[go: up one dir, main page]

WO2025049632A1 - Compositions et méthodes pour l'administration large d'arn dans un tissu - Google Patents

Compositions et méthodes pour l'administration large d'arn dans un tissu Download PDF

Info

Publication number
WO2025049632A1
WO2025049632A1 PCT/US2024/044269 US2024044269W WO2025049632A1 WO 2025049632 A1 WO2025049632 A1 WO 2025049632A1 US 2024044269 W US2024044269 W US 2024044269W WO 2025049632 A1 WO2025049632 A1 WO 2025049632A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
lipid
cells
lnp
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/044269
Other languages
English (en)
Inventor
John Ramunas
Glenn Jeremy MARKOV
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rejuvenation Technologies Inc
Original Assignee
Rejuvenation Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rejuvenation Technologies Inc filed Critical Rejuvenation Technologies Inc
Publication of WO2025049632A1 publication Critical patent/WO2025049632A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • RNA- and DNA-based biologies have expansive capacities to modulate cellular activities for treating inherited and acquired diseases.
  • the clinical success of LNPs has gained recent widespread attention.
  • Most lipid-based nucleic acid delivery platforms that are undergoing clinical studies or on the market consist of four or five lipid components. Recent studies have reported that not only the choice of lipid components, but also the relative proportions of the lipid ingredients in the formulation, greatly influence in vivo transfection efficiency and tissue-specific delivery.
  • lipid nanoparticle comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different
  • lipid nanoparticle comprising: (i) DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (ii) a cationic lipid at a molar percentage of between about 50% and about 70%, with a cationic lipid: ionizable lipid (C/I) ratio between 1 and 2, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types in the subject.
  • LNP lipid nanoparticle
  • the PEGylated lipid is DMG-PEG2000.
  • the cationic lipid is DOTAP.
  • the LNP of the methods described above and herein is comprised of the lipids set forth in Table 7.
  • the target cell types are located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 4 to 6 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 5 to 8 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 7 to 10 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 9 to 12 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 10 to 15 distinct organs of the subject.
  • the delivery is to a target cell.
  • target cell is often in any of a variety of tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, hematopoietic stem cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • the administration is intravenous, transcutaneous, intracutaneous injection via microneedle array, or topical delivery involving a step of permeabilizing the skin barrier using a mechanical means such as microneedling and/or application of a skin permeabilizing agent.
  • exemplary skin permeabilizing agents include solutions and mixtures containing one or more protease, one or more lipase, or other skin permeabilization agents known in the art.
  • the corresponding protein encoded by the synthetic RNA is expressed in two or more of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell. According to certain embodiments, the corresponding protein encoded by the synthetic RNA is expressed in each of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell. According to related embodiments, the bone marrow cell is a bone marrow stem cell and/or a progenitor cell.
  • the corresponding protein encoded by the synthetic RNA is expressed in two or more of a stem cell, progenitor cell, germ cell, differentiated cell, or terminally differentiated cell, a cancer cell, endothelial cell, and/or bone cell.
  • the corresponding protein encoded by the synthetic RNA is expressed in each of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell.
  • the bone marrow cell is a bone marrow stem cell and/or a progenitor cell and/or stem cell such as a hematopoietic stem cell.
  • the polynucleotide in the LNP will generally comprise a sequence encoding any therapeutic protein or protein useful in a diagnostic that can be expressed in a target cell, such as, for example, telomerase reverse transcriptase (TERT) protein or part thereof, selected from human TERT (hTERT), mouse TERT (mTERT), or TERT of another mammalian species.
  • telomerase reverse transcriptase e.g., mRNA
  • the polynucleotide often comprises SEQ ID NO: 1 or a fragment thereof.
  • the synthetic ribonucleic acid encodes telomerase reverse transcriptase (TERT), wherein optionally the TERT mRNA comprises a nucleic acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOS: 1-5, or SEQ ID NOS: 38-40 of PCT/US22/22642.
  • TERT telomerase reverse transcriptase
  • methods of delivering a polynucleotide to a bone marrow cell comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2%, and a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1; and/or (ii) DLin-MC3- DMA at a molar percentage of between about 30% and about 50%, DMG-PEG2000 at a molar percentage of between about 0.5% and about 2%, and a cationic lipid at a molar percentage of between about 50% and about 70%, with
  • methods of delivering a polynucleotide to one or more of a bone marrow cell, spleen cell, a hepatocyte, and/or a kidney cell comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2%, and a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1; and/or (ii) DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, a PEGylated lipid at a molar percentage of between about 0.5% and about
  • LNP lipid nanoparticle
  • the bone marrow cell is a bone marrow stem cell and/or a progenitor cell.
  • the polynucleotide often comprises a sequence encoding a diagnostic or therapeutic protein that can be expressed in a target cell.
  • the LNP further comprises a targeting ligand adapted to specifically target the cell.
  • the adaptation to specifically target the cell is often a binding or other interaction of the LNP with the cell.
  • the targeting ligand includes a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody adapted to bind the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer- antibody conjugate.
  • a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody adapted to bind the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer- antibody conjugate.
  • the method is adapted for diagnosis, prevention or treatment of a condition or disease involving the cell.
  • the delivery of the polynucleotide is for the diagnosis, prevention and/or treatment of conditions or disease of various tissues and cell types throughout the mammalian body.
  • conditions or diseases include influenza, asthma, diabetes mellitus type 1, diabetes mellitus type 2, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer’s disease, Parkinson’s disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn’s disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testi
  • COPD chronic obstructive
  • the method of delivering the polynucleotide is for the modulation of a condition or disease of various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • the presently described LNP composition is administered at a time before the subject has or is suspected of having the condition or disease.
  • the presently contemplated LNPs are capable of targeting the cell type and tissue where the condition or disease manifests.
  • the presently described LNPs are shown to provide for the ability to transfect cells with the encapsulated polynucleotide throughout the whole body of the subject, including cell types that exist throughout the body.
  • the use of targeting ligands or moieties provides for more specific targeted cell type and tissue transfection, which is often desired based on the objective of diagnosis, prevention and/or treatment and the specific type or types of condition or disease that is the subject of the administration to a subject.
  • the polynucleotide encapsulated in the LNP will vary based on the condition or disease and the objective of the delivery (diagnosis, prevention or treatment), the presently described LNPs are provided as an enabling delivery vehicle for such polynucleotide such that it can be delivered to areas that have been difficult or not possible to deliver a polynucleotide in vivo in a subject (e.g., mammal) previously.
  • a subject e.g., mammal
  • the LNP is selected from an LNP of Table 7.
  • the LNP has only a single charged lipid, for example DOTAP.
  • LNPs e.g., 3-MC3 and/or PDS
  • the LNPs are manufactured in a manner that forms the LNP prior to addition of nucleic acid.
  • such LNPs are 2, 3 or 5 lipids LNPs described herein in Table 7 and the method employs vortex mixing during the addition of the nucleic acid.
  • the LNP is composed of 2 or 3 lipid LNPs described herein.
  • FIG. 2 depicts the ratio of bioluminescence signal from the indicated organs, produced from luciferase protein translated from a formulation of mRNA in LNPs (“mRNA LNPs”) injected intravenously, at the indicated time points post intravenous injection.
  • mRNA LNPs mRNA in LNPs
  • FIG. 3 shows that LNPs formulated with TERT mRNA (“TERT mRNA LNPs”) extend telomeres in lung cells in vivo, where telomere length was measured in lung tissue sections using Q-FISH, and the telomere signal intensity (green/lighter) is proportional to signal length.
