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WO2022206816A1 - Système d'administration de plasmide d'arn pour le traitement de la maladie de parkinson - Google Patents

Système d'administration de plasmide d'arn pour le traitement de la maladie de parkinson Download PDF

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Publication number
WO2022206816A1
WO2022206816A1 PCT/CN2022/083979 CN2022083979W WO2022206816A1 WO 2022206816 A1 WO2022206816 A1 WO 2022206816A1 CN 2022083979 W CN2022083979 W CN 2022083979W WO 2022206816 A1 WO2022206816 A1 WO 2022206816A1
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sequence
rna
targeting
plasmid
delivery system
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Chinese (zh)
Inventor
张辰宇
陈熹
付正
李菁
张翔
周心妍
张丽
余梦超
郭宏源
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Nanjing University
Nanjing Tech University
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Nanjing University
Nanjing Tech University
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    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA plasmid delivery system for treating Parkinson's.
  • Parkinson's disease also known as tremor palsy, is a common neurodegenerative disease of the middle-aged and elderly people. The main lesions are in the substantia nigra and striatum. Parkinson's disease is the fourth most common neurodegenerative disease in older adults.
  • RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but many problems have been encountered during clinical practice, and the development of this therapy has lagged far behind expectations.
  • RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
  • the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
  • the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
  • the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
  • Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
  • the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
  • the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
  • exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes.
  • Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery.
  • the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
  • the embodiments of the present application provide an RNA plasmid delivery system for treating Parkinson's and its application, so as to solve the technical defects existing in the prior art.
  • One of the inventions of the present application is to provide an RNA plasmid delivery system for treating Parkinson's, the system comprising a plasmid carrying an RNA fragment that can be used for treating Parkinson's, and the plasmid can be used in the host's organs and tissues It is enriched in the host organ tissue, and endogenously and spontaneously forms a complex structure containing the RNA fragment in the host organ tissue, and the complex structure can bind to the target tissue, and the RNA fragment is sent into the target tissue to achieve par Treatment of Kinson's disease.
  • the RNA fragment comprises one, two or more specific RNA sequences of medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences of medical significance that can inhibit or hinder the development of Parkinson's disease .
  • the plasmid also includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the plasmid includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; in each of the plasmids, at least An RNA fragment and a targeting tag are included, the RNA fragment and targeting tag being in the same circuit or in different circuits.
  • the plasmid also includes a flanking sequence, a compensation sequence and a loop sequence that can fold the circuit into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the plasmid includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag Or 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the purpose of deleting bases 1-5 of the RNA reverse complement sequence is to make the sequence unexpressed.
  • siRNA and miRNA precursors due to deletion of the 9th and 10th bases of the reverse complementary strand of the active strand (such as the reverse complementary strand of siRNA), the active strand of siRNA and miRNA will form a bulge. structure, which facilitates more efficient silencing of gene expression. To sum up, it is an accepted conclusion that the silencing efficiency can be improved by deleting the reverse complementary sequence of the 9th and 10th bases.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);
  • sequence 1 is CAGATC
  • sequence 2 is a sequence consisting of 5-80 bases
  • sequence 3 is TGGATC.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • the organ tissue is liver
  • the composite structure is exosome
  • the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  • the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
  • the targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
  • the targeting tag is preferably an RVG targeting peptide or an RVG-LAMP2B fusion protein.
  • the RNA sequence is 15-25 nucleotides in length.
  • the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • the RNA sequence is 18-22 nucleotides in length.
  • the RNA capable of treating Parkinson's is selected from: siRNA of the LRRK2 gene, or an RNA sequence with more than 80% homology to the above sequence, or a nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequences in the "nucleic acid molecules encoding the above RNA sequences" here also include RNA sequences with a homology of more than 80% of each RNA.
  • the siRNA of the LRRK2 gene includes AUUAACAUGAAAAUAUCACUU, UUAACAAUAUCAUAUAAUCUU, AUCUUUAAAAUUUGUUAACGC, UUGAUUUAAGAAAAUAGUCUC, UUUGAUAACAGUAUUUUUCUG, other sequences that inhibit the expression of the LRRK2 gene, and sequences with more than 80% homology to the above sequences.
  • sequences with more than 80% homology may be 85%, 88%, 90%, 95%, 98%, etc. homology.
