhead Research Corp., Pasadena, CA) formulation by Madison (Maison, WI), without limitation, SMARTT POLYMER TECHNOLOGY ™ (Trademark) (PHASERX ™, etc. ) Polymer formulations, DMRI / DOPE, Poroxamer, VAXFECTIN® adjuvant with Visual (San Diego, CA), Chitosan, Cyclodextrins with Calando Pharmaceuticals (Pasadena, CA), Dendrimer and Poly (lactic acid-co-glycolic acid) (Lactic acid-co-glycolic acid). PH response including, but not limited to, PLGA) polymers, RONDEL ™ (RNAi / oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corporation, Pasadena, CA), and PHASERX® (Suite, WA). Sex block polymers (co-block polymers) can be mentioned.

Non-limiting examples of chitosan formulations include the core of positively charged chitosan, as well as the outer portion of the negatively charged substrate (US Publication No. 20120258176, which is incorporated herein by reference in its entirety). Chitosan includes N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof. Not limited.

In one embodiment, the polymer used in the present invention has undergone processing to reduce and / or inhibit the binding of unwanted substances, such as, but not limited to, to the surface of the polymer. Polymers may be processed by the methods described in WO 2012150467, which are known in the art and / or described in the art and / or incorporated herein by reference in their entirety. ..

Non-limiting examples of PLGA formulations include, but are not limited to, PLGA injectable depots (eg, formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP)). ELIGARD®, the rest of which is an aqueous solvent and leuprolide. Once injected, PLGA and leuprolide peptides precipitate in the subcutaneous cavity).

Many of these polymer approaches have demonstrated efficacy in delivering oligonucleotides into the cytoplasm of cells in vivo (deFougelles Hum Gene Ther. 2008 19, which is incorporated herein by reference in its entirety). : Outlined in 125-132). The two polymer approaches that have resulted in robust in vivo delivery of nucleic acids (using small interfering RNAs (siRNAs) in this example) are dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches has been shown to use dynamic polycomplexes to effectively deliver siRNA in vivo in mice and silence endogenous target mRNAs within hepatocytes. (Rosema et al., Proc Natl Acad Sci USA. 2007 104: 12982-1287, which is incorporated herein by reference in its entirety). This particular approach is a multi-component polymer system, the main feature of which is that the nucleic acid (siRNA in this example) is covalently linked via a disulfide bond, a PEG (for charge shielding) group and N-acetylgalactosamine (for charge shielding). Includes membrane-active polymers in which both groups are linked via pH-sensitive bonds (for hepatocyte targeting), Rosema et al., Proc Natl Acad Sci USA, which is incorporated herein by reference in its entirety. 2007 104: 12982-12887). Upon binding to hepatocytes and entering the endosome, the polymer complex disintegrates in a low pH environment, exposing its positive charge, resulting in escape from the endosome and release of siRNA from the polymer into the cytoplasm. .. It has been shown that the replacement of N-acetylgalactosamine groups with mannose groups can alter targeting from hepatocytes expressing the asialoglycoprotein receptor to sinusoidal endothelial cells and Kupffer cells. Another polymer approach involves the use of cyclodextrin-containing polycationic nanoparticles that target transferrin. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in Ewing's sarcoma tumor cells expressing transferrin receptors (Hu-Rieskovan, which is incorporated herein by reference in its entirety). et al., Cancer Res. 2005 65: 8984-8882), siRNAs formulated in these nanoparticles were well tolerated in non-human primates (whole by reference incorporated herein by reference). , Heidelet al., Proc Natl Acad Sci USA 2007 104: 5715-21). Both of these delivery strategies incorporate rational approaches using both targeted delivery mechanisms and endosome escape mechanisms.

Polymer formulations may allow sustained or delayed release of polynucleotides, primary constructs, or mmRNAs (eg, after intramuscular or subcutaneous injection). Altered release profiles for polynucleotides, primary constructs, or mmRNAs can, for example, result in translation of the encoded protein over time. Polymer formulations can also be used to increase the stability of polynucleotides, primary constructs, or mmRNAs. Biodegradable polymers have previously been used to protect nucleic acids other than mmRNA from degradation and have been shown to result in sustained release of payload in vivo (each referred to herein in its entirety). Incorporated, Rosema et al., Proc Nucleic acid
Acad Sci USA. 2007 104: 12982-12887, Sullivan et al. , Expert Opin Drag Deliv. 2010 7: 1433-1446, Convertine et al. , Biomacromolecules. 2010 Oct 1, Chu et al. , Acc Chem Res. 2012 Jan 13, Mananiello et al. , Biomaterials. 2012 33: 2301-2309, Benoit et al. , Biomacromolecules. 2011 12: 2708-2714, Singha
et al. , Nucleic Acid Ther. 2011 2: 133-147, deFougeloles Hum Gene Ther. 2008 19: 125-132, Schaffert and Wagner, Gene Ther. 2008 16: 1131-1138, Chaturvedi et al. , Expert Opin Drag Deliv. 2011 8: 1455-1468, Davis, Mol
Pharm. 2009 6: 659-668, Davis, Nature 2010 464: 1067-1070).

In one embodiment, the pharmaceutical composition may be a sustained release formulation. In a further embodiment, the sustained release formulation may be for subcutaneous delivery. Sustained release formulations include PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Deerfield) (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc. Deerfield, IL), PEG-based sealants, and COSEAL® (Registered Trademarks) (Baxter International, Inc. Deerfield, IL) for surgery. Material may be included.

As a non-limiting example, modified mRNAs are prepared with adjustable release rates (eg, days and weeks) and PLGA microspheres are prepared to maintain the integrity of the modified mRNAs during the encapsulation process. However, it may be formulated in PLGA microspheres by encapsulating it in PLGA microspheres. EVAc is a non-biodegradable, biocompatible polymer widely used in preclinical sustained release implant applications (eg, continuous release of pilocarpine intraocular inserts for glaucoma). Product Ocusert or continuous release progesterone intrauterine device, Progesterone; transdermal delivery system Testoderm, Drugetic, and Selegiline; catheter). Poloxamer F-407 NF is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene, a hydrophilic nonionic surfactant having a low viscosity at temperatures below 5 ° C. at 15 ° C. Form a solid gel at temperatures above. A PEG-based surgical sealant comprises two synthetic PEG constituents mixed in a delivery device that can be prepared in 1 minute, sealed in 3 minutes and reabsorbed within 30 days. GELSITE® and natural polymers are capable of gelling insights at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates via ionic interactions to provide stabilizing effects.

Polymer formulations are exemplified by folate, transferrin, and N-acetylgalactosamine (GalNAc), but can also be selectively targeted through the expression of different ligands not limited by these (each by reference in whole). , Benoit et al., Biomacromolecules. 2011 12: 2708-2714, Rosema et al., Proc Natl Acad Sci USA. 2007 104: 12982-1287, Davis, Mol-Pharm. 668, Davis, Nature 2010 464: 1067-1070).

The modified nucleic acid of the present invention, and mmRNA, may be formulated with or in a polymer compound. Polymers include polyethenes, polyethylene glycol (PEG), poly (l-lysine) (PLL), PLL-grafted PEG, cationic lipopolymers, biodegradable cationic lipopolymers, polyethyleneimines (PEI). , Crosslinked branched poly (alkyleneimine), polyamine derivative, modified poroxamer, biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable Polyester block polymers, biodegradable polyester block random copolymers, multi-block polymers, linear biodegradable copolymers, poly [α- (4-aminobutyl) -L-glycolic acid) (PAGA), biodegradable crosslinked cationic mulch Block Copolymer, Polycarbonate, Polyanhydride, Polyhydroxyic Acid, Polypropyl Fumarate, Polycaprolactone, Polyamide, Polyacetal, Polyether, Polyester, Poly (Orthoester), Polycyanoacrylate, Polyvinyl Alcohol, Polyurethane, Polyphosphazene , Polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly (ethyleneimine), poly (serine ester), poly (L-lactide-co-L-lysine), poly (4-hydroxy) -L-Proline ester), acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof, and the like, but may include at least one polymer.

As a non-limiting example, the modified nucleic acid or mm mRNA of the invention is formulated with a PLL-grafted PEG polymer compound as described in US Pat. No. 6,177,274, which is incorporated herein by reference in its entirety. It may be converted. The pharmaceuticals may be used for transfection of cells in vitro or for in vivo delivery of modified nucleic acids and mmRNAs. In another example, modified nucleic acids and mmRNAs are incorporated herein by reference in whole in a solution or medium containing a cationic polymer, in a dry pharmaceutical composition, or by reference in their entirety. It may be suspended in a solution that can be dried as described in No. and No. 20090042825.

As another non-limiting example, the polynucleotides, primary constructs, or mmRNAs of the invention are PLGA-PEG block copolymers, US Publication No. US20110004293 and US Pat. No. 8, which are incorporated herein by reference in their entirety. It may be formulated with (see 236,330) or PLGA-PEG-PLGA block copolymers (see US Pat. No. 6,004,573, which is incorporated herein by reference in its entirety). .. As a non-limiting example, the polynucleotides, primary constructs, or mMVs of the invention may be formulated with PEG and PLA or diblock copolymers of PEG and PLGA (incorporated herein by reference in their entirety). See U.S. Pat. No. 8,246,968).

Polyamine derivatives may be used to deliver nucleic acids, or treat and / or prevent disease, or be included in implantable or injectable devices (incorporated herein by reference in their entirety, US publication). No. 2012rea0817). As a non-limiting example, the pharmaceutical composition may include modified nucleic acids and mmRNAs, as well as the polyamine derivatives described in US Publication No. 201260817, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the polynucleotides, primary constructs, and mmRNAs of the invention are prepared by combining carbohydrate diazide monomers with dilkyne units containing oligoamines, 1,3-bipolar addition polymers. May be delivered using polyamide polymers, including but not limited to polymers (US Pat. No. 8,236,280, which is incorporated herein by reference in its entirety).

In one embodiment, the polynucleotides, primary constructs, or mRNAs of the invention are incorporated herein by reference in their entirety, WO 20111,15862, WO 2012082574, and WO 2012068187, respectively. It may also be formulated with at least one polymer and / or derivative thereof as described in US Publication No. 20120283427. In another embodiment, the modified nucleic acid or mmRNA of the invention may be formulated with a polymer of formula Z as described in WO2011115862, which is incorporated herein by reference in its entirety. In yet another embodiment, the modified nucleic acids or polymers are formulated in Formula Z, according to WO2012082574 or WO2012868187 and US Publication No. 2012028342, each of which is incorporated herein by reference in its entirety. It may be formulated with a polymer of Z'or Z''. Polymers formulated with the modified RNA of the present invention may be synthesized by the methods described in WO2012082574 or WO201268187, each of which is incorporated herein by reference in its entirety.

The polynucleotide, primary construct, or mMV of the present invention may be formulated with at least one acrylic polymer. Acrylic polymers include acrylic acid, methacrylic acid, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), and polycyano. Acrylates, as well as combinations thereof, are included, but not limited to these.

The polynucleotide, primary construct, or mmRNA formulation of the present invention may contain at least one amine-containing polymer such as, but not limited to, polylysine, polyethyleneimine, poly (amide amine) dendrimer, or a combination thereof.

For example, the modified nucleic acid or mm mRNA of the present invention is a poly (alkyleneimine), biodegradable cationic lipopolymer, biodegradable block copolymer, biodegradable polymer, or biodegradable random copolymer, biodegradable polyester block copolymer, It may be formulated in a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable crosslinked cationic multiblock copolymer, or a pharmaceutical compound containing a combination thereof. Biodegradable cationic lipopolymers are methods known in the art and / or each of which is incorporated herein by reference in their entirety, U.S. Pat. No. 6,696,038, U.S. Application No. 20030073619. , And the method described in 20040142474. Poly (alkyleneimine) may be made using methods known in the art and / or the methods described in US Publication No. 20010004315, which is incorporated herein by reference in its entirety. Biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester block copolymers, biodegradable polyester polymers, or biodegradable polyester random copolymers are methods known in the art. And / or may be made using the methods described in US Pat. Nos. 6,517,869 and 6,267,987, the contents of which are incorporated herein by reference in their entirety. Linear biodegradable copolymers are known in the art and / or may be made using the method described in US Pat. No. 6,652,886. PAGA polymers may be made using methods known in the art and / or the methods described in US Pat. No. 6,217,912, which is incorporated herein by reference in its entirety. PAGA polymers can be copolymerized to include, but are not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamine, polylactide, and poly (lactide-co-glycolide). Copolymers or block copolymers may be formed with them. Biodegradable crosslinked cationic multi-block copolymers are methods known in the art and / or each of which is incorporated herein by reference in their entirety, U.S. Pat. No. 8,057,821 or U.S. Publication No. 2012009145. It may be produced by the method described in No. For example, multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks that have a distinctly different pattern compared to branched polyethyleneimine. In addition, compositions or pharmaceutical compositions are incorporated herein by methods known in the art, methods described herein, or each incorporated herein by reference in its entirety, U.S.A. No. 20010004315 or the United States. It may be produced by the methods described in Japanese Patent Nos. 6,267,987 and 6,217,912.

The polynucleotides, primary constructs, and mMVs of the present invention may be formulated with at least one degradable polyester that may contain a polycationic side chain. Degradable polyesters include poly (serine ester), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline ester), and combinations thereof. Not limited to. In another embodiment, the degradable polyester may include a PEG complex to form a PEGylated polymer.

The polynucleotide, primary construct, mmRNA of the present invention may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include the crosslinkable polyesters described in US Publication No. 20120269761, which are known in the art and are incorporated herein by reference in their entirety.

In one embodiment, the polymers described herein may be complexed with lipid-terminating PEG. As a non-limiting example, PLGA may be complexed to PEG with lipid ends to form PLGA-DSPE-PEG. As another non-limiting example, a PEG complex for use with the present invention is described in WO2008103276, which is incorporated herein by reference in its entirety. Polymers may be conjugated using ligand complexes, such as, but not limited to, the complexes described in US Pat. No. 8,273,363, which are incorporated herein by reference in their entirety.

In one embodiment, the modified RNA described herein may be complexed with another compound. Non-limiting examples of complexes are described in US Pat. Nos. 7,964,578 and 7,833,992, each of which is incorporated herein by reference in its entirety. In another embodiment, the modified RNAs of the invention are the formulas described in US Pat. Nos. 7,964,578 and 7,833,992, each of which is incorporated herein by reference in its entirety. It may be complexed with a complex of 1-122. The polynucleotides, primary constructs, and / or mMVs described herein may be complexed with metals such as, but not limited to, gold. (See, for example, Giljohann et al. John. Amer. Chem. Soc. 2009 131 (6): 2072-2073, which is incorporated herein by reference in its entirety). In another embodiment, the polynucleotides, primary constructs, and / or mMVs described herein may be complexed and / or encapsulated in gold nanoparticles. (International Publication No. WO201216269 and US Publication No. 20120302940, each of which is incorporated herein by reference in its entirety).

The gene delivery composition may comprise a nucleotide sequence and a poloxamer, as described in US Publication No. 20010004313, which is incorporated herein by reference in its entirety. For example, the modified nucleic acids and mmRNAs of the present invention may be used in gene delivery compositions comprising the poloxamers described in US Publication No. 20010004313.

In one embodiment, the polymeric formulations of the present invention may be stabilized by contacting the polymeric formulations, which may include cationic carriers, with cationic lipopolymers which may be covalently attached to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with the cationic lipopolymer using the method described in US Publication No. 20090042829, which is incorporated herein by reference in its entirety. Cationic carriers include polyethyleneimine, poly (trimethyleneimine), poly (tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermin, spermidin, poly (2-dimethylamino). Ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationized gelatin, dendrimer, chitosan, 1,2-diore oil-3-trimethylammonium-propane (DOTAP), N- [1- (2,2) 3-diore oil oxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 1- [2- (oleoyloxy) ethyl] -2-oleyl-3- (2-hydroxyethyl) imidazole Nium chloride (DOTIM), 2,3-diorailoxy-N- [2 (sperminecarboxamide) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), 3B- [N-( N', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-cholesterol HCl) diheptadecylamide glycylspermidine (DOGS), N, N-distealyl-N, N-dimethylammonium bromide (DDAB) , N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N, N-diorail-N, N-dimethylammonium chloride DODAC), And combinations thereof, but are not limited to these.

The polynucleotides, primary constructs, and / or mRNAs of the present invention may be formulated in a polyplex of one or more polymers (each of which is incorporated herein by reference in its entirety, US Publication No. 201202375565. No. and No. 20120270927). In one embodiment, the polyplex comprises two or more cationic polymers. Cationic polymers may include poly (ethyleneimine) (PEI) such as linear PEI.

The polynucleotides, primary constructs, and mmRNAs of the present invention can be formulated as nanoparticles using a combination of other biodegradable agents such as, but not limited to, polymers, lipids, and / or calcium phosphate. .. The components may be combined as a core-shell, hybrid, and / or alternating stacking architecture to allow fine tuning of nanoparticles to improve delivery of polynucleotides, primary constructs, and mmRNA. (Wang et al., Nat Mater. 2006 5: 791-796, Fuller et al., Biomaterials. 2008 29: 1526-1532, DeKoker, which is incorporated herein by reference in its entirety.
et al. , Adv Drag Deliv Rev. 2011 63: 748-761, Endres et al. , Biomaterials. 2011 32: 7721-7731, Su et al. , Mol Pharm. 2011 Jun 6; 8 (3): 774-87). As a non-limiting example, the nanoparticles include, but are not limited to, hydrophilic-hydrophobic polymers (eg, PEG-PLGA), hydrophobic polymers (eg, PEG), and / or hydrophilic polymers. Polymers may be included (International Publication No. WO20120225129, which is incorporated herein by reference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipids and / or polymers have been shown to deliver polynucleotides, primary constructs, and mmRNAs in vivo. In one embodiment, lipid-coated calcium phosphate nanoparticles, which may also contain a targeting ligand such as anisamide, may be used to deliver the polynucleotides, primary constructs, and mmRNAs of the invention. For example, lipid-coated calcium phosphate nanoparticles were used to effectively deliver siRNA in a mouse metastatic lung model (see Li et al., J Control Rel., Which is incorporated herein by reference in its entirety. 2010 142: 416-421, Li et al., J Control Rel. 2012 158: 108-114, Yang et al., Mol
The. 2012 20: 609-615). This delivery system combines both targeted nanoparticles with the component calcium phosphate to improve escape from endosomes in order to improve the delivery of siRNA.

In one embodiment, the polynucleotide, primary construct, and mmRNA may be delivered using calcium phosphate with PEG-polyanion block copolymers (Kazikawa et al., J Control Rel, which is incorporated herein by reference in its entirety). 2004 97: 345-356, Kazikawa et al., J.
Control Rel. 2006 111: 368-370).

In one embodiment, PEG charge-converting polymers (Pitella et al., Biomaterials. 2011 32: 3106-3114) are used to form nanoparticles for delivering the polynucleotides, primary constructs, and mmRNAs of the invention. You may. PEG charge-converting polymers can be cleaved into polycations at acidic pH and therefore can improve PEG-polyanion block copolymers by improving escape from endosomes.

The use of core-shell nanoparticles further focuses on high-treatment approaches for the synthesis of cationically crosslinked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011 108: 12996-13001). .. Complex formation, delivery, and internalization of polymer nanoparticles can be precisely controlled by altering the chemical composition of both core and shell constituents of the nanoparticles. For example, core-shell nanoparticles can efficiently deliver siRNA to mouse hepatocytes after covalently binding cholesterol to the nanoparticles.

In one embodiment, a hollow lipid core containing an intermediate PLGA layer and an outer neutral lipid layer containing PEG may be used to deliver the polynucleotides, primary constructs, and mmRNAs of the invention. As a non-limiting example, it was confirmed that lipid-polymer-lipid hybrid nanoparticles significantly suppressed luciferase expression in mice carrying a luciferase-expressing tumor as compared to conventional lipoplexes. (The whole is incorporated herein by reference, Shi et al, Angew Chem Int Ed. 2011 50: 7027-7031).

In one embodiment, the lipid nanoparticles may include a core of modified nucleic acid molecules disclosed herein, and a polymer shell. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, a polymer shell can be used to protect the modified nucleic acid in the core.

Core-shell nanoparticles for use with the modified nucleic acid molecules of the invention are described, which are formed by the method described in US Pat. No. 8,313,777, which is incorporated herein by reference in its entirety. You may.

In one embodiment, the core-shell nanoparticles may include a core of modified nucleic acid molecules disclosed herein, and a polymer shell. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, a polymer shell can be used to protect the modified nucleic acid molecule in the core. As a non-limiting example, core-shell nanoparticles can be used to treat eye diseases or disorders (see, eg, US Publication No. 20120321719, which is incorporated herein by reference in its entirety).

In one embodiment, the polymer used with the formulation described herein is the modified polymer described in WO2011120053, which is incorporated herein by reference in its entirety, such as, but limited to, modified polyacetals. It may not be).

Peptides and Proteins The polynucleotides, primary constructs, and mmRNAs of the invention can be formulated with peptides and / or proteins to increase transfection of cells with polynucleotides, primary constructs, or mmRNAs. In one embodiment, peptides such as, but not limited to, cell-permeable peptides and proteins and peptides that allow intracellular delivery may be used to deliver pharmaceutical formulations. Non-limiting examples of cell-penetrating peptides that can be used with the pharmaceutical formulations of the invention are cell-penetrating peptide sequences bound to polycations that facilitate delivery into the intracellular space, such as HIV-derived TAT. Peptides, penetratins, transportans, or hCT-derived cell-penetrating peptides (eg, Caron et al., Mol. Ther. 3 (3): 310-8, all of which are incorporated herein by reference in their entirety). 2001), Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Ratton FL, 2002), El-Andaloussi et al., Curr. Pharm. Des. 11 (200), Curr. Pharm. Des. See Deshayes et al., Cell. Mol. Life Sci. 62 (16): 1839-49 (2005)). The composition can also be formulated to include a cell permeable agent that enhances delivery of the composition to the intracellular space, such as liposomes. The polynucleotides, primary constructs, and mRNAs of the present invention, such as peptides and / or proteins by Aireron Therapeutics (Cambridge, MA) and Permeon Biologics (Cambridge, MA), are used to enable intracellular delivery. Complexes may be formed into peptides and / or proteins, including, but not limited to, the Cronican et, all incorporated herein by reference in its entirety.
al. , ACS Chem. Biol. 2010 5: 747-752, McNaughton et al. , Proc. Natl. Acad. Sci. USA 2009 106: 6111-6116, Sawyer, Chem Biol Drug Des. 2009 73: 3-6, Verdine and Hilinski, Methods
Enzymol. 2012; 503: 3-33).

In one embodiment, the cell permeable polypeptide may include a first domain and a second domain. The first domain may include a hypercharged polypeptide. The second domain may include protein binding partners. As used herein, "protein binding partners" include, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. Cell-permeable polypeptides may further include intracellular binding partners for protein binding partners. Cell-permeable polypeptides can be secreted from cells into which polynucleotides, primary constructs, or mmRNA can be introduced.

Formulations containing peptides or proteins are used to increase cell transfection with polynucleotides, primary constructs, or mmRNAs and alter the biological distribution of polynucleotides, primary constructs, or mmRNAs (eg, specific tissues or cell types). (By targeting) and / or increased translation of the encoded protein. (See, eg, International Publication No. WO2012110636, which is incorporated herein by reference in its entirety).

Cells The polynucleotides, primary constructs, and mmRNAs of the invention can be transfected into cells with Exvivo, which is then transplanted into the subject. As a non-limiting example, pharmaceutical compositions include erythrocytes for delivering modified RNA to liver and bone marrow cells, virosomes for delivering modified RNA in virus-like particles (VLPs), and MAXCYTE®. It may include cells according to (Gathersburg, MD), cells according to ERYTECH® (Lyon, France), and the like, but not limited to, electroporation-treated cells for delivering modified RNA. Examples of the use of erythrocytes, viral particles, and electroporated cells to deliver payloads other than mmRNA are documented (all incorporated herein by reference in their entirety, Godfrin et al. , Expert Opin Biol Ther. 2012 12: 127-133, Fang et al., Expert Opin Biol Ther. 2012 12: 385-389, Hu et
al. , Proc Natl Acad Sci USA. 2011 108: 10980-10985, Lund et al. , Pharm Res. 2010 27: 400-420, Huckriede et al. , J Liposomes Res. 2007; 17: 39-47, Cushi, Hum Vaccin. 2006 2: 1-7, de Jonge et al. , Gene Ther. 2006 13: 400-411).

Polynucleotides, primary constructs, and mmRNAs are delivered in synthetic VLPs, each synthesized by the methods described in WO2011085231 and US Publication 20110171248, each of which is incorporated herein by reference in its entirety. May be good.

Cell-based formulations of polynucleotides, primary constructs, and mmRNAs of the invention are used to ensure cell transfection (eg, in cell carriers) and alter the biodistribution of polynucleotides, primary constructs, or mmRNAs (eg, in cell carriers). For example, by targeting the cell carrier to a specific tissue or cell type) and / or increasing the translation of the encoded protein.

A variety of methods suitable for the intracellular introduction of nucleic acids, including viral and non-viral mediated techniques, are known in the art. Examples of typical non-viral mediated techniques include electroporation, calcium phosphate mediated transfer, nucleoffection, sonoporation, heat shock, magnetfection, liposome-mediated transfer, microinjection, microprojectile mediated. Transfer (nanoparticles), cationic polymer-mediated transfer (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.), or cell fusion, but is not limited to these.

Sonoporation, or cell sonication techniques, use sound (eg, ultrasound frequency) to modify the permeability of the protoplasmic membrane. Sonopulation methods are known to those of skill in the art and are used to deliver nucleic acids in vivo (all incorporated herein by reference in their entirety, Yoon and Park, Expert Opin Drug Dev. 2010 7: 321-330, Postema and Gilja, Curr Palm Biotechnol. 2007 8: 355-361, Newman and Better, Gene Ther. 2007 14: 465-475). The sonoporation method is known in the art and is also known in the art, for example, in US Pat. Taught in Publication No. 20010004224, each of these is incorporated herein by reference in their entirety.

Electroporation techniques are also well known in the art and are used to deliver nucleic acids in vivo and clinically (all incorporated herein by reference, Andre et al., Curr Gene). The. 2010 10: 267-280, Chiarella et al., Curr Gene Ther. 2010 10: 281-286, Hojman, Curr Gene Ther. 2010 10: 128-138). In one embodiment, the polynucleotide, primary construct, or mmRNA can be delivered by electroporation as described in Example 8.

Hyaluronidase Intramuscular or subcutaneous local injections of the polynucleotides, primary constructs, or mmRNAs of the invention can include hyaluronidases that catalyze the hydrolysis of hyaluronan. By catalyzing the hydrolysis of hyaluronan, a constituent of the intervening barrier, hyaluronidase reduces the viscosity of hyaluronan, thereby increasing tissue permeability (incorporated herein by reference in its entirety). Frost, Expert Opin. Drug Dev. (2007) 4: 427-440). It is useful to accelerate their dispersion and the systemic distribution of the encoded proteins produced by the transfected cells. Alternatively, hyaluronidase can be used to increase the number of cells exposed to the polynucleotides, primary constructs, or mRNA of the invention administered intramuscularly or subcutaneously.

Nanoparticle Mimetics The polynucleotides, primary constructs, or mmss of the invention can be encapsulated and / or absorbed in a nanoparticle mimic. Nanoparticle mimetics can mimic the delivery function of an organism or particle, including, but not limited to, pathogens, viruses, bacteria, fungi, parasites, prions, and cells. As a non-limiting example, the polynucleotides, primary constructs, or mMVs of the invention may be encapsulated in non-viron particles capable of mimicking the delivery function of the virus (see book in its entirety). See International Publication No. WO2012006376 incorporated herein).

Nanotubes The polynucleotides, primary constructs, or mmRNAs of the present invention include, but are not limited to, roset nanotubes, rosette nanotubes having twin bases using linkers, carbon nanotubes and / or single-walled carbon nanotubes. , Can be attached or bound to at least one nanotube. Polynucleotides, primary constructs, or mmRNAs can be attached to nanotubes via forces such as, but not limited to, steric forces, ionic forces, covalent bonds, and / or other forces.

In one embodiment, nanotubes can release one or more polynucleotides, primary constructs, or mmRNAs into cells. Changing the size and / or surface structure of at least one nanotube to control the interaction of the nanotubes in the body and / or to attach or bind to the polynucleotides, primary constructs, or mmRNAs disclosed herein. Can be done. In one embodiment, the building blocks of at least one nanotube and / or the functional groups attached to the building blocks can be varied to adjust the dimensions and / or properties of the nanotubes. As a non-limiting example, the length of the nanotubes prevents the nanotubes from passing through holes in the walls of normal blood vessels, but remains small enough to pass through larger holes in the blood vessels of tumor tissue. May be changed to.

In one embodiment, the at least one nanotube may be coated with a delivery-enhancing compound, including but not limited to polymers such as polyethylene glycol. In another embodiment, the at least one nanotube and / or polynucleotide, primary construct, or mmRNA may be mixed with a pharmaceutically acceptable excipient and / or delivery vehicle.

In one embodiment, the polynucleotide, primary construct, or mMV is attached and / or bound to at least one rosette nanotube. Rosette nanotubes can be formed by a process known in the art and / or by the process described in WO2011094304, which is incorporated herein by reference in its entirety. Even if at least one polynucleotide, primary construct, and / or mmRNA is attached and / or bound to at least one rosette nanotube by the process described in WO2011094304, which is incorporated herein by reference in its entirety. Well, here the rosette nanotubes or the modules that form the rosette nanotubes are at least one in an aqueous medium under conditions where at least one polynucleotide, primary construct, or mmRNA can become attached or attached to the rosette nanotubes. It is mixed with polynucleotides, primary constructs, and / or mmRNA.

In one embodiment, the polynucleotide, primary construct, or mMV may be attached and / or bound to at least one carbon nanotube. As a non-limiting example, a polynucleotide, primary construct, or mMV may be attached to a linking agent, and the linked agent may be attached to a carbon nanotube (eg, in its entirety by reference). See U.S. Pat. No. 8,246,995 incorporated in the book). The carbon nanotubes may be single-walled nanotubes (see, eg, US Pat. No. 8,246,995, which is incorporated herein by reference in its entirety).

Complex The polynucleotides, primary constructs, and mmRNAs of the invention contain two coding regions that produce fusion proteins covalently or together with carriers or targeting groups (eg, targeting groups and therapeutic proteins). (Or carrying a peptide), polynucleotides, primary constructs, or complexes such as mmRNA.

Complexes of the invention include proteins (eg, human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin), carbohydrates (eg, dextran, purulan, chitin, chitosan, etc.). Includes naturally occurring substances such as inulin, cyclodextran or hyaluronic acid), or lipids. The ligand may be a synthetic polymer, eg, a recombinant or synthetic molecule such as a synthetic polyamino acid, an oligonucleotide (eg, an aptamer). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymers, poly (L-lactide-co-glycolide) copolymers, Divinyl ether-malein anhydride copolymer, N- (2-hydroxypropyl) methacrylicamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N -Isopropylacryl amide polymer, or polyphosphazene can be mentioned. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide mimicking polyamines, dendrimer polyamines, arginine, amidin, protamine, cationic lipids, cationic porphyrins, and polyamines. Examples include grade salts and α-helix peptides.

Representative US patents that teach the preparation of polynucleotide complexes, especially to RNA, are incorporated herein by reference in their entirety, US Pat. No. 4,828,979, No. 4,948,882, No. 5,218,105, No. 5,525,465, No. 5,541,313, No. 5,545,730, No. 5,552 , 538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5 , 138,045, 5,414,077, 5,486,603, 5,512,439, 5,578,718, 5,608,046. , 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4, 4, 824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245 , 022, 5,254,469, 5,258,506, 5,262,536, 5,272,250, 5,292,873, No. 5,317,098, No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510, No. 475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. 5,574,142, No. 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928 No. 5,688,941, No. 6,294,664, No. 6,320,017, No. 6,576,752, No. 6,783,931, No. 6 , 900, 297 and 7,037,646, but are not limited thereto.

In one embodiment, the complex of the invention can serve as a carrier for the modified nucleic acids and mmRNAs of the invention. The complex may include cationic polymers such as, but not limited to, polyamines, polylysines, polyalkyleneimines, and polyethyleneimines that can be grafted with poly (ethylene glycol). As a non-limiting example, the complex may be similar to a polymer complex, and the method of synthesizing the polymer complex is incorporated herein by reference in its entirety, U.S. Pat. No. 6,586,524. It is described in.

The complex may also include a targeting group, eg, an antibody that binds to a cell or tissue targeting agent, eg, a lectin, glycoprotein, lipid, or protein, eg, a designated cell type such as kidney cells. Targeting groups are tyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mutin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosyl. Polyamino acids, polyvalent galactose, transferase, bisphosphonate, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptide mimetic, or aptamer. obtain.

The targeting group is designated as a molecule or antibody having a specific affinity for a protein, eg, a glycoprotein, or a peptide, eg, a co-ligand, eg, a cancer cell, an endothelial cell, or an osteoocyte. It can be an antibody that binds to a cell type. Targeting groups can also include hormones and hormone receptors. They include lipids such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, or non-peptides such as aptamers. Seeds may also be included. The ligand is, for example, lipopolysaccharide, or p38.
It can be an activator of MAP kinase.

The targeting group can be any ligand capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, apatamers, integrin receptor ligand, chemokine receptor ligand, transferase, biotin, serotonin receptor ligand, PSMA, endoserine, GCPII, Included are somatostatins, LDLs, and HDL ligands. In certain embodiments, the targeting group is an aptamer. Aptamers may be unmodified or may have any combination of modifications disclosed herein.

In one embodiment, the pharmaceutical composition of the present invention may include chemical modifications such as, but not limited to, modifications similar to, but not limited to, locked nucleic acids.

Representative US patents that teach the preparation of locked nucleic acids (LNAs), such as those by Santaris, are incorporated herein by reference in their entirety, U.S. Pat. Nos. 6,268,490. 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, and 7,399, 845 includes, but is not limited to.

Representative U.S. patents that teach the preparation of PNA compounds are incorporated herein by reference, U.S. Pat. Nos. 5,539,082, 5,714,331, and 5, respectively. , 719, 262, but not limited to these. Further teaching of PNA compounds can be found, for example, in Nielsen et al. , Science, 1991,254, 1497-1500.

Some embodiments that characterize the invention are polynucleotides, primary constructs, or mmRNAs having a phosphorothioate scaffold, as well as other modified scaffolds, in particular-US Pat. No. 5,489,677 as referenced above. _{-CH 2 --NH - CH 2 -,} - CH 2 --N (CH 3) - O - CH 2 - [ known as a methylene (methylimino) or MMI backbone], - _{CH 2 --O - N (CH 3} ) - CH 2 -, - CH 2 --N (CH 3) - N (CH 3) - CH 2 -, and --N (CH _{3) - CH 2 --CH 2 -} [ wherein the native phosphodiester backbone is _{represented, - O-P (O)} 2 --O - CH 2 - represented as, and the above-referenced Includes oligonucleosides having an amide skeleton of US Pat. No. 5,602,240. In some embodiments, the polynucleotides that characterize the specification have the morpholino skeletal structure of US Pat. No. 5,034,506 referenced above.

Modifications at the 2'position can also aid delivery. Preferably, the modification at the 2'position is not located in the polypeptide coding sequence, i.e. not in the translatable region. The modification at the 2'position may be located in the 5'UTR, 3'UTR, and / or the tail region. Modifications at the 2'position can include one of the following at the 2'position: H (ie, 2'-deoxy);F; O-, S-, or N-alkyl; O-, S. -Or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl (in the formula, alkyl, alkenyl, and alkynyl are substituted or unsubstituted C _1- C ₁₀ alkyl or C. _{2 to} C ₁₀ alkenyl and alkynyl may be used. Suitable modifications of the examples include O [(CH ₂ ) _n O] _m CH ₃ , O (CH ₂ ) _n OCH ₃ , O (CH ₂ ) _n NH. ₂ , O (CH ₂ ) _n CH ₃ , O (CH ₂ ) _n ONH ₂ , and O (CH ₂ ) _n ON [(CH ₂ ) _n CH ₃ )] _2, where n and m are included in the equation. , 1 to about 10. In other embodiments, the polynucleotide, the primary construct or MmRNA, is the 2 'position include one of the _following: C 1 _{-C 10} lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O- alkaryl Alternatively, O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocyclo Alkaline, aminoalkylamino, polyalkylamino, substituted silyls, RNA cleavage groups, reporter groups, intercalators, groups for improving pharmacodynamic properties, or groups for improving pharmacodynamic properties, and similar properties. Other substituents that have. In some embodiments, the modification is also known as 2'-methoxyethoxy (2'-O-CH ₂ CH ₂ OCH ₃ (2'-O- (2-methoxyethyl) or 2'-MOE)). (Martinet al., Helv. Chim. Acta, 1995, 78: 486-504) That is, it contains an alkoxy-alkoxy group. Another exemplary modification includes 2'-dimethylaminooxyethoxy described in the following Examples herein, _{i.e., O (CH 2) 2 ON} (CH 3) , also known as ₂ group (2'-DMAOE 2'-Dimethylaminoethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), as described in the following examples herein below. , _2'-O - is a _{CH 2 --N (CH 2) 2} - CH 2 --O. Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), and 2'-fluoro (2'-F). Similar modifications may be made at other positions, especially at the 3'position of the sugar on the 3'end nucleotide, or at the 2'-5'binding dsRNA, and at the 5'position of the 5'end nucleotide. The polynucleotide of the present invention may have a sugar mimic such as a cyclobutyl moiety instead of the pentoflanosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures are U.S. Pat. Nos. 4,981,957, 5,118,800, each incorporated herein by reference. No. 5,319,080, No. 5,359,044, No. 5,393,878, No. 5,446,137, No. 5,466,786, No. 5,514, No. 785, No. 5,519,134, No. 5,567,811, No. 5,576,427, No. 5,591,722, No. 5,597,909, No. 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633 No., and Nos. 5,700,920, but not limited to these.

Still in other embodiments, the polynucleotide, primary construct, or mmRNA is covalently complexed to the cell-permeable polypeptide. The cell-permeable peptide may also include a signal sequence. Complexes of the invention can be designed to have increased stability, increased cell transfection, and / or altered biological distribution (eg, targeted to a specific tissue or cell type). ..

In one embodiment, the polynucleotide, primary construct, or mRNA may be complexed with an agent to improve delivery. As a non-limiting example, the agent may be a monomer or polymer, such as a targeted monomer or polymer having a targeting block as described in WO2011062965, which is incorporated herein by reference in its entirety. In another non-limiting example, the agent may be a transport agent covalently linked to a polynucleotide, primary construct, or mmRNA of the invention (eg, each incorporated herein by reference in its entirety. See U.S. Pat. Nos. 6,835.393 and 7,374,778). In yet another non-limiting example, the agents are the agents described in US Pat. Nos. 7,737,108 and 8,003,129, each of which is incorporated herein by reference in its entirety. It may be a membrane barrier transport improving agent such as.

In another embodiment, the polynucleotide, primary construct, or mRNA may be complexed to SMARTT POLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle, WA).

Self-assembling nanoparticles Nucleic acid self-assembling nanoparticles Self-assembling nanoparticles have a well-defined size in which the nucleic acid chains can be precisely controlled so that they can be easily reprogrammed. For example, the optimal particle size for a cancer-targeted nanodelivery carrier is 20 because diameters greater than 20 nm avoid renal clearance with improved permeability and retention effect and improve delivery to certain tumors. ~ 100 nm. A single population of uniform size and shape with precisely controlled spatial localization and density of cancer targeting ligands for improved delivery using self-assembling nucleic acid nanoparticles. As a non-limiting example, oligonucleotide nanoparticles were prepared using short DNA fragments and programmable self-assembly of therapeutic siRNAs. These nanoparticles have a controllable particle size and target ligand location and density and are molecularly identical. DNA fragments and siRNAs self-assembled into a one-step reaction to produce DNA / siRNA tetrahedral nanoparticles for targeted in vivo delivery. (Lee et al., Nature Nanotechnology 2012 7: 389-393, which is incorporated herein by reference in its entirety).

In one embodiment, the polynucleotides, primary constructs, and / or mMVs disclosed herein can be formulated as self-assembling nanoparticles. As a non-limiting example, nucleic acids may be used to make nanoparticles that can be used in delivery systems for the polynucleotides, primary constructs, and / or mmRNAs of the invention (eg, the whole by reference). See International Publication No. WO2012125987, which is incorporated in the specification).

In one embodiment, nucleic acid self-assembling nanoparticles may comprise the polynucleotides, primary constructs, or mmRNA cores disclosed herein, and polymer shells. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, polymer shells may be used to protect polynucleotides, primary constructs, and mmRNAs in the core.

Polymer-based self-assembling nanoparticles Polymers may be used to form self-assembled sheets into nanoparticles. These nanoparticles may be used to deliver the polynucleotides, primary constructs, and mmRNAs of the invention. In one embodiment, these self-assembling nanoparticles are microsponges formed of a long polymer of RNA hairpins that form crystalline "folded" sheets before self-assembling into the microsponge. May be good. These microsponges are tightly packed sponge-like microparticles that can act as efficient carriers and deliver cargo to cells. The microsponge can have a diameter of 1 μm to 300 nm. Microsponges can be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, microsponges can be complexed with agents that form the outer layer to promote cell uptake, such as polycationic polyethyleneimine (PEI). This complex can form particles with a diameter of 250 nm that can remain stable at high temperatures (150 ° C.) (Grabow and Jaegar, Nature Materials 2012, 11 :, which are incorporated herein by reference in their entirety. 269-269). In addition, these microsponges may be capable of exhibiting extraordinary protection from degradation by ribonucleases.

In another embodiment, polymer-based self-assembling nanoparticles, such as, but not limited to, microsponges, can be fully programmable nanoparticles. The shape, size, and stoichiometry of nanoparticles can be precisely controlled to produce nanoparticles that are optimal for delivery of cargo, such as, but not limited to, polynucleotides, primary constructs, and / or mmRNA. can.

In one embodiment, polymer-based nanoparticles can include the polynucleotides, primary constructs, and / or mmRNA cores disclosed herein, and polymer shells. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, polymer shells may be used to protect polynucleotides, primary constructs, and / or mmRNAs in the core.

In yet another embodiment, the polymer-based nanoparticles may comprise a non-nucleic acid polymer comprising a plurality of heterogeneous monomers, such as the monomers described in WO201309736, which is incorporated herein by reference in its entirety. good.

Inorganic Nanoparticles The polynucleotides, primary constructs, and / or mMVs of the invention may be formulated in inorganic nanoparticles (US Pat. No. 8,257,745, which is incorporated herein by reference in its entirety). ). Inorganic nanoparticles may include, but are not limited to, water-swellable clay materials. As a non-limiting example, inorganic nanoparticles may include synthetic smectite clays made from simple silicates (eg, each of which is incorporated herein by reference in its entirety, US Pat. No. 5). , 585, 108 and 8,257,745).

In one embodiment, the inorganic nanoparticles may comprise a modified nucleic acid core and a polymeric shell disclosed herein. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, a polymer shell may be used to protect the modified nucleic acid in the core.

Semi-Conducting and Metallic Nanoparticles The polynucleotides, primary constructs, and / or mMVs of the invention are formulated in water-dispersible nanoparticles containing semi-conducting or metallic materials (whole by reference). US Publication No. 20120285655, incorporated herein by reference), or may be formed in magnetic nanoparticles (US Publications, 20120265001 and 20120283503, each of which is incorporated herein by reference in its entirety). .. The water-dispersible nanoparticles may be hydrophobic nanoparticles or hydrophilic nanoparticles.

In one embodiment, the semi-conducting and / or metallic nanoparticles may comprise the polynucleotides, primary constructs, and / or mRNA cores disclosed herein, and polymer shells. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, polymer shells may be used to protect polynucleotides, primary constructs, and / or mmRNAs in the core.

Gels and Hydrogels In one embodiment, the polynucleotides, primary constructs, and / or mMVs disclosed herein are encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. It may be enclosed. Hydrogels are networks of polymer chains that are hydrophilic and are sometimes found as colloidal gels in which water is the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymers (they can contain more than 99% water). Hydrogels also have a flexibility very similar to natural tissue due to their considerable water content. The hydrogels described herein may be used to encapsulate biocompatible, biodegradable, and / or porous lipid nanoparticles.

As a non-limiting example, the hydrogel may be an aptamer-functionalized hydrogel. Aptamer-functionalized hydrogels may be programmed to release one or more polynucleotides, primary constructs, and / or mmRNAs using nucleic acid hybridization. (Battig et al., J. Am. Chem. Society. 2012 134: 12410-12413), which is incorporated herein by reference in its entirety.

As another non-limiting example, the hydrogel may be molded as an inverted opal. Opal hydrogels show higher swelling rates, and their swelling rate is also an order of magnitude faster. A method for producing opal hydrogels and a description of opal hydrogels are described in WO2012148648, which is incorporated herein by reference in its entirety.

In yet another non-limiting example, the hydrogel may be an antibacterial hydrogel. Antibacterial hydrogels may include pharmaceutically acceptable salts or organic materials such as, but not limited to, pharmaceutical grade and / or medical grade silver salts and aloe vera gels or extracts. (International Publication No. WO2012151438, which is incorporated herein by reference in its entirety).

In one embodiment, the modified mRNA may be encapsulated in lipid nanoparticles, which in turn may be encapsulated in hydrogel.

In one embodiment, the polynucleotides, primary constructs, and / or mMVs disclosed herein may be encapsulated in any gel known in the art. As a non-limiting example, the gel may be a fluorouracil injection gel or a fluorouracil injection gel containing chemical compounds and / or drugs known in the art. As another example, polynucleotides, primary constructs, and / or mRNAs may be encapsulated in fluorouracil gels containing epinephrine (eg, Smith et al. Cancer, which is incorporated herein by reference in its entirety). Chemotherapty and Pharmacology, 1999 44 (4): 267-274).

In one embodiment, the polynucleotides, primary constructs, and / or mMVs disclosed herein may be encapsulated in fibrin gel, fibrin hydrogel, or fibrin glue. In another embodiment, the polynucleotide, primary construct, and / or mRNA is formulated into lipid nanoparticles or rapidly excreted lipid nanoparticles before being encapsulated in fibrin gel, fibrin hydrogel, or fibrin glue. You may. In yet another embodiment, the polynucleotide, primary construct, and / or mRNA may be formulated as a lipoplex prior to being encapsulated in fibrin gel, hydrogel, or fibrin glue. Fibrin gels, hydrogels, and glues contain two components: a fibrinogen solution and a calcium-rich thrombin solution (eg, Spicer and Mikos, Journal of, each of which is incorporated herein by reference in its entirety. Controlled Release 2010.148: 49-55, Kidd et al. Journal
of Controlled Release 2012.157: 80-85). Concentrations of the constituents of fibrin gels, hydrogels, and / or glues include, but are not limited to, altering the release properties of fibrin gels, hydrogels, and / or glues. Can be varied to alter the properties, network mesh size, and / or decomposition properties of. (For example, Spicer and Mikos, Journal of Control Release 2010.148: 49-55, Kidd et al. Journal of Control Release 2012.157: 80-85, each of which is incorporated herein by reference in its entirety. et al. Tissue Engineering 2008.14: 119-128). This feature can be advantageous when used to deliver the modified mRNAs disclosed herein. (For example, Kidd et al. Journal of Controlled Release 2012.157: 80-85, Catalyst et al. Tissue Engineering, each of which is incorporated herein by reference in its entirety.
2008.14: 119-128).

Cations and anions The polynucleotides, primary constructs, and / or mmRNA formulations disclosed herein may contain cations or anions. In one embodiment, the pharmaceutical product contains metal cations such as, but not limited to, Zn2 +, Ca2 +, Cu2 +, Mg +, and combinations thereof. As a non-limiting example, the pharmaceutical product may contain a polymer and a polynucleotide complexed with a metal cation, a primary construct, and / or m mRNA (eg, each of which is incorporated herein by reference in its entirety. , US Pat. Nos. 6,265,389 and 6,555,525).

Molded Nanoparticles and Fine Particles The polynucleotides, primary constructs, and / or mMVs disclosed herein may be formulated into nanoparticles and / or fine particles. These nanoparticles and / or fine particles can be molded into any size form and chemical composition. As an example, nanoparticles and / or microparticles can be made using PRINT® technology by LIQUIDA TECHNOLOGIES® (Morrisville, NC) (eg, incorporated herein by reference in their entirety). , International Publication No. WO2007024323).

In one embodiment, the molded nanoparticles may comprise the polynucleotides, primary constructs, and / or mmRNA cores disclosed herein, and polymer shells. The polymer shell can be any of the polymers described herein and known in the art. In a further embodiment, polymer shells may be used to protect polynucleotides, primary constructs, and / or mmRNAs in the core.

Nano Jackets and Nano Liposomes
The polynucleotides, primary constructs, and / or mMVs disclosed herein may be formulated in nanojackets and nanoliposomes with Keystone Nano (State College, PA). Nanojackets are made of compounds that are found naturally in the body and can contain calcium, phosphates, and even small amounts of silicates. Nanojackets can range in size from 5 to 50 nm and can be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs, and / or mRNA. can.

Nanoliposomes are made of lipids that are naturally occurring in the body, but are not limited to these. Nanoliposomes can range in size from 60 to 80 nm and can be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs, and / or mRNA. can. In one aspect, the polynucleotides, primary constructs, and / or mmRNAs disclosed herein are formulated into nanoliposomes such as, but not limited to, ceramide nanoliposomes.

Excipients Pharmaceutical formulations, when used herein, are any solvent, dispersion medium, diluent, or other liquid vehicle, dispersion or suspension aid suitable for the particular dosage form desired. , A pharmaceutically acceptable excipient, including a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a solid binder, a lubricant and the like. Remington's The Science and Practice of Pharmacy, 21st Edition, A. ^{et al., Which is incorporated herein by reference in its entirety.} R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006 discloses various excipients used to formulate pharmaceutical compositions and known techniques for their preparation. With the substance or derivative thereof, the excipient medium of the drug causes any undesired biological effects or otherwise interacts with any other component (s) of the pharmaceutical composition in a detrimental manner. Its use is intended within the scope of this disclosure, except where it is non-conforming.

In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, excipients are approved by the United States Food and Drug Administration. In some embodiments, the excipient is medicinal grade. In some embodiments, the excipients are United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia (British Pharmacopoeia), and / or It meets the criteria of International Pharmacopoeia).

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders. , Preservatives, buffers, lubricants, and / or oils, but not limited to these. Such excipients may optionally be included in the pharmaceutical composition.

Examples of diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol. , Sodium chloride, dried starch, corn starch, powdered sugar and / or combinations thereof, but is not limited thereto.

Examples of granulators and / or dispersants include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cations. Replacement resin, calcium carbonate, silicate, sodium carbonate, cross-linked poly (vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose , Pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and / or combinations thereof. Includes, but is not limited to.

Examples of surfactants and / or emulsifiers include natural emulsifiers (eg, acacia, agar, alginic acid, sodium alginate, tragant, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol. , Wax, and lecithin), colloidal clay (eg, bentonite [aluminum silicate] and VEEGUM® [magnesium silicate]), long chain amino acid derivatives, high molecular weight alcohols (eg, stearyl alcohol, cetyl alcohol, etc.) Oleyl alcohol, triacetin monostearate
Monosteate), ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomer (eg, carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives. (For example, sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid ester (for example, polyoxyethylene sorbitan monolaurylate [TWEEN® 20], polyoxyethylene Solbitan [TWEENn® 60], Polyoxyethylene sorbitan monooleate [TWEEN® 80], Solbitan monopalmitate [SPAN® 40], Solbitan monostearate [SPAN® 60] ], Solbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene ester (eg, polyoxyethylene monostearate [MYRJ (eg) 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (eg, CREMOPHOR (eg, CREMOPHOR). Registered trademark)), polyoxyethylene ether, (eg, polyoxyethylene lauryl ether [BRIJ® 30]), poly (vinyl-pyrrolidone), diethylene glycol monolaurylate, triethanolamine oleate, sodium oleate, Potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC® F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate Includes, but is not limited to, sodium and the like, and / or combinations thereof.

Examples of binders include starch (eg, corn starch and starch paste); gelatin; sugars (eg, sucrose, glucose, dextrose, dextrin, sugar honey, lactose, lactitol, mannitol, etc.); natural and synthetic gums (eg, eg, mannitol). Acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol huk mucus, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl Methyl cellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone), magnesium aluminum silicate (Vegum®), and larch arabogalactan; alginate; polyethylene oxide; polyethylene glycol; inorganic calcium Salts; silicic acids; polymethacrylates; waxes; water; alcohols, etc .; and combinations thereof are included, but not limited to these.

Illustrated preservatives may include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. .. Examples of antioxidants include α-tocopherol, ascorbic acid, ascorbic palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl ascorbic acid, sodium ascorbate. , Sodium bisulfite, sodium metabisulfite, and / or sodium sulphate, but not limited to these. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetonic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and. / Or contains trisodium edetate. Examples of antibacterial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimid, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidi. , Imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal, but not limited to these. Examples of antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Included, but not limited to. Illustrated alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenols, phenolic compounds, bisphenols, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohols. Illustrated acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, β-carotene, citric acid, acetic acid, dihydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include tocopherol, tocopherol acetate, trademark meshylate, cetrimid, butylated hydroxyanisole (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), Sodium lauryl ether sulfate (SLES), sodium bisulfate, sodium metabisulfite, potassium sulfate, potassium metabisulfite, GLYDANT PLUS (registered trademark), PHENONIP (registered trademark), methylparaben, GERMALL (registered trademark) 115, GERMABEN (registered) Includes, but is not limited to, II, NEOLONE ™, KATHON ™, and / or EUXYL®.

Illustrated buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluvionate, calcium gluceptate, calcium gluconate, d-gluconic acid. , Calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, calcium dibasic phosphate, phosphoric acid, calcium tertiary phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, potassium dibasic phosphate , Primary potassium phosphate, potassium phosphate mixture, sodium acetate, sodium dicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, sodium monophosphate, sodium phosphate mixture, tromethamine, hydroxide Includes, but is not limited to, magnesium, aluminum hydroxide, alginic acid, exothermic acid removing water, isotonic saline, Ringer's solution, ethyl alcohol and / or combinations thereof.

Examples of lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, behanate glyceryl, hydride vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, Includes, but is not limited to, magnesium lauryl sulphate, sodium lauryl sulphate, and combinations thereof.

Examples of oils include almond oil, apricot oil, avocado oil, babas oil, bergamot oil, black currant seed oil, kade oil, chamomile oil, canola oil, caraway oil, carnauba oil, castor oil, katsura oil, cocoa butter. Oil, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, flaxseed oil, geraniol oil, gourd oil, grape seed oil, hazelnut oil, hisop oil, isopropyl myristate Oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange raffy oil, palm oil, palm kernel oil, peach seed oil, peanut oil, poppy oil, pumpkin seed oil, rapeseed oil, rice bran oil , Rosemary oil, Benibana oil, Byakudan oil, sasquaana oil, sevory, seaback thorn oil, sesame oil, shea butter oil, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, camellia oil, vetiver Includes, but is not limited to, oils, peach oils, and wheat germ oils. Illustrated oils include butyl stearic acid, caprylic acid triglyceride, capric acid triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof. Includes, but is not limited to.

Excipients such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavors, and / or fragrances may be present in the composition at the discretion of the formulator.

Delivery The present disclosure presents the disclosure of polynucleotides, primary constructs, or mRNAs for therapeutic, pharmaceutical, diagnostic, or imaging purposes by any suitable route that takes into account the prospect of scientific progress in drug delivery. Also includes delivery. The delivery may be naked or formulated.

Naked Delivery The polynucleotides, primary constructs, or mMVs of the invention may be delivered naked to cells. As used herein, "naked" refers to delivering a polynucleotide, primary construct, or mmRNA without the use of agents that promote transfection. For example, polynucleotides, primary constructs, or mRNAs delivered to cells may contain no modifications. Naked polynucleotides, primary constructs, or mmRNAs may be delivered to cells using routes of administration known in the art and described herein.

Formulated Delivery The polynucleotides, primary constructs, or mMVs of the invention may be formulated using the methods described herein. The pharmaceutical product may contain a polynucleotide, a primary construct, or mmRNA that may be modified and / or unmodified. The pharmaceutical product can further include, but is not limited to, a cell penetrant, a pharmaceutically acceptable carrier, a delivery agent, a biodegradable or biocompatible polymer, a solvent, and a sustained release delivery depot. The formulated polynucleotide, primary construct, or mmRNA may be delivered to cells using the route of administration known in the art and described herein.

The composition is a substrate such as a textile or biodegradable material coated or impregnated with the composition by direct immersion or immersion, via a catheter, gel, powder, ointment, cream, gel, lotion, and / or droplets. Can also be formulated for direct delivery to an organ or tissue in any of several methods in the art, including, but not limited to, by using.

Administration The polynucleotides, primary constructs, or mmRNAs of the invention may be administered by any route that produces therapeutically effective outcomes. These include enteral, transgastrointestinal, epidural, oral, percutaneous, epidural (intravitreal), intracerebral (into the cerebral), intraventricular (into the ventricle), and on the skin (into the ventricles). Intradermal application), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (via the nose), intravenous (into the vein), intraarterial (in the artery) Intravitreal (into the muscle), in the heart (into the heart), intraosseous injection (into the bone marrow), intrathecal cavity (into the spinal canal), intraperitoneal (into the abdomen) Infusion or injection), intravesical infusion, intravitreal (via the eye), intracavitary injection (into the base of the penis), intravaginal administration, intrauterine, extralamellar administration, transdermal (for systemic distribution) Intact skin (inject)
Includes those in sublingual (sin) (diffusion), transmucosa (diffusion through mucosa), gas infusion (nasal suction), sublingual, sublip, enema, eye drops (on the conjunctiva), or ear drops. , Not limited to these. In certain embodiments, the compositions may be administered in such a manner as to allow them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. Non-limiting routes of administration for polynucleotides, primary constructs, or mmRNAs of the invention are described below.

Parenteral and Injectable Administration Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. .. In addition to the active ingredient, liquid dosage forms include, for example, water or other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl. Solubilizers and emulsifiers such as formamide, oils (specifically cotton seed oil, lacquer oil, corn, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, Also, inert diluents commonly used in the art, such as mixtures thereof, may be included. In addition to the Inactive Diluent, the oral composition can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or fragrances. In certain embodiments for parenteral administration, the composition is a solubilizer such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. Is mixed with.

Injectable preparations, such as sterile injectable aqueous or oily suspensions, may be formulated according to known techniques with suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations are in non-toxic parenterally acceptable diluents and / or solvents in sterile injectable solutions, suspensions, and / or emulsions, such as 1,3-butanediol. It may be as a solution. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, U.S.A. S. P. , And isotonic sodium chloride solution. Non-irritating fixed oils are customarily used as solvents or suspension media. Any sterile fixed oil can be used for this purpose, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

The injectable formulation is, for example, a sterile agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium by filtration through a bacterial retention filter and / or prior to use. By incorporating, it can be sterilized.

It is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection in order to prolong the effect of the active ingredient. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with low solubility in water. The rate of absorption of the drug then depends on its rate of dissolution, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of the drug form administered parenterally is accomplished by dissolving or suspending the drug in an oily vehicle. The injectable depot form is made by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the drug-to-polymer ratio and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoester) and poly (anhydride). Depot injectable formulations are prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissue.

Rectal and Intravaginal Administration Compositions for rectal or intravaginal administration are typically suppositories, which are solid at ambient temperature but liquid at body temperature and thus in the rectal or vaginal cavity. It can be prepared by mixing with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol, or suppository wax that dissolves and releases the active ingredient.

Oral Administration Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms include, for example, water or other solvents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, Solubilizers and emulsifiers such as dimethylformamide, oils (particularly cottonseed oil, lacquer oil, corn, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and these. May include inactive diluents commonly used in the art, such as mixtures of. In addition to the Inactive Diluent, the oral composition can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or fragrances. In certain embodiments for parenteral administration, the composition is a solubilizer such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. Is mixed with.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is at least one inert pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or a filler or bulking agent (eg, starch). , Lactose, sucrose, glucose, mannitol, and silicic acid), binders (eg, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), water retention agents (eg, glycerol), disintegrants (eg, glycerol). , Agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (eg, paraffin), absorption enhancers (eg, quaternary ammonium compounds). ), Wetting agents (eg cetyl alcohol and glycerol monostearate), absorbents (eg kaolin and bentonite clay), and lubricants (eg starch, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate). ), As well as a mixture of these. For capsules, tablets, and pills, the dosage form may also include a buffer.

Topical or transdermal administration As described herein, compositions containing the polynucleotides, primary constructs, or mMVs of the invention may be formulated for topical administration. The skin can be an ideal target for delivery because it is easily accessible. Gene expression is restricted only to the skin, which may not only avoid non-specific toxicity, but may also be restricted to specific layers and cell types within the skin.

The site of skin expression of the delivered composition depends on the pathway of nucleic acid delivery. The following three pathways are considered for delivering polynucleotides, primary constructs, or mmRNAs to the skin: (i) topical application (eg, for topical / local therapeutic and / or cosmetic use), (ii). Percutaneous injection (eg, for topical / local treatment and / or cosmetic use), and (iii) systemic delivery (eg, for the treatment of skin disorders that affect both the skin and extradermal areas). Polynucleotides, primary constructs, or mmRNAs can be delivered to the skin by several different approaches known in the art. Non-cationic liposome-DNA complex, topical application of cationic liposome-DNA complex, particle-mediated (gene gun), puncture-mediated gene transfection, and virus delivery approaches, but not limited to most. Local delivery approach has been shown to be effective in delivering DNA. After delivery of the nucleic acid, the gene product has been detected in several different skin cell types, including but not limited to basal keratinocytes, sebaceous gland cells, dermal fibroblasts, and dermal macrophages.

In one embodiment, the invention provides a variety of dressings (eg, wound dressings) or bandages (eg, adhesive bandages) for conveniently and / or effectively performing the methods of the invention. Typically, the dressing or bandage is a sufficient amount of the pharmaceutical composition and / or polynucleotide described herein to allow the user to perform multiple treatments of the subject (s). , Primary constructs, or mmRNA can be included.

In one embodiment, the invention provides a polynucleotide, primary construct, or mmRNA composition delivered in more than one infusion.

In one embodiment, prior to topical and / or transdermal administration, an area of at least one tissue, such as skin, may be subjected to a device and / or solution that may increase permeability. In one embodiment, the tissue may be subjected to a polishing device to increase skin permeability (see US Patent Publication No. 20080275468, which is incorporated herein by reference in its entirety). In another embodiment, the tissue may be subjected to an ultrasound-enhanced device. Ultrasound-enhanced devices are the devices described in US Publication No. 20040236268 and US Pat. Nos. 6,491,657 and 6,234,990, each of which is incorporated herein by reference in its entirety. May include, but are not limited to. Methods for improving tissue permeability are described in US Publications 20040171980 and 20040236268 and US Pat. Nos. 6,190,315, each of which is incorporated herein by reference in its entirety. ..

In one embodiment, the device may be used to increase tissue permeability prior to delivery of the modified mRNA preparation described herein. Skin permeability can be measured by methods known in the art and / or by the methods described in US Pat. No. 6,190,315, which is incorporated herein by reference in its entirety. As a non-limiting example, the modified mRNA preparation may be delivered by the drug delivery method described in US Pat. No. 6,190,315, which is incorporated herein by reference in its entirety.

In another non-limiting example, the tissue may also be treated with a local anesthetic eutectic mixture (EMLA) cream before, during and / or after the tissue may be subjected to a device capable of increasing permeability. good. Katz et al. (Anest Analg (2004); 98: 371-76, which is incorporated herein by reference in its entirety) used EMLA cream in combination with low energy to reduce the occurrence of superficial skin analgesia. It was shown that it was seen as early as 5 minutes after pretreatment with ultrasound.

In one embodiment, the accelerator may be applied to the tissue before, during, and / or after the tissue is treated to increase permeability. Accelerators include, but are not limited to, transport promoters, physical promoters, and cavitation promoters. Non-limiting examples of accelerators are described in US Pat. No. 6,190,315, which is incorporated herein by reference in its entirety.

In one embodiment, the device may be used to increase tissue permeability prior to delivery of the modified mRNA formulation described herein, which may further contain a substance that evokes an immune response. In another non-limiting example, the formulations containing substances that evoke an immune response are by the methods described in US Publications 20040171980 and 20040236268, each of which is incorporated herein by reference in its entirety. It may be delivered.

Dosage forms for topical and / or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. In general, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any required preservatives and / or buffers as required.

In addition, the invention contemplates the use of transdermal patches, which can often have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the compound in a suitable medium. Alternatively, or further, the rate can be controlled either by providing a rate control membrane and / or by dispersing the compound in a polymer matrix and / or gel.

Suitable formulations for topical administration include liquid and / or semi-liquid preparations such as liniments, lotions, oil-in-water and / or water-in-oil emulsions such as creams, ointments, and / or pastes, and / or solutions. And / or suspensions are included, but not limited to these.

Topically administrable formulations may contain, for example, about 0.1 w / w% to about 10 w / w% active ingredient, but the concentration of active ingredient is up to the limit of solubility of the active ingredient in the solvent. It may be expensive. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Depot Administration As described herein, in some embodiments, the composition is formulated during depot for sustained release. Generally, a specific organ or tissue (“target tissue”) is targeted for administration.

In some embodiments of the invention, the polynucleotide, primary construct, or mmRNA is spatially retained within or proximal to the target tissue. The target tissue (containing one or more target cells) and the composition, in particular the nucleic acid constituents of the composition (s) are substantially retained within the target tissue, i.e. at least the composition. 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or more than 99.99% retained in the target tissue By contacting under such conditions, a method of providing the composition to a target tissue of a mammalian subject is provided. Advantageously, retention is determined by measuring the amount of nucleic acid present in the composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, of nucleic acids administered to a subject after a period of administration. 99.9, 99.99, or more than 99.99% is present intracellularly. For example, intramuscular injection into a mammalian subject is performed with an aqueous composition containing ribonucleic acid and a transfection reagent, and retention of the composition is performed by measuring the amount of ribonucleic acid present in the muscle cells. It is determined.

Aspects of the invention are to contact a composition with a target tissue (containing one or more target cells) under conditions such that the composition is substantially retained within the target tissue. The target is a method of providing to a target tissue of a mammalian target. The composition contains an effective amount of polynucleotide, primary construct, or mmRNA such that the polypeptide of interest is produced in at least one target cell. Compositions generally contain cell penetrants and pharmaceutically acceptable carriers, but "naked" nucleic acids (such as nucleic acids that do not contain cell penetrants or other agents) are also contemplated.

In some situations, the amount of protein produced by cells in the tissue is preferably increased. Preferably, this increase in protein production is spatially restricted to cells within the target tissue. Therefore, there is provided a method of increasing the production of the protein of interest in the tissue of a mammalian subject. A polynucleotide, primary, characterized in that the unit amount of composition is determined to produce the polypeptide of interest in a substantial proportion (%) of cells contained within a defined volume of target tissue. Constructs, or compositions containing mmRNA, are provided.

In some embodiments, the composition comprises a plurality of different polynucleotides, primary constructs, or mmRNAs, wherein one or more of the polynucleotides, primary constructs, or mmRNAs encode the polypeptide of interest. Optionally, the composition also contains a cell permeabilizer to assist in the intracellular delivery of the composition. Produces the desired polypeptide in a significant proportion (%) of cells contained within a defined volume of target tissue (generally, in tissue adjacent to or distal to the defined volume, the target poly. Determining the dose of the composition required (without inducing significant production of the peptide) is made. Following this determination, the determined dose is introduced directly into the tissue of the mammalian subject.

In one embodiment, the invention provides a polynucleotide, primary construct, or mMr of interest delivered in more than one infusion or by divided dose infusion.

In one embodiment, the invention may be retained in the vicinity of the target tissue using a small disposable drug reservoir, patch pump, or osmotic pump. Non-limiting examples of patch pumps include BD® (Franklin Lakes, NJ), Insule Corporation (Bridgewater, MA), SteadyMed Therapeutics (San Francisco, CA), Medtronic (Minneapolis), Medtronic (Minneapolis, etc.) ), UniLife (York, PA), Valeritas (Bridgewater, NJ), and SpringLeaf Therapeutics (Boston, MA). Non-limiting examples of osmotic pumps include those manufactured by DURECT® (Cupertino, CA) (eg, DUROS® and ALZET®).

Pulmonary administration The pharmaceutical composition may be prepared, packaged and / or sold in a formulation suitable for pulmonary administration via the cavity. Such a formulation may contain dry particles containing the active ingredient and having a diameter in the range of about 0.5 nm to about 7 nm or about 1 nm to about 6 nm. Such compositions preferably use a device comprising a dry powder reservoir in which the flow of propellant can be directed to disperse the powder and / or dissolve in a low boiling point propellant in a sealed container. And / or in the form of a dry powder for administration using a self-injection solvent / powder dispensing container such as a device containing a suspended active ingredient. Such powders include particles in which at least 98% by weight of the particles have a diameter greater than 0.5 nm and at least 95% (quantity) of the particles have a diameter less than 7 nm. Alternatively, at least 95% by weight of the particles have a diameter greater than 1 nm and at least 90% (quantity) of the particles have a diameter less than 6 nm. The dry powder composition may comprise a solid fine powder diluent such as sugar, which is conveniently provided in unit dose form.

Low boiling point propellants generally include liquid propellants having a melting point below 65 ° F. (about 18.3 ° C.) at atmospheric pressure. In general, the propellant may constitute 50 w / wv% to 99.9 w / wv% of the composition and the active ingredient may constitute 0.1 w / w% to 20 w / w% of the composition. The propellant may further comprise additional components such as a liquid nonionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same size as the particles containing the active ingredient).

As a non-limiting example, the polynucleotides, primary constructs, and / or mMVs described herein are lunged by the method described in US Pat. No. 8,257,685, which is incorporated herein by reference in its entirety. It may be formulated for delivery.

Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of solution and / or suspension. Such formulations may be optionally prepared, packaged and / or sold as aqueous and / or diluted alcohol solutions and / or suspensions containing the active ingredient, conveniently. It can be administered using any spray and / or atomization device. Such formulations include, but are not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methyl benzoate. It may be included. The droplets provided by this route of administration can have an average diameter in the range of about 0.1 nm to about 200 nm.

Nasal, Nasal, and Buccal Doses The formulations described herein as useful for pulmonary delivery are useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a crude powder containing the active ingredient and having an average particle of about 0.2 μm to 500 μm. Such a preparation is administered in such a manner that inhalation is performed through the nose, that is, by rapid inhalation through the nostrils from a container of powder held close to the nose.

A suitable formulation for nasal administration may include, for example, an active ingredient of a minimum of about 0.1 w / w% to a maximum of 100 w / wv%, or may include one or more of the additional ingredients described herein. good. The pharmaceutical composition may be prepared, packaged and / or sold in a formulation suitable for buccal administration. Such a formulation may be in the form of tablets and / or lozenges made using conventional methods, for example, and may be, for example, 0.1 w / w% to 20 w / w% active ingredient. The rest may comprise an orally soluble and / or degradable composition, and optionally one or more of the additional components described herein. Alternatively, a suitable formulation for buccal administration may include powders and / or aerosolized and / or atomized solutions and / or suspensions containing the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle size and / or droplet diameter in the range of about 0.1 nm to about 200 nm, as described herein. It may further comprise one or more of any of the additional components described.

Intraocular administration The pharmaceutical composition may be prepared, packaged and / or sold in a formulation suitable for intraocular administration. Such formulations are in the form of eye drops containing, for example, a solution and / or suspension of 0.1 / 1.0 w / w% active ingredient in an aqueous or oily liquid excipient. May be good. Such droplets may further contain a buffer, a salt, and / or one or more of any additional components described herein. Other intraocularly administrable formulations that are useful include those that contain the active ingredient in microcrystalline form and / or in liposome preparations. Ear drops and / or eye drops are intended to be within the scope of the present invention. Multilayer thin film devices may be prepared to contain the pharmaceutical composition for delivery to the eye and / or surrounding tissue.

Payload Administration: Detectable Agents and Therapeutic Agents The polynucleotides, primary constructs, or mMVs described herein are detectable for delivery of a substance (“payload”) to a biological target, eg, detection of a target. It can be used in several different scenarios where delivery of a substance or delivery of a therapeutic agent is desired. Detection methods include both in vitro imaging and in vivo imaging, such as immunohistochemistry, bioluminescence imaging (BLI), magnetic resonance imaging (MRI), positron-emitting tomography (PET), and electron microscopy. X-ray computed tomography, Raman imaging, optical interference tomography, absorption imaging, thermal imaging, fluorescence reflection imaging, fluorescence microscopy, fluorescence molecular tomography imaging, magnetic resonance imaging, X-ray imaging , Ultrasonic imaging, photoacoustic imaging, laboratory assays, or methods in any situation where tagging / staining / imaging is required, but not limited to these.

The polynucleotide, primary construct, or mmRNA can be designed to contain both the linker and payload at any useful localization. For example, using a linker with two ends, one end is at the C-7 or C-8 position of deaza-adenosine or deaza-guanosine, or at the N-3 or C-5 position of cytosine or uracil. The payload and the other end are attached to the nucleobase. The polynucleotides of the invention can include more than one payload (eg, labels and transcription inhibitors), as well as a cleavable linker. In one embodiment, the modified nucleotide has one end of the cleaving linker attached to the C7 position of 7-deraza-adenine and the other end of the linker to the inhibitor (eg, the C5 position of the nucleobase on cytidine). A modified 7-deraza-adenosine triphosphate that is bound and the label (eg, Cy5) is bound to the center of the linker (eg, US Pat. No. 7,994,304, incorporated herein by reference). See Compound 1 of A * pCp C5 Parg Capless in FIG. 5 and columns 9 and 10). When modified 7-deaza-adenosine triphosphate is incorporated into the coding region, the resulting polynucleotide with a cleavable linker is bound to a label and inhibitor (eg, a polymerase inhibitor). When the linker is cleaved (eg, under reducing conditions for reducing the linker with a cleavable disulfide moiety), the label and inhibitor are released. Additional linkers and payloads (eg, therapeutic agents, detectable labels, and cell-permeable payloads) are described herein.

Scheme 12 below illustrates an exemplary modified nucleotide in which the nucleobase adenine is attached to a linker at the C-7 carbon of 7-deazaadenine. In addition, Scheme 12 depicts a linker and payload, eg, a modified nucleotide in which a detectable agent is integrated on the 3'end of the mRNA. Detectable agent is released by disulfide cleavage and 1,2-addition of thiol groups on propargyl ester. The remaining structure (eg, described as pApC5Parg in Scheme 12) is an inhibitor. The rationale for the structure of modified nucleotides is that the moored inhibitor sterically interferes with the ability of the polymerase to incorporate the second base. Therefore, the mooring is long enough to affect this function, and inhibits or prohibits the inhibitor from joining the extending polynucleotide chain of the second and subsequent nucleotides. It is essential to be in stereochemical localization.
Scheme 12

For example, the polynucleotides, primary constructs, or mMVs described herein can be used to reprogram induced pluripotent stem cells (iPS cells), thereby comparing to total cells in a cluster. , The cells to be transfected can be tracked directly. In another example, a drug that can be attached to a polynucleotide, primary construct, or mMV via a linker and that can be fluorescently labeled can be used to track the drug in vivo, eg, intracellularly. Other examples include, but are not limited to, the use of polynucleotides, primary constructs, or mmRNAs in intracellular reversible drug delivery.

The polynucleotides, primary constructs, or mMVs described herein can be used in the intracellular targeting of payloads, such as detectable agents or therapeutic agents, into specific organelles. Illustrated intracellular targets can include, but are not limited to, nuclear localization for advanced mRNA processing, or nuclear localization sequences (NLS) linked to mRNA containing an inhibitor.

In addition, the polynucleotides, primary constructs, or mMVs described herein can be used to deliver therapeutic agents to cells or tissues, for example in living animals. For example, the polynucleotides, primary constructs, or mMVs described herein can be used to deliver highly polar chemotherapeutic agents to kill cancer cells. A polynucleotide, primary construct, or mMV that is bound to a therapeutic agent via a linker may allow the therapeutic agent to migrate intracellularly and reach an intracellular target by facilitating permeation of the member. ..

In one example, the linker is attached at the 2'position of the ribose ring and / or at the 3'and / or 5'position of the polynucleotide, the primary construct mmRNA (eg, incorporated herein by reference in its entirety, internationally. See Publication No. WO2012030683). The linker may be any linker disclosed herein, known in the art, and / or disclosed in WO2011030683, which is incorporated herein by reference in its entirety.

In another example, the polynucleotide, primary construct, or mmRNA can be attached to the polynucleotide, primary construct, or mmRNA virus inhibitory peptide (VIP) via a cleavable linker. Cleavable linkers can release VIPs and dyes intracellularly. In another example, a polynucleotide, primary construct, or mMV can be linked via a linker to ADP-ribosylate, which is involved in the action of several bacterial toxins such as cholera toxin, diphtheria toxin, and pertussis toxin. These toxin proteins are ADP-ribosyltransferases that modify the target protein in human cells. For example, the cholera toxin ADP-ribosylate G protein denatures human cells by causing large amounts of fluid secretion from the lining of the small intestine, resulting in fatal diarrhea.

In some embodiments, the payload may be a therapeutic agent such as a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, or another therapeutic agent. Cytotoxic or cytotoxic agents include any agent that can be harmful to cells. Examples include taxol, cytocaracin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, teniposide, vincristine, vinblastine, corhitin, doxorubicin, daunorubicin, dihydroxyanthracendione, dihydroxyanthracinedione, mitoxantrone. Mycin D, 1-dehydrotestosterone, sugar corticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, mitanorubicin, eg, mitancinol, a US patent that is incorporated herein by reference in its entirety. 5,208,020), Raquermycin (CC-1065, all incorporated herein by reference, US Pat. Nos. 5,475,092, 5,585,499, and 5). , 846, 545), as well as analogs or homologues thereof, but not limited to these. Radioactive ions include, but are not limited to, iodine (eg, iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and placeodimium. .. Other therapeutic agents include metabolic antagonists (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildacarbazine), alkylating agents (eg, mechloretamine, thiotepachlorambusyl, raquel). Mycin (CC-1065), melphalan, carmustin (BSNU), romustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), Anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antifibrinolytic agents (For example, vincristine, vinblastine, taxol, and mytancinoids), but are not limited to these.

In some embodiments, the payload is a variety of small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent substances, luminescent substances (eg, luminol), bioluminescent substances (eg, luciferase, luciferin, and equolin). ), Chemically luminescent substances, radioactive substances (for example, ¹⁸ F, ⁶⁷ Ga, ^{81 m} Kr, ⁸² Rb, ¹¹¹ In, ¹²³ I, ¹³³ Xe, ²⁰¹ Tl, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³ H, or r ^{99 m.} tc (e.g., pertechnetate (pertechnetate (VII), TcO ₄ - as), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxide (e.g. , Pertechnetate iron oxide (SPIO), single crystal iron oxide nanoparticles (MION), and micro-pertechnetate iron oxide (USPIO), manganese chelate (eg, Mn-DPDP), barium sulfate, iodo-based contrast agent medium (iohexol (iohexol) It can be a detectable agent such as Iohexol)), microbubbles, or perfluorocarbon). Such photodetectable labels include, for example, without limitation, 4-acetamide-4'-stilben-2 isothiocyanate, 2'disulfonic acid; aclysine and derivatives (eg aclysine and aclysine isothiocyanate); 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N- [3-vinylsulfonyl) Phenyl] naphthalimide-3,5 disulfonate; N- (4-anilino-1-naphthyl) maleimide; anthranilamide; BODIPY; brilliant yellow; AMC, coumarin 120), and 7-amino-4-trifluoromethyl coumarin (cumarin 151); cyanine dye; cyanocin; 4', 6-diamidino-2-phenylindole (DAPI); 5'5 "-dibromopyrogalol- Sulfonaphthalene (naphthalein) (bromopyrogalol red); 7-diethylamino-3- (4'-phenyl isothiocyanate) -4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-dihydro-stilben-2 diisothiocyanate, 2'-disulfonic acid; 4,4'-diisothiocyanate stillben-2,2'-disulfonic acid; 5- [dimethylamino]- Naphthalene-1-sulfonyl chloride (DNS, Dansil Lolide); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives (eg, eosin and eosin isothiocyanate); erythrosin and derivatives (eg, erythrosin) B and erythrosin isothiocyanate); ethidium; fluorosane and derivatives (eg 5-carboxyfluorosane (FAM), 5- (4,6-dichlorotriazine-2-yl) aminofluorosane (DTAF), 2', 7 '-Dimethoxy-4'5'-dichloro-6-carboxyfluorosane, fluorosane, fluorosane isothiocyanate, X-rhodamine-5-(and -6) -isothiocyanate (QFITC or XRITC), and fluorescamine) 2- [2- [3-[[1,3-Dihydro-1,1-dimethyl-3- (3-sulfopropyl) -2H-benz [e] indol-2-iriden] etiliden] -2- [ 4- (ethoxycarbonyl) -1-piperazinyl] -1-cyclopentene-1-yl] ethenyl] -1,1-dimethyl-3- (3-sulfopropyl) -1H-benz [e] indolium water Oxide, intramolecular salt, compound with n, n-diethylethaneamine (1: 1) (IR144); 5-chloro-2- [2- [3-[(5-chloro-3-ethyl-2) 3H) -benzothiazole-iriden) etiliden] -2- (diphenylamino) -1-cyclopenten-1-yl] ethenyl] -3-ethylbenzothiazolium perchlorate (IR140); malakite green isothiocyanate; 4 -Methylumveriferon orthocresol phthalein; nitrotyrosine; pararosaniline; phenol red; B-ficoerythrin; o-phthaldialdehyde; pyrene and derivatives (eg pyrene, pyrene butyrate, and succinimidyl 1-pyrene); butyrate Quantum Dot; Reactive Red 4 (CIBACRON ™ Brilliant Red 3B-A); Rhodamine and Derivatives (eg 6-carboxy-X-Rhodamine (ROX), 6-carboxyrhodamine (R6G), Lisamin Rhodamine Bsulfonyl Chloride) Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Isothiocyanate, Sul Holodamin B, Sulforhodamine 101, Sulfyldamine Derivatives of Sulforhodamine 101 (Texas Red), N, N, N', N'Tetramethyl-6-carboxyrhodamine (TAMRA) Tetramethyl Rhodamine, and Tetramethyl Rhodamine Isothiocyanate (TRITC) ); Rhodamine; Rhodamine; Terbium chelate derivative; Cyanine-3 (Cy3); Cyanine-5 (Cy5); Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalocyanine; as well as naphthalocyanine.

In some embodiments, the detectable agent is an undetectable precursor (eg, a fluorescent tetrazine-fluorophore construct (eg, tetrazine-BODIPY FL, tetrazine-olegon) that becomes detectable when activated. It may be Green 488, or Tetrazine-BODIPY TMR-X), or an enzyme-activated fluorescent agent (eg, PROSENSE® (VisEn Medical)). In vitro where the enzyme labeled composition can be used. Assays include, but are not limited to, enzyme-bound immunoadsorption assay (ELISA), immunoprecipitation assay, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and western blot analysis.

Combination polynucleotides, primary constructs, or mmRNAs may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. Although "in combination" is not intended to imply that the agents need to be co-administered and / or formulated for delivery together, these delivery methods are disclosed herein. Is within the range of. The composition can be administered prior to or thereafter in parallel with one or more other desired therapeutic agents or medical procedures. In general, each drug will be administered at the dose determined for that drug and / or on a time schedule. In some embodiments, the disclosure discloses pharmaceutical, prophylactic, diagnostic, or imaging compositions that improve their bioavailability, reduce and / or modify their metabolism, and excrete them. Includes delivery in combination with agents that can inhibit and / or modify their biodistribution. As a non-limiting example, nucleic acids or mmRNAs may be used in combination with pharmaceuticals for the treatment of cancer or for controlling hyperproliferative cells. In US Pat. No. 7,964,571, which is incorporated herein by reference in its entirety, a pharmaceutical composition comprising a DNA plasmid encoding interleukin-12 with a lipopolymer is used and at least one anti-cancer agent or chemistry. Combination therapies for the treatment of primary or metastatic solid tumors that administer therapeutic agents are described. In addition, the nucleic acids and mRNAs of the invention encoding antiproliferative molecules may be present in pharmaceutical compositions with lipopolymers (eg, pharmaceutical compositions comprising DNA plasmids and lipopolymers encoding antiproliferative molecules). (See US Publication No. 20110218231), which claims the material, which is incorporated herein by reference in its entirety), it can be administered with at least one chemotherapeutic or anti-cancer agent.

It is further understood that the therapeutic, prophylactic, diagnostic, or imaging activators used in combination may be administered together in a single composition or separately in different compositions. NS. In general, drugs used in combination are expected to be used at levels that do not exceed the levels at which they are used individually. In some embodiments, the levels used in combination will be lower than the levels used individually. In one embodiment, the combinations may be administered individually or together according to the split dosing regimen described herein.

Dosing The present invention provides methods comprising administering modified mRNAs according to the present invention and their encoded proteins or complexes to subjects in need thereof. Nucleic acid, protein, or complex, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof, are diseases, disorders, and / or conditions (eg, diseases, disorders, and / related to memory impairment efforts. Or the condition) can be administered to the subject using any amount and any route of administration that is effective for prevention, treatment, diagnosis, or imaging. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, disease severity, specific composition, method of administration thereof, method of activation thereof, and the like. Compositions according to the invention are typically formulated in unit dosage forms for ease of administration and uniform dosage. However, it is understood that the total daily use of the compositions of the present invention may be determined by the attending physician within the scope of appropriate medical judgment. The particular therapeutically effective, prophylactically effective, or appropriate imaging dose level of any particular patient is the disorder being treated and the severity of the disorder; the activity of the particular compound used; the particular composition used; Patient's age, weight, overall health, gender, and diet; time of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; in combination with or with the particular compound used Drugs used at the same time; depends on various factors including those well known in the medical field.