  • FIG. 3 shows that LNPs formulated with TERT mRNA (“TERT mRNA LNPs”) extend telomeres in lung cells in vivo, where telomere length was measured in lung tissue sections using Q-FISH, and the telomere signal intensity (green/lighter) is proportional to signal length.
  • TERT mRNA LNPs extend telomeres in lung cells in vivo, where TERT mRNA LNPs extend median and average telomere length in AT2 cells. At least 50 pro- SPC-positive cell nuclei were quantified per mouse. The error bars are standard error of the mean (SEM) for the telomere length between mice.
  • FIG. 5 shows that TERT mRNA LNPs extend telomeres in lung cells in vivo, where TERT mRNA LNPs extend median and average telomere length in lung alveolar cells. Telomeres of at least 100 alveolar cells were quantified per mouse. The error bars are standard error of the mean (SEM) for the telomere length between mice.
  • FIG. 6A shows experimental design and timeline of dosing for data of the present disclosure presented in FIGS 3-12 and FIGS 20-21.
  • FIG. 6B shows experimental design and timeline of dosing for data of the present disclosure presented in FIGS 13-16.
  • FIG. 7 shows that TERT mRNA LNPs reduce fibrosis in lung in vivo, where high- density fibrotic foci (yellow/darker) are mapped using picrosirius red staining in lungs in bleomycin-treated mice treated with either control mRNA LNPs (carrying luciferase mRNA) or TERT mRNA LNPs. “No bleo” indicates a lung section from a normal healthy mouse, not treated with bleomycin.
  • FIG. 8 shows that TERT mRNA LNPs reduce fibrosis in lung in vivo, as measured by quantification of high-density fibrotic foci in Picrosirius Red stained tissue sections.
  • FIG. 9 shows that TERT mRNA LNPs increase the amount of useful lung volume in vivo, as determined by quantification of the change in normo-aerative lung ratio from baseline using 3D quantification of fibrosis detected by signal intensity in x-ray computed tomography scan data.
  • FIG. 10 shows that TERT mRNA LNPs improve tissue elastance in vivo, as determined by quantification of measurement of tissue elastance in mice at the end of the study using the Flexivent system.
  • FIG. 11 shows that TERT mRNA LNPs improve lung function in vivo, as determined by measurement of forced expiratory volume in mice at the end of the study using the Flexivent system.
  • FIG. 12 shows that TERT mRNA LNPs improve lung function in vivo as determined by measurement of forced vital capacity in mice at the end of the study using the Flexivent system.
  • FIG. 13 shows that TERT mRNA ENPs improve lung architecture in vivo, where Morpho-Quant Fung software was used to analyze digitized images of tissue sections to quantify alveolar number per unit area.
  • FIG. 14 shows that TERT mRNA LNPs improve lung architecture in vivo, where Morpho-Quant Lung software was used to analyze digitized images of tissue sections to quantify alveolar diameter.
  • FIG. 15 shows that TERT mRNA LNPs improve lung architecture in vivo, where Morpho-Quant Lung software was used to analyze digitized images of tissue sections to quantify alveolar circularity.
  • FIG. 16 shows that TERT mRNA LNPs improve lung architecture in vivo, where representative images of alveoli false colored by Morpho-Quant Lung software during the digitized analysis of alveolar architecture are shown.
  • FIG. 17 shows pharmacodynamic data of TERT mRNA in vivo following intravenous infusion in a formulation of TERT mRNA LNPs, where in the level of telomerase activity in lung tissue is quantified at the indicated time points following infusion.
  • FIG. 18 shows an experimental schema for quantifying the effect of TERT mRNA LNPs on human primary epithelial cell colony formation capacity.
  • FIG. 19 shows that TERT mRNA LNPs increase the colony formation capacity of human primary epithelial cells in the experimental model shown in FIG. 18.
  • FIG. 20 depicts levels of P21+ senescent lung alveolar cells involving the LNP of Table 3, based on the experimental model shown in FIG. 6A.
  • FIG. 21 is a representative immunohistochemistry image of anti-P21 staining of lung sections, as quantified in FIG. 20.
  • FIGS. 22A, 22B, 22C and 22D provide lung delivery data obtained using 2 lipid LNP compositions.
  • FIG. 23 depicts the results of titrating lipid : mRNA ratio in 2 lipid LNP.
  • FIG. 24 depicts the time course of protein expression after delivery of lung-targeting mRNA-LNP encoding firefly luciferase using the LNPs of Table 3.
  • FIGS. 25 A, 25B and 25C provide lung radiance, encapsulation efficiency, and body weight variance in titrating SS-OP lipid into the ‘DOTAP + PEG’ LNP using LNPs of FIG. 25D.
  • FIGS. 26A and 26B lung and liver delivery data using the LNP of Table 4.
  • FIGS. 27A and 27B provide exemplary LNPs and encapsulation efficiency thereof.
  • FIGS. 27C and 27D provide lung delivery data and body weight variance data in titrating SS-OP lipid into the ‘DOTAP + PEG’ LNP using LNPs of FIGS. 27A.
  • FIGS. 28A and 28B provide comparative fresh versus long term cryopreserved lung transfection and weight data for LNPs of Table 3.
  • FIGS. 29A and 29B provide comparative fresh versus short term cryopreserved lung transfection and particle size data for LNPs of Table 3.
  • FIG. 30A provides exemplary 2, 3 and 5 lipid LNPs.
  • FIGS. 30B and 30C provide lung delivery data and body weight variance data in titrating N/P ratios and comparing 2, 3, and 5 lipid formulations of FIG. 30A.
  • FIGS. 31A and 3 IB provide exemplary 3 and 5 lipid LNPs and encapsulation efficiencies thereof.
  • FIGS. 31C provides lung delivery data in titrating N/P ratio (mRNA to lipid ratio) with 3 and 5 lipid formulations 30A.
  • FIGS. 32A and 32B provide lung transfection data and LNP size and encapsulation efficiency data for LNPS having lipid ratios of Table 3 prepared using vortex or microfluidic mixing.
  • FIG. 33A provides exemplary 3 and 5 lipid LNPs.
  • FIG. 33B provides lung transfection data using the LNPs of FIG. 33A.
  • FIG. 34A provides exemplary 2, 3 and 5 lipid LNPs.
  • FIG. 34B and 34C provide lung transfection data using, and particle size and encapsulation efficiency of the LNPs of FIG. 34A.
  • FIG. 35A provides exemplary 5 lipid LNPs prepared using different flow rates.
  • FIGS. 38B and 38C provide encapsulation percentage and lung transfection data using the LNPs of FIG. 38 A.
  • FIG. 39A provides exemplary LNPs prepared by swapping out SS-OP for DLin-MC3- DMA in 5 lipid LNPs.
  • FIGS. 39B and 39C provide encapsulation percentage and lung transfection data using the LNPs of FIG. 39A.
  • FIG. 40A provides a depiction of titrating time that mRNA is adsorbed to form LNP in a 2-lipid formulation.
  • FIG. 40B provides body weight change data using the LNPs of FIG. 40A.
  • FIGS. 41 A and 4 IB provide lung transfection and encapsulation percentage data of 2- lipid LNPs made while titrating mRNAdipid ratio.
  • FIGS. 42A and 42B provide lung transfection and encapsulation percentage data of 3- lipid LNPs made while titrating N/P ratio.
  • FIGS. 43 A and 43B provide lung transfection and body weight change data using the listed 2-lipid LNPS titrated with DMG-PEG2000.