  • the RNA fragment includes an RNA sequence ontology and a modified RNA sequence obtained by modifying the RNA sequence ontology with ribose sugar. That is, the RNA fragment can be composed of only at least one RNA sequence ontology, or only at least one modified RNA sequence, and can also be composed of RNA sequence ontology and modified RNA sequence.
  • the isolated nucleic acid also includes its variants and derivatives.
  • the nucleic acid can be modified by one of ordinary skill in the art using general methods. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification Segment modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like.
  • the modification is an internucleotide linkage, for example selected from: phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, phosphine Acid alkyl esters, phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • phosphorothioate 2'-O methoxyethyl (MOE), 2'-fluoro
  • phosphine Acid alkyl esters phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • the modification is a modification of nucleotides, such as selected from: peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinose-nucleic acid (FANA), analogs, derivatives objects and their combinations.
  • the modification is a 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on RNA with 2'-F.
  • 2'-F can make RNA not easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment transmission in vivo. sex.
  • the present application also provides an application of the RNA delivery system for treating Parkinson's in medicine.
  • the drug is a drug for the treatment of Parkinson's disease and its related diseases
  • the related diseases here refer to the associated diseases or complications, sequelae, etc. that occur during the formation or development of Parkinson's disease, or have a certain relationship with Parkinson's disease. related other diseases.
  • the administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection. Intravenous injection is preferred.
  • the medicine includes the above-mentioned plasmids, specifically, the plasmids here represent plasmids that carry RNA fragments, or carry RNA fragments and targeting tags, and can enter the host body and can be enriched in the liver, self-contained. Assembled to form composite exosomes, the composite structure can deliver RNA fragments to the lungs, so that the RNA fragments are expressed in the brain of the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating Parkinson's disease.
  • the plasmids here represent plasmids that carry RNA fragments, or carry RNA fragments and targeting tags, and can enter the host body and can be enriched in the liver, self-contained. Assembled to form composite exosomes, the composite structure can deliver RNA fragments to the lungs, so that the RNA fragments are expressed in the brain of the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating Parkinson's disease.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • RNA delivery system for treating Parkinson's uses plasmid as a carrier and plasmid as a mature injection. Its safety and reliability have been fully verified, and its druggability is very good. The final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of Parkinson's provided in this application can be tightly combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and facilitates uptake by recipient cells, intracytoplasmic release and lysosomal escape, and requires a low dose.
  • RNA delivery system for the treatment of Parkinson's disease provided in this application is applied to medicine, that is, it provides a drug delivery platform, which can greatly improve the therapeutic effect of Parkinson's disease, and can also form a research and development basis for more RNA drugs through this platform. , which greatly promotes the development and use of RNA drugs.
  • Fig. 1 is a comparison diagram of plasmid distribution and metabolism in mice provided by an embodiment of the present application
  • Fig. 2 is a comparison diagram of protein expression levels in mice provided by an embodiment of the present application.
  • FIG. 3 is a comparison diagram of related siRNA levels in mice provided by an embodiment of the present application.
  • FIG. 4 is a comparison diagram of absolute siRNA levels in various tissues of mice provided in an embodiment of the present application.
  • Figure 5 is a comparison diagram of the effect of plasmid doses on mouse siRNA levels provided by an embodiment of the present application.
  • Fig. 6 is the metabolic situation comparison diagram of the precursor and the mature body in the mouse liver after injecting the plasmid provided by an embodiment of the present application;
  • FIG. 7 is a comparison diagram of siRNA kinetics and distribution in different tissues of mice provided by an embodiment of the present application.
  • Figure 8 is a comparison diagram of the influence of different promoters on siRNA provided by an embodiment of the present application.
  • FIG. 9 is a comparison diagram of the fluorescence intensity of eGFP in different tissues of mice provided by an embodiment of the present application.
  • Figure 10 is a comparison diagram of mouse alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, creatinine content, and thymus gland weight, spleen weight, and peripheral blood cell percentage provided by an embodiment of the present application;
  • Fig. 11 is a comparison diagram of Parkinson's treatment in transgenic mice provided in an embodiment of the present application.
  • FIG. 12 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains 6 kinds of RNA alone.
  • FIG. 13 is a graph of in vivo enrichment data when plasmid 2 provided by another embodiment of the present application contains 6 kinds of RNAs alone.