In certain embodiments, the compositions according to the invention are from about 0.0001 mg / kg to about 100 mg / kg, about 0. 001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / Kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg , About 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg (body weight of the subject) can be administered at a dosage level sufficient to deliver at least once daily. The desired dosage is 3 times a day, 2 times a day, once a day, every other day, every 2 days, once a week, once every 2 weeks, once every 3 weeks, or 4 weeks. May be delivered once. In certain embodiments, the desired dosage is multiple doses (eg, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, It may be delivered by 12 times, 13 times, 14 times or more administration). When multiple doses are used, a split dosing regimen, such as the split dosing regimen described herein, may be used.

According to the present invention, administration of mmRNA in a divided dose regimen has been found to produce higher levels of protein in mammalian subjects. As used herein, a "split dose" is a single unit dose or a total daily dose divided into two or more doses, eg, a single unit dose in two or more doses. As used herein, a "single dose" is any treatment administered at a single dose / at a time / in a single route / at a single point of contact, ie in a single dose event. The dose of the drug. As used herein, a "total daily dose" is an amount given or prescribed within 24 hours. It may be administered as a single unit dose. In one embodiment, the mmRNAs of the invention are administered to a subject in divided doses. The mRNA may be formulated in buffer alone or in the formulations described herein.

Dosage Form The pharmaceutical compositions described herein are for topical, intranasal, intratracheal, or injectable (eg, intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous) and the like. It can be formulated in the dosage form described herein.

Liquid Dosage Forms Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms include water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3. -Butylene glycol, dimethylformamide, oils (specifically cottonseed oil, lacquer oil, corn, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohols, sorbitan polyethylene glycols and fatty acid esters, and these. May include inactive diluents commonly used in the art, including but not limited to mixtures. In certain embodiments for parenteral administration, the composition comprises solubilizers such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. May be mixed with.

Injectables Injectable preparations, such as sterile injectable aqueous or oily suspensions, may be formulated according to known techniques and may also contain suitable dispersants, wetting agents, and / or suspending agents. good. Sterile injectable preparations are non-toxic parenteral acceptable diluents and / or solvents such as sterile injectable solutions, suspensions, and / or emulsions in 1,3-butanediol solutions. obtain. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, U.S.A. S. P. , And isotonic sodium chloride solution, but not limited to these. Non-irritating fixed oil is customarily used as a solvent or suspension medium. Any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used for this purpose. Fatty acids such as oleic acid can be used in the preparation of injectables. The injectable formulation is incorporated, for example, by filtration through a bacterial retention filter and / or in the form of a sterile solid composition in which the sterile agent can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Can be sterilized by. Longer In order to prolong the effect of the active ingredient, it may be desired to delay the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with low solubility in water. The rate of absorption of the polynucleotide, primary construct, or mmRNA can then depend on its rate of dissolution and then on the size and morphology of the crystal. Alternatively, delayed absorption of parenterally administered polynucleotides, primary constructs, or mmRNAs can be accomplished by dissolving or suspending the polynucleotides, primary constructs, or mmRNAs in an oil vehicle. Depot forms for injection are made by forming a microcapsule matrix of polynucleotides, primary constructs, or mmRNAs in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the polynucleotide, primary construct, or mmRNA to the polymer, and the nature of the particular polymer used, the rate of release of the polynucleotide, primary construct, or mmRNA can be adjusted. Examples of other biodegradable polymers include, but are not limited to, poly (orthoester) and poly (anhydride). Depot injection formulations may be prepared by encapsulating polynucleotides, primary constructs, or mmRNAs in liposomes or microemulsions compatible with body tissue.

The formulations described herein as useful for pulmonary pulmonary delivery can also be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration may be a crude powder containing the active ingredient and having an average particle of about 0.2 μm to 500 μm. Such a pharmaceutical product may be administered in such a manner that inhalation is performed through the nose, that is, by rapid inhalation through the nostrils from a container of powder held close to the nose.

A suitable formulation for nasal administration may include, for example, an active ingredient of a minimum of about 0.1 w / w% to a maximum of 100 w / w%, or may include one or more of the additional ingredients described herein. good. The pharmaceutical composition may be prepared, packaged and / or sold in a formulation suitable for buccal administration. Such a formulation may be, for example, in the form of tablets and / or lozenges made using conventional methods, containing, for example, about 0.1 w / w% to 20 w / w% of active ingredient. The equilibrium may comprise an orally soluble and / or degradable composition and optionally one or more of the additional components described herein. Alternatively, a suitable formulation for buccal administration may include powders and / or aerosolized and / or atomized solutions and / or suspensions containing the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle size and / or droplet diameter in the range of about 0.1 nm to about 200 nm, as described herein. It may further comprise one or more of any of the additional components described.

General considerations in the formulation and / or manufacture of pharmaceuticals are described, for example, in Remington: Science and Practice of Pharmacy21 ^sted . , Lippincott Williams & Wilkins, 2005.

Coatings or Shells Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation field. .. They may optionally contain opalescent agents, which optionally release only the active ingredient (s) or preferentially in certain parts of the intestinal tract, in a delayed manner. It can be a composition to be used. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions may be used as fillers in soft and hard packed gelatin capsules using such excipients as lactose or lactose, as well as high molecular weight polyethylene glycol and the like.

Characteristics of Pharmaceutical Compositions The pharmaceutical compositions described herein can be characterized by one or more of bioavailability, therapeutic range, and / or distribution.

Bioavailability When a polynucleotide, primary construct, or mMV is formulated into a composition with a delivery agent described herein, it is compared to a composition lacking the delivery agent described herein. , Can exhibit increased bioavailability. As used herein, the term "bioavailability" refers to the systemic availability of a given amount of polynucleotide, primary construct, or mmRNA administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or maximum serum or plasma concentration (C _{max) of the unchanged form of the compound after administration of the compound to a mammal.} AUC is the determination of the area under the curve in which the serum or plasma concentration of a compound along the Cartesian coordinates (Y-axis) is plotted against the time along the Cartesian coordinates (X-axis). In general, AUCs of a particular compound are incorporated herein by reference in their entirety by methods known to those of skill in the art. S. Banker, Modern Pharmaceuticals, Drugs and the Pharmaceutical Sciences, v. et al. 72, Marcel Dekker, New York, Inc. , 1996.

The C _max value is the maximum concentration of compound achieved in mammalian serum or plasma after administration of the compound to the mammal. _{The C max} value of a particular compound can be measured using methods known to those of skill in the art. The expression "increased bioavailability" or "improved pharmacokinetics" refers to the systemic availability of a first polynucleotide, primary construct, or mMV as measured as _{AUC, C max} , or C _{min in mammals.} However, when co-administered with the delivery agent described herein, it means that it is higher than when co-administered with the delivery agent described herein. In some embodiments, the bioavailability of the polynucleotide, primary construct, or mmRNA is at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about about. 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about It can increase by 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

Therapeutic Area A polynucleotide, primary construct, or mmRNA, when formulated into a composition with a delivery agent described herein, is an administered polynucleotide, primary construct, lacking the delivery agent described herein. Alternatively, it may exhibit an increase in the therapeutic area of the administered polynucleotide, primary construct, or mmRNA composition as compared to the therapeutic area of the mmRNA composition. As used herein, "therapeutic area" refers to the range of plasma concentrations that are likely to elicit a therapeutic effect, or the range of levels of therapeutically active substance at the site of action. In some embodiments, the therapeutic range of polynucleotides, primary constructs, or mmRNAs, when co-administered with the delivery agents described herein, is at least about 2%, at least about 5%, at least about 10%. At least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, It can increase by at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

Distribution Amount A polynucleotide, primary construct, or mm mRNA, when formulated into a composition with the delivery agent described herein, is a distribution amount compared to a composition lacking the delivery agent described herein. It may exhibit an improvement (eg, reduction or targeting) of (V- _dist). Vdist associates the amount of drug in the body with the concentration of drug in blood or plasma. As used herein, the term "distributed amount" refers to the amount of fluid that is required to contain the total amount of drug in the body at the same concentration as in blood or plasma, where V _dust refers to the body. Amount of drug / concentration of drug in blood or plasma. For example, for a dose of 10 mg and a plasma concentration of 10 mg / L, the distribution amount is 1 liter. The amount of distribution reflects the extent to which the drug is present in extravascular tissue. The large amount of distribution reflects the tendency of compounds to bind to tissue constituents compared to plasma protein binding. In clinical practice, V _dust can be used to determine loading doses and achieve steady-state concentrations. In some embodiments, the distribution of polynucleotides, primary constructs, or mmRNAs is at least about 2%, at least about 5%, at least about 10%, when co-administered with the delivery agents described herein. At least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, It can be reduced by at least about 65% and at least about 70%.

Biological effects In one embodiment, the biological effects of modified mRNAs delivered to animals can be classified by analyzing protein expression in animals. Protein expression can be determined by analyzing biological samples collected from mammals administered with the modified mRNAs of the invention. In one embodiment, at least 50 pg / mL of expressed protein encoded by the modified mRNA administered to the mammal may be preferred. For example, 50-200 pg / mL of expressed protein of the protein encoded by the modified mRNA delivered to the mammal can be considered as a therapeutically effective amount of protein in the mammal.

Detection of Modified Nuclei by Mass Spectrometry Mass spectrometry (MS) is an analytical technique that can provide structural mass and molecular weight / concentration information of a molecule after ionic conversion of the molecule. Molecules are first ionized to obtain positive or negative charge, then they travel through mass spectrometers and differ regions of the detector according to their mass / charge (m / z) ratio. To reach.

Mass spectrometry is performed using a mass spectrometer containing an ion source to ionize the fractionated sample and create charged molecules for further analysis. For example, sample ionization includes electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization fast atomic impact (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization ( It can be performed by MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the choice of positive and negative modes, and the like.

After the sample is ionized, the resulting positive or negative charge ions can be analyzed to determine the mass-to-charge ratio (ie, m / z). Suitable analyzers for determining the mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Ions can be detected using several detection modes. For example, the selected ions can be detected (ie, using selective ion monitoring mode (SIM)), or the ions can be scanned in a scanning mode, eg, multiple reaction monitoring (MRM) or selective reaction monitoring (ie, using selective ion monitoring mode (SIM)). Can be detected using SRM).

Liquid chromatography-multi-reaction monitoring (LC-MS / MRM) linked with stable isotope-labeled dilution of peptide standards has been shown to be an effective method for protein validation (eg, of these). Each is incorporated herein by reference in its entirety, Keshishian et al., Mol Cell Protocols 2009.
8: 2339-2349, Khun et al. , Clin Chem 2009 55: 1108-1117, Lopez et al. , Clin Chem 2010
56: 281-290). Unlike untargeted mass spectrometry, which is often used in biomarker discovery studies, targeted MS methods have selected tens to hundreds of full analytical capabilities of the instrument in complex mixtures. A mode based on the peptide sequence of MS that concentrates on the peptide. Sensitivity and reproducibility are dramatically improved compared to discovery mode MS methods by limiting to peptides derived only from proteins intended for detection and fragmentation. This mass spectrometric-based multiplex reaction monitoring (MRM) quantification method dramatically impacts the discovery and quantification of biomarkers through rapid targeted multiplexed protein expression profiling of clinical samples. Can be done.

In one embodiment, a biological sample that may contain at least one protein encoded by at least one modified mRNA of the invention can be analyzed by the MRM-MS method. Quantification of biological samples can further include, but is not limited to, isotope-labeled peptides or proteins as internal standards.

According to the present invention, the biological sample may be subjected to enzymatic digestion when it is obtained from the subject. As used herein, the term "digestion" means splitting into shorter peptides. As used herein, the phrase "treating a sample to digest a protein" means manipulating the sample in such a way as to disassemble the protein in the sample. These enzymes include, but are not limited to, trypsin, endoproteinase Glu-C, and chymotrypsin. In one embodiment, a biological sample that may contain at least one protein encoded by at least one modified mRNA of the invention can be digested with an enzyme.

In one embodiment, a biological sample that may contain the protein encoded by the modified mRNA of the invention can be analyzed for the protein using electrospray ionization. Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to assist the transfer of ions from solution to gas phase before they are analyzed by mass spectrometry. Samples can be analyzed using methods known in the art (eg, Ho et al., Clin Biochem Rev. 2003 24 (1): 3-12, which is incorporated herein by reference in its entirety). The ionic species contained in the solution move to the gas phase by dispersing a fine spray of droplets, evaporating the solvent, and expelling ions from the droplets to produce a spray of highly charged droplets. obtain. Spraying of highly charged droplets is analyzed using, but not limited to, a quadrupole mass spectrometer, but not limited to, at least one, at least two, at least three, or at least four mass spectrometers. obtain. In addition, mass spectrometry can include purification steps. As a non-limiting example, the first quadrupole may be set to select a single m / z ratio, thus filtering out other molecular ions with different m / z ratios. This can eliminate the complex and time-consuming sample purification procedure prior to MS analysis.

In one embodiment, a biological sample that may contain the protein encoded by the modified mRNA of the invention can be analyzed for the protein in a tandem ESIMS system (eg, MS / MS). As a non-limiting example, droplets can be analyzed using product scans (or daughter scans), precursor scans (parent scans), neutral loss, or multiple reaction monitoring.

In one embodiment, a biological sample that may contain the protein encoded by the modified mRNA of the invention can be analyzed using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides non-destructive evaporation and ionization of both macromolecules and small molecules such as proteins. In MALDI analysis, the analyte is initially co-crystallized with a high molar excess of matrix compounds, which may also include, but are not limited to, UV-absorbing weak organic acids. Non-limiting examples of matrices used for MALDI are α-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and 2,5-dihydroxybenzoic acid. Laser emission of the analyte-matrix mixture can result in evaporation of the matrix and analyte. Laser-induced desorption provides high ion yields for intact analytes, enabling highly accurate compound measurements. Samples can be analyzed using methods known in the art (eg, Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000: 5880-5894, which are incorporated herein by reference in their entirety). As a non-limiting example, mass spectrometers used for MALDI analysis may include linear time-of-flight (TOF), TOF reflectorrons, or Fourier transform mass spectrometers.

In one embodiment, the analyte-matrix mixture can be formed using the dry droplet method. The biological sample is mixed with the matrix to create a saturated matrix solution, with a matrix-to-sample ratio of approximately 5000: 1. A constant amount (approximately 0.5-2.0 μL) of saturated matrix solution is then dried to form an analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture can be formed using the thin layer method. A matrix homogeneous thin film is formed first, after which the sample can be applied and absorbed by the matrix to form an analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture can be formed using the thick layer method. The matrix homogeneous thin film is formed with a nitrocellulose matrix additive. When a uniform nitrocellulose matrix layer is obtained, the sample is applied and absorbed into the matrix to form an analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture can be formed using the sandwich method. A thin layer of matrix crystals is prepared as in the thin layer method, after which aqueous trifluoroacetic acid, a sample, and a droplet of matrix are added. The sample is then absorbed into the matrix to form an analyte-matrix mixture.

V. Use of the polynucleotides, primary constructs, and mmRNAs of the invention The polynucleotides, primary constructs, and mmRNAs of the invention are, in preferred embodiments, avoiding harmful biological responses such as immune responses and / or degradation pathways or Evasion, overcoming expression thresholds and / or improving protein production capacity, improved expression rate or translation efficiency, improved drug or protein half-life and / or protein concentration, optimized protein localization Provides stability and / or clearance in tissues, uptake and / or kinetics by receptors, cell arrival by composition, involvement with translational mechanisms, secretory efficiency (if applicable), reachability to blood circulation. It is designed to improve sex and / or one or more of the regulation of cell status, function, and / or activity.

Therapeutic Agents Therapeutic agents The polynucleotides, primary constructs, or mmRNAs of the invention, such as modified nucleic acids and modified RNAs, described herein, and proteins translated from them, may be used as therapeutic or prophylactic agents. can. They are provided for use in medicine. For example, the polynucleotides, primary constructs, or mmRNAs described herein can be administered to a subject, and the polynucleotides, primary constructs, or mmRNAs are translated in vivo and therapeutically or prophylactically translated in the subject. Produces a polypeptide. Compositions, methods, kits, and reagents for the diagnosis, treatment, or prevention of diseases or conditions in humans and other mammals are provided. The active therapeutic agent of the present invention comprises a polynucleotide, a primary construct, or a cell containing a polynucleotide, a polynucleotide, a primary construct, or mmRNA, or a polypeptide translated from a polynucleotide, a primary construct, or mmRNA.

In certain embodiments, one or more polynucleotides comprising a translatable region encoding a protein (s) that boost immunity in a mammalian subject, as well as a protein that induces antibody-dependent cellular cytotoxicity. Combination therapeutic agents containing primary constructs, or mRNAs, are provided herein. For example, therapeutic agents containing one or more nucleic acids encoding trastuzumab and granulocyte colony stimulating factor (G-CSF) are provided herein. Specifically, such combination therapeutics are useful in Her2 + breast cancer patients who develop induced resistance to trastuzumab. (See, for example, Albrecht, Immunotherapy. 2 (6): 795-8 (2010)).

Provided herein are methods of inducing translation of a recombinant polypeptide within a cell population using the polynucleotides, primary constructs, or mMVs described herein. Such translations can be in vivo, ex vivo, in culture, or in vitro. The cell population is contacted with an effective amount of composition containing a nucleic acid having a translatable region encoding at least one nucleoside modification and recombinant polypeptide. Populations are contacted under conditions such that the nucleic acid is localized within one or more cells of the cell population and the recombinant polypeptide is translated from the nucleic acid within the cell.

An "effective amount" of the composition is provided, at least in part, based on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid (eg, size, and degree of modified nucleoside), and other determinants. Will be done. In general, an effective amount of the composition provides efficient, preferably more efficient protein production than the composition containing the corresponding unmodified nucleic acid in the cell. Increased efficiency includes increased cell transfection (ie, percentage of cells transfected with nucleic acid (%)), increased protein translation from nucleic acid, decreased nucleic acid degradation (eg, increased protein translation from modified nucleic acid). It can be demonstrated by its duration) or by the reduced natural immune response of the host cell.

Aspects of the invention are directed to methods of inducing in vivo translation of recombinant polypeptides in mammalian subjects in need. Among them, an effective amount of a composition containing a nucleic acid having a translatable region encoding at least one structural modification or chemical modification and a recombinant polypeptide was subjected to a subject using the delivery method described herein. Be administered. The nucleic acid is provided in an amount and under other conditions such that the nucleic acid is localized in the cell of interest and the recombinant polypeptide is translated from the nucleic acid in the cell. The cell in which the nucleic acid is localized, or the tissue in which the cell resides, can be targeted by administration of the nucleic acid for one or more cycles.

In certain embodiments, the polynucleotide, primary construct, or mMV administered provides functional activity that is substantially absent in the cell, tissue, or organism to which the recombinant polypeptide is translated. Directs the production of one or more recombinant polypeptides. For example, the deficient functional activity can have the properties of enzymatic, structural, or gene regulatory activity. In a related embodiment, the polynucleotide, primary construct, or mm mRNA administered increases functional activity that is present in the cell to which the recombinant polypeptide is translated but is substantially deficient (eg,). , Synergistically) directs the production of one or more recombinant polypeptides.

In other embodiments, the polynucleotide, primary construct, or mMV administered replaces one polypeptide (or plurality of polypeptides) that is substantially absent in the cell into which the recombinant polypeptide is translated. Directs the production of one or more recombinant polypeptides. Such absence may be due to a gene mutation in the coding gene or its regulatory pathway. In some embodiments, the recombinant polypeptide increases the level of the endogenous protein in the cell to a desired level, such an increase from a subnormal level to a normal level. And, or can go from a normal level to an overnormal level.

Alternatively, the recombinant polypeptide functions to antagonize the activity of endogenous proteins present in the cell, on the top surface, or secreted by the cell. Usually, the activity of an endogenous protein is detrimental to the subject, for example, due to mutations in the endogenous protein resulting in altered activity or localization. In addition, recombinant polypeptides directly or indirectly antagonize the activity of biological parts present within the cell, on the top surface, or secreted by the cell. Examples of biological parts to be antagonized include lipids (eg, cholesterol), lipoproteins (eg, low specific gravity lipoproteins), nucleic acids, carbohydrates, protein toxins such as Shiga toxin and tetanus toxin, or botulinum toxin, cholera toxin, and Examples include small molecule toxins such as diphtheria toxin. In addition, the biological molecule to be antagonized may be an endogenous protein exhibiting undesired activity such as cytotoxic activity or cell proliferation inhibitory activity.

The recombinant proteins described herein may be engineered for localization intracellularly, possibly within specific compartments such as the nucleus, or secreted from cells or plasma membranes of cells. Manipulated for the transition to.

In some embodiments, the modified mRNAs according to the invention and their encoded polypeptides are any of a variety of diseases, disorders, and / or conditions including, but not limited to, one or more of the following: May be used therapeutically: autoimmune disorders (eg, diabetes, ulcer, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (eg, arthritis, pelvic inflammatory disease); infectious diseases (eg, eg) Viral infections (eg HIV, HCV, RSV), bacterial infections, fungal infections, septicemia; neuropathy (eg Alzheimer's disease, Huntington's disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (eg atherosclerotic arteries) Yellow spot degeneration such as sclerosis, hypercholesterolemia, thrombosis, coagulation disorder, angiogenesis disorder); proliferative disorder (eg, cancer, benign neoplasm); respiratory disorder (eg, chronic obstructive pulmonary disease); digestive organs Disorders (eg, inflammatory bowel disease, gastric ulcer); Musculoskeletal disorders (eg, fibromyalgia, arthritis); Endocrine, metabolic, and nutritional disorders (eg, diabetes, osteoporosis); Urological disorders (eg, renal disease); Mental disorders (eg, depression, schizophrenia); skin disorders (eg, wounds, eczema); blood and lymphatic disorders (eg, anemia, hemophilia), etc.

Diseases characterized by dysfunctional or abnormal protein activity include cystic fibrosis, sickle cell anemia, epidermolysis bullosa, amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenase deficiency. Includes illness. The present invention provides a method for treating such a condition or disease in a subject by introducing a nucleic acid or cell-based therapeutic agent containing the polynucleotide, primary construct, or mmRNA provided herein. However, this polynucleotide, primary construct, or nucleic acid encodes a protein that antagonizes or overcomes the aberrant protein activity present in the cells of interest. A specific example of a dysfunctional protein is a missense mutation variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene that produces a dysfunctional protein variant of the CFTR protein that causes cystic fibrosis. Can be mentioned.

Diseases characterized by deficient (or substantially reduced normal or physiological protein function) protein activity include cystic fibrosis, Niemann-Pick disease type C, and severe β Includes thalassemia, Duchenne muscular dystrophy, Harler syndrome, Hunter syndrome, and hemophilia A. Such proteins may be absent or are essentially non-functional. The present invention is described herein. A method for treating such a condition or disease in a subject by introducing a nucleic acid or cell-based therapeutic agent containing the polynucleotide, primary construct, or mMV provided in the book provides a method for treating such a condition or disease in a subject. The primary construct, or mmRNA, replaces the protein activity deficient in the target cell of interest. Specific examples of dysfunctional proteins produce non-functional protein variants of the CFTR protein that cause cystic fibrosis. , Nonsense mutant variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Therefore, the cells of interest are contacted with a polynucleotide, primary construct, or mmRNA having a translatable region encoding a functional CFTR polypeptide under conditions such that an effective amount of CTFR polypeptide is present in the cell. Provides a method of treating cystic fibrosis in mammalian subjects. Preferred target cells are epithelial, endothelial, and mesothelial cells such as lung, and the method of administration is determined in consideration of the target tissue, i.e., for lung delivery, RNA molecules are formulated for administration by inhalation. Will be done.

In another embodiment, the invention is introduced into a cell population of interest with a modified mRNA molecule encoding a protein, Sortilin, recently characterized by genomic studies, thereby ameliorating hyperlipidemia in the subject. To provide a method for treating hyperlipidemia in a subject. The SORT1 gene encodes a trans-Golgi network (TGN) transmembrane protein called Sortilin. Genetic studies have shown that one in five individuals predispose to low levels of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) at the 1p13 locus of the SORT1 gene, rs12740374. It shows that it has. Each copy of this minor allele present in about 30% of people alters LDL cholesterol by 8 mg / dL, while two copies of this minor allele present in about 5% of the population change LDL cholesterol. Decrease 16 mg / dL. Carriers of this minor allele have also been shown to have a 40% reduced risk of myocardial infarction. Functional in vivo studies in mice have shown that overexpression of SORT1 in mouse liver tissue results in significantly lower LDL-cholesterol levels, up to less than 80%, and that silencing of SORT1 increases LDL cholesterol by approximately 200%. (Musunuru K et al. From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466: 714-721).

In another embodiment, the invention is for treating hematopoietic disorders, cardiovascular disorders, oncology, diabetes, cystic fibrosis, neurological disorders, congenital metabolic disorders, skin and systemic disorders, and blindness. Provide a method. Entirety by molecular targets for the treatment of these specific diseases have been described (reference is incorporated herein, Templeton ed, Gene and Cell Therapy :. Therapeutic Mechanisms and Strategies, 3 rd Edition, Bota Raton , FL: CRC Press).

Methods for preventing infection and / or sepsis in subjects at risk of developing infection and / or sepsis are provided herein, and the methods provide infection and / or methods for subjects in need of such prevention. / Or in an amount sufficient to prevent sepsis, an antibacterial polypeptide (eg, an antibacterial polypeptide), or a polynucleotide, primary construct, or mRNA precursor encoding a partially or fully processed form thereof. Includes administration of a composition comprising the body. In certain embodiments, the subject at risk of developing infection and / or sepsis may be a cancer patient. In certain embodiments, cancer patients may have undergone a pretreatment regimen. In some embodiments, regiments include, but are not limited to, chemotherapy, radiation therapy, or both. As a non-limiting example, a polynucleotide, primary construct, or mmRNA may encode protein C, its tymogen or preproprotein, or an activated form of protein C (APC) or a variant of protein C, which are the same. Known in the technical field. Polynucleotides, primary constructs, or mmRNAs may be chemically modified and delivered to cells. Non-limiting examples of polypeptides that can be encoded within chemically modified mRNAs of the invention are U.S. Pat. Nos. 7,226,999, the same, each of which is incorporated herein by reference in its entirety. Those taught in Nos. 7, 498, 305 and Nos. 6, 630, 138 can be mentioned. These patents teach protein C-like molecules, variants, and derivatives, any of which can be encoded within the chemically modified molecules of the invention.

Further provided herein are methods for treating infection and / or sepsis in a subject, the method being sufficient to treat infection and / or sepsis in a subject in need of such treatment. And an antibacterial polypeptide (eg, an antibacterial polypeptide), eg, an antibacterial polypeptide described herein, or a polynucleotide, a primary construct, or a primary construct encoding a partially or fully processed form thereof. Includes administration of a composition comprising an mmRNA precursor. In certain embodiments, the subject in need of treatment is a cancer patient. In certain embodiments, the cancer patient has undergone a pretreatment regimen. In some embodiments, regiments include, but are not limited to, chemotherapy, radiation therapy, or both.

In certain embodiments, the subject may exhibit an acute or chronic microbial infection (eg, a bacterial infection). In certain embodiments, the subject may or may have been treated. In certain embodiments, treatment may include, but is not limited to, radiation therapy, chemotherapy, steroids, UV radiation, or a combination thereof. In certain embodiments, the patient may suffer from microangiopathy. In some embodiments, the microangiopathy can be diabetes. In certain embodiments, the patient may have a wound. In some embodiments, the wound can be an ulcer. In a specific embodiment, the wound can be a diabetic foot ulcer. In certain embodiments, the subject may have one or more burn wounds. In certain embodiments, administration may be local or systemic. In certain embodiments, administration may be subcutaneous. In certain embodiments, administration may be intravenous. In certain embodiments, administration may be oral. In certain embodiments, administration may be topical. In certain embodiments, administration may be by inhalation. In certain embodiments, the administration may be rectal. In certain embodiments, administration may be intravaginal.

Another aspect of the disclosure relates to transplantation of cells containing a polynucleotide, primary construct, or mmRNA into a mammalian subject. Administration of cells to mammalian subjects is known to those skilled in the art and includes topical (topical) transplantation (eg, topical or subcutaneous administration), organ delivery or systemic implantation (eg, intravenous injection or inhalation), And including, but not limited to, the preparation of cells in a pharmaceutically acceptable carrier. Such compositions containing polynucleotides, primary constructs, or mmRNAs are intramuscular, transarterial, intraperitoneal, intravenous, intranasal, subcutaneous, endoscopic, and transdermal. It can be formulated for administration either, or intrathecally. In some embodiments, the composition may be formulated for sustained release.

Subjects to whom the therapeutic agent can be administered may suffer from or be at risk of developing a disease, disorder, or adverse condition. Based on these, methods of identifying, diagnosing, and classifying subjects are provided, including clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art. good.

Rare liver disease or liver damage Progressive familial intrahepatic cholestasis (PFIC)
In one embodiment, the rare liver disease or disorder polynucleotides, primary constructs or mmRNAs of the invention can be used to treat progressive familial intrahepatic cholestasis (PFIC). As used herein, the term "progressive familial intrahepatic cholestasis" or "PFIC" refers to liver damage that can lead to liver failure. PFIC is characterized by autosomal recessive bile production defects and hepatocellular cholestasis. As used herein, "hepatocyte" is a term used to describe things that affect or relate to hepatocytes (also called hepatocytes). As used herein, the term "cholestasis (symptom)" refers to a condition characterized by slowing or impeding the flow of bile from the liver. As used herein, the term "bile" refers to a liquid substance produced by the liver that contains water, bile salts, mucus, fat, inorganic salts and cholesterol and aids in the emulsification and digestion of dietary fat.

Three types of PFIC are known (PFIC-1, PFIC-2 and PFIC-3), all of which are caused by mutations in genes encoding proteins involved in the hepatocyte transport system and bile production. It is said that.

PFIC-1 and PFIC-2 are usually diagnosed in early childhood, but may also be diagnosed in early birth or neonatal period. As used herein, the term "prenatal" refers to a period of the life of an organism before birth. As used herein, the term "newborn" refers to a period of life of an organism after birth. In humans, the neonatal period can include the period from birth to about 1 month, about 3 months or about 6 months after birth. As used herein, the term "infant" refers to a period of life of an organism from birth to childhood. In humans, early childhood can include the period from birth to about 1 year, about 2 years, about 3 years or about 4 years.

PFIC-3 can be diagnosed in early birth, neonatal period, or early childhood. In some cases, PFIC-3 may escape diagnosis until childhood or adolescence. As used herein, the term "childhood" refers to the period of an organism's life from infancy to adolescence. In humans, childhood is from about 2 years to about 10 years, from about 3 years to about 11 years, from about 4 years to about 12 years, or from about 5 years to about 13 years. May include a period. As used herein, the term "adolescence" refers to the period of life of an organism from childhood to adulthood. In humans, adolescence is from about 10 years to about 16 years, from about 11 years to about 17 years, from about 12 years to about 18 years, from about 13 years to about 19 years. It may include periods from about 14 years old to about 20 years old.

Clinical symptoms of PFIC include, but are not limited to, pruritus, cholestasis and jaundice. As used herein, the term "itch" refers to an unpleasant sensation that creates the urge to scratch or rub the affected area of the body. As used herein, the term "jaundice" refers to a condition characterized by yellowed skin, eyeballs and / or mucous membranes due to the accumulation of bilirubin.

Most patients with PFIC develop fibrosis by adulthood and develop liver failure. Individuals affected by PFIC can be diagnosed by clinical symptom observation, cholangiography, hepatic ultrasonography, hepatic histology examination, and genetic testing. Other tests may be done to rule out other disorders that cause cholestasis in children.

Failure of one of three different cell transporters results in the development of PFIC types 1, type 2, and type 3, respectively. Each cell transporter is involved in lipid transport, and each plays a vital role in bile secretion from the liver. Type PFIC1 is due to a genetic mutation that causes a dysfunction of the ATPase aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1). ATP8B1 functions to transfer phosphatidylserine and phosphatidylethanolamine from one side of the phospholipid bilayer to the other. Both PFIC-2 and PFIC-3 are caused by mutations that affect the function of ATP-binding cassette (ABC) transporters. The ABC, subfamily B, member 11 (ABCB11) transporter assists bile production by transporting cholic acid-binding compounds, including taurocholic acid, from hepatocytes into bile. Deficiency in ABCB11 function leads to PFIC-2. ABC, subfamily B, member 4 (ABCB4) transports phospholipids, preferably phosphatidylcholine, across the hepatic cell membrane for uptake into bile. A genetic mutation that causes ABCB4 dysfunction causes PFIC-3 (Davit-Spral et al., Progressive familial intrahepatic cholestasis. Orphanet JRare).
Dis. 2009 Jan 8; 4: 1; Desiorgio, D. et al. Et al., Molecular characterization and conditional impedances of 25 new ABCB4 mutations in Progressive femilial intrahepatic chocolate type 3 (PFIC). Eur J Hum Genet. 2007 Dec; 15 (12): 1230-8; each of which is incorporated herein by reference in its entirety).

In one embodiment, a patient with PFIC may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or liver damage polypeptides, primary constructs or mmRNAs are, but are not limited to, ATP-binding cassettes, subfamilies (MDR / TAP), member 4 (ABCB4), ATP-binding cassettes, subfamilies (MDR / TAP). , Member 11 (ABCB11) and ATPase, aminophopholipid transporter, class I, type 8B, peptides such as member 1 (ATP8B1), proteins or fragments thereof.

In one embodiment, PFIC-1 is administered by administering a composition of the invention comprising at least one ATP8B1 peptide, protein or fragment thereof, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. Can be treated. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NO: 855 or 856, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. This can treat PFIC-1.

In one embodiment, PFIC-2 is administered by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof of ABCB11. Can be treated. In another embodiment, PFIC-2 is administered by administering a composition of the invention comprising a peptide comprising SEQ ID NO: 865, a rare liver disease or injury polynucleotide encoding a protein or fragment thereof, a primary construct or mmRNA. Can be treated.

In one embodiment, PFIC-3 is administered by administering a composition of the invention comprising at least one polynucleotide, primary construct or mmRNA of a rare liver disease or disorder encoding a peptide, protein or fragment thereof of ABCB4. Can be treated. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NOs: 866-871, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. This can treat PFIC-3.

Familial hypercholesterolemia In one embodiment, familial hypercholesterolemia (FH) can be treated with the rare liver disease or disorder polynucleotides, primary constructs or mmRNAs of the invention. As used herein, the term "familial hypercholesterolemia" or "FH" refers to a hereditary disorder characterized by elevated levels of low-density lipoprotein (LDL) -related cholesterol in plasma. Patients or subjects with FH may have an increased risk of cardiovascular disease at an early age. In some embodiments, elevated blood LDL levels in such individuals may be due to mutations in the gene encoding the LDL receptor. By no means limited, it is believed that the LDL receptor binds to LDL in circulating blood and promotes endocytosis of LDL into the intracellular expression of that receptor. When this receptor becomes dysfunctional, circulating LDL levels remain elevated, promoting the development of atherosclerosis. Individuals with FH can have either heterozygous or homozygous FH-related gene mutations. Symptoms of homozygous individuals can be more severe. Diagnosis can be made in childhood or adolescence by methods known in the art, including physical examination to reveal xanthoma (growth of fat-rich skin). FH can be diagnosed at a relatively early stage by family history and genetic analysis (Sjouke, B. et al., Familial hypercholesterolemia: present and future management. Curr Cardiol Rep. 2011 Dec; 13-36): 5 Avis, HJ et al., A systematic review and meta-analysis of statin therapy in children with familial hypercholesterolemia. Incorporated into).

In one embodiment, a patient with FH may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or injury polypeptides, primary constructs or mmRNAs are, but are not limited to, low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subproteinin / kexin type 9 (PCSK9). ) And other peptides, proteins or fragments thereof.

In one embodiment, FH is treated by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mm mRNA that encodes a peptide, protein or fragment thereof of LDLR. obtain. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NO: 1145-1151, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. This can treat FH.

In one embodiment, FH is treated by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mMV encoding a peptide, protein or fragment thereof of APOB. obtain. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NO: 819 or 819, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. This can treat FH.

In one embodiment, FH is treated by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof of PCSK9. obtain. In another embodiment, the composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide, protein or fragment thereof, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA is administered. This can treat FH.

Ornithine transcarbamylase deficiency In one embodiment, ornithine transcarbamylase deficiency (OTCD) can be treated with the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. As used herein, the term "ornithine transcarbamylase deficiency" or "OTCD" refers to a hereditary disorder of urea synthesis resulting from a mutation in the gene encoding the enzyme ornithine transcarbamylase (OTC). Mutations can lead to hyperammonemia, neurological problems and increased mortality. OTC is an important component of the urea circuit and catalyzes the production of citrulin from carbamoyl phosphate and ornithine. This process is carried out by the body. It is extremely important to remove excess ammonia from the OTCD because the gene for OTC is on the X chromosome. OTCD is an X-linked disorder. Like most X-linked disorders, OTCD is predominantly male. Affected and females become carriers. The severity of OTCD depends on the nature of the genetic mutation. Genetic mutations that result in non-functional OTC usually cause the individual to die within the first month of life. Such mutations can be detected by genetic analysis of individuals suspected of having OTCD. Individuals with genetic mutations that provide varying degrees of enzymatic function have a relatively long survival time. Treatments that reduce the effects of the disease may be effective. OTCD can be diagnosed after observing symptoms and detecting high levels of ammonia and ornithine in the urine (Brunetti-Pierri, N. et al., Et al. Phenotypic direction of ornithine transcarbamylase deficiency using low dose helper-dependent genetic vectors. J Gene Med. 2008
Aug; 10 (8): 890-6; Wilmslow, U.S.A. K. , Ornithine
transcarbamyrace defecty: urea cycle defect. Eur J Paediator Neurol. 2003; 7 (3): 115-21; each in its entirety is incorporated herein by reference).

In one embodiment, a patient with OTC may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or damage polynucleotides, primary constructs or mmRNAs can encode peptides, proteins or fragments thereof, such as, but not limited to, ornithine carbamoyl transferase (OTC).

In one embodiment, the OTCD is treated by administering a composition of the invention comprising at least one of a rare liver disease or disorder polynucleotide, a primary construct or mm mRNA that encodes an OTC peptide, protein or fragment thereof. obtain. In another embodiment, by administering a composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide comprising SEQ ID NO: 1192, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. OTCD can be treated.

Crigler-Najer Syndrome In one embodiment, the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention can be used to treat Crigler-Najer syndrome. As used herein, the term "Crigler-Najer syndrome" refers to a congenital anomaly that affects the function of the 1A1 isoform (UGT1A1) of the enzyme bilirubin-uridine diphosphate glucuronosyltransferase. UGT1A1 is extremely important for the detoxification of hydrophobic bilirubin, a toxic heme-degrading by-product. UGT1A1 is believed to function by binding hydrophobic bilirubin and glucuronic acid and excreting it in the bile. Individuals suffering from Crigler-Najer syndrome develop unconjugated hyperbilirubinemia (or excess hydrophobic bilirubin in the circulating blood), resulting in neurological disorders and benefit with aging of the patient. Decreased regular phototherapy may be required. Diagnosis of Crigler-Najer syndrome usually begins with the observation of jaundice in infants, and then enzyme function can be assessed by enzymes known in the art and liver assays (Lysy, PA et al., Live cell transplantation for Crigler). -Najjar syndrome type I: update and perspectives.World J Gastroenterol.2008 Jun 14; 14 (22): 3464-70; Sugatani, J., Function, genetic polymorphism, and transcriptional regulation of human UDP-glucuronosyltransferase (UGT) 1A1. Drag Metab Pharmacokine. 2012 Oct 23. [Pre-Print Electronic Publishing]; each of which is incorporated herein by reference in its entirety).

In one embodiment, a patient with Crigler-Najer syndrome may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs can encode peptides, proteins or fragments thereof, such as, but not limited to, UGT1A1.

In one embodiment, Crigler-Najer syndrome by administering a composition of the invention comprising at least one UGT1A1 peptide, protein or fragment thereof, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. Can be treated. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NO: 1357 or 1358, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. This can treat Crigler-Najer syndrome.

Ceruloplasminemia In one embodiment, the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention can be used to treat ceruloplasminemia. As used herein, the term "ceruloplasminemia" refers to a disease in which the CP is defective and / or the CP is dysfunctional due to a genetic mutation in the ceruloplasmin (CP) gene. CP is thought to be a transport protein involved in transporting iron from cells into capillaries. Once inside the capillaries, iron can bind ferritin and enter the bloodstream. In the absence of a functioning CP, iron accumulates in the cells and serum ferritin levels increase (Ogimoto, M. et al., Criteria for early identification of aceruloplasminemia. Internet Med. 2011; 50 (13): 1415-8; Miyajima, H., Aceruloplasminemia. GeneReviews [Internet]. Seattle (WA): Universality of Washington, Seattle; 2003 Aug 12 [Updated February 17, 2011];

Iron accumulation can be particularly high in the liver as well as in the eye, brain and pancreas. Acelluloplasminemia caused by a genetic defect in the CP gene is an autosomal recessive disorder. As used herein, the term "autosomal recessive" refers to a disease, disorder, trait or phenotype in which the causative gene can affect homozygous individuals. Symptoms of this disease usually develop between the ages of 25 and 60 years. Serum analysis of ceruloplasmin and iron levels or MRI analysis of the brain to search for iron accumulation can make a diagnosis of individuals suspected of having the disease.

In one embodiment, a patient with acelluloplasminemia may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs can encode peptides, proteins or fragments thereof, such as, but not limited to, CP.

In one embodiment, ceruloplasmin-free blood by administration of a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mMV encoding a peptide, protein or fragment thereof of CP. Can treat the disease. In another embodiment, by administering a composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide containing SEQ ID NO: 891, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. Can treat ceruloplasminemia.

α-Mannose Disease In one embodiment, α-mannose disease can be treated with the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. As used herein, the term "α-mannosidase" refers to a disorder that causes lysosomal storage disease due to defective and / or dysfunctional α-D-mannosidase enzyme activity. Α-Mannosidase caused by a genetic defect in the MAN2B1 gene (encoding α-D-mannosidase) is an autosomal recessive disorder. Characteristics of persons suffering from α-mannose disease include, but are not limited to, immunodeficiency, facial and skeletal abnormalities, hearing loss and intellectual disability. Diagnosis can be made as early as early childhood using methods known in the art, including, but not limited to, phenotypic features and analysis of liver and splenic dysfunction. Relatively mild illnesses may not be diagnosed during childhood and adolescence. Diagnosis of the disease is usually confirmed by measurement of α-D-mannosidase enzyme activity in whole blood cells (Malm, D. et al., Alpha-mannosidase. Orphanet JRare).
Dis. 2008 Jul 23; 3:21; the whole of which is incorporated herein by reference).

In one embodiment, a patient with α-mannose disease may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs can encode peptides, proteins or fragments thereof, such as, but not limited to, MAN2B1.

In one embodiment, α-mannose disease by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA encoding a peptide, protein or fragment thereof of MAN2B1. Can be treated. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NOs: 1156 to 1158, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. This can treat α-mannose disease.

Tyrosinemia In one embodiment, the rare liver disease or disorder polynucleotides, primary constructs or mmRNAs of the invention can be used to treat tyrosinemia. As used herein, the term "tyrosinemia" refers to disorders and / or conditions characterized by elevated levels of blood tyrosine and / or tyrosine by-products. In some embodiments, tyrosinemia is due to a genetic mutation that results in a defect and / or dysfunction of the enzyme required for tyrosine degradation. Symptoms are not particularly limited, but are growth failure, diarrhea, vomiting, jaundice, increased epistaxis, microcephaly, tremor, ataxia, self-injury, fine coordination disorder, speech disorder, spasm and / or A cabbage-like odor can be mentioned (Nakamura, K. et al., Animal models of tyrosinemia. J Nutr. 2007 Jun; 137 (6 Suppl 1): 1556S-1560S; discussion 1573S-1575S; ? endoplasmic reticulum stress disorder Med Sci (Paris) .2003 Oct; 19 (10):. 976-80; Mehere, P, et al., Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations.Protein Cell.2010 Nov; 1 (11 ): 1023-32; each of which is incorporated herein by reference in its entirety).

Type 1 tyrosinemia refers to a disordered form caused by a lack of fumarylacetoacetic acid hydrolase (FAH) activity. The lack of FAH activity in type 1 tyrosinemia causes the accumulation of the metabolite fumarylacetoacetic acid, which causes cell activity such as apoptosis, mutagenesis, aneuploidy and mitosis.

Type 2 tyrosinemia refers to a disordered form caused by a lack of tyrosine aminotransferase (TAT) activity. TAT is involved in the reversible transamination of tyrosine and other aromatic amino acids, including but not limited to p-hydroxyphenylpyruvic acid (pHPP).

In one embodiment, a patient with tyrosinemia may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs can encode peptides, proteins or fragments thereof, such as, but not limited to, FAH or TAT.

In one embodiment, type 1 tyrosinemia is administered by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or mMV encoding a peptide, protein or fragment thereof of FAH. Can treat the disease. In another embodiment, type 1 tyrosinemia is administered by administering a composition of the invention comprising at least one of the rare liver disease or liver damage polynucleotides encoding a peptide, protein or fragment thereof comprising SEQ ID NO: 985-987. Can treat bloodemia.

In one embodiment, type 2 tyrosine blood is administered by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or m mRNA encoding a peptide, protein or fragment thereof of TAT. Can treat the disease. In another embodiment, by administering a composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide containing SEQ ID NO: 1356, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. Can treat type 2 tyrosinemia.

Hemochromatosis In one embodiment, hemochromatosis can be treated with the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. As used herein, the term "hemochromatosis" refers to a disorder or condition characterized by iron excess that can result from a genetic defect. Symptoms of hemochromatosis include, but are not limited to, liver cirrhosis, hyperpigmentation, hypopituitary function, diabetes and / or arthritis. The genetic defect that causes hemochromatosis is used to characterize the type of disease. Hemochromatosis type 1 is caused by a gene mutation in the HFE gene. Types 2A and 2B are due to mutations in the HFE2 and HAMP genes, respectively. Symptoms of hemochromatosis type 1 are usually seen until adulthood, whereas symptoms of hemochromatosis types 2A and 2B are usually seen in childhood (Papanikolaou, G. et al., Hepcidin).
in iron overload disorders. Blood. 2005 May 15; 105 (10): 4103-5; Nanda, W. et al. Et al., HFE gene
variants affect iron in the brain. J Nutr. 2011 Apr 1; 141 (4): 729S-739S; Wallace, D.I. F. Et al., Non-HFE hemochromatosis. World J Gastroenterol. 2007 Sep 21; 13 (35): 4690-8; each in its entirety is incorporated herein by reference).

In one embodiment, a patient with hemochromatosis may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or injury polynucleotides, primary constructs or mmRNAs are not particularly limited, but are peptides, proteins such as HFE2, HAMP, solute transporter family 40, member 1 (SLC40A1) and transferrin receptor 2 (TFR2). Or it can code for that fragment.

In one embodiment, hemochromatosis 1 by administering a composition of the invention comprising at least one HFE peptide, protein or fragment thereof, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. The mold can be treated. In another embodiment, the composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide containing SEQ ID NO: 1038-1050, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA is administered. This can treat hemochromatosis type 1.

In one embodiment, hemochromatosis is administered by administering a composition of the invention comprising at least one HFE2 or HAMP peptide, protein or fragment thereof, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA. Tosis type 2 can be treated. In another embodiment, the composition of the invention comprises at least one peptide, protein or fragment thereof encoding a peptide, protein or fragment thereof, a rare liver disease or disorder polynucleotide, a primary construct or m mRNA comprising SEQ ID NO: 1051-1057, 1067 or 1068. Hemochromatosis type 2 can be treated by administering the substance.

In one embodiment, hemochromatosis is administered by administering a composition of the invention comprising at least one TFR2 or SLC40A1 peptide, a rare liver disease or liver disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mRNA. Can treat tosis. In another embodiment, the composition of the invention comprises at least one peptide, protein or fragment thereof encoding a peptide containing SEQ ID NO: 1290 to 1296 or 1323-1326, a rare liver disease or disorder polynucleotide, primary construct or mmRNA. Hemochromatosis can be treated by administering the substance.

Glycogenosis In one embodiment, the rare liver disease or hepatic disorder polynucleotides, primary constructs or mmRNAs of the invention can be used to treat type IV glycogenosis. As used herein, the term "glycogen storage disease" or "GSD" refers to a disorder of an organism characterized by defects in glycogen synthesis and / or degradation. In some embodiments, GSD includes, but is not limited to, type 4 GSD, also known as Anderson's disease, branching enzyme deficiency, amylopectinosis and glycogen branching enzyme deficiency. Type IV GSD results from a deficiency of the glycogen branching enzyme (GBE) encoded by the GBE1 gene, causing the production of abnormal glycogen that can be cytotoxic. Patients with classical liver type IV GSD appear healthy at birth, although there are variations due to the expression of tissue-specific isoforms, but cirrhosis develops shortly after birth and usually develops liver failure by 5 months of age ( Ozen, H., Glycogen liver diseases: new perceptives. World J Gastroenterol. 2007 May 14; 13 (18): 2541-53; the entire body is incorporated herein by reference).

In one embodiment, a patient with GSD may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or damage polynucleotides, primary constructs or mmRNAs encode peptides, proteins or fragments thereof, such as, but not limited to, glucan (1,4-α-), branching enzyme 1 (GBE1). Can be.

In one embodiment, type IV GSD is administered by administering a composition of the invention comprising at least one of a rare liver disease or liver disorder polynucleotide, primary construct or m mRNA encoding a peptide, protein or fragment thereof of GBE1. Can be treated. In another embodiment, the composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide containing SEQ ID NO: 1010-1012, a rare liver disease or liver disorder polynucleotide, primary construct or mmRNA is administered. This can treat type IV GSD.

Funconicystinosis In one embodiment, funconicystinosis can be treated with the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. As used herein, the term "cystinosis" refers to a disease characterized by the abnormal accumulation of cystine in lysosomes. Renal disease, which inhibits the reabsorption of metabolites into the bloodstream and instead allows them to pass through the urine, is the most common cause of Funconi syndrome. Cystine is produced via a disulfide bond between two cysteine residues. Lysosomes are the sites where proteins are digested by hydrolysis, and cystine accumulation within lysosomes depends on transporter-mediated transport that releases cystine. The cystinosin encoded by the CTNS gene is a 7-transmembrane protein that binds to cystine within lysosomes and works with other proteins to promote cystine removal from lysosomes. Individuals with severe deficiency in cystinosin function suffer between 6 and 12 months of age, resulting in renal and metabolic complications, including loss of water and electrolytes. Renal failure is usually present by the age of 10. Currently used treatments are cysteamine, a drug that can cleave cystine molecules and remove them from lysosomes (Kalatzis, V. et al., Cystinosis: from gene to disease. Nephrol Dial Transplant. 2002 Nov. 17 (11): 1883-6; the entire body is incorporated herein by reference).

In one embodiment, a patient with hemochromatosis may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or damage polynucleotides, primary constructs or mmRNAs can encode peptides, proteins or fragments thereof, such as, but not limited to, cystinocin, lysosomal cysteine transporter (CTNS).

In one embodiment, funconicystinosis is caused by administration of a composition of the invention comprising at least one of a rare liver disease or disorder polynucleotide, a primary construct or mmRNA that encodes a peptide, protein or fragment thereof of CTNS. Can be treated. In another embodiment, the composition of the invention comprising at least one peptide containing SEQ ID NOs: 938-941, a rare liver disease or disorder polynucleotide encoding a protein or fragment thereof, a primary construct or mm mRNA is administered. By doing so, funconicystinosis can be treated.

Farber Fat Granulomatosis In one embodiment, the rare liver disease or liver damage polynucleotides, primary constructs or mMVs of the present invention can be used to treat Farber fatty granulomatosis. As used herein, "Farber lipogranulomatosis" or "Farber's lipogranulomatosis" or "Farber's disease" refers to lysosomal storage disease identified by Sidney Farber in 1957. The disease is caused by a defect in the acid ceramidese, an enzyme encoded by the N-acylsphingosine amide hydrolase (acidic ceramidese) 1 (ASAH1) gene. When this enzyme is deficient, intracellular ceramide is not normally decomposed into sphingosine and fatty acids, and abnormal accumulation of ceramide occurs, resulting in disease symptoms. Symptoms are usually seen early in early childhood, but may be seen later. Typical forms of the disease develop symptoms within the first few weeks of life, with joint malformations, the presence of subcutaneous nodules, and hoarseness. Patients may also have other neurological disorders and usually die in early childhood. Patients with mild neurological disorders may survive up to their 40s. There is currently no treatment available for Farber lipogranulomatosis (Ekici, B. et al., Farber disease: A pediatric diagnosis. J Pediatric Neurosci. 2012 May; 7 (2): 154-5; Farber, S. et al. Et al., Lipogranulomatosis; a new lipo-glycoprotein therapy diagnosis. J Mt Sinai Hosp N Y. 1957 Nov-Dec; 24 (6): 816-37; each of which is incorporated herein by reference in its entirety).

In one embodiment, a patient with hemochromatosis may be administered a composition comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs of the invention. Rare liver disease or liver damage polynucleotides, primary constructs or mMVs encode peptides, proteins or fragments thereof, such as, but not limited to, N-acylsphingosine amidohydrolase (acidic ceramidase) 1 (ASAH1). could be.

In one embodiment, Faber fatty granulomatosis by administering a composition of the invention comprising at least one of the rare liver disease or liver damage polynucleotides, primary constructs or mmRNAs encoding a peptide, protein or fragment thereof of ASAH1. Can treat the disease. In another embodiment, a composition of the invention comprising at least one peptide, protein or fragment thereof encoding a peptide containing SEQ ID NO: 1171-1175, a rare liver disease or disorder polynucleotide, a primary construct or mm mRNA is administered. By doing so, funconicystinosis can be treated.

Wound Management The polynucleotides, primary constructs, or mMVs of the present invention may be used in wound healing, eg, in the treatment of wounds that exhibit delayed healing. Methods are provided herein that include administration of polynucleotides, primary constructs, or mmRNA to manage wound healing. The methods herein may further include steps performed either before, at the same time, or after administration of the polynucleotide, primary construct, or mmRNA. For example, the wound bed may need to be cleaned and prepared to facilitate wound healing and preferably obtain wound closure. Several strategies can be used to promote wound healing and achieve wound closure, including but not limited to: (i) Necrotic tissue removal (optionally repeated), sharpening. Necrotic tissue removal (surgical removal of dead or infected tissue from the wound), optionally a chemical necrotic tissue remover such as an enzyme for removing the necrotic tissue, (ii) providing a moist and warm environment for the wound Wound dressing to promote tissue repair and healing.

Examples of materials used in formulating wound dressings include, but are not limited to: hydrogels (eg, AQUASORB®; DUODERM®), hydrocolloids (eg, AQUASORB®), hydrocolloids (eg, AQUASORB®; For example, AQUACEL®; COMFEEL®), foaming material (eg, LYOFOAM®; SPYROSORB®), and alginate (eg, ALGISITE®; CURASORB®). )), (Iii) Further growth factors to stimulate cell division and proliferation and promote wound healing, eg, human recombinant platelet-derived growth approved by the FDA for the treatment of neuropathy foot ulcers. The factor, becaplermin (REGRANEX Gel®), (iv) may require a soft tissue wound dressing, i.e. a skin implant, to obtain a clean, unhealed wound dressing. Examples of skin grafts that can be used for soft tissue coating include, but are not limited to: autologous skin grafts, carcass skin grafts, bioengineered skin grafts (eg, APLIGRAF). Registered trademark); DERMAGRAFT®).

In certain embodiments, the polynucleotides, primary constructs, or mmRNAs of the invention include hydrogels (eg, AQUASORB®; DUODERM®), hydrocolloids (eg, AQUACEL®; COMFEEL). (Registered Trademark)), foam material (eg, LYOFOAM®; SPYROSORB®), and / or alginate (eg, ALGISITE®; CURASORB®) are further included. May be good. In certain embodiments, the polynucleotides, primary constructs, or mmRNAs of the invention are autologous skin grafts, carcass skin grafts, or bioengineered skin grafts (eg, APLIGRAF®; DERMAGRAFT). It may be used with skin grafts including, but not limited to, registered trademarks)). In some embodiments, the polynucleotide, primary construct, or mmRNA may be applied with a wound dressing formulation and / or a skin graft, or it may be, soaked or sprayed, etc. It may be applied separately except in a non-limiting method.

In some embodiments, the composition for wound management comprises an antibacterial polypeptide (eg, an antibacterial polypeptide) and / or a polynucleotide encoding an antiviral polypeptide, a primary construct, or an mRNA. But it may be. Precursors of antibacterial polypeptides or partially or fully processed forms may be encoded. The composition may be formulated for administration with a bandage (eg, adhesive bandage). Antibacterial and / or antiviral polypeptides may be intermixed with the dressing composition or may be applied separately, eg, by dipping or spraying.

Production of Antibodies In one embodiment of the invention, polynucleotides, primary constructs, or mmRNAs can encode antibodies and fragments of such antibodies. These may be produced by any one of the methods described herein. Antibodies may be any of different subclasses or isotypes of immunoglobulins, or any of the other subclasses, such as, but not limited to, IgA, IgG, or IgM. Exemplified antibody molecules and fragments that can be prepared in accordance with the present invention include, but are not limited to, immunoglobulin molecules, substantially intact immunoglobulin molecules, and portions of immunoglobulin molecules that may contain paratopes. Such moieties of the antibody that may contain the paratope include, but are not limited to, Fab, Fab', F (ab') ₂ , F (v) and moieties known in the art.

The polynucleotides of the invention encode variant antibody polypeptides that can have certain identities with the reference polypeptide sequence or have binding characteristics that are similar or dissimilar to the reference polypeptide sequence. Can be.

The antibody obtained by the method of the present invention may be a chimeric antibody containing a non-human antibody-derived variable region (s) sequence derived from an immunized animal and a human antibody-derived constant region (s) sequence. .. In addition, they are also humanized antibodies, including the complementary determination region (CDR) of non-human antibodies derived from immunized animals and the framework region (FR) and constant region derived from human antibodies. obtain. In another embodiment, the methods provided herein can be useful for improving the yield of antibody protein products in the cell culture process.

Controlling Infection In one embodiment, by administering a polynucleotide, a primary construct, or mmRNA encoding an antibacterial polypeptide, the subject is associated with a microbial infection (eg, a bacterial infection) and / or a microbial or viral infection. A method for treating or preventing an infected disease, disorder, or condition, or its symptoms is provided. The administration may be combined with an antibacterial agent (eg, an antibacterial agent), eg, an antibacterial polypeptide or small molecule antibacterial compound described herein. Antibacterial agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents, antiparasitic agents, and antiprion agents.

The agents can be administered simultaneously, for example, in combined unit doses (eg, providing simultaneous delivery of both agents). The drug may be administered at specified time intervals, such as, but not limited to, minutes, hours, days, or weeks. In general, the agent can be biologically available, eg, detectable, in parallel in the subject. In some embodiments, the agent may be administered essentially simultaneously, eg, in two unit dosages administered simultaneously, or in combination of the two agents. In other embodiments, the drug may be delivered in separate unit dosages. The agents may be administered in any order or by one or more preparations comprising the two or more agents. In a preferred embodiment, at least one dose of one of the agents, eg, the first agent, is several minutes, 1, 2, 3, or 4 hours of the other agent, eg, the second agent. It may be done within a day or even within a day or two. In some embodiments, the combination exceeds synergistic results, eg, additive outcomes, eg, at least 25, 50, 75, 100, 200, 300, 400, or more than 500% of additive outcomes. Can be achieved.

Conditions Related to Bacterial Infection Diseases, disorders, or conditions that may be associated with bacterial infection include abscesses, necroticism, acute prostatic inflammation, Eromonas hydrophila, perennial ryegrass poisoning, charcoal scab, rod fungal purpura, fungi. Hememia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginal disease, bacterial-related skin conditions, Baltonella disease, BCG tumor, botriomise disease, botulinum disease, Brazilian purpura, Brodie abscess, Brucella disease , Bruli ulcer, campirobacta disease, caries, carion disease, cat scratch disease, bee follitis, chlamydia infection, cholera, chronic bacterial pneumonia, chronic recurrent multifocal myelitis, crustridium necrotizing enteritis, periodontal-endodontic Combined periodotic-endodontic lesions, bovine lung epidemic, diphtheria, diphtheria stomatitis, erythrosis, toxicosis, ligalottitis, toxicosis, Fitz-Hugh Curtis syndrome, flea-mediated spot fever, rotting infection Foot dermatitis), Garre sclerosing myelitis, gonorrhea, inguinal granulomas, human granulocytic anaplasmosis, human monocytic erythrocytosis, hundred days' infection, pyoderma, late congenital syphilis Sexual eye disease, regionerosis, Remière syndrome, Rye's disease (Hansen's disease), Leptospyrosis, Listeriosis, Lime's disease, lymphadenitis, nasal ulcer, meningitis bacillus disease, meningitis bacillus septicemia, methicillin-resistant yellow staphylococcus ( MRSA) infection, mycobacterial complex (MAI), mycoplasma pneumonia, necrotizing myocarditis, nocardiosis, water cancer (noma) (water cancer (cancrum)
Oris) or necrotizing stomatitis), umbilitis, orbital honeycomb inflammation, myelitis, severe infection after splenectomy (OPSI), sheep serrata, pasturella disease, periorbital honeycomb inflammation, pertussis (hooping cowh) , Pest, pneumococcal pneumonia, pot disease, rectal inflammation, pseudomonas infection, parrot disease, purulence, purulent myitis, Q fever, recurrent fever (return fever), rheumatic fever, Rocky mountain spotted fever (RMSF), rickettosis , Salmonerosis, Red fever, septicemia, Seratia infection, bacterial erythema, southern tick-associated rash illness, staphylococcal exfoliation syndrome, streptococcal pharyngitis, pool granuloma, porcine granulosis, syphilis, Plum toxic aortitis, tetanus, toxic shock syndrome (TSS), tracoma, pit fever, tropical ulcer, tuberculosis, rabbit disease, intestinal typhoid, rash typhoid, urinary tract tuberculosis, urinary tract infection, vancomycin-resistant yellow staphylococcal infection, water One or more, but not limited to, Haus-Friedrichsen syndrome, pseudotuberculosis (Elsina), and Ersina. Other diseases, disorders, and / or conditions associated with bacterial infections include, for example, Alzheimer's disease, neuropathic loss of appetite, asthma, atherosclerosis, attention deficit hyperactivity disorder, autism, autoimmune disease. , Bipolar disorder, cancer (eg, colonic rectal cancer, bile sac cancer, lung cancer, pancreatic cancer, and gastric cancer), chronic fatigue syndrome, chronic obstructive pulmonary disease, Crohn's disease, coronary heart disease, dementia, depression, Gillan Valley syndrome, visceral fat syndrome, multiple sclerosis, myocardial infarction, obesity, compulsive disorder, panic disorder, psoriasis, rheumatoid arthritis, sarcoidosis, schizophrenia, stroke, thromboangiitis obliterans (Burger's disease), and Tourette's syndrome Can include.

Bacterial Pathogens The bacteria described herein can be Gram-positive or Gram-negative. Bacterial pathogens include Acinetobacta Baumanni, Bacillus anthracis, Staphylococcus butiris, Bordetera partasis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella meritensis, Brucella Switzerland, Campylobacter jejuni, Chlamydia Pneumoniae, Chlamydia trachomatis, Cramidophila citassi, Crostridium botulinum, Crostridium difficile, Crostridium perfringens, Crostridium tetani, Coagulase-negative staphylococci, Corinebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli Sexual Escherichia coli (ETEC), enteropathogenic Escherichia coli, Escherichia coli O157: H7, Enterobacter species, Francicella staphylococcus, Hemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Regionella pneumophila, Leptospyra interrogans, Listeria monosite Genes, Moraxella catararis, Staphylococcus repre, Staphylococcus tuberculosis, Mycoplasma pneumoniae, Niseria gonoley, Niseria meningitidis, Preteus mirabilis, Proteus mirabilis Pseudomonas erginosa, liquettia liquetti, salmonella chifi, salmonella typhyllium, serratia marcesens, sigera flexneri, sigera sonei, staphylococcus aureus, staphylococcus epidelmides, staphylococcus epidelmides , Streptococcus agaractia, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema paridum, Vibrio cholerae, and Elsina pestis. Bacterial pathogens include bacteria that cause resistant bacterial infections, such as clindamycin-resistant Crostridium-difficile, fluoroquinolone-resistant Crostridium-difficile, methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Enterococcus faecalis, multidrug-resistant. Enterococcus faecium, multidrug-resistant Pseudomonas erginosa, multidrug-resistant Acinetobacter Baumanni, and vancomycin-resistant Staphylococcus aureus (VRSA) may also be included.

Antibiotic Combinations In one embodiment, the modified mRNAs of the invention can be administered in combination with one or more antibiotics. These include acnilocs, ambisome, amoxifloxacin, ampicillin, augmentin, avelox, azithromycin, bactroban, betazine, betonovet, levofloxacin, cefchlorol, cefadroxil, cefdinir, cefprozil, cefadroxil, cefdinir, cefprozil Cel Lindamycin, Clindatech, cloxacillin, colistin, cotrimoxazole, demecrocycline, diclocil, dicloxacillin, doxicycline, dulicef, erythromycin, flamazine, floxin, floxin Fucidin, fladantin, fusidic, gachifloxacin, gemifloxacin, gemifloxacin, iroson, iodo, levofloxacin, romefloxacin, maxaquin, mefomixin, mefomixin (Myambutol), mycostatin, neosporin, netromycin, nitrofrantoin, norfloxacin, norilet, floxacin, omnicef, ospamox, oxytetracycline, paraxin, paraxin, paraxin Polyfax, Povidone, Rifazine, Rifampin, Rifaximin, Rifinah, Limactan, Rosefin, Loxythromycin, Cefprozil, Soframycin, Sparfloxacin, Staflex, Tagoside Tetradox, Tetralysal, tobramycin, Tobramycin, Trecator, Taigacil, Bancosin, Velosef, Vibramycin, Xifaxan, Zagan, Zitrotek, Zoderm, Zoderm, Ziderm, Ziderm However, it is not limited to these.

Antibacterial agents Examples of antibacterial agents include aminoglycosides (eg, AMIKIN (registered trademark)), gentamycin (GARAMYCIN (registered trademark)), kanamycin (KANTREX (registered trademark)), and neomycin (MYCIFRADIN (registered trademark)). , Netyrmycin (NETROMYCIN®), Tobramycin (NEBCIN®), Palomomycin (HUMATIN®), Ansamycin (eg, geldanamycin, harbimycin), Carbacepheme (eg, LORABID (eg, LORABID) (Registered Trademarks)), Carbapenem (eg, Eltapenem (INVANZ®), Dripenem (DORIBAX®), ImiPenem / Silastatin (PRIMAXIN®), Melopenem (MERREM®), Cephalosporins Classes (1st generation) (eg, cephalosporin (DURICEF®), cephazoline (ANCEF®), cephalosporin or cephalosin (KEFLIN®), cephalexin (KEFLEX®), Cephalosporins (2nd generation) (eg, cephalosporin (CECLOR®), cephamandol (MANDOL®), cefuroxime (MEFOXIN®), cefprodil (CEFZIL®), Cefuroxime (CEFTIN (registered trademark), ZINNAT (registered trademark)), cephalosporins (3rd generation) (for example, cefuroxime (SUPRAX (registered trademark)), cefdinyl (OMNICEF (registered trademark), CEFDIEL (registered trademark)) , Cephalosporin (SPECTRACEF (registered trademark)), Cephoperazone (CEFOBID (registered trademark)), Cefotaxim (CLAFORAN (registered trademark)), Cefupodoxime (VANTIN (registered trademark)), Ceftazim (FORTAZ (registered trademark)), Ceftibten (CEDAX (registered trademark)) (Registered Trademarks)), Cefuroxime (CEFIZOX®), Ceftriaxon (ROCEPHIN®), Cephalosporins (4th generation) (eg, Cefepim (MAXIPIME®), Cephalosporins (Fifth generation) (eg, cefuroxime (ZEFTERA®), glycopeptides (eg, TARGOCID) (Registered Trademark)), Vancomycin (VANCOCIN®), Terravancin (VIBATIV®), Lincomycin (eg, CLEOCIN®), Lincomycin (LINKOCIN®), Lipopeptides (eg, daptomycin (CUBICIN®), macrolides (eg, azithromycin (ZITHROMAX®, SUMAMED®, ZITROCIN®), clarislomycin (BIAXIN®) ), Girithromycin (DYNABAC®), Erythromycin (ERYTHOCIN®, ERYTHROPED®), Loxythromycin, Trolleyandomycin (TAO®), Teristromycin (KETEK) (Registered Trademark)), Lincomycin (TROBICIN®), Monovactam (eg, Aztreonam (AZACTAM®)), Nitrofuran (eg, FUROXONE®), Nitrofurantoin (MACRODANTIN) (Registered Trademark), MACROBID (Registered Trademark)), Penicillin (eg, Amoxicillin (NOVAMOX®, AMOXIL®), Ampicillin (PRINCIPEN®), Aztreonam, Calvenicillin (GEOCILLIN®) ), Chloxacillin (TEGOPEN®), Dicloxacillin (DYNAPEN®), Fluxacillin (FLOXAPEN®), Mezrosillin (MEZLIN®), Meticillin (STAPHCILLIN®), Nafcillin (UNIPEN®), Oxacillin (PROSTAPHLIN®), Penicillin G (PENTIDS®), Penicillin V (PEN-VEE-K®), Piperacillin (PIPRACIL®) , Temocillin (NEGABAN®), Tikarcillin (TICAR®), Penicillin combination (eg, amoxicillin / clavolic acid (AUGMENTIN®), Ampicillin / sulvactam (UNASYN®), Piperacillin / Tazobactam (ZOSYN®), Tikarcillin / Clublanate (TIMENTI) N®), polypeptides (eg, bacitracin, colistin (COLY-MYCIN-S®), polymixin B, quinolones (eg, ciprofloxacin (CIPRO®, CIPROXIN®)) ), Ciprofloxacin (registered trademark)), enoxacin (PENETREX (registered trademark)), gachifloxacin (TEQUIN (registered trademark)), levofloxacin (LEVAQUIN (registered trademark)), romefloxacin (MAXAQUIN (registered trademark)), moxifloxacin Syn (AVELOX®), Naridix acid (NEGGRAM®), Norfloxacin (NOROXIN®), Ofloxacin (FLOXIN®, OCUFLOX®), Trovafloxacin (TROVAN®) (Registered Trademarks)), Grepafloxacin (RAXAR®), Sparfloxacin (ZAGAM®), Temafloxacin (OMNIFLOX®), Symphonamides (eg, SULFAMYLON®) )), Sulfonamide chrysoydin (PRONTOSIL®), sulfacetamide (SULAMYD®, BLEPH-10®), sulfaziazine (MICRO-SULFON®), silver sulfoxacin (SILVADENE®). )), THIOSULFIL
FORTE®), Sulfamethoxazole (GANTANOL®), Sulfanylimide, Sulfamethoxazole (AZULFIDINE®), Sulfamethoxazole (GANTRISIN®), Trimethoprim (PROLOPRIM) (Registered Trademarks)), TRIMPEX®), Trimethoprim-Sulfamethoxazole (Cotrimoxazole) (TMP-SMX) (BACTRIM®, SEPTRA®), Tetracycline (eg, Registered Trademarks) Demecrocycline (DECLOMYCIN®), Doxycycline (VIBRAMYCIN®), Minocycline (MINOCIN®), Oxytetracycline (TERRAMYCIN®), Tetracycline (SUMMYCIN®), ACHROMYCIN® V, STECLIN®), drugs against mycobacteria (eg, clofadimine (LAMPRENE®), Dapson (AVLOSULFON®), capreomycin (CAPASTAT®), cycloserine (SEROMYCIN) (Registered Trademarks)), Etambutol (MYAMBUTOR®), Ethionamide (TRECATOR®), Isoniazide (INH®), Pyrazineamide (ALDINAMIDE®), RIFADIN (Registered Trademarks), RIMACTANE®), Rifabutin (MYCOBUTIN®), Rifapentin (PRIFTIN®), Streptomycin), and others (eg, ALVARSAN®), Kuro Ramphenicol (CHLOROMYCETIN®), Phosphormycin (MONUROL®), Fucidic acid (FUCIDIN®), Linezolide (ZYVOX®), Metronidazole (FLAGYL®), Mupyrosine ( BACTROBAN®), platensimycin, quinupristin / dalhopristin (SYNERCID®), rifaxamine (XIFAXAN®), thianphenicol, tigecycline (TIGACYL®), tinidazole (TIN) DAMAX® (registered trademark), FASIGYN® (registered trademark), but not limited to these.

Conditions Related to Viral Infection In another embodiment, an antiviral agent, eg, a polynucleotide encoding a primary construct in combination with an antiviral polypeptide or small molecule antiviral agent described herein, or an mRNA anti. By administering a viral polypeptide, eg, an antiviral polypeptide described herein, a subject is treated with a viral infection and / or a disease, disorder, or condition associated with the viral infection, or symptoms thereof. A method of doing or preventing is provided.

Diseases, disorders, or conditions associated with viral infections include acute febrile pharyngitis, pharyngeal conjunctivitis, epidemic keratoconjunctivitis, infant gastroenteritis, coxsackie infection, infectious mononuclear disease, barkit lymphoma, acute hepatitis, chronic. Hepatitis, liver cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (eg, gingival stomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (eg, lip herpes and herpes simplex), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononuclear disease, giant cell encapsulation, Kaposi sarcoma, multicentric Castleman's disease, primary humoral lymphoma, AIDS, influenza , Rye syndrome, measles, post-infection encephalomyelitis, epidemic parotid inflammation, hyperplastic epithelial lesions (eg, vulgaris, flattened, sole and anal genital vulgaris, laryngeal papilloma, vulgar epidermis development Abnormalities), cervical cancer, herpes squamous cell carcinoma, group, pneumonia, herpes bronchitis, sickness, gray myelitis, mad dog disease, bronchitis, pneumonia, influenza-like syndrome, severe bronchitis with pneumonia, German measles, congenital Examples include, but are not limited to, sexual eczema, varicella and herpes zoster.

Viral pathogens Viral pathogens include adenovirus, coxsackie virus, dengue virus, encephalitis virus, Epstein bar virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, simple herpesvirus type 1, and simple herpesvirus type 2. , Cytomegalovirus, human herpesvirus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza, poliovirus, mad dog disease virus, respiratory polynuclear virus, wind rash virus, varicella-belly zoster Viruses, Western Nile virus, and yellow fever virus include, but are not limited to. Viral pathogens also include viruses that cause resistant viral infections.

Antiviral Agents Examples of antiviral agents include amprenavir (ZIAGEN®), abacavir / ramibdin / zidobudin (trizivir®), acyclovir or aciclovir (CYCLOVER®), HERPEX. (Registered Trademarks), ACIVIR® (Registered Trademarks), ACIVIRAX® (Registered Trademarks), ZOVIRAX® (Registered Trademarks), ZOVIR® (Registered Trademarks), Adefovir (Preveon®, Hepsera®), Amprenavir (SYMMETREL®) (Trademarks)), Amprenavir (AGENERASE®), amprenavir, Albidol, Atazanavir (REYATAZ®), Boseprevir, Sidfovir, Darnavir (PREZISTA®), RESCRIPTOR® Trademarks)), didanosin (VIDEO®), docosanol (ABREVA®), edoxdin, efavirents (SUSTIVA®, STOCRIN®), EMTRIVABIN (EMTRIVA®), EMTRIcitabine / Tenohovir / Effavirents (ATRIPLA®), Enfbiltide (FUZEON®), Entecavir (BARACLUDE®, ENTAVIR®), Famcyclovir (FAMVIR®), Homivirsen (VITRAVENE) Registered Trademarks)), Hosamprenavir (LEXIVA®, TELZIR®), Foscalnet (FOSCAVIR®), Phosphonet, Gancyclovir (CYTOVENE®, CYMEVENE®) , VITRASERT®), GS9137 (ELVITEGRAVIR®), Imikimod (ALDARA®, ZYCLARA®, BESELNA®), Indinavir (CRIXIVAN®), Inosin, Inosin Planovex (IMUNOVIR®), Type I Interferon, Type II Interferon, Type III Interferon, Kutapressin (NEXAVIR®), Lamibudin (ZEFFIX®, HEPTOVIR®, EPI) VIR (registered trademark)), ramibudine / zidovudine (COMBIVIR (registered trademark)), lopinavir, robiride, malavirok (SELZENTRY (registered trademark), CELSENTRI (registered trademark)), methisazone, MK-2048, moroxydin, nerfinavir (registered trademark). VIRACEPT®), Nevirapin (VIRAMUNE®), Osertamivir (TAMIFLU®), Peginterferon α-2a (PEGASYS®), Pencyclovir (DENAVIR®), Peramivir, Preconalyl , Podophilotoxin (CONDYLOX®), Lartegrabir (ISENTRESS®), Ribavirin (CODEGUs®, REBETOL®, RIBASPHERE®, VILONA® ANDVIRAZOLE® (Trademarks)), Ribavirin (FLUMADINE®), Ritnavir (NORVIR®), pyramidine, Sakuinavir (INVIRASE®, FORTOVASE®), stubzine, tea tree oil (Merareuka (registered trademark)) Melaleuca oil), tenohovir (VIREAD®), tenohobil / EMTRIcitabine (TRUVADA®), tipranavir (APTIVUS®), trifluridine (VIROPTIC®), tromantagine (VIRU-MERZ (registered trademark)) (Registered Trademarks)), Baracyclovir (VALTREX®), Valgancyclovir (VALCYTE®), Bicliviroc, Vidalabine, Ribavirin, Zarcitabine, Zanamivir (RELENZA®), and Zidovudine (AZT). ), RETROVIR (registered trademark), RETROVIS (registered trademark)), but is not limited thereto.

Conditions Related to Fungal Infection Diseases, disorders, or conditions associated with fungal infections include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycosis, paracoccidioidomycosis, and tinea pedis. However, it is not limited to these. In addition, people with immunodeficiency are particularly susceptible to diseases caused by fungi such as Aspergillus, Candida, Cryptococcus, Histoplasma, and Pneumocystis. Other fungi are so-called dermatophytic fungi and keratophytic fungi, which can attack the eyes, nails, hair, and especially the skin, which are diverse in which ringworms such as ringworm are common. Cause a condition. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxa can induce an allergic reaction in some people.

Fungal pathogens Fungal pathogens include Ascomycota (eg, Fusarium oxysporum, Pneumocystis girobesi, Aspergillus, Coccidiides imitis / Posadashi, Candida albicans), Microsporidia (eg, Philovaziera neoformans, Tricosporon), Microsporidia (eg, encephalitozone kunikuri, enterrositozone bienusi), and Kekabi subphylum (eg, Mucor cilcinelloides, lysopus olise, Richtheimia corymbi) Not limited to these.

Antifungal agents Examples of antifungal agents include polyene antifungal agents (eg, natamicin, rimocidin, Philippines, Nystatin, amphotelicin B, candicin, hammycin), imidazole antifungal agents (eg, miconazole (MICATIN)). (Registered Trademark), DAKTALIN (Registered Trademark)), Ketoconazole (NIZORAL® (Registered Trademark), FUNGORAL® (Registered Trademark), SEBIZOLE (Registered Trademark)), Clotrimazole (LOTRIMIN® (Registered Trademark), LOTRIMIN® AF, CANESTEN®), Econazole, Omoconazole, Bihonazole, Butconazole, Fenticonazole, Isoconazole, Oxyconazole, Sertaconazole (ERTACZO®), Sulconazole, Thioconazole), Triazole antifungal agents (eg, Albaco) Nazole, fluconazole, itraconazole, isabconazole, labconazole, posaconazole, boriconazole, terconazole), thiazole antifungal agents (eg, abafungin), allylamine (eg, tervinafin (LAMISIL®), naphthin (registered trademark)) ), Butenafin (LOTRIMIN® Ultra), Equinocandin (eg, Anidura Fungin, Caspo Fungin, Mika Fungin), and others (eg, Polygodial, benzoic acid, Cyclopyrox, Tornaftate (TINACTIN®), DESENEX®, AFTATE®), undecyleneic acid, flucitocin or 5-fluorocitosine, glyceofrubin, haloprozin, sodium hydrogencarbonate, alicin), but not limited to these.

Conditions associated with protozoan infections Diseases, disorders, or conditions associated with protozoan infections include amoeba, diardiosis, trichomonas, African sleeping sickness, American sleeping sickness, leishmaniasis (Kalaazal), babesiosis, toxoplasmosis, Malaria, Acanto amoeba keratitis, and Babesiosis include, but are not limited to.

Protozoan pathogens Protozoan pathogens include diarrhea amoeba, Giardia lambila, vaginal trypanosoma, trypanosoma crusey, trypanosoma cruzi, donovanlyshmania, balantidium coli, toxoplasma gondii, genus Plasmodium, and murine babecia. , Not limited to these.

Antiprotozoal agents Examples of antiprotozoal agents include eflornitine, furazolidone (FUROXONE®, DEPENDAL-M®), meralsoprol, metronidazole (FLAGYL®), ornidazole, paromomycin sulfate. (HUMATIN®), pentamidine, pyrimethamine (DARAPRIM®), and tinidazole (TINDMAX®, FASIGYN®), but are not limited thereto.

Conditions Related to Parasitic Infection Diseases, disorders, or conditions associated with parasitic infections include Acanto amoeba keratitis, amoeba disease, taeniasis, babesia disease, valantidiasis, baylisascariasis, shagas disease, Hepatic worm disease, cochleomiasis, cryptospolidium disease, diphyllobothriasis, medina worm disease, echinococcus disease, elephantiasis, worm disease, hepatic worm disease, hypertrophic worm disease, filariasis, diardiasis, gnathostomiasis, membranous streak Insect disease, isosporosis, Katayama fever, Leishmania disease, Lime disease, malaria, Yokogawa insect disease, fly larvae, onchocerciasis, shirami parasite, gnathostomiasis, diphyllobothriasis, sleep disease, fecal nematode disease, taeniasis , Onchocerciasis, onchocerciasis, diphyllobothriasis, and gnathostomiasis, but not limited to these.

Parasitic pathogens Parasitic pathogens include Akanto amoeba, Anisakis, roundworm, bovine fly, colon valanchedium, Nanjing worm, tapeworm, tapeworm, Cochliomyia hominivorax, diarrhea amoeba, liver worm rumble whip Mania, nasal tongue, liver sucker, loa loa, lung sucker, worm, falciparum malaria protozoa, blood-sucking worm, fecal nematode, tick, tapeworm, toxoplasma protozoa, tripanosoma, whipworm, bancroft filamentous worm. , Not limited to these.

Antiparasitics Examples of antiparasitic agents include antiprotozoals (eg, mebendazole, pyranterpamoate, thiabendazole, diethylcarbamazine, ibermectin), antiprotozoals (eg, nicrosamide, praziquantel, etc.). Albendazole), antiprotozoals (eg praziquantel), antiamoebics (eg riffampin, amphotericin B), and antiprotozoal drugs (eg mebendazole, eflornitin, metronidazole, tinidazole) However, it is not limited to these.

Creutzfeldt-Jakob disease (CJD), iatrogenic Creutzfeldt-Jakob disease (iCJD), atypical Creutzfeldt-Jakob disease (vCJD) , Familiar Creutzfeldt-Jakob disease (fCJD), sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Stroisler-Scheinker syndrome (GSS), lethal familial insomnia (FFI), Cour disease, scrapy , Bovine spongiform encephalopathy (BSE), mad cow disease, transmissive mink encephalopathy (TME), chronic wasting disease (CWD), cat spongiform encephalopathy (FSE), exogenous hoof encephalopathy (EUE), and spongy encephalopathy However, it is not limited to these.

Anti-prion agents Examples of anti-prion agents include, but are not limited to, flupiltin, pentosan polysulfate, quinacrine, and tetracyclic compounds.

Regulation of immune response Avoidance of immune response
As described herein, a useful feature of the polynucleotides, primary constructs, or mmRNAs of the invention is the ability to reduce, escape, or avoid the innate immune response of cells. In one embodiment, when delivered to a cell, it is induced by a reference compound, eg, a polynucleotide of the invention, a primary construct, or mmRNA, or an unmodified polynucleotide corresponding to a different polynucleotide, primary construct, or mmRNA of the invention. Provided herein are polynucleotides, primary constructs, or mmRNAs encoding the polypeptide of interest that result in a reduced immune response from the host as compared to the response. As used herein, a "reference compound" is any molecule or substance that, when administered to a mammal, results in an innate immune response with a known degree, level, or amount of immune stimulation. The reference compound does not have to be a nucleic acid molecule, nor does it have to be either the polynucleotide, primary construct, or mmRNA of the invention. Thus, measurement of evasion, evasion, or failure of the induction of an immune response by a polynucleotide, primary construct, or mmRNA is any compound or substance known to elicit such a response. Can be shown by comparison with.

The term "innate immune response" generally includes a cellular response to exogenous single-stranded nucleic acids of viral or bacterial origin, which includes the expression and release of cytokines, especially interferon, as well as the induction of cell death. .. As used herein, the innate immune response or interferon response is cytokine expression, cytokine release, general inhibition of protein synthesis, general disruption of cellular RNA, upregulation of major histocompatibility molecules, and / or. It functions at the single-cell level, which induces apoptosis death, apoptosis, anti-growth, and gene transcription of genes involved in innate and adaptive immune cell activation. Some genes induced by type I IFN include PKR, ADAR (adenosine deaminase acting on RNA), OAS (2', 5'-oligoadenylate synthetase), RNase L, and Mx protein. PKR and ADAR result in inhibition of translation initiation and RNA editing, respectively. OAS is a dsRNA-dependent synthetase that activates the endoribonuclease RNase L to degrade ssRNA.