  • FIG. 44 provides lung mean radiance in a comparison of microfluidic vs manual mixing of lipids in ethanol with malic acid buffer.
  • FIGS. 45A-45D provide formulation, transfection and IHC data comparing an MC3 formulation with a 5 lipid LNP composition of the present specification, demonstrating transfection of endothelial and epithelial cells of the lung alveolar region.
  • FIG. 46A provides exemplary 2, 3 and 5 lipid LNPs.
  • FIGS. 46B, 46C and 46D provide lung, liver and spleen cell transfection data using the LNPs of FIG. 46A.
  • FIG. 46E provides IHC data related to use of exemplary 2, 3 and 5 lipid LNPs in lung, liver and spleen tissue.
  • FIGS. 47A, 47B and 47C depict the pharmacokinetics of specific 2-lipid LNPs in plasma, lung and liver.
  • FIG. 48 depicts the pharmacokinetics of specific 5-lipid LNPs from Table 1.
  • FIGS. 49A and 49B depict biodistribution of exemplary 5-lipid LNPs from Table 1 when administered to a mammal.
  • FIGS. 50A, 50B and 50C depict biodistribution of 3-lipid LNP as described in FIG. 46A when administered to a mammal.
  • FIGS. 50D, 50E and 50F depict organ level bioluminescence related to the use of 3- lipid LNPs in mammals as described in Example 33.
  • FIG. 50G depicts biodistribution of exemplary 3-lipid LNPs as described in FIG. 46A when administered to a mammal.
  • FIGS. 51 A and 5 IB provide data related to telomere extension and telomerase activity in lung epithelial cells using the LNP of Table 3.
  • FIG. 52 provides data related to telomerase activity in lung fibroblasts using the LNP of Table 3.
  • FIGS. 53A and 53B provide lung transfection and encapsulation efficiency data using the LNPs of Example 38.
  • FIGS. 54A and 54B provide lung transfection and encapsulation efficiency data using the LNPs of Example 39.
  • FIG. 55 demonstrates lung mean radiance of the freeze-dried (lyophilized) LNPs of Example 36.
  • FIG. 56 models optimal cationic lipid to ionizable lipid ratio (“C/I Ratio”) and optimal lipid nitrogen : polynucleotide phosphate ratio (“N/P ratio”) for LNPs containing either the SS-OP family or DLin-MC3-DMA family of ionizable lipids.
  • the grey boxes indicate the optimal ranges of C/I ratio and N/P ratio.
  • the horizontal dashed lines indicate thresholds of relative lung radiance selected so that the convex peak(s) of relative lung radiance in each graph is/are substantially above the dashed lines.
  • the horizonal dotted line is the threshold of relative yield such that the convex curve of relative yield is substantially above the dashed line.
  • the limits of the optimal ranges are determined by the intersections of the curves of relative lung radiance and relative yield with the horizontal dashed or dotted threshold lines, respectively.
  • FIG. 57 demonstrates an exemplary flow cytometry gating strategy including DAPI to gate for live bone marrow cells.
  • FIGS. 58A, 58B and 58C provide flow cytometric results of bone marrow cells collected six days after administration of mRNA-LNPs formulated with mRNA encoding Cre recombinase and lipids in the exemplary PDS and 3-MC3 LNPs.
  • FIG. 58D refers to the results of FIGS. 58A, 58B and 58C and provides results of the bone marrow transfection efficiency for the exemplary PDS and 3-MC3 LNPs.
  • FIGS. 59A and 59B show fluorescent imaging data in Ail4 LdTomato flox/flox mice that have received an injection of exemplary 3-MC3 and PDS LNPs containing Cre mRNA (compared with control).
  • FIG. 59A shows the test Cy3 channel and
  • FIG. 59B shows the control Cy7 channel.
  • FIG. 60A shows fluorescent imaging data, with whole body gating, of the dorsal side of Ail4 tdTomato flox/flox mice that have received an injection of exemplary 3-MC3 and PDS LNPs containing Cre mRNA (compared with control).
  • FIG. 60B shows the quantification of the Cy3 image in FIG. 60A.
  • FIG. 61 A shows fluorescent imaging data, with gating around limbs, tail and pelvis, of the dorsal side of Ail4 tdTomato flox/flox mice that have received an injection of exemplary 3-MC3 and PDS LNPs containing Cre mRNA (compared with control).
  • FIG. 61B shows the quantification of the Cy3 image in FIG. 61A.
  • FIG. 62A shows the results of an assay where bone marrow cells were isolated from mice in the previous slide at 126 days post-dosing.
  • the gating structure is shown in FIGS. 57 and 58A-58C.
  • the percentage of tdTomato-i- cells in the bone marrow for PBS control, 3- MC3, and PDS is shown.
  • FIG. 62B is representative of the same data of FIG. 62A, but the percent tdTomato+ cells in the PBS control was subtracted from all samples at each time point to allow direct comparison.
  • FIG. 63A shows quantification of the percentage of transfected cells in liver hepatocytes, spleen white pulp cells, spleen red pulp cells, kidney cells, brain tissue cells, and cells obtained from the foot and leg tissues of the same Ail4 mice discussed in connection with the data of FIGS. 58-62, taken on day 126 post-delivery.
  • FIGS. 63B-63F show IHC data related to use of exemplary 3-MC3 and PDS LNPs in liver (FIG. 63B), spleen (FIG. 63C), kidney tissue (FIG. 63D), leg (FIG. 63E) and tail (FIG. 63F) of the same Ail4 mice discussed in connection with the data of FIGS. 58-63.
  • FIGS. 64A-64B show a flow cytometric experiment demonstrating hematopoietic stem cell targeting in a mammal (Lineage-, Scal+, cKit-i- bone marrow cells) using a lipid nanoparticle targeted with an anti-cKit antibody conjugated to a pegylated lipid.
  • FIG. 65 shows transfection of hematopoietic stem cells (Lineage-, Scal+, cKit+ bone marrow cells) using PDS LNP at multiple doses.
  • the term “approximately” or “about” may refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the term “and/or” may mean “and,” it may mean “or,” it may mean “exclusive-or,” it may mean “one,” it may mean “some, but not all,” it may mean “neither,” and/or it may mean “both.”
  • the terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, prevention, or treatment is desired.
  • the subject may be a mammalian subject.
  • Mammalian subjects include, e. g., humans, non-human primates, rodents, (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc.
  • the subject is a human.
  • the subject is a non-human primate, for example a cynomolgus monkey.
  • the subject is a companion or service animal (e.g.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating nucleic acid sequences which, upon being translated (if RNA) or transcribed and translated (if DNA), produce the same sequence of amino acid residues as the original nucleic acid sequence; or, in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • nucleotide sequence refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • ribonucleic acid means a polymer of ribonucleotides.
  • mRNA species means mRNA molecules with identical ribonucleotide lengths and sequences.
  • compositions capable of delivery of a polynucleotide to traditionally hard to reach places (tissues and cells) in a subject. This includes, for example, transfection of stem cells (e.g., hematopoietic stem cells, progenitor cells), tumor cells or areas receiving low level of circulation.
  • stem cells e.g., hematopoietic stem cells, progenitor cells
  • the broad transfection ability demonstrated by the present compositions provides for the ability to deliver a nucleic acid payload to any of a variety of body tissues and cells with or without specific targeting ligands (which are also contemplated for inclusion in the present LNPs (e.g., 3-MC3 and/or PDS)), including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNPs e.g., 3-MC3 and/or PDS
  • Such LNPs have very diverse use opportunities since they have been demonstrated as capable of transfecting a broad variety of cells and tissue types.