  • FIG. 14 is a graph of the in vivo self-assembly data of the exosomes provided in an example of the present application when each of the exosomes individually contains 6 kinds of RNAs.
  • Fig. 15 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression results of LRRK2 gene when the plasmids provided in an example of the present application contain 6 kinds of RNA alone.
  • FIG. 16 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains any two RNA sequences respectively.
  • Figure 17 is a graph of the in vivo self-assembly data of exosomes provided in an embodiment of the present application when they contain any two RNA sequences.
  • Fig. 18 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when plasmid 1 provided in an embodiment of the present application contains any two RNA sequences respectively.
  • Fig. 19 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided by another embodiment of the present application contains any two RNA sequences respectively.
  • Fig. 20 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when the plasmid 1 provided in an embodiment of the present application contains any three RNA sequences respectively.
  • Fig. 21 is a diagram showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided in another embodiment of the present application contains any three RNA sequences respectively.
  • Figure 22 is a graph of the in vivo enrichment data of the plasmid provided in an example of the present application when the sequences in the plasmid contain siRNA and RVG.
  • Figure 23 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when the sequence in the plasmid provided in an example of the present application contains siRNA and RVG.
  • Figure 24 is a data comparison diagram of the therapeutic effect of RVG-LAMP2B fusion protein and other fusion proteins on a plasmid vector for Parkinson's disease provided in an example of the present application, and the data is reflected by the relative level of LRRK2 mRNA.
  • FIG. 25 is a graph of in vivo enrichment data of the gene circuit provided in an example of the present application when three RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene are included.
  • Figure 26 is a data comparison diagram of the therapeutic effect on Parkinson's disease when the gene circuit provided by an embodiment of the present application includes 3 RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene. level to reflect.
  • HE staining Hematoxylin-eosin staining, referred to as HE staining.
  • HE staining is one of the most basic and widely used technical methods in histology and pathology teaching and research.
  • the hematoxylin staining solution is alkaline and can stain the basophilic structure of the tissue (such as ribosome, nucleus and ribonucleic acid in the cytoplasm) into blue-violet; eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
  • the basophilic structure of the tissue such as ribosome, nucleus and ribonucleic acid in the cytoplasm
  • eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
  • HE staining include: sample tissue fixation and sectioning; tissue sample dewaxing; tissue sample hydration; tissue section hematoxylin staining, differentiation and anti-blue; tissue section eosin staining and dehydration; tissue sample section air-drying and sealing; Observe and photograph under the microscope.
  • Masson staining renders collagen fibers blue (stained by aniline blue) or green (stained by bright green) and muscle fibers red (stained by acid fuchsin and Ponceau), which is consistent with the size and organization of the anionic dye molecules of permeability.
  • the fixed tissue is stained sequentially or mixed with a series of anionic water-soluble dyes. It can be found that red blood cells are stained with the smallest molecular anionic dyes, muscle fibers and cytoplasm are stained with medium-sized anionic dyes, and collagen fibers are stained with macromolecular anionic dyes. Dyeing with anionic dyes.
  • red blood cells have the least permeability to anionic dyes, followed by muscle fibers and cytoplasm, and collagen fibers have the largest permeability.
  • Type I and III collagens are green (GBM, TBM, mesangial matrix and renal interstitium are green), and erythropoietin, tubular cytoplasm, and erythrocytes are red.
  • Masson staining The specific steps of Masson staining include:
  • Tissues were fixed in Bouin's solution, rinsed with running water overnight, and embedded in conventional dehydration; sections were deparaffinized to water (deparaffinized in xylene for 10 min ⁇ 3 times, and the liquid was blotted dry with absorbent paper; 100% ethanol 5 min ⁇ 2 times, with water absorption Dry the liquid with paper; 95% ethanol for 5min ⁇ 2 times, blot the liquid with absorbent paper; run water for 2min, blot dry with absorbent paper); Weiger's iron hematoxylin staining for 5-10min; ; Rinse with running water for 3min; Stain with Ponceau red acid fuchsin solution for 8min; Rinse slightly with distilled water; Treat with 1% phosphomolybdic acid aqueous solution for about 5min; Do not wash with water, directly counterstain with aniline blue solution or bright green solution for 5min; Treat with 1% glacial acetic acid 1min; dehydrated in 95% ethanol for 5min ⁇ 2 times,
  • Western Blot (Western Blot) is to transfer the protein to the membrane, and then use the antibody for detection.