In some embodiments, the innate immune response comprises expression of type I or type II interferon, the expression of type I or type II interferon not in contact with the polynucleotides, primary constructs, or mRNA of the invention. It does not increase more than twice as much as the reference from the cell.

In some embodiments, the innate immune response comprises the expression of one or more IFN signature genes, the one or more IFN signature genes not in contact with the polynucleotides, primary constructs, or mmRNAs of the invention. It does not increase by more than 3 times compared to references from cells.

In some situations, eliminating the intracellular innate immune response may be beneficial, but the present invention was substantially reduced when administered, without completely eliminating such response (). It provides polynucleotides, primary constructs, and mmRNAs that provide an immune response that includes (significantly lower) interferon signaling.

In some embodiments, the immune response is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, as compared to an immune response induced by a reference compound. 95%, 99%, 99.9%, or more than 99.9% lower. The immune response itself can be measured by determining the level of expression or activity of type 1 interferon, or the expression of interferon regulatory genes such as Toll-like receptors (eg, TLR7 and TLR8). Reduction of the innate immune response can also be measured by measuring the level of reduced cell death with one or more administrations to the cell population. For example, cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or more than 95% less frequent than the cell death frequency observed with the reference compound. In addition, cell death is 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, of cells in contact with polynucleotides, primary constructs, or mmRNA. Or it can affect less than 0.01%.

In another embodiment, the polynucleotides, primary constructs, or mMVs of the invention are significantly less immunogenic than unmodified in vitro synthetic RNA molecular polynucleotides or primary constructs, or reference compounds having the same sequence. As used herein, "significantly low immunogenicity" refers to a detectable reduction in immunogenicity. In another embodiment, the term refers to a multiple reduction in immunogenicity. In another embodiment, the term refers to a reduction such that an effective amount of polynucleotide, primary construct, or mmRNA can be administered without inducing a detectable immune response. In another embodiment, the term reduces a polynucleotide, primary construct, or mMV so that it can be repeatedly administered without eliciting an immune response sufficient to detectably reduce the expression of the recombinant protein. Point. In another embodiment, the reduction is such that the polynucleotide, primary construct, or mmRNA can be repeatedly administered without eliciting an immune response sufficient to eliminate detectable expression of the recombinant protein. be.

In another embodiment, the polynucleotide, primary construct, or mmRNA is twice as immunogenic as its unmodified counterpart or reference compound. In another embodiment, immunogenicity is reduced by a factor of three. In another embodiment, immunogenicity is reduced by a factor of 5. In another embodiment, immunogenicity is reduced 7-fold. In another embodiment, immunogenicity is reduced by a factor of 10. In another embodiment, immunogenicity is reduced by a factor of 15. In another embodiment, immunogenicity is reduced by a multiple. In another embodiment, immunogenicity is reduced by a factor of 50. In another embodiment, immunogenicity is reduced 100-fold. In another embodiment, immunogenicity is reduced by a factor of 200. In another embodiment, immunogenicity is reduced by a factor of 500. In another embodiment, immunogenicity is reduced 1000-fold. In another embodiment, immunogenicity is reduced 2000-fold. In another embodiment, immunogenicity is reduced by another multiple.

Methods for determining immunogenicity are well known in the art and include, for example, cytokines (eg IL-12, IFNα, TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β. , Or measuring the secretion of IL-8), measuring the expression of DC activation markers (eg, CD83, HLA-DR, CD80, and CD86), or measuring the ability to act as an adjuvant for the adaptive immune response. To do.

The polynucleotides, primary constructs, or mMVs of the invention, including the combinations of modifications taught herein, may have excellent properties that make them more suitable as therapeutics.

The "all-or-none" model in the art has been established to be severely inadequate to explain the biological phenomena associated with the therapeutic utility of modified mRNAs. In order to improve protein production, the present inventors consider the nature of the modification or combination of modifications and the rate of modification (%) so as to determine the effect and risk profile of a specific modified mRNA. It has been confirmed that more cytokines or metrics can be tested.

In one aspect of the invention, a method of determining the effectiveness of a modified mRNA as compared to unmodified comprises the measurement and analysis of one or more cytokines whose expression is induced by administration of the exogenous nucleic acid of the invention. .. These values are compared with the administration of unmodified nucleic acids or with standard metrics such as cytokine response, poly IC, R-848 or other standards known in the art.

One example of standard metrics developed herein is the administration or modification nucleic acid of a modified nucleic acid at the level or amount of encoded polypeptide (protein) produced in a cell, tissue, or organism. A measurement of the ratio of cytokines whose expression is induced in a cell, tissue, or organism to one or more levels or amounts (or panels) as a result of contact with. Such ratios are referred to herein as protein: cytokine ratios or "PC" ratios. The higher the PC ratio, the more effective the modified nucleic acid (polynucleotide encoding the protein being measured). The preferred PC ratio with the cytokines of the invention can be greater than 1, greater than 10, greater than 100, greater than 1000, greater than 10,000, or greater. Modified nucleic acids having a higher PC ratio than modified nucleic acids of different or unmodified constructs are preferred.

The PC ratio can be further assessed by the percentage of modifications present in the polynucleotide. For example, normalized to 100% modified nucleic acid, protein production as a cytokine (or risk) function or cytokine profile can be determined.

In one embodiment, the present invention compares the PC ratio of a modified nucleic acid (polynucleotide, primary construct, or mmRNA) to any particular modified polynucleotide over a chemical, cytokine, or percentage of modification. , Primary constructs, or methods for determining the relative effects of nucleic acids.

MmRNAs containing various levels of nucleobase substitutions can be produced that maintain increased protein production and reduced immunostimulatory potential. The relative proportion (%) of any modified nucleotide to its naturally occurring nucleotide counterpart can vary during the IVT reaction (eg, 100, 50, 25, 10, 5, 2.5, 1, 0.1). , 0.01% 5-methylcytidine used vs. cytidine; 100, 50, 25, 10, 5, 2.5, 1, 0.1, 0.01% pseudouridine or N1-methyl-pseudouridine used vs. uridine ). It is also possible to make mmRNA that utilizes different ratios (eg, different ratios of pseudouridine and N1-methyl-pseudouridine) by using two or more different nucleotides for the same base. The mm mRNA can also be produced simultaneously in a mixed ratio at more than one "base" position, such as the ratio of 5-methylcytidine / cytidine and pseudouridine / N1-methyl-pseudouridine / uridine. The use of modified mRNA in varying proportions of modified nucleotides can be beneficial in reducing possible exposure to chemically modified nucleotides. Finally, the positional introduction of modified nucleotides into mmRNA that regulates protein production and / or immunostimulatory potential is also possible. The ability of such m mRNAs to demonstrate these improved properties can be assessed in vitro (using assays such as the PBMC assays described herein) and of proteins encoded by mmRNAs. It can also be assessed in vivo through measurements of both production and mediators of innate immune recognition such as cytokines.

In another embodiment, the relative immunogenicity of a polynucleotide, primary construct, or mMV and its unmodified counterpart is of the above response to the same extent as a given amount of unmodified nucleotide or reference compound. It can be determined by determining the amount of polynucleotide, primary construct, or mmRNA required to induce one. For example, if a polynucleotide, primary construct, or mmRNA that is twice as much as a polynucleotide, primary construct, or mmRNA is required to elicit the same response, the polynucleotide, primary construct, or mmRNA is unmodified. It is twice as immunogenic as a nucleotide or reference compound.

In another embodiment, the relative immunogenicity of a polynucleotide, primary construct, or mmRNA to its unmodified counterpart is the administration of the polynucleotide, primary construct, or mmRNA to the same amount of unmodified nucleotide or reference compound. Determined by determining the amount of cytokines secreted in response to (eg, IL-12, IFNα, TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β, or IL-8). Will be done. For example, if a polynucleotide, primary construct, or mmRNA is secreted with half the cytokine, the polynucleotide, primary construct, or mmRNA is twice as immunogenic as the unmodified nucleotide. In another embodiment, the background level of stimulation is subtracted before calculating immunogenicity in the above method.

Methods for performing titration, reduction, or elimination of an immune response intracellularly or in a population of cells are also provided herein. In some embodiments, cells are contacted with different doses of the same polynucleotide, primary construct, or mm mRNA and dose response is assessed. In some embodiments, cells are contacted with several different polynucleotides, primary constructs, or mmRNAs at the same or different doses to determine the optimal composition to produce the desired effect. With respect to the immune response, the desired effect may be to avoid, escape, or reduce the immune response of the cell. The desired effect can also be to alter the efficiency of protein production.

The polynucleotides, temporary constructs and / or mmRNAs of the invention may be used to reduce an immune response using the method described in WO2013003475, which is incorporated herein by reference in its entirety.

Immune Response Activation: Vaccines In addition, certain modified nucleosides, or combinations thereof, activate the innate immune response when introduced into the polynucleotides, primary constructs, or mmRNAs of the invention. Such activating molecules are useful as adjuvants when combined with polypeptides and / or other vaccines. In certain embodiments, the activating molecule contains a translatable region that encodes a polypeptide sequence that is useful as a vaccine, thus providing the ability to be a self-assistant.

In one embodiment, the polynucleotides, primary constructs, and / or mRNAs of the invention can encode immunogens. Delivery of polynucleotides, primary constructs, and / or mRNAs encoding immunogens can activate an immune response. As a non-limiting example, polynucleotides encoding immunogens, primary constructs, and / or mmRNAs can be delivered to cells to induce multiple natural response pathways (incorporated herein by reference in their entirety). , International Publication No. WO2012006377). As another non-limiting example, the polynucleotides, primary constructs, and mmRNAs of the invention encoding immunogens can be delivered to vertebrates at doses large enough to be immunogenic to vertebrates ( See WO2012006372 and WO2012006369, each of which is incorporated herein by reference in its entirety).

The polynucleotide, primary construct, or mMV of the present invention may encode a polypeptide sequence for a vaccine, which may further comprise an inhibitor. Inhibitors can impair antigen presentation and / or inhibit various pathways known in the art. As a non-limiting example, the polynucleotides, primary constructs, or mmRNAs of the invention may be used in vaccines in combination with inhibitors that can impair antigen presentation (each of which, by reference in its entirety, is described herein. See International Publications WO2012089225 and WO2012089338 incorporated in the book).

In one embodiment, the polynucleotide, primary construct, or mmRNA of the invention can be a self-replicating RNA. Self-replicating RNA molecules can improve the efficiency of RNA delivery and the expression of encapsulated gene products. In one embodiment, the polynucleotide, primary construct, or mMV may contain at least one modification described herein and / or known in the art. In one embodiment, the self-replicating RNA can be designed so that the self-replicating RNA does not induce the production of infectious viral particles. As a non-limiting example, self-replicating RNAs may be designed by the methods described in US Publication No. US201110300205 and International Publication No. WO2011005799, each of which is incorporated herein by reference in its entirety.

In one embodiment, the self-replicating polynucleotide, primary construct, or mMV of the invention can encode a protein that can elicit an immune response. As a non-limiting example, a polynucleotide, primary construct, or mRNA can be a self-replicating mRNA that can encode at least one antigen (each of which is incorporated herein by reference in its entirety, U.S.A. See US201110300205, and International Publications WO2011005799, WO2013006838, and WO2013006842).

In one embodiment, the self-replicating polynucleotides, primary constructs, or mMVs of the invention can be formulated as described herein or using methods known in the art. As a non-limiting example, self-replicating RNA may be formulated for delivery by the method described in Gear et al (Nonvarial delivery of self-amplifying RNA vaccines, PNAS 2012; PMID: 229082894).

In one embodiment, the polynucleotide, primary construct, or mMV of the invention may encode an amphipathic and / or immunogenic amphipathic peptide.

In one embodiment, the polynucleotide, primary construct, or m mRNA formulation of the invention may further comprise an amphipathic and / or immunogenic amphipathic peptide. As a non-limiting example, polynucleotides, primary constructs, or mRNAs containing amphipathic and / or immunogenic amphipathic peptides, each of which is incorporated herein by reference in its entirety, published in the United States. It may be formulated as described in US 201010250237, and WO20100009277 and WO201009065.

In one embodiment, the polynucleotide, primary construct, or mMV of the invention can be immunostimulatory. As a non-limiting example, polynucleotides, primary constructs, or mMVs may encode all or part of the positive-sense or negative-sense strand RNA virus genome (each incorporated herein by reference in its entirety). , International Publication No. WO2011092569 and US Publication No. US20120177701). In another non-limiting example, the immunostimulatory polynucleotides, primary constructs, or mMVs of the present invention are formulated using the excipients described herein and / or known in the art. (Refer to International Publication No. WO2012868295 and US Publication No. US20120213812, each of which is incorporated herein by reference in its entirety).

In one embodiment, the response of a vaccine formulated by the method described herein can be enhanced by the addition of various compounds to induce a therapeutic effect. As a non-limiting example, the vaccine formulation may include a MHCII-binding peptide or a peptide having a sequence similar to the MHCII-binding peptide (each of which is incorporated herein by reference in its entirety, WO 2012027365. , WO2011031298, and US Publications US20120070493, US20110110965). As another example, the vaccine formulation may contain modified nicotine compounds capable of producing an antibody response to the nicotine residue in the subject (each of which is incorporated herein by reference in its entirety, WO 20120671717 and See US Publication No. US201102114677).

Naturally occurring mutants In another embodiment, improved disease modifications using polynucleotides, primary constructs, and / or mmRNAs, including increased biological activity, improved patient prognosis, or protective function, etc. It is capable of expressing variants of active, naturally occurring proteins. Many such modified genes have been described in mammals (Nadeau, Curent Opinions in Genetics & Development 2003 13: 290-295; Hamilton and Yu, PLos Genet, all incorporated herein by reference in their entirety. 2012; 8: e1002644; Corder et al., Nature Genetics 1994 7: 180-184). Examples in humans include apoE2 protein, apoAI variant protein (ApoAI Milano, ApoAI Paris), hyperfunctional factor IX protein (Factor IX Padua Arg338Lys), transthyretin mutations. Body (TTR
Thr119Met). Expression of apoE2 (cys112, cys158) reduces susceptibility to other possible conditions such as Alzheimer's disease and cardiovascular disease, thereby reducing other apoE isoforms (apoE3 (cys112, arg158)). And apo E4 (arg112, arg158)) have been shown to provide protection (all of which are incorporated herein by reference in their entirety, Corder.
et al. , Nature Genetics 1994 7: 180-184, Seripa et al. , Rejuvenation Res. 2011 14: 491-500, Liu et al. Nat Rev Neurol. 2013 9: 106-118). Expression of the apo AI variant was accompanied by reduced cholesterol (all incorporated herein by reference, deGoma and Rader, 2011 Nature Rev Cardiol 8: 266-271, Nissen et al.,. 2003 JAMA 290: 2292-2300). The amino acid sequence of apo AI in a particular population was changed to cysteine in Apo AI Milano (Arg173 was changed to Cys) and in Apo AI Paris (Arg151 was changed to Cys). Was done. Factor IX mutations at position R338L (FIX Padua) result in factor IX proteins with approximately 10-fold increased activity (all incorporated herein by reference in their entirety, Simioni et al., N. Engl J Med. 2009 361: 1671-1675, Finn
et al. , Blood. 2012 120: 4521-4523, Cantor
et al. , Blood. 2012 120: 4517-20). Mutations in transthyretin at position 104 or 119 (Arg104His, Thr119Met) have also been shown to provide protection for patients who also have a disease-causing Val30Met mutation (all incorporated herein by reference in their entirety). Saraiva, Hum Mutat. 2001 17: 493-503, DATA BASE ON TRANSTYRETIN MUTATIONS http: //www.ibmc.up.pt/mjsaraviva/trmut.html). Differences in clinical findings and symptom severity in Portuguese and Japanese Met30 patients with Met119 and His104 mutants, respectively, are apparently protective effects expressed by non-pathogenic variants that provide additional stability to the molecule. Observed with (all incorporated herein by reference, Coelho et al. 1996 Neuromuscular).
Diseases (Suppl) 6: S20, Terrazaki et al. 1999. Biochem Biophyss Res Commun 264: 365-370). Modified mRNAs encoding these protected TTR alleles can be expressed in patients with TTR amyloidosis, thereby reducing the effect of pathogenic mutant TTR proteins.

Main Groove Interaction Partner As described herein, the expression "main groove interaction partner" detects an RNA ligand through interaction with the surface of the main groove of a nucleotide or nucleic acid, eg, binding, to it. Refers to the responding RNA recognition receptor. Thus, RNA ligands containing the polynucleotides, primary constructs, or modified nucleotides or nucleic acids such as mmRNA described herein reduce interaction with the main groove binding partner and thus reduce the innate immune response.

Illustrated main groove interactions, such as binding partners, include, but are not limited to, the following nucleases and helicases. In the membrane, TLRs (Toll-like receptors) 3, 7, and 8 can respond to single- and double-stranded RNA. In the cytoplasm, DEX (D / H). Members of the superfamily 2 class of helicases and ATPases can sense RNA that initiates an antiviral response. These helicases include RIG-I (retinoic acid-inducible genes). I) and MDA5 (melanoma differentiation-associated gene 5). Other examples include laboratory and physiology laboratory (laboratory ofgenics and physiology) 2 (LGP2), HIN- Examples of the protein to be used, or a helicase domain-containing protein.

Targeting Pathogenic Organisms or Diseased Cells Using polynucleotides, primary constructs, or mMVs encoding cell growth inhibitory or cytotoxic polypeptides, pathogenic microorganisms such as bacteria, yeasts, protozoans, worms, or cancer cells, etc. Methods for targeting diseased cells of the disease are provided herein. Preferably, the mRNA introduced comprises a modified nucleoside or other nucleic acid sequence modification that has been exclusively or preferentially translated in the target pathogenic organism to reduce the expected off-target effect of treatment. Such methods are useful for removing pathogenic organisms found in blood, semen, eggs, and any biomaterial, including transplant materials including embryos, tissues, and organs, or killing diseased cells. be.

Bioprocessing The methods provided herein can be useful in improving protein product yields in cell culture processes. In cell cultures containing multiple host cells, the introduction of polynucleotides, primary constructs, or mMVs described herein results in increased protein production efficiency compared to the corresponding unmodified nucleic acid. Such increased protein production efficiencies include, for example, increased protein translation from increased cell transfection, polynucleotides, primary constructs, or mm mRNA, decreased nucleic acid degradation, and / or reduced innate immune response of the host cell. Can be demonstrated by showing. Protein production can be measured by enzyme-linked immunosorbent assay (ELISA) and protein activity can be measured by a variety of functional assays known in the art. Protein production can be produced in a continuous or batch-fed mammalian process.

In addition, it is useful to optimize the expression of a particular polypeptide, especially a protein variant of a reference protein having a known activity, in a potential target cell line or collection of cell lines. be. In one embodiment, by providing multiple target cell types and independently, each of the multiple target cell types is contacted with a polynucleotide, primary construct, or mMr encoding the polypeptide of interest. This provides a method of optimizing the expression of the polypeptide of interest in the target cell. Cells can be transfected with two or more polynucleotides, primary constructs, or mmRNAs simultaneously or sequentially.

In certain embodiments, multiple rounds of the methods described herein can be used to obtain cells with increased expression of one or more nucleic acids or proteins of interest. For example, cells may be transfected with one or more polynucleotides, primary constructs, or mmRNAs encoding the nucleic acid or protein of interest. Cells are isolated according to the methods described herein before being subjected to further rounds of transfection with one or more other nucleic acids encoding the nucleic acid or protein of interest prior to being isolated again. May be good. This method involves a complex of proteins, a nucleic acid or protein in the same or related biological pathway, a nucleic acid or protein that acts upstream or downstream of each other, a nucleic acid or protein that has regulatory, activating, or inhibitory functions with each other, a function, or It may be useful to generate cells with increased expression of nucleic acids or proteins that are dependent on each other in terms of activity, or nucleic acids or proteins that share homology.

Furthermore, the culture conditions can be changed to increase the protein production efficiency. Subsequently, the presence and / or level of the polypeptide of interest in multiple target cell types is detected and / or quantified and optimized by selecting efficient target cells and cell culture conditions for polypeptide expression. to enable. Such methods are particularly useful when the polypeptide contains one or more post-translational modifications or has substantial tertiary structure, which is often a situation that complicates efficient protein production. ..

In one embodiment, the cells used in the methods of the invention can be cultured. Cells can be cultured in suspension or as adherent cultures. Cells can be cultured in a variety of containers, including, but not limited to, bioreactors, cell bags, wave bags, culture plates, flasks, and other containers known to those of skill in the art. Cells can be cultured in any other suitable medium, including but not limited to IMDM (Invitrogen, Catalog No. 12440-53) or known composition medium formulations. Further, ambient conditions suitable for cell culture such as temperature and atmospheric composition are well known to those skilled in the art. The method of the invention can be used in any cell suitable for use in protein production.

The present invention provides, for example, repeated introduction (eg, transfection) of modified nucleic acids into a target cell population in vitro, ex vivo, insights, or in vivo. For example, contact with the same cell population can be repeated more than once (more than 2, 3, 4, 5, or 5 times, etc.). In some embodiments, the step of contacting the cell population with a polynucleotide, primary construct, or mmRNA is repeated several times to be sufficient to achieve the predetermined efficiency of protein translation in the cell population. Assuming that nucleic acid modification provided the frequently reduced cytotoxicity of the target cell population, repeated transfections, as provided herein, in a diverse set of cell types and within diverse tissues. Achievable.

In one embodiment, the bioprocessing methods of the invention can be used to produce antibodies or functional fragments thereof. Functional fragments may include Fab, Fab', F (ab') ₂ , Fv domain, scFv, or bispecific antibody. They can be variable in any region, including complementarity determining regions (CDRs). In one embodiment, there is complete diversity in the CDR3 region. In another embodiment, the antibody is substantially conserved except within the CDR3 region.

Antibodies that bind to or associate with any biomolecule, whether of human pathogenic or non-human origin, can be made. The pathogen can be present in non-human mammals, clinical specimens, or commercial products such as cosmetic or pharmaceutical materials. They can also bind to any specimen or sample, including clinical specimens or tissue samples from any organism.

In some embodiments, the contact step is at a frequency selected from the group consisting of 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 84 hours, 96 hours, and 108 hours, and Repeated multiple times at concentrations of less than 20 nM, less than 50 nM, less than 80 nM, or less than 100 nM. The composition may be administered in less than 1 mM, less than 5 mM, less than 10 mM, less than 100 mM, or less than 500 mM.

In some embodiments, the polynucleotide, primary construct, or mMV is 50 molecules / 100 molecules / cell, 200 molecules / cell, 300 molecules / cell, 400 molecules / cell, 500 molecules / cell, 600 molecules / cell per cell. It is added in an amount of cells, 700 molecules / cell, 800 molecules / cell, 900 molecules / cell, 1000 molecules / cell, 2000 molecules / cell, or 5000 molecules / cell.

In other embodiments, the polynucleotide, primary construct, or mMV is 0.01 fmol / 106 cells, 0.1 fmol / 106 cells, 0.5 fmol / 106 cells, 0.75 fmol / 106 cells, 1 fmol / 106 cells, 2 fmol. / 106 cells, 5fmol / 106 cells, 10fmol / 106 cells, 20fmol / 106 cells, 30fmol / 106 cells, 40fmol / 106 cells, 50fmol / 106 cells, 60fmol / 106 cells, 100fmol / 106 cells, 200fmol / 106 cells, 300fmol It is added at a concentration selected from the group consisting of / 106 cells, 400 fmol / 106 cells, 500 fmol / 106 cells, 700 fmol / 106 cells, 800 fmol / 106 cells, 900 fmol / 106 cells, and 1 pmol / 106 cells.

In some embodiments, the production of biological products there is one or more measurable, such as parameters selected from the group consisting of cell density, pH, oxygen level, glucose level, lactate level, temperature, and protein production. Detected by monitoring various bioprocess parameters. Protein production can be measured as specific productivity (SP) (concentration of products such as polypeptides expressed asymmetrically in solution), can be expressed in mg / L or g / L, and is another. As a method, the specific productivity may be indicated by pg / cell / day. Increased SP can refer to an absolute or relative increase in the concentration of product produced under two predetermined conditions (eg, when compared to a control untreated with modified mRNA (s)). ..

Cells In one embodiment, cells are selected from the group consisting of mammalian cells, bacterial cells, plants, microorganisms, algae and fungal cells. In some embodiments, the cells are mammalian cells such as, but not limited to, human, mouse, rat, goat, horse, rabbit, hamster, or bovine cells. In a further embodiment, the cells are HeLa, NS0, SP2 / 0, KEK 293T, Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jarkat, CHO-K1, DG44, CHOK1SV, CHO-S, Hubec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2, IMR-90, MCF-7, U-20S, Per. It can be derived from cell lines that include, but are not limited to, C6, SF9, SF21, or Chinese hamster ovary (CHO) cells.

In certain embodiments, the cells are, but are not limited to, chrysosporium cells, aspergillus cells, trichoderma cells, dictosterium cells, candida cells, saccharomyces cells, schizosaccharomyces cells, penicillium cells, and the like. It is a fungal cell.

In certain embodiments, the cell is a bacterial cell, such as, but not limited to, Escherichia coli, Bacillus subtilis, or BL21 cells. Primary and secondary cells transfected by the methods of the invention can be obtained from a variety of tissues, including but not limited to all cell types that can be maintained in culture. For example, primary and secondary cells that can be transfected by the methods of the invention include fibroblasts, keratinocytes, epithelial cells (eg, mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, nerve cells, blood. Formation elements (eg, lymphocytes, bone marrow cells), muscle cells, and precursors of these somatic cell types include, but are not limited to. Primary cells can also be obtained from donors of allogeneic or other species (eg, mice, rats, rabbits, cats, dogs, pigs, cows, birds, sheep, goats, horses).

Purification and Isolation One of ordinary skill in the art should be able to determine the method used to purify or isolate the protein of interest from cultured cells. Generally, this is done through a capture method that uses affinity binding or non-affinity purification. If the protein of interest is not secreted by the cultured cells, then lysis of the cultured cells should be performed prior to purification or isolation. Contains the cell culture medium constituents in the present invention, cell culture additives such as antifoaming compounds and other nutrients and nutritional supplements, cells, cell debris, host cell proteins, DNA, viruses, and the like. Uncleaned cell culture medium may be used. The process can be carried out in the bioreactor itself. The fluid may either be pre-adjusted to the desired stimulus, such as pH, temperature, or other stimulus characteristics, or the fluid may be adjusted in response to the addition of the polymer (s). , Or the polymer (s) may be added to the carrier liquid appropriately adjusted to the parameters required for the stimulus conditions required to solubilize the polymer in fluid. The polymer can be completely circulated using a fluid, then a stimulus can be applied (changes in pH, temperature, salt concentration, etc.) to remove the desired protein and polymer (s) precipitates from the solution. can. The polymer and the desired protein (s) can be separated from the remaining fluid and may optionally be washed one or more times to remove any trapped or loosely bound contaminants. The desired protein is then recovered from the polymer (s), for example by elution or the like. Preferably, the elution can be carried out under conditions such that during the selected elution of the desired protein, the polymer remains in its precipitated form and retains any impurities therein. Polymers and proteins as well as any impurities may be solubilized in new fluids such as water or buffers, the proteins having affinity, ions that have protein preference and selectivity over those of the polymers or impurities. It can be recovered by exchange, hydrophobicity, or some other type of chromatography or the like. The eluted protein is then recovered and, where appropriate, can be subjected to a further processing step, either a batch like step or a continuous flow-through step.

In another embodiment, the expression of a particular polypeptide, particularly a protein variant of a reference protein having a known activity, is optimized in a cell line of potential interest or a collection of cell lines of interest. That can be useful. In one embodiment, by providing a plurality of target cell types, and independently, by contacting each of the plurality of target cell types with a modified mRNA encoding a polypeptide, the target is in the target cell. A method of optimizing the expression of a polypeptide is provided. Furthermore, the culture conditions can be changed to increase the protein production efficiency. Subsequently, the presence and / or level of the polypeptide of interest in multiple target cell types is detected and / or quantified, and by selecting efficient target cells and cell culture conditions for the expression of the polypeptide of interest. Allows optimization. Such methods can be useful when the polypeptide of interest, which often complicates efficient protein production, contains one or more post-translational modifications or has substantial tertiary structure. ..

Protein Recovery The protein of interest can preferably be recovered from the culture medium as a secreted polypeptide or, if expressed without a secretory signal, from the host cell lysate. It may be necessary to purify the protein of interest from other recombinant proteins and host cell proteins in a manner that provides a substantially homogeneous regulation of the protein of interest. Cell and / or granular cell debris can be removed from the culture medium or lysate. Then, for example, fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC (RP-HPLC), SEPHADEX® chromatography, chromatography on silica or on a cation exchange resin such as DEAE. Allows the product of interest to be purified from contaminant-soluble proteins, polypeptides and nucleic acids. Methods for purifying proteins that are non-homologously expressed by host cells are well known in the art.

The methods and compositions described herein are used to produce proteins in mammalian subjects that can attenuate or block the endogenous agonist biological response and / or antagonize a receptor or signaling molecule. can do. For example, IL-12 and IL-23 receptor signaling can be associated with chronic autoimmune disorders such as multiple sclerosis and rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis, and Crohn's disease. Can be enhanced in inflammatory diseases (Kickly K, Liu)
L, Na S, Sedgwich JD (2006) Cur. Opin. Immunol. 18 (6): 670-5). In another embodiment, the nucleic acid encodes an antagonist of the chemokine receptor. Chemokine receptors CXCR-4 and CCR-5 are required for HIV to enter the host cell (Arenza-Seisdedos F et al, (1996) Nature. Oct 3; 383 (6599): 400).

Gene silencing The polynucleotides, primary constructs, and mmRNAs described herein are useful for arresting (ie, preventing or significantly reducing) the expression of one or more target genes in a cell population. Is. A polynucleotide, primary construct, or mMV encoding a polypeptide of interest capable of directing sequence-specific histone H3 methylation is targeted through histone H3 methylation and subsequent heterochromatin formation after the polypeptide has been translated. It is introduced into cells in a population under conditions that reduce gene transcription of the gene. In some embodiments, silencing mechanisms are performed on cell populations present in mammalian subjects. As a non-limiting example, a useful target gene is a mutant Janus kinase-2 family member in which a mammalian subject expresses a mutant target gene that suffers from a myeloproliferative disorder that is the result of abnormal kinase activity. be.

Co-administration of polynucleotides, primary constructs, and mmRNA with RNAi agents is also provided herein.

Regulation of biological pathways Rapidly translated polynucleotides, primary constructs, and mmRNAs introduced into cells provide the desired mechanism for regulating biological pathways. Such regulation includes antagonism or agonism of a given pathway. In one embodiment, the polynucleotide, primary construct, and mmRNA are localized intracellularly, and the polypeptide can be translated intracellularly from the polynucleotide, primary construct, and mmRNA, which polypeptide. Intracellular by contact with an effective amount of composition containing a polynucleotide, a primary construct, or mmRNA encoding a polypeptide of interest in a cell under conditions that inhibit the activity of the polypeptide function in the biological pathway. Methods for antagonizing the biological pathways of the organism are provided. An exemplary biological pathway is one that is deficient in autoimmune or inflammatory disorders such as polysclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis, colitis, or Crohn's disease. In particular, the antagonism of the IL-12 and IL-23 signaling pathways is particularly useful (Kickly K, Liu L, Na S, Sedgwick).
JD (2006) Curr. Opin. Immunol. 18 (6): 670-5)).

In addition, polynucleotides, primary constructs, or mmRNAs encoding chemokine receptor antagonists are provided. Chemokine receptors CXCR-4 and CCR-5 are required, for example, for HIV to enter the host cell (Arenza-Seisdedos F et al, (1996) Nature. Oct 3; 383 (6599): 400). ).

Alternatively, the nucleic acid is localized intracellularly, the recombinant polypeptide can be translated intracellularly from the nucleic acid, and the recombinant polypeptide induces the activity of the polypeptide function in the biological pathway. Under conditions, a method of stimulating an intracellular biological pathway is provided by contacting the cell with an effective amount of a polynucleotide, a primary construct, or nucleic acid encoding a recombinant polypeptide. Illustrated stimulated biological pathways include pathways that regulate cell fate determination. Such aggregation is reversible or irreversible.

Expression of Ligands or Receptors on Cell Surface In some embodiments and embodiments described herein, the polynucleotides, primary constructs, or mmRNAs described herein are used on the surface of cells. A ligand or ligand receptor can be expressed (eg, the homing moiety). The ligand or ligand receptor moiety bound to the cell surface allows the cell to have the desired biological interaction with the tissue or drug in vivo. Ligands include antibodies, antibody fragments, aptamers, peptides, vitamins, carbohydrates, proteins or polypeptides, receptors such as cell surface receptors, adhesion molecules, glycoproteins, sugar residues, therapeutic agents, drugs, glycosaminoglycans, Or it can be any combination of these. For example, the ligand may be an antibody that recognizes a cancer cell-specific antigen, giving cells capable of preferentially interacting with tumor cells and allowing tumor-specific localization of modified cells. Since the preferred ligand can interact with the target molecule on the outer surface of the tissue to be treated, the ligand can confer the ability of the cell composition to accumulate in the tissue to be treated. Ligands with limited cross-reactivity with other tissues are generally preferred.

In some cases, the ligand can act as a homing moiety that allows the cell to target a particular tissue or interact with a particular ligand. Such homing moieties include: Fv fragment, single chain Fv (scFv) fragment, Fab'fragment, F (ab') 2 fragment, single domain antibody, camelized antibody and antibody fragment, humanized antibody and antibody. Fragments, and any member of a specific binding pair, antibody, monoclonal antibody, or derivative or analog thereof, including, but not limited to, the polyvalent alternatives described above: disulfide-stabilized Fv fragment, scFv tandem. Unispecific or bispecific antibodies such as ((SCFV) 2 fragments), typically covalently bound or stabilized (ie, leucine zipper or helix stabilized) scFv Fragments of, but not limited to, bispecific antibodies, trispecific antibodies, or tetrabodies; and, for example, aptamers, receptors, and fusion proteins. It may include, but is not limited to, other homing moieties including.

In some embodiments, the homing moiety can be a surface-binding antibody that can allow the regulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be produced against the epitope of interest at the desired targeting site. In one embodiment, multiple antibodies are expressed on the surface of the cell, and each antibody can have different specificity for the desired target. Such techniques can increase the cohesiveness and specificity of homing interactions.

Those skilled in the art can select any homing moiety based on the desired localization or function of the cell, for example, estrogen receptor ligands such as tamoxyphene have increased estrogen receptor on the cell surface. It can be targeted to estrogen-dependent breast cancer cells that have a body count. Other non-limiting examples of ligand / receptor interactions include CCRI (eg, for the treatment of inflamed joint tissue or brain in rheumatoid arthritis and / or polysclerosis), CCR7, CCR8 (eg, lymph). CCR6, CCR9, CCR10 (eg, to target intestinal tissue), CCR4, CCR10 (eg, to target skin), CXCR4 (eg, generally improved play) HCELL (eg, for the treatment of inflammation and inflammatory disorders, bone marrow), α4β7 (eg, for targeting the intestinal mucosa), VLA-4 / VCAM-1 (eg, targeting the endothelium) Can be mentioned. In general, any receptor involved in targeting (eg, cancer metastasis) can be utilized in the applications of the methods and compositions described herein.

Regulation of Cell Lineage A method of inducing changes in cell fate in target mammalian cells is provided. Target mammalian cells may be progenitor cells, and alterations may include promoting differentiation within the lineage or blocking such differentiation. Alternatively, the target mammalian cell may be a differentiated cell, and the cell fate change promotes dedifferentiation into pluripotent precursor cells, or such dedifferentiation such as dedifferentiation of the cancer cell into cancer stem cells. Includes blocking differentiation. In situations where changes in cell fate are desired, an effective amount of mRNA encoding a cell fate-inducing polypeptide is introduced into the target cell under conditions that induce changes in cell fate. In some embodiments, the modified mRNA is useful for reprogramming a subpopulation of cells from the first phenotype to the second phenotype. Such reprogramming may be temporary or permanent. Optionally, reprogramming induces target cells to pretend to be an intermediate phenotype.

In addition, the methods of the invention depend on highly efficient transfection, the ability to retransfect cells, and the adequacy of the amount of recombinant polypeptide produced within the target cells, induced pluripotent stem cells (iPS cells). Is especially useful for producing. Moreover, the use of iPS cells generated using the methods described herein is expected to have a reduced incidence of teratoma formation.

Also provided are methods of reducing cell differentiation in the target cell population. For example, an effective amount of polynucleotide encoding a polypeptide, a target cell population containing one or more progenitor cell types, under conditions such that the polypeptide is translated and reduces progenitor cell differentiation. Contact with the primary construct, and the composition with the mMr. In a non-limiting embodiment, the target cell population comprises injured or surgically treated tissue in a mammalian subject. Progenitor cells are, for example, stromal progenitor cells, neural progenitor cells, or mesenchymal progenitor cells.

In specific embodiments, polynucleotides, primary constructs, or mmRNAs encoding one or more differentiation factors Gata4, Mef2c, and Tbx4 are provided. These mRNA-producing factors are introduced into fibroblasts and promote reprogramming into cardiomyocytes. Such reprogramming can be performed in vivo by contacting the mRNA-containing patch or other material with damaged heart tissue to facilitate cardiac redifferentiation. Such a process promotes cardiomyocyte development as opposed to fibrosis.

Mediation of Cell Death In one embodiment, cells (eg, by increasing the expression of cell death receptors, cell death receptor ligands, or combinations thereof, using polynucleotides, primary constructs, or mmRNA compositions. It can induce apoptosis in cancer cells). This method can be used to induce cell death in any desired cell and is particularly useful in the treatment of cancer in which cells escape the natural apoptotic signal.

Apoptosis converges according to the final effector mechanism, which consists of multiple interactions between several "cell death receptors" and their ligands, which belong to the tumor necrosis factor (TNF) receptor / ligand superfamily. Can be induced by an independent signaling pathway. The most well-characterized cell death receptors are CD95 (“Fas”), TNFRI (p55), death receptor 3 (DR3 or Apo3 / TRAMO), DR4, and DR5 (Apo2-TRAIL-R2). ). The final effector mechanism of apoptosis can be the activation of a series of proteinases designed as caspases. Activation of these caspases results in the cleavage and cell death of a series of living cell proteins. The molecular mechanism of cell death receptor / ligand-induced apoptosis is well known in the art. For example, Fas / FasL-mediated apoptosis is induced by the binding of three FasL molecules that induce fas receptor trimer formation via the C-terminal death domain (DD), which in turn leads to the adapter protein FADD (death). Recruit Fas-related proteins with a domain) and caspase-8. The oligomerization of this trimolecular complex, Fas / FAIDD / caspase 8, results in proteolytic cleavage of the zymogen caspase 8 into active caspase 8, which in turn results in other downstream containing caspase 3 through proteolysis. By activating the caspase, it initiates the apoptotic process. Death ligands are generally apoptotic when formed within trimers or higher-order structures. With respect to monomers, they can serve as anti-apoptotic agents by competing with trimers for binding to cell death receptors.

In one embodiment, the polynucleotide, primary construct, or mRNA composition encodes a cell death receptor (eg, Fas, TRAIL, TRAMO, TNFR, TLR, etc.). Cells made to express a cell death receptor by transfection of polynucleotides, primary constructs, and mmRNAs are susceptible to cell death induced by ligands that activate the receptor. Similarly, for example, cells made to express death ligand on their surface induce cell death at the receptor when the transfected cells are contacted with the target cells. In another embodiment, the polynucleotide, primary construct, and mmRNA composition encode a cell death receptor ligand (eg, FasL, TNF, etc.). In another embodiment, the polynucleotide, primary construct, and mmRNA composition encode a caspase (eg, caspase 3, caspase 8, caspase 9, etc.). In another embodiment, synthetic polynucleotides, primary constructs, and mmRNA compositions are cell death receptors and theirs, where cancer cells often exhibit failure to properly differentiate into non-proliferative or controlled proliferative forms. It encodes both suitable activation ligands. In another embodiment, synthetic polynucleotides, primary constructs, and mmRNA compositions have reduced cell non-pathogenic or non-self-renewal phenotype (eg, reduced) when expressed in cancer cells such as cancer stem cells. cell growth rate, or induced to differentiate into reduced cell division, etc.), or the cells dormant cell stage (e.g., encodes a differentiation factor that induces in G ₀ telogen).

For those of skill in the art, the use of apoptosis-inducing techniques may require polynucleotides, primary constructs, or mmRNAs to be adequately targeted, for example, to tumor cells to prevent unwanted widespread cell death. Let's understand that. Therefore, a delivery mechanism that recognizes a cancer antigen (eg, a binding ligand or antibody that targets a liposome or the like) can be used such that the polynucleotide, primary construct, or mmRNA is expressed only in the cancer cell.

Cosmetological Applications In one embodiment, polynucleotides, primary constructs, and / or mmRNAs can be used to treat, ameliorate, or prevent cosmetological conditions. Such conditions include contusion, rosacea, scars, wrinkles, eczema, herpes zoster, psoriasis, age spots, mother's spots, dry skin, octopus, rash (eg, dandruff, heat rash), scabies, warts. Common signs of rash, warts, insect bites, chloasma, dandruff, freckles, and aging include.

VI. Kits and Devices Kits The present invention provides a variety of kits for conveniently and / or effectively performing the methods of the invention. Typically, the kit is an amount and / or number of components sufficient to allow the user to perform multiple treatments (s) of interest and / or multiple experiments of interest. including.

In one aspect, the invention provides a kit comprising the molecule of the invention (polynucleotide, primary construct, or mmRNA). In one embodiment, the kit comprises one or more functional antibodies or functional fragments thereof.

The kit can be for protein production, including a first polynucleotide containing a translatable region, a primary construct, or mmRNA. The kit may further include packaging and instructions for forming the pharmaceutical composition and / or delivery agent. The delivery agent may include saline solution, buffer, lipidoid, or any delivery agent disclosed herein.

In one embodiment, the buffer may contain sodium chloride, calcium chloride, phosphate, and / or EDTA. In another embodiment, the buffer is saline, saline containing 2 mM calcium, 5% sucrose, 5% sucrose containing 2 mM calcium, 5% mannitol, 5% mannitol containing 2 mM calcium, Ringer. It may include, but is not limited to, sodium chloride containing lactate, sodium chloride, 2 mM calcium and mannitol (see, eg, US Publication No. 20120258046, which is incorporated herein by reference in its entirety). In a further embodiment, the buffer may be precipitated or lyophilized. The amount of each component can be changed to allow a consistent, reproducible high-concentration saline solution or simple buffered formulation. The components can also be modified to increase the stability of the modified RNA over a period of time and / or in buffer under a variety of conditions. In one aspect, the invention comprises a polynucleotide comprising a translatable region, which, when introduced into a target cell, is provided in an amount effective to produce a protein encoded by the desired amount of translatable region. A protein comprising a primary construct, or mmRNA, a second polynucleotide containing an inhibitory nucleic acid, provided in an amount effective in substantially inhibiting the cell's natural immune response, and packaging and instructions. Kits for production are provided.

In one aspect, the invention is for protein production, comprising a polynucleotide containing a translatable region, a primary construct, or mmRNA, packaging and instructions, wherein the polynucleotide exhibits reduced degradation by cellular nucleases. Provide a kit.

In one aspect, the invention is suitable for translating a translatable region containing a translatable region, a primary construct, or mmRNA and a translatable region of a first nucleic acid, wherein the polynucleotide exhibits reduced degradation by a cellular nuclease. Kits for protein production, including mammalian cells, are provided.

In one embodiment, the level of protein C can be measured by an immunoassay. Assays may be purchased from BioMérieux, Inc. (Durham, NC), Abbott Laboratories (Abbott Park, IL), Siemens Medical Solutions USA, Inc. (Malvern, PA), BIOPORTO® Diagnostics A / S (Gentofte, Denmark), USCN® Life Science Inc. (Houston, TX), or available from any number of sources, including Roche Scientific Corporation (Indianapolis, IN). In this embodiment, the assay can be used to assess the level of protein C or active or variant thereof delivered, either as a modified mRNA molecule or in response to its administration.

Device The present invention provides a device capable of incorporating a polynucleotide, a primary construct, or mmRNA encoding a polypeptide of interest. These devices contain reagents for synthesizing polynucleotides in formulations that can be immediately delivered to subjects in need thereof, such as human patients, in stable formulations. Non-limiting examples of such targeted polypeptides include growth factors and / or angiogenic stimulators for wound healing, peptide antibacterial agents for facilitating infection control, and newly identified. Examples include antigens for rapidly stimulating an immune response against the virus.

The device can also be used with the present invention. In one embodiment, the device is used to assess the level of protein administered in the form of modified mRNA. The device may include a blood, urine, or other biofluid test. It can be a large or small decentralized desktop device that includes an automated central lab platform. It can be a point of care or a handheld device. In this embodiment, for example, protein C or APC can be quantified before, during, or after treatment with modified mRNA encoding protein C (its thymogen), APC, or any variant thereof. .. Autoprothrombin IIA and protein C, also known as blood coagulation factor XIV, are zymogens or precursors of serine proteases that play important roles in the regulation of blood coagulation and the development of fibrinolytic activity in vivo. It is synthesized in the liver as a single-stranded polypeptide, but undergoes post-translational processing that yields a double-stranded intermediate. Its intermediate form of protein C is converted to an active form known as "activated protein C" (APC) via thrombin-mediated cleavage of a 12-residue peptide from the amino terminus of the heavy chain to the amino terminus of the molecule. .. The device may be useful in drug discovery efforts as a companion diagnostic test associated with protein C or APC treatment such as sepsis or severe sepsis. Early studies suggested that APC had the ability to reduce mortality in severe sepsis. Following this line of study, clinical studies have led to FDA approval of one compound, activated drotrecogin alfa (recombinant protein C). However, at the end of 2011, following the results of the PROWESS-SHOCK trial, which showed that the 28-day all-cause mortality rate in patients with septic shock did not meet the primary endpoint of a statistically significant reduction. The drug was withdrawn from all markets. The present invention provides modified mRNA molecules that can be used in the diagnosis and treatment of sepsis, severe sepsis, and sepsis, overcoming previous challenges or problems associated with increased protein expression efficiency in mammals.

In some embodiments, the device is self-contained and optionally capable of wireless telecommunications to obtain instructions for the synthesis and / or analysis of the generated polynucleotide, primary construct, or mRNA. be. The device is capable of mobile synthesis of at least one polynucleotide, primary construct, or mmRNA, and preferably an unlimited number of different polynucleotides, primary constructs, or mmRNAs. In certain embodiments, the device can be transported by one or a small number of individuals. In other embodiments, the device is tuned to fit a desk or desk. In other embodiments, the device is tuned to fit in a suitcase, backpack, or object of similar size. In another embodiment, the device may be a point of care or handheld device. In a further embodiment, the device is tuned to fit in a vehicle such as a passenger car, carrier, or ambulance, or a military vehicle such as a tank or troop transport vehicle. The information required to generate the modified mRNA encoding the polypeptide of interest resides in the computer-readable medium present on the device.

In one embodiment, the device can be used to assess the level of protein administered in the form of a polynucleotide, primary construct, or mmRNA. The device may include a blood, urine, or other biofluid test.

In some embodiments, the device is capable of communicating (eg, wireless communication) using a database of nucleic acid and polypeptide sequences. The device includes at least one sample block for inserting one or more sample containers. Such a sample container is capable of accepting liquids or other forms of any number of materials such as template DNA, nucleotides, enzymes, buffers, and other reagents. The sample container can also be heated or cooled by contact with the sample block. The sample block generally communicates with a device base having one or more electronic controls for at least one sample block. The sample block preferably comprises a heating module, which is a heating module capable of heating and / or cooling the sample container and its components to temperatures above about −20 ° C. to + 100 ° C. The device base communicates with a voltage source such as a battery or an external voltage source. The device also includes means for storing and distributing materials for RNA synthesis.

Optionally, the sample block comprises a module for separating the synthesized nucleic acid. Alternatively, the device includes a separation module that is operably coupled to the sample block. Preferably, the device comprises means for analyzing the synthesized nucleic acid. Such analyzes include sequence identity (demonstrated by hybridization, etc.), absence of unwanted sequences, measurement of the integrity of the synthesized mRNA (eg, by microfluidic viscosity measurement in combination with spectrophotometry). , And the concentration and / or potency of the modified RNA (such as by spectrophotometry).

In certain embodiments, the device is combined with a means for detecting pathogens present in the biomaterial obtained from the subject, eg, the IBIS PLEX-ID system for microbial identification (Abbott, Abbott Park, IL). Be done.

Suitable devices for use in the intradermal delivery of the pharmaceutical compositions described herein are US Pat. Nos. 4,886,499, each of which is incorporated herein by reference in its entirety. , No. 5,190,521, No. 5,328,483, No. 5,527,288, No. 4,270,537, No. 5,015,235, No. 5, Examples thereof include short-hand devices such as the devices described in 141, 496 and 5, 417, 662. The intradermal composition limits the effective penetration length of the needle into the skin, such as the device described in PCT Publication No. WO 99/34850, which is incorporated herein by reference in its entirety, and its functional equivalents. Can be administered by the device. Jet injection devices that deliver the liquid composition to the dermis and produce jets that reach the dermis are preferred via a liquid jet injector and / or through a needle that punctures the stratum corneum. Jet injection devices are, for example, US Pat. Nos. 5,480,381, 5,599,302, 5,334,144, each of which is incorporated herein by reference in its entirety. , No. 5,993,412, No. 5,649,912, No. 5,569,189, No. 5,704,911, No. 5,383,851, No. 5, 893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,064,413, No. 5,520,639, No. 4,596,556, No. 4,790,824, No. 4,941,880, No. 4,940,460, and PCT Publication No. WO97. It is described in / 37705 and WO97 / 13537. Ballistic powder / particle delivery devices that use compressed gas to accelerate the vaccine in powder form and reach the dermis through the outer layer of skin are preferred. Alternatively, or in addition, conventional syringes can be used in the classical Mantu method of intradermal administration.

In some embodiments, the device may be a pump or comprises a catheter for administration of a compound or composition of the invention across the blood-brain barrier. Such devices include, but are not limited to, pressurized or olfactory delivery devices, iontophoresis devices, multi-layer microfluidic devices, and the like. Such devices may be portable or fixed. They may be implantable in the body, moored externally in the body, or a combination thereof.

The administration device may be used to deliver the polynucleotides, primary constructs, or mmRNAs of the invention according to the single, multiple, or divided dosing regimens taught in the present invention. Such devices are described below.

Methods and devices known in the art for multiple doses to cells, organs, and tissues are contemplated for use with the methods and compositions disclosed herein as embodiments of the present invention. NS. These include, for example, methods and devices having hybrid devices that utilize multiple needles, such as lumens or catheters, and devices that utilize heat, current, or radiation drive mechanisms.

In accordance with the present invention, these multi-dose devices may be utilized to deliver single-dose, multi-dose, or divided doses as contemplated herein.

Methods for delivering therapeutic agents to solid tissues have been described by Bahrami et al., For example, taught in US Patent Publication No. 20112030839, the contents of which are incorporated herein by reference in their entirety. According to Bahrami, a series of needles are incorporated into the device to deliver substantially the same amount of fluid anywhere in its solid tissue along the length of each needle.

Devices for delivering biomaterials across biological tissues are described by Kodgule et al., For example, in US Patent Publication No. 20110172610, the contents of which are incorporated herein by reference in their entirety. Be incorporated. According to Kodgule, multiple hollow microneedles made from one or more metals and having an outer diameter of about 200 microns to about 350 microns and a length of at least 100 microns are incorporated into the device to incorporate peptides, proteins, Deliver carbohydrates, nucleic acid molecules, lipids, and other pharmaceutically effective ingredients or combinations thereof.

Delivery probes for delivery of therapeutic agents to tissues are described by Gunday et al., For example, are taught in US Patent Publication No. 20112070184, the contents of which are incorporated herein by reference in their entirety. .. According to Gunday, multiple needles are incorporated into the device, which moves the added capsule between the active and non-acting positions and allows the drug to exit the capsule through the needle.

Multi-injection medical devices are described by Assaf, for example, as taught in US Pat. No. 4,2011,218497, the contents of which are incorporated herein by reference in their entirety. According to Assaf, multiple needles are incorporated into the device, and the device provides a chamber that connects to one or more of the needles and a means of continuously replenishing the chamber with medical fluid after each infusion. Have.

In one embodiment, the polynucleotide, primary construct, or mmRNA is administered subcutaneously or intramuscularly via at least three needles, simultaneously or within 60 minutes to three different, optionally adjacent sites (eg,). , Simultaneously or within 60 to 4, 5, 6, 7, 8, 9, or 10 sites). The divided doses can be administered simultaneously to adjacent tissues using the devices described in U.S. Patent Publication Nos. 2011230839 and 20110218497, each of which is incorporated herein by reference in its entirety.

A system that is at least partially implantable for injecting a substance into the patient's body, specifically a penile erection stimulating system, is described by Forsell, for example, as taught in US Pat. No. 20110196198. Are incorporated herein by reference in their entirety. According to Forsel, multiple needles are incorporated within the device, which is implanted with one or more enclosures adjacent to the patient's left and right clitoral corpus cavernosum. A reservoir and pump are also implanted to supply the drug through the needle.

Methods for transdermal delivery of therapeutically effective amounts of iron have been described by Berenson, eg, are taught in US Patent Publication No. 20010130910, the contents of which are incorporated herein by reference in their entirety. Is done. According to Berenson, multiple needles can be used to create multiple microchannels within the stratum corneum to enhance transdermal delivery of ionic iron on an iontophoretic patch.

Methods for the delivery of biomaterials across biological tissues have been described by Kodgule et al., For example, in US Patent Publication No. 20111996308, the contents of which are incorporated herein by reference in their entirety. Is done. According to Kodgule, multiple biodegradable microneedles containing therapeutically active ingredients are incorporated into the device to deliver proteins, carbohydrates, nucleic acid molecules, lipids, and other pharmaceutical active ingredients, or combinations thereof.

Dermal patches containing the botulinum toxin composition are described by Donovan, for example, taught in US Patent Publication No. 20080220020, the contents of which are incorporated herein by reference in their entirety. According to Donovan, multiple needles are incorporated into the patch to deliver botulinum toxin under the stratum corneum through the needles that protrude through the stratum corneum of the skin without rupturing the blood.

A small disposable drug reservoir, or patch pump, capable of holding approximately 0.2-15 mL of liquid formulation is placed on the skin and the formulation is continuously subcutaneously placed using a fine needle (eg, 26-34 gauge). Can be delivered to. As a non-limiting example, the patch pump is a spring-loaded 50 mm x 76 mm x 20 mm (BD ™, Microinfoser, Franklin Lakes NJ) with 30-34 gauge needles, a 2 mL reservoir used for drug delivery such as insulin. 41 mm x 62 mm x 17 mm (OMNIPOD®, Insulin Corporation Bedford, MA), or 43-60 mm diameter with 0.5-10 mL reservoir, 10 mm thick (PATCHPUMP®, SteadyMed Therapeutics, San Francisco, CA) may be used. In addition, the patch pump can be battery-powered and / or rechargeable.

Frozen probes for administration of activators to sites of cryotherapy are described by Toubia, eg, U.S. Patent Publication No. 20080140061, the contents of which are incorporated herein by reference in their entirety. Be incorporated. According to Toubia, multiple needles are incorporated into the probe, which receives the activator into the chamber and administers the drug to the tissue.

Methods for treating or preventing inflammation or promoting healthy joints have been described by Stock et al., For example, in US Patent Publication No. 2009015518, the content of which is described by reference in these. The whole is incorporated herein. According to Stock, multiple needles are incorporated into the device to administer a composition containing a signaling regulator compound.

Multisite injection systems have been described by Kimmell et al., For example, are taught in US Patent Publication No. 2012025694, the contents of which are incorporated herein by reference in their entirety. According to Kimmel, multiple needles are incorporated into the device to deliver the drug into the stratum corneum through the needles.

Methods for delivering interferon to the intradermal compartment are described by Dekker et al., For example, taught in US Patent Publication No. 20050181033, the contents of which are incorporated herein by reference in their entirety. According to Dekker, multiple needles with outlets with an exposed height of 0 to 1 mm are incorporated into the device, which delivers the substance at a depth of 0.3 mm to 2 mm for pharmacokinetics and Improve bioavailability.

Methods for delivering genes, enzymes, and biopharmaceuticals to histiocytes are described by Desai, for example, as taught in US Patent Publication No. 20030073908, the contents of which are incorporated herein by reference in their entirety. Incorporated in. According to Desai, multiple needles are incorporated into a device that is inserted into the body and deliver the drug fluid through the needles.

Methods for treating arrhythmias with fibroblasts have been described by Lee et al., For example, taught in US Pat. No. 2,400,500,295, the contents of which are incorporated herein by reference in their entirety. Is done. According to Lee, multiple needles are integrated into the device to deliver fibroblasts to local areas of tissue.

Methods for treating brain tumors using magnetically controlled pumps have been described by Shachar et al., Eg. Are incorporated herein by reference in their entirety. According to Shachar, multiple needles are incorporated into the pump, which pushes the drug therapeutic agent through the needles at a controlled speed.

Methods for treating bladder dysfunction in female mammals have been described by Versi et al., For example, in US Pat. No. 8,029,496, the contents of which are incorporated herein by reference in their entirety. Incorporated in. According to Versi, a series of microneedles are incorporated into the device to deliver the therapeutic agent directly into the trigone of the bladder through the needles.

Microneedle percutaneous transport devices are described by Angel et al., For example, are taught in US Pat. No. 7,364,568, the contents of which are incorporated herein by reference in their entirety. According to Angel, multiple needles are incorporated into the device, which transports material into the body surface through the needles, which are inserted into the surface from different directions. Micro The needle percutaneous transport device may be a solid microneedle system or a hollow microneedle system. As a non-limiting example, a solid microneedle system has a volume of up to 0.5 mg with ^{a drug-coated height of about 150-700 μm and 300-1500 solid microneedles per cm 2.} Can be. The microneedles penetrate the stratum corneum and remain on the skin for a short duration (eg, 20 to 15 minutes). In another example, the hollow microneedle system delivers the liquid formulation using 15-20 microneedles per ^{cm 2} with a volume of up to 3 mL and a height of approximately 950 μm. The microneedles penetrate the skin and allow the liquid formulation to flow from the device into the skin. The hollow microneedle system can be consumed in 1 to 30 minutes, depending on the volume and viscosity of the formulation.

Subcutaneous infusion devices are described by Dalton et al., For example, in US Pat. No. 7,150,726, the contents of which are incorporated herein by reference in their entirety. According to Dalton, multiple needles are incorporated into the device and deliver fluid to the subcutaneous tissue through the needles.

Devices and methods for intradermal delivery of vaccines and gene therapy agents through microcannula are described by Mikszta et al., For example, in US Pat. No. 7,473,247, the contents of which are described by reference. Is incorporated herein in its entirety. According to Mitzzta, at least one hollow microneedle is incorporated into the device to deliver the vaccine to the subject's skin at a depth of 0.025 mm to 2 mm.

Methods of delivering insulin are described by Pettis et al., For example, in US Pat. No. 7,722,595, the contents of which are incorporated herein by reference in their entirety. According to Pettis, the two needles are integrated into the device, the first needle is less than 2.5 mm deep to deliver insulin to the intradermal compartment, and the second needle delivers insulin to the intradermal compartment. At a depth greater than 2.5 mm and less than 5.0 mm, both needles are basically inserted into the skin at the same time.

Skin infusion delivery under aspiration is described by Kochamba et al., For example, is taught in US Pat. No. 6,896,666, the contents of which are incorporated herein by reference in their entirety. According to Kochamba, multiple needles that are relatively adjacent to each other are incorporated into the device to inject fluid under the skin layer.

Devices for removing or delivering substances through the skin have been described by Down et al., For example, in US Pat. No. 6,607,513, the contents of which are incorporated herein by reference in their entirety. Incorporated into the book. According to Down, multiple skin permeation members are incorporated within the device, have a length of about 100 to about 2000 microns, and are about 30 to 50 gauge.

Devices for delivering substances to the skin have been described by Palmer et al., For example, in US Pat. No. 6,537,242, the contents of which are incorporated herein by reference in their entirety. .. According to Palmer, a series of microneedles are incorporated into the device and use a pulling assembly to enhance the contact between the needle and the skin, resulting in a more uniform delivery of material.

Perfusion devices for topical drug delivery are described by Zamoiski, eg, US Pat. No. 6,468,247, the contents of which are incorporated herein by reference in their entirety. According to Zamoiski, multiple hypodermic needles are incorporated into a device that injects the contents of the subcutaneous syringe into the tissue as the subcutaneous syringe retracts.

A method for enhanced transport of drugs and biomolecules across tissues by improving the interaction between microneedles and human skin has been described by Prausnitz et al., For example, US Pat. No. 6, 6. As taught in No. 743,211 the contents are incorporated herein by reference in their entirety. According to Prusice, multiple microneedles are incorporated within the device, allowing the device to exhibit a stronger, less deformable surface to which the microneedles are applied.

Devices for organ administration of pharmaceutical agents are described by Ting et al., For example, taught in US Pat. No. 6,077,251, the contents of which are incorporated herein by reference in their entirety. Is done. According to Ting, multiple needles with side openings for augmented administration are incorporated into the device, and the device extends and retracts the needle from the needle chamber and into the needle chamber to extend the drug from the reservoir. Push it into the needle and inject the drug into the target organ.

Multiple needle holders and subcutaneous multichannel infusion ports are described by Brown, for example, as taught in US Pat. No. 4,695,273, the contents of which are incorporated herein by reference in their entirety. Is done. According to Brown, multiple needles on the needle holder are inserted through the septum of the infusion port and communicate with an isolated chamber within the infusion port.

Double subcutaneous syringes are described by Horn and are taught, for example, in US Pat. No. 3,552,394, the contents of which are incorporated herein by reference in their entirety. According to the horn, the two needles incorporated within the device are separated by less than 68 mm and may be of different styles and lengths, which allows the injection to be performed at different depths.

Syringes with multiple needles and multiple fluid compartments are described by Hershberg, for example, as taught in US Pat. No. 3,572,336, the contents of which are incorporated herein by reference in their entirety. Is done. According to Hershberg, multiple needles can be incorporated into a syringe with multiple fluid compartments to simultaneously administer incompatible drugs that cannot be mixed into a single infusion.

Surgical instruments for intradermal injection of fluids have been described by Eliscu et al., For example, in US Pat. No. 2,588,623, the contents of which are incorporated herein by reference in their entirety. Incorporated in. According to Eliscu, multiple needles are incorporated into the device to inject fluid into the skin with a wider dispersion.

A device for the simultaneous delivery of substances to ducts within multiple breasts is described by Hung, eg, is taught in European Patent No. 1818017, the contents of which are incorporated herein by reference in their entirety. Incorporated in. According to Hung, multiple lumens are incorporated into the device and inserted through ductal network orifices to deliver fluid to the ductal network.

Catheter for the introduction of the drug into the tissues of the heart or other organs is described by Tkebuchava, for example, as taught in WO 200136138109, the contents of which are incorporated herein by reference in their entirety. Be incorporated. According to Tkebuchava, two curved needles that enter the organ wall in a flat trajectory are incorporated.

Devices for delivering pharmaceutical agents have been described by McCay et al., For example, as taught in WO2006118804, the contents of which are incorporated herein by reference in their entirety. According to McCay, multiple needles with multiple orifices on each needle are incorporated into the device to facilitate local delivery to tissues such as the internal disc space of the spinal disc.

Methods for delivering immunomodulatory substances directly into the intradermal space within mammalian skin have been described by Pettis, for example, taught in WO2004020014, which is described in its entirety by reference. Incorporated into the specification. According to Pettis, multiple needles are incorporated into the device to deliver material through the needles to a depth of 0.3 mm to 2 mm.

Methods and devices for administering substances into at least two compartments of the skin for systemic absorption and improved pharmacokinetics have been described by Pettis et al., For example, as taught in WO 200309945. This content is incorporated herein by reference in its entirety. According to Pettis, multiple needles with a length of about 300 μm to about 5 mm are incorporated into the device and delivered simultaneously to the intradermal and subcutaneous tissue compartments.

Drug delivery devices with needles and rollers have been described by Zimmerman et al., For example, as taught in WO 2012006259, the contents of which are incorporated herein by reference in their entirety. According to Zimmerman, a plurality of hollow needles located within the rollers are incorporated into the device, and as the rollers rotate, the contents in the reservoir are delivered through the needles.

Drug delivery devices such as stents known in the art are taught, for example, in U.S. Pat. Nos. 8,333,799, U.S. Publication No. US2006002039, U.S. Publication No. US2004017212, and U.S. Pat. No. 6,2010,1032, respectively. The content is incorporated herein by reference in its entirety. The polynucleotides, primary constructs, and mRNA formulations described herein can be delivered using stents. In addition, the stents used herein can deliver multiple polynucleotides, primary constructs, and / or mRNAs and / or formulations at the same or different delivery rates. Non-limiting examples of stent manufacturers include CORDIS® (Miami, FL) (CYPHER®), Boston Scientific Corporation (Natick, MA) (TAXUS®), Medtronic (Minneapolis). , MN) (ENDAVOUR®), and Abbott (Abbott Park, IL) (XIENCE V®).

Catheter and / or Lumen Utilization Methods and Devices Catheter and lumen utilization methods and devices can be used to administer the mmRNA of the invention in single, multiple, or divided dosing schedules. Such methods and devices are described below.

Catheter-based delivery from skeletal myoblasts to the myocardium of the injured heart has been described by Jacoby et al. Incorporated into the specification. According to Jacoby, multiple needles are integrated into the device, at least a portion of which is inserted into a blood vessel, through which the cell composition is delivered to a local region of the heart of interest.

Devices for treating asthma with neurotoxins have been described by Deem et al., For example, in US Patent Publication No. 20060225742, the contents of which are incorporated herein by reference in their entirety. .. According to Deem, multiple needles are integrated into the device, through which the neurotoxin is delivered into the bronchial tissue.

Methods for performing multi-component therapies are described by Nayak, for example, as taught in US Pat. No. 7,699,803, the contents of which are incorporated herein by reference in their entirety. According to Nayak, multiple infusion cannulas may be incorporated within the device and may include depth slots to control the depth, where the therapeutic material is delivered into the tissue.

Surgical devices for cleaving channels and delivering at least one therapeutic agent into the desired area of tissue have been described by McIntyre et al., For example, as taught in US Pat. No. 8,012,096. This content is incorporated herein by reference in its entirety. According to McIntyre, multiple needles are integrated into the device, which dispenses the therapeutic agent into the area of tissue surrounding the channel and is particularly well suited for transmyocardial revascularization surgery.

Methods for treating bladder dysfunction in female mammals have been described by Versi et al., For example, in US Pat. No. 8,029,496, the contents of which are incorporated herein by reference in their entirety. Incorporated in. According to Versi, a series of microneedles are incorporated into the device to deliver the therapeutic agent directly into the trigone of the bladder through the needles.

Devices and methods for delivering fluids to flexible biological barriers have been described by Yeshurun et al., For example, US Pat. Nos. 7,998,119 (devices) and 8,007,466 (methods). As taught in, their contents are incorporated herein by reference in their entirety. According to Yeshurun, the microneedles on the device permeate and extend into a flexible biological barrier, and the fluid is injected through the holes in the hollow microneedle.

A method of being placed in the torso for injecting a substance into an area of heart tissue having an epicardial surface and having an epicardium has been described by Bonner et al., For example, U.S. Pat. No. Teached in 7,628,780, this content is incorporated herein by reference in its entirety. According to Bonner, the device has an elongated shaft and a distal injection head for maneuvering the needle into the tissue and injecting the drug into the tissue through the needle.

Devices for sealing puncture wounds have been described by Nielsen et al., For example, in US Pat. No. 7,972,358, the contents of which are incorporated herein by reference in their entirety. According to Nielsen, multiple needles are incorporated into the device to deliver the closure agent into the tissue surrounding the puncture tract.

Methods for myogenesis and angiogenesis are described by Chiu et al., For example, taught in US Pat. No. 6,551,338, the contents of which are incorporated herein by reference in their entirety. .. According to Chiu, 5 to 15 needles with a maximum diameter of at least 1.25 mm and a length effective to provide a puncture depth of 6 to 20 mm are incorporated into the device and inserted near the myocardium and extrinsic. Sexual angiogenesis or myogenic factors are supplied to the myocardium through conduits within at least part of the needle.

Methods for the treatment of prostate tissue are described by Bolmsj et al., For example, in US Pat. No. 6,524,270, the contents of which are incorporated herein by reference in their entirety. According to Bolmsj, the device containing the catheter inserted through the urethra has at least one hollow end that can be extended into the surrounding prostate tissue. Astringents and analgesics are administered into the prostate tissue through its ends.

A method of injecting a fluid into an intraosseous site is described by Findlay et al., For example, taught in US Pat. No. 6,761,726, the contents of which are incorporated herein by reference in their entirety. .. According to Findlay, multiple needles capable of penetrating the hard shell of the material covered with a layer of soft material are incorporated within the device to deliver the fluid down from the hard shell of the material at a defined distance.

Devices for injecting drugs into the walls of blood vessels are described by Vigil et al., For example, in US Pat. No. 5,713,863, the contents of which are incorporated herein by reference in their entirety. Be incorporated. According to Vigil, multiple injectors are placed on each of the flexible tubes within the device, through a multi-lumen catheter into the flexible tube for infusion into the vessel wall, and outside the injector. Introduce the drug fluid into.

Catheter for delivering therapeutic and / or diagnostic agents to tissues surrounding the body passage is described by Faxon et al., For example, taught in US Pat. No. 5,464,395, which is referenced. All of these are incorporated herein by. According to Faxon, at least one needle cannula is incorporated into the catheter and delivers the desired drug to the tissue through the needle that projects out of the catheter.

Balloon catheters for delivering therapeutic agents are described by Orr and are taught, for example, in WO20110024871, the contents of which are incorporated herein by reference in their entirety. According to Orr, multiple needles are incorporated into the device to deliver the therapeutic agent to different depths within the tissue. In another embodiment, the drug-eluting balloon may be used to deliver the formulations described herein. Drug-eluting balloons are used in applications to target lesions such as, but not limited to, intrastent restenosis treating lesions within tortuous vessels, bifurcation lesions, femoral / popliteal lesions, and lesions below the knee. obtain.

Devices for delivering therapeutic agents (eg, polynucleotides, primary constructs, or mmRNAs) to tissues placed around the lumen have been described by Perry et al., For example, as taught in US Patent Publication No. US2010125239. These contents are incorporated herein by reference in their entirety. According to Perry, the catheter has a balloon, which can be coated with a therapeutic agent by methods known in the art and described by Perry. When the balloon inflates, the therapeutic agent comes into contact with surrounding tissue. The device may further have a heat source for varying the temperature of the coating on the balloon to release the therapeutic agent into the tissue.

Current-Utilized Methods and Devices The current-utilized methods and devices can be used to deliver the mmRNAs of the invention according to the single, multiple, or divided dosing regimens taught herein. Such methods and devices are described below.

Electrocollagen-induced therapeutic devices are described by Marquez and are taught, for example, in US Patent Publication No. 20090137945, the contents of which are incorporated herein by reference in their entirety. According to Marquez, multiple needles are incorporated into the device, repeatedly puncturing the skin and pulling some of the material that is initially applied to the skin into the skin area.

Electrodynamic systems have been described by Etheredge et al., For example, are taught in US Patent Publication No. 20070185432, the contents of which are incorporated herein by reference in their entirety. According to Etheredge, microneedles are incorporated into the device and an electric current propels the drug through the needle to the targeted treatment site.

The iontophoresis device is described by Matsumura et al., For example, is taught in US Pat. No. 7,437,189, the contents of which are incorporated herein by reference in their entirety. According to Matsumura, multiple needles can be incorporated into the device to deliver the ionizable drug in vivo at a faster rate or with higher efficiency.

Intradermal delivery of bioactive agents by needleless injection and electroporation has been described by Hoffmann et al., For example, in US Pat. No. 7,171,264, which is described in its entirety by reference. Incorporated herein. According to Hoffmann, one or more needleless injectors are integrated into the electroporation device, and the combination of needleless injection and electroporation is sufficient to introduce the drug into the cells of the skin, muscle, or mucous membranes. Is.

Methods for intracellular delivery via electroporation have been described by Lundkvist et al., For example, in US Pat. No. 6,625,486, the contents of which are described by reference. The whole is incorporated herein. According to Lundkvist, a pair of needle electrodes are incorporated into the catheter. The catheter is located within the lumen of the body and is followed by its needle electrode for permeation into the tissue surrounding the lumen. The device then introduces the drug through at least one of its needle electrodes and applies an electric field at the needle electrode, allowing the drug to pass through the cell membrane into the cell at the treatment site.

Delivery systems for percutaneous immunization have been described by Levin et al., For example, are taught in WO 2006003659, the contents of which are incorporated herein by reference in their entirety. According to Levin, multiple electrodes are incorporated within the device to apply electrical energy between the electrodes to create microchannels in the skin to facilitate transdermal delivery.

Methods for delivering RF energy into the skin have been described by Schomacker, for example, as taught in WO 2011163264, the contents of which are incorporated herein by reference in their entirety. According to Schomacker, multiple needles are incorporated into the device and decompressed to attract the skin into contact with the plate so that the needles insert and deliver RF energy through holes on the plate.

VII. Definitions In various places herein, substituents of the compounds of the present disclosure are disclosed in groups or in scope. The present disclosure is specifically intended to include any individual partial combination of members of such groups and ranges. For example, _{the term "C 1-6} alkyl" is specifically intended to disclose methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C _{6 alkyl individually.}

About: As used herein, the term "about" means +/- 10% of the listed values.

Administered in combination: As used herein, the terms "administered in combination" or "combined dosing" mean that two or more drugs are administered simultaneously or with overlapping effects of each drug on a patient. It means that it is administered to a subject within a certain interval. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the administration of the agents is spaced close enough to each other so that a combined (eg, synergistic) effect is achieved.

Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human being at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and helminths. In some embodiments, the animal is a transgenic animal, genetically modified animal, or clone.

Antigen of interest or desired antigen: As used herein, the term "antigen of interest" or "desired antigen" refers to the antibodies described herein, as well as fragments, variants thereof. Includes the proteins and other biomolecules provided herein that are immunospecifically bound by variants and variants. Examples of target antigens include insulin, insulin-like growth factor, hGH, tPA, interleukin (IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-. 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, Tumor necrosis factor (TNF) such as interferon (IFN) α, IFNβ, IFNγ, IFNω, or IFNτ, TNFα and TNFβ, TNFγ, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1, and VEGF, etc. Cytokines include, but are not limited to.

Approximately: As used herein, the term "approximately" or "approximately" applied to one or more values of interest refers to a value that is similar to a defined reference value. In certain embodiments, the term "approximately" or "about" is defined in any direction (greater than or less than that) of the defined reference value, unless otherwise defined or otherwise apparent from the context. ), 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%. Refers to the range of applicable values within, 5%, 4%, 3%, 2%, 1%, or less (unless such numbers exceed 100% of the possible values).

Meeting with: When used herein, when used with respect to two or more parts, "to be associated with," "complexed," "linked," and "combined." , And the terms "moored", either directly or through one or more additional parts that act as ligators, which parts are physically associated or bound to each other. It is meant that the moiety forms a sufficiently stable structure so that it remains physically associated under the conditions under which the structure is used, eg, physiological conditions. The "association" does not have to go through strictly direct covalent chemical bonds. It may also suggest sufficiently stable ionic or hydrogen bonding or hybridization-based binding so that the "associated" entities remain physically associated.

Bifunctionality: As used herein, the term "bifunctionality" refers to any substance, molecule, or moiety that is capable of maintaining at least two functions. This feature may achieve the same or different results. The structures that provide this function may be the same or different. For example, the bifunctional modified RNA of the present invention may encode a cytotoxic peptide (first function), while the nucleoside containing the coding RNA is itself cytotoxic (second function). be. In this example, delivery of the bifunctional modified RNA to cancer cells not only results in a peptide or protein molecule that can ameliorate or treat the cancer, but in the unlikely event that degradation occurs instead of translation of the modified RNA. If there is, it will deliver the cytotoxic payload of the nucleoside to the cells.

Biocompatibility: As used herein, the term "biocompatibility" poses little or no risk of injury, toxicity, or rejection by the immune system and is a living cell, tissue, organ, or system. Means that it is compatible with.

Biodegradability: As used herein, the term "biodegradable" means that it can be degraded to harmless products by the action of living organisms.

Biologically active: As used herein, the expression "biologically active" refers to the characteristics of any substance that is active in a biological system and / or organism. For example, a substance that has a biological effect on an organism when administered to the organism is considered biologically active. In certain embodiments, the polynucleotide, primary construct, or mmRNA of the invention is biologically active or biologically relevant, even in a small portion of the polynucleotide, primary construct, or mmRNA. If it mimics the activity that is considered to be, it can be considered biologically active.

Chemical terms: The following provide definitions of various chemical terms from "acyl" to "thiol".

As used herein, the term "acyl" refers to a hydrogen or alkyl group as defined herein (eg, an alkyl group) that is attached to a parent molecular group via a carbonyl group as defined herein. It represents a haloalkyl group) and is shown as an example by formyl (ie, a carboxylaldehyde group), acetyl, propionyl, butanoyl and the like. The exemplified unsubstituted acyl group contains 1 to 7, 1 to 11, or 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "acylamino" refers to an acyl group as defined herein, which is attached to a parent molecular group via an amino group as defined herein. -N ( ^RN1 ) -C (O) -R, where R is an H or optionally substituted C _1-6 , C _1-10 , or C _1-20 alkyl group. And ^RN1 is as defined herein). An exemplary unsubstituted acylamino group contains 1 to 41 carbons (eg, 1 to 7, 1 to 13, 1 to 21, 2 to 7, 2 to 13, 2 to 21, or 2 to 41 carbons). include. In some embodiments, the alkyl group is further substituted with 1, 2, 3 or 4 substituents described herein and / or the amino group is -NH ₂ or -NHR ^N1 . , ^{wherein, R N1} is _{^{independently, OH, NO 2, NH 2}} , NR N2 2, SO 2 oR N2, SO 2 R N2, SOR N2, alkyl or aryl, and each ^{R N2} is H , Alkyl, or aryl.

As used herein, the term "acyloxy" refers to an acyl group as defined herein that is attached to a parent molecular group via an oxygen atom (ie, -OC (O)). -R, where R is an H or optionally substituted C _1-6 , C _1-10 , or C _1-20 alkyl group). An exemplary unsubstituted acyloxy group contains 1 to 21 carbons (eg, 1 to 7 or 1 to 11 carbons). In some embodiments, the alkyl group is further substituted with 1, 2, 3 or 4 substituents described herein and / or the amino group is -NH ₂ or -NHR ^N1 . , ^{wherein, R N1} is ^{_{independently, OH, NO 2, NH 2}} , NR N2 2, SO 2 oR N2, SO 2 R N2, SOR N2, alkyl or aryl, and each ^{R N2} is H , Alkyl, or aryl.

As used herein, the term "alkary" refers to an aryl group as defined herein, which is attached to a parent molecular group via an alkylene group as defined herein. show. An exemplary unsubstituted alkaline group is 7 to 30 carbons (eg, C _1-6 alk-C _6-10 aryl, C _1-10 alk-C _6-10 aryl, or C _1-20 alk-C. 7 to 16 or 7 to 20 carbons, such as _{6-10 aryl).} In some embodiments, the alkylene and aryl can be further substituted with 1, 2, 3, or 4 substituents as defined herein for each group, respectively. Other groups following the prefix "Arc-" are defined in the same manner, where "Arc" _{refers to C 1-6} alkylene, unless otherwise noted, and the chemical structure to which it is attached is herein. As defined in.

The term "alkcycloalkyl" is used via an alkylene group as defined herein (eg, an alkylene group of 1-4, 1-6, 1-10, or 1-20 carbons). Represents a cycloalkyl group as defined herein that is attached to a parent molecular group. In some embodiments, the alkylene and cycloalkyl can be further substituted with 1, 2, 3, or 4 substituents as defined herein for each group, respectively.

As used herein, the term "alkenyl" refers to 2 to 20 carbons (eg, 2 to 6 or 2 to 2) containing one or more carbon-carbon double bonds, unless otherwise specified. Represents a monovalent linear or branched chain group of 10 carbons) and is illustrated by, for example, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. .. Alkenyl contains both cis and trans isomers. The alkenyl group is an amino, aryl, cycloalkyl, or heterocyclyl (eg, heteroaryl) independently selected 1, 2, 3, or 4 substituents, or herein. It may be optionally substituted with any of the exemplified alkyl substituents described in.

The term "alkenyloxy" refers to a chemical substituent of formula-OR, where R is a C _2-20 alkenyl group (eg, C _2-6 or C _2-10 alkenyl) unless otherwise specified. be. Illustrated alkenyloxy groups include ethenyloxy, propenyloxy and the like. In some embodiments, the alkenyl group can be further substituted with 1, 2, 3, or 4 substituents (eg, hydroxy groups) as defined herein.

The term "alkheteroaryl" refers to a heteroaryl group as defined herein, which is attached to a parent molecular group via an alkylene group as defined herein. An exemplary unsubstituted arc heteroaryl group may have 2-32 carbons (eg, C _1-6 arc-C _1-12 heteroaryl, C _1-10 arc-C _1-12 heteroaryl, or C _1-20. 2-22, 2-18, 2-17, 2-16, 3-15, 2-14, 2-13, or 2-12 carbons, such as ALC-C _{1-12 heteroaryl).} In some embodiments, the alkylene and heteroaryl can be further substituted with 1, 2, 3, or 4 substituents as defined herein for each group, respectively. An arc heteroaryl group is a subset of an arc heterocyclyl group.

The term "alkheterocyclyl" refers to a heterocyclyl group as defined herein, which is attached to a parent molecular group via an alkylene group as defined herein. An exemplary unsubstituted alkheterocyclyl group comprises 2-32 carbons (eg, C _1-6 alk-C _1-12 heterocyclyl, C _1-10 alk-C _1-12 heterocyclyl, or C _1-20 alk-C. 2-22, 2-18, 2-17, 2-16, 3-15, 2-14, 2-13, or 2-12 carbons, such as _{1-12 heterocyclyl).} In some embodiments, the alkylene and heterocyclyl can be further substituted with 1, 2, 3, or 4 substituents as defined herein for each group, respectively.

The term "alkoxy" refers to a chemical substituent of formula-OR, where R is a C _1-20 alkyl group (eg, C _1-6 or C _1-10 alkyl) unless otherwise specified. .. Examples of alkoxy groups include methoxy, ethoxy, propoxy (eg, n-propoxy and isopropoxy), t-butoxy and the like. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents (eg, hydroxy or alkoxy) as defined herein.

The term "alkoxyalkoxy" refers to an alkoxy group substituted with an alkoxy group. An exemplary unsubstituted alkoxyalkoxy group may have 2 to 40 carbons (eg, C _1-6 alkoxy-C _1-6 alkoxy, C _1-10 alkoxy-C _1-10 alkoxy, or C _1-20 alkoxy-C. _Contains 2-12 or 2-20 carbons, such as 1-20 alkoxy. In some embodiments, each alkoxy group can be further substituted with 1, 2, 3, or 4 substituents as defined herein.

The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group. An exemplary unsubstituted alkoxyalkyl group may have 2 to 40 carbons (eg, C _1-6 alkoxy-C _1-6 alkyl, C _1-10 alkoxy-C _1-10 alkyl, or C _1-20 alkoxy-C. _Contains 2-12 or 2-20 carbons, such as 1-20 alkyl). In some embodiments, alkyl and alkoxy can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for each group.

As used herein, the term "alkoxycarbonyl" refers to an alkoxy defined herein that is attached to a parent molecular group via a carbonyl atom (eg, -C (O) -OR). And in the formula, R is an H or optionally substituted C _1-6 , C _1-10 , or C _1-20 alkyl group). An exemplary unsubstituted alkoxycarbonyl contains 1 to 21 carbons (eg, 1 to 11 or 1 to 7 carbons). In some embodiments, the alkoxy group is further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "alkoxycarbonylalkoxy" refers to an alkoxy group defined herein that is substituted with an alkoxycarbonyl group defined herein (eg, -O-). Alkoxy-C (O) -OR, where R is an optionally substituted C _1-6 , C _1-10 , or C _1-20 alkyl group). The exemplified unsubstituted alkoxycarbonylalkoxy is 3 to 41 carbons (eg, C _1-6 alkoxycarbonyl-C _1-6 alkoxy, C _1-10 alkoxycarbonyl-C _1-10 alkoxy, or C _1-20 alkoxy. 3-10, 3-13, 3-17, 3-21, or 3-31 carbons, such as carbonyl-C _{1-20 alkoxy).} In some embodiments, each alkoxy group is independently further substituted with 1, 2, 3, or 4 substituents (eg, hydroxy groups) described herein.

As used herein, the term "alkoxycarbonylalkyl" refers to an alkyl group as defined herein (eg, -alkyl-, which is substituted with an alkoxycarbonyl group as defined herein. C (O) -OR, where R is an optionally substituted C _1-20 , C _1-10 , or C _1-6 alkyl group). The exemplified unsubstituted alkoxycarbonylalkyl is 3 to 41 carbons (eg, C _1-6 alkoxycarbonyl-C _1-6 alkyl, C _1-10 alkoxycarbonyl-C _1-10 alkyl, or C _1-20 alkoxy. Includes 3-10, 3-13, 3-17, 3-21, or 3-31 carbons, such as carbonyl-C _{1-20 alkyl.} In some embodiments, each alkyl and alkoxy group is independently further substituted with 1, 2, 3, or 4 substituents (eg, hydroxy groups) described herein.