  • LNPS LNPS
  • stem cells progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells are specifically demonstrated, these are merely examples.
  • the presently described LNPs can further include attachment of ligands to the liposome surface.
  • the lipid particle comprises a targeting agent, such as a targeting lipid described herein.
  • the targeting is often moiety specific to a cell type or tissue.
  • the targeting of LNPs (e.g., 3-MC3 and/or PDS) using various targeting moieties such as ligands, cell surface receptors, glycoproteins, vitamins (eg riboflavin) and monoclonal antibodies is a known concept.
  • the targeting moiety can include the entire protein or a fragment thereof.
  • the targeting mechanism usually requires that the targeting agent be positioned on the surface of the lipid particle in such a way that the targeting moiety is available for interaction with the target (e.g., a cell surface receptor).
  • the targeting agent e.g., a cell surface receptor
  • a ligand that targets lipid particles is bound to the polar head group of the lipid that forms the lipid particle.
  • Standard methods for binding the target agent can be used.
  • targeting moieties may include other proteins specific for cellular components, including antigens associated with stem cells, progenitor cells, germ cells, and differentiated and terminally-differentiated cells, neoplasms, and tumors. Proteins used as targeting moieties can be covalently attached to liposomes (see Heath, Covalent Attachment of Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)).
  • Other targeting methods include the biotin-avidin system, maleimide-thiol reactions, and succinimidyl ester-amine reactions.
  • moieties are ligands that are bound either directly or indirectly via an intervening tether, preferably covalently.
  • the ligand alters the distribution, targeting or lifetime of the molecule in which it is incorporated.
  • the ligand is selected from a target (e.g., molecule, cell or cell type, compartment (e.g., cell compartment or organ compartment), tissue, Enhance affinity for organs or body regions).
  • a ligand that enhances affinity for a selected target is also referred to as a targeting ligand.
  • a preferred ligand for conjugation to the lipids of the present invention is a targeting ligand.
  • Some ligands can have endosomal lytic properties. Endosomal lytic ligands promote endosomal lysis and / or transport of the compositions of the invention, or components thereof, from the endosome to the cytoplasm of the cell.
  • the endosomal lytic ligand can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenic properties. In certain embodiments, the endosomal lytic ligand assumes its active conformation at endosomal pH.
  • an “active” conformation is a conformation in which the endosomal lytic ligand facilitates endosomal lysis and / or transport of the composition of the invention, or components thereof, from the endosome to the cytoplasm of the cell.
  • exemplary endosomal lytic ligands include GALA peptides, and derivatives thereof.
  • the endosomal lytic component can include chemical groups (e.g., amino acids) that change charge or protonation in response to changes in pH.
  • the endosomal lytic component may be linear or branched.
  • Preferred ligands can improve transport properties, hybridization properties, and specificity properties, and the resulting natural or modified oligoribonucleotides, or monomers and / or natural ribonucleotides described herein. It may also improve the nuclease resistance of polymer molecules containing any combination of nucleotides or modified ribonucleotides.
  • Ligands generally include, for example, therapeutic modifiers to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; crosslinkers; and moieties that confer nuclease resistance, it can. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.
  • Ligands include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids.
  • the ligand may be a recombinant molecule or a synthetic molecule, such as a synthetic polymer (e.g., a synthetic polyamino acid), an oligonucleotide (e.g., an aptamer).
  • polyamino acids examples include polylysine (PLL), poly L-aspartic acid, poly L- glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer, polyamino acids that are N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphadine.
  • PLL polylysine
  • poly L-aspartic acid examples include poly L-aspartic acid, poly L- glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer,
  • polyamines examples include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine Grade salts, or alpha helix peptides.
  • the ligand can also include a targeting group, such as a cell targeting agent or tissue targeting agent (e.g., a lectin, glycoprotein, lipid or protein (e.g., an antibody that binds to a particular cell type such as a stem cell, progenitor cell, germ cell, differentiated cell, terminally-differentiated cell, or a specific cell type, for example a kidney cell)).
  • a targeting group such as a cell targeting agent or tissue targeting agent (e.g., a lectin, glycoprotein, lipid or protein (e.g., an antibody that binds to a particular cell type such as a stem cell, progenitor cell, germ cell, differentiated cell, terminally-differentiated cell, or a specific cell type, for example a kidney cell)).
  • Type of targeting groups include antibodies, peptides, aptamers, small molecules, carbohydrates, lipids, nanobodies, and aptamer- antibody conjugates.
  • targeting groups include thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, Glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B 12, biotin, RSG peptide, RSG peptide mimic, or can be an aptamer.
  • antibodies, nanobodies, aptamers targeting clusters of differentiation on cell surfaces for example, CDla, CDlb, CDlc, CDld, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CDlla, CDllb, CDllc, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49d, CD49e, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62L, CD63, CD64, CD65, CD66, CD67,
  • ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, suffirin), polycyclic aromatic hydrocarbons (e.g., phenazine), Dihydrophenazine), artificial endonucleases (eg EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1 -pyrenebutyric acid, dihydrotestosterone, l,3-bis-0 (hexadecyl) glycerol, geranyloxy Hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoy
  • a ligand can be a protein (e.g., a glycoprotein), or a peptide (e.g., a molecule with specific affinity for a co-ligand), or an antibody (e.g., with specificity to specific types of cells, for example a stem cell, progenitor cell, germ cell, differentiated cell, or terminally differentiated cell, a cancer cell, endothelial cell, or bone cell).
  • Ligands can also include hormones and hormone receptors.
  • Ligand includes non-peptide such as lipid, lectin, carbohydrate, vitamin, cofactor, polyvalent lactose, polyvalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, or aptamer Species can also be included.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
  • a ligand is a substance that increases the uptake of a polynucleotide of the LNP into a cell, for example, by increasing the affinity of the LNP for a molecule on the surface of the cell, or increasing the affinity of the LNP for a molecule that in turn binds to a molecule on the surface of the cell, or by disrupting the cytoskeleton of the cell (e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell, e.g., a drug).
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanosine, or myoserbin.
  • the ligand can increase the uptake of the polynucleotide of the LNP into the cell, for example, via endocytosis, or by activating an inflammatory response.
  • exemplary ligands having such effects include tumor necrosis factor a (TNFa), interleukin- 113, or y interferon.
  • the ligand is a lipid or lipid-based molecule.
  • lipids or lipid-based molecules preferably bind to serum proteins such as human serum albumin (HSA).
  • HSA binding ligands allow the conjugate to be distributed to target tissues (e.g., target tissues other than the body's kidneys).
  • target tissues e.g., target tissues other than the body's kidneys.
  • the target tissue can be bone marrow containing hematopoietic stem cells.
  • Other molecules that can bind to a target tissue can also be used as ligands.
  • Lipid based ligands can be used, for example, to control the binding of the conjugate to the target tissue.
  • the inclusion of one or more targeting ligand with the LNPs of the present disclosure is provided for diagnosis, prevention and/or treatment of the suspected or actual underlying disease or condition (including those listed above in the Summary) in the specifically targeted cell type and/or tissue.
  • the presently contemplated LNPs are capable of targeting the cell type and tissue where the condition or disease manifests.
  • the presently described LNPs are shown to provide for the ability to transfect cells with the encapsulated polynucleotide throughout the whole body of the subject, including cell types that exist throughout the body.