  • the corresponding antibody can be used as the primary antibody for detection, and the expression product of the new gene can be detected by the fusion part of the antibody. .
  • Western Blot uses polyacrylamide gel electrophoresis, the detected object is protein, the "probe” is an antibody, and the "color development” is a labeled secondary antibody.
  • the protein sample separated by PAGE is transferred to a solid phase carrier (such as nitrocellulose membrane), and the solid phase carrier adsorbs proteins in the form of non-covalent bonds, and can keep the types of polypeptides separated by electrophoresis and their biological activities unchanged.
  • the protein or polypeptide on the solid phase carrier is used as an antigen, which reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled secondary antibody to detect the specific target gene separated by electrophoresis through substrate color development or autoradiography.
  • expressed protein components The steps mainly include: protein extraction, protein quantification, gel preparation and electrophoresis, membrane transfer, immunolabeling and development.
  • Immunohistochemistry using antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody, determines the antigen (polypeptide) in tissue cells by developing the color of the chromogenic reagent (fluorescein, enzyme, metal ion, isotope) labeled antibody through chemical reaction. and protein), the localization, qualitative and relative quantitative research, called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).
  • chromogenic reagent fluorescein, enzyme, metal ion, isotope
  • the main steps of immunohistochemistry include: section soaking, overnight drying, xylene dewaxing, gradient alcohol dewaxing (100%, 95%, 90%, 80%, 75%, 70%, 50%, 3min each time) , double-distilled water, dropwise addition of 3% hydrogen peroxide solution to remove catalase, water washing, antigen retrieval, dropwise addition of 5% BSA, blocking for 1 h, dilution of primary antibody, washing with PBS buffer, incubation with secondary antibody, washing with PBS buffer , color developing solution, washing with water, hematoxylin staining, dehydration with gradient ethanol, and sealing with neutral gum.
  • the detection of the siRNA level, the protein content and the mRNA content involved in the present invention is to establish the mouse stem cell in vitro model by injecting the RNA delivery system into the mouse.
  • the expression levels of mRNA and siRNA in cells and tissues were detected by qRT-PCR. Absolute quantification of siRNA was determined by plotting a standard curve using the standards.
  • the internal reference gene is U6snRNA (in tissue) or miR-16 (in serum, exosomes)
  • the gene is GAPDH or 18s RNA.
  • Western blotting was used to detect protein expression levels in cells and tissues, and ImageJ software was used for protein quantitative analysis.
  • This embodiment provides an RNA plasmid delivery system for treating Parkinson's, the system comprising a plasmid carrying an RNA fragment that can be used for treating Parkinson's, and the plasmid can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing the RNA fragment in the host organ tissue, and the composite structure can send the RNA fragment into the target tissue to achieve the treatment of Parkinson's disease.
  • the plasmid also includes a promoter and a targeting tag.
  • the plasmid includes any one of the following circuits or a combination of several circuits: promoter-RNA sequence, promoter-targeting tag, promoter-RNA sequence-targeting tag, and each of the plasmids includes at least one RNA fragment and a targeting tag, the RNA fragment and targeting tag are located in the same line or in different lines.
  • the plasmid can include only a promoter-RNA sequence-targeting tag, or a combination of a promoter-RNA sequence, a promoter-targeting tag, or a promoter-targeting tag, a promoter- A combination of RNA-seq-targeting tags.
  • the plasmid can also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the plasmid includes the following Any one line or combination of several lines: 5'-promoter-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5'-promoting sub-targeting tag-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3'flanking sequence.
  • the plasmid vector contains 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensation sequence-3'flanking sequence
  • its RNA delivery system has in vivo enrichment and Parkinson's treatment
  • the effect is shown in Figure 22-23, the targeting tag is RVG, and the RNA fragment is siRNA with therapeutic effect.
  • the Parkinson's treatment effect of the RVG-LAMP2B fusion protein on the plasmid vector is shown in Figure 24, and its treatment effect is reflected by the level of HTT mRNA.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
  • flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequences of composition such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • Any sequence may be used, preferably a sequence consisting of 10-50 bases, more preferably a sequence consisting of 20-40 bases, and sequence 3 is preferably TGGATC.
  • Sequence 2 is specifically shown in Table 1 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • sequence 4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-1 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • RNA fragments comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the RNA sequence can be a siRNA sequence, a shRNA sequence or a miRNA sequence, preferably a siRNA sequence.