As used herein, the term "alkyl" refers to saturated groups of 1 to 20 (eg, 1 to 10 or 1 to 6) carbons, both straight chain and branched chain, unless otherwise specified. include. Alkyl groups are shown as examples by methyl, ethyl, n- and iso-propyl, n-, sec-, iso-, and tert-butyl, neopentyl, etc. and are independently selected from the group consisting of: It may be optionally substituted with 1, 2 or 3 substituents or, in the case of 2 or more carbon alkyl groups, with 4 substituents: (1) C _1-6 alkoxy; (2). ) C _1-6 alkylsulfinyl; (3) Amino (eg, unsubstituted amino (ie, -NH ₂ ) or substituted amino (ie, -N ( ^RN1 ) ₂ ) as defined herein, in the formula, R. ^N1 is as defined for amino); (4) C _6-10 aryl-C _1-6 alkoxy; (5) azide; (6) halo; (7) (C _2-9 heterocyclyl) oxy; (8) Hydroxy; (9) Nitro; (10) Oxo (eg, carboxylaldehyde or acyl); (11) C _1-7 Spirocyclyl; (12) Thioalkyl; (13) Thiol; (14) -CO ₂ R ^{A '(wherein,} R ^A' is, _{(a) C 1-20} alkyl _{(e.g., C 1-6 alkyl), (b) C 2-20} alkenyl _{(e.g., C 2-6} alkenyl), (c) C _6-10 aryl, (d) hydrogen, (e) C _1-6 alk-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g)-(CH ₂ ) _s2 (OCH ₂ CH) ₂ ) _s1 (CH ₂ ) _{s3 OR'polyethylene} glycol (in the formula, s1 is an integer of 1-10 (eg 1-6 or 1-4), and each of s2 and s3 is independent. Integers from 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), where R'is H or C _1-20 alkyl), and (h). ) -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 amino-polyethylene glycol (in the formula, s1 is 1-10 (eg, 1-6 or 1-4)). Each of s2 and s3 is independently an integer of 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R is ^N1 is independently a hydrogen or _{C 1-6} alkyl optionally substituted) is selected from the group consisting of); (15) -C (O ) NR B 'R C' ( wherein each of, R ^{B ',} and R ^C' is independently, (a (Selected from the group consisting of hydrogen, (b) C _1-6 alkyl, (c) C _6-10 aryl, and (d) C _1-6 arc-C _6-10 aryl); (16) -SO ₂ ^RD' (In the formula, ^RD' is (a) C _1-6 alkyl, (b) C _6-10 aryl, (c) C _1-6 arc-C _6-10 aryl, and (d). It is selected from the group consisting of hydroxy); (each ^{_{17) -SO 2 NR E 'R}} F' ( wherein, R ^{E 'and} R ^F' is independently, (a) hydrogen, (b) C _{Selected from the group consisting of 1-6} alkyl, (c) C _6-10 aryl, and (d) C _1-6 arc-C _6-10 aryl); (18) -C (O) ^RG' ( wherein, R ^{G 'is,} _{(a) C 1-20} alkyl _{(e.g., C 1-6 alkyl), (b) C 2-20} alkenyl _{(e.g., C 2-6 alkenyl),} (c) _{C 6- 10} aryl, (d) hydrogen, (e) C _1-6 arc-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g)-(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _{Polyethylene glycol of s3} OR'(in the formula, s1 is an integer of 1-10 (eg, 1-6 or 1-4), and each of s2 and s3 is 0-10 independently. (For example, 0-4, 0-6, 1-4, 1-6, or 1-10), where R'is H or C _1-20 alkyl), and (h) -NR. ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 amino-polyethylene glycol (in the formula, s1 is an integer of 1 to 10 (eg, 1 to 6 or 1 to 4). There, each of s2 and s3 are, independently, 0 (e.g., 0～4,0～6,1～4,1～6 or 10) is an integer, and each ^{R N1} is independently, is hydrogen, or _{C 1-6} alkyl optionally substituted) is selected from the group consisting ^{of); (19) -NR H '} C (O) R I' ( wherein, R ^{H 'is} selected from the group consisting of (a1) hydrogen and _{(b1) C 1-6} alkyl, R ^I' is _{(a2) C 1-20} alkyl _{(e.g., C 1-6} alkyl), (b2 ) C _2-20 alkenyl (eg C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 arc-C _{6-10 a} Reel, (f2) amino-C _1-20 alkyl, (g2)-(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _{s3 OR'polyethylene} glycol (in the formula, s1 is 1-10 (in the formula) For example, it is an integer of 1 to 6 or 1 to 4), and each of s2 and s3 is independently 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1). -10), where _R'is H or C 1-20 alkyl), and (h2) -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 (In the formula, s1 is an integer of 1 to 10 (eg, 1 to 6 or 1 to 4), and each of s2 and s3 is independently 0 to 10 (eg, 0 to 0). 4,0～6,1～4,1～6, or 10) is an integer, and each ^{R N1} is independently a hydrogen, or _{C 1-6} alkyl optionally substituted is selected from the group consisting of ^{certain)); (20) -NR J} 'C (O) oR K' ( wherein, R ^{J 'is} a group consisting of (a1) hydrogen and _{(b1) C 1-6} alkyl is selected from, R ^{K 'is,} _{(a2) C 1-20} alkyl _{(e.g., C 1-6 alkyl), (b2) C 2-20} alkenyl _{(e.g., C 2-6} alkenyl), (c2) _{C 6 -10} aryl, (d2) hydrogen, (e2) C _1-6 arc-C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2-)-(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _{The polyethylene glycol of s3} OR'(in the formula, s1 is an integer of 1 to 10 (eg, 1 to 6 or 1 to 4), and each of s2 and s3 is 0 to 0 independently. An integer of 10 (eg, 0-4, 0-6, 1-4, 1-6, or 1-10), where R'is H or C _1-20 alkyl), and (h2)-. NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 amino-polyethylene glycol (in the formula, s1 is an integer of 1-10 (eg, 1-6 or 1-4)). , and the each of s2 and s3 are, independently, 0 (e.g., 0～4,0～6,1～4,1～6 or 10) is an integer, and each ^{R N1} is , Independently hydrogen or optionally substituted C _1-6 alkyl) (Selected from the group); and (21) amidine. In some embodiments, each of these groups can be further substituted as described herein. For example, C 1 _- alkylene group alkaryl, be further substituted by an oxo group, can result in the respective aryloyl (aryloyl) substituent.

As used herein, the terms "alkylene" and the prefix "alk-" refer to saturated divalent hydrocarbon groups derived from linear or branched saturated hydrocarbons by the removal of two hydrogen atoms. It is represented and is shown as an example by methylene, ethylene, isopropylene and the like. _{The terms "C xy} alkylene" and the prefix "C _xy alk-" represent an alkylene group having xy carbons. The exemplary values for x are 1, 2, 3, 4, 5, and 6, and the exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 , 16, 18, or 20 (eg, C _1-6 , C _1-10 , C _2-20 , C _2-6 , C _2-10 , or C _2-20 alkylene). In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituents as defined herein for the alkyl group.

As used herein, the term "alkylsulfinyl" refers to an alkyl group attached to a parent molecular group via an -S (O) -group. An exemplary unsubstituted alkylsulfinyl group is 1-6, 1-10, or 1-20 carbons. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents as defined herein.

As used herein, the term "alkylsulfinylalkyl" refers to an alkyl group as defined herein, substituted with an alkylsulfinyl group. An exemplary unsubstituted alkyl sulfinyl alkyl group is 2-12, 2-20, or 2-40 carbons. In some embodiments, each alkyl group can be further substituted with 1, 2, 3, or 4 substituents as defined herein.

As used herein, the term "alkynyl" refers to 2 to 20 carbon atoms (eg, 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond. ), Represents a monovalent straight chain or branched chain group, and is represented by ethynyl, 1-propynyl, etc. as an example. The alkynyl group is an aryl, cycloalkyl, or heterocyclyl (eg, heteroaryl) independently selected 1, 2, 3, or 4 substituents as defined herein, or described herein. Any of the above-exemplified alkyl substituents may be optionally substituted.

The term "alkynyloxy" refers to a chemical substituent of formula-OR, where R is a C _2-20 alkynyl group (eg, C _2-6 or C _2-10 alkynyl) unless otherwise specified. be. Illustrated alkynyloxy groups include ethynyloxy, propynyloxy and the like. In some embodiments, the alkynyl group can be further substituted with 1, 2, 3, or 4 substituents (eg, hydroxy groups) as defined herein.

As used herein, the term "amidine" refers to _two -C (= NH) NHs.

As used herein, the term "amino", ^{-N (R} _N1) represents _2, wherein each ^{R N1} is _{independently, H, OH, NO 2,} N (R N2) ₂ , SO ₂ OR ^N2 , SO ₂ ^RN2 , SOR ^N2 , N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkalil, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl, heterocyclyl (eg, Heteroaryl), or alkheterocyclyl (eg, alkheteroaryl), each of these listed ^RN1 groups can be optionally substituted as defined herein for each group, or 2 One of ^{R N1} combine to form a heterocyclyl or N-protecting group, each ^{R N2} is independently H, alkyl or aryl. The amino group of the present invention can be an unsubstituted amino (ie, -NH ₂ ) or a substituted amino (ie, -N ( ^RN1 ) ₂ ). In a preferred embodiment, amino is -NH ₂ or -NHR ^{N1, wherein, R N1} is _{^{independently, OH, NO 2, NH 2}} , NR N2 2, SO 2 OR N2, SO 2 R N2 , SOR ^N2 , alkyl, carboxyalkyl, sulfoalkyl, or aryl, and each ^RN2 can be H, C _1-20 alkyl (eg, C _1-6 alkyl), or C _6-10 aryl.

The term "amino acid" as used herein refers to a molecule having a side chain, an amino group, and an acidic group (eg, a carboxy group of _{-CO 2 H or a sulfo group of -SO 3 H).} , Side chain, amino group, or acidic group (eg, side chain) to attach to the parent molecular group. In some embodiments, the amino acid is attached to the parent molecular group by a carbonyl group, and this side chain or amino group is attached to the carbonyl group. Illustrated side chains include optionally substituted alkyls, aryls, heterocyclyls, alkalines, alkheterocyclyls, aminoalkyls, carbamoylalkyls, and carboxyalkyls. Examples of amino acids include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolidine, selenocysteine, Includes serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid group is optionally 1, 2 or 3 substituents selected independently from the group consisting of the following, or 4 substituents in the case of 2 or more carbon amino acid groups. May be substituted: (1) C _1-6 alkoxy; (2) C _1-6 alkylsulfinyl; (3) amino (eg, unsubstituted amino (ie, -NH ₂ )) as defined herein or Substituted amino (ie, -N ( ^RN1 ) ₂ (where ^RN1 is defined for amino in the formula)); (4) C _6-10 aryl-C _1-6 alkoxy; (5) azide (6) Halo; (7) (C _2-9 heterocyclyl) oxy; (8) hydroxy; (9) nitro; (10) oxo (eg, carboxylaldehyde or acyl); (11) C _1-7 spirocyclyl; (12) thioalkoxy; (13) ^{_{thiol; (14) -CO 2 R A}} '( wherein, R ^A' is, _{(a) C 1-20} alkyl _{(e.g., C 1-6} alkyl), (b) C _2-20 alkenyl (eg C _2-6 alkenyl), (c) C _6-10 aryl, (d) hydrogen, (e) C _1-6 arc-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g)-(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _{s3 OR'polyethylene} glycol (in the formula, s1 is 1-10 (eg 1-6 or 1-). 4), each of s2 and s3 is independently an integer of 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). _R'is H or C 1-20 alkyl), and (h) -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 amino-polyethylene glycol (in the formula). , S1 are integers from 1 to 10 (eg, 1 to 6 or 1 to 4), and each of s2 and s3 is independently 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 1). 4,1～6 or 10) is an integer of, each ^{R N1} is independently selected from the group consisting of is hydrogen or a _{C 1-6} alkyl optionally substituted) that); (15) -C (O ) NR B 'R C' ( wherein each of R ^{B 'and} R ^C' is independently, (a) hydrogen, _{(b) C 1-6} alkyl, _Is it a group consisting of (c) C _6-10 aryl and (d) C _1-6 alk-C 6-10 aryl? (16) -SO ₂ ^RD' (in the formula, ^RD' is (a) C _1-6 alkyl, (b) C _6-10 aryl, (c) C _1-6 arc. -C _6-10 aryl, and (d) is selected from the group consisting of _{hydroxy); (17) -SO 2 NR} E 'R F' ( wherein each of R ^{E 'and} R ^F' is independently (A) selected from the group consisting of (a) hydrogen, (b) C _1-6 alkyl, (c) C _6-10 aryl, and (d) C _1-6 arc-C _{6-10 aryl);} 18) -C (O) ^RG' (in the formula, ^RG' is (a) C _1-20 alkyl (eg C _1-6 alkyl), (b) C _2-20 alkenyl (eg C _{2). -6} alkenyl), (c) C _6-10 aryl, (d) hydrogen, (e) C _1-6 arc-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g)-( CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _{s3 OR'polyethylene} glycol (in the formula, s1 is an integer of 1-10 (eg 1-6 or 1-4), s2 and s3 Each of these is independently an integer from 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), where R'is H or C _1-20. (Alkyl), and (h) -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 amino-polyethylene glycol (in the formula, s1 is 1-10 (eg, eg) 1 to 6 or 1 to 4), each of s2 and s3 independently 0 to 10 (eg 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). ), And each ^RN1 is independently selected from the group consisting of ( _{C 1-6} alkyl, which is either hydrogen or optionally substituted ^{); (19) -NR H'.} C (O) R ^{I '(wherein,} R ^H' is selected from the group consisting of (a1) hydrogen and _{(b1) C 1-6} alkyl, R ^{I 'is} _{(a2) C 1-20} alkyl ( For example, C _1-6 alkyl), (b2) C _2-20 alkenyl (eg C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 arc- C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2)-(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂₎ ) _{Polyethylene glycol of s3} OR'(in the formula, s1 is an integer of 1-10 (eg, 1-6 or 1-4), and each of s2 and s3 is independently 0-10 (eg, eg). 0-4, 0-6, 1-4, 1-6, or 1-10), where R'is H or C _1-20 alkyl), and (h2) -NR ^N1 (CH). ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 amino-polyethylene glycol (in the formula, s1 is an integer of 1-10 (eg, 1-6 or 1-4), s2 and each s3, independently, 0 (e.g., 0～4,0～6,1～4,1～6 or 10) is an integer, and each ^{R N1} is independently is hydrogen or _{C 1-6} alkyl optionally substituted) is selected from the group consisting ^{of); (20) -NR J '} C (O) oR K' ( wherein, R ^{J '} is, (a1) is selected from the group consisting of hydrogen and _{(b1) C 1-6} alkyl, R ^{K 'is,} _{(a2) C 1-20} alkyl _{(e.g., C 1-6} alkyl), (b2) _{C 2 -20} alkenyl (eg C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 arc-C _6-10 aryl, (f2) amino-C _{1- 20} Alkyl, (g2)-(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _{s3 OR'polyethylene} glycol (in the formula, s1 is 1-10 (eg 1-6 or 1-4)) Each of s2 and s3 is an integer of 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10) independently of R'. Is an H or C _1-20 alkyl), and (h2) -NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH _{2 ) s3} NR ^N1 amino-polyethylene glycol (s1 in the formula). Is an integer of 1 to 10 (eg, 1 to 6 or 1 to 4), and each of s2 and s3 is independently 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1-6, or 1-10), each ^RN1 is independently selected from the group consisting of hydrogen or optionally substituted C _{1-6 alkyl).} And (21) Amidin. In some embodiments, each of these groups can be further substituted as described herein.

As used herein, the term "aminoalkoxy" refers to an alkoxy group as defined herein, substituted with an amino group as defined herein. Each alkyl and amino, 1, 2, and 3 described herein for each of the groups or 4 substituents, (e.g., _CO 2 R ^{A '(wherein,} R ^A' is (a) _{C 1 (Selected from the group consisting of -6} alkyl, (b) C _6-10 aryl, (c) hydrogen, and (d) C _1-6 arc-C _6-10 aryl, eg, carboxy)). obtain.

As used herein, the term "aminoalkyl" refers to an alkyl group as defined herein, substituted with an amino group as defined herein. Each alkyl and amino, 1, 2, and 3 described herein for each of the groups or 4 substituents, (e.g., _CO 2 R ^{A '(wherein,} R ^A' is (a) _{C 1 (Selected from the group consisting of -6} alkyl, (b) C _6-10 aryl, (c) hydrogen, and (d) C _1-6 arc-C _6-10 aryl, eg, carboxy)). obtain.

As used herein, the term "aryl" represents a monocyclic, bicyclic, or polycyclic carbocyclic ring system with one or two aromatic rings, phenyl, naphthyl. , 1,2-Dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthrasenyl, phenylanthrenyl, fluorenyl, indanyl, indenyl, etc., as examples and independently selected from the group consisting of: 1, 2, 3, 4 or 5 optionally may be substituted with a substituent,: _{(1) C 1-7} acyl (e.g., _{carboxaldehyde); (2) C 1-20} alkyl _{(e.g., C 1- 6-} alkyl, C _1-6 alkoxy-C _1-6 alkyl, C _1-6 alkyl sulfinyl-C _1-6 alkyl, amino-C _1-6 alkyl, azide-C _1-6 alkyl, (carboxyaldehyde) -C _1-6 alkyl, halo-C _1-6 alkyl (eg, perfluoroalkyl), hydroxy-C _1-6 alkyl, nitro-C _1-6 alkyl, or C _1-6 thioalkoxy-C _1-6 alkyl; (3) C _1-20 alkoxy (eg, C _1-6 alkoxy such as perfluoroalkoxy); (4) C _1-6 alkylsulfinyl; (5) C _6-10 aryl; (6) amino; (7) C _1-6 Alc-C _6-10 Aryl; (8) Azide; (9) C _3-8 Cycloalkyl; (10) C _1-6 Alc-C _3-8 Cycloalkyl; (11) Halo; (12) C _1-12 heterocyclyl (eg, C _1-12 heteroaryl); (13) (C _1-12 heterocyclyl) oxy; (14) hydroxy; (15) nitro; (16) C _1-20 thioalkoxy (eg, eg) C _{1-6 thioalkoxy); (17) - (CH} 2) q CO 2 R a '( wherein, q is an integer of 0 to 4, R ^a' is _{(a) C 1-6} alkyl , (B) C _6-10 aryl, (c) hydrogen, and (d) C _1-6 arc-C _6-10 aryl selected from the group); (18)-(CH ₂ ) _q CONR ^{B. 'R C'} (wherein, q is an integer of 0 to 4, R ^{B ',} and R ^C' is (a) hydrogen, _{(b) C 1-6} alkyl, _{(c) C 6-10} aryl , And (d) _{selected independently from the group consisting of C 1-6} arc-C _6-10 aryl); (19)-(CH ₂ ) _q SO ₂ R ^{D '(wherein,} q is an integer of 0 to 4, R ^D' is (a) alkyl, _{(b) C 6-10} aryl, and (c) the group consisting of alk _{-C 6-10} aryl is selected _{from); (20) - (CH} 2) q SO 2 NR E 'R F' ( wherein, q is an integer of 0 to 4, each R ^{E 'and} R ^F' is independently (A) selected from the group consisting of (a) hydrogen, (b) C _1-6 alkyl, (c) C _6-10 aryl, and (d) C _1-6 alkyl-C _{6-10 aryl);} (21) Thiols; (22) C _6-10 Aryloxy; (23) C _3-8 Cycloalkoxy; (24) C _6-10 Aryl-C _1-6 Alkoxy; (25) C _1-6 Arc-C _1-12 heterocyclyls (eg, C _1-6 arc-C _1-12 heteroaryl); (26) C _2-20 alkenyl; and (27) C _2-20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, _{the alkylene group of C 1} -alkalyl or C _1- alkheterocyclyl can be further substituted with an oxo group to give the respective allylloyl and (heterocyclyl) oil substituents.

As used herein, the term "arylalkoxy" refers to an alkalil group as defined herein, which is attached to a parent molecular group via an oxygen atom. An exemplary unsubstituted alkoxyalkyl group may have 7 to 30 carbons (eg, C _6-10 aryl-C _1-6 alkoxy, C _6-10 aryl-C _1-10 alkoxy, or C _6-10 aryl-C. Contains 7-16 or 7-20 carbons, such as _{1-20 alkoxy.} In some embodiments, the arylalkoxy groups can be substituted with 1, 2, 3, or 4 substituents as defined herein.

The term "aryloxy" refers to a chemical substituent of formula-OR', where R'is an aryl group of 6-18 carbons, unless otherwise stated. In some embodiments, the aryl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.

As used herein, the term "allyloyl" refers to an aryl group as defined herein that is attached to a parent molecular group via a carbonyl group. The exemplified unsubstituted allyloyl group is of 7 to 11 carbons. In some embodiments, the aryl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.

The term "azide" refers to _three -N groups, which can also be expressed as -N = N = N.

As used herein, the term "bicyclic" refers to a structure with two rings that can be aromatic or non-aromatic. Bicyclic structures include a spirocyclyl group as defined herein, and two rings that share one or more crosslinks, such as one atom, or two, three. , Or can include chains containing more atoms. Illustrated bicyclic groups include bicyclic carbocyclyl groups (the first and second rings thereof are carbocyclyl groups as defined herein); bicyclic aryl groups (the first and second rings thereof). The ring is an aryl group as defined herein); a bicyclic heterocyclyl group (the first ring of which is a heterocyclyl group and the second ring is a carbocyclyl (eg, aryl) or heterocyclyl (eg, aryl)). (For example, a heteroaryl) group; and a bicyclic heteroaryl group (the first ring of which is a heteroaryl group and the second ring is carbocyclyl (eg, aryl) or heterocyclyl (eg, heteroaryl). ) Is included). In some embodiments, bicyclic groups can be substituted with 1, 2, 3, or 4 substituents as defined herein for cycloalkyl, heterocyclyl, and aryl groups.

_{As used herein, the terms "cyclic" and "carbocyclyl" are optionally substituted C 3-12} monocycles in which a ring, which can be aromatic or non-aromatic, is formed by carbon atoms. Refers to ring, bicyclic, or tricyclic structures. Carbocyclic structures include cycloalkyl, cycloalkenyl, and aryl groups.

As used herein, the term "carbamoyl" stands for -C (O) -N ( ^RN1 ) ₂ , and in the formula, ^{the meaning of each RN1} is the "amino" provided herein. Is found in the definition of.

As used herein, the term "carbamoylalkyl" refers to an alkyl group as defined herein, substituted with a carbamoyl group as defined herein. Alkyl groups can be further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "carbamyl" ^{refers to a carbamate group having the structure -NR N1} C (= O) OR or -OC (= O) N ( ^RN1 ) ₂ , and in the formula, each ^{The meaning of RN1} is found in the definition of "amino" provided herein, where R is an alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (eg,) as defined herein. , Heteroaryl), or alkheterocyclyl (eg, alkheteroaryl).

As used herein, the term "carbonyl" represents a C (O) group, which can also be represented as C = O.

The term "carboxyaldehyde" refers to an acyl group having a structure-CHO.

As used herein, the term "carboxy" means _{-CO 2 H.}

As used herein, the term "carboxyalkoxy" refers to an alkoxy group as defined herein, substituted with a carboxy group as defined herein. Alkoxy groups can be further substituted with 1, 2, 3, or 4 substituents described herein for alkyl groups.

As used herein, the term "carboxyalkyl" refers to an alkyl group as defined herein, substituted with a carboxy group as defined herein. Alkyl groups can be further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "cyano" represents a -CN group.

The term "cycloalkoxy" refers to a chemical substituent of formula-OR, where R is a C _3-8 cycloalkyl group as defined herein, unless otherwise stated. Cycloalkyl groups can be further substituted with 1, 2, 3, or 4 substituents described herein. The exemplified unsubstituted cycloalkoxy group is 3 to 8 carbons. In some embodiments, the cycloalkyl group can be further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "cycloalkyl" represents a monovalent saturated or unsaturated non-saturated non-aromatic cyclic hydrocarbon group of 3-8 carbons, unless otherwise specified, cyclopropyl, cyclobutyl. , Cyclopentyl, Cyclohexyl, Cycloheptyl, Bicyclo [2.2.1. ] Shown as an example by Heptil et al. When a cycloalkyl group contains one carbon-carbon double bond, the cycloalkyl group can be referred to as a "cycloalkenyl" group. Illustrated cycloalkenyl groups include cyclopentenyl, cyclohexenyl and the like. The cycloalkyl groups of the present invention can be optionally substituted with: (1) C _1-7 acyl (eg, carboxylaldehyde); (2) C _1-20 alkyl (eg, C _1-6 alkyl, C _1-6 alkoxy _{-C 1-6 alkyl, C 1-6} alkylsulfinyl _{-C 1-6} alkyl, amino _{-C 1-6} alkyl, azido _{-C 1-6} alkyl, _{(carboxaldehyde) -C 1-6} Alkyl, halo-C _1-6 alkyl (eg, perfluoroalkyl), hydroxy-C _1-6 alkyl, nitro-C _1-6 alkyl, or C _1-6 thioalkoxy-C _1-6 alkyl); (3) C _1-20 alkoxy (e.g., perfluoroalkoxy, etc. _{C 1-6} alkoxy); _{(4) C 1-6} alkylsulfinyl, _{(5) C 6-10} aryl; (6) amino, _{(7) C 1-6} Alkoxy _6-10 aryl; (8) azide; (9) C _3-8 cycloalkyl; (10) C _1-6 alcohol C _3-8 cycloalkyl; (11) halo; (12) C _{1- 12} heterocyclyl _{(e.g., C 1-12} heteroaryl); _{(13) (C 1-12} heterocyclyl) oxy; (14) hydroxy; (15) nitro; _{(16) C 1-20} thioalkoxy _{(e.g., C 1- 6 thioalkoxy); (17) - (CH} 2) q CO 2 R a '( wherein, q is an integer of 0 to 4, R ^a' is _{(a) C 1-6} alkyl, (b ) _{C 6-10} aryl is selected from the group consisting of (c) hydrogen, and _{(d) C 1-6} alk _{-C 6-10 aryl); (18) - (CH} 2) q CONR B 'R C ^'(wherein, q is an integer of 0 to 4, R ^B', and R ^{C 'is} (a) hydrogen, _{(b) C 6-10} alkyl, _{(c) C 6-10} aryl, and ( _{d) C 1-6} is independently selected from the group consisting of alk _{-C 6-10 aryl); (19) - (CH} 2) q SO 2 R D '( wherein, q is 0 to 4 is an integer, R ^{D 'is} selected from _{(a) C 6-10} alkyl, _{(b) C 6-10} aryl, and _{(c) C 1-6} group consisting alkaryl _{-C 6-10} aryl) _{; (20) - (CH 2} ) q SO 2 NR E 'R F' ( wherein, q is an integer of 0 to 4, each R ^{E 'and} R ^F' is independently, (a ) Hydrogen, (b) C _{6-1 0} alkyl is selected from _{(c) C 6-10} aryl, and _{(d) C 1-6} group consisting alkaryl _{-C 6-10} aryl); (21) thiol; _{(22) C 6-10} aryloxy (23) C _3-8 cycloalkoxy; (24) C _6-10 aryl-C _1-6 alkoxy; (25) C _1-6 alk-C _1-12 heterocyclyl (eg, C _1-6 alk-C) _1-12 heteroaryl); (26) oxo; (27) C _2-20 alkoxy; and (28) C _2-20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, _{the alkylene group of C 1} -alkalyl or C _1- alkheterocyclyl can be further substituted with an oxo group to give the respective allylloyl and (heterocyclyl) oil substituents.

As used herein, the term "diastereomers" means stereoisomers that are not mirror images of each other and cannot be superimposed on each other.

As used herein, the "effective amount" of the agent used herein is an amount sufficient to produce a beneficial or desired result, eg, a clinical result, and thus an "effective amount". Depends on the context in which it is applied. For example, in the context of administering a drug that treats cancer, an effective amount of drug, for example, treats cancer as defined herein as compared to a response that would be obtained without administration of the drug. Enough amount to achieve.

As used herein, the term "enantiomer" is at least 80% (ie, at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least at least. It means each individual optically active form of a compound of the invention having an optical purity of 90%, more preferably at least 98% or an enantiomer excess (as determined by standard methods in the art).

As used herein, the term "halo" refers to a halogen selected from bromine, chlorine, iodine, or fluorine.

As used herein, the term "haloalkoxy" refers to an alkoxy group as defined herein, substituted with a halogen group (ie, F, Cl, Br, or I). The haloalkoxy may be substituted with 1, 2 or 3 halogens, or 4 halogens in the case of alkyl groups of 2 or more carbons. The haloalkoxy groups include perfluoroalkoxy (eg, -OCF ₃ ) _{, -OCHF 2} , -OCH ₂ F, -OCCl ₃ , -OCH ₂ CH ₂ Br, -OCH ₂ CH (CH ₂ CH ₂ Br) CH ₃ , And -OCHICH ₃ are included. In some embodiments, the haloalkoxy group can be further substituted with 1, 2, 3, or 4 substituents described herein for the alkyl group.

As used herein, the term "haloalkyl" refers to an alkyl group as defined herein, substituted with a halogen group (ie, F, Cl, Br, or I). The haloalkyl may be substituted with 1, 2 or 3 halogens, or 4 halogens in the case of alkyl groups of 2 or more carbons. The haloalkyl groups include perfluoroalkyl (eg, -CF ₃ ), -CHF ₂ , -CH ₂ F, -CCl ₃ , -CH ₂ CH ₂ Br, -CH ₂ CH (CH ₂ CH ₂ Br) CH ₃ , and -CHICH ₃ is included. In some embodiments, the haloalkyl group can be further substituted with 1, 2, 3, or 4 substituents described herein for the alkyl group.

As used herein, the term "heteroalkylene" is defined herein as an alkylene in which one or two of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur, respectively. Refers to the group. In some embodiments, the heteroalkylene group may be further substituted with 1, 2, 3, or 4 substituents described herein for the alkylene group.

As used herein, the term "heteroaryl" refers to a subset of heterocyclyls as defined herein that are aromatic, i.e. they are within a monocyclic or polycyclic ring system. Contains 4n + 2 π electrons. Illustrated unsubstituted heteroaryl groups may also include 1 to 12 (eg, 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10 or 2 to 9) carbons. Is. In some embodiments, the heteroaryl is substituted with 1, 2, 3, or 4 substituents defined for the heterocyclyl group.

の基も含み、式中、 As used herein, the term "heterocyclyl" refers to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, unless otherwise stated. Represents a 5, 6, or 7-membered ring containing. The 5-membered ring has 0 to 2 double bonds, and the 6 and 7 membered rings have 0 to 3 double bonds. The exemplified unsubstituted heterocyclyl groups are those of 1 to 12 carbons (for example, 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10 or 2 to 9). be. The term "heterocyclyl" refers to a heterocyclic compound having a crosslinked polycyclic structure, eg, a quinuclicinyl group, in which one or more carbons and / or heteroatoms crosslink two non-adjacent members of a monocyclic ring. Also represents. The term "heterocyclyl" refers to any one of the above heterocyclic rings being one, two, or three carbocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentane ring, or Includes bicyclic, tricyclic, and tetracyclic groups that are fused to another monocyclic heterocyclic ring such as indrill, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, etc. Examples of condensed heterocyclyls include tropane and 1,2,3,5,8,8a-hexahydroindolizine. Heterocycles include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolydinyl, imidazolyl, imidazolynyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, pyridazinyl, oxazolyl, oxazolidinyl. , Thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indrill, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, sinolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzimidazolyl, benzothiazolyl , Frill, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl (eg 1,2,3-oxadiazolyl), prynyl, thiadiazolyl (eg 1,2,3-thiazolizolyl), tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl , Dihydrothienyl, dihydroindrill, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, etc. Includes their dihydro and tetrahydro forms in which the above double bonds are reduced and replaced with hydrogen. Yet other exemplary heterocyclyls include 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl; 2,3,4,5-tetrahydro-5. -Oxo-1H-pyrazolyl (eg, 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H- Imidazolyl (eg, 2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl); 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (For example, 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-1H-triazolyl (eg, 4,5-dihydro-3-3). Methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (eg 1,2,3,4-tetrahydro-2,4-dioxo) -3,3-diethylpyridinyl); 2,6-dioxo-piperidinyl (eg, 2,6-dioxo-3-ethyl-3-phenylpiperidinyl); 1,6-dihydro-6-oxopyridimi Nyl; 1,6-dihydro-4-oxopyrimidinyl (eg, 2- (methylthio) -1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro -2,4-dioxopyrimidinyl (eg 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (eg 1,6) −Dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (eg, 1,6-dihydro-5-isopropyl-6-oxo-) 1,2,4-Triazinyl); 2,3-dihydro-2-oxo-1H-indrill (eg, 3,3-dimethyl-2,3-dihydro-2-oxo-1H-indrill and 2,3-dihydro) -2-oxo-3,3'-spiropropane-1H-indole-1-yl);1,3-dihydro-1-oxo-2H-iso-indrill; 1,3-dihydro-1,3-dioxo- 2H-iso-indrill; 1H-benzopyrazolyl (eg, 1- (ethoxycarbonyl) -1H-benzopyrazolyl); 2,3-dihydro-2-oxo-1H-benz Imidazolyl (eg, 3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl); 2,3-dihydro-2-oxo-benzoxazolyl (eg, 5-chloro-2,3-dihydro) -2-oxo-benzoxazolyl); 2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl; 1,4-benzodioxanyl; 1,3-benzodioxaly Nyl; 2,3-dihydro-3-oxo, 4H-1,3-benzthiadinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (eg, 2-methyl-3,4-dihydro-4-oxo) -3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (eg, 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H- Kinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-prinyl (eg 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-prinyl) ); 1,2,3,6-tetrahydro-2,6-dioxo-1H-prinyl (eg, 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-prinyl) Includes 2-oxobenz [c, d] indyl; 1,1-dioxo-2H-naphtho [1,8-c, d] isothiazolyl; and 1,8-naphthylene carboxamide. Further heterocycles include 3,3a, 4,5,6,6a-hexahydro-pyrrolo [3,4-b] pyrrol- (2H) -yl, and 2,5-diazabicyclo [2.2.1]. ] Includes heptane-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiocanyl. The heterocyclic group is expressed in the formula:

Including the group of

E 'is selected from the group consisting of -N- and -CH-, F' _{is, -N = CH -, - NH} -CH 2 -, - NH-C (O) -, - NH -, - CH = N-, -CH _2- NH-, -C (O) -NH-, -CH = CH-, -CH _2- , -CH ₂ CH _2- , -CH ₂ O-, -OCH ₂ -,- Selected from the group consisting of O- and -S-, G'is selected from the group consisting of -CH- and -N-. Any of the heterocyclyl groups referred to herein may be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: Good: (1) C _1-7 acyl (eg, carboxylaldehyde); (2) C _1-20 alkyl (eg, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, C _1-6) Alkyl sulfinyl-C _1-6 alkyl, amino-C _1-6 alkyl, azido-C _1-6 alkyl, (carboxyaldehyde) -C _1-6 alkyl, halo-C _1-6 alkyl (eg perfluoroalkyl), Hydroxy-C _1-6 alkyl, nitro-C _1-6 alkyl, or C _1-6 thioalkoxy-C _1-6 alkyl); (3) C _1-20 alkoxy (eg, C _{1-6 such as perfluoroalkoxy).} Alkoxy); (4) C _1-6 alkylsulfinyl; (5) C _6-10 aryl; (6) amino; (7) C _1-6 arc-C _6-10 aryl; (8) azide; (9) C _3-8 cycloalkyl; (10) C _1-6 arc-C _3-8 cycloalkyl; (11) halo; (12) C _1-12 heterocyclyl (eg, C _2-12 heteroaryl); (13) (C _1-12 heterocyclyl) oxy; (14) hydroxy; (15) nitro; (16) C _1-20 thioalkoxy (eg, C _1-6 thioalkoxy); (17)-(CH ₂ ) _q CO ₂ R ^{a '(wherein,} q is an integer of 0 to 4, R ^a' is _{(a) C 1-6} alkyl, _{(b) C 6-10} aryl, (c) hydrogen, and (d) C _1-6 selected from the group consisting of alk _{-C 6-10 aryl); (18) - (CH} 2) q CONR B 'R C' ( wherein, q is an integer of 0 to 4, R ^{B 'and} R ^C' is, (a) hydrogen, _{(b) C 1-6} alkyl, _{(c) C 6-10} aryl, and _{(d) C 1-6} group consisting alkaryl _{-C 6-10} aryl (19)-(CH ₂ ) _q SO ₂ R ^D' (In the equation, q is an integer from 0 to 4, and R ^D' is (a) C _1-6. alkyl, _{(b) C 6-10} aryl, and _{(c) C 1-6} is selected from the group consisting of alk _{-C 6-10 aryl); (20) - (CH} 2) q SO 2 NR E 'R ^F' (in the formula, q Is an integer from 0 to 4, each R ^{E 'and} R ^F' is independently, (a) hydrogen, _{(b) C 1-6} alkyl, _{(c) C 6-10} aryl, and ( d) _{Selected from the group consisting of C 1-6} arc-C _6-10 aryl); (21) thiol; (22) C _6-10 aryloxy; (23) C _3-8 cycloalkoxy; (24) Arylalkoxy; (25) C _1-6 arc-C _1-12 heterocyclyl (eg, C _1-6 arc-C _1-12 heteroaryl); (26) oxo; (27) (C _1-12 heterocyclyl) imino (28) C _2-20 Alkoxy; and (29) C _2-20 Alkinyl. In some embodiments, each of these groups can be further substituted as described herein. For example, _{the alkylene group of C 1} -alkalyl or C _1- alkheterocyclyl can be further substituted with an oxo group to give the respective allylloyl and (heterocyclyl) oil substituents.

As used herein, the term "(heterocyclyl) imino" refers to a heterocyclyl group as defined herein that is attached to a parent molecular group via an imino group. In some embodiments, the heterocyclyl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.

As used herein, the term "(heterocyclyl) oxy" refers to a heterocyclyl group as defined herein that is attached to a parent molecular group via an oxygen atom. In some embodiments, the heterocyclyl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.

As used herein, the term "(heterocyclyl) oil" refers to a heterocyclyl group as defined herein that is attached to a parent molecular group via a carbonyl group. In some embodiments, the heterocyclyl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.

As used herein, the term "hydrocarbon" refers to a group consisting only of carbon and hydrogen atoms.

As used herein, the term "hydroxy" refers to a -OH group.

As used herein, the term "hydroxyalkenyl" refers to an alkenyl group as defined herein, substituted with 1-3 hydroxy groups, provided that there is more than one hydroxy group. Is not attached to a single carbon atom of the alkyl group, which is illustrated by dihydroxypropenyl, hydroxyisopentenyl, etc. as an example.

As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein, substituted with 1-3 hydroxy groups, provided that there is more than one hydroxy group. However, provided that it does not bond to a single carbon atom of the alkyl group, it is illustrated by hydroxymethyl, dihydroxypropyl, etc. as an example.

As used herein, the term "isomer" means any tautomer, stereoisomer, enantiomer, or diastereomer of any of the compounds of the invention. The compounds of the invention can have one or more chiral centers and / or double bonds and are therefore double bond isomers (ie, geometric E / Z isomers) or diastereomers (eg, mirror isomers). It is recognized that it can exist as a steric isomer such as a body (ie, (+) or (−)) or cis / trans isomer). According to the present invention, the chemical structures described herein, and thus the compounds of the present invention, are all of the corresponding three isomers, i.e. stereoisomerically pure forms (eg, geometrically pure, enantiomerically). Includes both pure or diastereomerically pure) and enantiomer and stereoisomer mixtures, such as racemates. The mirror image isomer mixture and the steric isomer mixture of the compound of the present invention can be used for chiral phase gas chromatography, chiral phase high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, crystallization of the compound in a chiral solvent, etc. By well-known methods of, they can typically be decomposed into chiral or steric isomers of their constituents. Enantiomers and steric isomers can also be obtained from sterically or mirror isomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthesis methods.

As used herein, the term "N-protected amino" refers to an amino group as defined herein that is attached to one or two N-protecting groups as defined herein. ..

As used herein, the term "N-protecting group" refers to a group intended to protect an amino group against undesired reactions during a synthetic procedure. N-protecting groups that are commonly used, are incorporated herein by reference, Greene, "Protective Groups in Organic Synthesis," 3 rd Edition (John Wiley & Sons, New York, 1999) which is incorporated herein by reference. N-protecting groups include formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutylyl, benzoyl, Acyl, allyloxy, or carbamil groups such as 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L, or D, alanine, leucine, phenylalanine. L-amino acids such as benzenesulfonyl, sulfonyl-containing groups such as p-toluenesulfonyl; benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl , P-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5- Dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1- (p-biphenylyl) -1-methylethoxycarbonyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryl Oxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9 -Includes carbamate-forming groups such as methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, alkaline groups such as benzyl, triphenylmethyl and benzyloxymethyl, and silyl groups such as trimethylsilyl. Preferred N protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

As used herein, the term "nitro" refers to _two -NOs.

As used herein, it represents the term "oxo", = O.

As used herein, the term "perfluoroalkyl" refers to an alkyl group as defined herein in which each hydrogen radical attached to an alkyl group has been replaced by a fluoride radical. The perfluoroalkyl group is shown as an example by trifluoromethyl, pentafluoroethyl and the like.

As used herein, the term "perfluoroalkoxy" refers to an alkoxy group as defined herein in which each hydrogen radical attached to an alkoxy group is replaced by a fluoride radical. The perfluoroalkoxy group is shown as an example by trifluoromethoxy, pentafluoroethoxy and the like.

_{As used herein, the term "spirocyclyl" refers to a C 2-7} alkylene diradical, both ends of which bond to the same carbon atom of the parent group to form a spirocyclic group, and also the same at both ends. _{Represents a C 1-6} heteroalkylene diradical attached to a carbon atom. The heteroalkylene radical forming a spirocyclyl group can contain 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl group contains 1 to 7 carbons, except for the carbon atom to which the diradical is attached. The spirocyclyl group of the present invention may be optionally substituted with 1, 2, 3, or 4 substituents provided herein as any substituents on cycloalkyl groups and / or heterocyclyl groups.

As used herein, the term "stereoisomer" refers to all possible heteroisomers as well as sterics that a compound (eg, a compound of any formula described herein) may have. Refers to conformational forms, especially all possible stereochemical and conformational isomers, all diastereomers of basic molecular structure, mirror isomers, and / or conformational isomers. Some compounds of the invention may exist as different tautomeric forms, all of the latter being within the scope of the invention.

As used herein, the term "sulfoalkyl" means an alkyl group as defined substituted by sulfo group -SO 3 _H, herein. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "sulfonyl" refers to the -S (O) ₂ -group.

As used herein, the term "thioalkal" represents a chemical substituent of formula-SR, where R is an alkaline group. In some embodiments, the alkaline reel group can be further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "thioalkheterocyclyl" represents a chemical substituent of formula-SR, where R is an archeterocyclyl group. In some embodiments, the archeterocyclyl group can be further substituted with 1, 2, 3, or 4 substituents described herein.

As used herein, the term "thioalkoxy" represents a chemical substituent of formula-SR, where R is an alkyl group as defined herein. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents described herein.

The term "thiol" represents a -SH group.
Compounds: As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

The compounds described herein can be asymmetric (eg, have one or more stereocenters). Unless otherwise specified, all stereoisomers such as enantiomers and diastereomers are intended. The compounds of the present disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic form. Methods relating to how to prepare optically active forms from optically active starting materials are known in the art, such as by decomposition of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, such as C = N double bonds, can also be present in the compounds described in the present invention, and all such stable isomers are contemplated in the present disclosure. Sith and trans geometric isomers of the compounds of the present disclosure are described, which can be isolated as a mixture of isomers or as a separated isomer.

The compounds of the present disclosure also include tautomeric forms. The tautomeric form results from the exchange of single bonds with adjacent double bonds and the accompanying transfer of protons. Tautomers include proton tautomers, which are isomer protonated states with the same empirical formula and total charge. Illustrated proton tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imide acid pairs, enamine-imine pairs, and protons at two or more positions in a heterocyclic system. Includes cyclic forms capable of occupying, eg, 1H- and 3H-imidazoles, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Is done. The tautomeric form can be equilibrated or sterically fixed into one form by appropriate substitution.

The compounds of the present disclosure also include all isotopes of atoms that occur in intermediates or final compounds. "Isotope" refers to an atom that has the same atomic number but a different mass number due to the different number of neutrons in the nucleus. For example, hydrogen isotopes include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared by conventional methods by combining with solvents or water molecules to form solvates and hydrates.

Conserved: As used herein, the term "conserved" is a residue that occurs unchanged at the same position in two or more sequences being compared, each polynucleotide sequence. Alternatively, it refers to a nucleotide residue or an amino acid residue of a polypeptide sequence. Relatively conserved nucleotides or amino acids are those that are more conserved among sequences that are more relevant than nucleotides or amino acids that appear elsewhere in the sequence.

In some embodiments, two or more sequences are said to be "fully conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. Will be. In some embodiments, "two or more sequences are" about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. It is highly preserved. " In some embodiments, two or more sequences are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% to each other. If they are identical, or at least 95% identical, they are said to be "preserved." In some embodiments, two or more sequences are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% to each other. If they are identical, about 95% identical, about 98% identical, or about 99% identical, they are said to be "preserved." Sequence preservation may be applied to the full length of the oligonucleotide or polypeptide, or to a portion, region, or feature thereof.

Controlled Release: As used herein, the term "controlled release" refers to a release profile of a pharmaceutical composition or compound that fits a particular release pattern to produce a therapeutic outcome.

Circular or cyclized: As used herein, the term "circular" refers to the presence of a continuous loop. Cyclic molecules do not have to be circular, they simply need to be linked to form an uninterrupted chain of subunits. The engineered cyclic molecules such as RNA or mRNA of the present invention may be single units or multimers, or may contain one or more components of a complex or higher order structure.

Cell proliferation inhibitory: As used herein, "cell proliferation inhibitory" refers to cells (eg, mammalian cells (eg, human cells), bacteria, viruses, fungi, protozoa, parasites, prions, or these. Refers to inhibiting, reducing, or suppressing the growth, division, or proliferation of the combination of.

Cytotoxicity: As used herein, "cytotoxic" refers to cells (eg, mammalian cells (eg, human cells), bacteria, viruses, fungi, protozoans, parasites, prions, or combinations thereof. To kill or cause harmful, toxic, or fatal effects on them.

Delivery: As used herein, "delivery" refers to the action or mode of delivering a compound, substance, entity, part, cargo, or payload.

Delivery Agent: As used herein, "delivery agent" refers to any substance that at least partially facilitates in vivo delivery of a polynucleotide, primary construct, or mmRNA to a targeted cell. ..

Destabilized: As used herein, the terms "unstable", "destabilizing", or "destabilizing region" refer to the starting form, wild type, of the same region or molecule. Alternatively, it means a region or molecule that is less stable than the natural type.

Detectable Labels: As used herein, "detectable labels" are readily detected by methods known in the art including radiophotographic photography, fluorescence, chemiluminescence, enzymatic activity, absorption and the like. Refers to one or more markers, signals, or parts that are combined with, incorporated with, or associated with another entity to be. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots and the like. The detectable label may be located at any position in the peptides or proteins disclosed herein. They may be within amino acids, peptides, or proteins, or may be located at the N-terminus or C-terminus.

Digestion: As used herein, the term "digestion" means breaking down into smaller pieces or constituents. When referring to a polypeptide or protein, digestion results in the production of the peptide.

Distal: As used herein, the term "distal" means located away from the center or away from a point or region of interest.

Dosing regimen: As used herein, a "dosing regimen" is a schedule of administration or a treatment, preventive, or palliative care regimen determined by a physician.

Dose Split Factor (DSF) -The ratio of the PUD for dose split treatment divided by the total daily dose or single unit dose PUD. Values are derived from a comparison of groups of dosing regimens.

Encapsulation: As used herein, the term "encapsulation" means encapsulating, enclosing, or enveloping.

Encoded protein cleavage signal: As used herein, "encoded protein cleavage signal" refers to a nucleotide sequence that encodes a protein cleavage signal.

Manipulation: As used herein, embodiments of the invention, whether structural or chemical, have different features or properties from starting point molecules, wild-type molecules, or natural molecules. When designed to be "manipulated".

Exosomes: As used herein, "exosomes" are vesicles secreted by mammalian cells, or complexes involved in RNA degradation.

Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) Generation of an RNA template from a DNA sequence (eg, by transcription), (eg, by transcription). 2) Processing of RNA transcription (eg, by splicing, editing, 5'cap formation, and / or 3'end processing), (3) translation of RNA into a polypeptide or protein, and (4) of a polypeptide or protein. Post-translation modification.

Feature: As used herein, "feature" refers to a feature, characteristic, or unique element.

Formulation: As used herein, a "formulation" comprises at least a polynucleotide, a primary construct, or mmRNA and a delivery agent.

Fragment: As used herein, "fragment" refers to a portion. For example, a fragment of a protein may contain a polypeptide obtained by digesting a full-length protein isolated from cultured cells.

Functional: As used herein, a "functional" biomolecule is a biomolecule that is in a form that exhibits the properties and / or activity in which it is characterized.

Homogeneity: As used herein, the term "homologity" refers to the whole between polymer molecules, eg, between nucleic acid molecules (eg, DNA and / or RNA molecules), and / or between polypeptide molecules. Refers to the relevance. In some embodiments, the polymer molecules have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 sequences thereof. %, 85%, 90%, 95%, or 99% are considered to be "homologous" to each other if they are identical or similar. The term "homology" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present invention, the two polynucleotide sequences are such that the polypeptide they encode is at least about 50%, 60%, 70%, 80%, 90%, 95%, or even over at least one sequence of at least about 20 amino acids. If it is 99%, it is considered to be homologous. In some embodiments, the homologous polynucleotide sequence is characterized by the ability to encode a sequence of at least 4-5 uniquely designated amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is characterized by the ability to encode a sequence of at least 4-5 uniquely designated amino acids. According to the present invention, two protein sequences are considered homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical over at least one sequence of at least about 20 amino acids. ..

Identity: As used herein, the term "identity" is used between polymer molecules, such as between oligonucleotide molecules (eg, DNA and / or RNA molecules), and / or between polypeptide molecules. Refers to the overall relevance. Calculation of the percent identity of two polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (eg, for optimal alignment of the first and second nucleic acids). Gap can be introduced into one or both of the sequences and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparative purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% of the length of the reference sequence. , At least 90%, at least 95%, or 100%. Nucleotides at the corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotides as the corresponding position in the second sequence, the molecule is identical at that position. The percent identity between the two sequences is the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. It is a function. Sequence comparisons and determination of percent identity between two sequences can be performed using mathematical algorithms. For example, the percent identity between two nucleotide sequences is Computational, each of which is incorporated herein by reference.
Molecular Biology, Lesk, A. et al. M. , Ed. , Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.M. W. , Ed. , Academic Press, New York, 1993, Sequence Analysis in Molecular Biology, von Heinje, G. et al. , Academic Press, 1987, Computer Analysis of Sequence Data, Part I, Griffin, A. et al. M. , And Griffin, H. et al. G. , Eds. , Humana Press, New Jersey, 1994, and Sequence Analysis Primer, Gribskov, M. et al. and Deverex, J. et al. , Eds. , M
It can be determined by using the method described in Stockton Press, New York, 1991 and the like. For example, the percent identity between two nucleotide sequences can be determined using the algorithms of Meyers and Miller (CABIOS, 1989, 4: 11-17), which is the PAM120 weight residue table, 12 gap length penalties. , And 4 are incorporated into the ALIGN program (version 2.0) with a gap penalty of 4. Percentage of identity between two nucleotide sequences, or NWS gapdna. It can be determined using the GAP program in the GCG software package that uses the CMP matrix. Commonly used methods for determining percent identity between sequences are described in Carillo, H. et al., Which are incorporated herein by reference. , And Lipman, D.I. , SIAM J Applied Math. , 48: 1073 (1988), including, but not limited to, the methods of disclosure. Techniques for determining identity are codified in publicly available computer programs. Illustrative computer software for determining homology between two sequences includes the GCG Program Package, Devereux, J. Mol. , Et al. , Nucleic Acids Research, 12 (1), 387 (1984)), BLASTP, BLASTN, and FASTA, Altschul, S. et al. F. et al. , J. Molec. Biol. , 215, 403 (1990)), but not limited to these.

Inhibiting Gene Expression: As used herein, the expression "inhibiting gene expression" means causing a reduction in the amount of gene expression product. The expression product can be RNA transcribed from the gene (eg, mRNA) or a polypeptide translated from mRNA transcribed from the gene. Typically, a reduction in the level of mRNA results in a reduction in the level of the polypeptide translated from it. The level of expression may be determined using standard techniques for measuring mRNA or protein.

In vitro: As used herein, the term "in vitro" is not used in an organism (eg, animal, plant, or microorganism), but in, for example, in a test tube or reaction vessel, in a cell culture, in a Petri dish, etc. Refers to an event that occurs in an artificial environment.

In vivo: As used herein, the term "in vivo" refers to an event that occurs within an organism (eg, an animal, plant, or microorganism, or its cells or tissues).

Isolated: As used herein, the term "isolated" is one of the components to which it is associated (whether natural or experimental). Refers to a substance or entity isolated from at least a portion of. The isolated material can have varying levels of purity with respect to the material with which it is associated. The isolated substances and / or entities are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70 of the other constituents with which they were first associated. It can be separated from%, about 80%, about 90%, or more. In some embodiments, the isolated agent is greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, about. More than 95%, more than 96%, more than about 97%, more than about 98%, more than about 99%, or more than about 99% pure. As used herein, a substance is "pure" in the absence of substantially any other constituent. Substantially isolated: By "substantially isolated" is meant that the compound is substantially isolated from the environment in which it was formed or detected. The partial separation may include, for example, a composition rich in the compounds of the present disclosure. Substantial separation is at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or Compositions containing at least about 99% by weight of the compounds of the present disclosure, or salts thereof, may be included. Methods for isolating compounds and salts thereof are customary in the art.

Linker: As used herein, a linker refers to, for example, an atomic group of 10 to 1,000 atoms, such as carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. However, it may consist of atoms or groups, not limited to these. The linker can be attached to a modified nucleoside or nucleotide on a nucleobase or sugar moiety at the first end and to a payload, eg, a detectable agent or therapeutic agent, at the second end. The linker can also be long enough so that it does not interfere with integration into the nucleic acid sequence. The linker is used for any useful purpose, for example, to form an mm mRNA multimer (eg, via a chain of two or more polynucleotides, a primary construct, or an mm mRNA molecule) or an mm mRNA complex, and herein. It can be used to administer the payload described in the book. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl. Each of the above can be optionally substituted as described herein. Examples of linkers include unsaturated alkanes, polyethylene glycol (eg, ethylene or propylene glycol monomer units, such as diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran. Polymers and derivatives thereof include, but are not limited to. Other examples include cleaveable moieties within the linker, such as disulfide bonds (-SS-) or azo bonds (-N = N-), which can be cleaved using a reducing agent or photolysis. However, it is not limited to these. Non-limiting examples of selectively cleavable bonds include, for example, tris (2-carboxyethyl) phosphine (TCEP), or amide bonds, which can be cleaved by the use of other reducing agents and / or photolysis. Also include, for example, ester bonds that can be cleaved by acidic or basic hydrolysis.

MicroRNA (miRNA) Binding Site: As used herein, a microRNA (miRNA) binding site represents a nucleotide location or region of a nucleic acid transcript to which at least a "seed" region of miRNA binds.

Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the invention. Molecules may be modified in many ways, including chemical, structural, and functional modifications. In one embodiment, the mRNA molecule of the invention is modified by the introduction of unnatural nucleosides and / or nucleotides, for example when it involves native ribonucleotides A, U, G, and C. Non-standard nucleotides such as cap structures are not considered to be "modified" even if they differ from the chemical structures of the A, C, G and U ribonucleotides.

Mucus: As used herein, "mucus" refers to a natural substance that is viscous and contains mucin glycoproteins.

Naturally occurring: As used herein, "naturally occurring" means present in nature without artificial assistance.

Non-human vertebrates: As used herein, "non-human vertebrates" includes all vertebrates except Homo sapiens, including wild and domesticated species. Examples of non-human vertebrates include alpaca, banteng, bison, camel, cat, cow, deer, dog, donkey, gaur, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep, buffalo, and Mammals such as donkeys can be mentioned, but are not limited thereto.

Off-target: As used herein, "off-target" refers to any unintended effect on any one or more targets, genes, or cell transcripts.

Open Reading Frame: As used herein, "open reading frame" or "ORF" refers to a sequence that does not contain a stop codon within a given reading frame.

Operatively linked: As used herein, the expression "operably linked" refers to a functional bond between two or more molecules, constructs, transcripts, entities, parts, etc. Point to.

Arbitrarily substituted: In the present specification, the phrase in the form of "arbitrarily substituted X" (for example, optionally substituted alkyl) is "X, where X is arbitrarily substituted in the formula. It is intended to be equivalent to "is" (eg, "alkyl, in the formula, the alkyl is optionally substituted"). Features It is not intended to mean that the "X" (eg, alkyl) itself is optional.

Peptide: As used herein, a "peptide" is 50 amino acids or less in length, eg, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

Paratope: As used herein, "paratope" refers to the antigen-binding site of an antibody.

Patient: As used herein, a "patient" may seek or require treatment, is in need of treatment, is being treated, will be treated, or has a particular disease or condition. Refers to a subject who is under the care of a professional trained for.

Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" herein refers to excessive toxicity, irritation, allergic reactions, or other problems or complications, within sound medical judgment. Suitable for use in contact with human and animal tissues without accompaniment, and used to refer to compounds, materials, compositions, and / or dosage forms commensurate with a reasonable benefit / risk ratio.

Pharmaceutically Acceptable Excipients: As used herein, the expression "pharmaceutically acceptable excipients" is other than the compounds described herein (eg, activity). A vehicle in which a compound can be suspended or dissolved), and refers to any ingredient that has the property of being substantially non-toxic and non-inflammatory in the patient. Excipients include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), softeners, emulsifiers, fillers (diluents). Agents), thin film forming agents or coating agents, flavoring agents, flavoring agents, flow promoters (fluidity promoters), lubricants, preservatives, printing inks, adsorbents, suspending agents or dispersants, sweetening Agents and water of hydration may be included. Excipients exemplified include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (second), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, Citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, martitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, α Chemicalized starch, propylparaben, retinyl palmitate, shelac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamins E, vitamin C, and xylitol.

Pharmaceutically Acceptable Salts: The disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, a "pharmaceutically acceptable salt" is the conversion of an existing acid or base moiety to its salt form (eg, by reacting a free base with a suitable organic acid). Refers to a derivative of a disclosed compound whose parent compound is modified by. Examples of pharmaceutically acceptable salts include, but are limited to, mineral salts or organic salts of basic residues such as amines, alkaline salts or organic salts of acidic residues such as carboxylic acids, and the like. Not done. Typical acid addition salts include acetate, adipate, alginate, ascorbate, asparagate, benzenesulfonate, benzoate, bicarbonate, borate, butyrate, and cerebral acid salt. , Cerebral sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptone, hexane Hydrochloride, hydrobromide, hydrochloride, hydrogen iodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurylate, laurylsulfate, malate, maleate, malonic acid Salt, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- Phenylpropionate, phosphate, picphosphate, pivalate, propionate, stearic acid, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. included. Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. , But not limited to, non-toxic ammonium, quaternary ammonium, and amine cations. The pharmaceutically acceptable salts of the present disclosure include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts react the free acid or free base form of these compounds with a chemical amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. In general, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or butanol), or acetonitrile is preferred. The list of suitable salts, each of which are incorporated by reference in their entireties ^{herein, Remington's Pharmaceutical Sciences, 17 th} ed. , Mack Publishing Company, Easton, Pa. , 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. et al. H. Stahl and C.I. G. Vermus (eds.), Wiley-VCH, 2008, and Berge et al. , Journal of Pharmaceutical Science, 66, 1-19 (1977).

Pharmaceutically Acceptable Solvates: As used herein, the term "pharmaceutically acceptable solvates" refers to compounds of the invention in which molecules of a suitable solvent are incorporated into the crystal lattice. Means. Suitable solvents are physiologically resistant to the dosage administered. For example, the solvate may be prepared from a solution containing an organic solvent, water, or a mixture thereof by crystallization, recrystallization, or precipitation. Examples of suitable solvents include ethanol, water (eg, 1, 2, and trihydrate), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N, N'-dimethylformamide (DMF), N, N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2- (1H) -pyrimidinone ( DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate and the like. When water is the solvent, the solvate is referred to as the "hydrate".

Pharmacokinetics: As used herein, "pharmacokinetics" refers to any one or more properties of a molecule or compound that are involved in determining the fate of a substance administered to a living organism. Pharmacokinetics is divided into several regions, including the degree and rate of absorption, distribution, metabolism, and excretion. This is commonly referred to as ADME, where (A) absorption is the process by which a substance enters the blood circulation and (D) distribution is the dispersion or dispersal of the substance throughout the body's fluids and tissues. Yes, (M) metabolism (or biotransformation) is the irreversible conversion of the parent compound to the daughter metabolite, and (E) excretion (or exclusion) refers to the elimination of the substance from the body. In rare cases, some drugs irreversibly accumulate in body tissues.

Physical Chemistry: As used herein, "physical chemistry" means that of or is associated with physical and / or chemical properties.

Polypeptides per unit drug (PUD): As used herein, PUD or product per unit drug is usually in concentration units such as pmol / mL, mmol / mL, etc. divided by measurements in body fluids. It is defined as the subdivided portion (usually 1 mg, pg, kg, etc.) of the total daily dose of the product (polypeptide, etc.) as measured in body fluids or tissues.

Prevent: As used herein, the term "prevent" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; a particular infection, disease, disorder, Partially or completely delaying the onset of one or more symptoms, features, or clinical signs of and / or a condition; one or more symptoms, features, or of a particular infection, disease, disorder, and / or condition. Partially or completely delaying the onset of symptoms; partially or completely delaying the progression of a particular disease, disorder, and / or condition from an infection; and / or infection, disease, disorder, and / Alternatively, it refers to reducing the risk of developing a condition-related pathology.

Prodrugs: The disclosure also includes prodrugs of the compounds described herein. As used herein, a "prodrug" refers to any substance, molecule, or entity in the form of a substance, molecule, or entity that acts as a therapeutic agent upon undergoing chemical or physical alteration. Point to. The prodrug can be covalently bound or sequestered in some way, releasing the active drug or being converted to an active drug moiety before, after, or after administration to the mammalian subject. NS. Prodrugs can be prepared by modifying the functional groups present in the compound in such a way that the modified moiety is cleaved into the parent compound either by routine procedures or in vivo. A prodrug is a compound in which a hydroxyl, amino, sulfhydryl, or carboxyl group is attached to any group, which when administered to a mammalian subject is cleaved to form a free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively. do. The preparation and use of prodrugs are both incorporated herein by reference in their entirety. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A. C. S. Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical
It is discussed in Association and Pergamon Press, 1987.

Proliferate: As used herein, the term "proliferate" means grow, expand, or increase, or cause rapid growth, expansion, or increase. "Proliferative" means having the ability to proliferate. "Anti-proliferative" means having properties that are contrary to or inappropriate for the proliferative properties.

Protein cleavage site: As used herein, "protein cleavage site" refers to the site where controlled cleavage of an amino acid chain is performed by chemical, enzymatic, or photochemical means.

Protein cleavage signal: As used herein, "protein cleavage signal" refers to at least one amino acid that flags or marks a polypeptide for cleavage.

Protein of interest: As used herein, the terms "protein of interest" or "desired protein" are those provided herein, as well as fragments, variants, variants, etc. thereof. And include variants.

Proximal: As used herein, the term "proximal" means located at the center or closer to the point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycoside isomer of nucleoside uridine. A "pseudouridine analog" is any modification, variant, isoform, or derivative of pseudouridine. For example, pseudopseudouridine analogs include 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine (m ^1). ψ), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1- Methyl-pseudouridine, 1-methyl-1-desa-pseudouridine, 2-thio-1-methyl-1-desa-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- Methoxy-4-thio-pseudouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 1-methyl-3- (3-amino-3-carboxypropyl) pseudo Includes, but is not limited to, uridine (acp ³ ψ) and 2'-O-methyl-pseudouridine (ψm).

Purified: As used herein, "purified", "purified", "purified" is either substantially purified or undesired constituents, contaminants, admixtures, or It means removing imperfections.

Sample: As used herein, the term "sample" or "biological sample" refers to a subset of its tissue, cells, or components (eg, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, etc.) Refers to body fluids including, but not limited to, saliva, amniotic fluid, umbilical cord blood, urine, vaginal fluid, and semen. Samples include, for example, plasma, serum, spinal fluid, lymph, external sections of skin, respiratory tract, intestinal tract, and genitourinary tract, tears, saliva, milk, blood cells, tumors, organs. Further may include homogenates, solubilizers, or extracts prepared from, but not limited to, a subset of the whole organism, or tissues, cells, or components thereof, or fractions or parts thereof. Samples also refer to media such as nutrient broths or gels that may contain cell constituents such as proteins or nucleic acid molecules.

Signal Sequence: As used herein, the expression "signal sequence" refers to a sequence that can direct protein transport or localization.

Single unit dose: As used herein, a "single unit dose" is administered in a single dose / one time / single pathway / single contact point, i.e., in a single dosing event. The dose of any therapeutic agent.

Similarity: As used herein, the term "similarity" is used between polymer molecules, eg, between polynucleotide molecules (eg, DNA and / or RNA molecules), and / or between polypeptide molecules. Refers to the overall relevance. The calculation of the percent similarity to each other of the polymer molecules can be performed in the same way as the calculation of the percent identity, but as is understood in the art, the calculation of the percent similarity takes into account conservative substitutions. Except that.

Divided dose: As used herein, a "divided dose" is a single unit dose or a total daily dose divided into two or more doses.

Stable: When used herein, "stable" is strong enough to remain after isolation at a useful purity from the reaction mixture and is preferably pharmaceuticalable into an effective therapeutic agent. Refers to a compound.

Stabilized: As used herein, the terms "stabilized," "stabilized," and "stabilized region" mean to be stabilized or stabilized.

Subject: As used herein, the term "subject" or "patient" is arbitrary, where a composition according to the invention can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Refers to the creatures of. Typical subjects include animals (eg, mice, rats, rabbits, non-human primates, and mammals such as humans) and / or plants.

Substantially: As used herein, the term "substantially" refers to a qualitative condition that indicates the full range or degree or near the full range or degree of a feature or property of interest. Experts in the field of biological technology rarely complete and / or progress to completion, or achieve or avoid absolute consequences of biological and chemical phenomena. You will understand that. The term "substantially" is therefore used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

Substantially Equal: When used herein as with respect to the time difference between doses, the term means plus / minus 2%.

Substantially Simultaneously: As used herein, and when it relates to multiple doses, the term means within 2 seconds.

Suffering from: Disease, Disorder, and / or Condition An individual "affected" has been diagnosed with the disease, disorder, and / or condition, or has one or more symptoms of the disease, disorder, and / or condition. Present.

Susceptible to: Disease, Disorder, and / or Condition An individual who is "susceptible to" is not diagnosed with the disease, disorder, and / or condition, and / or the disease, disorder, and / or condition. It may not show the symptoms of, but has an internal tendency to develop the disease or its symptoms. In some embodiments, an individual susceptible to a disease, disorder, and / or condition (eg, cancer) can be characterized by one or more of the following: (1) disease, disorder, and / Or gene mutations associated with the development of the condition, (2) gene polymorphisms associated with the development of the disease, disorder, and / or condition, (3) proteins and / or or / or conditions associated with the disease, disorder, and / or condition. Increased and / or decreased expression and / or activity of nucleic acids, (4) habits and / or lifestyles associated with the development of diseases, disorders, and / or conditions, (5) diseases, disorders, and / or conditions. Family history, and (6) exposure to and / or infection with microorganisms associated with the development of diseases, disorders, and / or conditions. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

Sustained Release: As used herein, the term "sustained release" refers to a pharmaceutical composition or compound release profile that is compatible with a release rate over a particular period of time.

Synthesis: The term "synthesis" means produced, prepared, and / or manufactured by human hands. The synthesis of the polynucleotides or polypeptides or other molecules of the invention may be chemical or enzymatic.

Targeted Cell: As used herein, "targeted cell" refers to any one or more cells of interest. The cells can be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. The organism can be an animal, preferably a mammal, more preferably a human, most preferably a patient.

Therapeutic agent: The term "therapeutic agent" has therapeutic, diagnostic, and / or prophylactic effects and / or induces the desired biological and / or pharmacological effect when administered to a subject. , Refers to any drug.

Therapeutically effective amount: As used herein, the term "therapeutically effective amount" is used when administered to a subject who suffers from or is susceptible to infections, diseases, disorders, and / or conditions. Drugs to be delivered (eg, nucleic acids) that are sufficient to treat, ameliorate, diagnose, prevent, and / or delay the onset of infections, diseases, disorders, and / or conditions. , Drugs, therapeutic agents, diagnostic agents, preventive agents, etc.).

Therapeutic Outcomes: As used herein, the term "therapeutically effective outcomes" refers to infections, diseases in subjects who suffer from or are susceptible to infections, diseases, disorders, and / or conditions. , Disorders, and / or conditions that are sufficient to treat, ameliorate, diagnose, prevent, and / or delay their onset.

Total Daily Dose: As used herein, "total daily dose" is the amount given or prescribed over a 24-hour period. It may be administered as a single unit dose.

Transcription Factors: As used herein, the term "transcription factor" refers to a DNA-binding protein that regulates the transcription of DNA into RNA, for example by activating or repressing transcription. Some transcription factors provide transcriptional regulation alone, while other transcription factors work in concert with other proteins. Some transcription factors can both activate and suppress transcription under certain conditions. In general, transcription factors bind to a particular target sequence (s) that closely resembles a particular consensus sequence within the regulatory region of the target gene. Transcription factors can regulate transcription of a target gene alone or in combination with other molecules.

Treat: As used herein, the term "treat" partially or completely alleviates one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. It refers to ameliorating, ameliorating, ameliorating, delaying its onset, inhibiting its progression, reducing its severity, and / or reducing its incidence. For example, "treating" cancer can mean inhibiting tumor survival, growth, and / or metastasis. Treatment is for subjects who do not show signs of disease, disorder, and / or condition, and / or disease, disorder, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. And / or may be administered to subjects who present only early signs of the condition.

Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule before it has been altered in any manner. Unmodified can refer to, but is not always, the wild-type or natural-type of a biomolecule. The molecule may undergo a series of modifications, whereby each modified molecule can serve as an "unmodified" starting molecule for subsequent modifications.

Equivalents and Scope One of ordinary skill in the art can recognize or elucidate many equivalents to the specific embodiments according to the invention described herein, using only routine experiments. The scope of the present invention is not intended to be limited to the above description, but rather to the scope of the appended claims.

In the claims, articles such as "a", "an", and "the" may mean one or more, unless otherwise specified or otherwise apparent from the context. .. A claim or statement containing "or" between one or more members of a group is one of the members of that group unless otherwise specified or otherwise apparent from the context. If two or more, or all, are present in, used in, or related to a given product or process, they are considered to be satisfied. The present invention includes embodiments in which exactly one member of the group is present in, is used in, or is associated with a given product or process. The present invention includes embodiments in which more than one or all of the members of the group are present in, are used in, or are associated with a given product or process.

It should also be noted that the term "contains" is intended to be unrestricted and allows the incorporation of additional elements or steps, but does not require it. When the term "contains" is used herein, the term "consisting of" is therefore also included and disclosed.

If a range is given, the endpoints are included. Moreover, unless otherwise specified or otherwise apparent from the context and understanding of one of ordinary skill in the art, the values represented as ranges are in different embodiments of the invention unless there is explicit contextual indication that they are not. It should be understood that within a defined range, any concrete value or subrange up to one tenth of the lower limit of the range can be taken.

Further, it should be understood that any particular embodiment of the invention that falls within the scope of the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are known to those of skill in the art, their exclusions may or may not be explicitly stated herein. Whether any particular embodiment of the composition of the invention (eg, any nucleic acid or protein encoded by it, any method of production, any method of use, etc.) is relevant to the presence of the prior art. Nevertheless, it may be excluded from any one or more claims for any reason.

All cited sources, such as references, publications, databases, database entries, and techniques cited herein are incorporated herein by reference, even if not explicitly stated in the citation. Is done. In the event of conflicting statements of the sources cited and this application, the statements in this application shall prevail.

Section and table headings are not intended to be limiting.

Example 1. Production of Modified mRNA Modified mRNA (mRNA) according to the present invention can be prepared using standard laboratory methods and materials. The open reading frame (ORF) of the gene of interest provides a 5'untranslated region (UTR) and / or oligo (dT) sequence that may contain a strong Kozak translation initiation signal for template addition of the poly A tail. It may be adjacent to the α-globin 3'UTR which may be included. Modified mRNA can be modified to reduce the innate immune response of the cell. Modifications to reduce the response of cells, pseudouridine ([psi) and 5-methyl - cytidine (5meC, 5mC or ^m 5 C,) may include (respectively, which are incorporated by reference in their entireties herein, Kariko See K et al. Immunoty 23: 165-75 (2005), Kariko K et al. Mol Ther 16: 1833-40 (2008), Anderson BR et al. NAR (2010)).

The ORF is DNA 2.0 (Menlo Park, CA), etc., but is limited to various upstream or downstream additions (β-globin, tags, etc.) that can be purchased from optimization services not limited to this. Can also include multiple cloning sites that may have XbaI recognition. Upon acceptance of the construct, it can be reconstituted and converted into chemically competent E. coli.

NEB DH5-α competent Escherichia coli is used in the present invention. Conversion is performed using 100 ng of plasmid according to NEB's instructions. The protocol is as follows.
1 Thaw the tube of NEB 5-α competent E. coli cells on ice for 10 minutes.
21 5 μL containing 1 pg to 100 ng of plasmid DNA is added to the cell mixture. Shake the tube carefully 4-5 times to mix the cells and DNA. Do not vortex.
3 Place the mixture on ice for 30 minutes. Do not mix.
4 Apply heat shock at 42 ° C for exactly 30 seconds. Do not mix.
Place on ice for 55 minutes. Do not mix.
6 Place the room temperature SOC into the mixture with a 950 μL pipette.
7 Place at 37 ° C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8 Warm the selection plate to 37 ° C.
9 Shake the tube and turn it upside down to completely mix the cells.

Spread 50-100 μL of each dilution on a selection plate and incubate overnight at 37 ° C. Alternatively, incubate at 30 ° C. for 24-36 hours or 25 ° C. for 48 hours.

The single colony is then inoculated with 5 mL of LB growth medium with the appropriate antibiotic and grown for 5 hours (250 RPM, 37 ° C.). It is then used to inoculate 200 mL of culture medium and grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), maxiprep is performed using the Invitrogen PURELINK ™ HiPure Maxiprep Kit (Carlsbad, CA) according to the manufacturer's instructions.

To generate cDNA for in vitro transcription (IVT), the plasmid (an example of which is shown in FIG. 3) is first linearized with a restriction enzyme such as XbaI. Typical restriction digestion with XbaI includes: plasmid 1.0 μg; 10-fold buffer 1.0 μL; XbaI 1.5 μL; dH ₂₀ up to 10 μL; incubation at 37 ° C. for 1 hour. When performed on a laboratory scale (less than 5 μg), the reactants are purified using Invitrogen's PURELINK ™ PCR Micro Kit (Carlsbad, CA) according to the manufacturer's instructions. Larger scale purification may need to be performed using products with larger filling volumes, such as Invitrogen's standard PURELINK ™ PCR Kit (Carlsbad, CA). After purification, the linearized vector is quantified using NanoDrop and analyzed using agarose gel electrophoresis to confirm linearization.

The modified mRNA production methods described herein can be used to produce molecules of all sizes, including long molecules. Acidic α-glucosidase (GAA) (3.2 kb), cystic fibrosis membrane conductance regulator (CFTR) (4.7 kb), factor VII (7.3 kb), lysosomal acid lipase (45) using the methods described. Modified mRNAs of different sized molecules were generated, including .4 kDa), glucoserebrosidase (59.7 kDa), and isulonic acid 2-sulfatase (76 kDa).

As a non-limiting example, G-CSF may represent the polypeptide of interest. The sequences used for the steps outlined in Examples 1-5 are shown in Table 11. Note that the start codon (ATG) is underlined in each sequence of Table 11.
Table 11. G-CSF sequence

Example 2: PCR for cDNA production
The PCR procedure for the preparation of cDNA is performed using 2xKAPA HIFI ™ HotStart ReadyMix by Kappa Biosystems (Woburn, MA). The system includes 12.5 μL of 2 × KAPA ReadyMix, 0.75 μL of forward primer (10 uM), 0.75 μL of reverse primer (10 uM), 100 ng of template cDNA, and dH ₂₀ diluted to 25.0 μL. The reaction conditions are 95 ° C. for 5 minutes, 98 ° C. for 20 seconds for 25 cycles, then 58 ° C. for 15 seconds, then 72 ° C. for 45 seconds, then 72 ° C. for 5 minutes, and then 4 ° C. until completion.

The reverse primers of the invention incorporate poly T _{120 for poly A 120 in mRNA.} Other reverse primers with longer or shorter poly (T) pathways can be used to regulate the length of the poly (A) tail in mRNA.

The reaction is purified using Invitrogen's PURELINK ™ PCR Micro Kit (Carlsbad, CA) according to the manufacturer's instructions (up to 5 μg). Larger reactions need to be purified with larger volumes of products. After purification, the cDNA is quantified using NANODROP ™ and analyzed by agarose gel electrophoresis to confirm that the cDNA is of the predicted size. The cDNA is then submitted for sequencing analysis before proceeding with an in vitro transcription reaction.

Example 3. In vitro transcription (IVT)
In vitro transcriptional reaction produces mRNA containing modified nucleotides or modified RNA. The nucleotide triphosphate (NTP) mixture to be charged is self-prepared using natural and non-natural NTP.

Typical in vitro transcriptional reactants include:
1 Template cDNA 1.0 μg
2 10-fold transfer buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, 10 mM spermidine) 2.0 μL
3 Custom NTP (25 mM each) 7.2 μL
4 RNase inhibitor 20U
5 T7 RNA polymerase 3000U
6 dH20 Maximum 20.0 μL
7 Incubation at 37 ° C for 3 to 5 hours.

The crude IVT mixture can be stored overnight at 4 ° C. for next day purification. The original template is then digested using 1 U of RNase-free DNase. After incubation at 37 ° C. for 15 minutes, mRNA is purified using Ambion's MEGACLEAR ™ Kit (Austin, TX) according to the manufacturer's instructions. This kit can purify up to 500 μg of RNA. After purification, RNA is quantified using NanoDrop and analyzed by agarose gel electrophoresis to confirm that the RNA is of the proper size and that RNA has not been degraded.

Example 4. Enzyme capping of mRNA Capping of mRNA is performed as follows, and the mixture contains 60 μg to 180 μg of IVT RNA and up to 72 _{μL of dH 20.} The mixture is incubated at 65 ° C. for 5 minutes to denature the RNA and then immediately transferred to ice.

This protocol is then followed by 10-fold capping buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl ₂ ) (10.0 μL), 20 mM GTP (5.0 μL), 20 mM S-. Adenosylmethionine (2.5 μL), RNase inhibitor (100 U), 2'-O-methyltransferase _{(400 U), vaccinia capping enzyme (guanylyl transferase) (40 U), dH 20} (up to 28 μL), and 60 μg. RNA is incubated at 37 ° C. for 30 minutes or 180 μg RNA with up to 2 hours of incubation.

The mRNA is then purified using Ambion's MEGACLEAR ™ Kit (Austin, TX) according to the manufacturer's instructions. After purification, RNA was quantified using NANODROP ™ (Thermo Fisher, Waltham, MA) and analyzed by agarose gel electrophoresis to result in RNA size and RNA degradation. Make sure it is not. In addition, reverse transcription PCR may be performed on the RNA product for sequencing in order to generate cDNA for sequencing.

Example 5. Poly-A tailing reaction If the cDNA is free of poly-T, a poly-A tailing reaction must be performed before purifying the final product. This is capped IVT RNA (100 μL), RNase inhibitor (20 U), 10-fold tailing buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl ₂ ) (12.0 μL). ), 20mM ATP (6.0μL), poly a polymerase (20 U), a mixture of dH ₂ 0 up 123.5MyuL, carried out by incubating at 37 ° C. 30 min. If the poly A tail is already present in the transcript, the tailing reaction may be omitted and purification may proceed directly with Ambion's MEGACLEAR ™ kit (Austin, TX) (up to 500 μg). The poly A polymerase is preferably a recombinant enzyme expressed in yeast.

In the studies performed and described herein, the poly A tail is encoded in an IVT template to include 160 nucleotides in length. However, it should be understood that the progression or integrity of the polyA tailing reaction does not always result in exactly 160 nucleotides. Thus, a poly A tail of about 160 nucleotides, eg, about 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, or 165, is within the scope of the invention.

Example 6. 5'capping of native 5'caps and 5'cap analogs modified RNA may be concomitantly completed during the in vitro transcription reaction with the following chemical RNA cap analogs according to the manufacturer's protocol. 'Guanosine cap structures are produced: 3'-O-Me-m7G (5') ppp (5') G [ARCA cap], G (5') ppp (5') A, G (5') ppp (5') G, m7G (5') ppp (5') A, m7G (5') ppp (5') G (New England BioLabs, Ipsich, MA). 5'capping of modified RNA can be completed post-transcriptional using a vaccinia virus capping enzyme to produce a "cap 0" structure: m7G (5') ppp (5') G (New England BioLabs, Ipswich, MA). .. The cap 1 structure can be produced using both the vaccinia virus capping enzyme and the 2'-O methyl tranceferase to give m7G (5') ppp (5') G-2'-O-methyl. The cap 2 structure can be generated from the cap 1 structure after 2'-O-methylation of the 3rd nucleotide from the 5'end using a 2'-O methyl-transferase. The cap 3 structure can be generated from the cap 2 structure after 2'-O-methylation of the 4th nucleotide from the 5'end using a 2'-O methyl-transferase. The enzyme is preferably derived from a recombinant source.

When transfecting mammalian cells, the modified mRNA has stability of greater than 12-18 hours, or greater than 18 hours, eg, 24, 36, 48, 60, 72, or greater than 72 hours.

Example 7. Capping A. Protein Expression Assay A synthetic mRNA encoding human G-CSF containing an ARCA (3'O-Me-m7G (5') ppp (5') G) cap analog or cap 1 structure (cDNA at SEQ ID NO: 16099). The mRNA sequence shown and fully modified with 5-methylcytosine in each cytosine and pseudouridine substitution at each uridine site is shown in SEQ ID NO: 21438, with a poly A tail of approximately 160 nucleotides (nucletoide) length in the sequence. (Not shown) can be transfected into human primary keratinocytes at equal concentrations. The amount of G-CSF secreted into the culture medium can be assayed by ELISA 6, 12, 24, and 36 hours after transfection. Synthetic mRNAs that secrete higher levels of G-CSF in the medium correspond to synthetic mRNAs with a more highly translationally competent cap structure.

B. Purity Analytical Synthesis Synthetic mRNA encoding human G-CSF containing an ARCA cap analog or a crude synthetic product of cap 1 structure (the cDNA is shown in SEQ ID NO: 16099, at 5-methylcytosine in each cytosine and at each uridine site. The mRNA sequence fully modified with pseudouridine substitution is shown in SEQ ID NO: 21438 and the poly A tail of about 160 nucleotides is not shown in the sequence), using modified agarose-urea gel electrophoresis or HPLC analysis. Can be compared. Synthetic mRNAs with a single aggregated band by electrophoresis correspond to products of higher purity compared to synthetic mRNAs with multiple bands or streaky bands. Synthetic mRNAs with a single HPLC peak also correspond to higher purity products. A more efficient capping reaction results in a purer mRNA population.

C. Cytosine Analysis Synthetic mRNA encoding human G-CSF containing an ARCA cap analog or cap 1 structure (the cDNA is shown in SEQ ID NO: 16099 and is complete by 5-methylcytosine at each cytosine and pseudouridine substitution at each uridine site. The mRNA sequence modified to is shown in SEQ ID NO: 21438 and the poly A tail, which is about 160 nucleotides in length, is not shown in the sequence) can be transfected into human primary keratinocytes at multiple concentrations. The amount of pro-inflammatory cytokines such as TNF-α and IFN-β secreted into the culture medium can be assayed by ELISA 6, 12, 24, and 36 hours after transfection. Synthetic mRNAs that secrete higher levels of pro-inflammatory cytokines in the medium correspond to synthetic mRNAs with an immunostimulatory cap structure.

D. Capping reaction efficiency Synthetic mRNA encoding human G-CSF containing an ARCA cap analog or cap 1 structure, fully modified by 5-methylcytosine and pseudouridine substitution at each uridine site, cDNA is shown in SEQ ID NO: 16099. The mRNA sequence that was obtained is shown in SEQ ID NO: 21438 in each cytosine, and the poly A tail, which is about 160 nucleotides in length, is not shown in the sequence), and the efficiency of the capping reaction by LC-MS after nuclease treatment of the capped mRNA. Can be analyzed. Nuclease treatment of the capped mRNA results in a mixture of free nucleotides detectable by LC-MS and a capped 5'-5 triphosphate cap structure. The amount of capped product on the LC-MS spectrum can be expressed as a percentage of total mRNA from the reaction and will correspond to the efficiency of the capping reaction. Cap structures with higher capping reaction efficiencies will have higher amounts of capped products by LC-MS.

Example 8. Agarose Gel Electrophoresis of Modified RNA or RT PCR Product Individual modified RNA (200-400 ng in 20 μL volume) or reverse-transcribed PCR product (200-400 ng), undenatured 1.2% agarose E-gel (Invitrogen, Carlsbad, CA) wells are filled and run for 12-15 minutes according to the manufacturer's protocol.