  • the polynucleotide encapsulated in the LNP will vary based on the condition or disease and the objective of the delivery (diagnosis, prevention or treatment), the presently described LNPs are provided as an enabling delivery vehicle for such polynucleotide such that it can be delivered to areas that have been difficult or not possible to deliver a polynucleotide in vivo in a subject (e.g., mammal) previously.
  • telomere extension offers potential benefits for mitigating some age- related changes, it is crucial to consider the associated risks such as cellular immortalization, which can occur with delivery of telomerase DNA to cells.
  • telomere extension in various tissues and cell types throughout the mammalian body including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells using TERT mRNA or TERT circular RNA delivered in vivo using the delivery vehicles disclosed herein provides a way to provide a variety of benefits.
  • One such benefit includes boosting replicative and regenerative capacity of a wide range of different cell populations, where increased telomerase activity can delay replicative senescence in a specifically targeted cell population, promoting tissue renewal and homeostasis. Another benefit is to help preserve or enhance tissue structure in a variety of organs and tissues, wherein improved structural integrity through telomere extension can protect against or slow the rate of degradation of that tissue. Still another such benefit includes the ability to accelerate wound healing in tissues, where promoting cellular proliferation and migration can enhance the tissue’s regenerative capacity.
  • telomere extension strategies might offer a potential approach to reduce the risk of a variety of cancers associated with aging.
  • the delivery of diagnostic and/or therapeutic agents such as mRNAs (including TERT mRNA) using LNPs described herein to cells can be provided in a method to treat, ameliorate or prevent a condition or disease that can be modulated by an engineered polynucleotide such as a modified mRNA.
  • an engineered polynucleotide such as a modified mRNA.
  • contemplated a variety of conditions or diseases where the condition or disease itself or its symptoms that may be modulated (e.g., treated, ameliorated or prevented) by the presently described and encompassed LNP compositions and formulations are contemplated.
  • These include preventing, treating or ameliorating various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells conditions or diseases.
  • the data provided herein demonstrate or support, for example, pharmacokinetics of the exemplified LNPs in vivo, and particularly information and data related to the time course of concentration levels of a lipid from the LNPs in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • L P compositions and formulations are provided that are rapidly cleared from the circulatory system of the subject but have a longer half-life in the body tissues.
  • the data provided herein also demonstrate or support, for example, the use of LNPs in which the mRNA encodes telomerase.
  • LNPs for use in introducing nucleic acids to body tissues including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • the LNPs are manufactured in a manner that forms the LNP prior to addition of nucleic acid.
  • such LNPs are 2, 3 or 5 lipids LNPs described herein and the method often employs vortex mixing during the addition of the nucleic acid.
  • the present inventors have found that in LNPs adapted to work according to the presently contemplated methods and uses, the ratios of cationic lipid to ionizable lipid (e.g. SSOP : DOTAP or MC3 : DOTAP) are unpredictable based on the specific constituents of the LNP. Specifically, while not intending to be bound by any specific theory, experimentally it appears that the particular ionizable lipid in the LNP affects the cationic lipid to ionizable lipid ratio in a manner that requires experimentation to confirm.
  • LNPs are used in the presently described methods to introduce a genetic pay load (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic pay load e.g., mRNA, including TERT mRNA
  • the cationic lipid to ionizable lipid ratio is at or about 1.8 and the ionizable lipid is SS-OP.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.8 and is composed of 5 lipids.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.8 and is composed of 5 lipids, including DOTAP and DOPC.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.8 and is composed of 5 lipids, including a pegylated lipid (e.g., DMG-PEG2000), DOPC and DOTAP.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • mRNA including TERT mRNA
  • the LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • mRNA including TERT mRNA
  • the cationic lipid to ionizable lipid ratio is at or about 1.5 and the ionizable lipid is DLin-MC3-DMA.
  • the LNP has a cationic lipid to ionizable lipid ratio is at or about 1.5 and is composed of 3 lipids.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.5 and is composed of 3 lipids, including DOTAP.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.5 and is composed of 3 lipids, including a pegylated lipid (e.g., DMG-PEG2000) and DOTAP.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • mRNA including TERT mRNA
  • the LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • mRNA including TERT mRNA
  • LNP compositions of the present disclosure include and encompass a variety of lipid ratios and lipid : mRNA ratios (lipid : mRNA ratios are also referred to herein as “N/P ratio”).
  • LNP compositions refers to either or both the final LNP composition or the formulation used to produce the LNP which will be evident based on the context in which the phrase is used.
  • Specific examples of contemplated compositions are provided in the Examples, including the supporting Figures.
  • Particularly preferred LNP compositions of the present disclosure are provided, for example, in Table 7 and other tables, Figures and descriptions provided herein.
  • nucleic acid containing LNP compositions composed of 5 different lipids, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 10 and 30 are provided.
  • Table 7 provides some such exemplary compositions.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of between 0.8 to 1.8.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of at or about 0.8.
  • such LNP compositions include SS-OP as the ionizable lipid in the composition.
  • LNPs are used in the presently described methods to introduce a genetic pay load (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic pay load e.g., mRNA, including TERT mRNA
  • such LNP compositions are used in vivo in a mammal subject and target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a manner that provides for transfection of the nucleic acid payload in the various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions are contemplated herein as adapted to various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNP compositions preferentially target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • such LNP compositions have an increased half-life in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • such LNP compositions have a half-life in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells or over 30 hours, or between 30 hours and 40 hours, or about 37 hours.
  • nucleic acid containing LNP compositions composed of 3 different lipids, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 4 and 24 are provided, or an N/P ratio of between 4.5 and 12.
  • Table 7 provides some such exemplary compositions.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of between 0.8 to 1.5.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of at or about 0.8.
  • such LNP compositions include SS-OP as the ionizable lipid in the composition and a cationic lipid to ionizable lipid ratio of at or about 0.8. Also according to such embodiments such LNP compositions include DLin-MC3-DMA as the ionizable lipid in the composition and a cationic lipid to ionizable lipid ratio of at or about 1.5, and optionally an N/P ratio of at or about 4.5.
  • such LNP compositions are used in vivo in a mammal subject and target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a manner that provides for transfection of the nucleic acid payload in the various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions are contemplated herein as adapted to target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNP compositions preferentially target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • nucleic acid containing LNP compositions composed of 3 or 5 different lipids including DOTAP, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 4 and 24 are provided, or an N/P ratio of at or about 12. Also according to preferred embodiments herein, nucleic acid containing LNP compositions composed of 3 different lipids including DLin-MC3-DMA, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 4 and 24 are provided, or an N/P ratio of at or about 4.5.
  • LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • nucleic acid containing LNP compositions composed of 2 different lipids having a N/P ratio of at or about 4 are provided.
  • one of the two lipids is or comprises DOTAP.
  • such LNP compositions are used in vivo in a mammal subject and target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a manner that provides for transfection of the nucleic acid payload in the various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions are contemplated herein as adapted to target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • sucrose is a surprisingly good cryoprotectant for the presently contemplated LNP compositions. Specifically, it has been found that sucrose included at a range of between 5% to 25% of the final composition provides for preferred levels of cryoprotection. In specific embodiments the cryoprotectant (e.g., sucrose) is included at or about 15% of the final composition.
  • LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • telomere delivery of synthetic nucleoside-modified mRNA encoding TERT enables transient elevation of telomerase activity in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells which extends telomeres in hours sufficiently to reverse years of telomere shortening.