  • the length of an RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, and 22nt. This range of sequence lengths was not chosen arbitrarily, but was determined through trial and error. A large number of experiments have proved that when the length of the RNA sequence is less than 18nt, especially less than 15nt, the RNA sequence is mostly invalid and will not play a role. The cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into consideration, and the effect is the best.
  • nt nucleotides
  • the RNA capable of treating Parkinson's disease is selected from: siRNA of LRRK2 gene or nucleic acid molecule encoding the above RNA.
  • the number of required delivery RNA effective sequences is one, two or more.
  • the functional structural region of the plasmid vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
  • the functional structural region of the plasmid vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connecting sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3' flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'
  • the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F.
  • 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
  • the liver will phagocytose exogenous plasmids, and up to 99% of the exogenous plasmids will enter the liver. Therefore, when plasmids are used as vectors, they can be enriched in liver tissue without specific design.
  • the plasmid is opened to release RNA molecules (siRNA, shRNA, or miRNA), and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
  • RNA delivery mechanism in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance”, we designed a targeting tag in the plasmid injected into the body, and the targeting tag will also be assembled into exosomes by liver tissue , especially when certain specific targeting tags are selected, the targeting tags can be inserted into the surface of exosomes, thereby becoming a targeting structure that can guide exosomes, which greatly improves the RNA delivery mechanism of the present invention.
  • the amount of exogenous plasmids that need to be introduced can be greatly reduced, and on the other hand, the efficiency of potential drug delivery can be greatly improved.
  • the targeting tag is selected from one of the peptides, proteins or antibodies with targeting function.
  • the selection of the targeting tag is a process that requires creative work. On the one hand, it is necessary to select the available targeting tags according to the target tissue. It is ensured that the targeting label can stably appear on the surface of exosomes, so as to achieve the targeting function.
  • Targeting peptides that have been screened so far include, but are not limited to, RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) SEQ ID No: 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6) : 7), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucle
  • RNAs comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the plasmid vector can also be composed of multiple plasmids with different structures, one of which contains a promoter promoter and targeting tags, other plasmids contain promoters and RNA fragments. Loading the targeting tag and RNA fragment into different plasmid vectors, and injecting the two plasmid vectors into the body, the targeting effect is no worse than the targeting effect produced by loading the same targeting tag and RNA fragment into one plasmid vector .
  • the plasmid vector containing the RNA sequence can be injected first, and then the plasmid vector containing the targeting tag can be injected after 1-2 hours, so that a better target can be achieved. to the effect.
  • the delivery systems described above can all be used in mammals, including humans.
  • FIG. 1A in order to understand the distribution of plasmids in the body, we carried out a plate test on mice. 720h) sampling, using the plasmid extracted by spectinomycin for transformation, observing the number of clones in liver, plasma, lung, brain, kidney, spleen, the results are shown in Figure 1B, Figure 1C, Figure 1D, it can be seen that the plasmid It is most distributed in the liver of mice, and reaches the peak at about 3 hours after injection, and is basically metabolized at 12 hours after injection.
  • the CMV eGFP siRE circuit co-expressing eGFP protein and EGFR siRNA was injected intravenously into C57BL/6J mice. The results are shown in Figure 2.
  • the eGFP fluorescence in the mouse liver gradually increased over time, reaching a peak at about 12 hours. 48 After hours, it dropped to the background level, and no obvious eGFP signal was seen in other tissues.
  • CMV-scrR The control plasmid
  • CMV-siR E the plasmid expressing EGFR siRNA
  • Figure 3A The related siRNA levels in exosomes, the results are shown in Figure 3A, it can be seen that there is siRNA expression in the exosomes of mouse hepatocytes injected with CMV-siRNA.
  • FIG. 4A After intravenous injection of plasmids into mice, the distribution of mature siRNA in different tissues is shown in Figure 4. It can be seen from Figure 4A that the levels of EGFR-siRNA in plasma, exosomes, and exosome-free plasma show time-dependent changes; from Figure 4B, it can be seen that mouse EGFR-siRNAs in the liver, lung, pancreas, and spleen , The accumulation in the kidney is time-dependent.