Example 9. Quantification of Nanodrop Modified RNA and UV Spectral Data The modified RNA in TE buffer (1 μL) is used for Nanodrop UV absorption reading to quantify the yield of each RNA from the in vitro transcription reaction.

Example 10. Formulation of Modified mRNA with Lipidoid The modified mRNA (m mRNA) is formulated for in vitro experiments by mixing the m mRNA and lipidoid at a set ratio before adding to the cells. In vivo formulations may require the addition of additional ingredients to promote circulation throughout the body. To test the ability of these lipidoids to form particles suitable for working in vivo, the standard formulation process used for siRNA-lipidoid formulations was used as the starting point. The initial mmRNA-lipidoid preparation may consist of particles composed of 42% lipidoid, 48% cholesterol, and 10% PEG, allowing further optimization of the ratio. After particle formation, mmRNA is added and integrated with the complex. Encapsulation efficiency is determined using a standard dye exclusion assay.

Materials and Methods of Examples 11-15 A. Lipid Synthesis To formulate six lipids, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, and DLin-MC3-DMA with modified RNA, the art. Synthesized by the method outlined in. DLin-DMA and precursors were described in Heies et. al, J. Synthesized as described in Control Release, 2005, 107, 276-287. DLin-K-DMA and DLin-KC2-DMA, as well as precursors, were presented in Simple et. It was synthesized as described in al, Nature Biotechnology, 2010, 28, 172-176. 98N12-5 and precursors were added to Akinc et. al, nature
It was synthesized as described in Biotechnology, 2008, 26, 561-569.

C12-200 and precursors were added to Love et. It was synthesized according to the method outlined in al, PNAS, 2010, 107, 1864-1869. 2-Epoxy dodecane (5.10 g, 27.7 mmol, 8.2 eq) was added to a vial containing Amine 200 (0.723 g, 3.36 mmol, 1 eq) and a stir bar. The vial was sealed and warmed to 80 ° C. The reaction was stirred at 80 ° C. for 4 days. The mixture was then purified from pure dichloromethane (DCM) by silica gel chromatography using a DCM: MeOH 98: 2 gradient. The target compound was further purified by RP-HPLC to obtain the desired compound.

DLin-MC3-DMA and precursors were synthesized according to the procedure described in WO20100054401, which is incorporated herein by reference in its entirety. Dilinoleylmethanol (1.5 g, 2.8 mmol, 1 eq), N, N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1 eq), DIPEA (0.73 mL, 4. Eq) in 10 mL DMF. A mixture of 2 mmol, 1.5 eq) and TBTU (1.35 g, 4.2 eq, 1.5 eq) was stirred at room temperature for 10 hours. The reaction mixture was then diluted in ether and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified from DCM by silica gel chromatography using a DCM: MeOH 98: 2 gradient. Subsequently, the target compound was subjected to further RP-HPLC purification (which was performed using a YMC-Pack C4 column) to obtain the target compound.

B. Formulation of Modified RNA Nanoparticles Synthetic lipids, 1,2-distearoyl-3-phosphatidylcholine (DSPC) (Avanti Polar Lipids, Alabaster, AL), cholesterol (Sigma-Aldrich, Taufkirchen, Germany), and α- [3' -(1,2-Dimiristyl-3-propanoxy) -carboxamide-propyl] -ω-methoxy-polyoxyethylene (PEG-c-DOMG) (NOF, Bowwelven, Bergium) solution at a concentration of 50 mM in ethanol. It was prepared and stored at −20 ° C. The lipids were combined to give a molar ratio of 50:10: 38.5: 1.5 (lipid: DSPC: cholesterol: PEG-c-DOMG) and diluted with ethanol to a final lipid concentration of 25 mM. A solution of modified mRNA at a concentration of 1-2 mg / mL in water was diluted in 50 mM sodium citrate buffer at pH 3 to form a stock solution of modified mRNA. The lipid and modified mRNA preparations were prepared by combining the synthetic lipid solution and the modified mRNA solution in a weight ratio of 10: 1, 15: 1, 20: 1, and 30: 1 total lipid to modified mRNA. The lipid ethanol solution was rapidly injected into the modified mRNA aqueous solution to obtain a suspension containing 33% ethanol. The solution was injected either manually (MI) or using a syringe pump (SP) (Harvard Pump 33 Dual Syringe).
Pump Harvard Apparatus Holliston, MA).

To remove ethanol and achieve buffer exchange, the formulation is made of the primary product at a molecular weight cutoff (MWCO) of 10 kDa, using a Slide-A-Lyzer cassette (Thermo Fisher Scientific Inc. Rockfort, IL). It was dialyzed twice against phosphate buffered saline (PBS) at a pH of 7.4 in a volume of 200 times. The first dialysis was performed at room temperature for 3 hours, then the formulation was dialyzed overnight at 4 ° C. The resulting nanoparticle suspension was filtered through a 0.2 μm sterilization filter (Sarthtedt, N, high b-nitrogen, mo, soot CGermany), placed in a glass vial, and pressure-sealed.

C. Formulation Characteristics Using Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK), the particle size, polydispersity index (PDI), and zeta potential of modified mRNA nanoparticles were determined to be 1 when determining the particle size. Judgment was made in double PBS and in 15 mM PBS when determining the zeta potential.

Ultraviolet-visible spectroscopy was used to determine the concentration of the modified mRNA nanoparticle formulation. 100 μL of the pharmaceutical product diluted in 1-fold PBS was added to 900 μL of a 4: 1 (v / v) mixture of methanol and chloroform. After mixing, the absorbance spectra of the solutions were recorded at 230 nm to 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of modified RNA in the nanoparticle formulation was calculated based on the attenuation coefficient of the modified RNA used in the formulation and the difference between the absorbance at a wavelength of 260 nm and the baseline at a wavelength of 330 nm.

Encapsulation of modified RNA with nanoparticles was evaluated using the QUANT-IT ™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA). Samples were diluted to a concentration of approximately 5 μg / mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). A 50 μL diluted sample was transferred to a polystyrene 96-well plate and then either 50 μL TE buffer or 50 μL 2% Triton X-100 solution was added. The plate was incubated at a temperature of 37 ° C. for 15 minutes. The RIBOGREEN® reagent was diluted 1: 100 in TE buffer and 100 μL of this solution was added to each well. Fluorescence intensity was measured at an excitation wavelength of about 480 nm and an emission wavelength of about 520 nm using a fluorescence plate reader (Wallac Victor 1420 Multibull Counter; Perkin Elmer, Waltham, MA). The fluorescence value of the reagent blank was subtracted from each sample, and the percentage of free modified RNA was the sample (Triton X-10) in which the fluorescence intensity of the intact sample (without the addition of Triton X-100) was divided. It was determined by dividing by the fluorescence value of) (caused by the addition of).

D. In vitro Incubation Human fetal kidney epithelium (HEK293) and hepatocellular carcinoma epithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) were seeded on 96-well plates (Greener Bio-one GmbH, Frickenhausen, Germany). The plate was pre-coated with type 1 collagen. In 100 μL of cell culture medium, HEK293 was seeded at a density of 30,000 cells per well and HepG2 was seeded at a density of 35,000 cells. For HEK293, the cell culture medium was 2 mM L-glutamine, 1 mM sodium pyruvate, and 1x non-essential amino acids (Biochrom AG, Berlin, Germany), and 1.2 mg / mL sodium bicarbonate (Sigma-Aldrich,). DMEM, 10% FCS supplemented with Munich, Germany), and for HepG2, the culture medium was supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 1x non-essential amino acids (Biochrom AG, Berlin, Germany). MEM (Gibco Life Technologies, Damstat, Germany) was 10% FCS. MCherry mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) was quadrupled immediately after seeding the cells and incubated. MCherry cDNA with T7 promoter, 5'untranslated region (UTR), and 3'UTR used for in vitro transcription (IVT). , SEQ ID NO: 21440. MCherry mRNA was modified with 5 meC in each cytosine and pseudouridine substitution at each uridine site.

Cells were collected by transferring the culture medium supernatant to a 96-well Pro-Bind U bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with half the amount of trypsin / EDTA (Biochrom AG, Berlin, Germany), pooled with their respective supernatants, and single doses of PBS / 2% FCS (all Biochrom AG, Berlin, Germany) / 0. It was fixed by adding 5% formaldehyde (Merck, Darmstadt, Germany). Samples were then subjected to flow cytometer measurements on an LSRII cytometer (Beckton Dickinson GmbH, Heidelberg, Germany) using a 532 nm excitation laser and a 610/20 filter for PE-Texas Red. The mean fluorescence intensity (MFI) of all events and the standard deviation of the four independent wells are presented for the samples analyzed.

表１３．ＨＥＫ２９３およびＨｅｐＧ２、２４ウェル、２５０ｎｇの修飾ＲＮＡ／ウェル

Example 11. Purification of Nanoparticle Formulations DLin-KC2-DMA and 98N12-5 nanoparticle formulations were tested in HEK293 and HepG2 to determine if average fluorescence intensity (MFI) was dependent on lipid to modified RNA ratio and / or purification. Judged. Three formulations of DLin-KC2-DMA and two formulations of 98N12-5 were produced using a syringe pump to the specifications shown in Table 12. Purified samples were purified by SEPHADEX ™ G-25 DNA grade (GE Healthcare, Sweden). The pre-purification and post-purification (aP) formulations were tested in 24-well plates at a concentration of 250 ng of modified RNA per well. Table shows the percentage of cells positive for FL4 channel markers (FL4 positive%) when analyzed by flow cytometer for each formulation and background sample, and the MFI of FL4 channel markers for each formulation and background sample. It is shown in 13. The purified formulation had a slightly higher MFI than the formulation tested prior to purification.
Table 12. pharmaceutical formulation

Table 13. HEK293 and HepG2, 24 wells, 250 ng of modified RNA / well

Example 12. Concentration Response Curves 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003) nanoparticle formulations were tested at various concentrations over a wide range of concentrations, FL4 or mCherry (mRNA sequence is SEQ ID NO: SEQ ID NO: The MFI of (shown in 21439; the polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) was determined. The formulations tested are outlined in Table 14. To determine the optimum concentration of 98N12-5 nanoparticle formulation, various concentrations of formulated modified RNA (100 ng, 10 ng, 1.0 ng, 0.1 ng, and 0.01 ng per well) were added. Tested in HEK293 on a 24-well plate, the FL4 MFI results for each dose are shown in Table 15. Similarly, various concentrations of formulated modified RNA (250 ng 100 ng, 10 ng, 1.0 ng, 0.1 ng, and per well) are used to determine the optimal concentration of DLin-KC2-DMA nanoparticle formulations. 0.01 ng) was tested on a 24-well plate HEK293 and the results of FL4 MFI for each dose are shown in Table 16. Nanoparticle formulations of DLin-KC2-DMA were also tested in 24-well plates of HEK293 with various concentrations of formulated modified RNA (250 ng, 100 ng, and 30 ng per well) and the FL4 MFI results are shown in Table 17. show. A dose of 1 ng / well for 98N12-5 and a dose of 10 ng / well for DLin-KC2-DMA were found to be similar to the background FL4 MFI.

表１５．ＨＥＫ２９３、ＮＰＡ−００５、２４ウェル、ｎ＝４

表１６．ＨＥＫ２９３、ＮＰＡ−００３、２４ウェル、ｎ＝４

表１７．ＨＥＫ２９３、ＮＰＡ−００３、２４ウェル、ｎ＝４

表１８．濃度およびＭＦＩ

To determine how similar the concentrations were to the background, we used a flow cytometer with a filter set optimized for the detection of mCherry expression and results with increased sensitivity compared to background levels. I was able to get. Dose of 25 ng / well, 0.25 ng / well, 0.025 ng / well, and 0.0025 ng / well were analyzed for 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003). The MFI of mCherry was determined. As shown in Table 18, concentrations of 0.025 ng / well and below are similar to mCherry's background MFI levels of about 386.125.
Table 14. pharmaceutical formulation

Table 15. HEK293, NPA-005, 24 wells, n = 4

Table 16. HEK293, NPA-003, 24 wells, n = 4

Table 17. HEK293, NPA-003, 24 wells, n = 4

Table 18. Concentration and MFI

Example 13. Manual Infusion and Syringe Pump Formulas Two formulations, DLin-KC2-DMA and 98N12-5, are prepared by manual infusion (MI) and syringe pump infusion (SP) and analyzed with background samples to mCherry (different formulations). The mRNA sequence is shown in SEQ ID NO: 21439; the poly A tail of about 160 nucleotides is not shown in the sequence; the MFI of 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) was compared. Table 19 shows that the syringe pump formulation had a higher MFI compared to the manual infusion formulation of the same lipid and lipid / RNA ratio.
Table 19. Formulation and MFI

表２１．ＨＥＫ２９３、９６ウェル、６０ｎｇの修飾ＲＮＡ／ウェル

表２２．ＨＥＫ２９３、６２．５ｎｇ／ウェル

表２３．ＨＥＫ２９３、６２．５ｎｇ／ウェル

表２４．ＨｅｐＧ２、６２．５ｎｇ／ウェル

表２５．ｅｐＧ２、６２．５ｎｇ／ウェル

Example 14. Lipid nanoparticle preparations The preparations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, and DLin-MC3-DMA are 60 ng / well or 62.5 ng / well or 62.5 ng / well on the plate of HEK293. Incubate for 24 hours at a concentration of wells, at a concentration of 62.5 ng / well on a plate of HepG2 cells, for each formulation mCherry (sequence number 21439; poly A tail of approximately 160 nucleotides not shown in the sequence; The MFI of 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) was determined. The formulations tested are outlined in Table 20 below. As shown in Table 21 for 60 ng / well and Tables 22, 23, 24, and 25 for 62.5 ng / well, the NPA-003 and NPA-018 formulations have the highest mCherry MFI. The formulations of NPA-008, NPA-010, and NPA-013 are most similar to the mCherry MFI value of the background sample.
Table 20. pharmaceutical formulation

Table 21. HEK293, 96 wells, 60 ng of modified RNA / well

Table 22. HEK293, 62.5 ng / well

Table 23. HEK293, 62.5 ng / well

Table 24. HepG2, 62.5 ng / well

Table 25. epG2, 62.5 ng / well

Example 15. In vivo drug product study A single dose of a drug product containing modified mRNA and lipid is administered intravenously, subcutaneously, or intramuscularly to rodents (n = 5). The modified mRNA administered to the rodent is G-CSF (mRNA sequence is shown in SEQ ID NO: 21438; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), erythropoetin (EPO). (MRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), factor IX (mRNA sequence is shown in SEQ ID NO: 1622; about 160 nucleotides. Poly A tail is not shown in the sequence; 5'cap, cap 1), or mCherry (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, Selected from cap 1). The erythropoietin cDNA having the T7 promoter, 5'untranslated region (UTR), and 3'UTR used for in vitro transcription (IVT) is shown in SEQ ID NO: 21441 and SEQ ID NO: 21442.

The respective formulations are also among DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, reLNP, ATUPLEX®, DACC, and DBTC. Contains a lipid selected from one. Rodents are infused with 100 μg, 10 μg, or 1 μg of formulated modified mRNA and samples are collected at specified time intervals.

Serum from rodents administered with a preparation containing human G-CSF modified mRNA was measured by specific G-CSF ELISA, and serum from mice administered with human Factor IX modified RNA was obtained with specific IX. Analyze by factor ELISA or color assay. Liver and spleen from mice treated with mCherry-modified mRNA are analyzed by immunohistochemistry (IHC) or fluorescence activated cell selection (FACS). As a control, serum and tissues thereof are collected and analyzed by ELISA, FACS, and / or IHC without injecting any preparation into one mouse group.

A. Time change A rodent is administered with a preparation containing at least one modified mRNA, and the change over time in protein expression of the administered preparation is studied. Blood is collected from rodents at specified time intervals before and after administration of the modified mRNA preparation to determine protein expression and total blood cell count. Samples are also collected from the administration site of rodents to which the modified mRNA preparation is subcutaneously or intramuscularly administered to determine protein expression in tissues.

B. Dose Response Rodents are administered with a formulation containing at least one modified mRNA to determine the dose response of each formulation. Blood is collected from rodents at specified time intervals before and after administration of the modified mRNA preparation to determine protein expression and total blood cell count. In addition, rodents are slaughtered and the effects of modified mRNA preparations on internal tissues are analyzed. Samples are also collected from the administration site of rodents to which the modified mRNA preparation is subcutaneously or intramuscularly administered to determine protein expression in tissues.

C. Toxicity Rodents are administered with preparations containing at least one modified mRNA to study the toxicity of each preparation. Blood is collected from rodents at specified time intervals before and after administration of the modified mRNA preparation to determine protein expression and total blood cell count. In addition, rodents are slaughtered and the effects of modified mRNA preparations on internal tissues are analyzed. Samples are also collected from the administration site of rodents to which the modified mRNA preparation is subcutaneously or intramuscularly administered to determine protein expression in tissues.

Example 16. PLGA Microsphere Formulation Optimizing the parameters used to formulate PLGA microspheres allows for adjustable release rates and high encapsulation efficiency while maintaining the integrity of modified RNA encapsulated in microspheres. can do. Optimal preparation can be achieved by optimizing parameters such as particle size, recovery rate, and encapsulation efficiency, but not limited thereto.

A. Synthesis of PLGA Microspheres Polygalactic glycolic acid (PLGA) microspheres, PLGA (Lactel, Catalog No. B6010-2, Intrinsic Viscosity 0.55 to 0.75, LA: GA at 50:50), Polyvinyl Alcohol (PVA) (Sigma, Catalog No. 348406-25G, MW 13-23k), dichloromethane, and water were used to synthesize using the water / oil / water double emulsification method known in the art. Briefly, 0.1 mL of water (W1) was added to 2 mL of PLGA dissolved in dichloromethane (DCM) (O1) over a PLGA concentration range of 50-200 mg / mL. The W1 / O1 emulsion was homogenized at a rate of 4 (about 15,000 rpm) for 30 seconds (IKA Ultra-Turrax Homogenizer, T18). The W1 / O1 emulsion was then added to 100-200 mL of 0.3-1% PVA (W2) and homogenized at various rates for 1 minute. The pharmaceutical product was stirred as it was for 3 hours and then washed by centrifugation (20-25 minutes, 4,000 rpm, 4 ° C.). The supernatant was discarded and the PLGA pellets were resuspended in 5-10 mL of water, which was repeated twice. The average particle size (representing 20 to 30 particles) of each pharmaceutical product was determined by microscopic examination after washing. Table 26 shows that the increase in PLGA concentration resulted in larger particle size microspheres. A PLGA concentration of 200 mg / mL resulted in an average particle size of 14.8 μm, an average particle size of 8.7 μm at 100 mg / mL, and an average particle size of 4.0 μm at 50 mg / mL PLGA.
Table 26. Changes in PLGA concentration

Table 27 shows that the decrease in homogenization rate from 5 (about 20,000 rpm) to speed 4 (about 15,000 rpm) resulted in an increase in particle size from 14.8 μm to 29.7 μm.
Table 27. Change in homogenization rate

Table 28 shows that increasing the volume of W2 (ie, increasing the W2: O1 ratio from 50: 1 to 100: 1) resulted in a slight decrease in average particle size. The change in PVA concentration from 0.3 to 1% by weight had little effect on the size of the PLGA microspheres.
Table 28. Changes in W2 volume and concentration

B. Encapsulation of modified mRNA Modified G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; with 5'cap, cap 1; 5-methylcytosine and pseudouridine. Fully modified) was dissolved in water at a concentration of 2 mg / mL (W3). Three batches of PLGA microspheres were prepared as described above according to the following parameters: 0.1 mL W3 at 2 mg / mL, 1.6 mL O1 at 200 mg / mL, 160 mL W2 at 1%, and the first. The emulsion of 1 (W3 / O1) is homogenized at a speed of 3, and the second emulsion (W3 / O1 / W2) is homogenized at a speed of 5. After washing by centrifugation, the formulations were frozen in liquid nitrogen and then lyophilized for 3 days. To test the encapsulation efficiency of the formulation, the lyophilized material was deformed in DCM for 6 hours and then extracted into water overnight. The concentration of modified RNA in the sample was then determined by OD260. Encapsulation efficiency was calculated by taking the actual amount of modified RNA and dividing by the starting amount of modified RNA. The three batches tested had encapsulation efficiencies of 59.2, 49.8, and 61.3.

C. Completeness of Modified mRNA Encapsulated in PLGA Microspheres Modified Factor IX mRNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; (Completely modified with 5-methylcytosine and pseudouridine) was dissolved in water at various concentrations (W4) to change the packing weight percent (mg modified RNA / mg PLGA * 100) in the formulation to increase encapsulation efficiency. Judged. Using the parameters in Table 29, the first emulsion (W4 / O1) has a homogenization rate of 4, the second emulsion (W4 / O1 / W2) has a homogenization rate of 5, and four different batches of PLGA micro. A sphere preparation was prepared.
Table 29. Parameters of Factor IX PLGA Microspheres

Ｄ．ＰＬＧＡマイクロスフェアにカプセル封入された修飾ｍＲＮＡの放出研究 After lyophilization, PLGA microspheres were weighed into 2 mL Eppendorf tubes to correspond to approximately 10 μg of modified RNA. Freeze-drying was found not to destroy the overall structure of PLGA microspheres. Increasing amounts of modified RNA were added to the sample to increase the packing weight percent (% by weight) of PLGA microspheres. PLGA microspheres were deformulated by adding 1.0 mL of DCM to each tube and then shaking the sample for 6 hours. For extraction of modified RNA, 0.5 mL of water was added to each sample, the sample was shaken overnight, and then the concentration of modified RNA in the sample was determined by OD260. To determine the recovery of the extraction process, unformulated factor IX modified RNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) (depreparation control) was added to the DCM and subjected to the depreparation process. Table 30 shows the efficiency of sample filling and encapsulation. All encapsulation efficiency samples were normalized to degeneralized controls.
Table 30. Filling weight percent and encapsulation efficiency

D. Release study of modified mRNA encapsulated in PLGA microspheres

With factor IX modified RNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine). The formulated PLGA microspheres were deprepared as described above and the completeness of the extracted modified RNA was determined by automated electrophoresis (Bio-Rad Experion). The extracted modified mRNA was compared to the unformulated modified mRNA and the depreparized control to study the integrity of the encapsulated modified mRNA. As shown in FIG. 4, the majority of the modified RNA has batch identifiers A, B, C, and D, a deformated control (deformated control), and a non-formulated control (non-formulated control). Was intact.

E. Protein expression of modified mRNA encapsulated in PLGA microspheres Factor IX modified RNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; PLGA microspheres formulated with 5-methylcytosine and pseudouridine) were deprepared as described above and the protein expression of the extracted modified RNA was determined by in vitro transfection assay. HEK293 cells were triple-backtransfected with 250 ng of Factor IX-modified RNA complexed with RNAiMAX (Invitrogen).

Factor IX-modified RNA was diluted to a concentration of 25 ng / μL in nuclease-free water, and RNAiMAX was diluted 13.3 times in serum-free EMEM. Equal volumes of diluted RNAiMAX and diluted RNAiMAX were mixed together and allowed to stand at room temperature for 20-30 minutes. Subsequently, 20 μL of the transfection mixture containing 250 ng of Factor IX modified RNA was added to an 80 μL cell suspension containing 30,000 cells. The cells were incubated for 16 hours in a humidified 37 ° C./5% CO2 cell culture incubator and then the cell culture supernatant was collected. The protein expression of Factor IX in the cell supernatant was analyzed, especially with the Elisa kit for Factor IX (Molecular Innovations, Catalog No. HFIXKT-TOT), and the protein expression is shown in Table 31 and FIG. In all PLGA microsphere batches tested, factor IX modified RNA remained active and expressed factor IX protein after formulation into PLGA microspheres and subsequent deformation.
Table 31. Protein expression

F. Release study of modified mRNA encapsulated in PLGA microspheres Factor IX modified RNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; PLGA microspheres formulated with 5-methylcytosine and pseudouridine) were resuspended in water to a PLGA microsphere concentration of 24 mg / mL. After resuspension, 150 μL of PLGA microsphere suspension was aliquoted into Eppendorf tubes. Samples were continuously incubated and shaken at 37 ° C. during the study process. Triple samples were removed at 0.2, 1, 2, 8, 14, and 21 days. To determine the amount of modified RNA released from PLGA microspheres, the sample was centrifuged, the supernatant was removed, and the concentration of modified RNA in the supernatant was determined by OD260. The percent release shown in Table 32 was calculated based on the total amount of modified RNA in each sample. After 31 days, 96% of Factor IX modified RNA was released from the PLGA microspheres.
Table 32. Emission percentage

G. Particle size reproducibility of PLGA microspheres Three batches of factor IX modified RNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5 -PLGA microspheres (fully modified with methylcytosine and pseudouridine) were made using the same conditions as described for batch D shown in Table 29 (at 4 mg / mL 0.4 mL W4, at 200 mg / mL). 2.0 mL O1, 1% 200 mL W2, and W4 / O1 / W2 emulsion homogenized at rate 5). Filtration was incorporated prior to centrifugation to improve the homogeneity of the PLGA microsphere suspension. After stirring for 3 hours and prior to centrifugation, all pharmaceutical materials were passed through a 100 μm nylon mesh sieve (Fisherbrand Cell Strainer, Catalog No. 22-363-549) to remove large aggregates. After washing with water and resuspending, 100-200 μL of PLGA microspheres sample was used to measure the particle size of the pharmaceutical by laser diffraction (Marvern Mastersizer 2000). The particle size of the sample is shown in Table 33.
Table 33. Summary of particle size

The results of three PLGA microsphere batches with filtration were compared to PLGA microsphere batches made under the same conditions without filtration. Incorporating a filtration step prior to washing reduced the average particle size and showed a consistent particle size distribution among the three PLGA microsphere batches.

H. Serum Stability of Factor IX PLGA Microspheres Factor IX mRNA mRNA in buffer (TE) or 90% serum (Se) (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of approximately 160 nucleotides is within the sequence Not shown in; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), or buffer, 90% serum, or factor IX mRNA in PLGA in 1% serum, buffer, Incubated in 90% serum, or 1% serum, at an mRNA concentration of 50 ng / μL in a total volume of 70 μL. Samples were removed in 0, 30, 60, or 120 minutes. 25 μL 4-fold proteinase K buffer (0.4 mL 1M TRIS-HCl pH 7.5, 0.1 mL 0.5M EDTA, 0.12 mL 5M NaCl, and 0.4 mL 10% SDS) and 8 μL proteinase By adding K at 20 mg / mL, RNase was inactivated by proteinase K digestion at 55 ° C. for 20 minutes. Prior to analysis with a bioanalyzer, factor IX mRNA was precipitated (250 μL of 95% ethanol was added for 1 hour, centrifuged at 13 kHz for 10 minutes, the supernatant was removed and 200 μL of 70% ethanol was pelleted. Was added to and centrifuged again at 13 krpm for 5 minutes, the supernatant was removed and the pellet was resuspended in 70 μL of water) or extracted from PLGA microspheres (centrifuged at 13 krpm for 5 minutes and above. Remove the clarification, wash the pellet with 1 mL of water, centrifuge at 13 kHz for 5 minutes, remove the supernatant, add 280 μL dichloromethane to the pellet, shake for 15 minutes, add 70 μL of water, then Shake for 2 hours to remove aqueous phase). PLGA microspheres protect factor IX-modified mRNA from degradation in 90% and 1% serum for 2 hours. Factor IX-modified mRNA is completely degraded in 90% of serum at the start.

Example 17. In vivo Study of Lipid Nanoparticles A cDNA having the T7 promoter, 5'untranslated region (UTR), and 3'UTR used for in vitro transcription is shown in SEQ ID NO: 21437. The mRNA sequence is SEQ ID NO: 21438. Shown in; Poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) and factor IX (used for in vitro transcription, T7). The cDNA with the promoter, 5'UTR, and 3'UTR is shown in SEQ ID NO: 21443. The mRNA sequence is shown in SEQ ID NO: 1622; the poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap. 1; Completely modified with 5-methylcytosine and pseudouridine) modified mRNA was formulated as lipid nanoparticles (LNPs) using the syringe pump method. LNP has a final lipid molar ratio of 50:10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-c-DOMG) with a weight ratio of total lipid to modified mRNA of 20: 1. It was formulated with. The formulations listed in Table 34 were characterized by particle size, zeta potential, and encapsulation.
Table 34. pharmaceutical formulation

The LNP preparation was intravenously administered to mice (n = 5) at a modified mRNA dose of 100, 10, or 1 μg. Mice were sacrificed 8 hours after dosing. Serum was collected from mice treated with G-CSF or Factor IX modified mRNA preparation by cardiac puncture. Protein expression was determined by ELISA.

表３６．第ＩＸ因子タンパク質発現

There was no significant weight loss (<5%) in the G-CSF or Factor IX group. Protein expression in the G-CSF or Factor IX administration group was determined by ELISA from a standard curve. Serum samples were diluted (about 20-2500 times for G-CSF and about 10-250 times for Factor IX) to ensure that the samples were within the linear range of the standard curve. As shown in Table 35, the G-CSF protein expression as determined by ELISA was about 17, 1200, and 4700 ng / mL for the 1, 10, and 100 μg dose groups, respectively. As shown in Table 36, Factor IX protein expression as determined by ELISA was about 36,380, and 3000-11000 ng / mL for the 1, 10, and 100 μg dose groups, respectively.
Table 35. G-CSF protein expression

Table 36. Factor IX protein expression

As shown in Table 37, the LNP preparations described above are administered with the same dose of modified mRNA intravenous (IV) lipoplex preparation, and the same dose of modified mRNA intramuscularly (IM) or subcutaneously in physiological saline. It has about 10,000-100,000-fold increase in protein production as compared to (SC) administration. When used in Table 37, the symbol "about (~)" means about.
Table 37. Protein production

Materials and Methods of Examples 18-23 G-CSF (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methyl Fully modified with cytosine and pseudouridine) and EPO (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and pseudouridine The modified mRNA was formulated as lipid nanoparticles (LNP) using the syringe pump method. LNP has a final lipid molar ratio of 50:10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-c-DOMG) with a weight ratio of total lipid to modified mRNA of 20: 1. It was formulated with. The formulations listed in Table 38 were characterized by particle size, zeta potential, and encapsulation.
Table 38. pharmaceutical formulation

Example 18. In vivo Study of Lipid Nanoparticles Using Modified mRNA The LNP preparation shown in Table 38 (above) was injected intravenously (IV), muscle into rats (n = 5) at a single modified mRNA dose of 0.05 mg / kg. It was administered intramuscularly (IM) or subcutaneously (SC). Control rats (n = 4) were untreated. Blood was drawn from rats after administration of 2 hours, 8 hours, 24 hours, 48 hours, and 96 hours, and G-CSF or EPO-modified mRNA preparations to them, and protein expression was determined by ELISA. Rats to which EPO-modified mRNA was intravenously administered were collected on the 7th day.

As shown in Table 39, EPO protein expression in rats intravenously administered with modified EPO mRNA was detectable up to 5 days. G-CSF in rats intravenously administered with modified G-CSF mRNA was detectable up to 7 days. Subcutaneous and intramuscular administration of EPO-modified mRNA was detectable for at least 24 hours, and G-CSF-modified mRNA was detectable for at least 8 hours. In Table 39, "OSC" refers to a value that was outside the standard curve and "NT" means untested.
Table 39. G-CSF and EPO protein expression

Example 19. In vivo Studies over Time The LNP preparations shown in Table 38 (above) were intravenously (IV) in mice (n = 5) at a single modified mRNA dose of 0.5, 0.05, or 0.005 mg / kg. It was administered. Blood was collected from mice 8 hours, 24 hours, 72 hours, and 6 days after administration of the G-CSF or EPO-modified mRNA preparation, and protein expression was determined using ELISA.

As shown in Table 40, EPO and G-CSF protein expression in mice intravenously administered with modified mRNA reaches 72 hours in mice administered with 0.005 mg / kg and 0.05 mg / kg of modified mRNA. And, in the mice to which EPO-modified mRNA was administered, it was detectable until 6 days. In Table 40, ">" means that it is larger than that, and "ND" means that it was not detected.
Table 40. Protein expression

Example 20. In vivo study of LNP preparations in rodents A. LNP Preparations in Mice The LNP preparations shown in Table 38 (above) were administered intravenously (IV) to mice (n = 4) at a single modified mRNA dose of 0.05 mg / kg or 0.005 mg / kg. There were also three untreated control mouse groups (n = 4). Blood was collected from mice 2 hours, 8 hours, 24 hours, 48 hours, and 72 hours after administration of the G-CSF or EPO-modified mRNA preparation to determine protein expression. Protein expression of G-CSF and EPO was determined using ELISA.

As shown in Table 41, protein expression of EPO and G-CSF in mice reaches at least 48 hours in mice receiving a dose of 0.005 mg / kg of modified RNA, and a dose of 0.05 mg / kg. In mice that received the modified RNA of, it was detectable for up to 72 hours. In Table 41, "OSC" refers to a value that was outside the standard curve and "NT" means untested.
Table 41. Protein expression in mice

B. LNP Preparations in Rodent Animals The LNP preparations shown in Table 38 (above) are administered intravenously (IV) to rats (n = 4) at a single modified mRNA dose of 0.05 mg / kg. There is also an untreated control rat group (n = 4). Blood is collected from rats 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 14 days after administration of the G-CSF or EPO-modified mRNA preparation to determine protein expression. Protein expression of G-CSF and EPO is determined using ELISA.

Example 21. Early Time Study of LNP The LNP preparations shown in Table 38 (above) were injected intravenously into mammals at a single modified mRNA dose of 0.5 mg / kg, 0.05 mg / kg, or 0.005 mg / kg. IV), intramuscularly (IM), or subcutaneously (SC). Control mammals are untreated. Blood was drawn 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, and / or 2 hours after administration of the modified mRNA LNP preparation to the mammal, and using ELISA. Determine protein expression. In addition, blood is collected from mammals to determine the total blood cell count such as granulocyte level and red blood cell count.

Example 22. In vivo studies of non-human primates.
Non-human primates (NHP) as intravenous bolus infusion (IV) over approximately 30 seconds using the LNP preparations shown in Table 38 (above) using a hypodermic needle that can be attached to a syringe / abbocath or butterfly as needed. ) (Crab-eating monkey) (n = 2). NHP was administered a single modified mRNA IV dose of 0.05 mg / kg EPO or G-CSF, or 0.005 mg / kg EPO at a dosage of 0.5 mL / kg. Blood was collected 5 to 6 days before the NHP was dosed with the modified mRNA LNP preparation to determine serum protein expression and baseline total blood cell count. After administration of the modified mRNA preparation, blood was collected from NHP at 8, 24, 48, and 72 hours to determine protein expression. Twenty-four and 72 hours after dosing, the total blood cell count of NHP was also determined. Protein expression of G-CSF and EPO was determined by ELISA. NHP urine was collected and analyzed throughout the course of the experiment to assess clinical stability. After administering G-CSF or EPO-modified mRNA preparations to NHP, samples were taken from them and protein expression was determined using ELISA. We also analyzed clinical chemistry, hematology, urinalysis, and cytokines in non-human primates.

As shown in Table 42, EPO protein expression in NHP administered at 0.05 mg / kg is detectable up to 72 hours, and with administration of 0.005 mg / kg of EPO formulation, up to 48 hours. It is detectable. In Table 42, "<" means less than a given value. G-CSF protein expression was observed up to 24 hours after administration of the modified mRNA preparation. Preliminarily, increased levels of granulocytes and reticulocytes were seen in the NHP after administration of the modified mRNA preparation.
Table 42. Protein expression in non-human primates

Example 23. In vivo Study of Non-Human Primates on G-CSF and EPO The LNP preparation shown in Table 38 (above) was administered intravenously (IV) to non-human primates (NHP) (cynomolgus monkeys) (n = 2). bottom. NHP was administered a single modified mRNA IV dose of G-CSF or EPO at a dosage of 0.5 mL / kg, 0.5 mg / kg, 0.05 mg / kg, or 0.005 mg / kg. Blood was collected before administering the modified mRNA LNP preparation to NHP to determine serum protein expression and baseline total blood cell count. After administration of the G-CSF modified mRNA preparation, blood was collected from NHP at 8, 24, 48, and 72 hours to determine protein expression. After administration of the EPO-modified mRNA preparation, blood was collected from NHP at 8, 24, 48, 72 hours, and 7 days to determine protein expression.

Samples collected after administration of G-CSF or EPO-modified mRNA preparation to NHP were analyzed by ELISA to determine protein expression. The number of neutrophils and reticulocytes was also determined before, 24 hours, 3, 7, 14, and 18 days after administration of the G-CSF or EPO preparation.

As shown in Table 43, G-CSF protein expression was not detected beyond 72 hours. In Table 43, “<39” refers to a value lower than the lower limit of detection of 39 pg / mL.
Table 43. G-CSF protein expression

As shown in Table 44, EPO protein expression was not detected beyond 7 days. In Table 44, “<7.8” refers to a value lower than the lower limit of detection of 7.8 pg / mL.
Table 44. EPO protein expression

As shown in Table 45, there was an increase in neutrophils in all G-CSF groups compared to pre-dose levels.
Table 45. Pharmacological effect of G-CSF mRNA on NHP

As shown in Table 46, there was an increase in reticulocytes 3 to 14/18 days after dosing compared to reticulocyte levels 24 hours after dosing in all EPO groups.
Table 46. Pharmacological effect of EPO mRNA on neutrophil count

表４８．ヘマトクリットに対するＥＰＯ ｍＲＮＡの薬理学的効果

表４９．赤血球に対するＥＰＯ ｍＲＮＡの薬理学的効果

As shown in Tables 47-49, administration of EPO-modified RNA had an effect on other erythropoiesis parameters, including hemoglobin (HGB), hematocrit (HCT), and red blood cell (RBC) counts.
Table 47. Pharmacological effect of EPO mRNA on hemoglobin

Table 48. Pharmacological effect of EPO mRNA on hematocrit

Table 49. Pharmacological effect of EPO mRNA on erythrocytes

表５１．アスパラギン酸トランスアミナーゼに対するＥＰＯ ｍＲＮＡの薬理学的効果

As shown in Tables 50 and 51, administration of modified RNA had an effect on serum chemical parameters including alanine transaminase (ALT) and aspartate transaminase (AST).
Table 50. Pharmacological effect of EPO mRNA on alanine transaminase

Table 51. Pharmacological effect of EPO mRNA on aspartate transaminase

As shown in Table 52, administration of lipid nanoparticles-formulated modified RNA at high doses (0.5 mg / kg) caused an increase in cytokine or interferon α (IFN-α) after administration of modified mRNA. rice field.
Table 52. Pharmacological effect of EPO mRNA on alanine transaminase

Example 24. Study of intramuscular and / or subcutaneous administration in non-human primates Modified EPO mRNA in physiological saline (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) or G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of approximately 160 nucleotides is not shown in the sequence; 5'cap, cap 1; Completely modified with 5-methylcytosine and pseudouridine) was administered intramuscularly (IM) or subcutaneously (SC) to non-human primates (cracked monkeys) (NHP). The single modified mRNA dose of 0.05 mg / kg or 0.005 mg / kg was at a dosage of 0.5 mL / kg. Non-human primates are bled 5-6 days prior to dosing to determine serum protein levels and baseline total blood cell counts. After administration of the modified mRNA preparation, blood is collected from NHP at 8 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days to determine protein expression. Protein expression of G-CSF and EPO is determined by ELISA. The total blood cell count of NHP is also determined 24 hours, 72 hours, 7 days, and 14 days after administration. Urine is collected from NHP and analyzed throughout the entire experimental process to assess clinical stability. Tissue near the injection site is also collected and analyzed to determine protein expression.

Example 25. Transport of Modified RNA To determine the localization and / or transport of modified RNA, studies can be performed as follows.

LNP formulations of siRNA and modified mRNAs are formulated according to the methods known and / or described herein in the art. The LNP preparation may include proteins such as G-CSF, EPO, Factor VII, and / or at least one modified mRNA that can encode any of the proteins described herein. The formulation can be administered topically intramuscularly in mammals using intramuscular or subcutaneous injection. The dose of modified mRNA and the size of LNP are diverse to determine transport to the body of mammals and / or to assess effects on biological responses such as, but not limited to, inflammation. obtain. Mammals can be bled at different times to determine the expression of the protein encoded by the administered mRNA present in the serum and / or the total blood cell count in the mammal.

For example, modified mRNA encoding Factor VII expressed in the liver and secreted into serum can be administered intramuscularly and / or subcutaneously. SiRNA is administered at the same time as or prior to the administration of the modified mRNA to knock out endogenous Factor VII. Factor VII produced by intramuscular and / or subcutaneous injection of modified mRNA in blood is administered and measured. In addition, the level of Factor VII in the tissue near the injection site is also measured. When Factor VII is expressed in the blood, there is a transport of modified mRNA. If factor VII is expressed in tissue but not in blood, only local expression of factor VII is present.

Example 26. Preparation of Multiple Modified mRNAs LNP preparations of modified mRNAs are formulated according to methods known in the art and / or described herein or known in the art. The LNP preparation may include proteins such as G-CSF, EPO, thrombopoietin, and / or at least one modified mRNA that can encode any of the proteins described herein. At least one modified mRNA may include 1, 2, 3, 4, or 5 modified mRNA molecules. Formulations containing at least one modified mRNA can be administered intravenously, intramuscularly, or subcutaneously in a single or multiple dosing regimen. Biological samples such as, but not limited to, blood and / or serum can be collected and analyzed at various time points before and / after administration of at least one modified mRNA preparation. Expression of the protein in a 50-200 pg / mL biological sample after administration of a formulation containing at least one modified mRNA encoding the protein to the mammal would be considered biologically effective.

Example 27. Study of polyethylene glycol ratio A. Formulation and Characterization of PEG LNP Lipid nanoparticles (LNP) were formulated using the syringe pump method. LNP with total lipids and modified G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and pseudouridine Completely modified with 20: 1 by weight. The molar ratio range of the formulation is shown in Table 53.
Table 53. Molar ratio

Ｂ．ＰＥＧ ＬＮＰのインビボスクリーニング Two PEG lipids, 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DMG, NOF catalog number SUNBRIGHT® GM-020) and 1,2-distearoyl-sn-glycerol, methoxy Polyethylene glycol (PEG-DSG, NOF Catalog No. SUNBRIGHT® GS-020) was tested at 1.5 or 3.0 mol%. After LNP formation and encapsulation of modified G-CSF mRNA, LNP preparations are characterized by particle size, zeta potential, and encapsulation rate, and the results are shown in Table 54.
Table 54. Characterization of LNP preparation

B. In vivo screening of PEG LNP

The PEG LNP preparations shown in Table 55 were intravenously administered to mice (n = 5) at a dose of 0.5 mg / kg. Serum was collected from mice 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 8 days after administration of the drug. Serum was analyzed by ELISA to determine G-CSF protein expression and expression levels are shown in Table 55. The LNP preparation using PEG-DMG exhibited substantially higher levels of protein expression than the LNP preparation using PEG-DSA.
Table 55. Protein expression

Example 28. Research on cationic lipid preparations A. Formulation and Characterization of Cationic Lipid Nanoparticles Lipid nanoparticles (LNP) were formulated using the syringe pump method. LNP was formulated with a weight ratio of 20: 1 total lipid to modified mRNA. The molar ratio range of the final lipids of cationic lipids, DSPC, cholesterol, and PEG-c-DOMG is outlined in Table 56.
Table 56. Molar ratio

A 25 mM lipid solution in ethanol and modified RNA in 50 mM citrate at pH 3 were mixed to result in spontaneous vesicle formation. After stabilizing the vesicles in ethanol, ethanol was removed and buffer exchange was performed by dialysis. LNP was then characterized by particle size, zeta potential, and encapsulation rate. Table 57 shows EPO-modified mRNA using DLin-MC3-DMA, DLin-DMA, or C12-200 as the cationic lipid (mRNA sequence is about 160 nucleotides poly A tail shown in SEQ ID NO: 1638 in the sequence. Not shown in; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) or G-CSF modified mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of approximately 160 nucleotides (nucleotdie). Is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) is described for the characterization of LNP encapsulated.
Table 57. Characterization of cationic lipid preparations

B. In-vivo screening of cationic LNP preparation The preparation of the cationic lipid preparation shown in Table 57 was intravenously administered to mice (n = 5) at a dose of 0.5 mg / kg. Serum was collected from mice 2 hours, 24 hours, 72 hours, and / or 7 days after administration of the drug. Serum was analyzed by ELISA to determine EPO or G-CSF protein expression, and expression levels are shown in Table 58.
Table 58. Protein expression

Toxicity was confirmed in mice treated with LNP preparations using the cationic lipids C12-200 (NPA-075-1 and NPA-076-1), which showed dwarf coat, atrophic behavior, and greater than 10%. Since he showed symptoms such as weight loss, he was sacrificed in 24 hours. C12-200 was predicted to be more toxic, but also had high levels of expression in a short period of time. The cationic lipid DLin-DMA (NPA-073-1 and NPA-074-1) had the lowest expression of the three cationic lipids tested. DLin-MC3-DMA (NPA-071-1 and NPA-072-1) showed good expression until day 3, and EPO preparation exceeded the background sample until day 7.

Example 29. Screening method for protein expression A. Electrospray Ionization Prepare a biological sample that may contain the protein encoded by the modified RNA administered to the subject and use 1, 2, 3, or 4 mass spectrometers for electrospray ionization (ESI). Analyze according to the manufacturer's protocol. Biological samples can also be analyzed using a tandem ESI mass spectrometry system.
The pattern of protein fragments, or total protein, is compared to known controls for a given protein and the identity is determined by comparison.

B. Matrix-assisted laser desorption / ionization Prepare biological samples that may contain proteins encoded by the modified RNA administered to the subject and analyze according to the manufacturer's protocol for matrix-assisted laser desorption / ionization (MALDI).

The pattern of protein fragments, or total protein, is compared to known controls for a given protein and the identity is determined by comparison.

C. Liquid Chromatography Tandem Mass Spectrometry-Mass Spectrometry-Mass Spectrometry
A biological sample that may contain the protein encoded by the modified RNA is treated with a trypsin enzyme to digest the protein contained therein. The resulting peptide is analyzed by liquid chromatography tandem mass spectrometry (LC / MS / MS). Peptides are fragmented and placed in a mass spectrometer to generate characteristic patterns that can be matched to a database of protein sequences via computer algorithms. The digested sample can be diluted to give less than 1 ng of starting material for a given protein. Biological samples containing a simple buffer background (eg, water or volatile salts) are suitable for digestion in direct solution and have a more complex background (eg, surfactant, non-volatile). Salts, glycerol) require additional purification steps to facilitate sample analysis.

The pattern of protein fragments, or total protein, is compared to known controls for a given protein and the identity is determined by comparison.

Example 30. In vivo study of lipid nanoparticles mCherry mRNA (mRNA sequence is shown in SEQ ID NO: 21444; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and pseudouridine completely. Modified) was formulated as lipid nanoparticles (LNP) using the syringe pump method. LNP has a final lipid molar ratio of 50:10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-c-DOMG) with a weight ratio of total lipid to modified mRNA of 20 :. It was formulated in 1. The mCherry formulations listed in Table 59 were characterized by particle size, zeta potential, and encapsulation.
Table 59. mCherry preparation

The LNP preparation was intravenously administered to mice (n = 5) at a modified mRNA dose of 100 μg. Mice were sacrificed 24 hours after dosing. Liver and spleen from mice treated with mCherry modified mRNA preparation were analyzed by immunohistochemistry (IHC), Western blotting, or fluorescence activated cell sorting (FACS).

Liver histology showed uniform mCherry expression throughout the section, but untreated animals did not express mCherry. In addition, Western blot was used to confirm the expression of mCherry in treated animals, but mCherry was not detected in untreated animals. Tubulin was used as a control marker, which was detected in both treated and untreated mice, indicating that normal protein expression in hepatocytes was unaffected.

In addition, FACS and IHC were also performed on mCherry and untreated mouse spleens. FACS analysis showed that all leukocyte cell populations were negative for mCherry expression. Also, by IHC, there was no observable difference in spleen between mCherry-treated and untreated mice.

Example 31. Syringe pump in vivo study mCherry-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), lipid nanoparticles using the syringe pump method. It is formulated as (LNP). LNP has a final lipid molar ratio of 50:10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-c-DOMG) with a weight ratio of total lipid to modified mRNA of 20 :. Formulate in 1. The mCherry formulation is characterized by particle size, zeta potential, and encapsulation.

The LNP preparation is intravenously administered to mice (n = 5) at a modified mRNA dose of 10 or 100 μg. Mice are sacrificed 24 hours after dosing. Liver and spleen from mice treated with mCherry modified mRNA preparation are analyzed by immunohistochemistry (IHC), Western blotting, and / or fluorescence activated cell sorting (FACS).

Example 32. In vitro and in vivo expression A. In vitro Expression in Human Cells Using Lipidoid Formula The ratio of mmRNA to lipidoid used to test for in vitro transfection is experimentally tested with different lipidoid: mmRNA ratios. Previous studies with siRNA and lipidoids used weight: weight ratios of 2.5: 1, 5: 1, 10: 1, and 15: 1 lipidoids: siRNA. Considering that the length of mmRNA is longer compared to siRNA, a lower weight-to-weight ratio of lipidoid to mmRNA may be effective. In addition, for comparison, mRNA using RNAIMAX ™ (Invitrogen, Carlsbad, CA) or TRANSIT-mRNA (Mirus Bio, Madison, WI) cationic lipid delivery vehicle was also formulated.

Lipidoid-formulated luciferases expressing the desired protein product (IVT cDNA sequence as shown in SEQ ID NO: 21445; mRNA sequence shown in SEQ ID NO: 21446, poly A tail of approximately 160 nucleotides not shown in the sequence, 5'cap, cap 1, fully modified with 5-methylcytosine in each cytosine and pseudouridine substitution at each uridine site), green fluorescent protein (GFP) (IVT cDNA sequence as shown in SEQ ID NO: 21447; mRNA sequence The poly-A tail of approximately 160 nucleotides, shown in SEQ ID NO: 21448, is not shown in the sequence and is fully modified with 5'cap, cap 1, 5-methylcytosine at each cytosine and pseudouridine substitution at each uridine site), G. -CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), and EPO mRNA (mRNA sequence is shown in SEQ ID NO: 1638; A poly A tail of approximately 160 nucleotides is not shown in the sequence; 5'cap, cap 1) capacity, luminescence for luciferase expression, flow cytometry for GFP expression, and secretion of G-CSF and erythropoetin (EPO). Can be confirmed by ELISA.

B. In vivo expression after intravenous injection Systemic intravenous administration of the pharmaceutical product is performed with a variety of different lipidoids, including but not limited to 98N12-5, C12-200, and MD1.

The lipidoid preparation containing mmRNA is injected intravenously into the animal. Expression of the protein encoded by the modified mRNA (m mRNA) is evaluated in samples of blood and / or other organs such as liver and spleen taken from animals. Performing a single intravenous dose study will allow assessment of the scale of expression, dose responsiveness, and shelf life of the desired product.

In one embodiment, 98N12-5, C12-200, MD1, and other lipidoid formulations of lipidoids are administered to animals with luciferase, green fluorescent protein (GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP), etc. Used for delivery of human G-CSF, human factor IX, or human erythropoetin (EPO) m mRNA. As described above, after formulating mmRNA with lipids, the animals are divided into groups and from saline or luciferase, GFP, mCherry, SAP, human G-CSF, human factor IX, and human EPO. Accept any of the lipidoid formulations containing one of the various mmRNAs selected. Before injecting into animals, the m mRNA-containing lipidoid product is diluted in PBS. The animals are then administered a single dose of the formulated mmRNA, ranging from a dose of 10 mg / kg to as low as 1 ng / kg, preferably in the range of 10 mg / kg to 100 ng / kg. 20 grams of mice are dependent on the weight of the animal, such as accepting up to 0.2 mL of the drug (dose is based on mmRNA per kg of body weight). After administration of the mmRNA-lipidoid preparation, serum, tissue, and / or tissue lysate is obtained and the level of product encoded by mmRNA is determined at single and / or range of time intervals. Lipidoid-formulated luciferase, GFP, mCherry, SAP, G-CSF, factor IX, and EPO mmRNA ability to express the desired protein product, luminescence for luciferase expression, flow cytometry for GFP and mCherry expression For metric, SAP, enzyme activity, or secretion of G-CSF, factor IX, and / or EPO is confirmed by ELISA.

Further studies on multi-dose regimens were performed to determine maximal expression of mmRNA and to assess the saturation of mmRNA-driven expression (by feeding control and active mmRNA preparations simultaneously or sequentially). Determining the feasibility of repeated drug administration (by giving the mRNA at doses weeks or months apart and then determining whether expression levels are affected by factors such as immunogenicity). Assessment of the physiological function of proteins such as G-CSF and EPO is also determined by analysis of samples from the animals tested and detection of increased granulocyte and red blood cell counts, respectively. The activity of expressed protein products such as Factor IX in animals can also be assessed through analysis of the effects of Factor IX enzyme activity (activated partial thromboplastin time assay) and coagulation time.

C. The use of lipidoid formulations to deliver oligonucleotides containing mRNA expressed in vitro after intramuscular and / subcutaneous injection via the intramuscular or subcutaneous injection route has not been previously reported and will be evaluated. There is a need. Intramuscular and / or subcutaneous injection of mmRNA is evaluated to determine if the mmRNA-containing lipidoid formulation can result in both local and systemic expression of the desired protein.

98N12-, containing mmRNA selected from luciferase, green fluorescent protein (GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF, human factor IX, or human erythropoietin (EPO) mmRNA. 5, C12-200, and MD1 lipidoid formulations are injected intramuscularly and / or subcutaneously into animals. Expression of the protein encoded by mmRNA is assessed both in muscle or subcutaneous tissue and systemically in blood and other tissues such as liver and spleen. Single dose studies allow assessment of the scale of expression, dose responsiveness, and shelf life of the desired product.

Animals are grouped to accept either saline or modified mRNA-containing formulations. Prior to infusion, the mmRNA-containing lipidoid product is diluted in PBS. Animals are administered a single intramuscular dose of formulated mmRNA in the low dose range of 50 mg / kg to 1 ng / kg, preferably in the range of 10 mg / kg to 100 ng / kg. The maximum dose of intramuscular administration in mice is approximately 1 mg m mRNA or as low as 0.02 ng m mRNA for intramuscular injection into the hind limbs of mice. For subcutaneous administration, animals are administered a single subcutaneous dose of the formulated mRNA in the low dose range of 400 mg / kg to 1 ng / kg, preferably in the range of 80 mg / kg to 100 ng / kg. The maximum dose for subcutaneous administration in mice is approximately 8 mg of mm mRNA or as low as 0.02 ng.

In a 20 gram mouse, the volume of a single intramuscular injection is up to 0.025 mL and a single subcutaneous injection is up to 0.2 mL. The optimal dose of mmRNA administered is calculated from the body weight of the animal. At various time points after administration of mmRNA-lipidoid, serum, tissue, and tissue lysates are obtained to determine the level of product encoded by mmRNA. Lipidoid-formulated luciferase, green fluorescent protein (GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF, human factor IX, or human erythropoetin (EPO) mMr, which expresses the desired protein product. The ability is confirmed by luminescence for luciferase expression, flow cytometry for GFP and mCherry expression, enzymatic activity for sAP, and ELISA for G-CSF, Factor IX, and erythropoetin (EPO) secretion.

In addition, further studies on multiple dose regimens were performed to determine maximal expression with mmRNA and to assess the saturation of mmRNA-driven expression (by giving control and active mmRNA preparations simultaneously or sequentially). Determining the feasibility of repeated drug administration (achieved), giving the mRNA at doses weeks or months apart, and then determining whether expression levels are affected by factors such as immunogenicity. By). Studies using multiple subcutaneous or intramuscular injections in a single dose can also be used to further increase drug exposure to mmRNA and improve protein production. Evaluation of the physiological function of proteins such as GFP, mCherry, SAP, human G-CSF, human factor IX, and human EPO was performed by analyzing samples from the animals tested and changes in granulocytes and / or red blood cell count. Is determined by detecting. The activity of expressed protein products such as Factor IX in animals can also be assessed through analysis of the effects of Factor IX enzyme activity (activated partial thromboplastin time assay) and coagulation time.

Example 33. Bifunctional mmRNA
Using the teachings and synthetic methods described herein, modified RNA is bifunctional, thereby encoding one or more cytotoxic protein molecules, as well as synthesized with cytotoxic nucleosides. Design and synthesize to be done.

Administration of bifunctional modified mRNA is achieved using either saline or lipid carriers. Upon administration, the bifunctional modified mRNA is translated to produce the encoded cytotoxic peptide. Degradation of the delivered modified mRNA releases cytotoxic nucleosides, which also provide therapeutic benefits to the subject.

Example 34. Transfection of modified mRNA A. For experiments conducted in the reverse transfection 24-well collagen-coated tissue culture plates are seeded with keratinocytes at a cell density of 1 × 10 ^5. For carried out experiments with a collagen-coated 96-well tissue culture plates are seeded with keratinocytes at a cell density of 0.5 × 10 ^5. For each modified mRNA (m mRNA) to be transfected, a modified mRNA: RNAIMAX ™ is prepared as described and within the cell number period, eg, within 6 hours, before the cells attach to the tissue culture plate. In the multi-well plate, mix with the cells.

B. In collagen-coated tissue culture plates in the forward transfection 24-well, seeded keratinocytes at a cell density of 0.7 × 10 ^5. The collagen-coated 96-well tissue culture plates are seeded at a cell density of keratinocytes 0.3 × 10 ^5. Grow keratinocytes to over 70% confluency over a 24-hour period. For each modified mRNA (m mRNA) to be transfected, modified mRNA: RNAIMAX ™ was prepared as described and placed on the cells in a multiwell plate for 24 hours after seeding and attachment to the tissue culture plate. Transfect.

C. Translational screening of modified mRNA: G-CSF ELISA
Keratinocytes are grown with greater than 70% confluence in EPILIFE medium with Supplement S7 from Invitrogen (Carlsbad, CA). Keratinocytes were backtransfected with 300 ng of chemically modified mRNA (m mRNA) complexed with RNAIMAX ™ from Invitrogen. Another set of keratinocytes was sequentially transfected with 300 ng of modified mRNA complexed with RNAIMAX ™ from Invitrogen. The modified mRNA: RNAIMAX ™ complex is first formed by incubating RNA in 5-fold volume dilution with supplement-free EPILIFE® medium at room temperature for 10 minutes.

In a second vial, RNAIMAX ™ reagent was incubated in 10-fold volume dilution with supplement-free EPILIFE® medium at room temperature for 10 minutes. The RNA vials were then mixed with RNAIMAX ™ vials, incubated at room temperature for 20-30 minutes, and then added to the cells in a dropping manner. The concentration of human granulocyte colony stimulating factor (G-CSF) secreted into the culture medium is triple measured 18 hours after transfection for each of the chemically modified mRNAs.

Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R & D Systems (Minneapolis, MN) according to manufacturer-recommended instructions.
D. Dose and duration of modified mRNA: G-CSF ELISA

Keratinocytes are grown with greater than 70% confluence in EPILIFE® medium with Supplement S7 from Invitrogen. Reverse transtransfection of keratinocytes with any of 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng modified mRNA complexed with RNAIMAX ™ from Invitrogen (Carlsbad, CA). Fact. Modified mRNA: RNAIMAX ™ complex is formed as described. The concentration of human G-CSF secreted into the culture medium is triple measured at 0, 6, 12, 24, and 48 hours after transfection for each concentration of each modified mRNA. Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R & D Systems according to the manufacturer's recommended instructions.

Example 35. Split-dose studies Designed and performed studies using multiple subcutaneous or intramuscular injection sites in a single dose to investigate means of increasing drug exposure to mmRNA and improving protein production. In addition to the detection of expressed protein products, the assessment of the physiological function of the protein was also determined through analysis of samples derived from the animals tested.

Surprisingly, split dosing of mmRNA was found to result in higher protein production and phenotypic response than those provided by single or multiple dosing schemes.

The design of single-dose, multi-dose, and split-dose experiments was for human erythropoietin (EPO) mRNA (mRNA shown in SEQ ID NO: 1638; about 160 nucleotides of polyA tail sequenced in buffer alone. Not shown within; with the use of 5'caps, caps 1). The dosing vehicle (F buffer) was 150 mM NaCl, 2 mM CaCl ₂ , 2 mM Na ⁺ -phosphate (1.4 mM sodium monophosphate; 0.6 mM sodium dibasic), and 0.5 mM. EDTA, pH 6.5. The pH was adjusted with sodium hydroxide and the final solution was filtered sterilized. The mmRNA was modified with 5 meC at each cytosine and pseudouridine substitution at each uridine site.

Animals (n = 5) were injected IM (intramuscular) with a single unit dose of 100 μg. For multiple doses, two schedules were used: 3 doses of 100 μg and 6 doses of 100 μg. For the split dosing scheme, two schedules were used: 3 doses at 33.3 μg and 6 doses at 16.5 μg mmRNA. Control dosing was accompanied by the use of buffer alone at 6 doses. The control mRNA is a luciferase mRNA (IVT cDNA sequence is shown in SEQ ID NO: 21445; the mRNA sequence is shown in SEQ ID NO: 21446, the polyA tail of about 160 nucleotides is not shown in the sequence, 5 dosed at 100 μg 6 times. With the use of'cap, cap 1, fully modified with 5-methylcytosine in each cytosine and pseudouridine substitution at each uridine site). Blood and muscle tissue were evaluated 13 hours after infusion.

Human EPO protein was measured in mouse serum 13 hours after single, multiple, or split intramuscular dosing of EPO mRNA in buffer. Seven groups of mice (n = 5 mice per group) were treated and evaluated. The results are shown in Table 60.
Table 60. Split dose study

The split factor is defined as the product per unit drug divided by the single dose product per unit drug (PUD). For example, in treatment group 2, the product (EPO) value of 0.28 per unit drug (m mRNA) is divided by the single dose product 0.14 per unit drug. The result is 2. Similarly, for treatment group 4, for example, the product (EPO) value of 1.1 per unit drug (m mRNA) is divided by the single dose product 0.14 per unit drug. The result is 7.9. As a result, the dose split factor (DSF) can be used as an indicator of the effectiveness of the split dose regimen. The DSF should be equal to 1 for any single dose of the total daily dose. Therefore, any DSF greater than this value in the split dose regimen shows improved efficacy.

Studies will be conducted to determine dose response trends, injection site effects, and injection timing effects. In these studies, dose response results are determined using various doses of 1 μg, 5 μg, 10 μg, 25 μg, 50 μg, and values between them. A total dose of 100 μg divided doses includes 1.6 μg, 4.2 μg, 8.3 μg, 16.6 μg, or 3 or 6 doses of the total dose equal to the dose of the selected total dose.

The injection site is selected from the limbs or any body surface that exhibits sufficient area suitable for injection. This may also include selection of injection depth to target the dermis (intradermal), epithelium (epidermis), subcutaneous tissue (SC), or muscle (IM). Injection angles vary based on the targeted delivery site, targeting intradermal injections is at an angle of 10 to 15 degrees from the surface of the skin, and subcutaneous injection is 20 from the surface of the skin. It is ~ 45 degrees and is substantially an angle of 60-90 degrees for intramuscular injection.

Example 36. Quantification in Exosomes The number and localization of mmRNAs of the invention can be determined by measuring the amount (on an early, temporal, or residual basis) in isolated exosomes. In this study, mRNA is typically codon-optimized and the sequence is distinctly different from that of endogenous mRNA, so the level of mRNA is defined in Gibbings, the contents of which are incorporated herein by reference in their entirety. The method of PCT / IB2009 / 00478 is quantified compared to the endogenous level of natural or wild-type mRNA.

In these studies, the method first isolates exosomes or vesicles from the body fluids of patients who have preferably already been treated with the polynucleotides, primary constructs, or mRNAs of the invention, and then mRNA microarrays, qRT-. Levels of polynucleotides, primary constructs, or mRNA in said exosomes are measured by PCR, or one of the other methods for RNA measurement in the art, including suitable antibody or immunohistochemical methods. It is done by.

Example 37. Effect of Modified mRNA on Cell Viability, Cytotoxicity, and Apoptosis This experiment shows the cell viability, cytotoxicity, and apoptosis of distinctly different modified mRNAs transfected into human keratinocyte cells in vitro. Keratinocytes are grown with greater than 70% confluence in EPILIFE® medium with hydrocortisone-free human keratinocyte growth supplement from Invitrogen (Carlsbad, CA). Keratinocytes are backtransfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of modified mRNA complexed with RNAIMAX ™ from Invitrogen. Modified mRNA: Forming an RNAIMAX ™ complex. The concentration of human G-CSF secreted into the culture medium is triple measured at 0, 6, 12, 24, and 48 hours after transfection for each concentration of each modified mRNA. Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R & D Systems according to the manufacturer's recommended instructions.

Cell viability, cytotoxicity, and apoptosis at 0, 12, 48, 96, and 192 hours of transfection using the APOTOX-GLO ™ kit from Promega (Madison, WI) according to the manufacturer's instructions. Will be measured later.

Example 38. Detection of the innate immune response of cells to modified mRNA using the ELISA assay Human tumor necrosis factor α (TNF-α), human interferon β (IFN-β), and human interferon β (IFN-β) secreted from in vitro transfected human keratinocytes. The enzyme-bound immunoadsorption assay (ELISA) of human granulocyte colony stimulator (G-CSF) is tested for the detection of the innate immune response of cells. Keratinocytes are grown with greater than 70% confluence in EPILIFE® medium with hydrocortisone-free human keratinocyte growth supplement from Invitrogen (Carlsbad, CA). As described in the secreted TNF-α keratinocytes, 0 ng, 93.75 ng, l87.5 ng, 375 ng, 750 ng, 1500 ng, or 3000 ng of chemically modified mRNA complexed with RNAIMAX ™ from Invitrogen. mmRNA) is triple-backtransfected. TNF-α secreted into culture medium is measured for each of the chemically modified mRNAs 24 hours after transfection using an ELISA kit from Invitrogen according to the manufacturer's protocol.

IFN-β secreted into the same culture medium is measured for each of the chemically modified mRNAs 24 hours after transfection using an ELISA kit from Invitrogen according to the manufacturer's protocol. Concentrations of human G-CSF secreted into the same culture medium are measured for each of the chemically modified mRNAs 24 hours after transfection. Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R & D Systems (Minneapolis, MN) according to manufacturer-recommended instructions. These data show which modified mRNA (m mRNA) is compared to native and other chemically modified polynucleotides or reference compounds by measuring the exemplary type 1 cytokines TNF-α and IFN-β. Shows whether it can induce a decrease in the innate immune response of cells.

Example 39. Assay for cell proliferation induced by human granulocyte colony-stimulating factor (G-CSF) -modified mRNA Human keratinocytes are coated with 24-well collagen coating TRANSWELL® in EPILIFE® medium with Supplement S7 from Invitrogen. (Coming, Lowell, MA) Growing in co-cultured tissue culture plates with greater than 70% confluence. As described in keratinocytes, 750 ng of the indicated chemically modified mRNA (m mRNA) complexed with RNAIMAX from Invitrogen is backtransfected. Modified mRNA: RNAIMAX complex is formed as described. The medium of keratinocytes is changed 6-8 hours after transfection. 42 hours after transfection, a 24-well TRANSWELL® plate insert with a translucent polyester membrane with 0.4 μm pores was placed on a culture plate containing keratinocytes transfected with human G-CSF modified mRNA. put in.

Kasumi-1 cells or KG-1 is a human myeloblast cells (0.2 × 10 ⁵ cells) were seeded in insert wells, cell growth, in 100~120μL volume in 96-well plates, CyQuant Direct Cell Proliferation Assay Quantify using (Invitrogen, Cellsbad, CA) 42 hours after the start of co-culture. Myeloblast proliferation induced by modified mRNA encoding human G-CSF is expressed as the percent cell proliferation normalized to the co-culture control wells of untransfected keratinocytes / myeloblasts. The concentration of human G-CSF secreted in both keratinocyte and myeloblast insertion co-culture wells is doubly measured 42 hours after the start of co-culture for each modified mRNA. Human G-CSF secretion is quantified using an ELISA kit from Invitrogen according to the manufacturer's recommended instructions.

Human G-CSF-modified mRNA and untransfected human myeloblast cells transfected into human keratinocyte feeder cells are detected by RT-PCR. Total RNA from sample cells is extracted and lysed using the RNEASY® kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The extracted total RNA is modified mRNA using human G-CSF-specific primers and PROTOSCRIPT® M-MuLV Taq RT-PCR kit (New England BioLabs, Ipsich, MA) according to the manufacturer's instructions. -Perform RT-PCR for specific amplification of G-CSF. The RT-PCR product is visualized by 1.2% agarose gel electrophoresis.

Example 40: Co-culture assay Modified mRNA, which encodes human granulocyte colony stimulating factor (G-CSF) and is composed of chemically distinct nucleotides, is a cell proliferation of transfected non-competent cells in a co-culture environment. Can stimulate. Co-cultures include highly transfectable cell types such as human keratinocytes and non-transfectable cell types such as leukocytes (WBC). The modified mRNA encoding G-CSF is transfected into highly transfectable cells, allowing the production of G-CSF protein and its secretion into the extracellular environment, where G-CSF is paraclin-like. In a manner, it acts to stimulate and proliferate leukocytes expressing the G-CSF receptor. Use an expanded WBC population to treat immunocompromised patients or partially reconstruct the WBC population of immunosuppressed patients, thereby reducing the risk of opportunistic infections. Can be done. In another example, a particular example that supports and stimulates the growth, maintenance, or differentiation of refractory embryonic stem cells or induced pluripotent stem cells into highly transfectable cells such as fibroblasts. Transfect growth factors.

Example 41: Human IgG antibody detection assay A. ELISA detection of human IgG antibody This example is for ELISA of human IgG derived from Chinese hamster ovary (CHO) and human fetal kidney (HEK, HER-2 negative) 293 cells transfected with human IgG-modified mRNA (m mRNA). explain. Human fetal fetal kidney (HEK) 293 is grown in CD293 medium with a replacement for L-glutamine from Invitrogen until it reaches 80-90% confluence. CHO cells are grown in CD CHO medium with supplements for L-glutamine, hypoxanthine, and thymidine. In one embodiment, 2 x 106 cells are transfected with 24 μg of modified mRNA complexed with RNAIMAX ™ from Invitrogen in a 75 cm2 culture flask from Corning in 7 mL of medium. In another embodiment, 80,000 cells are transfected with 1 μg of modified mRNA complexed with RNAIMAX ™ from Invitrogen in a 24-well plate. Modified mRNA: RNAIMAX ™ complex is formed by incubating mmRNA in a 5-fold volume dilution with either CD293 or CDCHO medium in a vial for 10 minutes at room temperature. In a second vial, RNAIMAX ™ reagent is incubated with CD293 or CD CHO medium in 10-fold volume dilution for 10 minutes at room temperature. A vial of mmRNA is then mixed with a vial of RNAIMAX ™, incubated at room temperature for 20-30 minutes, and then added to CHO or HEK cells by drop method. The culture supernatant is stored at 4 degrees Celsius. For 24 μg m mRNA transfection, the concentration of human IgG secreted into the culture medium is measured 12, 24, 36 hours after transfection, and 1 μg m mRNA transfection is measured at 36 hours. Trastuzumab secretion from transfected HEK293 cells is quantified using an ELISA kit from Abcam (Cambridge, MA) according to the manufacturer's recommended instructions. The data show that mmRNAs of humanized IgG antibodies (such as trastuzumab) can be translated in HEK cells, and that trastuzumab is secreted from the cells and released into the extracellular environment. In addition, the data show that transfection of cells with trastuzumab-encoding mmRNA for the production of secretory proteins can be extended to bioreactor or large cell culture conditions.

B. Western Detection of Human IgG Antibody Produced by Modified mRNA Western blot CHO-K1 cells are co-transfected with 1 μg each of heavy and light chains of trastuzumab-modified mRNA (m mRNA). CHO cells are grown in 24-well plates using standard protocols. Cell supernatants or cell lysates are collected 24 hours after transfection, separated on a 12% SDS-Page gel and transferred onto a nitrocellulose membrane using IBOT® by Invitrogen (Carlsbad, CA). A first complex of rabbit polyclonal antibody and human IgG conjugated to DYLIGHT594 (ab96904, abcam, Cambridge, MA), and a second complex of goat polyclonal antibody and Rb IgG conjugated to alkaline phosphatase. Incubate with the body. After incubation, antibodies are detected using Novex® alkaline phosphatase chromogenic substrate by Invitrogen (Carlsbad, CA).

C. Cell immunostaining of trastuzumab and rituximab produced by modified mRNA CHO-K1 cells are co-transfected with 10 ng each of heavy and light chains of either trastuzumab or rituximab. Cells are grown in F-12K medium and 10% FBS from GIBCO® (Grand Island, NY). Cells are fixed with 4% paraformaldehyde in PBS, permeable with 0.1% Triton X-100 in PBS for 5-10 minutes at room temperature, and the cells are washed 3 times with PBS at room temperature. Staining of trastuzumab and rituximab is performed with a rabbit polyclonal antibody against human IgG conjugated to DYLIGHT® 594 (ab96904, abcam, Cambridge, MA) according to the dilution recommended by the manufacturer. Nuclear DNA staining is performed with DAPI dye from Invitrogen (Carlsbad, CA). Trastuzumab and rituximab proteins are translated and localized in the cytoplasm after transfection of modified mRNA. Photographs are taken 13 hours after transfection.

D. Trastuzumab and rituximab bound immunoblot assay produced by modified mRNA Trastuzumab and rituximab are detected using a bound immunoblot detection assay. Various concentrations (100 ng / μL to 0 ng / μL) of ErB2 peptide (ab400048, abeam, Cambridge, MA), trastuzumab and CD20 peptide antigens (ab97360, abeam, Cambridge, MA), rituximab antigens, 12% SDS- It is run on a Page gel and transferred to a membrane using iBlot from Invitrogen. Membranes are incubated with their respective cell supernatants from CHO-K1 cells co-transfected with 500 ng each of heavy and light chains of either trastuzumab or rituximab for 1 hour. Membranes are blocked with 1% BSA and a secondary anti-human IgG antibody conjugated with alkaline phosphatase (abcam, Cambridge, MA) is added. Antibody detection is performed using a NOVEX alkaline phosphatase chromogenic substrate by Invitrogen (Carlsbad, CA). The data show that humanized IgG antibodies generated from modified mRNA can recognize and bind to their respective antigens.

E. Cell proliferation assay Using the SK-BR-3 cell line, which is an adherent cell derived from human breast cancer that overexpresses the HER2 / neu receptor, the antiproliferative properties of trastuzumab produced by modified mRNA (m mRNA) are compared. can do. Various concentrations of purified trastuzumab and trastuzumab produced from modified mRNAs are added to cell cultures and their effect on cell proliferation is evaluated by a triple cytotoxicity and viability assay.

Example 42: Bulk transfection of modified mRNA into cell culture medium A. Cationic Lipid Delivery Vehicle RNA transfection is performed using RNAIMAX ™ (Invitrogen, Carlsbad, CA) or TRANSIT-mRNA (Mirus Bio, Madison, WI) cationic lipid delivery vehicle. RNA and reagents are first diluted in Opti-MEM basal medium (Invitrogen, Carlsbad, CA). 100 ng / μL RNA is diluted 5-fold and 5 μL RNAIMax per 1 μg of RNA is diluted 10-fold. The diluted ingredients are pooled, incubated at room temperature for 15 minutes, and then dispensed into culture medium. For transfection of TRANSIT-mRNA, 100 ng / μL RNA is diluted 10-fold in Opti-MEM, BOOST reagent is added (at a concentration of 2 μL per 1 μg RNA), and TRANSIT-mRNA is added (2 μL per 1 μg RNA). (At concentration), then the RNA-lipid complex is incubated at room temperature for 2 minutes before delivery to culture medium. RNA transfection was performed in Nutristem xenofree hES medium (Stemment, Cambridge, MA) for induction of RiPS, and in Dermal Cell Basic Medium Plus Keratinocyte Cell Cell (Cambridge, MA) for keratinocyte experiments. All other experiments are performed in Opti-MEM with 2% FBS added. Successful introduction of modified mRNA (m mRNA) into host cells can be monitored using a variety of known methods such as fluorescent markers, such as green fluorescent protein (GFP). Successful transfection of modified mRNAs can also be determined, for example, by measuring protein expression levels of the target polypeptide by Western blot or immunocytochemistry. Similar methods can be applied to large scales expanded to multiple liter (5 to 10,000 L) culture formats according to similar RNA-lipid complex ratios.

B. Electroporation Delivery of Extrinsic Synthetic mRNA Transcripts Electroporation parameters are designed to transfect MRC-5 fibroblasts with in vitro synthetic modified mRNA (m mRNA) transcripts, especially to detect exogenous transcripts. Optimize by measuring transfection efficiency by quantitative RT-PCR with the primers. In a standard electroporation cuvette with a gap of 2 mm, discharging the capacitor 150μF was charged in the F Opti-MEM in 50μL (Invitrogen, Carlsbad, CA) to 2.5 × 10 ⁶ cells suspended in Is sufficient for repeated delivery of modified mRNA transcripts over 10,000 copies per cell as determined using standard curve method, while maintaining high viability (> 70%). Further experiments may reveal that the voltage required to efficiently transfect cells with mmRNA transcripts may depend on the cell density during electroporation. Cell densities range from 1x10 ⁶ cells l / 50 μL to 2.5x10 ⁶ cells / 50 μL, with 110V to 145V to transfect cells with similar efficiencies as measured by transcript copy per cell. is necessary. Large-scale multi-liter (5-10,000 L) electroporation similar to the large-scale flow electroporation strategy and similar to the method described in the constraints above can be performed (Li et al. 2002). , Strategy et al., 2010).

Example 43: In vivo delivery with lipoplex A. 100 μg of modified human erythropoetin mRNA (mRNA is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) (EPO; fully modified 5). Formulations containing-methylcytosine; N1-methyl-pseudouridine) were lipoplexed with 30% by volume RNAIMAX ™ in 50-70 μL (lipoplex-h-Epo-46; 2nd generation or Gen2). It was delivered intramuscularly to 4 C57 / BL6 mice. The other group served as a control group containing 100 μg of modified luciferase mRNA, lipoplexed modified luciferase mRNA (lipoplex-luc) (IVT cDNA sequence sequenced) lipoplexed with 30% by volume RNAiMAX ™. Shown in No. 21445; the mRNA sequence is shown in SEQ ID NO: 21446, the poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, 5-methylcytosine in each cytosine and pseudo in each uridine site. It consisted of mice that received infusions (fully modified with uridine substitutions) or mice that received infusions of formulation buffer as a negative control at a dosage of 65 μL. Thirteen hours after intramuscular injection, serum was collected from each mouse and the amount of human EPO protein in mouse serum was measured by human EPO ELISA and the results are shown in Table 61.
Table 61. Human EPO production (intramuscular injection route)

B. Human G-CSF modified RNA lipoplex Modified human G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) (5-methyl Two forms: G-CSF (G-CSF) fully modified with cytosine and pseudouridine or G-CSF (G-CSF-N1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine. A preparation containing 100 μg of one of them was lipoplexed with 30% by volume RNAIMAX ™, and 150 μL was intramuscular (IM) and 150 μL was subcutaneous (SC) in C57 / BL6 mice. , And 225 μL were delivered intravenously (IV).

In three control groups, 100 μg of modified luciferase mRNA (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is shown in SEQ ID NO: 21446, poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap , Cap 1, complete replacement with 5-methylcytosine in each cytosine and pseudouridine substitution at each uridine site) intramuscularly (Luc-unsp IM) or 150 μg of modified luciferase mRNA intravenously (Luc-unsp) IV) or 150 μL of formulation buffer was administered either intramuscularly (buffer IM). Six hours after administration of the drug, serum was collected from each mouse, and human G-CSF protein in mouse serum was measured by human G-CSF ELISA, and the results are shown in Table 62.

These results show that modified human G-CSF mRNA for both 5-methylcytosine / pseudouridine and 5-methylcytosine / N1-methyl-pseudouridine is administered in lipoplex preparations via the route of administration intravenously or intramuscularly. It is shown that when delivered, it can result in specific human G-CSF protein expression in serum.
Table 62. Human G-CSF in serum (IM, IV, SC infusion route)

C. Comparison of human G-CSF modified RNA lipoplex 5-Methylcytosine (5 mc) and pseudouridine (ψ) modified 30% by volume RNAIMAX ™ and modified human G-CSF mRNA (G-CSF-) lipoplexed. Gen1-lipoplex), modified human G-CSF mRNA (G-CSF-Gen1-physiological saline) with 5 mc and ψ modifications in physiological saline, N1-5 lipoplexed with 30% by volume RNAIMAX ™. Modified human G-CSF mRNA with -methylcytosine (N1-5mc) and ψ modification (G-CSF-Gen2-lipoplex), modified human G-CSF mRNA with N1-5mc and ψ modification in saline (G) -CSF-Gen2-Saline), modified luciferase (Luc-lipoplex) with 5 mc and ψ modifications lipoplexed with 30% by volume RNAIMAX ™, or modifications with 5 mc and ψ modifications in physiological saline. A preparation containing 100 μg of any of luciferase mRNA (Luc-saline) was delivered intramuscularly (IM) or subcutaneously (SC), and a dose of 80 μL was given to the control group of each administration method. C57 / BL6 mice were fed with the formulation buffer (F buffer) of. Thirteen hours after injection, serum and tissue from the injection site were collected from mice and analyzed by G-CSF ELISA to compare human G-CSF protein levels. The results of human G-CSF protein in mouse serum obtained from intramuscular administration and the results of subcutaneous administration are shown in Table 63.

These results show that 5-methylcytosine / pseudouridine and 5-methylcytosine / N1-methyl-pseudouridine-modified human G-CSF mRNA are intramuscular or subcutaneous, regardless of whether they are saline or lipoplex preparations. It is shown that when delivered by route of administration, it can result in specific human G-CSF protein expression in serum. As shown in Table 63, 5-methylcytosine / N1-methyl-pseudouridine-modified human G-CSF mRNA was generally increased compared to 5-methylcytosine / pseudo-pseudouridine-modified human G-CSF mRNA. Shows G-CSF protein production.
Table 63. Human G-CSF protein in mouse serum

D. Comparison of mCherry-modified RNA lipoplex Intramuscular and subcutaneous administration 30% by volume of RNAIMAX ™ and lipoplex-formed modified mCherry mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly-A tail of approximately 160 nucleotides is within the sequence. Not shown; formulations containing 100 μg of either 5'cap, cap 1) or modified mCherry mRNA in physiological saline are delivered intramuscularly and subcutaneously to mice. The product buffer is also administered to the control mouse group either intramuscularly or subcutaneously. The site of injection into the mouse is harvested 17 hours after injection for sectioning to determine the cell type (s) involved in protein production.

Intravitreal administration RNAIMAX ™ and lipoplex-formed modified mCherry mRNA, modified mCherry mRNA in the drug buffer, RNAMAX ™ and lipoplex-formed modified luciferase mRNA, 10 μg of any of the modified luciferase in the drug buffer. The containing formulation can be administered to rats by intravitreal injection (IVT) at a dosage of 5 μL / eye. The product buffer is also administered by IVT to the control rat group at a dosage of 5 μL / eye. Eyes of treated rats were harvested 18 hours after injection for sectioning and lysis to determine if mmRNA could be effectively delivered to the eye in vivo and result in protein production, and further. , Cell types (s) involved in protein production in vivo can be determined.

Nasal administration 30% by volume of RNAIMAX ™ and lipoplex-formed modified mCherry mRNA, 30% by volume of RNAIMAX ™ and lipoplex-formed modified luciferase mRNA, or in physiological saline A preparation containing 100 μg of any of the modified luciferase mRNAs is delivered intranasally. The product buffer is also administered intranasally to the control group. Approximately 13 hours after infusion, lungs can be harvested for sectioning (for those receiving mCherry mRNA) or homogenization (for those receiving luciferase mRNA). These samples are used to determine if mmRNA can be effectively delivered to the lungs in vivo and result in protein production, as well as the cell type (s) involved in protein production in vivo. can do.

Example 44: In vivo Delivery Using Different Lipid Ratios Modified mRNAs were delivered to C57 / BL6 mice to evaluate different lipid ratios and resulting protein expression. 100 μg of modified human EPO mRNA lipoplexed with 10%, 30%, or 50% RNAIMAX ™ (mRNA is shown in SEQ ID NO: 1638; polyA tail of about 160 nucleotides is not shown in the sequence; 5 'Cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), 100 μg of modified luciferase mRNA lipoplexed with 10%, 30%, or 50% RNAIMAX ™ (IVT cDNA sequence is SEQ ID NO: Shown in 21445; the mRNA sequence is shown in SEQ ID NO: 21446, the poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, 5-methylcytosine in each sitosin and pseudouridine in each uridine site. Completely modified by substitution), or formulation of formulation buffer, was intramuscularly administered to mice in a single 70 μL dose. Serum was collected 13 hours after injection and human EPO ELISA was performed to determine human EPO protein levels in each mouse. The results of the human EPO ELISA shown in Table 64 show that the modified human EPO expressed in the muscle is secreted into the serum for each of the different proportions of RNAIMAX ™.
Table 64. Human EPO protein in mouse serum (IM infusion route)

Example 45: Intramuscular and Subcutaneous In vivo Delivery in Mammalian Modified human EPO mRNA formulated in product buffer (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence. 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) was delivered to either C57 / BL6 mice or Sprague dolly rats to assess their dose dependence on human EPO production. .. Rats are shown in 50 μL of modified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is SEQ ID NO: as set forth in the dosing table of Table 65. A poly A tail of approximately 160 nucleotides, shown in 121446, is not shown in the sequence (completely modified with 5'cap, cap 1, 5-methylcytosine at each cytosine and pseudouridine substitution at each uridine site), or formulation buffer. The solution (F buffer) was injected intramuscularly.