  • LNP lipid nanoparticle
  • IV intravenous
  • Administration may also be provided transcutaneously, intracutaneous injection via microneedle array, topical delivery (e.g., in conjunction with a skin barrier-permeabilizing step such as microneedling or application of a permeabilizing agent such as a mixture containing proteases, lipases, or others known in the field).
  • RNA ribonucleic acid
  • RNA may refer to any RNA sequence comprising a mutation (point or deletion) or additional nucleotides not found in the wild type sequence.
  • a messenger RNA mRNA
  • mRNA messenger RNA
  • the nucleotides themselves may encode amino acids distinct from the wild type, or be modified to reduce immunogenicity in the cell or tissue.
  • An mRNA sequence in some embodiments may comprise any of the following modifications, including but not limited to an untranslated region (UTR), a 5' cap, and a poly-adenosine tail.
  • the RNA may be circular and/or self-replicating.
  • a mRNA may comprise a codon-optimized sequence. In some embodiments, a mRNA may comprise a uridine depleted sequence.
  • the 5' cap of the ribonucleic acid is a non-immunogenic cap.
  • the 5' cap may increase the translation of the ribonucleic acid.
  • the 5' cap may be treated with phosphatase to modulate the innate immunogenicity of the ribonucleic acid.
  • the 5' cap is an anti-reverse cap analog (“ARCA”), such as a 3'-O-Me-m7G(5')ppp(5')G RNA cap structure analog.
  • the 5' cap is m7G(5')ppp(5')(2'OmeA)pG (also known as CleanCap® AG).
  • the 5' cap is m7(3'OmeG)(5')ppp(5')(2'OmeA)pG (also known as CleanCap® AG (3' OMe)).
  • the above features, or others may increase translation of the protein encoded by the ribonucleic acid, may increase or decrease the stability of the ribonucleic acid itself in a cell type-specific or cell type-independent manner, or may do both.
  • the 5' UTR and/or the 3' UTR are from a gene that has a very stable mRNA and/or an mRNA that is rapidly translated, for example, a-globin or P-globin, c-fos, or tobacco etch virus.
  • the 5' UTR and 3' UTR are from different genes or are from different species than the species into which the compositions are being delivered.
  • the UTRs may also be assemblies of parts of UTRs from the mRNAs of different genes, where the parts are selected to achieve a certain combination of stability and efficiency of translation.
  • the UTRs may also comprise designed sequences that confer properties to the RNA such as cell type-specific stability or cell type-independent stability.
  • the ribonucleic acids of the present disclosure may comprise one or more modified nucleosides, and/or comprise primary sequences of nucleosides, that modulate translation, stability, or immunogenicity of the RNA.
  • Most mature RNA molecules in eukaryotic cells contain nucleosides that are modified versions of the canonical unmodified RNA nucleosides, adenine, cytidine, guanosine, and uridine.
  • the 5' cap of mature RNA comprises a modified nucleoside, and other modified nucleosides often occur elsewhere in the RNA. Those modifications may prevent the RNA from being recognized as a foreign RNA.
  • Synthetic RNA molecules made using certain nucleosides are much less immunogenic than unmodified RNA.
  • the immunogenicity can be reduced even further by purifying the synthetic mRNA, for example by using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the modified nucleosides may be, for example, chosen from the nucleosides listed below.
  • the nucleosides are, in some embodiments, pseudouridine, 1 -methylpseudouridine, 2- thiouridine, 5-methoxyuridine, or 5 -methylcytidine.
  • the primary sequence may be modified in ways that increase or decrease immunogenicity. Under some circumstances, it may be desirable for the modified RNA to retain some immunogenicity.
  • the ribonucleic acids of the instant compositions comprise a 1 -methylpseudouridine, pseudouridine, a 5-methoxyuridine (5-moU), a 2-thiouridine, a 5 -methylcytidine, or another modified nucleoside.
  • Modified nucleosides found in eukaryotic cells include mlA 1 -methyladenosine, m6A N6-methyladenosine, Am 2'- O-methyladenosine, i6A N6-isopentenyladenosine, io6A N6-(cis- hydroxyisopentenyl)adenosine, ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, g6A N6-glycinylcarbamoyladenosine, t6A N6-threonylcarbamoyladenosine, ms2t6A 2-methylthio-N6-threonyl carbamoyladenosine, Ar(p) 2'-O-ribosyladenosine (phosphate), mb 2A N6,N6-dimethyladenosine, m6Am N6,2
  • the presence of the modified nucleosides, and/or sequences of nucleosides that alter secondary structure of the RNA and/or binding of RNA to RNA binding proteins or microRNA, may enable mRNA to avoid activation of an immune response mediated by various receptors, including the Toll-like receptors and RIG-1.
  • Non-immunogenic mRNA has been used as a therapeutic agent in mice via topical delivery. Kormann et al. (2011) Nature Biotechnology 29:154-157.
  • the ribonucleic acids comprise more than one of the above nucleosides or combination of the above nucleosides.
  • the ribonucleic acids comprise 1- methylpseudouridine, 5-methoxyuridine, or pseudouridine and 5 -methylcytidine.
  • an immune response to the mRNA may be desired, and the RNA may be modified to induce an optimal level of innate immunity. In other embodiments, an immune response to the mRNA may not be desired, and the RNA may be modified in order to minimize such a reaction.
  • the RNA can be modified for either situation.
  • the ribonucleic acid molecules can be synthetic ribonucleic acids.
  • the term “synthetic”, as used herein, can mean that the ribonucleic acids are in some embodiments prepared using the tools of molecular biology under the direction of a human, for example as described below.
  • the synthetic ribonucleic acids may, for example, be prepared by in vitro synthesis using cellular extracts or purified enzymes and nucleic acid templates.
  • the synthetic ribonucleic acids may in some embodiments be prepared by chemical synthesis, either partially or completely.
  • the synthetic ribonucleic acids may in some embodiments be prepared by engineered expression in a cell, followed by disruption of the cell and at least partial purification of the ribonucleic acid.
  • the ribonucleic acids of the present disclosure may be prepared using a variety of techniques, as would be understood by one of ordinary skill in the art.
  • the ribonucleic acids may be prepared by in vitro synthesis.
  • the ribonucleic acids may be prepared by chemical synthesis.
  • the ribonucleic acids may be prepared by a combination of in vitro synthesis and chemical synthesis.
  • synthetic should be understood to include ribonucleic acids that are prepared either by chemical synthesis, by in vitro synthesis, by expression in vivo and at least partial purification, or by a combination of such, or other, chemical or molecular biological methods.
  • the ribonucleic acids may, in some embodiments, be purified. As noted above, purification may reduce immunogenicity of the ribonucleic acids and may be advantageous in some circumstances. In some embodiments, the ribonucleic acids are purified by one or more of HPLC, DNAse treatment, protease treatment, or by affinity capture and elution.
  • an mRNA sequence may be synthesized as an unmodified or modified mRNA.
  • An mRNA may be modified to enhance stability and/or evade immune detection and degradation.
  • a modified mRNA may include, for example, one or more of a nucleotide modification, a nucleoside modification, a backbone modification, a sugar modification, and/or a base modification.
  • the modified nucleoside is pseudouridine or a pseudouridine analog.
  • the pseudouridine analog is N-l-methylpseudouridine.
  • the modified nucleoside is 5- methoxyuridine.
  • a modified nucleoside as used herein may comprise any of the moieties listed in Table A.