  • mice were injected with control plasmid (CMV-scrR), 0.05mg/kg CMV-siR E plasmid, 0.5mg/kg CMV-siR E plasmid, 5mg/kg CMV-siR E plasmid, and detected the liver, Absolute siRNA (EGFR siRNA) levels in spleen, heart, lung, kidney, pancreas, brain, skeletal muscle, CD4 + cells, the results are shown in Figure 5A, it can be seen that there is no siRNA expression in the tissues of mice injected with the control plasmid , in each tissue of mice injected with CMV-siR E plasmid, the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid.
  • CMV-scrR control plasmid
  • EGFR siRNA Absolute siRNA
  • fluorescence in situ hybridization assay FISH also confirmed that the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid, that is, the tissue distribution of EGFR siRNA was dose-dependent.
  • the plasmid After the plasmid enters the body, it will express the precursor (Precursor) and then process it into the mature body (siRNA), so we tested the metabolism of the precursor (Precursor) and the mature body (siRNA) in the liver after the plasmid was injected into mice. , the results are shown in Figure 6. It can be seen that the expression levels of precursor (Precursor) and mature body (siRNA) in the mouse liver reached a peak at the time point of 6 hours after the injection of the plasmid. Metabolism of the precursor (siRNA) was complete, and the metabolism of the precursor (Precursor) in the mouse liver was complete 48 hours after the injection of the plasmid.
  • siRNA with albumin ALB as the promoter siRNA with CMV as the promoter
  • siRNA without any promoter were injected into mice intravenously.
  • the absolute siRNA levels in the mice were detected at 48 h, and the results are shown in Figure 8. It can be seen that the level of siRNA with CMV as the promoter in mice is the highest, that is, the effect of CMV as the promoter is the best.
  • mice were intravenously injected with PBS or 5 mg/kg CMV-siR G or CMV-RVG-siR G plasmid, and treated for 24 hours After the mice were sacrificed, their eGFP fluorescence levels were detected in cryosections.
  • Figure 9A shows a representative fluorescence microscope image, in which green indicates positive eGFP signal, blue indicates DAPI-stained nuclei, scale bar: 100 ⁇ m, CMV is visible - RVG-siR G plasmid has a more obvious inhibitory effect on mouse eGFP; eGFP transgenic mice were intravenously injected with PBS or CMV-scrR or CMV-siR E plasmid, and the mice were sacrificed after 24 hours of treatment, and they were detected in frozen sections.
  • the fluorescence level of eGFP is a bar graph of the fluorescence intensity (Fluorescence intensity) of the mouse heart, lung, kidney, pancreas, brain, and skeletal muscle injected with PBS, CMV- siRE , and CMV-RVG- siRE . It can be seen that, The contrast of fluorescence intensity in liver, spleen, lung and kidney of mice was more obvious.
  • mice injected with PBS, CMV-scrR, and CMV-siR E their alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), serum alkaline phosphatase (ALP), creatinine (CREA) content, thymus weight, spleen weight, and percentage of peripheral blood cells were detected.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • BUN blood urea nitrogen
  • ALP serum alkaline phosphatase
  • CREA creatinine
  • Figure 10G is a comparison chart of mouse liver, lung, spleen, and kidney tissue
  • Figure 10H -I is a comparison chart of mouse thymus and spleen tissue
  • FIG. 10J is a comparison chart of percentage in peripheral blood cells of mice.
  • mice injected with PBS, CMV- scrR , and CMV-siRE were almost the same.
  • the mice injected with CMV-siRE were compared with those injected with PBS.
  • the liver, lung, spleen, kidney also have no tissue damage.
  • RNA delivery system for treating Parkinson's uses plasmid as a carrier and plasmid as a mature injection, its safety and reliability have been fully verified, and its druggability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of Parkinson's disease provided in this example can be tightly combined with AGO 2 and enriched into a complex structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and is beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.
  • the medicament comprises a plasmid carrying an RNA fragment that can be used to treat Parkinson's, the plasmid can be enriched in the organ tissue of a host, and endogenously and spontaneously form in the host organ tissue containing the A composite structure of RNA fragments capable of delivering the RNA fragments into target tissues for Parkinson's disease treatment.
  • the RNA fragment comprises one, two or more specific RNA sequences of medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences of medical significance that can inhibit or hinder the development of Parkinson's disease .
  • a plasmid as a vector, which carries one or more RNA fragments, it has in vivo enrichment, self-assembly and Parkinson's treatment effects.