表６６．マウス研究

Mice were injected intramuscularly or subcutaneously with 50 μL of modified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc), or pharmaceutical buffer (F buffer) as described in the dosing table of Table 66. .. Thirteen hours after injection, blood was collected and serum was analyzed to determine the amount of human EPO in each mouse or rat. Means and geometric mean in pg / mL for rat studies are also shown in Table 65.
Table 65. Rat study

Table 66. Mouse research

Example 46: Duration of activity after intramuscular in vivo delivery Modified human EPO mRNA formulated in product buffer (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence. 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) was delivered to the sprag dolly rat to determine the duration of the dose response. In rats, 50 μL of modified human EPO mRNA (h-EPO), modified luciferase mRNA (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is shown in SEQ ID NO: 21446) as set forth in the dosing table of Table 67. The polyA tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, fully modified with 5-methylcytosine at each cytosine and pseudouridine substitution at each uridine site) (Luc), or formulation buffer. The solution (F buffer) was injected intramuscularly. Rats were bled 2, 6, 12, 24, 48, and 72 hours after intramuscular injection to determine the concentration of human EPO in serum at a given time point. Means and geometric mean in pg / mL for this study are also shown in Table 67.
Table 67. Dosing table

表６９．マウス血清中のヒトＥＰＯタンパク質（Ｉ．Ｍ．注入経路）

Example 47: Route of Administration Further studies were conducted to investigate dosing using different routes of administration. According to the protocol outlined in Example 35, 4 mice per group were treated intramuscularly (IM), intravenously (IV), or subcutaneously (SC) according to the dosing table outlined in Table 68. Dosing was given. Serum was collected from all mice 13 hours after injection, tissues were collected from the injection site in the intramuscular and subcutaneous administration groups, and spleen, liver, and kidneys were collected from the intravenous group. Results from the intramuscular and subcutaneous groups are shown in Table 69.
Table 68. Dosing table

Table 69. Human EPO protein in mouse serum (IM injection route)

Example 48. Research on Rapidly Ejected Lipid Nanoparticles (reLNP) A. Formulation of Modified RNA reLNP Synthetic Lipids, 1,2-Distearoyl-3-phosphatidylcholine (DSPC) (Avanti Polar Lipids, Alabaster, AL), Cholesterol (Sigma-Aldrich, Taufkirchen, Germany), and α- [3'- Prepare a solution of (1,2-dimiristoyl-3-propanoxy) -carboxamide-propyl] -ω-methoxy-polyoxyethylene (PEG-c-DOMG) (NOF, Taufkirchen, Belgium) and store at -20 ° C. do. Synthetic lipids are selected from DLin-DMA with internal esters, DLin-DMA with terminal esters, DLin-MC3-DMA-internal esters, and DLin-MC3-DMA with terminal esters. The reLNP is adjusted to give a molar ratio of 50:10: 38.5: 1.5 (reLNP: DSPC: cholesterol: PEG-c-DOMG). A preparation of reLNP and modified mRNA is prepared by combining the lipid solution and the modified mRNA solution in a weight ratio of 10: 1, 15: 1, 20: 1, and 30: 1 total lipid to modified mRNA.

B. Formulation Characteristics Using Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK), the particle size, polydispersity index (PDI), and zeta potential of modified mRNA nanoparticles were multiplied by 1 when determining the particle size. Judgment is made in PBS and in 15 mM PBS when determining the zeta potential.

Ultraviolet-visible spectroscopy is used to determine the concentration of the modified mRNA nanoparticle formulation. After mixing, the absorbance spectrum of the solution is recorded at 230 nm to 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of modified RNA in the nanoparticle formulation is calculated based on the attenuation coefficient of the modified RNA used in the formulation and the difference between the absorbance at a wavelength of 260 nm and the baseline at a wavelength of 330 nm.

QUANT-IT ™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA) is used to evaluate encapsulation of modified RNA with nanoparticles. Samples are diluted and transferred to polystyrene 96-well plates, then either TE buffer or 2% Triton X-100 solution is added. The plate is incubated, the RIBOGREEN® reagent is diluted in TE buffer and this solution is added to each well. Fluorescence intensity is measured using a fluorescence plate reader (Wallac Victor 1420 Multibull Counter; Perkin Elmer, Waltherm, MA) The fluorescence value of the reagent blank is subtracted from each of the samples, and the proportion of free modified RNA is determined for the intact sample. Judgment is made by dividing the fluorescence intensity by the fluorescence value of the divided sample.

C. In vitro Incubation Human fetal kidney epithelium (HEK293) and hepatocellular carcinoma epithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) were seeded on 96-well plates (Greener Bio-one GmbH, Frickenhausen, Germany), seeded in 96-well plates (Greener Bio-one GmbH, Frickenhausen, Germany). The plate is precoated with type 1 collagen. In 100 μL of cell culture medium, HEK293 is seeded at a cell density of about 30,000 per well and HepG2 is seeded at a cell density of about 35,000. mCherry mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) is added and incubated immediately after seeding the cells. The mCherry cDNA having the T7 promoter, 5'untranslated region (UTR), and 3'UTR used for in vitro transcription (IVT) is shown in SEQ ID NO: 21440.

Cells are collected by transferring the culture medium supernatant to a 96-well Pro-Bind U bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with half the amount of trypsin / EDTA (Biochrom AG, Berlin, Germany), pooled with their respective supernatants, and single doses of PBS / 2% FCS (all Biochrom AG, Berlin, Germany) / 0. Fix by adding 5% formaldehyde (Merck, Darmstadt, Germany). The sample is then subjected to flow cytometer measurements on an LSRII cytometer (Beckton Dickinson GmbH, Heidelberg, Germany) using an excitation laser and a filter for PE-Texas Red. The mean fluorescence intensity (MFI) of all events and the standard deviation of the four independent wells are presented for the samples analyzed.

D. In vivo drug product study Mice are intravenously administered with a single dose of a drug product containing modified mRNA and reLNP. The modified mRNA administered to the mouse is G-CSF (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), factor IX (mRNA is Shown in SEQ ID NO: 1622; a poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), or mCherry (mRNA sequence is shown in SEQ ID NO: 21439; a poly A tail of about 160 nucleotides Not shown in the sequence; selected from 5'caps, caps 1).

Mice are infused with 100 μg, 10 μg, or 1 μg of formulated modified mRNA and sacrificed 8 hours after administration of the drug. Serum from mice administered with a preparation containing human G-CSF modified mRNA was measured by specific G-CSF ELISA, and serum from mice administered with human Factor IX modified RNA was measured by specific factor IX ELISA. Or analyze by color assay. Liver and spleen from mice treated with mCherry-modified mRNA are analyzed by immunohistochemistry (IHC) or fluorescence activated cell selection (FACS). As a control, serum and tissues thereof are collected and analyzed by ELISA, FACS, and / or IHC without injecting any preparation into one mouse group.

Example 49. In vitro Transfection of VEGF-A Human Vascular Endothelial Growth Factor Isoform A (VEGF-A) Modified mRNA (mRNA sequence is shown in SEQ ID NO: 1672; poly A tail of approximately 160 nucleotides is not shown in the sequence; 5'cap , Cap 1) was transfected into human keratinocyte cells via reverse transfection in a 24-well plate. Human keratinocyte cells were grown in EPILIFE® medium with Supplement S7 from Invitrogen (Carlsbad, CA) until reaching 50-70% confluence. Modified mRNAs encoding 0, 46.875, 93.75, 187.5, 375, 750, and 1500 ng of VEGF-A complexed with RNAIMAX ™ from Invitrogen (Carlsbad, CA) in cells. (MmRNA) was transfected. RNA: RNAIMAX ™ complex was first formed by incubating RNA in 5-fold volume dilution with supplement-free EPILIFE® medium at room temperature for 10 minutes. In a second vial, RNAIMAX ™ reagent was incubated in 10-fold volume dilution with supplement-free EPILIFE® medium at room temperature for 10 minutes. The RNA vials were then mixed with RNAIMAX ™ vials, incubated at room temperature for 20-30 minutes, and then added to the cells in a dropping manner.

A fully optimized VEGF-A-encoding mRNA transfected into human keratinocyte cells (mRNA sequence is shown in SEQ ID NO: 1672; poly-A tail of approximately 160 nucleotides is not shown in the sequence; 5'cap, Cap 1) includes translational modifications such as natural nucleoside triphosphate (NTP), pseudouridine at each uridine site and 5-methylcytosine at each cytosine site (pseud-U / 5 mC), and N1 at each uridine site. -Methyl-pseudouridine and 5-methylcytosine (N1-methyl-pseud-U / 5mC) at each cytosine site were included. Cells are transfected with mmRNA encoding VEGF-A and the concentration of VEGF-A secreted into the culture medium (ρg / mL) is determined by ELISA from Invitrogen (Carlsbad, CA) according to the manufacturer's recommended instructions. Using the kit, concentrations were measured 6, 12, 24, and 48 hours after transfection, respectively. These data, shown in Table 70 and FIGS. 6A, 6B, and 6C, show that the modified mRNA encoding VEGF-A can be translated in human keratinocyte cells, and that VEGF-A is transported from the cell to the extracellular environment. Indicates that it will be released.
Table 70. VEGF-A dosing and protein secretion

Example 50. In vivo study of factor IX Human factor IX m mRNA (gen1; fully modified with 5-methylcytosine and pseudouridine) formulated in the product buffer was delivered to mice via intramuscular injection. The results show that factor IX protein in serum was elevated when measured 13 hours after administration.

In this study, 50 μL of factor IX mRNA (mRNA sequence is shown in SEQ ID NO: 1622) in mice (N = 5 for factor IX, N = 3 for luciferase or buffer control) at 2x100 μg / mouse; A poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), luciferase (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is shown in SEQ ID NO: 21446, poly of about 160 nucleotides. A tail is not shown in the sequence, 5'cap, cap 1, fully modified with 5-methylcytosine in each cytosine and pseudouridine substitution at each uridine site), or intramuscular injection of formulation buffer (F buffer) bottom. Blood was collected from mice 13 hours after intramuscular injection to determine the concentration of human polypeptide in serum in pg / mL units. The results reveal that administration of factor IX mmRNA resulted in levels of 1600 pg / mL at 13 hours compared to factor IX less than 100 pg / mL by administration of either luciferase or buffer control. I made it.

Example 51. Multisite administration: intramuscular and subcutaneous Gen1 or Gen2 (5-methylcytosine (5 mc) and pseudouridine (ψ) modifications, G-CSF-Gen1; or N1-5-methylcytosine (N1-5 mc) and ψ modifications, Human G-CSF-modified mRNA formulated in formulation buffer, modified as any of G-CSF-Gen2) (mRNA sequence is shown in SEQ ID NO: 21438; polyA tail of approximately 160 nucleotides is within the sequence. Not shown in; 5'cap, cap 1) was delivered to mice via intramuscular (IM) or subcutaneous (SC) injection. Injections were performed at 4 doses or 2 x 50 μg (2 sites) daily for 3 days (24 hour intervals). Four doses were administered 6 hours prior to blood sampling and CBC analysis. In control, luciferase (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is shown in SEQ ID NO: 121446, poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, each cytosine. Completely modified with 5-methylcytosine and pseudouridine substitutions at each uridine site), or formulation buffer (F buffer) was included. Blood was collected from mice 72 hours after the first mRNA injection (6 hours after the last modified mRNA administration) to determine the effect of mRNA-encoded human G-CSF on neutrophil count. The dosing regimen is shown in Table 71, as is the resulting neutrophil count (1000 units / μL). In Table 71, an asterisk (*) indicates statistical significance at p <0.05.

For intramuscular administration, the data show a 4-fold increase in neutrophil count over the control at day 3 for Gen1 G-CSF mRNA and a 2-fold increase in Gen2 G-CSF mRNA. For subcutaneous administration, the data show a 2-fold increase in neutrophil count over the control at day 3 with Gen2 G-CSF mRNA.

These data show that both 5-methylcytidine / pseudouridine and 5-methylcytidine / N1-methyl-pseudouridine-modified mRNA are biological, as evidenced by a specific increase in blood neutrophil count. Shows that it can be active.
Table 71. Dosing regimen

Example 52. Intravenous administration Human G-CSF modification formulated in 10% lipoplex (RNAiMax) modified with 5-methylcytosine (5 mc) and pseudouridine (ψ) modification (Gen1) or without modification. mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) is injected intravenously (IV) at a dose of 50 μg RNA and 100 μL volume. Was delivered to mice on days 0, 2, and 4. Neutrophils were measured on days 1, 5, and 8. Controls included non-specific mammalian RNA or pharmaceutical buffer alone (F buffer). Blood was drawn from mice on days 1, 5, and 8 to determine the effect of human G-CSF encoded by modified mRNA that increases neutrophil count. The dosing regimen is shown in Table 72, as is the resulting neutrophil count (1000 units / μL, K / μL).

For intravenous administration, the data show a 4- to 5-fold increase in neutrophil count in G-CSF modified mRNA over the control at day 5, but unmodified G-CSF mRNA or non-specific control. Will not be shown. Blood cell counts returned to baseline 4 days after the last infusion. No other changes were observed in the leukocyte population.

In Table 72, an asterisk (*) indicates statistical significance at p <0.001 as compared to buffer.

These data are obtained when lipoplex-formulated 5-methylcytidine / pseudouridine-modified mRNA is delivered via an intravenous route of administration, as evidenced by a specific increase in blood neutrophil count. Shows that it can be biologically active. Other cell subsets were not significantly altered. Similarly administered unmodified G-CSF mRNA showed no pharmacological effect on neutrophil count.
Table 72. Dosing regimen

Example 53. Saline preparation: Intramuscular administration A. Protein Expression Human G-CSF modified mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) and human EPO mRNA (mRNA sequence is SEQ ID NO: Shown in 1638; a poly A tail of about 160 nucleotides is not shown in the sequence; with 5'cap, cap 1), G-CSF modified mRNA (modified with 5-methylcytosine (5 mc) and pseudouridine (ψ)). EPO-modified mRNA (modified with N1-5-methylcytosine (N1-5mc) and ψ modification) was added to the formulation buffer (150 mM sodium chloride, 2 mM calcium chloride, 2 mM phosphate, 0.5 mM EDTA, pH 6). Formulated in (.5) and delivered to mice via intramuscular (IM) infusion at a dose of 100 μg.

In control, luciferase (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is shown in SEQ ID NO: 21446, poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, each cytosine. Completely modified with 5-methylcytosine and pseudouridine substitution at each uridine site) or formulation buffer (F buffer) was included. Blood was collected from mice 13 hours after injection to determine the concentration of human polypeptide in serum in pg / mL units. (In the G-CSF group, human G-CSF in mouse serum was measured, and in the EPO group, human EPO in mouse serum was measured). The data are shown in Table 73.
Table 73. Dosing regimen

B. Dosage Response Human EPO-modified mRNA (mRNA sequence is shown in SEQ ID NO: 1638; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) Was formulated in formulation buffer and delivered to mice via intramuscular (IM) infusion.

In control, luciferase (mRNA sequence shown in SEQ ID NO: 21446, poly A tail of about 160 nucleotides not shown in the sequence, fully modified with 5'cap, cap 1,5-methylcytosine and pseudouridine) or Formulation buffer (F buffer) was included. Blood was collected from mice 13 hours after injection to determine the concentration of human polypeptide in serum in pg / mL units. The dose and expression are shown in Table 74.
Table 74. Dosing regimen and expression

Example 54. Multiple doses of EPO / Multiple doses A study was designed and performed using multiple intramuscular injection sites at one time.

The design of a single multi-dose experiment was for human erythropoetin (EPO) mRNA administered in formulation buffer (mRNA sequence is shown in SEQ ID NO: 1638; polyA tail of approximately 160 nucleotides is not shown in the sequence; 5 With the use of'cap, cap 1) or G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1). A dosing vehicle (F buffer) was used as a control. EPO and G-CSF modified mRNAs were modified with 5-methylcytosine at each cytosine and pseudouridine substitution at each uridine site.

Animals (n = 5), namely Prague dolly rats, were injected IM (intramuscularly) with a single unit dose of 100 μg (delivered to one thigh). For multiple doses, 6 doses of 100 μg (delivered to two thighs) were used for both EPO and G-CSF mmRNA. Dosing to controls included the use of buffer in a single dose. Blood levels of human EPO were evaluated 13 hours after infusion.

Human EPO protein was measured in rat serum 13 hours after intramuscular injection. Five groups of rats were treated and evaluated. The results are shown in Table 75.
Table 75. Multiple dose studies

Example 55. Signal Sequence Exchange Study m mRNA encoding human granulocyte colony stimulator (G-CSF) (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of approximately 160 nucleotides is not shown in the sequence; 5'cap, cap 1 ) Was synthesized using the modified nucleotide pseudouridine and 5-methylcytosine (pseud-U / 5mC). These variants include either a wild-type N-terminal secretory signal peptide sequence (MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 95), a non-secretory signal peptide sequence, or a G-CSF encoding a secretory signal peptide sequence obtained from another mRNA. The construct was included. These have a wild-type G-CSF signal peptide sequence of human α-1-antitrypsin (AAT) (MMPSSVSWGILLAGLCCLVPVSLA; SEQ ID NO: 94), human factor IX (FIX) (MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILINRPKR; It contained a sequence substituted with either the signal peptide sequence of (MKGSLLLLLLVSNLLLCQSVAP; SEQ ID NO: 97) or human albumin (Alb) (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 98).

250 ng of modified mRNA encoding each G-CSF variant was used in 1 μL of Lipofectamine 2000 (Life Technologies) in a 24-well plate containing 300,000 cells in each well, HEK293A (293A in the table), mice. Cell lines of myoblasts (MM in the table) (C2C12, CRL-1772, ATCC) and rat myoblasts (RM in the table) (L6 strain, CRL-1458, ATCC) were transfected. The supernatant was collected after 24 hours and the secreted G-CSF protein was analyzed by ELISA using a human G-CSF ELISA kit (Life Technologies). The data shown in Table 76 show that cells transfected with the G-CSF m mRNA encoding the albumin signal peptide secrete at least 12-fold more G-CSF protein than its wild-type counterpart.
Table 76. Signal peptide exchange

Example 56. Cytokine research: PBMC
A. Isolation and culture of PBMCs 50 mL of human blood from two donors was received in a heparin sodium tube from Research Blood Components (lots KP30928 and KP30931). For each donor, blood was pooled, diluted with DPBS (SAFC Bioscience 59331C, lot 071M8408) to 70 mL and divided equally into two 50 mL conical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot 10074400) was gently dispensed under the blood layer. The tube was centrifuged at 2000 rpm for 30 minutes with low acceleration and braking. The tube was removed and the buffy PBMC layer was gently transferred to a new 50 mL conical tube and washed with DPBS. The tube was centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended in 50 mL DPBS and washed. The tube was centrifuged at 1250 rpm for 10 minutes. This wash step was repeated and PBMC pellets were resuspended in 19 mL Optimem I (Gibco 11058, lot 1072088) and counted. The cell suspension was adjusted to live cells at a concentration of 3.0 × 10 ^ 6 cells / mL.

These cells were then seeded in 5 96-well tissue culture treated round bottom plates (Costar 3799) per donor at 50 μL per well. Within 30 minutes, the transfection mixture was added to each well in an amount of 50 μL per well. Four hours after transfection, the medium was supplemented with 10 μL of fetal bovine serum (Gibco 10082, lot 1012368).

B. Transfection Preparation mmRNA encoding human G-CSF (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) ((1) native M mRNA (IVT) encoding luciferase, containing either NTP, (2) 100% substitution with 5-methylcytidine and pseudouridine, or (3) 100% substitution with 5-methylcytidine and N1-methyl-pseudouridine. The cDNA sequence is shown in SEQ ID NO: 21445; the mRNA sequence is shown in SEQ ID NO: 21446, the poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, 5-methylcytidine in each cytosine and each Completely modified with pseudouridine substitutions at the uridine site) (containing either (1) native NTP or (2) 100% substitution with 5-methylcytidine and pseudouridine), and the TLR agonist R848 (Invivogen trll-r848). ) Was diluted to 38.4 ng / μL in Optimem I having a final volume of 2500 μL.

Separately, 432 μL Lipofectamine 2000 (Invitrogen)
11668-027, lot 1070962) was diluted with 13.1 mL of Optimem I. In a 96-well plate, nine 135 μL aliquots of each mmRNA, positive control (R-848), or negative control (Optimem I) were added to 135 μL diluted Lipofectamine 2000. The plate containing the substance to be transfected was incubated for 20 minutes. The transfection mixture was then transferred to each human PBMC at 50 μL per well. The plate was then incubated at 37 ° C. At 2, 4, 8, 20, and 44 hours, each plate was removed from the incubator and the supernatant was frozen.

After removing the last plate, the supernatant was assayed using a human G-CSF ELISA kit (Invitrogen KHC2032) and a human IFN-α ELISA kit (Thermo Scientific 41105-2). Each condition was done twice.

C. Results Assess the ability of unmodified and modified mRNA (m mRNA) to produce the encoded protein over time (G-CSF production), as well as the ability of mRNA to induce innate immune recognition as measured by interferon α production. bottom. The use of in vitro PBMC cultures is a common method for measuring the immunostimulatory potential of oligonucleotides (Robbins et al., Oligonucleotides 2009 19: 89-102, which is incorporated herein by reference in its entirety).

The results were interpolated against the standard curve of each ELISA plate using a 4-parameter logistic curve fit. The averages of G-CSF and IFN-α production over time measured by a particular Elisa from two separate PBMC donors are shown in Tables 77 and 78.

表７８．ＩＦＮ−αシグナル

In G-CSF ELISA, the background signal due to the Lipofectamine 2000 untreated condition was subtracted at each time point. The data show that specific production of human G-CSF protein by human peripheral blood mononuclears is 100% substituted with native NTP, 5-methylcytidine and pseudouridine, or 5-methylcytidine and N1-methyl-pseudouridine. It was shown to be found in G-CSF mRNA containing 100% substitution of. G-CSF production was significantly increased by the use of modified mRNA compared to unmodified mRNA, with G-CSF m mRNA containing 5-methylcytidine and N1-methyl-pseudouridine having the highest G-CSF production. showed that. For innate immune recognition, unmodified mRNA resulted in significant IFN-α production, whereas modified mRNA significantly blocked interferon α production. G-CSF mRNA fully modified with 5-methylcytidine and N1-methyl-pseudouridine did not substantially increase cytokines, but was fully modified with 5-methylcytidine and pseudo-pseudouridine. mRNA induced IFN-α, TNF-α, and IP10. Many other cytokines were unaffected by any modification.
Table 77. G-CSF signal

Table 78. IFN-α signal

Example 57. Scope of chemical modification of modified mRNA Modified nucleotides such as, but not limited to, chemically modified 5-methylcytosine and pseudouridine have been shown to reduce the innate immune response and increase RNA expression in mammalian cells. ing. Surprisingly, and what has not been known so far, when the amount of chemical modification is less than 100%, the effect of the chemical modification can be titrated. Previously, it was thought that complete modification was necessary and sufficient to elicit the beneficial effects of chemical modification, and modification of less than 100% of mRNA had little effect. However, it has now been shown that the benefits of chemical modification can be induced by less than complete modifications, the effects of which depend on the target, concentration, and modification.

表８０．ＩＦＮ−αおよびＴＮＦ−αの発現

A. Modified RNA transfected into PBMC
Three normal blood donors using 960 ng of 5-methylcytosine (5 mC) and pseudouridine (pseudouridine) modified G-CSF mRNA or unmodified G-CSF mRNA with 0.8 μL of Lipofectamine 2000 ( Peripheral blood mononuclear cells (PBMC) from D1, D2, D3) were transfected. G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) was fully modified with 5 mC and pseudo U (100%). Modified), not modified with 5 mC and Pseudo U (0% modified), or the mRNA contains 50% modified, 25% modified, 10% modified, 5% modified, 1% modified, or 0.1% modified. Partially modified with 5 mC and Pseudo U. A control sample, luciferase (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5 meC and pseudo U) is also G-CSF. Expression was analyzed. Control samples of TNF-α and IFN-α, Lipofectamine 2000, LPS, R-848, luciferase (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, Cap 1; fully modified with 5 mC and pseudo), and P (I) P (C) were also analyzed. The supernatant was collected 22 hours after transfection and ELISA was performed to determine protein expression. The expression of G-CSF is shown in Table 79, and the expression of IFN-α and TNF-α is shown in Table 80. Expression of IFN-α and TNF-α can be a secondary effect of transfection of G-CSF mRNA. Tables 79 and 80 show that the amount of chemical modification of G-CSF, IFN-α, and TNF-α can be titrated when the mRNA is not completely modified, and the titrability trends are not the same for each subject. Indicates that there is no such thing.
Table 79. G-CSF expression

Table 80. Expression of IFN-α and TNF-α

B. Modified RNA transfected into HEK293
Human fetal kidney epithelial (HEK293) cells were seeded in 96-well plates at a density of 30,000 cells per well in 100 μL cell culture medium. 250 ng of modified G-CSF mRNA formulated with RNAiMAX ™ (Invitrogen, Carlsbad, CA) (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; A 5'cap, cap 1) was added to the wells. G-CSF was completely modified with 5 mC and pseudo U (100% modified), not modified with 5 mC and pseudo U (0% modified), or mRNA was 75% modified, 50% modified, or 25% modified. Was partially modified with 5 mC and Pseudo U to contain. Control samples (AK 5/2, mCherry (shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5 mC and pseudo U), and not yet. Processing) was also analyzed. The half-life of G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine is about 8-10 hours. The supernatant is collected after 16 hours and the secreted protein is analyzed by ELISA. Table 81 shows that the amount of chemical modification of G-CSF can be titrated when the mRNA is not completely modified.
Table 81. G-CSF expression

Example 58: In vivo delivery of modified RNA (m mRNA) Modified RNA was delivered intramuscularly, subcutaneously, or intravenously to C57 / BL6 mice and the biodistribution of the modified RNA was evaluated using luciferase. The formulation buffer used in all delivery methods was 150 mM sodium chloride, 2 mM calcium chloride, 2 mM Na + -phosphate (1.4 mM sodium monobasic phosphate and 0.6 mM sodium dibasic phosphate). Included), as well as 0.5 mM ethylenediamine tetraacetic acid (EDTA), adjusted with sodium hydroxide to reach final pH 6.5, then filtered and sterilized. 1x concentration was used as delivery buffer. To make a lipoplexized solution for delivery to mice, 50 μg of RNA was equilibrated in delivery buffer at room temperature for 10 minutes in one vial and 10 μL of RNAiMAX ™ in the second vial at room temperature. Equilibrated in delivery buffer for 10 minutes. After equilibration, the vials were combined, delivery buffer was added until a final volume of 100 μL was reached, which was then incubated at room temperature for 20 minutes. Luciferin was administered intraperitoneally (IP) to each mouse at 150 mg / kg and then imaged during the plateau phase of the luciferin exposure curve, which was 15-30 minutes. To make luciferin, 1 g of D-luciferin potassium or sodium salt was dissolved in 66.6 mL of distilled phosphate buffer (DPBS) without Mg2 + or Ca2 + to give a 15 mg / mL solution. .. The solution was gently mixed, passed through a 0.2 μm syringe filter, then nitrogen purged, aliquoted and stored at −80 ° C. while blocking light as much as possible. On the day of dosing, the solution was thawed using a warm bath, mixed gently if luciferin was not dissolved, and kept on ice.

Full-body images of each mouse were taken 2, 8, and 24 hours after dosing. Tissue images and serum were collected from mice 24 hours after dosing. Mice that received the dose intravenously imaged the liver, spleen, kidneys, lungs, heart, perineal adipose tissue, and thymus. Mice to which the dose was administered intramuscularly or subcutaneously had liver, spleen, kidney, lung, perirenal adipose tissue, and muscle at the injection site. From whole body images, bioluminescence was measured in photon counts per second for each route of administration and dosing regimen.

A. Intramuscularly administered mice fully modified with 5-methylcytosine and pseudouridine (naked-Luc), fully modified lipoplexized modified luciferase mRNA with 5-methylcitosin and pseudouridine (lipoplex) -Luc) (IVT cDNA sequence is shown in SEQ ID NO: 21445; mRNA sequence is shown in SEQ ID NO: 21446, poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, 5 in each cytosin -Completely modified with methylcitosine and pseudouridine substitutions at each uridine site), lipoplexed modified granulocyte colony stimulating factor (G-CSF) mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of approximately 160 nucleotides Is not shown in the sequence; either 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) (lipoplex-cytocytogene), or formulation buffer at an infusion volume of 50 μL for each formulation. A single modified RNA dose of 50 μg was administered intramuscularly (IM) to the right hind limb, and a single modified RNA dose of 5 μg at an infusion volume of 50 μL was administered intramuscularly (IM) to the left hind limb. The bioluminescent averages of the luciferase expression signals in each group 2, 8, and 24 hours after dosing are shown in Table 82. Bioluminescence showed positive signals for lipoplex-containing and non-lipoplex-containing 5 μg and 50 μg modified RNA formulations at the injection site.
Table 82. In vivo biophoton imaging (IM injection pathway)

B. Subcutaneous administration of modified luciferase mRNA (naked-Luc), lipoplexation-modified luciferase mRNA (lipoplex-luc), lipoplexation-modified G-CSF mRNA (lipoplex-G-CSF), or preparation buffer. Was administered subcutaneously (SC) at a single modified mRNA dose of 50 μg at an infusion volume of 100 μL for each preparation. The bioluminescent averages of luciferase expression signals in each group 2, 8, and 24 hours after dosing are shown in Table 83. Bioluminescence showed a positive signal at the injection site for 5 μg and 50 μg modified mRNA formulations with and without lipoplex.
Table 83. In vivo biophoton imaging (SC injection pathway)

C. Intravenously administered mice with modified luciferase mRNA (naked-Luc), lipoplexation-modified luciferase mRNA (lipoplex-luc), lipoplexation-modified G-CSF mRNA (lipoplex-G-CSF), or formulation buffer. Either was administered intravenously (IV) at a single modified mRNA dose of 50 μg at an infusion volume of 100 μL for each formulation. The bioluminescent averages of spleen luciferase expression signals from each group 2 hours after dosing are shown in Table 84. Bioluminescence showed a positive signal in the spleen of 50 μg of modified mRNA preparation containing lipoplex.
Table 84. In vivo biophoton imaging (IV injection pathway)

Example 59. Buffer formulation study G-CSF modified mRNA in buffer solution (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; N1-pseudouridine and Fully modified with 5-methylcitosine or factor IX modified mRNA (mRNA sequence is shown in SEQ ID NO: 1622; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; N1-pseud Completely modified with uridine and 5-methylcytosine) is intramuscularly administered to the rats at an infusion volume of 50 μL and a modified mRNA dose of 200 μg per rat (n = 5), as shown in Table 85. Modified mRNA is lyophilized in water for 1-2 days. It is then reconstituted to a target concentration of 6 mg / mL in the buffers listed below. The concentration is determined by OD 260. Samples are diluted to 4 mg / mL in appropriate buffer prior to dosing.

To precipitate the modified mRNA, 3M sodium acetate and pure ethanol at pH 5.5 are added at 1/10 and 4 times the total volume of the modified mRNA, respectively. Place the material at -80 ° C for at least 1 hour. The material is then centrifuged at 4000 rpm, 4 ° C. for 30 minutes. The supernatant is removed, the pellet is centrifuged and washed 3 times with 75% ethanol. Finally, the pellet is reconstituted with buffer to a target concentration of 6 mg / mL. The concentration is determined by OD 260. Samples are diluted to 4 mg / mL in appropriate buffer prior to dosing. All samples are prepared by lyophilization unless otherwise specified below.
Table 85. Buffer dosing group

Serum samples are taken from rats at various time intervals and analyzed for G-CSF or Factor IX protein expression using G-CSF and Factor IX ELISA.

Example 60. Multiple dose study Sprague dolly rats (n = 8) receive 8 intravenous injections (twice a week) for 28 days. Human G-CSF-modified mRNA of 0.5 mg / kg, 0.05 mg / kg, 0.005 mg / kg, or 0.0005 mg / kg luciferase-modified mRNA formulated in lipid nanoparticles in rats, physiological saline. Inject 0.5 mg / kg human G-CSF modified mRNA in water, 0.2 mg / kg human G-CSF protein Neupogen formulated in lipid nanoparticles or non-translatable human G-CSF modified mRNA. .. Serum is collected at predetermined time intervals for G-CSF protein expression (8, 24, and 72 hours after the first dose of the week), total blood cell count and white blood cell count (first dose of the week). 24 and 72 hours after), as well as clinical chemistry (24 and 72 hours after the first dose of the week). Rats are sacrificed on day 29, 4 days after the last dose, to assess total blood cell count, white blood cell count, clinical science, protein expression, and histopathology and anatomy to assess effects on major organs. In addition, on day 29, mice were subjected to antibody assay.

Example 61. LNP In vivo Study Luciferase-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence, fully modified with 5'cap, cap 1; 5-methylcytosine and pseudouridine. , Formulated as lipid nanoparticles (LNP) using the syringe pump method. LNP is the final lipid molar ratio of 50:10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-DMG) was formulated with a weight ratio of total lipid to modified mRNA of 20: 1. As shown in Table 86, the luciferase LNP formulation was characterized by particle size, zeta potential, and encapsulation.
Table 86. Luciferase preparation

As outlined in Table 87, the luciferase LNP preparation was administered intramuscularly, intravenously, and subcutaneously to Balb-C mice (n = 3), and the luciferase-modified RNA formulated in PBS was administered to the mice. It was administered intravenously.
Table 87. Luciferase preparation

Mice to which the luciferase LNP preparation was intravenously and intramuscularly administered were imaged at 2, 8, 24, 48, 120, and 192 hours, and mice to which the luciferase LNP preparation was subcutaneously administered were 2, 8, 24, 48, and. Imaging was performed at 120 hours to determine the luciferase expression shown in Table 88. In Table 88, "NT" means untested. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice.
Table 88. Luciferase expression

One mouse intravenously administered with the LNP preparation was sacrificed at 8 hours to determine luciferase expression in the liver and spleen. In addition, one mouse intramuscularly administered with the LNP preparation is sacrificed at 8 hours to determine luciferase expression in the muscles around the injection site as well as in the liver and pancreas. Expression was seen in both the liver and spleen after intravenous and intramuscular administration, as well as in the muscle around the intramuscular injection site, as shown in Table 89.
Table 89. Tissue luciferase expression

Example 62. Cytokine research: PBMC
A. Isolation and culture of PBMCs 50 mL of human blood from two donors was received in a heparin sodium tube from Research Blood Components (lots KP30928 and KP30931). For each donor, blood was pooled, diluted with DPBS (SAFC Bioscience 59331C, lot 071M8408) to 70 mL and divided equally into two 50 mL conical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot 10074400) was gently dispensed under the blood layer. The tube was centrifuged at 2000 rpm for 30 minutes with low acceleration and braking. The tube was removed and the buffy PBMC layer was gently transferred to a new 50 mL conical tube and washed with DPBS. The tube was centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended in 50 mL DPBS and washed. The tube was centrifuged at 1250 rpm for 10 minutes. This wash step was repeated and PBMC pellets were resuspended in 19 mL Optimem I (Gibco 11058, lot 1072088) and counted. The cell suspension was adjusted to live cells at a concentration of 3.0 × 10 ^ 6 cells / mL.

These cells were then seeded in 5 96-well tissue culture treated round bottom plates (Costar 3799) per donor at 50 μL per well. Within 30 minutes, the transfection mixture was added to each well in an amount of 50 μL per well. Four hours after transfection, the medium was supplemented with 10 μL of fetal bovine serum (Gibco 10082, lot 1012368).

B. Transfection Preparation Modified mRNA encoding human G-CSF (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) ((1) native NTP, containing either (2) 100% substitution with 5-methylcytidine and pseudouridine, or (3) 100% substitution with 5-methylcytidine and N1-methyl-pseudouridine, mRNA encoding luciferase (The IVT cDNA sequence is shown in SEQ ID NO: 21445; the mRNA sequence is shown in SEQ ID NO: 21446, the poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1, 5-methylcytidine in each cytosine. And fully modified with pseudouridine substitutions at each uridine site) (containing either (1) natural NTP or (2) 100% substitution with 5-methylcytidine and pseudouridine), and TLR agonist R848 (Invivogen tllll-). r848) was diluted to 38.4 ng / μL in Optimem I in a final volume of 2500 μL.

Separately, 110 μL Lipofectamine 2000 (Invitrogen)
11668-027, lot 1070962) was diluted with 6.76 mL Optimem I. In a 96-well plate, nine 135 μL aliquots of each mRNA, positive control (R-848), or negative control (Optimem I) were added to 135 μL diluted Lipofectamine 2000. The plate containing the substance to be transfected was incubated for 20 minutes. The transfection mixture was then transferred to each human PBMC at 50 μL per well. The plate was then incubated at 37 ° C. At 2, 4, 8, 20, and 44 hours, each plate was removed from the incubator and the supernatant was frozen.

After removing the last plate, the supernatant was assayed using a human G-CSF ELISA kit (Invitrogen KHC2032) and a human IFN-α ELISA kit (Thermo Scientific 41105-2). Each condition was done twice.

C. Protein and innate immune response analysis The ability of unmodified and modified mRNAs to produce encoded proteins is assessed over time (G-CSF production), as is the ability of mRNAs to induce innate immune recognition as measured by interferon α production. Evaluated to. The use of in vitro PBMC cultures is a common method for measuring the immunostimulatory potential of oligonucleotides (Robbins et al., Oligonucleotides 2009 19: 89-102).

The results were interpolated against the standard curve of each ELISA plate using a 4-parameter logistic curve fit. Tables 90 and 91 show the average of three separate PBMC donors producing G-CSF, interferon α (IFN-α), and tumor necrosis factor α (TNF-α) over time as measured by specific ELISA. ..

In G-CSF ELISA, the background signal due to the Lipofectamine 2000 (LF2000) untreated condition was subtracted at each time point. The data show that specific production of human G-CSF protein by human peripheral blood mononuclears is 100% substituted with native NTP, 5-methylcytidine and pseudouridine, or 5-methylcytidine and N1-methyl-pseudouridine. It was shown to be found in G-CSF mRNA containing 100% substitution of. G-CSF production was significantly increased by the use of 5-methylcytidine and N1-methyl-pseudouridine modified mRNA compared to 5-methylcytidine and pseudouridine modified mRNA.

For innate immune recognition, the chemical composition of both modified mRNAs significantly blocked IFN-α and TNF-α production compared to positive controls (R848, p (I) p (C)), but between chemical compositions. There was a significant difference in. 5-Methylcytidine and pseudouridine-modified mRNAs resulted in low but detectable levels of IFN-α and TNF-α production, while 5-methylcytidine and N1-methyl-pseudouridine-modified mRNAs were detectable. It did not result in the production of IFN-α and TNF-α.

As a result, in addition to the need to consider cytokine markers that exceed one of the activations of the innate immune response, surprisingly, the combination of modifications may result in different levels of cellular response (protein production and immune activation). It has been determined that it has been found. The modification, N1-methyl-pseudouridine, provides enhanced protection in this study over the standard 5-methylcytidine / pseudouridine combination, resulting in twice as many proteins and nearly 150 times more immune response. It has been shown to result in reduction (TNF-α).

Given that PBMCs contain a large number of innate immune RNA recognition sensors and are capable of protein translation, this provides a useful system for testing the interdependence of these two pathways. It is known that mRNA translation can be adversely affected by activation of such innate immune pathways (Karika et al. Immunocity (2005) 23: 165-175, Warren et al. Cell Stem Cell (2010)). 7: 618-630). Correlation between translation (in this case, G-CSF protein production) and cytokine production (in this case, exemplified by IFN-α and TNF-α protein production) by using PBMCs as an in vitro assay system. Can be established. Better protein production correlates with the induction of lower innate immune activation pathways, and new chemical compositions can be advantageously determined based on this ratio (Table 92).

表９１．ＩＦＮ−αおよびＴＮＦ−α

表９２．Ｇ−ＣＳＦとサイトカインの比率

In this study, the PC ratio of the two chemical modifications, pseudouridine and N1-methyl-pseudouridine, both with 5-methylcytosine, was 4742/141 compared to 9944/1 = 9944 of the cytokine IFN-α. = 34. For the cytokine TNF-α, the two chemical compositions have PC ratios of 153 and 1243, suggesting that N1-methyl-pseudouridine is a better modification for both cytokines, respectively. In Tables 90 and 91, "NT" means untested.
Table 90. G-CSF

Table 91. IFN-α and TNF-α

Table 92. Ratio of G-CSF to cytokines

Example 63. In vitro PBMC study: G-CSF mRNA or unmodified G-CSF mRNA modified with modified percent 480 ng of 5-methylcytosine (5 mC) and pseudouridine (pseudouridine) in normal blood with 0.4 μL Lipofectamine 2000. Peripheral blood mononuclear cells (PBMC) from donors (D1, D2, and D3) were transfected. G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) was fully modified with 5 mC and pseudo U (100%). Modified), not modified with 5 mC and pseudo U (0% modified), or partially modified with 5 mC and pseudo U so that the mRNA contains 75% modified, 50% modified, or 25% modified. A control sample, luciferase (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5 meC and pseudo U) is also G-CSF. Expression was analyzed. Control samples of TNF-α and IFN-α, Lipofectamine 2000, LPS, R-848, luciferase (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, Cap 1; fully modified with 5 mC and pseudo), and P (I) P (C) were also analyzed. The supernatant was collected 22 hours after transfection and ELISA was performed to determine protein expression. The expression of G-CSF is shown in Table 93, and the expression of IFN-α and TNF-α is shown in Table 94. Expression of IFN-α and TNF-α can be a secondary effect of transfection of G-CSF mRNA. Tables 93 and 94 allow the amount of chemical modification of G-CSF, interferon α (IFN-α), and tumor necrosis factor α (TNF-α) to be titrated when the mRNA is not completely modified. , Indicates that the titrable tendencies are not the same for each subject.

表９４．ＩＦＮ−αおよびＴＮＦ−αの発現

表９５．ＰＣ比および修飾率の影響

By using PBMCs as an in vitro assay system, it is possible to establish a correlation between translation (in this case, G-CSF protein production) and cytokine production (in this case, exemplified by IFN-α protein production). It is possible. Better protein production correlates with the induction of lower innate immune activation pathways, and the modification rate of the chemical composition can be advantageously determined based on this ratio (Table 95). Complete modifications with 5-methylcytidine and pseudouridine, calculated from Tables 93 and 94, are better proteins than those without either modification (natural G-CSF mRNA), as shown in Table 95. / Shows cytokine production ratio (100 times for IFN-α and 27 times for TNF-α). Partial modifications show a linear relationship that results in lower protein / cytokine ratios with less modification.
Table 93. G-CSF expression

Table 94. Expression of IFN-α and TNF-α

Table 95. Effect of PC ratio and modification rate

Example 64. Modified RNA transfected into PBMC
G-CSF mRNA or unmodified G-CSF mRNA modified with 500 ng of 5-methylcytosine (5 mC) and pseudouridine (pseudouridine) using 0.4 μL of Lipofectamine 2000 with normal blood donors (D1, D2, And peripheral blood mononuclear cells (PBMC) from D3) were transfected. G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) was fully modified with 5 mC and pseudo U (100%). Modified), not modified with 5 mC and Pseudo U (0% modified), or the mRNA contains 50% modified, 25% modified, 10% modified, 5% modified, 1% modified, or 0.1% modified. Partially modified with 5 mC and Pseudo U. Control sample mCherry (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5 meC and pseudouridine), 5-methyl G-CSF fully modified with cytosine and pseudouridine (control G-CSF), as well as untreated controls also express G-CSF, tumor necrosis factor α (TNF-α), and interferon α (IFN-α). Was analyzed. Supernatants were collected 6 and 18 hours after transfection and ELISA was performed to determine protein expression. The expression of G-CSF, IFN-α, and TNF-α of donor 1 is shown in Table 96, donor 2 is shown in Table 97, and donor 3 is shown in Table 98.

表９７．ドナー２

表９８．ドナー３

Complete 100% modification with 5-methylcytidine and pseudouridine resulted in the highest protein translation (G-CSF) and the lowest amount of cytokines produced across all three human PBMC donors. Reducing the amount of modification results in higher cytokine production (IFN-α and TNF-α), and therefore in order to reduce cytokines and improve protein translation (G- herein). It is further emphasized that complete modification is important (as evidenced by CSF production).
Table 96. Donor 1

Table 97. Donor 2

Table 98. Donor 3

Example 65. Study of innate immune response in BJ fibroblasts A. Single Transfection Human primary capsule fibroblasts (BJ fibroblasts) were obtained from the American Type Culture Collection (ATCC) (Cat. No. CRL-2522) at 5% CO ₂ at 37 ° C. and 10% bovine. It was grown in Eagle's Minimal Essential Medium (ATCC, Catalog Nos. 30-2003) supplemented with fetal serum. BJ fibroblasts were seeded in 24-well plates at a cell density of 300,000 per well in 0.5 mL culture medium. Completely modified with 5-methylcytosine and pseudouridine with or without cap 0 (Gen1) or complete with 5-methylcytosine and N1-methyl-pseudouridine (Gen2), 250 ng of modified G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; polyA tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1). Transfections were performed using Lipofectamine 2000 (Invitrogen, Catalog No. 11668-019) according to the vendor's protocol. Control samples poly I: C (PIC), Lipofectamine 2000 (Lipo), native luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, Cap 1) and native G-CSF mRNA were also transfected. Cells were harvested after 18 hours, total RNA was isolated and DNASE® treated with the RNeasy microkit (catalog number 74004) according to the manufacturer's protocol. 100 ng of total RNA was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Catalog No. 4368814) according to the manufacturer's protocol. The cDNA is then biorad according to the manufacturer's protocol.
Expression of innate immune response genes was analyzed by quantitative real-time PCR using SYbrGreen in a CFX 384 instrument. Table 99 shows the housekeeping gene HPRT (hypoxanthine).
It shows the expression level of the innate immune response transcript compared to phosphoribosistranfase)), which is expressed as a doubling compared to HPRT. In this table, the panel of standard metrics includes: RIG-I is retinoic acid-inducing gene 1, IL6 is interleukin-6, and OAS-1 is oligoadenylate synthase 1. IFNb is interferon β, AIM2 is the absence of melanoma-2, IFIT-1 is an interferon-induced protein 1 with tetralycopeptide repeats, PKR is protein kinase R, and TNFa is tumor necrosis factor. Factor α, IFNa is interferon α.
Table 99. Innate immune response transcript level

B. Repeated Transfection Human primary capsule fibroblasts (BJ fibroblasts) were obtained from the American Type Culture Collection (ATCC) (Cat. No. CRL-2522) and obtained from 10% fetal bovine serum at 37 ° C. under _{5% CO 2.} Was grown in Eagle's Minimal Essential Medium (ATCC, Catalog No. 30-2003) supplemented with. BJ fibroblasts were seeded in 24-well plates at a cell density of 300,000 per well in 0.5 mL culture medium. 250 ng of modified G-CSF mRNA, unmodified, fully modified with 5-methylcytosine and pseudouridine (Gen1), or fully modified with 5-methylcytosine and N1-methyl-pseudouridine (Gen2). The mRNA sequence is shown in SEQ ID NO: 21438; the polyA tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) was transfected daily for 5 days according to the manufacturer's protocol. Control samples Lipofectamine 2000 (L2000) and mCherry mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytidine and pseudo Fully modified with uridine) was also transfected daily for 5 days. The results are shown in Table 100.

Unmodified mRNA showed a cytokine response in interferon β (IFN-β) and interleukin-6 (IL-6) after day 1. At least pseudouridine-modified mRNAs showed a cytokine response after 2-3 days, whereas 5-methylcytosine and N1-methyl-pseudouridine-modified mRNAs showed a reduced response after 3-5 days. showed that.
Table 100. Cyclic response

Example 66. In vivo Detection of Spontaneous Immune Responses Female BALB / C mice (n = 5) with a 5'cap cap 1 for the purpose of distinguishing the importance of various chemical modifications of mRNA to in vivo protein production and in vivo cytokine responses. -Fullally modified with -CSF mRNA (G-CSF mRNA unmodified) (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence), 5-methylcytosine and pseudouridine. G-CSF mRNA with 5'cap, fully modified with G-CSF mRNA (G-CSF mRNA 5 mc / pU), 5-methylcytosine and N1-methyl-pseudouridine (G-CSF mRNA 5 mc / N1 pU) or A control that is either 5'uncapped G-CSF mRNA (G-CSF mRNA 5 mc / N1 pU uncapped) or R848 or 5% sucrose is injected intramuscularly as shown in Table 101. ..
Table 101. Dosing table

Blood is collected 8 hours after dosing. Using ELISA, protein levels of G-CSF, TNF-α, and IFN-α are determined by ELISA. Eight hours after dosing, muscle is harvested from the injection site and intramuscularly RIG-I, PKR, AIM-2, IFIT-1, OAS-2, MDA- using quantitative real-time polymerase chain reaction (QPCR). 5. Determine the mRNA levels of IFN-β, TNF-α, IL-6, G-CSF, CD45.

Example 67. In vivo Detection Study of Spontaneous Immune Response In female BALB / C mice (n = 5), G-CSF mRNA with a 5'cap of cap 1 (G-CSF mRNA unmodified) (mRNA sequence is shown in SEQ ID NO: 21438; A poly A tail of approximately 160 nucleotides is not shown in the sequence), G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine (G-CSF mRNA 5 mc / pU), 5-methylcytosine and N1-methyl. -G-CSF mRNA with a 5'cap fully modified with pseudouridine (G-CSF mRNA 5 mc / N1 pU) or G-CSF mRNA without a 5'cap (G-CSF mRNA 5 mc / N1 pU without cap), Alternatively, a control, either R848 or 5% sucrose, was injected intramuscularly as shown in Table 102. Blood was collected 8 hours after dosing and the protein levels of G-CSF and interferon-α (IFN-α) were determined by ELISA using ELISA and are shown in Table 102.

As shown in Table 102, unmodified 5 mc / pU and 5 mc / N1 pU modified G-CSF mRNA resulted in human G-CSF expression in mouse serum. Uncapped 5mC / N1pU-modified G-CSF mRNA does not show human G-CSF expression in serum, coordinating that having a 5'cap structure is important for protein translation.

As expected, only R848, 5% sucrose, and no human G-CSF protein was expressed in the untreated group. Importantly, there were significant differences in cytokine production as measured by mouse IFN-α in serum. As expected, the unmodified G-CSF mRNA showed a strong cytokine response in vivo (higher than the R848 positive control). 5 mc / pU unmodified G-CSF mRNA showed a low but detectable cytokine response in vivo, whereas 5 mc / N1 pU modified mRNA did not show detectable IFN-α in serum (vehicle and unmodified). The same was true for processed animals).

Moreover, the response of 5mc / N1pU modified mRNA was the same with or without a cap. These in vivo results are: 1) unmodified mRNA produces a strong innate immune response, 2) this is reduced by 100% incorporation of 5 mc / pU modification, but not abolished, and 3) 5 mc / It reinforces the conclusion that the incorporation of N1pU modifications does not result in a detectable cytokine response.

Finally, given that these injections are in 5% sucrose (which itself has no effect), these results should accurately reflect the immunostimulatory potential of these modifications. Is.

From this data, it is clear that the N1pU modified molecule produces more protein but has little or no effect on IFN-α expression. For this chemical modification, it is also clear that capping is required for protein production. The protein: cytokine ratio of 748 compared to the PC ratio of unmodified mRNA (PC = 9) means that this chemical modification is far superior in terms of action or biological significance associated with IFN-α. do.
Table 102. Human G-CSF and mouse IFN-α in serum

Example 68: In vivo Delivery of Modified RNA Protein production of modified mRNA was evaluated by delivering modified G-CSF mRNA or modified Factor IX mRNA to female Prague dolly rats (n = 6). Fully modified G-CSF mRNA in 100 μL of 400 μg 5-methylcytosine and pseudouridine reconstructed from lyophilized form in 5% sucrose in rats (mRNA sequence is shown in SEQ ID NO: 21438; about The 160 nucleotide poly A tail is not shown in the sequence; G-CSF mRNA (G) fully modified with 5'cap, cap 1) (G-CSF Gen1), 5-methylcytosine and N1-methyl-pseudouridine. -CSF Gen2), or factor IX mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5' Cap, cap 1) (IX factor Gen1) was injected. Blood was collected 8 hours after infusion and serum G-CSF protein levels were measured by ELISA. Table 103 shows the levels of G-CSF protein in serum after 8 hours.

These results show that both G-CSF Gen1 and G-CSF Gen2-modified mRNAs can result in human G-CSF protein in rats after a single intramuscular injection, and that human G-CSF protein production is a Gen1 chemical composition. It is shown that it is improved when the Gen2 chemical composition is used.
Table 103. G-CSF protein in rat serum (IM injection route)

Example 69. Chemical modification: In vitro studies A. In vitro screening in PBMCs Fully modified with the chemical modifications outlined in Tables 104 and 105, 500 ng of G-CSF (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is shown in the sequence. Not; 5'cap, cap 1) mRNA was transfected into peripheral blood mononuclear cells (PBMC) from 3 normal blood donors using 0.4 μL Lipofectamine 2000. Control samples LPS, R848, P (I) P (C), and mCherry (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence, 5'cap, cap 1 Completely modified with 5-methylcytosine and pseudouridine) were also analyzed. Supernatants were collected and cryopreserved until analyzed by ELISA to determine G-CSF protein expression and induction of cytokine interferon α (IFN-α) and tumor necrosis factor α (TNF-α). The protein expression of G-CSF is shown in Table 104, and the expression of IFN-α and TNF-α is shown in Table 105.

The data in Table 104 show that many, but not all, chemical modifications can be used for the productive production of human G-CSF in PBMCs. Notably, 100% N1-methyl-pseudouridine substitution shows the highest levels of human G-CSF production (almost 10-fold higher than pseudouridine itself). The use of N1-methyl-pseudouridine in combination with 5-methylcytidine also produces high levels of human G-CSF protein (also higher than the combination of pseudo-pseudouridine with 5-methylcytidine).

Considering the inverse correlation between protein production and cytokines in PBMCs, a similar tendency was confirmed in Table 105, where 100% substitution with N1-methyl-pseudouridine did not result in any cytokine induction (transfection). Pseudouridine exhibits higher detectable cytokine induction than the background only).

表１０５．化学修飾およびサイトカイン発現

Other modifications such as N6-methyladenosine and α-thiocitidine appear to increase cytokine stimulation.
Table 104. Chemical modification and G-CSF protein expression

Table 105. Chemical modification and cytokine expression

B. In vitro screening in HeLa cells The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY) and 96. In well cell culture plates (Corning, Manassas, VA), each well was inoculated into 100 μL of EMEM medium (supplemented with 10% FCS and 1-fold Glutamax). Cells were grown at 37 oG overnight in a 5% CO _{2 atmosphere.} The next day, 83 ng of luciferase-modified RNA with the chemical modifications set forth in Table 106 (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1). It was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY).

Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were combined and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The volume of lysate was adjusted or diluted until no more than 2 million relative luminescence (RLU) per well was detected for the sample producing the strongest signal, and each chemical composition tested is shown in Table 106. The plate reader is BioTek Synergy
It was H1 (BioTek, Winooski, VT). The background signal of the reagent-free plate was about 200 relative luminescence per well.

These results indicate that many, but not all, chemical modifications can be used for the productive production of human G-CSF in HeLa cells. Notably, 100% N1-methyl-pseudouridine substitutions show the highest levels of human G-CSF production.
Table 106. Relative luminescence of luciferase

C. In vitro screening of rabbit reticulocyte lysates The luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) are listed in Table 107. It was modified with modifications and diluted to a final amount of 250 ng in 10 μL in sterile nuclease-free water. Diluted luciferase was added to 40-k of freshly prepared rabbit reticulocyte lysate and an in vitro translation reaction was performed in a dry heating block at 30 ° C. in a standard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific, Waltham, Waltham, MA) I went inside. Translation assays were performed using the Rabbit Reticulocyte Lysate (nuclease treatment) kit (Promega, Madison, WI) according to the manufacturer's instructions. The reaction buffer is supplemented with a one-to-one mixture of the provided amino acid stock solutions deficient in either leucine or methionine, resulting in an effective in vitro translation of the reaction mixture containing sufficient amounts of both amino acids. I made it possible to do.

After 60 minutes of incubation, the reaction was stopped by placing the reaction tube on ice. Aliquots of the in vitro translation reaction containing luciferase-modified RNA were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The volume of the in vitro translation reaction was adjusted or diluted until no more than 2 million relative luminescence (RLU) per well was detected for the sample producing the strongest signal, and the RLU of each chemical composition tested is shown in Table 107. Shown in. The plate reader was BioTek Synergy H1 (BioTek, Winooski, VT). The background signal of the reagent-free plate was about 200 relative luminescence per well.

The results of these cell-free translations correlate very well with the results of protein production in HeLa cells, and the same modifications usually work or do not work in any system. One notable exception was 5-formylcytidine-modified luciferase mRNA, which worked in cell-free translation systems but not in HeLa cell-based transfection systems. Similar differences between the two assays were found with 5-formylcytidine-modified G-CSF mRNA.
Table 107. Relative luminescence of luciferase

Example 70. Chemical modification: In vivo studies A. In vivo Screening of G-CSF Modified mRNA Balb-C mice (n = 4) were fully modified with the chemical modifications outlined in Table 108 and formulated in 1-fold PBS (modified G-CSF mRNA). The mRNA sequence is shown in SEQ ID NO: 21438; the poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) is injected intramuscularly into each leg. Control luciferase-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with pseudouridine and 5-methylcytosine) And PBS controls are also tested. After 8 hours, serum is collected and ELISA is used to determine G-CSF protein level and cytokine level.
Table 108. G-CSF

B. In vivo Screening of Luciferase-Modified mRNA 42-103 μg of modified luciferase mRNA (mRNA) formulated in 1-fold PBS fully modified with the chemical modifications outlined in Table 109 in Balb-C mice (n = 4). The sequence is shown in SEQ ID NO: 21446; a poly A tail of about 160 nucleotides is not shown in the sequence; 200 μL containing 5'cap, cap 1) was subcutaneously injected. PBS controls were also tested. Dosings of modified luciferase mRNA are also outlined in Table 109. Eight hours after dosing, mice were imaged to determine luciferase expression. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice.

As shown in Table 109, with the exception of 2'-fluorouridine, all luciferase mRNA-modified chemical compositions showed in vivo activity. In addition, the 1-methylpseudouridine modified mRNA showed very high luciferase expression (5-fold higher expression than the pseudopseudouridine-containing mRNA).
Table 109. Luciferase screening

Example 71. In vivo Screening of Combination Luciferase-Modified mRNA 100 μg of modified luciferase mRNA (mRNA) formulated in 1-fold PBS fully modified with the chemical modifications outlined in Table 110 in Balb-C mice (n = 4). The sequence is shown in SEQ ID NO: 21446; a poly A tail of about 160 nucleotides is not shown in the sequence; 200 μL of 5'cap, cap 1) was subcutaneously injected. PBS controls were also tested. Eight hours after dosing, mice were imaged to determine luciferase expression. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice.

As shown in Table 110, all luciferase mRNA modified chemical compositions (in combination) showed in vivo activity. In addition, the presence of N1-methyl-pseudouridine in the modified mRNA (with N4-acetylcytidine or 5-methylcytidine) showed higher expression than the same combination tested with those with pseudouridine. Taken together, these data show whether N1-methyl-pseudouridine-containing luciferase mRNA is used alone (Table 109) or in combination with other modified nucleotides (Table 110). It is shown to result in improved protein expression in vivo.
Table 110. Combination of luciferase screening

Example 72. Spontaneous immune response in BJ fibroblasts Human primary capsule fibroblasts (BJ fibroblasts) were obtained from the American Type Culture Collection (ATCC) (Cat. No. CRL-2522) and 10% at 37 ° C. under 5% CO2. The cells are grown in Eagle's Minimal Essential Medium (ATCC, Catalog Nos. 30-2003) supplemented with fetal bovine serum. BJ fibroblasts are seeded in 24-well plates at a cell density of 130,000 per well in 0.5 mL culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence) completely modified with 5-methylcytosine and pseudouridine (Gen1) or fully modified with 5-methylcytosine and N1-methyl-pseudouridine (Gen2) Is shown in SEQ ID NO: 21438; a polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) using Lipofectamine 2000 (Invitrogen, Catalog No. 11668-019) according to the manufacturer's protocol. Transfect. Control samples Lipofectamine 2000 and unmodified G-CSF mRNA (natural G-CSF) are also transfected. Cells are transfected for 5 consecutive days. The transfection complex is removed at 4 hours of each transfection.

Culture supernatants are assayed daily by ELISA according to the manufacturer's protocol for secreted G-CSF (R & D Systems, Catalog DCS50), tumor necrosis factor α (TNF-α), and interferon α (IFN-α). Cells are analyzed for viability using CELL TITER GLO® (Promega, Catalog No. G7570) 6 and 18 hours after initial transfection, and every other day thereafter. At the same time, total RNA is isolated and treated with DNASE® using the RNAEASY microkit (Cat. No. 74004) according to the manufacturer's protocol. 100 ng of total RNA is treated with the High Capacity cDNA Reverse Transfection Kit according to the manufacturer's protocol. (Applied Biosystems, Catalog No. 4368814) is used for cDNA synthesis. The cDNA is then analyzed for expression of spontaneous immune response genes by quantitative real-time PCR using SibrGreen in a Biorad CFX 384 instrument according to the manufacturer's protocol. ..

Example 73. In vitro transcription luciferase mRNA by wild-type T7 polymerase (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) and G-CSF mRNA (mRNA sequence is Shown in SEQ ID NO: 21438; a poly A tail of about 160 nucleotides is not shown in the sequence; 5'caps, caps 1) are different listed in Tables 111-114 using wild T7 polymerase as described above. Completely modified with a chemical composition and a combination of chemical compositions.

The yield of the translation reactant was determined by spectrophotometric measurement (OD260), the yield of luciferase is shown in Table 111, and G-CSF is shown in Table 113.

表１１２．ルシフェラーゼ修飾ｍＲＮＡのキャッピング化学組成および収量

表１１３．Ｇ−ＣＳＦ修飾ｍＲＮＡのインビトロ転写化学組成および収量

表１１４．Ｇ−ＣＳＦ修飾ｍＲＮＡのキャッピング化学組成および収量

Luciferase and G-CSF modified mRNA were subjected to an enzyme capping reaction, the capping reaction of each modified mRNA was evaluated for yield by spectrophotometric measurement (OD260), and the exact size was evaluated using a bioanalyzer. The yield of luciferase by capping reaction is shown in Table 112, and G-CSF is shown in Table 114.
Table 111. In vitro transcriptional chemical composition of luciferase

Table 112. Capping chemical composition and yield of luciferase-modified mRNA

Table 113. In vitro transcriptional chemical composition and yield of G-CSF modified mRNA

Table 114. Capping chemical composition and yield of G-CSF modified mRNA

Example 74. In vitro transcription luciferase mRNA by mutant T7 polymerase (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) and G-CSF mRNA (mRNA sequence is Shown in SEQ ID NO: 21438; Poly A tail of approximately 160 nucleotides is not shown in the sequence; 5'cap, cap 1) with variant T7 polymerase (Durascribe® T7 Transcription Kit (Cat. No. DS010925) (Epicentre). (Registered Trademarks), Madison, WI) were fully modified with different chemical compositions and combinations of chemical compositions listed in Tables 115-118.

The yield of translational reactants was determined by spectrophotometric measurement (OD260), the yield of luciferase is shown in Table 115, and the G-CSF is shown in Table 117.

表１１６．ルシフェラーゼ修飾ｍＲＮＡのキャッピング化学組成および収量

表１１７．Ｇ−ＣＳＦ修飾ｍＲＮＡインビトロ転写化学組成および収量

表１１８．Ｇ−ＣＳＦ修飾ｍＲＮＡのキャッピング化学組成および収量

Luciferase and G-CSF modified mRNA were subjected to an enzyme capping reaction, the capping reaction of each modified mRNA was evaluated for yield by spectrophotometric measurement (OD260), and the exact size was evaluated using a bioanalyzer. The yield of luciferase from the capping reaction is shown in Table 116, and G-CSF is shown in Table 118.
Table 115. In vitro transcriptional chemical composition and yield of luciferase-modified mRNA

Table 116. Capping chemical composition and yield of luciferase-modified mRNA

Table 117. G-CSF Modified mRNA In Vitro Transcription Chemical Composition and Yield

Table 118. Capping chemical composition and yield of G-CSF modified mRNA

Example 75.2'O-methyl and 2'fluoro compound luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1). It was generated as a fully modified form with the chemical composition of Table 119 and transcribed using a mutant T7 polymerase (Durascribe® T7 Transcription Kit (Cat. No. DS010925) (Epicentre®, Madison, WI). . 2'fluoro-containing mRNA was prepared using Durascribbe T7, but 2'O-methyl-containing mRNA could not be transcribed using Durascribbe T7.

Integration of 2'O methyl-modified mRNA may be achieved using other mutant T7 polymerases (Nat Biotechnol. (2004), each of which is incorporated herein by reference in its entirety. ) 22: 1155-1160, Nuclear Acids Res. (2002) 30: e138), or US Pat. No. 7,309,570). Alternatively, the 2'OMe modification can be introduced post-transcriptional using enzymatic means.

The introduction of modifications to the 2'group of sugars has a number of potential advantages. 2'OMe substitutions, such as 2'fluoro substitutions, are known to protect against nucleases and have been shown to abolish innate immune recognition when incorporated into other nucleic acids such as siRNA and antisense. (entirely ^{incorporated, Crooke, ed.Antisense Drug Technology, 2} nd edition; Boca Raton: CRC press).

HeLa cells were then transfected with 2'fluoromodified mRNA to assess protein production in the cellular environment and the same mRNA in cell-free rabbit reticulocytes. Unmodified luciferase (native luciferase) controls were used in both transcription experiments, untreated or quasi-transfected (Lipofectamine 2000 alone) controls were also evaluated for HeLa transfection, and RNA-free controls were used for rabbit reticular site. analyzed.

For HeLa transfection experiments, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with Tripsin-EDTA solution (LifeTechnologies, Grand Island, NY) the day before transfection. , 96-well cell culture plates (Corning, Manassas, VA) were inoculated into 100 μL EMEM medium (supplemented with 10% FCS and 1x Glutamax) for each well. Cells were grown at 37 oG overnight in a 5% CO _{2 atmosphere.} The next day, 83 ng of 2'fluoro-containing luciferase-modified RNA with the chemical modifications set forth in Table 119 (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap. 1) was diluted with 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were combined and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature. After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The volume of lysate was adjusted or diluted until no more than 2 million luminescence per well was detected for the sample producing the strongest signal, and the RLU for each chemical composition tested is shown in Table 119. The plate reader was BioTek Synergy H1 (BioTek, Winooski, VT). The background signal of the reagent-free plate was about 200 relative luminescence per well.

For the rabbit reticulocyte lysate assay, 2'-fluoro-containing luciferase mRNA was diluted to a final amount of 250 ng in 10 μL in sterile nuclease-free water and added to 40 μL of freshly prepared rabbit reticulocyte lysate for in vitro translation. The reaction was carried out in a dry heating block at 30 ° C. in a standard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific, Walsam, MA). Translation assays were performed using the Rabbit Reticulocyte Lysate (nuclease treatment) kit (Promega, Madison, WI) according to the manufacturer's instructions. The reaction buffer is supplemented with a one-to-one mixture of the provided amino acid stock solutions deficient in either leucine or methionine, resulting in an effective in vitro translation of the reaction mixture containing sufficient amounts of both amino acids. I made it possible to do. After 60 minutes of incubation, the reaction was stopped by placing the reaction tube on ice.

Aliquots of the in vitro translation reaction containing luciferase-modified RNA were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The amount of in vitro translation reactants was adjusted or diluted until no more than 2 million relative luminescence (RLU) per well was detected for the sample producing the strongest signal, and the RLU for each chemical composition is shown in Table 120. .. The plate reader was BioTek Synergy H1 (BioTek, Winooski, VT). The background signal of the reagent-free plate was about 160 relative luminescence per well.

表１２０．ウサギ網状ライセート

As can be seen in Tables 119 and 120, multiple 2'fluoro-containing compounds are active in vitro and produce luciferase proteins.
Table 119. HeLa cells

Table 120. Rabbit reticulated lysate

Example 76. Luciferase in HeLa cells with a combination of modifications To assess the use of 2'fluoro-modified mRNA in combination with other modifications, a series of mRNAs were added to the wild-type T7 polymerase (fluoro) as described in Example 75. It was transcribed using either a free compound) or a mutant T7 polymerase (a fluoro-containing compound). All modified mRNAs were tested by in vitro transfection in HeLa cells.

The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY) and collected in 96-well cell culture plates (ATCC No. CCL-2; Manassas, VA). In Corning, Manassas, VA), each well was seeded in 100 μL of EMEM medium (supplemented with 10% FCS and 1x Glutamax). Cells were grown at 37 oG overnight in a 5% CO _{2 atmosphere.} The next day, 83 ng of luciferase-modified RNA with the chemical modifications set forth in Table 121 (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1). It was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were combined and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The volume of lysate was adjusted or diluted until no more than 2 million luminescence per well was detected for the sample producing the strongest signal, and the RLU for each chemical composition tested is shown in Table 121. The plate reader is BioTek Synergy
It was H1 (BioTek, Winooski, VT). The background signal of the reagent-free plate was about 200 relative luminescence per well.

As is clear from Table 121, most combinations of modifications, including combinations containing all fluorofree compounds and numerous 2'fluoro modifications, resulted in the production of functional luciferase proteins by mRNA.
Table 121. Luciferase

Example 77. G-CSF In vitro Transcription In connection with the second open reading frame, we reproduced the experiments already performed with luciferase mRNA, human G-CSF mRNA, to assess the activity of all the different chemical modifications. G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1), wild T7 polymerase (all fluorofree compounds) Alternatively, it was completely modified with the chemical compositions of Tables 122 and 123 using mutant T7 polymerase (all fluoro-containing compounds). Mutant T7 polymerase is commercially available (Durascribe® T7 Transcription Kit (Cat. No. DS010925) (Epicenter®, Madison, WI).

The modified RNAs in Tables 122 and 123 were transfected into HeLa cells in vitro or added to rabbit reticular lysate (250 ng of modified mRNA) as shown. Control with untreated, pseudotransfect (transfection reagent only), G-CSF fully modified with 5-methylcytosine and N1-methyl-pseudouridine, or with 5-methylcytosine and N1-methyl-pseudouridine. A fully modified luciferase control (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) was also analyzed. Expression of the G-CSF protein was determined by ELISA and the values are shown in Tables 122 and 123. In Table 122, "NT" means untested.

As shown in Table 123, a number of, but not all, chemical modifications resulted in human G-CSF protein production. These results from cell-based and cell-free translation systems correlate very well, and the same modifications usually work or do not work in either system. One notable exception was 5-formylcytidine-modified G-CSF mRNA, which worked in cell-free translation systems but not in HeLa cell-based transfection systems. Similar differences between the two assays were found with 5-formylcytidine-modified luciferase mRNA.

表１２３．ＨｅＬａ細胞における組み合わせ化学組成

As shown in Table 123, a large number, but not all, of G-CSF mRNA-modified chemical compositions (when used in combination) showed in vivo activity. In addition, the presence of N1-methyl-pseudouridine in the modified mRNA (with N4-acetylcytidine or 5-methylcytidine) showed higher expression than the same combination tested with those with pseudouridine. Taken together, these data indicate that N1-methyl-pseudouridine-containing G-CSF mRNA results in improved protein expression in vitro.
Table 122. G-CSF expression

Table 123. Combined chemical composition in HeLa cells

Example 78. Chemical Composition Screening The tables listed below (Tables 124-126) summarize most of the in vitro and in vitro screening data using the various compounds presented in the previous examples. A good correlation exists between the cell-based translation assay and the cell-free translation assay. The same chemical substitutions usually show good agreement regardless of whether they have been tested for luciferase or G-CSF mRNA. Finally, N1-methyl-pseudouridine-containing mRNA exhibits very high levels of protein expression both in vitro and in vivo with little or no detectable cytokine stimulation, both in vitro and in vivo. Is superior to mRNA containing.

Luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1) and G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; A poly A tail of about 160 nucleotides is not shown in the sequence; 5'caps, caps 1) are modified with natural or non-natural chemical compositions shown in Tables 124 and 125, or a combination of chemical compositions shown in Table 126. And tested using the methods described herein.

表１２５．非天然

In Tables 125 and 126, ^"*" refers to in vitro transcription reaction using the mutant T7 polymerase (Durascribe (TM) T7 Transcription Kit (Cat. No. DS010925) (Epicentre (TM), Madison, WI); ^{"* * "mutant} T7 polymerase (Durascribe (TM) T7 transcription kit (Cat. No. DS010925) (Epicentre (TM), Madison, refers to a second result of the in vitro transcription reaction using ^{WI);" ***} the protein production HeLa, "+", "+/-", and "- checked by";^"cell-free translation refers to the production seen in (rabbit reticulocyte lysate) for G-CSF PBMC mentioned In this case, "++++" means G-CSF exceeding 6,000 pg / mL, "++++" means G-CSF exceeding 3,000 pg / mL, and "++" means 1,500 pg / mL. "+" Means G-CSF greater than 300 pg / mL, "+/-" means G-CSF greater than 150-300 pg / mL, and background is approximately 110 pg. Was / mL; when referring to the cytokine PBMC, "++++" means interferon α (IFN-α) above 1,000 pg / mL and "++++" means IFN-α above 600 pg / mL. , "++" means more than 300 pg / mL IFN-α, "+" means more than 100 pg / mL IFN-α, "-" means less than 100 pg / mL, background Was about 70 pg / mL; as well as "NT" means untested. In Table 125, protein production was shown in the variant T7 polymerase (Durascribe® T7 Transcription Kit (Cat. No. DS010925)) (Epicentre®. Evaluated using Trademarks), Madeson, WI).
Table 124. Natural

Table 125. Non-natural

In Table 126, HeLa protein production is determined by "+", "+/-", and "-"; when referring to G-CSF PBMC, "++++" is greater than 6,000 pg / mL G-CSF. "+++" means G-CSF exceeding 3,000 pg / mL, "++" means G-CSF exceeding 1,500 pg / mL, and "+" means G-CSF exceeding 300 pg / mL. -CSF means -CSF, "+/-" means 150-300 pg / mL G-CSF, background was about 110 pg / mL; when referring to the cytokine PBMC, "++++" means 1,000 pg "++" means interferon α (IFN-α) above / mL, "+++" means IFN-α above 600 pg / mL, "++" means IFN-α above 300 pg / mL, and "+" Means IFN-α above 100 pg / mL, "-" means less than 100 pg / mL, and the background was about 70 pg / mL; "WT" refers to wild-type T7 polymerase, "MT""Refers to the variant T7 polymerase (Durascribe® T7 Transcriction Kit (Cat. No. DS010925) (Epicentre®, Madison, WI)", where "NT" means untested.
Table 126. Combination chemical composition

Example 79. Innate immune response of 2'fluorochemical G-CSF modified mRNA in PBMC (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, not shown in cap 1) The ability to induce is determined by measuring interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α) production. The use of in vitro PBMC cultures is a common method for measuring the immunostimulatory potential of oligonucleotides (Robbins et al., Oligonucleotides 2009 19: 89-102), and transfection methods are described herein. Table 127 shows the average of interferon-α (IFN-α) and tumor necrosis factor α (TNF-α) production over time as measured by specific ELISA from 2 or 3 separate PBMC donors. Control R848, P (I) P (C), LPS, and Lipofectamine 2000 (L2000) were also analyzed.

For innate immune recognition, both modified mRNA chemical compositions significantly inhibited IFN-α and TNF-α production compared to positive controls (R848, P (I) P (C)), but 2'fluoro compounds. Reduced IFN-α and TNF-α production lower than the other combinations, and the N4-acetylcitidine combination increased the cytokine profile.
Table 127. IFN-α and TNF-α

Example 80. Modified mRNA with tobacco etch virus 5'UTR
The 5'untranslated region (UTR) can be provided as an adjacent region. Multiple 5'UTRs can be contained in adjacent regions and can be the same or different sequences. Any part of the contiguous region (including any part that does not include the contiguous region) can be codon-optimized, and any one of them can independently be one or more before and / or after codon optimization. It may contain different structures or chemical modifications.

The 5'UTR may include the 5'UTR from the Tobacco Etch Virus (TEV). Variants of the 5'UTR can be utilized in which one or more nucleotides, including A, T, C, or G, are added to or removed from the ends.

Example 81. Expression of PLGA-formulated mRNA A. Synthesis and Characteristics of Luciferase PLGA Microspheres 25% with 5-methylcytosine and 25% with 2-thiouridine-substituted uridine and 25% with 5-methylcytosine-substituted cytosine, either fully modified with 5-methylcytosine and N1-methyl-pseudouridine A% modified, fully modified N1-methyl-pseudouridine, or fully modified pseudouridine, luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of approximately 140 nucleotides is sequenced. Not shown within; 5'cap, cap 1) was reconstituted in 1x TE buffer and then formulated in PLGA microspheres. PLGA microspheres, PLGA ester cap (Lactel, catalog number B6010-2, intrinsic viscosity 0.55 to 0.75, LA: GA at 50:50), polyvinyl alcohol (PVA) (Sigma, catalog number 348406-25G, MW 13-23k), dichloromethane, and water were used to synthesize using the water / oil / water double emulsification method known in the art. Briefly, 0.4 mL of mRNA in 4 mg / mL TE buffer (W1) was added to 2 mL of PLGA dissolved in dichloromethane (DCM) (O1) at a concentration of 200 mg / mL PLGA. The W1 / O1 emulsion was homogenized at a rate of 5 (about 19,000 rpm) for 30 seconds (IKA Ultra-Turrax Homogenizer, T18). The W1 / O1 emulsion was then added to 250 mL of 1% PVA (W2) and homogenized at a rate of 5 (about 19,000 rpm) for 1 minute. The pharmaceutical product was stirred as it was for 3 hours, then passed through a 100 μm nylon mesh sieve (Fisherbrand Cell Strainer, Catalog No. 22-363-549) and finally washed by centrifugation (10 minutes, 9,250 rpm, 4 ° C.). The supernatant was discarded and the PLGA pellets were resuspended in 5-10 mL of water, which was repeated twice. After washing with water and resuspending, 100-200 μl of PLGA microspheres sample was used to measure the particle size of the pharmaceutical by laser diffraction (Marvern Mastersizer 2000). The washed formulation was frozen in liquid nitrogen and then lyophilized for 2-3 days.

After lyophilization, approximately 10 mg of PLGA MS was weighed into a 2 mL Eppendorf tube, deformated by adding 1 mL of DCM, and the sample was shaken for 2-6 hours. The mRNA was extracted from deformated PLGA microspheres by adding 0.5 mL of water and the sample was shaken overnight. For use as a control in a transfection assay, unformulated luciferase mRNA in TE buffer (non-formulated control) was added to the DCM and subjected to the deformation process (deformation control). The encapsulation efficiency, percentage of weight to be filled, and particle size are shown in Table 128. Encapsulation efficiency was calculated as mg of mRNA from PLGA microsphere deformation divided by the initial amount of mRNA added to the formulation. The weight percent filled in the formulation was calculated as mg of mRNA from the deformation of PLGA microspheres divided by the initial amount of PLGA added to the formulation.
Table 128. PLGA characteristics

B. Protein expression of modified mRNA encapsulated in PLGA microspheres The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were subjected to trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). ), And seeded in 96-well cell culture plates (Corning, Manassas, VA) in 100 μL of EMEM medium (supplemented with 10% FCS and 1x Glutamax) per well. Cells were grown overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, 83 ng of de-formulated luciferase mRNA PLGA microsphere sample, de-formulated luciferase mRNA control (de-formulated control), or non-formulated luciferase mRNA control (non-formulated control) in a final volume of 10 μL OPTI-MEM. It was diluted in (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. Then, 20 μL of the combined solution was added to 100 μL of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The background signal of the reagent-free plate was about 200 relative luminescence per well. The plate reader was BioTek Synergy H1 (BioTek, Winooski, VT).

Cells are collected and the bioluminescence (relative luminescence, in RLU) for each sample is shown in Table 129. Transfection of these samples confirmed that the various chemical compositions of luciferase mRNA were still capable of expressing the luciferase protein after PLGA microsphere formulation.
Table 129. Chemical modification

Example 82. In vitro study of Factor IX A. Serum-free medium Human Factor IX mRNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; completely with 5-methylcytosine and pseudouridine Transfected into (modified) serum-free medium. Cell culture supernatants were harvested and subjected to trypsin digestion prior to two-dimensional HPLC separation of the peptides. Peptides were detected using matrix-assisted laser desorption / ionization. Eight peptides were detected, and seven of the detected peptides were specific to Factor IX. This result indicates that the mRNA transfected into the serum-free medium was able to express the full-length factor IX protein.

B. Codon-optimized human factor IX mRNA (mRNA sequence shown in SEQ ID NO: 1622; fully modified with 5-methylcytosine and pseudouridine; polyA tail of approximately 160 nucleotides) of 250 ng of human fetal kidney (HEK) 293A cells Is not shown in the sequence; 5'cap, cap 1) was transfected into HEK293A cells (150,000 cells / well) using Lipofectamine 2000 in DMEM in the presence of 10% FBS. Three hours after transfection, the transfection complex was removed. Cells were collected 3, 6, 9, 12, 24, 48, and 72 hours after transfection. Total RNA was isolated and used for cDNA synthesis. The cDNA was subjected to quantitative real-time PCR analysis using a codon-optimized Factor IX-specific primer set. Human hypoxanthine phosphoribosyl transferase 1 (HPRT) levels were used for normalization. The data are plotted as the percentage of detectable mRNA and the mRNA level is considered to be 100% at 3 hours. The half-life of factor IX-modified mRNA fully modified with 5-methylcytosine and pseudouridine in human fetal kidney 293 (HEK293) cells is approximately 8-10 hours.

Example 83. Physiological saline preparation: Subcutaneous administration Human G-CSF modified mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and Fully modified with pseudouridine) and human EPO-modified mRNA (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and pseudo Completely modified with uridine) was formulated in physiological saline and delivered to mice via intramuscular (IM) infusion at a dose of 100 μg.

In control, luciferase (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine. ) Or its formulation buffer (F. Buffer). Blood was collected from mice 13 hours after injection to determine the concentration of human polypeptide in serum in pg / mL units. (In the G-CSF group, human G-CSF in mouse serum was measured, and in the EPO group, human EPO in mouse serum was measured). The data are shown in Table 130.

When not formulated, the mRNA is rapidly degraded in serum, suggesting that the best way to deliver a longer-lasting mRNA in the system is by formulating the mRNA. As shown in Table 130, mRNA may be delivered subcutaneously using only a buffered formulation.
Table 130. Dosing regimen

Example 84. Intravivical delivery mCherry-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21439; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine , And luciferase-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine. Was formulated in physiological saline and delivered into the rat vitreous as described in Table 131. Samples were compared to saline-only controls delivered intravitreal.
Table 131. Dosing chart

On day 1, the drug is administered to the left or right eye of each animal while the animal is being anesthetized. The day before administration, gentamicin intraocular ointment or solution was applied twice to both eyes. Gentamicin intraocular ointment or solution was applied immediately after infusion and the day after infusion. Prior to dosing, mydriatic eye drops (1% tropicamide and / or 2.5% phenylephrine) are applied to each eye.

Eighteen hours after dosing, eyes that received the mCherry dose and delivery buffer were removed and placed separately in tubes containing 10 mL of 4% paraformaldehyde for tissue fixation overnight at room temperature. The next day, the eyes were separately transferred to tubes containing 10 mL of 30% sucrose and stored at 21 ° C. until they were processed and sectioned. Slides made from different sections were evaluated by F-microscopy. Slides made from eyes treated with mCherry-modified mRNA showed positive expression and controls did not.

Example 85. In vivo cytokine expression studies 5'cap, unmodified with cap 1 (unmodified), 5-methylcytosine and pseudouridine and 5'cap, fully modified with cap 1 (Gen1), or 5-methylcytosine And N1-methyl-pseudouridine and 5'cap, fully modified with cap 1 (Gen2 cap) or uncapped (not Gen2 cap), or 200 μg of G-CSF modified mRNA (mRNA sequence is in SEQ ID NO: 21438). Mice were intramuscularly injected (shown; about 160 nucleotides of polyA tail not shown in the sequence). Control R-848, 5% sucrose, and untreated mice were also analyzed. After 8 hours, sera were collected from mice and analyzed for interferon-α (IFN-α) expression. The results are shown in Table 132.
Table 132. IFN-α expression

Example 86. In vitro expression of VEGF-modified mRNA Modified mRNA (m mRNA) VEGF-A (mRNA sequence is SEQ ID NO: 1672) complexed with HEK293 cells at the concentrations shown in Table 133 using Invitrogen (Carlsbad, CA) Lipofectamine 2000. Shown in; a polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) was transfected. Protein expression was detected by ELISA and the protein (pg / mL) is shown in Table 133 and FIG.
Table 133. Protein expression

Example 87. In vitro screening of GFP in HeLa cells The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Then, in 96-well cell culture plates (Corning, Manassas, VA), each well was inoculated into 100 μL of EMEM medium (supplemented with 10% FCS and 1-fold Glutamax). Cells were grown overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, 37.5 ng or 75 ng of green fluorescent protein (GFP) modified RNA with the chemical modifications listed in Table 134 (mRNA sequence is shown in SEQ ID NO: 21448; poly A tail of about 160 nucleotides is not shown in the sequence. 5'caps, caps 1) were diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Green Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The central fluorescence intensity (MFI) for each chemical composition was determined and shown in Table 134.