  • an RNA e.g. an mRNA
  • an RNA may be synthesized from naturally occurring nucleosides and/or nucleoside analogs (modified nucleosides) including, but not limited to, nucleosides comprising adenosine (A), guanosine (G)) or pyrimidines (thymine (T), cytidine (C), uridine (U)), and nucleoside comprising analogues and derivatives thereof, e.g., 3 '-deoxy adenosine (cordycepin), 3 '-deoxyuridine, 3'-deoxycytosine, 3'- deoxyguanosine, 3 '-deoxy thymine, 2',3'-dideoxynucleosides, 2', 3'- dideoxyadenosine, 2', 3'- dideoxyuridine, 2', 3 '-dideoxycytosine, 2', 3 '-dideoxycytosine, 2', 3
  • uracil nucleosides of the mRNA are about 80%, about 90%, 95%, 99%, or 100% depleted and replaced with a uracil nucleoside analog, e.g., pseudouridine, 5-methoxyuridine, or N-l-methyl-pseudouridine.
  • a uracil nucleoside analog e.g., pseudouridine, 5-methoxyuridine, or N-l-methyl-pseudouridine.
  • an RNA may contain an RNA backbone modification.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are chemically modified.
  • Exemplary backbone modifications may include, but are not limited to, modifications in which the phosphodiester linkage is replaced with a member from the group consisting of peptides, methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-0-(l- thiophosphate)), boranophosphates, and/or positively charged guanidimum groups, or other means of replacing the phosphodiester linkage.
  • an RNA may contain sugar modifications.
  • a sugar modification may include but is not limited to, 2' O-methyl sugar modifications, 2' fluoro sugar modifications (e.g. 2'-fluororibose), 3' amino sugar modifications, 2' thio sugar modifications, 2'-O-alkyl sugar modifications, 5 -methylthioribose, and sugar modifications of 2'-deoxy-2'- fluoro-ribonucleotide (2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine), 2'-deoxy-2'- deamine-ribonucleotide (2'-amino-2'-deoxycytidine, 2,-amino-2'-deoxyuridine), 2'-O- alkylribonucleotide, 2'-deoxy-2'-C-alkylribonucleotide (2'-O-methylcytidine, 2'- methyluridine), 2'-C-
  • an RNA may be synthesized from one or more of the nucleotide triphosphates comprising any of the nucleosides and nucleotides disclosed herein, or any of the following nucleoside triphosphates: 2'-Deoxyadenosine-5'-O-(l-Thiotriphosphate), 2'-Deoxycytidine-5'-O-(l -Thiotriphosphate), 2'-Deoxyguanosine-5'-O-(l-Thiotriphosphate), 2'-Deoxythymidine-5'-O-(l -Thiotriphosphate), Adenosine-5'-O-(l-Thiotriphosphate), Cytidine-5 '-O-( 1 -Thiotriphosphate) , Gu nosine-5 '-O-( 1 -Thiotriphosphate) , Uridine-5 '-O-( 1 - Thio triphosphate), 2',3'-Dide
  • an mRNA may include the addition of a “cap” on the N- terminal (5') end, and a “tail” on the C-terminal (3') end.
  • the presence of the cap may provide resistance to nucleases found in eukaryotic cells.
  • the presence of a “tail” may protect the mRNA from exonuclease degradation.
  • an mRNA may include a 5' cap structure.
  • a 5' cap may comprise for example, a triphosphate linkage and a guanine nucleotide in which the 7-nitrogen is methylated.
  • Examples of cap structures include, but are not limited to, m7G(5')ppp (5') A, G(5')ppp(5')A, and G(5')ppp(5')G.
  • Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5 '-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside.
  • the cap is added in the nucleus by the enzyme guanylyl transferase immediately after initiation of transcription.
  • a 5 'cap may comprise an m7(3'OmeG)(5')ppp(5')(2'OmeA)pG or (CleanCap® 3' OMe) structure.
  • a 5' cap may comprise a m7G(5')ppp(5')G.
  • the AntiReverse Cap Analog (“ARCA”) or modified ARCA is a 5' cap in which the 2' or 3' OH group is replaced with -OCH3.
  • the ARCA comprises an 3'-0-Me- m7G(5')ppp(5')G structure.
  • the 5' cap comprises m7G(5')ppp(5')(2'OmeA)pG.
  • Additional mRNA caps may include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated caps e.g., GpppG); a memethylated cap (e.g., m2'7GpppG), a trimethylated cap analog, or anti reverse cap analogs (e.g., ARCA; m7,2'0meGpppG, m72'dGpppG, m7’3'0meGpppG, m7,3 dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al, ‘Wove anti-reverse cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).
  • a suitable cap is a 7-methyl guanylate (“m7G”) linked via a triphosphate bridge to the 5 ‘-end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleoside.
  • An embodiment of an m7G cap utilized in embodiments of the disclosure is m7G(5')ppp(5')G.
  • the cap is a CapO structure. CapO structures lack a 2'-0- methyl residue of the ribose attached to bases 1 and 2.
  • the cap is a Capl structure. Capl structures have a 2'-0-methyl residue at base 2.
  • the cap is a Cap2 structure. Cap2 structures have a 2'-0-methyl residue attached to both bases 2 and 3.
  • m7G cap analogs are known in the art, many of which are commercially available. These include the m7 GpppG described above, as well as the ARCA 3'- OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the disclosure include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E.
  • RNA, 10: 1479-1487 (2004) phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al, RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Patent Nos. 8,093,367 and 8,304,529, incorporated by reference herein.
  • the 5' cap is inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, CleanCap®, m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.
  • the 5' cap comprises or consists of an internal ribosome entry site (IRES).
  • IRES is within the 5' UTR.
  • the 5' cap comprises or consists of a 2A self-cleavage peptide, e.g, one or more of P2A, T2A, E2A and F2A.
  • a “tail” may serve to protect an mRNA from exonuclease degradation.
  • the poly-A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly-A tail can be added to an mRNA molecule thus rendering the RNA more stable.
  • Poly-A tails can be added using a variety of art- recognized techniques. For example, long poly-A tails can be added to synthetic or in vitro transcribed RNA using poly-A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly-A tails. In addition, poly-A tails can be added by transcription directly from PCR products.
  • Poly-A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2 nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
  • an mRNA may include a 3' poly(A) tail structure.
  • the length of the poly-A tail may be at least about 10, 50, 100, 200, 300, 400 or at least about 500 nucleotides.
  • a poly-A tail on the 3' terminus of an mRNA may include about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • the poly A tail is 120 adenosine nucleotides.
  • an mRNA may include a 3' poly-C tail structure.
  • a poly- C tail on the 3' terminus of mRNA may include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • the length of the poly-A or poly C tail is associated with the stability of a modified sense mRNA and, therefore, the transcription of the protein.
  • the length of the poly-A tail may influence the half-life of a sense mRNA molecule, the length of the poly-A tail may be adjusted to modify the level of resistance of the mRNA to nucleases, thereby providing more control over the time course of polynucleotide expression and/or polypeptide production.
  • an mRNA may include 5' untranslated region (UTR) and/or a 3' UTR.
  • a 5' UTR may include one or more elements that affect the stability or translation of an mRNA.
  • the 5'UTR for example, may include an iron responsive element.
  • 5' UTR may be between about 50 to about 100, or from about 50 to about 500 nucleotides in length.
  • 3' UTR includes one or more of a poly-A signal, a binding site for proteins that may affect mRNA stability or localization, or one or more binding sites for miRNAs.
  • 3' UTR may be between about 0 and about 50 nucleotides, or about 50 to about 100 nucleotides in length
  • Example 3' an’ 5' UTR sequences may be derived from mRNAs with relatively long half-lives (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule.