  • Figure 12-15 shows that the vector carries RNA fragments of 6 separate RNA sequences Data results presented;
  • Figures 16-19 show data results presented by vectors carrying 4 sets of RNA fragments containing any 2 RNA sequences;
  • Figures 20-21 show vectors carrying 3 sets of RNA fragments containing any 3 RNA sequences The therapeutic data results presented by sequenced RNA fragments.
  • CTX stands for cortex and STR stands for Striatum.
  • the plasmid also includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly injected or intravenously injected into the human body, it can be delivered to the target tissue through the RNA delivery system described in Example 1 to exert a therapeutic effect.
  • the drug can be used in combination with other Parkinson's disease drugs to improve the treatment effect, such as anticholinergics (Antan, etc.), antihistamines (diphenhydramine, amantadine, etc.), levodopa Drugs (Dopa, Sining, Xilaimei, etc.), dopamine receptor agonists (Texida, Xie Liangxing, Crepa, bromocriptine, etc.), monoamine oxidase B inhibitors (Sgining, Jin Siping, etc.) ), catecholamine oxygen methyltransferase inhibitors (Entocapone, etc.).
  • Parkinson's disease drugs such as anticholinergics (Antan, etc.), antihistamines (diphenhydramine, amantadine, etc.), levodopa Drugs (Dopa, Sining, Xilaimei, etc.), dopamine receptor agonists (Texida, Xie Liangxing, Cre
  • the medicine of this embodiment may also include a pharmaceutically acceptable carrier, which includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • a pharmaceutically acceptable carrier includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • the dosage forms of the medicine provided in this embodiment can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.
  • the medicine provided in this example uses the plasmid as the carrier and the plasmid as the mature injection, and its safety and reliability have been fully verified, and the drugability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • the drug provided in this application can be closely combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also benefit the receptor.
  • Cellular uptake, intracytoplasmic release and lysosomal escape require low doses.
  • this embodiment provides an application of an RNA delivery system for treating Parkinson's disease in a drug, and the drug is a drug for treating Parkinson's disease.
  • the application of the RNA delivery system in the treatment of Parkinson's disease is specifically described in conjunction with the following experiments.
  • LRRK2R1441G transgenic mice were selected for the experiment when they were 3 months old, and the experiment set up LPS intervention group and LPS non-intervention group.
  • the LPS intervention group was treated with CMV-scrR/CMV-RVG-siR LRRK2 after 7 days of LPS intervention.
  • Figure 11A is a western bolt image of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2
  • Figure 11B is a LRRK2R1441G transgenic mouse injected with CMV-scrR/CMV-RVG-siR LRRK2
  • the grayscale analysis of the protein shows that the levels of LRRK2 protein and S935 protein in mice injected with CMV-RVG-si RLRRK2 decreased, indicating that CMV-RVG-siR LRR K2 can pass through the blood brain after the liver releases siRNA and assembles into exosomes
  • the barrier reduces the expression of proteins deep in the brain.
  • FIG. 11C is an immunofluorescence image of TH+ neurons in the substantia nigra of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 .
  • the results showed that mice injected with CMV-RVG-siR LRRK2 rescued TH
  • the loss of neurons indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.
  • this figure is an immunofluorescence image of the activation level of microglia in LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 .
  • the results show that injection of CMV-RVG-siR LRRK2 mice can inhibit microglia
  • the activation of CMV-RVG-siR LRRK2 indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.

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Abstract

L'invention concerne un système d'administration d'ARN pour le traitement de la maladie de Parkinson, caractérisé en ce que le système comprend un plasmide. Le plasmide porte un fragment d'ARN permettant de traiter la maladie de Parkinson et peut être enrichi en tissus d'organes d'un hôte. Dans les tissus d'organe de l'hôte, le plasmide peut former de manière endogène et spontanée une structure composite contenant le fragment d'ARN. La structure composite peut fournir le fragment d'ARN à des tissus cibles pour traiter la maladie de Parkinson. La sécurité et la fiabilité du système de distribution d'ARN pour traiter une colite ont été complètement vérifiées et le système a une bonne aptitude à la pharmacopotentialité, ainsi qu'une universalité élevée.
PCT/CN2022/083979 2021-03-30 2022-03-30 Système d'administration de plasmide d'arn pour le traitement de la maladie de parkinson Ceased WO2022206816A1 (fr)

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Citations (6)

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