This result demonstrates that GFP fully modified with N1-methyl-pseudouridine and 5-methylcytosine produces more protein in HeLa cells compared to other chemical compositions. In addition, higher doses of GFP administered to the cells resulted in higher MFI values.
Table 134. Average fluorescence intensity

Example 88. Homogenization Different luciferase mRNA solutions (shown in Table 135, where "X" refers to the solution containing that component) (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 160 nucleotides is shown in the sequence. Not evaluated; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), yield percent of different solutions, mRNA completeness by bioanalyzer, and mRNA protein expression by in vitro transfection. Was tested. As shown in Table 135, in water, 4 mg / mL 1x TE buffer, prepare the mRNA solution and to achieve a final mRNA concentration of 0.8 mg / mL, dichloromethane (DCM) or 200 mg / mL. Add to any of the DCMs containing mL of poly (lactic acid-co-glycolic acid) (PLGA) (Lactel, Catalog No. B6010-2, intrinsic viscosity 0.55-0.75, LA: GA at 50:50). bottom. Solutions requiring homogenization were homogenized at a rate of 5 (about 19,000 rpm) for 30 seconds (IKA Ultra-Turrax Homogenizer, T18). The mRNA samples in water, dichloromethane (dichloromethane), and poly (lactic acid-co-glycolic acid) (PLGA) were unrecoverable (NR). All samples except NR samples maintained mRNA integrity as determined by a bioanalyzer (Bio-rad Experion).

The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY) and collected in 96-well cell culture plates. In (Corning, Manassas, VA), each well was inoculated into 100 μL of EMEM medium (supplemented with 10% FCS and 1-fold Glutamax). Cells were grown overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, 250 ng of luciferase mRNA from a recoverable sample was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. Then, 20 μL of the combined solution was added to 100 μL of cell culture medium containing HeLa cells. The plates were then incubated as described above. Control luciferase mRNA (luciferase mRNA formulated in physiological saline) (control) and untreated cells (untreated) were also evaluated. Cells are collected and their bioluminescence mean (in photons / sec) (bioluminescence (p / s)) for each signal is also shown in Table 135. When analyzed, all recoverable samples showed activity of luciferase mRNA.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The background signal of the reagent-free plate was about 200 relative luminescence per well. The plate reader was BioTek Synergy H1 (BioTek, Winooski, VT).

Cells are collected and their bioluminescence average (relative luminescence, in RLU) (bioluminescence (RLU)) is also shown in Table 135. When analyzed, all recoverable samples showed activity of luciferase mRNA.
Table 135. solution

Example 89. Evaluation of TE buffer and water Luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; complete with 5'cap, cap 1; 5-methylcytosine and pseudouridine Was reconstituted in water or TE buffer as outlined in Table 136 and then formulated in PLGA microspheres. PLGA microspheres, PLGA (Lactel, catalog number B6010-2, intrinsic viscosity 0.55 to 0.75, LA: GA with 50:50), polyvinyl alcohol (PVA) (Sigma, catalog number 348406-25G, MW 13) -23k), dichloromethane, and water were used to synthesize using the water / oil / water double emulsification method known in the art. Briefly, 0.2-0.6 mL of mRNA in water or TE buffer (W1) at a concentration of 2-6 mg / mL is dissolved in dichloromethane (DCM) (O1) at a concentration of PLGA of 100 mg / mL. It was added to 2 mL of PLGA that had been allowed to grow. The W1 / O1 emulsion was homogenized at a rate of 5 (about 19,000 rpm) for 30 seconds (IKA Ultra-Turrax Homogenizer, T18). The W1 / O1 emulsion was then added to 250 mL of 1% PVA (W2) and homogenized at a rate of 5 (about 19,000 rpm) for 1 minute.

The pharmaceutical product was stirred as it was for 3 hours, then passed through a 100 μm nylon mesh sieve (Fisherbrand Cell Strainer, Catalog No. 22-363-549) and finally washed by centrifugation (10 minutes, 9,250 rpm, 4 ° C.). The supernatant was discarded and the PLGA pellets were resuspended in 5-10 mL of water, which was repeated twice. The washed formulation was frozen in liquid nitrogen and then lyophilized for 2-3 days. After lyophilization, approximately 10 mg of PLGA MS was weighed into a 2 mL Eppendorf tube, deformated by adding 1 mL of DCM, and the sample was shaken for 2-6 hours. The mRNA was extracted from deprepared PLGA microspheres by adding 0.5 mL of water and the sample was shaken overnight. For use as a control in a transfection assay, unformulated luciferase mRNA (deformated control) in water or TE buffer was added to the DCM and subjected to the deformation process.

The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY) and collected in 96-well cell culture plates. In (Corning, Manassas, VA), each well was inoculated into 100 μL of EMEM medium (supplemented with 10% FCS and 1-fold Glutamax). Cells were grown overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, 100 ng of deformated luciferase mRNA sample was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. Then, 20 μL of the combined solution was added to 100 μL of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The background signal of the reagent-free plate was about 200 relative luminescence per well. The plate reader was BioTek Synergy H1 (BioTek, Winooski, VT). To determine the activity of luciferase mRNA from each formulation, the relative luminescence (RLU) for each formulation was divided by the RLU of the appropriate mRNA deformation control (mRNA in water or TE buffer). Table 136 shows the activity of luciferase mRNA. The activity of luciferase mRNA in the PLGA microsphere preparation (formulation) was significantly improved by formulation in TE buffer as compared with water.
Table 136. pharmaceutical formulation

Example 90. Chemical modification on mRNA The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). In 96-well cell culture plates (Corning, Manassas, VA), each well was inoculated into 100 μL of EMEM medium (supplemented with 10% FCS and 1-fold Glutamax). Cells were grown overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, 83 ng of luciferase-modified RNA with the chemical modifications set forth in Table 137 (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1). It was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature.

After an incubation of 18-22 hours, cells expressing luciferase were lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI) according to the manufacturer's instructions. Aliquots of lysate were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, VA) and combined with 100 μL of complete luciferase assay solution (Promega, Madison, WI). The volume of lysate was adjusted or diluted until no more than 2 million relative luminescence (RLU) per well was detected for the sample producing the strongest signal, and the RLU for each chemical composition tested is shown in Table 137. show. The plate reader is BioTek
It was Synergy H1 (BioTek, Winooski, VT). The background signal of the reagent-free plate was about 200 relative luminescence per well.
Table 137. Chemical modification

表１３９．皮下投与

Example 91. Intramuscular and Subcutaneous Administration of Modified mRNA Completely modified with 5-methylcytosine and pseudouridine (5 mC / pU) formulated in PBS (pH 7.4) or 5-methylcytosine and N1-methyl-pseud Completely modified with uridine (5 mC / N1 mpU), completely modified with pseudouridine (pU), completely modified with N1-methyl-pseudouridine (N1 mpU), or replaced with 5-methylcytosine. 25% modified with cytosine and 25% with uridine substituted with 2-thiouridine (5 mC / s2U), luciferase-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of approximately 140 nucleotides is within the sequence. Not shown; 5'cap, cap 1) was administered to Balb-C mice intramuscularly or subcutaneously at a dose of 2.5 mg / kg. Mice were fed 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours for intramuscular delivery, and 2 hours, 8 hours, 24 hours, 48 for subcutaneous delivery. Imaging was taken at time, 72 hours, 96 hours, and 120 hours. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. The average total luminous flux (photons / sec) for intramuscular administration is shown in Table 138, and the average total luminous flux (photons / sec) for subcutaneous administration is shown in Table 139. The background signal was 3.79E + 05 (p / s). Peak expression for intramuscular administration was seen between 24 and 48 hours for all chemical compositions, and expression was still detected at 144 hours. For subcutaneous delivery, peak expression was seen between 2 and 8 hours, and expression was also detected at 72 hours.
Table 138. Intramuscular administration

Table 139. Subcutaneous administration

Example 92. Osmotic pump research Prior to implantation, osmotic pumps (ALZET® osmotic pump 2001D, DURECT Corp. Cupertino, CA) were added to 0.2 mL of 1x PBS (pH 7.4) (PBS filling pump) or 0.2 mL of luciferase-modified mRNA at 1 mg / mL in 1x PBS (pH 7.4) (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1; Fully modified with 5-methylcytosine and N1-methyl-pseudouridine) (luciferase-filled pump) is loaded and incubated overnight at 37 ° C. in 1-fold PBS (pH 7.4).

Either a PBS-filled pump or a luciferase-filled pump is subcutaneously implanted in a Balb-C mouse (n = 3), and imaging is performed at 2, 8, and 24 hours. As a control, either a PBS fill pump is subcutaneously implanted and mice are injected subcutaneously with luciferase modification in 1x PBS (PBS filling pump; SC luciferase), or mice are not implanted with a 1x PBS in 1x PBS. Luciferase-modified mRNA is injected subcutaneously (SC luciferase). The luciferase preparation is outlined in Table 140.
Table 140. Luciferase preparation

Example 93. External osmotic pump research External osmotic pump (ALZET® osmotic pump 2001D, DURECT Corp. Cupertino, CA) is 0.2 mL of 1x PBS (pH 7.4) (PBS filling pump) or 1x PBS ( 0.2 mL of luciferase-modified mRNA at 1 mg / mL in pH 7.4) (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1; 5- Fill in (fully modified with methylcytosine and N1-methyl-pseudouridine) (luciferase-filled pump) and incubated overnight at 37 ° C. in PBS (pH 7.4).

The formulation is administered to Balb-C mice (n = 3) using a catheter connected to an external PBS filling pump or a luciferase filling pump. Mice are imaged at 2, 8, and 24 hours. As a control, mice are injected subcutaneously with luciferase modifications in 1x PBS using an external PBS filling pump (PBS filling pump; SC luciferase), or mice are in 1x PBS without an external pump. Luciferase-modified mRNA is only injected subcutaneously (SC luciferase). Twenty minutes before imaging, mice are injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals are then anesthetized and images are acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence is measured as the total luminous flux (photons / sec) of all mice. The luciferase formulations are outlined in Table 141 and have an average total luminous flux (photons / sec).
Table 141. Luciferase preparation

Example 94. Fibrin Sealant Research Fibrin sealants such as Tisseel (Baxter Healthcare Corp., Deerfield, IL) consist of fibrinogen and thrombin in a dual syringe. When mixed, fibrinogen is converted to fibrin, forming a fibrin clot in about 10-30 seconds. This clot can mimic the body's natural blood clotting mechanism. In addition, fibrin hydrogel is a three-dimensional structure that may be used in sustained release delivery. Fibrin sealants are currently recognized as an alternative to traditional surgical techniques such as suturing, ligation, and cauterization for hemostatic and sealing applications.

表１４３．全光束

The thrombin and fibrinogen components were separately loaded into a dual syringe. 50 μL of fibrinogen and 50 μL of thrombin were subcutaneously injected into Balb-C mice (n = 3) and modified luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is sequenced at the same site. Not shown within; 5'cap, cap 1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) (Tissel + luciferase), 50 μL fibrinogen and 50 μL thrombin (Tissel), or modified Luciferase mRNA (luciferase) was injected. Fibrinogen and thrombin infusions were completed simultaneously using a dual syringe. SC injection of luciferase was completed 15 minutes after fibrinogen / thrombin injection to allow fibrin hydrogel to polymerize (Tisseel + luciferase group). A control group of untreated mice was also evaluated. Mice were imaged at 5 and 24 hours. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. The luciferase preparation is outlined in Table 142, and the average total luminous flux (photons / sec) is shown in Table 143. Fibrin sealant was found not to interfere with imaging, and injection of luciferase and Tissel showed expression of luciferase.
Table 142. Luciferase preparation

Table 143. Total luminous flux

Example 95. Research on mRNA-containing fibrin sealants A. Modified mRNA and Calcium Chloride Luciferase mRNA fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine prior to reconstitution (mRNA sequence is sequence) Shown in number 21446; a poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) is added to calcium chloride. The calcium chloride is then used to reconstruct thrombin. Fibrinogen is reconstituted with a fibrinolysis inhibitor solution according to the manufacturer's instructions. Reconstituted thrombin and fibrinogen containing modified mRNA are filled into a dual syringe. Mice were injected subcutaneously with 50 μL fibrinogen containing modified mRNA and 50 μL thrombin, or 50 μL PBS containing an equivalent dose of modified luciferase mRNA. Control groups of untreated mice are also evaluated. Mice are imaged at predetermined intervals to determine the average total luminous flux (photons / sec).

B. Modified mRNA and Calcium Chloride Formulated with Lipid Nanoparticles Fully modified with 5-methylcytosine and N1-methyl-pseudouridine, formulated in lipid nanoparticles prior to reconstruction, or N1- Completely modified methyl-pseudouridine, luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) is added to calcium chloride. do. The calcium chloride is then used to reconstruct thrombin. Fibrinogen is reconstituted with a fibrinolysis inhibitor solution according to the manufacturer's instructions. Reconstituted thrombin and fibrinogen containing modified mRNA are filled into a dual syringe. Mice were injected subcutaneously with 50 μL thrombin containing 50 μL fibrinogen and modified mRNA, or 50 μL PBS containing an equivalent dose of modified luciferase mRNA. Control groups of untreated mice are also evaluated. Mice are imaged at predetermined intervals to determine the average total luminous flux (photons / sec).

C. Modified mRNA and fibrinogen Completely modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine prior to reconstitution, luciferase mRNA (mRNA sequence is SEQ ID NO: Shown in 21446; a poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) is added to the fibrinolysis inhibitor solution. The fibrinogen solution is then used to reconstruct fibrinogen. Thrombin is reconstituted with calcium chloride solution according to the manufacturer's instructions. Reconstituted fibrinogen and thrombin containing modified mRNA are filled into a dual syringe. Mice were injected subcutaneously with 50 μL fibrinogen containing 50 μL thrombin and modified mRNA, or 50 μL PBS containing an equivalent dose of modified luciferase mRNA. Control groups of untreated mice are also evaluated. Mice are imaged at predetermined intervals to determine the average total luminous flux (photons / sec).

D. Modified mRNA and Fibrinogen Formulated with Lipid Nanoparticles Fully modified with 5-methylcytosine and N1-methyl-pseudouridine, formulated in lipid nanoparticles prior to reconstruction, or N1-methyl -Completely modified pseudouridine, luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) in fibrinolytic inhibitor solution. Add to. The fibrinogen solution is then used to reconstruct fibrinogen. Thrombin is reconstituted with calcium chloride solution according to the manufacturer's instructions. Reconstituted fibrinogen and thrombin containing modified mRNA are filled into a dual syringe. Mice were injected subcutaneously with 50 μL fibrinogen containing 50 μL thrombin and modified mRNA, or 50 μL PBS containing an equivalent dose of modified luciferase mRNA. Control groups of untreated mice are also evaluated. Mice are imaged at predetermined intervals to determine the average total luminous flux (photons / sec).

E. Modified mRNA and Trombin Completely modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine prior to reconstitution, luciferase mRNA (mRNA sequence is SEQ ID NO: Shown in 21446; a poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) to the reconstituted trombin after reconstitution with calcium chloride according to the manufacturer's instructions. Added. The fibrinogen solution is then used to reconstruct fibrinogen according to the manufacturer's instructions. Thrombin containing reconstituted fibrinogen and modified mRNA is filled into a dual syringe. Mice were injected subcutaneously with 50 μL thrombin containing modified mRNA and 50 μL fibrinogen, or 50 μL PBS containing an equivalent dose of modified luciferase mRNA. Control groups of untreated mice are also evaluated. Mice are imaged at predetermined intervals to determine the average total luminous flux (photons / sec).

F. Modified mRNA and Trombin Formulated with Lipid Nanoparticles Fully modified or N1-methyl with 5-methylcytosine and N1-methyl-pseudouridine formulated in lipid nanoparticles prior to reconstruction -Completely modified pseudouridine, luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1), manufacturer's instructions. After being reconstituted with calcium chloride according to, it is added to the reconstructed trombin. The fibrinogen solution is then used to reconstruct fibrinogen according to the manufacturer's instructions. Thrombin containing reconstituted fibrinogen and modified mRNA is filled into a dual syringe. Mice were injected subcutaneously with 50 μL thrombin containing modified mRNA and 50 μL fibrinogen, or 50 μL PBS containing an equivalent dose of modified luciferase mRNA. Control groups of untreated mice are also evaluated. Mice are imaged at predetermined intervals to determine the average total luminous flux (photons / sec).

Example 96.5 Cationic lipid preparation of methylcytosine and N1-methyl-pseudouridine modified mRNA Fully modified luciferase mRNA with 5-methylcytosine and N1-methyl-pseudouridine (mRNA sequence is shown in SEQ ID NO: 21446). A polyA tail of about 140 nucleotides is not shown in the sequence; 5'caps, caps 1) were formulated in cationic lipids as described in Table 144. The preparation was administered intravenously (IV), intramuscularly (IM), or subcutaneously (SC) to Balb-C mice at a dose of 0.05 mg / kg.
Table 144. Cationic lipid preparation

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, and 24 hours after dosing and average total luminous flux (photons / sec) was measured for each route of administration and cationic lipid preparation. The background luminous flux was about 4.17E + 05p / s. The results of imaging are shown in Table 145. In Table 145, "NT" indicates that it was not tested.
Table 145. Luminous flux

Example 97. Lipid Nanoparticle Intravenous Study Luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; completely with 5-methylcytosine and pseudouridine Modified) containing 50% DLin-MC3-DMA or DLin-KC2-DMA, 38.5% cholesterol, 10% DSPC, and 1.5% PEG as shown in Table 146. Formulated in lipid nanoparticles. The formulation was administered intravenously (IV) to Balb-C mice at doses of 0.5 mg / kg, 0.05 mg / kg, 0.005 mg / kg, or 0.0005 mg / kg. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice.
Table 146. pharmaceutical formulation

表１４８．臓器の光束

In the case of DLin-KC2-DMA, mice were imaged 2 hours, 8 hours, 24 hours, 72 hours, 96 hours, and 168 hours after dosing and the average total luminous intensity (photons / sec) was taken for each dosing route and cationic lipids. The formulation was measured. The background luminous flux was about 3.66E + 05p / s. The results of imaging are shown in Table 147. Organs were imaged at 8 hours and average total luminous flux (photons / sec) was measured for liver, spleen, lungs, and kidneys. Controls for each organ were also analyzed. The results are shown in Table 147. The peak signal for all dose levels was 8 hours after dosing. Distribution to various organs (liver, spleen, lungs, and kidneys) may also be controlled by increasing or decreasing LNP doses.
Table 147. Luminous flux

Table 148. Luminous flux of organs

表１５０．臓器の光束

For DLin-MC3-DMA, mice are imaged 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 144 hours after dosing, and the average total luminous intensity (photons / second) is measured for each route of administration and cationic lipids. The formulation was measured. The background light beam was about 4.51E + 05p / s. The results of imaging are shown in Table 149. Organs were imaged at 8 hours and average total luminous flux (photons / sec) was measured for liver, spleen, lungs, and kidneys. Controls for each organ were also analyzed. The results are shown in Table 150. The peak signal for all dose levels was 8 hours after dosing. Distribution to various organs (liver, spleen, lungs, and kidneys) may also be controlled by increasing or decreasing LNP doses.
Table 149. Luminous flux

Table 150. Luminous flux of organs

Example 98. Lipid Nanoparticle Subcutaneous Study Luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine Was formulated in lipid nanoparticles containing 50% DLin-KC2-DMA, 385% cholesterol, 10% DSPC, and 1.5% PEG as shown in Table 151. .. The formulation was administered subcutaneously (SC) to Balb-C mice at doses of 0.5 mg / kg, 0.05 mg / kg, or 0.005 mg / kg.
Table 151. DLin-KC2-DMA preparation

表１５３．臓器の光束

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 144 hours after dosing and average total luminous intensity (photons / sec) was measured for each route of administration and cationic lipid preparation. The lower limit of detection was about 3E + 05p / s. The results of imaging are shown in Table 152. Organs were imaged at 8 hours and average total luminous flux (photons / sec) was measured for liver, spleen, lungs, and kidneys. Controls for each organ were also analyzed. The results are shown in Table 153. The peak signal for all dose levels was 8 hours after dosing. Distribution to various organs (liver, spleen, lungs, and kidneys) may also be controlled by increasing or decreasing LNP doses. At high doses, high levels of luciferase expression were detected in the liver, spleen, lungs, and kidneys, so the LNP preparation moves outside the subcutaneous injection site.
Table 152. Luminous flux

Table 153. Luminous flux of organs

Example 99. Subcutaneous study of cationic lipid nanoparticles Luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 160 nucleotides is not shown in the sequence; complete with 5'cap, cap 1; 5-methylcytosine and pseudouridine Is formulated in lipid nanoparticles containing 50% DLin-MC3-DMA, 38.5% cholesterol, 10% DSPC, and 1.5% PEG. The formulation is administered subcutaneously (SC) to Balb-C mice at doses of 0.5 mg / kg, 0.05 mg / kg, or 0.005 mg / kg.

Mice were imaged 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 144 hours after dosing and average total luminous intensity (photons / sec) was measured for each route of administration and cationic lipid preparation. Organs are imaged at 8 hours and average total luminous flux (photons / sec) is measured for liver, spleen, lungs, and kidneys. Controls for each organ are also analyzed.

Example 100. Luciferase lipoplex study Lipoplex-formed luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; polyA tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1), 5-methylcytosine and pseudo. Fully modified with uridine (5 mC / pU), fully modified with 5-methylcytosine and N1-methyl-pseudouridine (5 mC / N1 mpU), or 25% with 5-methylcytosine-substituted cytosine and It was 25% modified with 2-thiouridine-substituted uridine (5 mC / s2U). The preparation was administered to Balb-C mice intravenously (IV), intramuscularly (IM), or subcutaneously (SC) at a dose of 0.10 mg / kg.

表１５５．臓器に対する光束

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 8 hours, 24 hours, and 48 hours after dosing and average total luminous flux (photons / sec) was measured for each route of administration and chemical modification. The background signal was about 3.91E + 05p / s. The results of imaging are shown in Table 154. Organs were imaged at 6 hours and average total luminous flux (photons / sec) was measured for liver, spleen, lungs, and kidneys. Controls for each organ were also analyzed. The results are shown in Table 155.
Table 154. Luminous flux

Table 155. Luminous flux to organs

Example 101. Cationic lipid preparation of modified mRNA 25% (5 mC / s2U) modified luciferase mRNA with 5-methylcytosine-substituted cytosine and 2-thiouridine-substituted uridine (mRNA sequence is in SEQ ID NO: 21446). Shown; a poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) was formulated in cationic lipids as shown in Table 156. The preparation was administered intravenously (IV), intramuscularly (IM), or subcutaneously (SC) to Balb-C mice at a dose of 0.05 mg / kg.
Table 156. Cationic lipid preparation

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, and 24 hours after dosing and average total luminous flux (photons / sec) was measured for each route of administration and cationic lipid preparation. The background luminous flux was about 3.31E + 05p / s. The results of imaging are shown in Table 157. In Table 157, "NT" indicates that it was not tested. The untreated mice exhibited an average luminous flux of 3.14E + 05 at 2 hours, 3.33E + 05 at 8 hours, and 3.46E + 05 at 24 hours. Peak expression was seen in all three pathways tested at 8 hours. DLin-KC2-DMA had better expression than DLin-MC3-DMA, and DODA showed expression in all evaluated pathways.
Table 157. Luminous flux

Example 102.5-Methylcytosine and N1-methyl-pseudouridine modified mRNA preparation Fully modified luciferase mRNA with 5-methylcytosine and N1-methyl-pseudouridine (mRNA sequence is shown in SEQ ID NO: 21446; A poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) was formulated in PBS (pH 7.4). The preparation was administered intramuscularly (IM) or subcutaneously (SC) to Balb-C mice at a dose of 2.5 mg / kg.

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 5 minutes, 30 minutes, 60 minutes, and 120 minutes after dosing and average total luminous flux (photons / sec) was measured for each route of administration and cationic lipid preparation. The background luminous flux was about 3.78E + 05p / s. The results of imaging are shown in Table 158. Expression of luciferase was already seen at 30 minutes in both pathways of delivery. Peak expression from subcutaneous administration appears between 30 and 60 minutes. Intramuscular expression was still increased at 120 minutes.
Table 158. Luminous flux

表１６０．皮下投与

Example 103. Intramuscular and Subcutaneous Administration of Chemically Modified mRNA Completely modified with N4-acetylcytidine, fully modified with 5-methoxyuridine, completely modified with N4-acetylcytidine and N1-methyl-pseudouridine, or Luciferase-modified mRNA fully modified with 5-methylcytidine and 5-methoxyuridine, formulated in PBS (pH 7.4) (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of approximately 140 nucleotides Not shown in the sequence; 5'cap, cap 1) was administered to Balb-C mice at a dose of 2.5 mg / kg intramuscularly or subcutaneously. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged at 2, 8 and 24 hours. The average total luminous flux (photons / sec) for intramuscular administration is shown in Table 159, and the average total luminous flux (photons / sec) for subcutaneous administration is shown in Table 160. The background signal was 3.84E + 05 (p / s). Peak expression for intramuscular administration was seen between 24 and 48 hours in all chemical compositions, and expression was still detected at 120 hours. For subcutaneous delivery, peak expression was seen between 2 and 8 hours, and expression was also detected at 72 hours.
Table 159. Intramuscular administration

Table 160. Subcutaneous administration

Example 104. In vivo Studies Lusiferase-modified mRNAs containing at least one chemical modification are formulated as lipid nanoparticles (LNPs) using the syringe pump method and characterized by particle size, zeta potential, and encapsulation.

As outlined in Table 161, luciferase LNP preparations are administered intramuscularly (IM), intravenously (IV), and subcutaneously (SC) to Balb-C mice. do. As a control, luciferase-modified RNA formulated in PBS is intravenously administered to mice.
Table 161. Luciferase preparation

Mice are imaged at 2, 8, 24, 48, 120, and 192 hours to determine bioluminescence (measured as total luminous flux (photons / second) for all mice). At 8 or 192 hours, the liver, spleen, kidneys, and injection sites of subcutaneous and intramuscular administration are imaged to determine bioluminescence.

Example 105. Cationic Lipid Preparation Study of Chemically Modified mRNA 5-Methylcytosine and Pseudouridine (pU), Pseudouridine (pU), or N1-Methyl-Pseudouridine (N1mpU) fully modified luciferase mRNA (mRNA sequence) Is shown in SEQ ID NO: 21446; a polyA tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) was formulated in cationic lipids as shown in Table 162. The preparation was administered intravenously (IV), intramuscularly (IM), or subcutaneously (SC) to Balb-C mice at a dose of 0.05 mg / kg.
Table 162. Cationic lipid preparation

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, and 24 hours after dosing and average total luminous flux (photons / sec) was measured for each route of administration and cationic lipid preparation. The background luminous flux was about 4.11E + 05p / s. The results of imaging are shown in Table 163. Peak expression was seen in all three pathways tested at 8 hours.
Table 163. Luminous flux

Example 106. Study of chemically modified mRNAs Whether fully modified with N4-acetylcytidine (N4-acetyl) or 5-methoxyuridine (5meth), N4-acetylcytidine and N1-methyl-pseudouridine (N4-acetyl) Luciferase mRNA (mRNA sequence is shown in SEQ ID NO: 21446; approximately 140 nucleotides) fully modified with / N1mpU) or fully modified with 5-methylcytidine and 5-methoxyuridine (5 mC / 5-meth). Poly A tail is not shown in the sequence; 5'cap, cap 1) was formulated in DLin-MC3-DMA as described in Table 164.

The preparation was administered to Balb-C mice intravenously (IV), intramuscularly (IM), or subcutaneously (SC) at a dose of 0.05 mg / kg.
Table 164. Cationic lipid preparation

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 6 hours, and 24 hours after dosing and average total luminous flux (photons / sec) was measured for each route of administration and cationic lipid preparation. The background luminous flux was about 2.70E + 05p / s. The results of imaging are shown in Table 165.
Table 165. Luminous flux

Example 107. Lipid nanoparticle EPO mRNA containing multiple modified mRNAs (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and N1 -Completely modified with methyl-pseudouridine) and G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1; Completely modified with 5-methylcytosine and N1-methyl-pseudouridine) and factor IX mRNA (mRNA sequence is shown in SEQ ID NO: 1622; poly A tail of about 140 nucleotides is not shown in the sequence; 5 'Cap, cap 1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) are formulated in DLin-MC3-DMA as described in Table 166. The drug is administered intravenously (IV), intramuscularly (IM), or subcutaneously (SC) to Balb-C mice at a dose of 0.05 mg / kg. A control LNP preparation containing only one mRNA is also administered at an equivalent dose.
Table 166. DLin-MC3-DMA product

Serum is collected from mice 8 hours, 24 hours, 72 hours, and / or 7 days after administration of the drug. Serum is analyzed by ELISA to determine protein expression of EPO, G-CSF, and Factor IX.

Example 108.5-Methylcytosine and N1-methyl-pseudouridine modified mRNA cationic lipid preparation study EPO mRNA (mRNA sequence is shown in SEQ ID NO: 1638; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) or G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 21438; poly A tail of approximately 140 nucleotides is sequence Not shown within; 5'cap, cap 1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine), DLin-MC3-DMA and DLin-KC2- as shown in Table 167. Formulate in DMA. The drug is administered intravenously (IV), intramuscularly (IM), or subcutaneously (SC) to Balb-C mice at a dose of 0.05 mg / kg.
Table 167. DLin-MC3-DMA and DLin-KC2-DMA preparations

Serum is collected from mice 8 hours, 24 hours, 72 hours, and / or 7 days after administration of the drug. Serum is analyzed by ELISA to determine EPO and G-CSF protein expression.

Example 109. In vitro VEGF PBMC study Completely modified with 5-methylcytosine and pseudouridine (VEGF 5mC / pU), fully modified with 5-methylcytosine and N1-methyl-pseudouridine (VEGF 5mC / N1mpU), or not Modified (VEGF unmod), 500 ng of VEGF mRNA (mRNA sequence is not shown in the sequence with a poly A tail of about 160 nucleotides shown in SEQ ID NO: 1672; 5'cap, cap 1), Lipofectamine 2000, 0.4 ul. Transfected peripheral blood mononuclear cells (PBMC) from normal blood donors (D1, D2, and D3) using. Cells were also not treated for each donor as a control. The supernatant was collected 22 hours after transfection and ELISA was performed to determine protein expression and cytokine induction. VEGF expression and IFN-α induction are shown in Table 168 and FIGS. 8A and 8B.
Table 168. Protein and cytokine levels

表１７０．ＴＧＦβタンパク質発現

表１７１．ｒＢＰＩ−２１タンパク質発現

Example 110. In vitro expression of modified mRNA In HEK293 cells, EPO-modified mRNA (mRNA sequence is shown in SEQ ID NO: 1638; polyA tail of about 160 nucleotides is not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and pseudo. Transfected (fully modified with uridine) into HeLa cells with transforming growth factor β (TGF-β) modified mRNA (mRNA sequence is shown in SEQ ID NO: 1668; poly A tail of approximately 160 nucleotides is within the sequence. Not shown in; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) were forward-transfected, and HepG2 cells were modified with bactericidal / permeability enhancing protein (rBPI-21). They were transfected with mRNA (SEQ ID NO: 21449; poly A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), and they were presented in the book. Using the procedures described herein, complexes were formed using Liponucleomine 2000 from Invitrogen (Carlsbad, CA) at the concentrations shown in Tables 169, 170, and 171. Protein expression is detected by ELISA and the protein (pg / mL) is also shown in Tables 169, 170, and 171. In Table 169, ">" means greater than that. In the case of TGFβ, control untreated cells and sham transfection of Lipofectamine 2000 were also tested.
Table 169. EPO protein expression

Table 170. TGFβ protein expression

Table 171. rBPI-21 protein expression

Example 111. Bicistron modified mRNA
Human fetal kidney epithelium (HEK293) was seeded on a 96-well plate (Greener Bio-one GmbH, Frickenhausen, Germany), 100 μl of HEK293 in a cell culture medium (2 mM L-glutamine, 1 mM sodium pyruvate, and 1-fold non-. Seeded at a density of 30,000 in DMEM, 10% FCS supplemented with essential amino acids (Biochrom AG, Berlin, Germany) and 1.2 mg / mL sodium bicarbonate (Sigma-Aldrich, Munic, Germany). 75 ng of bicistron-modified mRNA (mCherry-2A-GFP) (SEQ ID NO: 21450; polyA tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1; completely with 5-methylcytosine and pseudouridine Modified), mCherry-modified mRNA (mRNA SEQ ID NO: 21439; poly A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), Or green fluorescent protein (GFP) modified mRNA (mRNA sequence is shown in SEQ ID NO: 21448; poly A tail of about 160 nucleotides is not shown in the sequence; complete with 5'cap, cap 1; 5-methylcytosine and pseudouridine. (Modified to) was added after seeding the cells and incubated. Control untreated cells were also evaluated. mCherry-2A-GFP refers to a modified mRNA sequence containing the mCherry coding region, the 2A peptide, and the GFP coding region.

Culture medium supernatants were collected by transfer to a 96-well Pro-Bind U bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with half the amount of trypsin / EDTA (Biochrom AG, Berlin, Germany), pooled with their respective supernatants, and single doses of PBS / 2% FCS (all Biochrom AG, Berlin, Germany) / 0. It was fixed by adding 5% formaldehyde (Merck, Darmstadt, Germany). Samples were then subjected to flow cytometer measurements on an LSRII cytometer (Beckton Dickinson GmbH, Heidelberg, Germany) using a 532 nm excitation laser and a 610/20 filter for PE-Texas Red. The average fluorescence intensity (MFI) for all events is shown in Table 172. Cells transfected with bicistron modified mRNA were capable of expressing both mCherry and GFP.
Table 172. MFI of modified mRNA

Example 112. Study of cationic lipid preparation of 5-methylcytosine and N1-methyl-pseudouridine modified mRNA for producing antibody Herceptin heavy chain (HC) mRNA (mRNA sequence is shown in SEQ ID NO: 21451; poly A tail of about 140 nucleotides Is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) and Herceptin light chain (LC) (mRNA sequence is shown in SEQ ID NO: 21452; A poly A tail of approximately 140 nucleotides is not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine), DLin as described in Table 173. Formulated in -MC3-DMA and DLin-KC2-DMA. The formulation is administered intravenously (IV) to Balb-C mice at doses of 0.500, 0.050, and 0.005 mg / kg.
Table 173. DLin-MC3-DMA and DLin-KC2-DMA preparations

Serum was collected from mice 8 hours, 24 hours, 72 hours, and / or 7 days after administration of the drug. Serum was analyzed by ELISA to determine Herceptin protein expression.

Example 113. Pseudouridine and N1-methyl-pseudouridine subject SAR
With the recent attention to pyrimidine nucleoside pseudouridine, a series of structural activity studies have been designed to study mRNAs containing modifications to pseudouridine or N1-methyl-pseudouridine.

In this study, the effect of chain length, increased lipophilicity, presence of ring structure, and hydrophobicity when modifications were made at the N1, C6, 2-, and 4-positions, and on the phosphate skeleton. Or designed to investigate changes in hydrophilic interactions. Also study stability.

表１７５．シュードウリジンおよびＮ１−メチル−シュードウリジンＳＡＲ

For this purpose, alkylation, cycloalkylation, alkyl-cycloalkylation, arylation, alkyl-aryllation, alkylating moieties with amino groups, alkylating moieties with carboxylic acid groups, and alkyls containing amino acid charged moieties. Study modifications, including alkylated moieties. The degree of alkylation is generally C _{1 to} C ₆ . Examples of chemical modifications include those shown in Tables 174 and 175.
Table 174. Pseudouridine and N1-methyl pseudouridine SAR

Table 175. Pseudouridine and N1-methyl-pseudouridine SAR

表１７７．非天然ヌクレオシドトリホスフェート

Example 114. Incorporation of Natural and Non-Natural Nucleosides Natural and non-natural nucleosides are incorporated into the mRNA encoding the polypeptide of interest. Examples of these are shown in Tables 176 and 177. Certain commercially available nucleoside triphosphates (NTPs) are studied in the polynucleotides of the invention. These options are shown in Table 176. The resulting mRNA is then tested for its ability to produce proteins, induce cytokines, and / or produce therapeutic outcomes.
Table 176. Natural and non-natural nucleosides

Table 177. Unnatural nucleoside triphosphate

表１７９．天然に生じる組み合わせ

Example 115. Incorporation of modifications to nucleobases and carbohydrates (sugars) Natural and unnatural nucleosides are incorporated into the mRNA encoding the polypeptide of interest. Commercially available nucleosides and NTPs with modifications to both nucleobases and carbohydrates (sugars) are tested for their ability to integrate into mRNA, produce proteins, induce cytokines, and / or produce therapeutic outcomes. Examples of these nucleosides are shown in Tables 178 and 179.
Table 178. Combination modification

Table 179. Naturally occurring combination

In this table, "UTP" is an abbreviation for uridine triphosphate, "GTP" is an abbreviation for guanosine triphosphate, "ATP" is an abbreviation for adenosine triphosphate, and "CTP" is an abbreviation for cytosine triphosphate. "TP" is an abbreviation for triphosphate, and "Bz" is an abbreviation for benzyl.

Example 116. Protein production of vascular endothelial growth factor in HeLa cells The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were administered in trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Collected by treatment and seeded in 96-well cell culture plates (Corning, Manassas, VA) in 100 μL total volume of EMEM medium (supplemented with 10% FCS and 1x Glutamax) per well. Cells were grown overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, 250 ng of vascular endothelial growth factor (VEGF) modified RNA with the chemical modifications listed in Table 180 (mRNA sequence is shown in SEQ ID NO: 1672; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap. , Cap 1) was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Israel, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL of lipofectamine 2000 was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature. A second set of HeLa cells was also evaluated with VEGF mRNA according to the procedure described above. For the first set of HeLa cells, control untreated cells and cells treated only with lipofecatamine 2000 were also analyzed.

After 18-22 hours of incubation, cell culture supernatants of VEGF-expressing cells were harvested and centrifuged at 10.000 rcf for 2 minutes. The clear supernatant was then analyzed using a VEGF-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determined values were within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 180.

These results demonstrate that VEGF fully modified with 1-methylpseudouridine produced more protein in HeLa cells compared to other chemical compositions.
Table 180. Protein production

Example 117. Comparison of protein production of chemically modified vascular endothelial growth factor in HeLa cells The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were subjected to trypsin-EDTA solution (LifeTechnologies, Grand Island,). Collected by treatment in NY) and seeded in 96-well cell culture plates (Corning, Manassas, VA) in 100 μL total volume of EMEM medium (supplemented with 10% FCS and 1x Glutamax) per well. Cells were grown overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, 250 ng of vascular endothelial growth factor (VEGF) modified RNA with the chemical modifications set forth in Table 181 (mRNA sequence is shown in SEQ ID NO: 1672; poly A tail of about 160 nucleotides is not shown in the sequence; 5'cap. , Cap 1) was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Israel, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL of lipofectamine 2000 was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature. Control untreated cells were also analyzed.

After 18-22 hours of incubation, cell culture supernatants of VEGF-expressing cells were harvested and centrifuged at 10.000 rcf for 2 minutes. The clear supernatant was then analyzed using a VEGF-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determined values were within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 181 and FIG.

This result demonstrates that VEGF fully modified with 1-methylpseudouridine produced more protein in HeLa cells compared to other chemical compositions.
Table 181. Protein production

Example 118. Production of vascular endothelial growth factor protein in mammals Lipoplex-formed vascular endothelial growth factor (VEGF) mRNA (mRNA sequence is shown in SEQ ID NO: 1672; polyA tail of approximately 160 nucleotides is not shown in the sequence; 5'cap, Whether cap 1) was unmodified, completely modified with 5-methylcytosine and pseudopseudouridine (5 mC / pU), or completely modified with 5-methylcytosine and 1-methylseudouridine (5 mC / 1 mPU). 25% modified with 5-methylcytosine-substituted cytosine and 25% modified with 2-thiouridine-substituted uridine (5 mC / s2U), fully modified with 1-methylpseudouridine (1 mpU), or pseudo Completely modified with uridine (pU). The preparation was intravenously administered to mice at a dose of 2 μg mRNA per mouse. As a control, a group of mice was untreated. Serum from mice is measured by specific VEGF ELISA for VEGF protein expression 3 hours after dosing. The results are shown in Table 182 and FIG.
Table 182. Protein production

Example 119. Protein production of granulocyte colony stimulating factor in HeLa cells The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were subjected to trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Collected by treatment in, and seeded in 96-well cell culture plates (Corning, Manassas, VA) in 100 μL EMEM medium (supplemented with 10% FCS and 1x Glutamax) per well. Cells were grown overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, 250 ng of granulocyte colony-stimulating factor (G-CSF) -modified RNA with the chemical modifications set forth in Table 183 (mRNA sequence is shown in SEQ ID NO: 21438; polyA tail of about 160 nucleotides is not shown in the sequence; The 5'cap, cap 1) was diluted in 10 μL of final volume OPTI-MEM (Life Technologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL of lipofectamine 2000 was diluted in 10 μL of final volume OPTI-MEM. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature. Control untreated cells and cells treated only with lipofecatamine 2000 were also analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing G-CSF were harvested and centrifuged at 10.000 rcf for 2 minutes. The clear supernatant was then analyzed using a G-CSF-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determined values were within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 183 and FIG.
Table 183. Protein production

Example 120. Production of Granulocyte Colony Stimulator Protein in Mammalian 5-Methylcytosine and Psoiduridine Fully Modified Granulocyte Colony Stimulator (G-CSF) mRNA (mRNA sequence shown in SEQ ID NO: 21438; about not shown in the sequence 160 nucleotides of poly-A tail; 5'cap, cap 1) (5 mC / pU), fully modified mRNA with 5-methylcytosine and 1-methylcytosine urine (5 mC / 1 mpU), of cytosine The same mRNA (5 mC / s2U) in which 25% was replaced with 5-methylcytosine and 25% of urine was replaced with 2-thiouridine, and the same mRNA completely modified with 1-methylpsoid uridine (1 mpU). Or the same mRNA (pU) completely modified with pseudocytosine. The formulation is mRNA per mouse
Mice (CD1) (n = 3) were intravenously administered at doses of 2 ug and 2 ul of Lipofectamine 2000 (L2000). As a control, one group of mice was untreated. Six hours after administration, mouse sera were measured for G-CSF protein expression by specific G-CSF ELISA. The results are shown in Table 184 and FIG.
Table 184. Protein production

Example 121. Protein production of chemically modified Factor IX in HeLa cell supernatant The day before transfection, 15,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were subjected to trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies). , Grand Island, NY) and collected in 24-well cell culture plates (Corning, Manassas, VA) with a total volume of 100 μL of Eagle's Minimal Essential Medium (EMEM) (10% bovine fetal serum (FCS)) per well. It was sown in 1x Glutamax supplementation). Cells were grown overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, it was completely modified with 5-methylcytosine and pseudouridine (5 mC / pU), completely modified with 5-methylcytosine and 1-methylseudouridine (5 mC / 1 mpU), or replaced with 5-methylcytosine. It was either 25% modified with 5-methylcytosine and 25% modified with 2-thiouridine-substituted uridine (s2U / 5mC), fully modified with 1-methylpseudouridine (1mpU), or completely modified with pseudopseudouridine. (PU), 250 ng of factor IX modified RNA (mRNA sequence is shown in SEQ ID NO: 1622; polyA tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) in a final volume of 10 μL. It was diluted in OPTI-MEM® (LifeTechnologies, Grand Islands, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 μL of lipofectamine 2000 was diluted in 10 μL of a final volume of OPTI-MEM®. After a 5 minute incubation at room temperature, both solutions were mixed and incubated at room temperature for an additional 15 minutes. 20 μL of the combined solution was then added to 100 μL of cell culture medium containing HeLa cells and incubated at room temperature. Untreated controls were also analyzed.

After 18 hours of incubation, cell culture supernatants of cells expressing factor IX were harvested from cells lysed with an immunoprecipitation (IP) buffer of Pierce Biotechnology (Thermo Scientific, Rockford, IL) for 2 minutes and 10 minutes. Centrifugation was performed at .000 rcf. The clear supernatant is diluted 1: 2 (1 mL lysate / 2 wells / 24 well plate) or 1: 5 (1 mL lysate / 5 wells / 24 well plate) and then factor IX according to the manufacturer's instructions. Analysis was performed using a specific ELISA kit. All samples were diluted until the determined values were within the linear range of the ELISA standard curve. The amount of protein produced in both studies is shown in Table 185 and FIG.
Table 185. Protein production

Example 122. Protein production of apolypoprotein AI in HeLa cells The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 20, treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY), 000 cells were collected and placed in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 100 ul per well in a 24-well cell culture plate (Corning, Manassas, VA). Sown. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, apolypoprotein AI wild-type (APOA 1 wt) -modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21453; poly-A of approximately 160 nucleotides not shown in the sequence. Tail; apolyproprotein A1 Paris (APOA1 Paris) -modified RNA fully modified with 5'cap, cap 1), 5-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 21454; shown in the sequence. Poly-A tail of about 160 nucleotides not; 5'cap, cap 1) or apolyproprotein fully modified with 5-methylcytosine and pseudouridine A1 Milano (APOA1)
Milano) Modified RNA (mRNA sequence shown in SEQ ID NO: 21455; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) 250 ng in final volume of 10 ul OPTI-MEM® Diluted with (Life Technologies, Grand Island, NY). Lipofectamine
2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and Lipofectamine 2000 0.2 ul was diluted with OPTI-MEM® in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of cells treated with lipofecatamine 2000 alone were analyzed.

After 18 hours of incubation, cells expressing APOA 1 wt, APOA 1 Paris or APOA 1 Milano by lysing the cells in Immunoprecipitation (IP) buffer (Thermo Scientific, Rockford, IL) from Pierce Biotechnology. Qing was collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant is diluted 1: 2 (solubilized solution 1 ml / 2 wells / 24 well plate) or 1: 5 (solubilized solution 1 ml / 5 wells / 24 well plate) and then with an APOA1-specific ELISA kit. The analysis was performed according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. This test was performed again and the amount of protein produced in both tests is shown in Table 186 and FIG.

Table 186. Protein production

Example 123. Detection of apolypoprotein AI wild protein: Western blot The day before transfection, HeLa cells (ATCC number CCL-2; Manassass, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). 750,000 cells were collected and added with Eagle's Minimal Essential Medium (EMEM) (10% bovine fetal serum (FCS) and 1 × Glutamax) in a total volume of 3 ml per well of 6-well cell culture plates (Corning, Manassas, VA). ) Was sown. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fully modified apolyproprotein AI (APOA1) wild mRNA with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21453; of approximately 160 nucleotides not shown in the sequence). Poly-A tail; 5'cap, cap 1) (5 mC / pU), the same mRNA fully modified with 5-methylcytosine and 1-methylcytosine uridine (5 mC / 1 mPU), 25% of cytosine is 5- The same mRNA (s2U / 5mC) substituted with methylcytosine and 25% of urine substituted with 2-thiouridine, the same mRNA completely modified with 1-methylcytosine uridine (1 mpU) or completely with pseudouridine 1250 ng of the modified mRNA (pU) was diluted with a final volume of 250 ul of OPTI-MEM® (LifeTechnologies, Grand Israel, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. ApoA1 rabbit polyclonal antibody against APOA1 (Abcam, Cambridge, MA) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker for 3 hours at room temperature. It was reacted. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to ApoA1 rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. The results of Western blot detection in HeLa cell lysates of APOA1 protein derived from APOA1 wild-type mRNA containing various chemical modifications are shown in FIG.

Example 124. Apolipoprotein AI Paris protein detection: Western blot The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 750 by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Collect 000 cells and add 3 ml of Eagle's Minimal Essential Medium (EMEM) per well of 6-well cell culture plates (Corning, Manassas, VA) (10% bovine fetal serum (FCS) and 1 x Glutamax added). Sown in. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, Apolyproprotein (APOA1) Paris mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21454; poly-A tail of approximately 160 nucleotides not shown in the sequence. 5'cap, cap 1) (5 mC / pU), fully modified mRNA with 5-methylcytosine and 1-methylcytosine uridine (5 mC / 1 mpU), 25% of cytosine replaced with 5-methylcytosine Twenty-five percent of the urine was replaced with 2-thiourine and modified with the same mRNA (s2U / 5mC), fully modified with 1-methylcytosine urine (1 mpU) or completely modified with pseudourine (s2U / 5mC). pU) 1250 ng was diluted with OPTI-MEM® (Life Technologies, Grand Israel, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. ApoA1 rabbit polyclonal antibody against APOA1 (Abcam, Cambridge, MA) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker for 3 hours at room temperature. It was reacted. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to ApoA1 rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. The results of Western blot detection in HeLa cell lysates of APOA1 protein derived from APOA1 Paris mRNA containing various chemical modifications are shown in FIG.

Example 125. Apolipoprotein AI Milano protein detection: Western blot The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 750 by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Collect 000 cells and add 3 ml of Eagle's Minimal Essential Medium (EMEM) per well of 6-well cell culture plates (Corning, Manassas, VA) (10% bovine fetal serum (FCS) and 1 x Glutamax added). Sown in. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fully modified apolyproprotein (APOA1) Milano mRNA with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21455; poly-A tail of approximately 160 nucleotides not shown in the sequence. 5'cap, cap 1) (5 mC / pU), fully modified mRNA with 5-methylcytosine and 1-methylcytosine uridine (5 mC / 1 mpU), 25% of cytosine replaced with 5-methylcytosine Twenty-five percent of the urine was replaced with 2-thiourine and modified with the same mRNA (s2U / 5mC), fully modified with 1-methylcytosine urine (1 mpU) or completely modified with pseudourine. 1250 ng of mRNA (pU) was diluted with 250 ul of final volume OPTI-MEM® (LifeTechnologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of a variable volume protein solubilizer, a RIPA buffer with a volume of 26 ul, 10 x reducing agent 4 ul and 4 x SDS loading buffer 10 ul (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. ApoA1 rabbit polyclonal antibody against APOA1 (Abcam, Cambridge, MA) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker for 3 hours at room temperature. It was reacted. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to ApoA1 rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. The results of Western blot detection in HeLa cell lysates of APOA1 protein derived from APOA1 Milano mRNA containing various chemical modifications are shown in FIG.

Example 126. Detection of fibrinogen A protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fibrinogen A (FGA) mRNA fully modified with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 21456; poly-A tail of approximately 160 nucleotides not shown in the sequence. 1250 ng of 5'cap, cap 1) (5 mC / 1 mpU) was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. Fibrinogen A goat polyclonal antibody against fibrinogen A (Abcam, Cambridge, MA) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilutions at room temperature with gentle stirring in an orbital shaker. It was allowed to react for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Donkey anti-goat HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to fibrinogen A goat polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed portion of FIG. 18, the results of Western blot detection of fibrinogen A were close to the predicted size of 95 kd.

Example 127. Protein production of chemically modified plasminogen in HeLa cell supernatant HeLa cells (ATCC number CCL-2; Manassas, VA) 20 by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY) the day before transfection. 000 cells were collected and seeded in 96-well cell culture plates (Corning, Manassas, VA) in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, a plasminogen-modified RNA fully modified with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 21457; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1) 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamines 2000 (Life Technologies, Grand)
Island, NY) was used as a transfection reagent and Lipofectamine 2000 0.2 ul was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, erythropoetin (EPO) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; poly-A tail of about 160 nucleotides not shown in the sequence; complete with 5'cap, cap 1, 5-methylcytosine and pseudouridine. Control and untreated cells (modified to) were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing plasminogen were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a plasminogen-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 187 and FIG.

Table 187. Protein production

Example 128. Detection of plasminogen protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Was collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. .. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, plasminogen mRNA fully modified with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 21457; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5 'Cap, cap 1) (5 mC / 1 mpU) 1250 ng was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. Plasminogen goat polyclonal antibody against plasminogen (Abcam, Cambridge, MA) in 3 ml of 1 × TBS solution containing 5% BSA at a dilution of 1: 500 to 1: 2000 with gentle stirring in an orbital shaker. The reaction was carried out at room temperature for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The donkey HRP conjugate (Abcam, Cambridge, MA) is conjugated with horseradish peroxidase and bound to the plasminogen goat polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. FIG. 20 shows that plasminogen was detected near the predicted size of 95 kd.

Example 129. Detection of galactose-1-urate uridyl transferase protein: Western blot The day before transfection, HeLa cells (ATCC No. CCL-2; Manassass) were treated with a solution of trypsin-ethylenediaminetetraacetic acid (EDTA) (LifeTechnologies, Grand Island, NY). , VA) 750,000 were collected, and Eagle's Minimal Essential Medium (EMEM) (10% bovine fetal serum (FCS)) with a total volume of 3 ml per well of 6-well cell culture plates (Corning, Manassas, VA) and 1 × Glutamax was added). Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, galactose-1-phosphate uridyryl transferase (GALT) mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21458; poly of about 160 nucleotides not shown in the sequence. -A tail; 5'cap, cap 1) (5 mC / pU) 1250 ng was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. GALT mouse monoclonal antibody (Novus biological, Littleton CO) against GALT in 3 ml of 1 x TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker for 3 hours at room temperature. It was reacted. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Donkey anti-mouse HRP conjugates (Abcam, Cambridge, MA) are conjugated to horseradish peroxidase and bound to GALT mouse monoclonal antibodies. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed portion of FIG. 21, the result of Western blot detection of GALT was about 42 kd.

Example 130. Detection of argininoscusinase acid lyase protein: Western blot The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 750 by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Collect 000 cells and add 3 ml of Eagle's Minimal Essential Medium (EMEM) per well of 6-well cell culture plates (Corning, Manassas, VA) (10% bovine fetal serum (FCS) and 1 x Glutamax added). Sown in. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fully modified argininosuccinase acid lyase (ASL) mRNA with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21459; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1) (5 mC / pU) 1250 ng was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. ASL mouse monoclonal antibody against ASL (Novus biological, Littleton, CO) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker, 3 at room temperature. Reacted for time. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Donkey anti-mouse HRP conjugates (Abcam, Cambridge, MA) are conjugated to horseradish peroxidase and bound to ASL mouse monoclonal antibodies. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed portion of FIG. 22, the result of Western blot detection of ASL was about 50 kd.

Example 131. Detection of tyrosine aminotransferase protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with a solution of trypsin-ethylenediaminetetraacetic acid (EDTA) (Life Technologies, Grand Island, NY). Was collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. .. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, tyrosine aminotransferase (TAT) mRNA fully modified with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 21460; poly-A of approximately 160 nucleotides not shown in the sequence. Tail; 5'cap, cap 1) (5 mC / 1 mpU) 1250 ng was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. TAT rabbit polyclonal antibody against TAT (Novus biologicals) was reacted at room temperature for 3 hours at 1: 500 to 1: 2000 dilution in 3 ml of 1 × TBS solution containing 5% BSA, gently stirring with an orbital shaker. .. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to TAT rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed area of FIG. 23, the result of Western blot detection of TAT was about 50 kd of the chemiluminescent band shown in white.

Example 132.1, 4-α-Glucan-branching enzyme Protein detection: Western blot The day before transfection, HeLa cells (ATCC number) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). CCL-2; Manassas, VA) 750,000 were collected and Eagle's Minimum Essential Medium (EMEM) (10% bovine fetal serum) with a total volume of 3 ml per well of 6-well cell culture plates (Corning, Manassas, VA). FCS) and 1 × Glutamax added) were sown. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, a fully modified 1,4-α-glucan-branched enzyme (GBE1) mRNA with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 21461; about not shown in the sequence. A poly-A tail of 160 nucleotides; 5'cap, cap 1) (5 mC / 1 mpU) 1250 ng was diluted with a final volume of 250 ul of OPTI-MEM® (Life Technologies, Grand Islands, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. GBE1 rabbit polyclonal antibody against GBE1 (Novus biological, Littleton, CO) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker, 3 at room temperature. Reacted for time. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to GBE1 rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed portion of FIG. 24, the result of Western blot detection of GBE1 was a predicted size of around 70 kd.

Example 133. Protein production of prothrombin in HeLa cell supernatant The day before transfection, 20,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY), 96. Well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, a prothrombin-modified RNA fully modified with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 21462; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'. Cap, cap 1) 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, erythropoetin (EPO) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; poly-A tail of about 160 nucleotides not shown in the sequence; complete with 5'cap, cap 1, 5-methylcytosine and pseudouridine. Control and untreated cells (modified to) were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing plasminogen were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a prothrombin-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 188 and FIG.

Table 188. Protein production

Example 134. Protein production of chemically modified prothrombin in HeLa cell supernatant The day before transfection, HeLa cells (ATCC No. CCL-2; Manassas, ATCC number CCL-2; Manassas, by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY)). 15,000 VA) were collected and 100 ul of Eagle's Minimum Essential Medium (EMEM) (10% bovine fetal serum (FCS) and 1 × Glutamax) per well in a 24-well cell culture plate (Corning, Manassas, VA). Was added). Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, prothrombin-modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 21462; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1). (5 mC / pU), 5-methylcytosine and 1-methylpsoid uridine fully modified RNA (5 mC / 1 mpU), 25% of cytosine replaced with 5-methylcytosine and 25% of uridine 2- Final volume of 250 ng of thiouridine-substituted and modified RNA (s2U / 5mC), 1-methylcytosine fully modified RNA (1 mpU) or pseudocytosine fully modified RNA (pU). It was diluted with 10 ul of OPTI-MEM® (LifeTechnologies, Grand Israel, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM® in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, untreated controls were analyzed.

After 18 hours of incubation, cell culture supernatants of cells expressing prothrombin were collected by lysing the cells with Thermo Scientific, Rockford, IL (Pierce Biotechnology, Immunoprecipitation (IP) buffer). Centrifugation was performed at 10.000 rcf for 2 minutes. The clarified supernatant is diluted 1: 2 (solubilized solution 1 ml / 2 wells / 24 well plate) or 1: 5 (solubilized solution 1 ml / 5 wells / 24 well plate) and then with a prothrombin-specific ELISA kit. The analysis was performed according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced in both tests is shown in Table 189 and FIG.

Table 189. Protein production

Example 135. Detection of celluloplasmin protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were treated with a solution of trypsin-ethylenediaminetetraacetic acid (EDTA) (LifeTechnologies, Grand Island, NY). Collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fully modified celluloplasmin (CP or CLP) mRNA with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 1621; poly-of approximately 160 nucleotides not shown in the sequence. A-tail; 5'cap, cap 1) (5 mC / 1 mpU) 1250 ng was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. Ceruloplasmin rabbit polyclonal antibody against CP protein (Novus biological, Littleton, CO) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker at room temperature. Was reacted for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to ceruloplasmin rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed portion of FIG. 27, the result of Western blot detection of ceruloplasmin (CLP) was about 148 kd.

Example 136. Protein production of transforming growth factor β1 in HeLa cell supernatant The day before transfection, 20,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were treated with Trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Collected and seeded in 96-well cell culture plates (Corning, Manassas, VA) in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, transforming growth factor β1 (TGF-β1) -modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1668; poly-A of approximately 160 nucleotides not shown in the sequence. Tail; 5'cap, cap 1) 83 ng, 250 ng or 750 ng was diluted with OPTI-MEM (Life Technologies, Grand Islands, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, control (L2000) and untreated cells of lipofecatmine 2000 treated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing TGF-β1 were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a TGF-β1-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 190 and FIG.

Table 190. Protein production

Example 137. Detection of ornithine transcarbamoylase protein: Western blot The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 750, treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). 000 cells were collected and placed in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well of 6-well cell culture plates (Corning, Manassas, VA). Sown. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, ornithine transcarbamoylase (OTC) mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1659; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5 'Cap, cap 1) (5 mC / pU) 1250 ng was diluted with a final volume of 250 ul of OPTI-MEM® (LifeTechnologies, Grand Island, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. OTC rabbit polyclonal antibody against OTC protein (Novus biological, Littleton, CO) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker at room temperature. It was allowed to react for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to OTC rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed portion of FIG. 29, the result of OTC Western blot detection was a predicted size of around 40 kd.

Example 138. In vivo test of LDLR in mammals Low density lipoprotein (LDL) receptor (LDLR) mRNA (mRNA shown in SEQ ID NO: 21463; fully modified with 5-methylcytosine and pseudouridine; 5'cap, cap 1; in sequence By mixing 8.0 ug of poly-A tail of 160 nucleotides (not shown in 1) and Dalveco's modified Eagle's medium (DMEM) to a final volume of 0.2 mL, the complex of the mRNA and Lipofectamine 2000 can be obtained. It was formed.

Lipofectamine 2000 was diluted 12.5-fold with DMEM and mixed with the same volume of diluted LDL receptor mRNA solution. Samples were incubated at room temperature for 5 minutes and 0.1 mL of the complexed mRNA mixture was injected into each tail vein of each of the three C57BL / 6 mice. A total dose of 2.0 ug of LDL receptor mRNA was administered to each individual. After 6 hours, the mice were sacrificed and the spleen was removed. Spleen cells were isolated according to standard methods (without pre-dissolving red blood cells) and stained with equal amounts of human LDL receptor-specific IgG or control non-immune IgG.

LDL receptor expression was assessed by flow cytometry gated to the CD11b + splenocyte population. As shown in FIG. 30, three cells due to the presence of right-shifted peaks (LDLR IgG) stained with LDL receptor IgG compared to cells stained with non-immune IgG (non-immune IgG). Expression of the LDL receptor in the in vivo CD11b + splenocyte population was apparent in individual mice.

No LDL receptor-specific peaks were observed in mice treated with Lipofectamine alone, and staining was similar to that observed with non-immune IgG.

Example 139. Testing of Modified UGT1A1 mRNA According to standard methods, HEK293 cells were seeded in DMEM containing 10% fetal bovine serum at a density of 500,000 cells / 6 well plates, and the cells were cultured overnight. The next day, UDP glucuronosyl transferase 1 family, polypeptide A1 (UGT1A1) mRNA (mRNA shown in SEQ ID NO: 21464; completely modified with 5-methylcytosine and pseudouridine; 5'cap, cap 1; not shown in the sequence 160 800 ng of poly-A tail of nucleotides) is diluted with 0.3 mL of optimal buffer, and 4.0 uL of Lipofectamine 2000 sample is diluted with 0.3 mL of optimal buffer, and the two solutions are mixed. As a result, a complex of mRNA and Lipofectamine 2000 was formed.

After incubating for 15 minutes at room temperature, cells were transfected with the UGT1A1 mRNA / Lipofectamine 2000 mixture. After incubating at 37 ° C. for 18 hours, the medium was aspirated and the cells were washed with PBS. The cells were scraped off with a cell lifter and collected, and the cells were centrifuged at 3000 rpm for 3 minutes. The cell pellet was washed with PBS, centrifuged as described, and 0.25 mL of RIPA buffer containing the protease inhibitor cocktail was added to lyse the cell pellet. The cell extract was centrifuged at 12,210 rpm for 10 minutes, and the supernatant fraction (solubilized solution) was frozen at −80 ° C.

Western blotting was performed using a human UGT1A1-specific antibody using a Simple Simon Western capillary electrophoresis apparatus. 0.005 mg of a solubilized solution sample was run in triple iterations. As shown in FIG. 31, UGT1A1 was detected as a single band of about 60 kDa. In contrast, no band was detected using a solubilized solution of control cells transfected with irrelevant mRNA.

Example 140. In vivo expression of LDLR in mice LDLR − / − mice are used to test the in vivo expression of LDLR m mRNA. LDL mRNA is injected into LDLR − / − mice. Mouse tissues were examined for LDLR expression. Western blot analysis of mouse tissue was performed and LDLR
The presence or absence of LDLR protein expression resulting from mmRNA administration is examined. Real-time RT-PCR is performed on mouse tissues to check for LDLR gene expression.

Example 141. Production of LDLR in Mammalian Ornithine transcarbamoylase (OTC) is a mitochondrial protein encoded within the nuclear genome. OTC deficiency is characterized by toxic accumulation of ammonia, resulting in urea cycle failure.

Mice described in Example 140 are administered a modified mRNA encoding OTC. Serum, tissues and / or organs are collected at various time points to determine protein expression.

In addition, a test will be conducted in which the mice of Example 140 are administered with LDLR-modified mRNA. Serum, tissues and / or organs are collected at various time points to determine protein expression.

Example 142. Generation of chemically modified factor XI in HEK293 cells Human fetal renal epithelial cells (HEK293) (LGC standards GmbH, Wesel, Greener Bio-one GmbH, Frickenhausen, Germany) precoated with type 1 collagen. Germany) is sown. HEK293 is seeded in 100 ul of cell culture medium at a density of approximately 100,000 cells / well. After seeding the cells, factor XI mRNA (modRNA FXI) (mRNA sequence set forth in SEQ ID NO: 1625; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1) 350 ng or 750 ng Directly add the formulation containing the above and incubate. In addition, controls of untreated cells were evaluated.

Cells are collected by transferring the supernatant of the culture medium to a 96-well Pro-Bind U bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with 1/2 volume of trypsin / EDTA (Biochrom AG, Berlin, Germany), pooled with each supernatant and 1 volume of PBS / 2% FCS (both Biochrom AG, Berlin, Germany) / 0 .Fixed by addition of 5% formaldehyde (Merck, Darmstadt, Germany). After 12 hours of incubation, cell culture supernatants of cells expressing Factor XI were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed according to the manufacturer with a Factor XI specific ELISA kit (Innovative Research, Novi, MI). The amount of protein produced is shown in FIG.

Example 143. Detection of aquaporin-5 protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Was collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. .. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, aquaporin-5 mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 1617; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1 ) (5 mC / pU), 5-methylcytosine and 1-methylcytosine uridine fully modified mRNA (5 mC / 1 mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine 5- 1250 ng of the same mRNA modified with methylcytosine (s2U and 5 mC), the same mRNA completely modified with pseudourine (pU) or the same mRNA completely modified with 1-methylcytosine urine (1 mpU) in a final volume of 250 ul. It was diluted with OPTI-MEM® (LifeTechnologies, Grand Islands, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above. In addition, controls of untreated cells were evaluated.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. Aquaporin-5 rabbit polyclonal antibody (Abcam, Cambridge, MA) against aquaporin-5 protein in 3 ml of 1 × TBS solution containing 5% BSA at a dilution of 1: 500 to 1: 2000 with gentle stirring in an orbital shaker. , The reaction was carried out at room temperature for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit conjugates (Abcam, Cambridge, MA) are conjugated with horseradish peroxidase and bound to aquaporin-5 rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed area of FIG. 33, Western blots detected proteins in each of the evaluated chemical reactions.

Example 144. Protein production of factor VII in HeLa cells The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (Life Technologies, Grand Island, NY), 96. Well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, factor VII modified RNA with the chemical modifications set forth in Table 191 (mRNA sequence shown in SEQ ID NO: 1623; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1). 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing Factor VII were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a Hyperen Biomed Chromogenic kit (Aniara, West Chester OH) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 191 and FIG.

Table 191. Protein production

Example 145. Protein production of chemically modified insulin glargine in HeLa cell supernatant The day before transfection, HeLa cells (ATCC No. CCL-2; Manassass) were treated with a solution of trypsin-ethylenediaminetetraacetic acid (EDTA) (LifeTechnologies, Grand Island, NY). , VA) 15,000 were collected and 100 ul of Eagle's Minimum Essential Medium (EMEM) (10% bovine fetal serum (FCS) and 1x) per well in a 24-well cell culture plate (Corning, Manassas, VA). Glutamax was added). Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, insulin glargine-modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21465; poly-A tail of approximately 140 nucleotides not shown in the sequence; 5'cap, cap 1 ) (5 mC / pU), 5-methylcytosine and 1-methylpsoid uridine completely modified RNA (5 mC / 1 mpU), 25% of cytosine is replaced with 5-methylcytosine and 25% of uridine is 2. -Final 250 ng of the same RNA (s2U / 5mC) substituted and modified with thiouridine, the same RNA (1 mpU) completely modified with 1-methylcytosine uridine, or the same RNA (pU) completely modified with psoid urine. It was diluted with 10 ul of OPTI-MEM® (LifeTechnologies, Grand Israel, NY). Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. (Registered trademark). After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, untreated controls were analyzed.

After 18 hours of incubation, cell culture supernatants of insulin glargine-expressing cells were harvested by lysis with Thermo Scientific (Rockford, IL) immunoprecipitation (IP) buffer from Pierce Biotechnology. Centrifugation was performed at .000 rcf for 2 minutes. After diluting the clarified supernatant to 1: 2 (solubilizer 1 ml / 2 wells / 24 well plate) or 1: 5 (solubilizer 1 ml / 5 wells / 24 well plate), an insulin-specific ELISA kit ( Analysis was performed at Mercodia AB, Uppsala, Sweden) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced in both tests is shown in Table 192 and FIG. In Table 192, ">" means to exceed.

Table 192. Protein production

Example 146. Protein production of tissue factor (factor 3) in HeLa cells The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Then, they were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) having a total volume of 100 ul per well of 96-well cell culture plates (Corning, Manassas, VA). Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, a tissue factor (factor 3) -modified RNA with the chemical modifications set forth in Table 193 (mRNA sequence shown in SEQ ID NO: 21466; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, Cap 1) 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing tissue factor were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a Hyperen Biomed Chromogenic kit (Aniara, West Chester OH) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 193 and FIG. In Table 193, ">" means to exceed.

Table 193. Protein production

Example 147. Protein production of chemically modified factor XI in HeLa cells The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 15,000 were treated with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Individuals were collected and seeded in 96-well cell culture plates (Corning, Manassas, VA) in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, factor XI modified RNA with the chemical modifications set forth in Table 194 (mRNA sequence shown in SEQ ID NO: 1625; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1). 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing Factor XI were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a Factor XI specific ELISA kit (Innovative Research, Novi, MI) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 194 and FIG. 37. In Table 194, ">" means to exceed.

Table 194. Protein production

Example 148. Protein production of factor XI in HeLa cell supernatant The day before transfection, 20,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). , 96-well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, factor XI modified RNA fully modified with 5-methylcytosine and 1-methylpsoiduridine (mRNA sequence set forth in SEQ ID NO: 1625; poly-A tail of approximately 140 nucleotides not shown in the sequence; 5'cap, cap 1) 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and Lipofectamine was used.
2000 0.2 ul was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, erythropoietin (EPO) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; poly-A tail of about 160 nucleotides not shown in the sequence; fully modified with cap, cap 1,5-methylcytosine and pseudouridine. ) And controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing plasminogen were harvested and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a Factor XI specific ELISA kit (Innovative Research, Novi, MI) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 195 and FIG. 38.

Table 195. Protein production

Example 149. Protein production of insulin aspart in HeLa cells The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY), 96. Well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, insulin aspart-modified RNA with the chemical modifications set forth in Table 196 (mRNA sequence set forth in SEQ ID NO: 21467; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1). 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of insulin-expressing cells were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with an insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 196 and FIG. 39. In Table 196, ">" means to exceed.

Table 196. Protein production

Example 150. Protein production of insulin lispro in HeLa cells The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY) and 96 wells. Cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, 250 ng of insulin lispro-modified RNA with the chemical modifications set forth in Table 197 (mRNA sequence shown in SEQ ID NO: 21468; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1). Was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing insulin lispro were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with an insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 197 and FIG. 40. In Table 197, ">" means to exceed.

Table 197. Protein production

Example 151. Protein production of insulin gluridin in HeLa cells The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY), 96. Well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, insulin glulinin-modified RNA with the chemical modifications set forth in Table 198 (mRNA sequence set forth in SEQ ID NO: 21469; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1). 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing insulin lispro were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with an insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 198 and FIG. In Table 198, ">" means to exceed.

Table 198. Protein production

Example 152. Protein production of human growth hormone in HeLa cells The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY), and 96 Well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, a human growth hormone (hGH) -modified RNA with the chemical modifications set forth in Table 199 (mRNA sequence set forth in SEQ ID NO: 1648; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, Cap 1) 250 ng was diluted with OPTI-MEM (Life Technologies, Grand Island, NY) having a final volume of 10 ul. Lipofectamine
2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and Lipofectamine 2000 0.2 ul was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing human growth hormone were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a human growth hormone ELISA kit (Cat. No. DGH00; R & D SYSTEMS®, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced compared to the untreated sample is shown in Table 199 and FIG. In Table 199, ">" means more.

Table 199. Protein production

Example 153. Detection of Tumor Protein 53 Protein: Lipoplexed Tumor Protein 53 (TP53 or p53) mRNA fully modified with 5-methylcytosine and pseudouridine in Western blot CD1 mice (Harlan Laboratories, South Easton, MA) (with SEQ ID NO: 1670) MRNA sequence shown; poly-A tail of about 140 nucleotides not shown in the sequence; fully modified with 5'cap, cap 1) (5 mC / pU), 5-methylcytosine and 1-methylpsoiduridine The same mRNA (5 mC / 1 mC), 25% of uridine modified with 2-thiouridine and 25% of cytosine modified with 5-methylcytosine (s2U and 5 mC), the same mRNA completely modified with pseudouridine The mRNA (1 mpU) completely modified with (pU) or 1-methylcytosine uridine was administered intravenously. MRNA 2 ug complexed with 2 ul of Lipofectamine 2000 (Life Technologies, Grand Island, NY) was dissolved in 100 ul of sterile DMEM basal medium (w / o additive, Life Technologies, Grand Island, NY).

After 6 hours, the mice were sacrificed and serum and spleen were collected. The spleen was transferred to a 6-well plate and kept on ice in the presence of 1 ml of PBS. One spleen was cut several times with an anatomical scalpel and spleen cells were squeezed out with a rubber cell scraper until the PBS became cloudy due to cell release.

Cells were transferred to a 100 um cell strainer (BD Biosciences, San Jose, CA) on a 12-well cell culture plate, leaving the fibrous component. The cells were passed through a cell strainer by gravity and collected in the lower 12-well culture dish. 1 ml of PBS was transferred to an Eppendorf tube together with floating splenocytes and rotated at 2000 rpm for 5 minutes. The PBS was discarded and the cell pellet was combined with fresh PBS 500 ul. Spleen cells were resuspended by vortexing briefly at 2000 rpm for 5 minutes. PBS was discarded and 1 ml of BD Pharmalyse was added to the cell pellet. Spleen cells were resuspended by a brief vortex. The cells were incubated at room temperature for 3 minutes and then spun at 200 rpm for 5 minutes. The cells were washed twice with 500 ul of PBS and rotated as described above. Cells were resuspended in 500 ul PBS and spun as described.

250 ul of splenocytes were combined with 1 × Pharmalyse buffer, vortexed for a short time or resuspended with a pipette, and then rotated at 2000 rpm for 2 minutes.

Cell pellets are resuspended in 500 ul of RIPA buffer containing a protease inhibitor cocktail for mammalian cells (Boston Bioproducts, Ashland, MA) in one tube and the solubilized solution is frozen or subsequently subjected to the BCA assay. 250 ul of FACS staining kit fixative (4% formaldehyde; Rand D Systems, Minneapolis, MN) is added to the second tube and then incubated at room temperature for 10 minutes. The cells were washed twice with 500 ul of PBS and rotated as described above. Cell pellets were resuspended in 500 PBS and stored at 4 ° C.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transfer, the membrane was incubated with 1 × TBS containing 5% BSA for 15 minutes and then with 1 × TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. The primary antibody against the target protein was reacted in 3 ml of 1 × TBS solution containing 5% BSA at a dilution of 1: 500 to 1: 2000 at room temperature for 3 hours with gentle stirring in an orbital shaker. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Secondary antibody [[Which antibody? ?? ]] Is conjugated with horseradish peroxidase, and the primary antibody [[Which antibody?] ?? ]] To combine. The secondary antibody was diluted 1: 1000 to 1: 5000 with 1 × TBS containing 5% BSA and incubated at room temperature for 3 hours. Primary and secondary antibodies are Abcam (Cambridge, MA), Novus Biologicals (Littleton, CO), Thermo Fisher (Rockford, IL), Millipore (Billerica, MA) or Randemsd. ) Purchased from.

At the end of the incubation time, the membrane was washed 3 times with 1 × TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The membrane was luminescent in 5 ml of Pierce WestPico Chemiluminescence Subrate (Thermo Fisher, Rockford, IL) as instructed.

As shown in the squared parts of FIGS. 43A and 43B, Western blots detected proteins with a predicted size of around 53 kd in each of the two samples evaluated for each chemical reaction.

Example 154. Detection of tough terin 1 protein: Western blot Human fetal renal epithelial cells (HEK293) were seeded on 96-well plates (Greener Bio-one GmbH, Frickenhausen, Germany) and the plates for HEK293 cells were precoated with type 1 collagen. .. HEK293 in cell culture medium (DMEM, 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and 1 x non-essential amino acids (Biochrom AG, Berlin, Germany) and 1.2 mg / ml sodium bicarbonate (Sigma-Aldrich, Munich) , Germany) was added) and seeded at a density of 35,000 in 100 ul.

Transfected cells were lysed with 20 ul / well sample buffer (20% glycerol, 4% SDS, 100 mM Tris-HCl pH 6.8, 0.2% bromophenol blue, 5% β-mercaptoethanol). The supernatant was precipitated with 4 volumes of ice-cold acetone and dissolved in sample buffer. Samples were heated at 95 ° C. for 5 minutes and run on an SDS-polyacrylamide gel containing 10% acrylamide.

The protein was transferred to the nitrocellulose membrane by semi-blotting. Membranes were blocked with TBS containing 5% skim milk powder and then incubated with 1: 500 diluted TUFT1 rabbit polyclonal antibody (Novus Biologicals, Catalog No. NBP1-87446). Signals were detected using donkey anti-rabbit HRP-conjugated secondary antibody (St. Cruz Biotech, Heidelberg, Germany) and Super Signal West Pico detection reagent (Pierce). As shown in FIG. 44, Western blot detected proteins in 2 ug (lane 2) and 200 ng (lane 3) samples. In FIG. 44, lane 1 is a marker.

Example 155. Detection of galactoxine 1 protein: Western blot CD1 mice (Harlan Laboratories, South Easton, MA) fully modified with 5-methylcytosine and pseudouridine lipoplexized galactoxine 1 (GALK1) mRNA (presented by SEQ ID NO: 21470). mRNA sequence; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1) (5 mC / pU), fully modified with 5-methylcytosine and 1-methylpsoiduridine. The mRNA (5 mC / 1 mC), 25% of uridine modified with 2-thiouridine and 25% of cytosine modified with 5-methylcytosine (s2U and 5 mC), the same mRNA completely modified with pseudouridine (pU) ) Or the same mRNA (1 mpU) completely modified with 1-methylcytosine uridine was administered intravenously. Lipofectamines 2000 (Life Technologies, Grand)
Mice were administered with 2 ug of mRNA complexed with 2 ul of Island, NY) dissolved in 100 ul of sterile DMEM basal medium (w / o additive, Life Technologies, Grand Island, NY).

After 6 hours, the mice were sacrificed and serum and spleen were collected. The spleen was transferred to a 6-well plate and kept on ice in the presence of 1 ml of PBS. One spleen was cut several times with an anatomical scalpel and spleen cells were squeezed out with a rubber cell scraper until the PBS became cloudy due to cell release.

Cells were transferred to a 100 um cell strainer (BD Biosciences, San Jose, CA) on a 12-well cell culture plate, leaving the fibrous component. The cells were passed through a cell strainer by gravity and collected in the lower 12-well culture dish. The cells were passed through a cell strainer by gravity and collected in the lower 12-well culture dish. 1 ml of PBS was transferred to an Eppendorf tube together with floating splenocytes and rotated at 2000 rpm for 5 minutes. The PBS was discarded and the cell pellet was combined with fresh PBS 500 ul. Spleen cells were resuspended by vortexing briefly at 2000 rpm for 5 minutes. PBS was discarded and 1 ml of BD Pharmalyse was added to the cell pellet. Spleen cells were resuspended by a brief vortex. The cells were incubated at room temperature for 3 minutes and then spun at 200 rpm for 5 minutes. The cells were washed twice with 500 ul of PBS and rotated as described above. Cells were resuspended in 500 ul PBS and spun as described.

250 ul of splenocytes were combined with 1 × Pharmalyse buffer, vortexed for a short time or resuspended with a pipette, and then rotated at 2000 rpm for 2 minutes.

Cell pellets are resuspended in 500 ul of RIPA buffer containing a protease inhibitor cocktail for mammalian cells (Boston Bioproducts, Ashland, MA) in one tube and the solubilized solution is frozen or subsequently subjected to the BCA assay. 250 ul of FACS staining kit fixative (4% formaldehyde; Rand D Systems, Minneapolis, MN) is added to the second tube and then incubated at room temperature for 10 minutes. The cells were washed twice with 500 ul of PBS and rotated as described above. Cell pellets were resuspended in 500 PBS and stored at 4 ° C.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transfer, the membrane was incubated with 1 × TBS containing 5% BSA for 15 minutes and then with 1 × TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. GALK1 rabbit polyclonal antibody against GALK1 protein (Abcam, Cambridge, MA) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker, 3 at room temperature. Reacted for time. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Goat anti-rabbit HRP conjugate (Abcam, Cambridge MA) is conjugated with horseradish peroxidase and bound to GALK1 rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. Primary and secondary antibodies are Abcam (Cambridge, MA), Novus Biologicals (Littleton, CO), Thermo Fisher (Rockford, IL), Millipore (Billerica, MA) or Randemsd. ) Purchased from.

At the end of the incubation time, the membrane was washed 3 times with 1 × TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The membrane was luminescent in 5 ml of Pierce WestPico Chemiluminescence Subrate (Thermo Fisher, Rockford, IL) as instructed.

As shown in the squared sections of FIGS. 45A and 45B, Western blots detected proteins with predicted sizes of around 30 kd and 42 kd in the two samples evaluated for each chemical reaction, respectively.

Example 156. Detection of defensin, β103A protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Was collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. .. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fully modified with 5-methylcytosine and pseudouridine, β103A (DEFB103A) mRNA (mRNA sequence set forth in SEQ ID NO: 1631; poly-A tail of approximately 140 nucleotides not shown in the sequence. 1250 ng of 5'cap, cap 1) (5 mC / pU) was diluted with OPTI-MEM® (LifeTechnologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above. In addition, controls of untreated cells were evaluated.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. DEFB103A rabbit polyclonal antibody (Abcam, Cambridge MA) against DEFB103A protein in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 to 1: 2000 dilution, gently stirring with an orbital shaker for 3 hours at room temperature. It was reacted. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. Donkey anti-rabbit NL557 conjugate (R & D Systems, Minneapolis, MN) is conjugated with horseradish peroxidase and bound to DEFB103A rabbit polyclonal antibody. The conjugated antibody was diluted 1: 1000 to 1: 5000 with 1 x TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in the boxed area of FIG. 46, Western blots detected proteins in each of the evaluated chemical reactions.

Example 157. Confirmation of Peptide Identity In order to confirm the peptide identity, proteins can be evaluated by quantitative LC multiple reaction monitoring (MRM) using liquid chromatography-mass spectrometry and mass spectrometry (LC-MS / MS) in conjunction with each other. ..

Any protein target described herein by a quantitative LC multiplex reaction monitoring (MRM) method (Biognosys AG, Schlieren Switzerland) using liquid chromatography-mass spectrometry and mass spectrometry (LC-MS / MS) in conjunction. Can be evaluated. HeLa cell lysates containing proteins expressed from modified mRNAs are evaluated by a quantitative LC-MRM assay (Biognosis, Schlieren Switzerland) using LC-MS / MS to confirm the identity of the peptide in the cytolysate. .. Known and / or described methods in the art are used to compare identified peptide fragments with known proteins, including isoforms.

A. Sample Preparation Reduce the protein in each sample in lysis buffer by incubating with 5 mM Tris (2-carboxyethyl) phosphine (TCEP) at 37 ° C. for 1 hour. Alkylation is performed with 10 mM iodoacetamide in the dark at room temperature for 30 minutes. Trypsin (Sequence Grade, Promega Corporation, Madison, WI) is used to digest the protein into peptides using a protease to protein ratio of 1:50. Digestion is performed overnight at 37 ° C. (total digestion time is 12 hours). Peptides are cleaned up to perform mass spectrometry using a C18 spin column (The Nest Group, Southborough, MA) according to the manufacturer's instructions. The peptide is completely dried and resuspended in LC solvent A (1% acetonitrile, 0.1% formic acid (FA)). All solvents shall be HPLC grade from SIGMA-ALDRICH® (St. Louis, MO) and all chemical products shall be from SIGMA-ALDRICH® (St. Louis, MO) unless otherwise stated. To buy.

B. LC-MS / MS and LC-MRM
In any mass spectrometry, a packed C18 column (Magical AQ, particle size 3 um, pore size 200 Å, Microbiosurces, Inc (Auburn, CA); column length 11 cm, inner diameter 75 um, New Objective) of a Proxeon Easy nLC nanoliquid chromatography system. , MA)) inject the peptide. The LC solvent is A: a 1% aqueous acetonitrile solution containing 0.1% FA; B: a 3% aqueous acetonitrile solution containing 0.1% FA. The LC gradient of the shotgun analysis is 120 minutes with 5 to 35% solvent B, then 2 minutes with 35 to 100% solvent B, and 8 minutes with 100% solvent B (gradient time is 130 minutes in total). LC-MS / MS shotgun analysis for peptide discovery is performed on a Thermo Scientific (Billerica, MA) Q Active mass spectrometer equipped with a standard nanoelectrospray source. The LC gradient of LC-MRM is 30 minutes with 5 to 35% solvent B, then 2 minutes with 35 to 100% solvent B, and 8 minutes with 100% solvent B (gradient time is 40 minutes in total). The Thermo Scientific (Thermo Fisher Scientific) (Billerica, MA) TSQ Vantage triple quadrupole mass spectrometer is equipped with a standard nanoelectrospray source. In the unscheduled MRM mode of recalibration, it is operated with a residence time of 20 ms per transition. For relative quantification of peptides between samples, the TSQ Vantage is operated in a scheduled MRM mode with a acquisition window length of 4 minutes. The LC eluate is electrosprayed at 1.9 kV and MRM analysis is performed using a Q1 peak width of 0.7 Da. The collision energy of the TSQ Vantage is calculated by linear regression according to the vendor's specifications.

C. Assay Design, Data Processing and Analysis To create the LC-MRM assay, the 12 strongest fragment ions obtained by LC-MS / MS analysis were measured in unscheduled LC-MRM mode and mProphet (Reiter et al., MProphet: Automated data processing and statistical validation for range-scale SRM experiments, Nature Methods, 2011 (8), 430-435; Data was processed using Karlsruhe, Germany). Manually verify the efficacy of the assay, determine the exact fragment strength, and determine the iRT (indexed retention time) of Biognosis's iRT-peptide (Escher et al., Using iRT, a normalized retention time for source measurement). Assigned to peptides, Proteomics, 2012 (12), 1111-1121; the contents of which are incorporated herein by reference).

Relative quantification of peptides between sample series measures the eight strongest transitions in each assay. Data analysis is performed using SpectroDrive ™ (Biognosis, Schlieren Switzerland). The total peak area is compared for the selected peptides and a false discovery rate of 0.05 is applied. Peptides with a Q value of less than 0.05 are excluded and are considered undetected in each sample.

Example 158. Confirmation of the identity of peptides derived from modified mRNAs including chemical modifications Low-density lipoprotein receptor (LDLR) -modified mRNAs (mRNA sequence shown in SEQ ID NO: 21463; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), ornithine carbamoyl transferase (OTC) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1659; poly of about 160 nucleotides not shown in the sequence. A-tail; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), wild-type apolipoprotein AI (APOA 1 wt) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21453; about not shown in the sequence. Poly-A tail of 160 nucleotides; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine), hepcidin (HEPC) modified mRNA (mRNA sequence shown in SEQ ID NO: 21471; not shown in the sequence Approximately 160 nucleotides of poly-A tail; 5'cap, cap 1; fully modified with 5-methylcytosine and 1-methylpsoiduridine), hemochromatosis type 2 (HFE2) modified mRNA (with SEQ ID NO: 21472) The mRNA sequence shown; the poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and 1-methylpsoiduridine) or fumarylacetacetic acid hydrolyzate. (FAH) Modified mRNA (mRNA sequence shown in SEQ ID NO: 21473; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; with 5-methylcytosine and 1-methylpsoiduridine Cell lysates containing the protein produced from (fully modified) were evaluated quantitatively by LC-MRM using LC-MS / MS as described in Example 157. The peptide fragments identified in the evaluated proteins are shown in Table 200. Both peptides were specific for the parent protein, and LDLR and HFE2 were specific for the parent protein and its isoforms. In Table 200, "Uniprot ID" refers to the UniProt protein identifier when the peptide fragment sequence was blast searched for all browsed proteins in the database. The housekeeping proteins used to evaluate the proteins in the cell lysates are shown in Table 201.

Table 200. Sequence of protein and peptide fragments

Table 201. Housekeeping protein

Example 159. Confirmation of Peptide Identity Derived from Chemically Modified mRNA 5-Cytotoxic T lymphocyte-related protein 4 (CTLA4) -modified mRNA completely modified with methylcytosine and methylpsoiduridine (mRNA shown in SEQ ID NO: 21521) Sequence; Poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), serpin peptidase inhibitor, clade A (α-1 antiproteinase, antitrypsin), member 1 (serpina 1) Modified mRNA (mRNA sequence shown in SEQ ID NO: 21522; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), sphingomyelin phosphodiesterase 1 (SMPD1) modified mRNA (at SEQ ID NO: 21523). The mRNA sequence shown; the poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), hypoxanthin-guanine phosphoribosyl transferase (HPRT1) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21524). Poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), 4-hydroxyphenylpyruvate dioxygenase (HPD) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21525; in the sequence Poly-A tail of about 140 nucleotides not shown in; 5'cap, cap 1), osteogenic protein-7 (BMP7) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21526; about 140 not shown in the sequence. Nucleotide poly-A tail; 5'cap, cap 1), lecithin-cholesterol acyltransferase (LCAT) -modified mRNA (mRNA sequence set forth in SEQ ID NO: 21527; poly-A of approximately 140 nucleotides not shown in the sequence. Tail; 5'cap, cap 1), apoptosis-inducing factor short isoform 1 (AIFsh) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21528; poly-A tail of about 140 nucleotides not shown in the sequence; 5' Cap, cap 1), tumor protein 53 (TP53 or P53) modified mRNA (mRNA sequence shown in SEQ ID NO: 1670; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), Argininosuccinate synthase (ASS1) modified mRNA (SEQ ID NO: The mRNA sequence shown in No. 21259; the poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1), KIT ligand / stem cell factor (KITLG) modified mRNA (mRNA shown in SEQ ID NO: 21530). Sequence; Poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), neuregulin 1, β2a isoform (NRG1) -modified mRNA (DNA sequence shown in SEQ ID NO: 21531; in the sequence T7 promoter used in in vitro transcription (IVT) shown in, 5'untranslated regions (UTR) and 3'UTR), vasoactive intestinal peptide (VIP) modified mRNA (mRNA sequence shown in SEQ ID NO: 21532; sequence Poly-A tail of about 140 nucleotides not shown in; 5'cap, cap 1), arylsulfatase B (ARSB) modified mRNA (mRNA sequence shown in SEQ ID NO: 1618; about 140 not shown in the sequence Poly-A tail of nucleotides; 5'cap, cap 1), 6-pyrovoyltetrahydropterin synthase (PTS) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1609; about 140 not shown in the sequence Poly-A tail of nucleotides; 5'cap, cap 1), fibronectin type III domain-containing protein 5 (FNDC5 or irisine) -modified mRNA (DNA sequence set forth in SEQ ID NO: 21533; in vitro transcription shown in the sequence (in-vitro transcription) T7 promoter used in IVT), 5'untranslated regions (UTR) and 3'UTR), amerogenin (AMELY) modified mRNA (mRNA sequence shown in SEQ ID NO: 1613; poly of about 140 nucleotides not shown in the sequence. -A tail; 5'cap, cap 1), aldolase A (ALDOA) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21534; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), nerve growth factor (NGF) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21535; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), glycogen synthase 2 ( GYS2) Modified mRNA (mRNA sequence shown in SEQ ID NO: 21536; not shown in the sequence Poly-A tail of about 140 nucleotides; 5'cap, cap 1), GTP cyclohydrolase 1 (GCH1) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1649; about 140 nucleotides not shown in the sequence. Poly-A tail; 5'cap, cap 1), hepatocellular growth factor (HGF) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21537; poly-A tail of about 140 nucleotides not shown in the sequence; 5' Cap, cap 1), ectodisplacin A (EDA) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1636; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), arginase (ARG1) modified mRNA (mRNA sequence shown in SEQ ID NO: 21538; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), serum amyloid P (APCS) modified mRNA (SEQ ID NO: The mRNA sequence shown by 21539; the poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), phosphorylase kinase, γ2 (PHKG2) modified mRNA (mRNA sequence shown by SEQ ID NO: 21540; Poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), 685 exoxyribonuclease I (DNASE1) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1632; in the sequence Poly-A tail of about 140 nucleotides not shown; 5'cap, cap 1), exenatide-modified mRNA (mRNA sequence shown in SEQ ID NO: 21541; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), glycogenin 1 (PYGL) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21542; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), α -Galactosidase (GLA) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1640; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), α-L-izronidase (IDUA) modification mRNA (mRNA sequence shown in SEQ ID NO: 1652; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap Galactoxine 1 (GALK1) -modified mRNA fully modified with p, cap 1), 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21470; poly-A of approximately 140 nucleotides not shown in the sequence. Tail; 5'cap, cap 1) (5 mC and pU), the same mRNA fully modified with 5-methylcytosine and 1-methylpsoid uridine (5 mC and 1 mpU), 25% of urine modified with 2-thiouridine Twenty-five percent of the cytosine was modified with 5-methylcytosine and modified with the same mRNA (s2U and 5mC), completely modified with pseudourine (pU) or fully modified with 1-methylpsoidurine. Cell lysates containing the protein produced from the mRNA (1 mpU) were evaluated quantitatively by LC-MRM using LC-MS / MS as described in Example 157. The peptide fragments identified in the evaluated proteins are shown in Table 202.

Table 202. Sequence of protein and peptide fragments

Example 160. Confirmation of Peptide Identity and Comparison with Recombinant Protein Liquid Chromatography (LC) -Mass Spectrometry (MS) Analysis, LC-MS / MS Trypsine Peptide Mapping / Sequencing Analysis and Two-Dimensional Fluorescent Differential Gel Electrophoresis (2-D DIGE) ), And fully modified ornithine carbamoyl transferase (OTC) -modified mRNA with 2-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 1659; poly-A tail of about 140 nucleotides not shown in the sequence. 5'cap, cap 1) or 5-methylcytosine and pseudouridine or 1-methylpsoiduridine fully modified UDP glucuronosyl transferase 1 family, polypeptide A1 (UGT1A1) modified mRNA (with SEQ ID NO: 21464) The mRNA sequence shown; the poly-A tail of about 140 nucleotides not shown in the sequence; the expression of each transcript in HeLa cells transfected with 5'cap, cap 1 was confirmed. The following materials were used: HPLC grade water (EMD Proteins Inc., Gibbstone, NJ); formic acid (90%) (JT Baker, Philippsburg, NJ); iodoacetamide (Sigma Aldrich, St. Louis, MO); methanol (EMD Protein, MO). , Gibbstone, NJ); Tris (2-carboxyethyl) phosphine hydrochloride solution (TCEP), 0.5M (Sigma Aldrich, St. Louis, MO); Tris (hydroxymethyl) aminomethane buffer (Tris buffer) , PH 8.0 (Fluka-Sigma Aldrich, St. Louis, MO); anhydrous calcium chloride ACS reagent (CaCl ₂ ) (Sigma Aldrich, St. Louis, MO); OTC reference protein [purified recombinant full-length human OTC protein (Abcam) , Cambridge, MA)]; UGT1A1 reference protein [purified recombinant human UGT1A1 protein (OriGene Technologies, Rockville, MD).

Hela cells were transfected with mRNA encoding OTC and mRNA encoding UGT1A1. After transfection, cells were lysed and centrifuged to form cell pellets. The total protein concentration of each sample was determined by the bicinchoninic acid assay (BCA assay). The cell pellet of untransfected HeLa cells had a total protein concentration of 1707 μg / mL, whereas the cell pellet of OTC-transfected HeLa cells and UGT1A1-transfected HeLa cells had a total protein concentration of 1707 μg / mL, respectively. It was 3497 μg / mL and 2609 μg / mL.

In trypsin peptide mapping / sequencing, recombinant proteins and cell pellets were digested with trypsin. Recombinant OTC and UGT1A1 were prepared with 100 mM Tris buffer containing 3 mM calcium chloride. The protein (5 μg each) was reduced with 2 mM TCEP and then denatured at 100 ° C. for 5 minutes. The sample was cooled to room temperature and then alkylated with 2 mM iodoacetamide. Alkylated samples were further reduced with 0.5 mM TCEP. Samples were treated with trypsin overnight at 37 ° C. in a water bath. The digested sample was removed from the water bath and the reaction was stopped with a 2% aqueous formic acid solution. Cell pellets were prepared with 100 mM Tris buffer (pH 8.0) containing 3 mM calcium chloride. The reconstituted cell pellet and supernatant sample (50 μg each) were reduced with 2 mM TCEP and then denatured at 100 ° C. for 5 minutes. The sample was cooled to room temperature and then alkylated with 100 mM iodoacetamide. Alkylated samples were further reduced with 0.5 mM TCEP. Samples were treated with trypsin overnight at 37 ° C. in a water bath. The digested sample was removed from the water bath and the reaction was stopped with a 2% aqueous formic acid solution.

Recombinant protein trypsin peptides, cell pellets and supernatant samples were injected into a 150 x 2.1 mm Xbridge BEH300 C18 column. The column heater was set to 35 ° C. The mobile phase A was a 0.1% formic acid aqueous solution. The mobile phase B was 90/10 acetonitrile / water (v / v) containing 0.1% formic acid. Mass spectra of digested products were obtained with an ABSciex API QSTAR ELITE quadrupole time-of-flight mass spectrometer (AB Science, Foster City, CA). Data were acquired using positive electrospray ionization (ESI) in time-of-flight (TOF) MS and MS / MS modes. The Turbo IonSpray interface was set to 500 ° C. to maintain a turbospray of 5.0 kV and a declustering potential of 35 V. Ionization was assisted by a sprayer and ion spray gas (nitrogen) set at 40 and 35 (arbitrary unit). Data were acquired and processed using Analyst QS 2.0 software (AB Siex, Foster City, CA).

Using BioAnallyst software (AB Sciences, Foster City, CA), LC-MS data were compared to the predicted / theoretical tryptic digest products of the OTC and UGT1A1 proteins. Digested products of recombinant proteins were used as reference peptides for peptide matching of digested cell pellets and supernatants. In addition, LC-MS and LC-MS / MS data were mapped and sequenced for specific tryptic digest products of HeLa cell-derived proteins. The peptide sequence of trypsin pepti was confirmed based on the product ion spectrum by LC-MS / MS.

The OTC protein was present in the cell pellet of HeLa cells transfected with OTC mRNA. Peptide mapping identified 16 OTC peptides in the tryptic digest products of cell pellets. The product ion spectrum of the cell-derived peptide was in good agreement with the recombinant protein. A database search of human proteins showed that the sequences of 13 unmodified peptides were specific only to human OTC. The UGT1A1 protein was expressed by HeLa cells transfected with UGT1A1 mRNA. Seven UGT1A1 peptides were identified in the trypsin digested products of cell pellets based on accurate mass number determination and retention time agreement with recombinant UGT1A1 protein. The identity of the matched peptide was confirmed by LC-MS / MS. Based on the search for human proteins, 4 of the 7 trypsin peptides observed were specific for the human UGT1A protein, and 1 of the 7 trypsin peptides observed was specific only to UGT1A1. rice field. Table 203 shows peptides identified from HeLa cell lysates of UGT1A1 modified mRNA, and Table 204 shows peptides identified from OTC modified mRNA.

Table 203. UGT1A1 peptide fragment

Table 204. OTC peptide fragment

The protein profile of the cell pellet sample was further characterized by two-dimensional fluorescence differential gel electrophoresis. The test sample was treated with a different fluorescent dye. Sample inclusions were separated using isoelectric focusing (IEF) in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. The protein profiles of OTC cell pellet and UGT1A1 cell pellet were compared. DeCyder Differential Analysis software (GE Healthcare, Buckinghamshire, UK) was used for data analysis. The results of the analysis confirmed the presence of OTC and UGT1A1 in HeLa cells.

Example 161. Identification of Peptides Derived from Modified mRNAs, Including Chemical Modifications 5-Catelicidine antibacterial peptide (CAMP) -modified mRNAs fully modified with methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 1621; about not shown in the sequence. 140 nucleotide poly-A tail; 5'cap, cap 1), CAP18 modified mRNA (mRNA sequence shown in SEQ ID NO: 21638; approximately 140 nucleotide poly-A tail not shown in the sequence; 5'cap , Cap 1), Ciliary Neurotrophic Factor (CNTF) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21639; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), Follicular stimulating hormone, β-polypeptide (FSHB) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21640; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), interferon, α2 ( IFNA2) modified mRNA (mRNA sequence shown in SEQ ID NO: 1654; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), interferon, β1, fibroblast (IFNB1) modified mRNA (MRNA sequence shown in SEQ ID NO: 1655; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), methylmalonyl CoA mutase, mitochondrial (MUTA) -modified mRNA (at SEQ ID NO: 1658). The mRNA sequence shown; a poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), galectin 3 (LEG3) modified mRNA (mRNA sequence shown in SEQ ID NO: 21641; shown in the sequence. Poly-A tail of about 140 nucleotides not shown; 5'cap, cap 1), transforming growth factor β-3 (TGFB3) modified mRNA (mRNA sequence shown in SEQ ID NO: 21642; about 140 not shown in the sequence Poly-A tail of the nucleotides of Cap, cap 1) (5 mC and pU), fully modified mRN with 5-methylcytosine and 1-methylpsoiduridine A (5 mC and 1 mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine modified with 5-methylcytosine modified mRNA (s2U and 5 mC), completely modified with pseudouridine. Cellular lysates containing proteins produced from mRNA (pU) or 1-methylcytosine uridine-modified mRNA (1 mPU) using LC-MS / MS as described in Example 157. Was evaluated by quantitative LC-MRM. The peptide fragments identified in the evaluated proteins are shown in Table 205. In Table 205, "Uniprot ID" refers to the protein identifier of the UniProt database when the peptide fragment sequence was blast searched for all browsed proteins in the database.

Table 205. Sequence of protein and peptide fragments

Example 162. Detection of Low Density Lipoprotein Receptor Expression in Cell Culture A. Transfection 24-well dishes HeLa cells (Corning Life Sciences, Tewksbury, MA ) (7.5 × 10 4 cells / well) in 10% fetal calf serum (FCS, Life Technologies, Grand Island , NY) and 1 × glutamax reagent HeLa cells were seeded in Eagle's minimum essential medium (EMEM, Life Technologies, Grand Islands, NY) supplemented with (Life Technologies, Grand Islands, NY) and cultured overnight under standard cell culture conditions. Low density lipoprotein receptor (LDLR) -modified mRNA fully modified with 5-methylcytosine and pseudouridine in the first tube (mRNA sequence set forth in SEQ ID NO: 21463; poly of about 140 nucleotides not shown in the sequence. -A tail; 5'cap, cap 1), mCherry-modified mRNA (mRNA sequence shown in SEQ ID NO: 21439; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; 5- Fully modified with methylcytosine and pseudouridine) or luciferase-modified mRNA (mRNA sequence set forth in SEQ ID NO: 21446; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1; 5-methylcytosine (Completely modified with psoid uridine) 250 ng and 50 μl of Opti-MEM reagents (Life Technologies, Grand Isoland, NY) were combined, and in the second tube, L2000 transfection reagents (Life Technologies, Grand Israel, NY) By combining with 50 μl, a transfection solution was prepared for each well to be treated. After preparation, the first and second tubes were incubated at room temperature for 5 minutes, after which the respective contents were combined. The combined transfection solution was incubated at room temperature for 15 minutes. Then 100 μl of transfection solution was added to each well. The cells were cultured for an additional 16 hours before proceeding with the analysis.

B. Detection of LDLR by Flow Cytometry After transfection, medium was removed from cells and 60 μl of 0.25% trypsin (Life Technologies, Grand Island, NY) was added to each well. After treating the cells with trypsin for 2 minutes, 240 μl / well of trypsin inhibitors (Life Technologies, Grand Island, NY) were added. The resulting cell solution was transferred to a 96-well plate (Corning Life Sciences, Tewksbury, MA), the cells were pelleted by centrifugation (800 x gravity for 5 minutes) and the supernatant was discarded. The cell pellet was washed with PBS and resuspended in Foxp3 fixation / permeation solution (eBioscience, San Diego, CA) for 45 minutes. Cells were pelleted again by centrifugation (800 x gravity for 5 minutes) and resuspended in permeabilization buffer (eBiosciences, San Diego, CA) for 10 minutes. Cells were pelleted again by centrifugation (800 x gravity for 5 minutes) and washed with permeabilization buffer. Cells were then treated with a primary antibody against LDLR and then with a phycoerythrin-labeled secondary antibody. Labeled cells were then combined with FACS buffer (PBS containing 1% bovine serum albumin and 0.1% sodium azide) and transferred to cluster tubes. As shown in FIG. 47, labeled cells were then analyzed by flow cytometry using BD Accuri (BD Biosciences, San Jose, CA).

C. Detection of LDLR by Immunofluorescence Transfected cells were washed with PBS and treated with fixative (PBS containing 4% formaldehyde) for 20 minutes at room temperature. The cells were then washed with PBS and treated with permeation / blocking solution (Tris buffered saline containing 5% bovine serum albumin and 0.1% Tween-20). The cells were incubated at room temperature for 2 hours with gentle stirring and then washed 3 times with PBS containing 0.05% Tween-20. The cells are then treated with a primary antibody (goat anti-LDLR, R & D Systems, Minneapolis, MN) for 2 hours at room temperature, without a primary antibody, or with a conventional IgG control, 0.05% Tween. It was washed 3 times with PBS containing -20 and treated with a secondary antibody solution containing fluorescently labeled 1: 200 diluted donkey anti-goat IgG (R & D Systems, Minneapolis, MN). Cells were washed again with PBS containing 0.05% Tween-20 and observed by fluorescence microscopy imaging. Cells transiently expressing luciferase or mCherry were observed by fluorescence microscopy without fluorescence immunostaining.

Example 163. Detection of defensin, β103A by immunofluorescence A. Transfection 24-well dishes HeLa cells (Corning Life Sciences, Tewksbury, MA ) (7.5 × 10 4 cells / well) in 10% fetal calf serum (FCS, Life Technologies, Grand Island , NY) and 1 × glutamax reagent HeLa cells were seeded in Eagle's minimum essential medium (EMEM, Life Technologies, Grand Islands, NY) supplemented with (Life Technologies, Grand Islands, NY) and cultured overnight under standard cell culture conditions. Defensin, β103A (DEFB103A) -modified mRNA in the first tube (mRNA sequence shown in SEQ ID NO: 1631; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1) or mCherry-modified mRNA (MRNA sequence shown in SEQ ID NO: 21439; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) 250 ng and Opti-MEM reagents (Life Technologies, Grand Islands, NY) 50 ul. To prepare a transfection solution for each well to be treated by combining 1 ul of L2000 transfectional reagents (Life Technologies, Grand Islands, NY) and 50 ul of Opti-MEM in a second tube. After preparation, the first and second tubes were incubated at room temperature for 5 minutes, after which the respective contents were combined. The combined transfection solution was incubated at room temperature for 15 minutes. Then 100 ul of transfection solution was added to each well. The cells were cultured for an additional 16 hours before proceeding with the analysis.

B. Detection of DEFB103A by Immunofluorescence Transfected cells were washed with PBS and treated with fixative (PBS containing 4% formaldehyde) for 20 minutes at room temperature. The cells were then washed with PBS and treated with permeation / blocking solution (Tris buffered saline containing 5% bovine serum albumin and 0.1% Tween-20). The cells were incubated at room temperature for 2 hours with gentle stirring and then washed 3 times with PBS containing 0.05% Tween-20. The cells are then left untreated with the primary antibody for 2 hours at room temperature, treated with an anti-DEFB103A primary antibody (rabbit anti-DEFB103A, Abcam, Cambridge, MA), or treated with a conventional IgG control antibody. It was washed 3 times with PBS containing 05% Tween-20 and treated with a secondary antibody solution containing 1: 200 diluted donkey anti-goat IgG (R & D Systems, Minneapolis, MN) fluorescently labeled in red. Cells were washed again with PBS containing 0.05% Tween-20 and observed by fluorescence microscopy imaging. Cells transfected with modified mRNA encoding mCherry were visualized without immunofluorescence staining.

Example 164. Confirmation of protein expression To confirm the expression of the protein and the length of the expressed protein, modified mRNA (5 mC / pU) fully modified with 5-methylcytosine and pseudouridine encoding the proteins listed in Table 205, 5 -The mRNA completely modified with methylcytosine and 1-methylpsoiduridine (5 mC / 1 mpU), 25% of cytosine is replaced with 5-methylcytosine and 25% of uridine is substituted with 2-thiouridine. The same mRNA (s2U / 5mC), the same mRNA completely modified with 1-methylcytosine uridine (1 mpU) or the same mRNA completely modified with pseudocytosine (pU) was analyzed by fluorescence and / or western blot. Modified mRNA (poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1) or cDNA (T7 promoter used for in vitro transcription (IVT) shown in the sequence, 5'untranslated The sequences of the regions (UTRs) and 3'UTRs, the chemical modifications evaluated and the lengths (base pairs) if known are listed in Table 205.

Table 205. Protein and expression

Example 165. Expression of vascular endothelial growth factor and cell viability The day before transfection, 100,000 HeLa cells (ATCC No. CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (Life Technologies, Grand Island, NY). A 24-well cell culture plate (Corning, Manassas, VA) was seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, fully modified vascular endothelial growth factor (VEGF) -modified RNA with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1672; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5 'Cap, cap 1) (5 mC / pU), fully modified RNA with 5-methylcytosine and 1-methylpsoid uridine (5 mC / 1 mpU), fully modified with 1-methylpsoid uridine. RNA (1 mpU) or unmodified RNA (unmodified) 62 ng, 250 ng, 750 ng or 1500 ng was mixed with 2 ul of Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as a transfection aid and double-repeated into cells. added.

A. Protein Expression of Vascular Endothelial Growth Factor After a 24-hour incubation, cell culture supernatants of VEGF-expressing cells were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a VEGF-specific ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. In addition, untreated cells, fully modified green fluorescent protein (GFP) -modified mRNA with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1672; poly-of approximately 160 nucleotides not shown in the sequence. A-tail; 5'cap, cap 1) (5 mC / pU), fully modified with 5-methylcytosine and 1-methylpsoid uridine (5 mC / 1 mpU), completely with 1-methylpsoid uridine Control and mCherry-modified mRNA (mRNA sequence shown in SEQ ID NO: 21439; shown in the sequence) of cells treated with double repeats of the modified mRNA (1 mpU) or the unmodified mRNA (unmodified) 62 ng or 750 ng. Not about 160 nucleotides of poly-A tail; 5'cap, cap 1) 250 ng controls were analyzed. The amount of VEGF produced (pg / ml) is shown in Table 206 and FIG. In FIG. 48, "eGFP" refers to green fluorescent protein-modified mRNA, "MC" refers to mCherry-modified mRNA, "UNT-1" refers to untreated cells, and "G0" refers to modified mRNA without modification. Means that "G1" is fully modified with 5-methylcytosine and pseudouridine, and "G1" is fully modified with 5-methylcytosine and 1-methylpsoiduridine. Meaning, "G5" means fully modified with 1-methylcytosine uridine.

Highest VEGF expression in cells transfected with VEGF-modified mRNA fully modified with 5-methylcytosine and 1-methylpseduoruidine or the same mRNA fully modified with 1-methylpseuduridine. Was seen.

Table 206. VEGF protein production (pg / ml)

B. Vascular Endothelial Growth Factor After 24 hours of incubation, media was removed and cells were lysed with 200 ul of Cell Titter Glo (CTG) assay reagent (Promega Corporation, Madison, WI) per well. After incubating for 15 minutes at room temperature, two sets of 10 ul of each solubilizer were transferred to white opaque polystyrene 96-well plates (Corning, Tewksbury, MA). 100 ul of Cell Titter Glo assay reagent was added again to each aliquot of cell lysate. A total volume of 110 ul of reagent mixture was analyzed on a BioTek Synergy H1 plate reader using a common luminescence program. Mean values of double repeat measurements were plotted on a graph with reference to untreated HeLa cells (1.0 = 100% viability). In addition, double-repeat untreated cells and mCherry-modified mRNA (mRNA sequence shown in SEQ ID NO: 1672; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) treated with 250 ng. The control of the cells was evaluated. Normalized ATP levels / cell viability are shown in Table 207 and FIG. In FIG. 49, "MC" refers to mCherry-modified mRNA, "UNT-1" and "UNT-2" refer to untreated cells, "G0" means that the modified mRNA does not contain modification, and "G1". "" Means fully modified with 5-methylcytosine and pseudouridine, "G1" means fully modified with 5-methylcytosine and 1-methylpsoiduridine, "G5" Means that it is completely modified with 1-methylcytosine uridine, and " ^* " means that only one data point was obtained.

Cells transfected with VEGF-modified mRNA fully modified with 5-methylcytosine and pseudouridine or the same unmodified mRNA showed reduced luminescent activity, an indicator of loss of cell viability. Cells transfected with VEGF-modified mRNA fully modified with 5-methylcytosine and 1-methylpseudouridine or the same mRNA fully modified with 1-methylpsoiduridine No decrease in viability was observed.

Table 207. Normalized ATP amount / cell viability

Example 166. Protein production of erythropoetin in HeLa cell supernatant The day before transfection, 100,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY), 96. Well cell culture plates (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, erythropoetin (EPO) -modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1638; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, Cap 1) (5 mC / pU), 5-methylcytosine and 1-methylcytosine completely modified RNA (5 mC / 1 mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine 250 ng of 5-methylcytosine-modified and modified RNA (s2U and 5 mC), fully modified pseudouridine (pU) or 1-methylcytosine-modified RNA (1 mpU) Was diluted with OPTI-MEM (Life Technologies, Grand Israel, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of cells treated with lipofecatmine 2000 (L2000) and untreated cells were analyzed.

After incubation for 18-22 hours, cell culture supernatants of cells expressing EPO were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with an EPO ELISA kit (Stem Cell Technologies, Vancouver Canada) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 208.

Table 208. Protein production

Example 167. In vitro expression of insulin aspart-modified mRNA Fully modified insulin aspart-modified mRNA with 5-methylcytosine and pseudouridine complexed with Lipofectamine 2000 from Invitrogen (Carlsbad, CA) (presented in SEQ ID NO: 21468). mRNA sequence; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) (5 mC / pU), fully modified with 5-methylcytosine and 1-methylpsoiduridine. mRNA (5mC / 1mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine modified with 5-methylcytosine (s2U and 5mC), completely modified with pseudouridine Cells were transfected with the same mRNA (1 mPU) fully modified with mRNA (pU) or 1-methylcytosine uridine at the concentrations shown in Table 209. The total volume of the transfection mixture per 96 wells was 50 ul and was divided into two 25 ul aliquots, respectively. One 25 ul aliquot contained 1 ul of each mMr and Opti-MEM 24 ul, and the other 25 ul aliquot contained Lipofectamine 2000 0.4 ul and Opti-MEM 24.6 ul. If necessary, an aliquot containing modified mRNA and Opti-MEM was serially diluted with Opti-MEM. Both aliquots were incubated separately for 15 minutes at room temperature. Both aliquots were then mixed and incubated for an additional 20 minutes at room temperature before finally transferring a total of 50 ul of transfection mixture to 96 wells containing 100 ul of cell culture medium and 35,000 HEK293 cells. The cells were then incubated for 24 hours in a humidified 37 ° C./5% CO ₂ cell culture incubator before collecting cell culture supernatants or preparing cell lysates. Protein expression was detected by ELISA and the protein (pg / ml) is shown in Figure 50 and Table 209. In Table 209, ">" means to exceed.

Table 209. Protein expression

Example 168. In vitro expression of insulin glargine-modified mRNA Fully modified insulin glargine-modified mRNA with 5-methylcytosine and pseudouridine complexed with Lipofectamine 2000 from Invitrogen (Carlsbad, CA) (mRNA sequence shown in SEQ ID NO: 21465). Poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) (5 mC / pU), 5-methylcytosine and 1-methylpsoid uridine fully modified mRNA ( 5 mC / 1 mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine modified with 5-methylcytosine (s2U and 5 mC), the same mRNA completely modified with pseudouridine (5 mC / 1 mC) Cells were transfected with the same mRNA (1 mPU) fully modified with pU) or 1-methylcytosine uridine at the concentrations shown in Table 210. The total volume of the transfection mixture per 96 wells was 50 ul and was divided into two 25 ul aliquots, respectively. One 25 ul aliquot contained 1 ul of each mMr and Opti-MEM 24 ul, and the other 25 ul aliquot contained Lipofectamine 2000 0.4 ul and Opti-MEM 24.6 ul. If necessary, an aliquot containing modified mRNA and Opti-MEM was serially diluted with Opti-MEM. Both aliquots were incubated separately for 15 minutes at room temperature. Both aliquots were then mixed and incubated for an additional 20 minutes at room temperature before finally transferring a total of 50 ul of transfection mixture to 96 wells containing 100 ul of cell culture medium and 35,000 HEK293 cells. The cells were then incubated for 24 hours in a humidified 37 ° C./5% CO ₂ cell culture incubator before collecting cell culture supernatants or preparing cell lysates. Protein expression was detected by ELISA and the protein (pg / ml) is shown in FIG. 51 and Table 210.

Table 210. Protein expression

Example 169. In vitro expression of insulin glulisine-modified mRNA Fully modified insulin glulisine-modified mRNA with 5-methylcytosine and pseudouridine complexed with Lipofectamine 2000 from Invitrogen (Carlsbad, CA) (shown in SEQ ID NO: 21469). mRNA sequence; poly-A tail of about 160 nucleotides not shown in the sequence; the mRNA fully modified with 5'cap, Cap) (5 mC / pU), 5-methylcytosine and 1-methylpsoiduridine. (5mC / 1mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine modified with 5-methylcytosine (s2U and 5mC), the same mRNA fully modified with pseudouridine Cells were transfected with the same mRNA (1 mpU) fully modified with (pU) or 1-methylcytosine uridine at the concentrations shown in Table 211. The total volume of the transfection mixture per 96 wells was 50 ul and was divided into two 25 ul aliquots, respectively. One 25 ul aliquot contained 1 ul of each mMr and Opti-MEM 24 ul, and the other 25 ul aliquot contained Lipofectamine 2000 0.4 ul and Opti-MEM 24.6 ul. If necessary, an aliquot containing modified mRNA and Opti-MEM was serially diluted with Opti-MEM. Both aliquots were incubated separately for 15 minutes at room temperature. Both aliquots were then mixed and incubated for an additional 20 minutes at room temperature before finally transferring a total of 50 ul of transfection mixture to 96 wells containing 100 ul of cell culture medium and 35,000 HEK293 cells. The cells were then incubated for 24 hours in a humidified 37 ° C./5% CO ₂ cell culture incubator before collecting cell culture supernatants or preparing cell lysates. Protein expression was detected by ELISA and the protein (pg / ml) is shown in FIG. 52 and Table 211. In Table 211, ">" means greater.

Table 211. Protein expression

Example 170. Dose-Response and Injection Site Selection and Injection Timing Tests are performed according to the protocol outlined in Example 35 to determine dose-response trends, injection site effects, and injection timing effects. This test reveals the results of a dose response using various amounts of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug and values in between. A divided dose of a total dose of 100 ug comprises 3 or 6 doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6 ug or a value, the total dose equal to the dose of the selected total dose.

Select the injection site from the limbs or any body surface that provides sufficient area for injection. This may also include selection of injection depth targeting the dermis (intradermal), epidermis (epidermis), subcutaneous tissue (SC) or muscle (IM). The injection angle depends on the target delivery site, with injections targeting intradermal sites 10 to 15 degrees to the skin surface, subcutaneous injections 20 to 45 degrees to the skin surface, and substantial intramuscular injection. Then, the angle is 60 to 90 degrees.

Example 171. Protein production of transforming growth factor β1 in HeLa cell supernatant The day before transfection, 100,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Collected and seeded in 96-well cell culture plates (Corning, Manassas, VA) in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, transforming growth factor β1 (TGFB1) -modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1668; poly-A tail of approximately 140 nucleotides not shown in the sequence; 5'cap, cap 1) (5 mC / pU), fully modified RNA with 5-methylcytosine and 1-methylpsoid uridine (5 mC / 1 mpU), 25% of uridine modified with 2-thiouridine and cytosine 25% of the same RNA modified with 5-methylcytosine (s2U and 5mC), the same RNA fully modified with pseudouridine (pU) or the same RNA completely modified with 1-methylpsoidurine 250 ng (1 mpU) was diluted with OPTI-MEM (Life Technologies, Grand Israel, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of cells treated with lipofecatmine 2000 (L2000) and untreated cells were analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing TGFB1 were collected and centrifuged at 10.000 rcf for 2 minutes. The clarified supernatant was then analyzed with a TGFB1 ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 212.

Table 212. Protein production

Example 172. Protein production of transforming growth factor β1 in HeLa cell supernatant The day before transfection, 100,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY). Collected and seeded in 96-well cell culture plates (Corning, Manassas, VA) in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, transforming growth factor β1 (TGFB1) -modified RNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1668; poly-A tail of approximately 140 nucleotides not shown in the sequence; 5'cap, cap 1) (5 mC / pU), fully modified RNA with 5-methylcytosine and 1-methylpsoid uridine (5 mC / 1 mpU), 25% of uridine modified with 2-thiouridine and cytosine 25% of the same RNA modified with 5-methylcytosine (s2U and 5mC), the same RNA fully modified with pseudouridine (pU) or the same RNA completely modified with 1-methylpsoidurine 250 ng (1 mpU) was diluted with OPTI-MEM (Life Technologies, Grand Israel, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, controls of cells treated with lipofecatmine 2000 (L2000) and untreated cells were analyzed.

After incubation for 18-22 hours, cell culture solubilized solutions of cells expressing TGFB1 were collected and centrifuged at 10.000 rcf for 2 minutes. Cell lysates (7 ug / ELISA) were then analyzed with the TGFB1 ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were diluted until the determination was within the linear range of the ELISA standard curve. The amount of protein produced is shown in Table 213.

Table 213. Protein production

Example 173. Identification of Peptides Derived from Modified mRNA 5-Angiotensin-converting enzyme 2 (ACE2) -modified mRNA fully modified with methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 21678; approximately 140 nucleotides not shown in the sequence Poly-A tail; 5'cap, cap 1), activity-dependent neuroprotective factor (ADNP) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21679; poly-A tail of about 140 nucleotides not shown in the sequence. 5'cap, cap 1), septin-4 (ARTS) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21680; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1) , Bone-forming protein 2 (BMP2) -modified mRNA (mRNA sequence shown in SEQ ID NO: 21681; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), celluloplasmin (CP) modification. mRNA (mRNA sequence shown in SEQ ID NO: 1620; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1), COMM domain-containing protein 1 (COMMD1) -modified mRNA (in SEQ ID NO: 21682). The mRNA sequence shown; a poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1), diablo, IAP-binding mitochondrial protein (DIABLO or SMAC) -modified mRNA (mRNA shown in SEQ ID NO: 21683). Sequence; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1), coagulation factor XIII α (F13A1) modified mRNA (mRNA sequence shown in SEQ ID NO: 21684; shown in the sequence Poly-A tail of about 160 nucleotides not shown; 5'cap, cap 1), hepcidin (HEPC) modified mRNA (mRNA sequence shown in SEQ ID NO: 21471; poly-A of about 160 nucleotides not shown in the sequence Tail; 5'cap, cap 1), IgM heavy chain modified mRNA (mRNA sequence shown in SEQ ID NO: 21685; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1), inter Leukin-21 (IL21) modified mRNA (mRNA sequence set forth in SEQ ID NO: 21686; approximately 160 nu not shown in the sequence Cleotide poly-A tail; 5'cap, cap 1), N-acetylglutamate synthase (NAGS) -modified mRNA (mRNA sequence set forth in SEQ ID NO: 21687; poly-A of approximately 160 nucleotides not shown in the sequence Tail; 5'cap, cap 1) or E3 ubiquitin-protein ligase (XIAP) -modified mRNA (mRNA sequence set forth in SEQ ID NO: 21688; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, Cap 1) (5 mC and pU), the same mRNA fully modified with 5-methylcytosine and 1-methylpsoid uridine (5 mC and 1 mpU), 25% of uridine modified with 2-thiouridine and 25% of cytosine From the same mRNA modified with 5-methylcytosine (s2U and 5mC), the same mRNA completely modified with pseudouridine (pU) or the same mRNA completely modified with 1-methylpsoiduridine (1 mpU) Cell lysates containing the protein produced were evaluated quantitatively by LC-MRM using LC-MS / MS as described in Example 157. The peptide fragments identified in the evaluated proteins are shown in Table 214.

Table 214. Sequence of protein and peptide fragments

Example 174. Confirmation of protein expression To confirm the expression of the protein and the length of the expressed protein, modified mRNA (5 mC / pU) fully modified with 5-methylcytosine and pseudouridine encoding the proteins shown in Table 215, 5 -The mRNA completely modified with methylcytosine and 1-methylpsoiduridine (5 mC / 1 mpU), 25% of cytosine is replaced with 5-methylcytosine and 25% of uridine is substituted with 2-thiouridine. The same mRNA (s2U / 5mC), the same mRNA completely modified with 1-methylcytosine uridine (1 mpU) or the same mRNA completely modified with pseudocytosine (pU) was evaluated by fluorescence and / or western blot. Table 215 shows the sequence of the modified mRNA (poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1), the chemical modification evaluated and the length (base pair number) if known. Described in.

Table 215. Protein and expression

Example 175. Pulmonary delivery of 1-methylpseudouridine or 5-methylcytosine and 1-methylpseudouridine-modified luciferase mRNA formulated in cationic lipid nanoparticles Completely modified with 1-methylpseudouridine (Luc) -G5-LNP-KC2), or fully modified with 1-methylpseudouridine and 5-methylcytosine (Luc-G2-LNP-KC2), luciferase-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21446; about A 140 nucleotide poly A tail is not shown in the sequence; 5'cap, cap 1) was formulated in cationic lipid nanoparticles (LNP-KC2) as shown in Table 216.
Table 216. pharmaceutical formulation

The formulation was administered intranasally (IN) at a dose of 0.3 mg / kg to Balb-C mice. Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, 48, and 72 hours after dosing and average total luminous flux (photons / sec) was measured and is shown in Table 217. The background luminous flux was about 6.3 + 05p / s. The results of imaging Luc-G5-LNP-KC2 or Luc-G5-LNP-KC2 vs. vehicle are shown in Table 217, where "NT" means not tested.
Table 217. Luciferase expression after intranasal dosing

Example 176. Pulmonary delivery of lipoplex-formed 1-methylpseudouridine or 5-methylpseudouridine and 1-methylpseudouridine-modified luciferase mRNA in Lipofectamine 2000 by intranasal administration Completely modified with 1-methylpseudouridine (Luc-G5-) Luciferase-modified mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A of approximately 140 nucleotides), fully modified with 1-methylseudouridine and 5-methylcytosine (Luc-G2-lipoplex). The tail is not shown in the sequence; 5'cap, cap 1) by first step diluting G5 or G2-modified luciferase mRNA from 3 mg / mL to 16.6 μL into 108.5 μL DMEM. There, the second step was to dilute 100 μL of LF2000 into 25 μL of DMEM and then gently mix both together to make a total of 250 μL of mixer, in which the lipoplex was formed. It was incubated for 5-10 minutes at room temperature to form a lipid-mRNA complex prior to injection. Lipoplexes from 1-methylpseudouridine or both 1-methylpseudouridine and fully modified 5-methylcytosine luciferase mRNA in the nasal cavity (IN) at a dose of 0.5 mg / kg Balb -C mice were administered.

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, and 24 hours after dosing and average total luminous flux (photons / sec) was measured and shown in Table 218. The background signal was about 4.8 + 05p / s. The background luminous flux was about 6.3 + 05p / s. The results of imaging of Luc-G5-lipoplex or Luc-G2-lipoplex vs. vehicle are shown in Table 218.
Table 218. Luciferase expression after intranasal dosing

Example 177. Pulmonary delivery of 1-methylpseudouridine or 5-methylpseudouridine and 1-methylpseudouridine-modified luciferase mRNA formulated in PBS 1-methylpseud formulated in PBS (pH 7.4) Completely modified with uridine (Luc-G5-buffer (PBS)) or completely modified with 1-methylpseudouridine and 5-methylcytosine (Luc-G2-buffer (PBS)), luciferase modification mRNA (mRNA sequence is shown in SEQ ID NO: 21446; poly A tail of about 140 nucleotides is not shown in the sequence; 5'cap, cap 1) in the nasal cavity (IN) 7.5 mg / kg. The dose was administered to Balb-C mice.

Twenty minutes before imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg / kg. The animals were then anesthetized and images were acquired using the IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as the total luminous flux (photons / sec) of all mice. Mice were imaged 2 hours, 8 hours, and 24 hours after dosing and average total luminous flux (photons / sec) was measured and shown in Table 219. .. The background luminous flux was about 4.8 + 05p / s. The background luminous flux was about 6.3 + 05p / s. The results of imaging Luc-G5 vs. vehicle in buffer are shown in Table 219.
Table 219. Luciferase expression after intranasal dosing

Example 178. In vitro transfection of interleukin 7 In human keratinocyte cells of a 24-well plate, interleukin 7 (IL7) -modified mRNA (mRNA sequence shown by SEQ ID NO: 1656; poly-A of about 160 nucleotides not shown in the sequence. Tail; 5'cap, cap 1) was transfected by reverse transfection. Human keratinocyte cells were grown in Invitrogen (Carlsbad, CA) Supplement S7-added EPILIFE® medium until 50-70% confluence was reached. Modified mRNA (m mRNA) 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng and 750 ng encoding IL-7 in which cells were complexed with RNAIMAX ™ from Invitrogen (Carlsbad, CA). Transfected. RNA: RNAIMAX ™ complex was first formed by incubating RNA at room temperature with Supplement-free EPILIFE® medium at 5-fold volume dilution for 10 minutes. In the second vial, the RNAIMAX ™ reagent was incubated with Supplement-free EPILIFE® medium at room temperature for 10 minutes at 10-fold volume dilution. RNA vials and RNAIMAX ™ vials were then mixed, incubated at room temperature for 20-30 minutes, and then added dropwise to the cells.

A fully optimized mRNA encoding IL-7 (mRNA sequence set forth in SEQ ID NO: 1656; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1) 5- Completely modified with methylcytosine and pseudouridine. Cells are transfected with mmRNA encoding IL-7, and 6 and 12 hours after transfection, culture medium for each concentration according to the manufacturer's recommended instructions using the Invitrogen (Carlsbad, CA) ELISA kit. The IL-7 concentration (pg / ml) in the medium was measured. These data shown in FIG. 53 show that at 375 ng and 750 ng amounts, the modified mRNA encoding IL-7 is translatable within human keratinocyte cells.

Example 179. In vitro transfection of erythropoietin 24 Multiwell plates of erythropoietin (EPO) -modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; poly-A tail of about 160 nucleotides not shown in the sequence; 5' The cap and cap 1) were transfected by reverse transfection. Human keratinocyte cells were grown in Invitrogen (Carlsbad, CA) Supplement S7-added EPILIFE® medium until 50-70% confluence was reached. Cells were transfected with 46.875 ng, 93.75 ng, 187.5 ng, 375 ng and 750 ng of EPO-encoding modified mRNA (m mRNA) complexed with Invitrogen (Carlsbad, CA) RNAIMAX ™. RNA: RNAIMAX ™ complex was first formed by incubating RNA at room temperature with Supplement-free EPILIFE® medium at 5-fold volume dilution for 10 minutes. In the second vial, the RNAIMAX ™ reagent was incubated with Supplement-free EPILIFE® medium at room temperature for 10 minutes at 10-fold volume dilution. RNA vials and RNAIMAX ™ vials were then mixed, incubated at room temperature for 20-30 minutes, and then added dropwise to the cells.

Fully optimized mRNA encoding EPO (mRNA sequence shown in SEQ ID NO: 1638; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) is 5-methylcytosine. And fully modified with pseudouridine. Cells are transfected with mmRNA encoding EPO and 6 and 12 hours after transfection, in culture medium for each concentration according to the manufacturer's recommended instructions using the Invitrogen (Carlsbad, CA) ELISA kit. The EPO concentration (pg / ml) was measured. These data, shown in FIG. 54, indicate that the modified mRNA encoding EPO is translatable within human keratinocyte cells.

Example 180. Detection of lysosomal acid lipase protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Was collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. .. The cells were cultured overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, fully modified lysosomal acid lipase (LAL) mRNA with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1657; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap , Cap 1) 750 ng was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent, and 5.5 ul was OPTI-MEM® (Registered Trademark) with a final volume of 250 ul (Life Technologies, Grand).
It was diluted with Island, NY). After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. The primary antibody against the target protein (anti-lysosomal lipase antibody; Abcam, catalog number ab89771) was diluted 1: 500 to 1: 2000 in 3 ml of a 1 × TBS solution containing 5% BSA, gently stirring with an orbital shaker. , Reacted at room temperature for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The secondary antibody (Western Breeze, Invitrogen) is conjugated with horseradish peroxidase and bound to the primary antibody. The secondary antibody was diluted 1: 1000 to 1: 5000 with 1 × TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in FIG. 55, Western blot detection of LAL was observed near 45.4 kDa. In FIG. 55, lanes 1 and 2 show modified mRNA, and lanes 3 and 4 show only the dosing solvent.

Example 181. Detection of glucocerebrosidase protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Was collected and seeded in 6-well cell culture plates (Corning, Manassas, VA) in Eagle's Minimal Essential Medium (EMEM) (added 10% bovine fetal serum (FCS) and 1 x Glutamax) with a total volume of 3 ml per well. .. The cells were cultured overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, a glucocerebrosidase mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 1645; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1 ) 750 ng was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. The primary antibody against the target protein (anti-GBA antibody; Abcam, Catalog No. ab55080 and Sigma, Catalog No. G4046) is diluted 1: 500 to 1: 2000 in 3 ml of 1 × TBS solution containing 5% BSA and gently in an orbital shaker. The reaction was carried out at room temperature for 3 hours with stirring. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The secondary antibody (Western Breeze, Invitrogen) is conjugated with horseradish peroxidase and bound to the primary antibody. The secondary antibody was diluted 1: 1000 to 1: 5000 with 1 × TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in FIG. 56, Western blot detection of glucocerebrosidase was observed near 59.7 kDa. In FIG. 56, lanes 1 and 2 show modified mRNA, and lanes 3 and 4 show only the dosing solvent.

Example 182. Detection of isulonic acid-2-sulfatase protein: Western blot The day before transfection, HeLa cells (ATCC number CCL-2; Manassas, VA) 750 by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (LifeTechnologies, Grand Island, NY). Collected 000 cells and added 3 ml of Eagle's Minimal Essential Medium (EMEM) per well of 6-well cell culture plates (Corning, Manassas, VA) (10% bovine fetal serum (FCS) and 1 x Glutamax added). Sown in. The cells were cultured overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, iduronate-2-sulfatase mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence set forth in SEQ ID NO: 1652; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap , Cap 1) 750 ng was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent, and 5.5 ul was OPTI-MEM® (Registered Trademark) with a final volume of 250 ul (Life Technologies, Grand).
It was diluted with Island, NY). After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. The primary antibody against the target protein (anti-iduronate-2-sulfatase antibody; Abcam, Catalog No. ab70025 and R & D Systems, Catalog No. AF2449) was diluted 1: 500 to 1: 2000 in 3 ml of 1 × TBS solution containing 5% BSA. The reaction was carried out at room temperature for 3 hours with gentle stirring in an orbital shaker. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The secondary antibody (Western Breeze, Invitrogen) is conjugated with horseradish peroxidase and bound to the secondary antibody. The secondary antibody was diluted 1: 1000 to 1: 5000 with 1 × TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in FIG. 57, Western blot detection of iduronate-2-sulfatase was observed near 76 kDa. In FIG. 57, lanes 1 and 2 show modified mRNA, and lanes 3 and 4 show only the dosing solvent.

Example 183. Detection of luciferase protein: Western blot The day before transfection, 750,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Life Technologies, Grand Island, NY). Then, they were seeded in Eagle's minimum essential medium (EMEM) (10% bovine fetal serum (FCS) and 1 × Glutamax added) having a total volume of 3 ml per well of a 6-well cell culture plate (Corning, Manassas, VA). The cells were cultured overnight at 37 ° C. in a 5% CO2 atmosphere. The next day, 750 ng of luciferase mRNA fully modified with 5-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO: 21446; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1) Was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) having a final volume of 250 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 5.5 ul was diluted with OPTI-MEM® (Life Technologies, Grand Island, NY) in a final volume of 250 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 500 ul of the combined solution was added to 3 ml of cell culture medium containing HeLa cells. The plates were then incubated as described above.

After 16-18 hours of incubation, medium was removed and cells were washed with 1 ml PBS. After the PBS was completely removed, 500 ul of fresh PBS was added. Cells were scraped and collected with a cell scraper. The cells treated with the same mRNA and collected were then placed in a single 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. Remove PBS and dissolve cells in 250 ul of Radioimmunoprecipitation (RIPA) buffer (containing a mixture of PMSF and eukaryotic protease inhibitors) (BostonBioProducts (Ashland, MA)) by gentle pipetting. bottom. The solubilized solution was clarified by centrifugation at 10,000 rpm, 4 ° G. and 10 minutes. The clarified solubilized solution was transferred to an Amicon filter (Waters, Milford, MA) having a molecular weight cutoff of 10,000 kd and rotated at 12,000 rpm at 4 ° G. for 20 minutes. The reversed filter was placed in an unused 1.5 ml Eppendorf tube and rotated at 3,000 rpm for 1 minute to recover the concentrated protein solubilizer. The final volume of the solubilizer was 25-40 ul.

Protein concentrations were determined using a BCA kit for Microtiter plates from Pierce (Thermo Fisher, Rockford, IL). The standard protein of the titration curve was dissolved in RIPA buffer (as described for cell lysate preparation) rather than in dilution buffer.

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 4 ul of RIPA buffer with a volume of 26 ul, 10 ul of reducing agent and 10 ul of 4 x SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. The primary antibody against the target protein (anti-luciferase, AbCam, catalog number ab2176) is diluted 1: 500 to 1: 2000 in 3 ml of 1 × TBS solution containing 5% BSA at room temperature with gentle stirring in an orbital shaker. It was allowed to react for 3 hours. Membranes were washed 3 times with 1 x TBS / 0.1% Tween for 5 minutes with gentle stirring each time. The secondary antibody (Western Breeze, Invitrogen) is conjugated with horseradish peroxidase and bound to the primary antibody. The secondary antibody was diluted 1: 1000 to 1: 5000 with 1 × TBS containing 5% BSA and incubated at room temperature for 3 hours. As shown in FIG. 58, Western blot detection of luciferase was observed near 60.7 kDa. In FIG. 58, lanes 1 and 2 show modified mRNA, and lanes 3 and 4 show only the dosing solvent.

Example 184. Herceptin in vivo test Herceptin heavy chain (HC) modified mRNA (SEQ ID NO: 21451; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and 1-methylpu Completely modified with soidouridine) and Herceptin light chain (LC) modified mRNA (SEQ ID NO: 21452; poly-A tail of approximately 140 nucleotides not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and 1 -Completely modified with methylcytosine uridine) was formulated with DLin-MC3-DMA and DLin-KC2-DMA as shown in Table 220.

Table 220. DLin-MC3-DMA and DLin-KC2-DMA preparations

The Herceptin preparations shown in Table X1 were intravenously administered to mice (CD1) at a dose of 0.5 mg / kg. In addition, a control of luciferase mRNA was evaluated (Luc in FIG. 59). Blood was collected from mice at 8 hours, 24 hours, 48 hours, 96 hours, 168 hours (7 days), 336 hours (14 days), and 504 hours (21 days) after Herceptin administration, and ELISA (ABCAM®, Cambridge) was taken. The serum IgG concentration was determined by the whole human IgG detection kit by MA). Blood was drawn only at 8 hours for individuals injected with control luciferase. The results are shown in Table 222 and FIG. 59.

Table 222. Herceptin preparation

Example 185. Herceptin in vitro test The day before transfection, 15,000 HeLa cells (ATCC number CCL-2; Manassas, VA) were collected by treatment with trypsin-EDTA solution (LifeTechnologies, Grand Island, NY), and a 96-well cell culture plate. (Corning, Manassas, VA) were seeded in EMEM medium (added 10% FCS and 1 × Glutamax) with a total volume of 100 ul per well. Cells were cultured overnight at 37 ° C. in a 5% CO _{2 atmosphere.} The next day, Herceptin heavy chain (HC) mRNA (SEQ ID NO: 21451; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and 1-methylpus eduridine) ) 150 ng or Herceptin light chain (LC) (SEQ ID NO: 21452; poly-A tail of about 140 nucleotides not shown in the sequence; 5'cap, cap 1; 5-methylcytosine and 1-methyl 75 ng (fully modified with methylcytosine) or 500 ng of Herceptin HC / 250 ng of Herceptin LC was diluted with OPTI-MEM (Life Technologies, Grand Islands, NY) having a final volume of 10 ul. Lipofectamine 2000 (Life Technologies, Grand Island, NY) was used as a transfection reagent and 0.2 ul of Lipofectamine 2000 was diluted with OPTI-MEM in a final volume of 10 ul. After incubating at room temperature for 5 minutes, the two solutions were combined and incubated at room temperature for an additional 15 minutes. Then, 20 ul of the combined solution was added to 100 ul of cell culture medium containing HeLa cells, and the mixture was incubated at room temperature. In addition, as controls, culture medium (assay buffer + control medium) in untreated wells in which recombinant human IgG (Hu IgG control), assay buffer and assay buffer were present was analyzed.

After 18-22 hours of incubation, cell culture supernatants of cells expressing Herceptin heavy or light chains were collected and manufacturer's instructions were taken using an All Human IgG ELISA Kit (ABCAM®, Cambridge MA). Analyzed according to. The amount of IgG produced is shown in Table 223 and FIG. In Table X3, "OTC" means a value that does not fit in the graph.

Table 223. Herceptin test

Example 186. Herceptin in vitro test: in vitro by immunoblotting
Confirmation of antibody production of vitro Herceptin heavy chain and light chain mRNA (Herceptin heavy chain (HC) mRNA (SEQ ID NO: 21451; not shown in the sequence) in the absence of serum, as described in Example 185. Poly-A tail of 140 nucleotides; 5'cap, cap 1; fully modified with 5-methylcytosine and 1-methylpus eduridine), Herceptin light chain (LC) (SEQ ID NO: 21452; in sequence Approximately 140 nucleotides not shown in the poly-A tail; 5'cap, cap 1; 5-methylcytosine and 1-methylpus eduridine (fully modified) or Herceptin HC / LC (H & L)) Transfected in vivo. Twenty-four hours after transfection, the supernatant was collected and concentrated in the presence of reducing agent (lanes 1-5 in FIG. 61) and in the absence (lanes 6-10 of FIG. 61), 4-12% MOPS NuPAGE. Each lane of the gel was loaded with 4 ug of protein and electrophoretic separation was performed. The protein was then transcribed onto a PVDF membrane and the Ig heavy chain and Ig light chain were detected simultaneously by searching with an antibody.

As shown in FIG. 61, a light chain band was detected under both reducing and non-reducing conditions in the lane where the supernatant of cells transfected with only the light chain was run. Under non-reducing conditions, a relatively large band was also detected near 50 kDa. This fully supports the fact that only the light chain is secretory. No signal was observed in the lane containing the supernatant of cells transfected with heavy chain mRNA alone, with or without a reducing agent, confirming that heavy chain alone is not secreted. Two bands were found in the supernatant of cells transfected with both heavy and light chain mRNAs with and without reducing agents. A band specific to the light chain was observed in both conditions. It represented a light chain derived from a denatured Ig molecule under reducing conditions and a free light chain protein under non-reducing conditions. Under reducing conditions, heavy chains secreted only in association with the light chain were found at the 55 kDa position, whereas under non-reducing conditions, individual heavy chain bands were seen, as expected. There wasn't. In this lane, a band of all Ig of 160 kDa was clearly detected. This supports the molecular identity of both proteins produced when HeLa cells are transfected with chemically modified mRNAs encoding light and heavy chain proteins.

In FIG. 61, "MWM" represents a molecular weight marker, "LC" represents a Herceptin light chain, "HC" represents a Herceptin heavy chain, and "medium" represents culture medium only.

Example 187. Enzyme activity assay A. Enzyme activity of glucocerebrosidase Glucocerebrosidase-modified mRNA (mRNA sequence shown by SEQ ID NO: 1645; poly-A tail of about 160 nucleotides not shown in the sequence; poly-A tail, which is complexed with RNAiMAX (Invitrogen) in HEK293 cells; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) 0 ng, 625 ng, 125 ng, 250 ng, 500 ng, 1000 ng or 2000 ng was transfected. Cells were washed with PBS 6 hours, 12 hours, 24 hours and 48 hours after transfection. The cells were then lysed in 100 mM citrate-phosphate buffer (pH 5.4) containing 0.54% sodium taurodeoxycholate and 1% Triton X-100. The cell lysates were then incubated at 37 ° C. for 1 hour in the presence of 0.5 mM 4-methylumbelliferyl β-D-galactopyranoside (4-MUG) substrate. After 1 hour, the reaction was stopped with an equal volume of 200 mM sodium hydroxide. The conversion of 4-MU was measured with a plate reader (Ex = 370 nm; Em = 460 nm). FIG. 62 shows an mRNA control transfection complexed with RNAiMAX (Invitrogen) (iduronate-2-sulfatase (mRNA sequence set forth in SEQ ID NO: 1652; poly-A tail of approximately 160 nucleotides not shown in the sequence). It shows the enzymatic activity expressed as a percentage normalized to 5'cap, cap 1) fully modified with 5-methylcytosine and pseudouridine).

B. Enzymatic activity of lysosomal acid lipase Lysosomal acid lipase-modified mRNA (mRNA sequence shown by SEQ ID NO: 1657; poly-A tail of about 160 nucleotides not shown in the sequence; poly-A tail, which is complexed with RNAiMAX (Invitrogen) in HEK293 cells; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) 0 ng, 625 ng, 125 ng, 250 ng, 500 ng, 1000 ng or 2000 ng was transfected. Cells were washed with PBS 6 hours, 12 hours, 24 hours and 48 hours after transfection. The cells were then lysed in 100 mM citrate-phosphate buffer (pH 5.4) containing 0.54% sodium taurodeoxycholate and 1% Triton X-100. The cell lysates were then incubated at 37 ° C. for 1 hour in the presence of 0.5 mM 4-methylumbelliferyl β-D-galactopyranoside (4-MUG) substrate. After 1 hour, the reaction was stopped with an equal volume of 200 mM sodium hydroxide. The conversion of 4-MU was measured with a plate reader (Ex = 370 nm; Em = 460 nm). FIG. 63 shows the mRNA control transfection complexed with RNAiMAX (Invitrogen) (iduronate-2-sulfatase (mRNA sequence shown in SEQ ID NO: 1652; poly-A tail of about 160 nucleotides not shown in the sequence). It shows the enzymatic activity expressed as a percentage normalized to 5'cap, cap 1) fully modified with 5-methylcytosine and pseudouridine).

Example 188. In vitro transfection of factor VIII: protein detection Factor VIII modified mRNA (mRNA sequence shown in SEQ ID NO: 1623; poly-A tail of about 160 nucleotides not shown in the sequence in HepG2 cells in a 24-multiwell plate 5'cap, cap 1) was transfected by reverse transfection. HepG2 cells were grown in Invitrogen (Carlsbad, CA) Supplement S7-added EPILIFE® medium until 50-70% confluence was reached. Cells were transfected with 0 ng, 250 ng, 500 ng, 750 ng, 1000 ng of modified factor VIII-encoding mRNA (m mRNA) complexed with RNAIMAX ™ from Invitrogen (Carlsbad, CA). RNA: RNAIMAX ™ complex was first formed by incubating RNA at room temperature with Supplement-free EPILIFE® medium at 5-fold volume dilution for 10 minutes. In the second vial, the RNAIMAX ™ reagent was incubated with Supplement-free EPILIFE® medium at room temperature for 10 minutes at 10-fold volume dilution. RNA vials and RNAIMAX ™ vials were then mixed, incubated at room temperature for 20-30 minutes, and then added dropwise to the cells.

Fully optimized mRNA encoding factor VIII transfected into HepG2 cells (mRNA sequence shown in SEQ ID NO: 1623; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, At the time of translation, cap 1) was prepared for natural nucleoside triphosphate (NTP), psoiduridine at each urine site and 5-methylcytosine at each cytosine site (psoid-U / 5 mC), and N1-methyl-psoiduridine at each urine site and each cytosine. It contained modifications such as 5-methylcytosine (N1-methyl-psoid-U / 5mC) at the site. Cells are transfected with mmRNA encoding factor VIII and 18 hours after transfection, secreted into culture medium at each concentration according to the manufacturer's recommended instructions using the Invitrogen (Carlsbad, CA) ELISA kit. The concentration of factor VIII (pg / ml) was measured. These data, shown in Table 224 and FIG. 64, show that the modified mRNA encoding Factor VIII is translatable within HepG2 cells.

Table 224. Administration of factor VIII and protein secretion

Example 189. In vitro transfection of factor VIII: chromogenic activity Factor VIII-modified mRNA (mRNA sequence shown in SEQ ID NO: 1623; poly-A tail of about 160 nucleotides not shown in the sequence) in HepG2 cells of a 24-multiwell plate. 5'cap, cap 1) was transfected by reverse transfection. HepG2 cells were grown in Invitrogen (Carlsbad, CA) Supplement S7-added EPILIFE® medium until 50-70% confluence was reached. Cells were transfected with 0 ng, 250 ng, 500 ng, 750 ng, 1000 ng of modified factor VIII-encoding mRNA (m mRNA) complexed with RNAIMAX ™ from Invitrogen (Carlsbad, CA). RNA: RNAIMAX ™ complex was first formed by incubating RNA at room temperature with Supplement-free EPILIFE® medium at 5-fold volume dilution for 10 minutes. In the second vial, the RNAIMAX ™ reagent was incubated with Supplement-free EPILIFE® medium at room temperature for 10 minutes at 10-fold volume dilution. RNA vials and RNAIMAX ™ vials were then mixed, incubated at room temperature for 20-30 minutes, and then added dropwise to the cells.

Fully optimized mRNA encoding factor VIII transfected into HepG2 cells (mRNA sequence shown in SEQ ID NO: 1623; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, At the time of translation, cap 1) was composed of pseudouridine at each urine site, 5-methylcytosine (psoid-U / 5mC) at each cytosine site, N1-methyl-psoiduridine at each urine site, and 5-methylcytosine (N1-) at each cytosine site. It contained modifications such as methyl-psoid-U / 5mC). Cells were transfected with mmRNA encoding factor VIII. When factor VIII is activated by thrombin, it forms an enzyme complex with factor IXa, phospholipids and calcium, which activates factor X to factor Xa, which is excessively added to the assay at a constant concentration. The factor X is activated into the factor Xa. This activity is directly related to the amount of Factor VIII.

Eighteen hours after transfection, factor VIII activity was produced by activity on a factor Xa-specific chromogenic substrate (SXa-11) as measured by the amount of pNA released determined by color development at 405 nm. Factor Xa was measured.

Factor VIII activity was normalized to a control standard for Factor VIII 25-30% concentrate (% activity). These data, shown in Table 225 and FIG. 65, show that the modified mRNA encoding Factor VIII is translatable within HepG2 cells.

Table 225. Administration of factor VIII and protein secretion

Example 190. Expression of Low Density Lipoprotein Receptor (LDLR) A. In vitro LDLR expression Human fetal renal epithelial cell (HEK293) cells (LGC standards GmbH, Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose, USA). HEK293 is seeded in 3 mL of cell culture medium at a density of approximately 500,000 cells / well. After cell seeding, LDLR mRNA (mRNA set in SEQ ID NO: 21463; fully modified with 5-methylcytosine and 1-methylpsoiduridine; 5'cap, cap 1, poly-A tail of approximately 160 nucleotides (sequence). (Not shown in)) or control G-CSF mRNA (mRNA shown in SEQ ID NO: 21438; fully modified with 5-methylcytosine and pseudouridine; 5'cap, cap 1; about 160 not shown in the sequence Formulations containing (poly-A tail) of nucleotides from the above are added directly in amounts of 4000 ng, 800 ng, 400 ng, 40 ng and 4 ng per well and incubated. As a control, cells transfected with G-CSF mRNA were treated with anti-LDLR antibody and a pair of LDLR-transfected cells were treated with normal goat IgG. After treatment with PE-labeled secondary antibody, bound primary antibody was detected by FACS analysis.

As shown in FIG. 66A, the results of FACS analysis show that LDLR expression was detected in 74.8% of gated whole living cells at an LDLR mRNA dose of 800 ng. At a LDLR mRNA dose of 40 ng, expression of LDLR was detected in 11.6% of gated whole living cells. No staining was observed in cells treated with LDLR mRNA and stained with control non-immune IgG. No LDLR-positive cells were detected in the cells transfected with G-CSF mRNA.

B. Protein Accumulation Human fetal renal epithelial cell (HEK293) cells (LGC standards GmbH, Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose, USA). HEK293 is seeded in 3 mL of cell culture medium at a density of approximately 500,000 cells / well. After cell seeding, LDLR mRNA (mRNA set in SEQ ID NO: 21463; fully modified with 5-methylcytosine and 1-methylpsoiduridine; 5'cap, cap 1, poly-A tail of approximately 160 nucleotides (sequence). (Not shown in)) or control G-CSF mRNA (mRNA shown in SEQ ID NO: 21438; fully modified with 5-methylcytosine and pseudouridine; 5'cap, cap 1; about 160 not shown in the sequence Nucleotide poly-A tail) Formulation containing SEQ ID NO: 21438 is added directly per well and incubated. After 15 hours, the transfection medium is replaced with complete medium. Transfect cells are collected 0 hours, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours and 72 hours after the medium change. Transfected cells were treated with phycoerythrin (PE) -conjugated anti-LDLR antibody and, as a control, a pair of LDLR-transfected cells were treated with PE-conjugated normal goat IgG. The bound primary antibody was detected by FACS analysis as described above.

As shown in FIG. 66B, FACS analysis showed that at 0.0 hours after flushing the transfection medium (15.0 hours after transfection), approximately 65% of the gated whole cells had LDLR. It indicates that expression was detected. At 37 ° C., the percentage of positive cells decreased over time and LDLR was no longer detected by 24 hours after removal of transfection medium.

C. BODIPY® Label LDLR
BODIPY® labeled LDL was used to examine whether the expressed LDLR was functional. HEK293 cells were completely modified with LDLR-modified mRNA (mRNA shown in SEQ ID NO: 21463; 5-methylcytosine and 1-methylpsoiduridine; 5'cap, cap 1, poly-A tail of approximately 16 nucleotides (sequence). (Not shown in)) or G-CSF modified mRNA (mRNA shown in SEQ ID NO: 21438; fully modified with 5-methylcytosine and pseudouridine; 5'cap, cap 1; about 160 not shown in the sequence. The cells were transfected overnight with a poly-A tail of nucleotides), the cells were washed, and the amount of BODIPY-LDL was incrementally incubated. After incubation at 37 ° C. for 1.0 hour, cells were washed and BODIPY-LDL binding was evaluated by FACS. As shown in FIG. 66C, the binding of BODIPY-LDL to cells transfected with LDLR mRNA had high affinity (Kd about 60 ng / mL) and was saturated.

In a competitive study, unlabeled LDL was able to reduce BODIPY-LDL binding in a dose-dependent manner (Fig. 66D). The above data indicate that the binding between BODIPY-LDL and cells expressing LDLR mRNA is saturated, specific and highly compatible.

Example 191. Expression of UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) A. In vitro UGT1A1 expression Human fetal renal epithelial cell (HEK293) cells (LGC standards GmbH, Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose, USA). HEK293 is seeded in 3 mL of cell culture medium at a density of approximately 500,000 cells / well. After cell seeding, UDP glucuronosyl transferase 1 family, polypeptide A1 (UGT1A1) mRNA (mRNA shown in SEQ ID NO: 21463; fully modified with 5-methylcytosine and 1-methylpsoid uridine; 5'cap, cap 1 , Poly-A tail of about 160 nucleotides (not shown in the sequence) or control G-CSF mRNA (mRNA shown in SEQ ID NO: 21438; fully modified with 5-methylcytosine and pseudouridine; 5' Caps, caps 1; poly-A tails of about 160 nucleotides not shown in the sequence) are added directly in amounts of 4000 ng, 800 ng, 400 ng, 40 ng and 4 ng per well and incubated. As a control, cells transfected with G-CSF mRNA were treated with anti-UGT1A1 antibody and a pair of cells transfected with UGT1A1 was treated with normal goat Ig. After treatment with PE-labeled secondary antibody, bound primary antibody was detected by FACS analysis.

As shown in FIG. 67, the percentage of positive cells measured by FACS increased with the amount of UGT1A1 mRNA transfected into HEK293 cells, with 74.8% of gated whole cells at a UGT1A1 mRNA dose of 800 ng. The maximum value has been reached. No further increase in percent positive cells was observed at the maximum dose tested (4000 ng). These data indicate that mRNA delivery and / or expression in mammalian cells is saturated. No staining was observed in cells treated with UGT1A1 mRNA and stained with control non-immune IgG, and no UGT1A1-positive cells were detected in cells transfected with G-CSF mRNA.

B. In vitro UGT1A1 expression-protein accumulation Human fetal renal epithelial cell (HEK293) cells (LGC standards GmbH, Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose, USA). HEK293 is seeded in 3 mL of cell culture medium at a density of approximately 500,000 cells / well. After cell seeding, UGT1A1 mRNA (mRNA shown in SEQ ID NO: 2146; fully modified with 5-methylcytosine and 1-methylpsoid uridine; 5'cap, cap 1, poly-A tail of approximately 160 nucleotides (sequence) (Not shown in)) or control G-CSF mRNA (mRNA shown in SEQ ID NO: 21438; fully modified with 5-methylcytosine and pseudouridine; 5'cap, cap 1; about 160 not shown in the sequence The formulation containing the nucleotide poly-A tail) is added directly per well and incubated. After 15 hours, the transfection medium is replaced with complete medium. Transfect cells are collected 0 hours, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours and 72 hours after the medium change. Transfected cells were treated with phycoerythrin (PE) -conjugated anti-UGT1A1 antibody and, as a control, a pair of UGT1A1-transfected cells were treated with PE-conjugated normal goat IgG. The bound primary antibody was detected by FACS analysis as described above.

As shown in FIG. 68, UGT1A1 protein accumulation in cells transfected with UGT1A1 mRNA increased with incubation time after medium exchange, reached a maximum after 24 hours, and then decreased to baseline by 72 hours. .. Under the conditions listed here, the apparent half-life of UGT1A1 mRNA was about 40.0 hours.

Example 192. Detection of UGT1A1 and OTC by Western blotting [0001882] HEK293 Human fetal renal epithelial cell (HEK293) cells (LGC standards GmbH, Wesel, Germany) are seeded on a 6-well plate (BD Biosciences, San Jose, USA). HEK293 is seeded in 2.4 mL of cell culture medium at a density of approximately 500,000 cells / well. After cell seeding, UGT1A1 mRNA (mRNA shown in SEQ ID NO: 21464; fully modified with 5-methylcytosine and 1-methylpsoid uridine; 5'cap, cap 1, poly-A tail of approximately 160 nucleotides (sequence) (Not shown in)) or OTC mRNA (mRNA shown in SEQ ID NO: 1659; fully modified with 5-methylcytosine and 1-methylpsoid uridine; 5'cap, cap 1; about 160 not shown in the sequence. Formulations containing poly-A tails of nucleotides) are added directly in an amount of 800 ng per well and incubated for 16 hours. Cells were washed with PBS 500 uL, cells were scraped and collected in PBS 500 uL. Cells were centrifuged at 200 xg for 5 minutes and resuspended in 250 uL of RIPA buffer supplemented with a protease inhibitor. The tube was centrifuged at 14,000 xg for 10 minutes. The supernatant was transferred to a centrificon concentrated column, centrifuged at 14,000 xg for 30 minutes, and the solubilized solution was concentrated. BCA Protein Assay (Thermo)
The solubilized solution was quantified by Fisher Scientific Inc, Rockford, USA).

NuPage SDS-PAGE system with off-the-shelf 1.5 mm bis-Tris gel and 4-12% acrylamide gradient and containing MOPS buffer to aid migration (above, Life Technologies (Grand Island, NY)). ) Was loaded with a protein solubilizer. Each solubilized solution sample was prepared to a final volume of 40 ul. This sample contains 25 ug of variable volume protein solubilizer, 10 μl of RIPA buffer to a volume of 26 μl, 10 μl of 10 × reducing agent and 10 μl of 4 × SDS loading buffer (both from Life Technologies (Grand Island, NY)). It was. The sample was heated at 95 ° C. for 5 minutes and loaded onto the gel. The standard settings were chosen by the manufacturer and were 200V, 120mA and up to 25W. The migration time was 60 minutes, which was not longer than the migration stain reached the bottom edge of the gel.

After completion of the electrophoresis, the plastic case was divided and the gel in the case was transferred to a ready-made nitrocellulose membrane kit and power source (iBLOT; Life Technologies, Grand Island, NY). Using the initial settings, the protein solubilizer was transferred from the gel to the membrane by high amperage electricity.

After transcription, the membrane is incubated with 1 x Tris buffered saline (TBS) containing 5% bovine serum albumin (BSA) for 15 minutes and then with 1 x TBS + 0.1% Tween containing 5% BSA for an additional 15 minutes. bottom. The primary antibodies goat anti-human UGT1A1 (R & D systems) and rabbit anti-human OTC (NBP1) in 3 ml of 1 × TBS solution containing 5% BSA at 1: 500 dilution at room temperature with gentle stirring in an orbital shaker. It was allowed to react for 3 hours. OTC primary antibody was detected on the Western Breeze Chromogenic Kit-Anti-Labbit according to the manufacturer's protocol, and UGT1A1 primary antibody was detected on the Western Breeze Chromogenic Kit-Anti-Goat (Invitrogen) manufacturer according to the manufacturer's protocol.

As shown in FIG. 69, UGT1A1 was detected as a band of about 60 kDa by anti-UGT1A1 IgG in the solubilized solution of cells transfected with UGT1A1 mRNA (lane 4), but not in cells transfected with OTC mRNA. (Lane 3). OTC was detected as a band of about 39 kDa by anti-OTC IgG in the solubilized cells of cells transfected with OTC mRNA using the same solubilized solution (lane 7), but was detected in cells transfected with UGT1A1 mRNA. There was no (lane 8). These data support that exogenous UGT1A1 and OTC mRNAs are capable of directing allogeneic protein synthesis in mammalian cells.

Example 193. Expression and detection of PAHs and UGT1A1 A. Transfection of HEK293 cells, mouse myoblasts and rat myoblasts HEK293 (LGC standards GmbH, Wesel, Germany), C2C12 mouse myoblast line (ATCC) and L6 rat myoblast line (ATCC) 24 weldish (ATCC) BD Biosciences, San Jose, plated on 10% fetal calf serum USA) (ATCC) added DMEM (ATCC) (1.5 × 10 5 cells / well).

PAH-modified mRNA fully modified with 5-methylcytosine and pseudouridine in the first tube (SEQ ID NO: 1660; poly-A tail of approximately 160 nucleotides not shown in the sequence; 5'cap, cap 1), UGT1A1 Modified mRNA (SEQ ID NO: 21464; poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) or G-CSF modified mRNA (SEQ ID NO: 21438; Poly-A tail of about 160 nucleotides not shown in the sequence; 5'cap, cap 1; fully modified with 5-methylcytosine and pseudouridine) 250 ng and Opti-MEM reagents (Life Technologies, Grand Islands, NY) ) 50 ul and 1 ul of L2000 nucleotides (Life Technologies, Grand Islands, NY) and Opti-MEM 50 ul in a second tube to provide a transfection solution for each well to be treated. Prepared. After preparation, the first and second tubes were incubated at room temperature for 5 minutes, after which the respective contents were combined. The combined transfection solution was incubated at room temperature for 15 minutes. Then 100 ul of transfection solution was added to each well. The cells were cultured for an additional 14 hours before proceeding with the analysis.

B. Detection of PAH, UGT1A1 by Flow Cytometry After transfection, the medium was removed from the cells and 60 ul of 0.25% trypsin (Life Technologies, Grand Island, NY) was added to each well. After treating the cells with trypsin for 2 minutes, 240 ul / well trypsin inhibitors (Life Technologies, Grand Island, NY) were added. The resulting cell solution was transferred to a 96-well plate (Corning Life Sciences, Tewksbury, MA), the cells were pelleted by centrifugation (800 x gravity for 5 minutes) and the supernatant was discarded. The cell pellet was washed with PBS and resuspended in Foxp3 fixation / permeation solution (eBioscience, San Diego, CA) for 45 minutes. Cells were pelleted again by centrifugation (800 x gravity for 5 minutes) and resuspended in permeabilization buffer (eBiosciences, San Diego, CA) for 10 minutes. Cells were pelleted again by centrifugation (800 x gravity for 5 minutes) and washed with permeabilization buffer. PAH-transfected cells were treated with anti-PAH antibodies conjugated with phycoerythrin (PE). As a staining control, a set of PAH-transfected cells was treated with normal goat IgG conjugated with PE. As a transfection control, a pair of cells transfected with G-CSF was treated with an anti-PAH antibody conjugated to phycoerythrin (PE). The bound primary antibody was detected by FACS analysis as described above. Labeled cells were then combined with FACS buffer (PBS containing 1% bovine serum albumin and 0.1% sodium azide), transferred to cluster tubes, and then using BD Accuri (BD Biosciences, San Jose, CA). The labeled cells were analyzed by flow cytometry. As shown in FIG. 70, 11.9% PAH expression was detected in PAH-transfected HEK293 cells, and UGT1A1 expression was detected in 58.6% of UGT1A1-transfected HEK293 cells. For each mRNA, even with rat or mouse myoblasts, the percentage of positive cells was about the same as the results obtained with HEK293 cells.

Example 194. UGT1A1 expression in HEK293 cells using direct capture capillary electrophoresis UGT1A1 mRNA was transfected into HEK293 cells to prepare cell lysates as described above. Western analysis was performed using the Simple Simon Western system (Protein Simple). As shown in FIG. 71, UGT1A1 (a band of about 60 kDa) was detected by anti-UGT1A1 IgG in the solubilized solution of cells transfected with UGT1A1 mRNA (lanes 1, 4 and 6), but at the same concentration of non-immune IgG. Not detected by (lanes 2 and 5). Similarly, neither UGT1A1 was detected in the same amount of cell lysate of untransfected Hep3b cells (lane 3) nor in the solubilized solution of HEK293 cells transfected with OTC mRNA (lane 7).

Normal C57BL6 mice were injected with a single intravenous injection of LNP containing UGT1A1 mRNA at a dose of 0.5 mg / kg. After 24 hours, the mice were sacrificed and the liver was removed from each mouse. Microsome membrane extracts were prepared from each liver by homogenizing in 5.0 mL of ice-cold phosphate buffered saline (pH 7.4) containing a protease inhibitor using a Polytron homogenizer. Tissue homogenate is mixed with 40 ml of ice-cold microsome buffer (2.62 mM KH ₂ PO ₄ , 1.38 mM K2 HPO 4, 2% glycerol and 0.5 mM dithiothreitol) at 4 ° C., 12,000 xg for 20 minutes. Centrifuged. The supernatant fraction was then centrifuged at 100,000 xg for 60 minutes. The microsome pellet was resuspended in microsome buffer and the protein concentration was measured by the Bradford method. Microsome extracts were characterized by detecting calnexin bands on Western blots with anti-calnexin antibodies (assay Designs, Ann Arbor, MI). Approximately 2-5 μg of microsome extract was added to the capillary and electrophoresis and protein immobilization were performed as described by the manufacturer. As shown in FIG. 72, using an antibody specific for UGT1A1 (above), UGT1A1 was detected in microsome extracts of mice treated with LNP containing UGT1A1 mRNA (lane 2), but non-specific antibody. Was not detected (lane 4). The molecular weight of UGT1A1 in mouse liver was the same as that observed in the extract of HEK293 cells transfected with UGT1A1 mRNA and searched for with anti-UGT1A1 IgG (lane 3). No UGT1A1 band was detected in the extract of HEK293 cells transfected with OTC mRNA (lane 5). The above data show in vivo expression of UGT1A1 mRNA in mice injected with LNP containing UGT1A1 mRNA.

The words used are descriptive words, not limited, and in their broader aspects may be modified within the appended claims without departing from the true scope and spirit of the invention. Understood.

The present invention has been described in some detail and in some detail with respect to some of the described embodiments, but as this should be limited to any such details or embodiments, or any particular embodiment. The appended claims are unintentionally provided in view of the prior art to provide the broadest possible interpretation of such claims, thereby effectively embracing the intended scope of the invention. Should be interpreted with reference to the scope of.

All publications, patent applications, patents, and other references referred to herein are incorporated herein by reference in their entirety. In the event of inconsistency, the specification, including the definitions, shall prevail. In addition, section headings, materials, methods, and examples are illustration only and are not intended to be limiting.

Claims (9)

A composition containing a plurality of lipid nanoparticles having an average particle size of 80 nm to 160 nm, wherein the lipid nanoparticles are:
A open reading frame encoding (a) secreted proteins, modified uridine, cytidine, adenosine, and it consists nucleo shea de guanosine, wherein the modified uridine, Nl-methyl - a pseudouridine, the open reading With the frame
(B) 5'-UTR and
(C) At least one 5'cap structure and
(D) 3'-UTR and
(E) A composition encapsulating a polynucleotide comprising a 3'tail sequence of a bound nucleoside. When administered to mammalian cells, the polynucleotide is an increase in the encoded polypeptide as compared to a corresponding polynucleotide containing an open reading frame consisting of nucleotides containing uracil, cytosine, adenine, and guanine. and having the expression, composition according to claim 1. The plurality of lipid nanoparticles are 0 . The composition according to claim 1 or 2 , having a PDI of 02 to 0.2 and a lipid: polynucleotide ratio (weight / weight) of 10 to 20. The lipid nanoparticles are DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODA, DDMA, DLenDMA, reLNP, PLGA, or PEGylated lipid. The composition according to any one of claims 1 to 3 , which comprises. The composition according to any one of claims 1 to 4 , wherein the secreted protein is a cytokine. The composition according to any one of claims 1 to 5 , wherein the 3'tail sequence of the bound nucleoside is selected from the group consisting of a poly A tail of 160 nucleotides and a poly AG quadruplex. The at least one 5'terminal cap is cap 0, cap 1, ARCA, inosine, 1-methyl-guanosine, 2'fluoro-guanosine, 7-deraza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, The composition according to any one of claims 1 to 6 , selected from the group consisting of LNA-guanosine and 2-azido-guanosine. The composition according to any one of claims 1 to 7 , wherein the open reading frame is codon-optimized to bias the GC content. For use in a method of expressing a polypeptide in a mammalian cell, a composition according to any one of請Motomeko 1-8,
It said method comprises the polynucleotide or the composition be contacted with a cell, set Narubutsu.

Images