  • 5' UTR sequence may include a partial sequence of a cytomegalovirus (CMV) immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide.
  • CMV cytomegalovirus
  • IE1 immediate-early 1
  • the 5 ’ UTR could include the sequence of the tobacco etch virus (TEV).
  • these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides resistance to in vivo nuclease digestion.
  • a UTR may improve tissue specific expression, e.g., in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • the UTR may be any of, or functional variants of, those described in any of PCT Application No. WO2017053297A1 and Patent No. US10519189B2, both of which are incorporated herein in their entirety. Ionizable lipids
  • an LNP may comprise an ionizable lipid, e.g. SS-OP or analogs thereof, or MC3 or analogs thereof.
  • the charge of the lipid may depend on pH of the surrounding solution, making it an ionizable lipid.
  • the ionizable lipid may also be cleavable.
  • the ionizable lipid may be cationic at ranges of pH found in endosomes or lysosomes in mammalian cells.
  • An ionizable lipid may refer to any of a number of lipid species that have a net positive charge at a selected pH, such as a physiological pH.
  • an LNP may comprise an ionizable lipid as disclosed in either of WO 2010/053572 or WO 2012/170930, or variations thereof, both of which are incorporated herein by reference in their entirety.
  • an LNP may comprise one or more of MC3 (((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate), DLin- MC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yL 10,13-nonadecadien-l-yl ester), or analogs thereof including but not limited to LenMC3, y- LenMC3, MC3MC, MC2C, MC2MC, MC3 Thioester, MC3 Ether, MC4 Ether, MC3 Alkyne, MC3 Amide, Pan-MC3, Pan-MC4, Pan-MC5, CP-LenMC3, CP-y-LenMC3,
  • an LNP may comprise one or more of cKK-E12 (3,6- Bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione), DLinDAP (l,2-dilineoyl-3- dimethylammonium-propane), DLin-DMA, DLin-D-DMA, DLin-KC2-DMA (2,2-dilinoleyL 4-dimethylaminoethyl-[l,3]-dioxolane), and DODMA.
  • cKK-E12 3,6- Bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione
  • DLinDAP l,2-dilineoyl-3- dimethylammonium-propane
  • DLin-DMA DLin-D-DMA
  • DLin-KC2-DMA 2,2-dilinoleyL 4-dimethyl
  • the ionizable lipid may comprise SS-OP or analogs thereof. In some embodiments, the ionizable lipid is a compound of Formula (1):
  • R la and R lb each independently represents an alkylene group having 1 to 6 carbon atoms, and may be linear or branched.
  • the alkylene group may have 1 to 4 carbon atoms, or may have 1 to 2.
  • Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, and a neopentylene group.
  • R la and R lb may be each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group, or a tetramethylene group, and may be an ethylene group.
  • R la may be different or be the same as R lb .
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups, and/or each independently a cyclic alkylene having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups and an alkylene tertiary amino group.
  • the alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is branched even if it is linear.
  • the alkyl group may be annular.
  • the alkyl group may have 1 to 3 carbon atoms.
  • Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • Neopentyl group, t-pentyl group, 1 ,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, A cyclohexyl group etc. can be mentioned.
  • a specific structure of an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is represented by X 1 .
  • R 5 of X 1 represents an alkyl group having 1 to 6 carbon atoms and may be linear, branched or cyclic.
  • the alkyl group may have 1 to 3 carbon atoms.
  • Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec -butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • the number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups may be 4 to 5.
  • Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include aziridylene group, azetidylene group, pyrrolidylene group, piperidylene group, imidazolidylene group, a piperazylene group, optionally a pyrrolidylene group, a piperidylene group or a piperazylene group.
  • Number is 2 to 5 carbon atoms, and specific structure of alkylene tertiary amino groups containing 1 annular tertiary amino group represented by X 2 .
  • P of X 2 is 1 or 2.
  • X 2 is a pyrrolidylene group, and when p is 2,
  • X 2 is a piperidylene group.
  • a specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is represented by X 3 .
  • W of X 3 is 1 or 2.
  • X 3 is an imidazolidylene group, and when w is
  • X 3 is a piperazylene group.
  • X a may be different be identical to X b .
  • R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having 8 or less carbon atoms, optionally each independently an alkylene group having 8 or less carbon atoms.
  • the alkylene group having 8 or less carbon atoms may be linear or branched but is optionally linear.
  • the number of carbon atoms contained in the alkylene group is optionally
  • alkylene group having 8 or less carbon atoms include methylene group, ethylene group, propylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, and the like.
  • a methylene group, an ethylene group, a propylene group, and a tetramethylene group are included.
  • the oxydialkylene group having 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) via an ether bond, and the total number of carbon atoms of two alkylene groups is 8 or less.
  • the two alkylenes may be the same or different, but are optionally the same.
  • Specific examples of the oxydialkylene group having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydipropylene group, and an oxydibutylene group.
  • R 2a may be the same or different from R 2b .
  • Y a and Y b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, optionally each independently an ester bond, an amide bond or a carbamate bond. While Y binding orientation of Y a and Y b are not limited, if Y a and Y b is an ester bond, optionally, -Z a -CO — R 2a - and -Z b -CO-O-R 2b -structure. [00219] Y a may be different or identical to Y b .
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom.
  • the number of carbon atoms contained in the aromatic compound is optionally 6 to 12, or 6 to 7.
  • the number of aromatic rings contained in the aromatic compound is optionally one.
  • aromatic rings contained in the aromatic compound having 3 to 16 carbon atoms as for aromatic hydrocarbon rings, benzene ring, naphthalene ring, anthracene ring, and aromatic heterocycles as imidazole ring, pyrazole ring, oxazole ring, Isoxazole ring, thiazole ring, isothiazole ring, triazine ring, pyrrole ring, furanthiophene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, purine ring, pteridine ring, benzimidazole ring, indole ring, benzofuran ring, quinazoline ring, phthalazine ring, quinoline ring, isoquinoline ring, coumarin ring, chromone ring, benzodiazepine ring, phenoxazine
  • the aromatic ring may have a substituent.
  • substituents include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, and 2 to 2 carbon atoms.
  • acyloxy groups acylamino groups having 2 to 4 carbon atoms, alkoxycarbonylamino groups having 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylsulfanyl groups having 1 to 4 carbon atoms, 1 carbon atom Alkylsulfonyl group having 4 to 4, arylsulfonyl group having 6 to 10 carbon atoms, nitro group, trifluoromethyl group, cyano group, alkyl group having 1 to 4 carbon atoms, ureido group having 1 to 4 carbon atoms, 1 to carbon atoms 4 alkoxy groups, aryl groups having 6 to 10 carbon atoms, aryloxy groups having 6 to 10 carbon atoms, and the like.
  • Some examples include acetyl groups, methoxycarbonyl groups, methyl carbonate groups, and the like, moyl group, acetoxy group, acetamide group, methoxycarbonylamino group, fluorine atom, chlorine atom, bromine atom, iodine atom, methylsulfanyl group, phenylsulfonyl group, nitro group, trifluoromethyl group, cyano group, methyl group, ethyl group Propyl group, isopropyl group, t-butyl group, ureido group, methoxy group, ethoxy group, propoxy group, isopropoxy group, t-butoxy group, phenyl group and phenoxy group.
  • a specific structure of Z a and Z b includes Z 1 .
  • s represents an integer of 0 to 3
  • t represents an integer of 0 to 3
  • u represents an integer of 0 to 4
  • S in Z 1 is optionally an integer of 0 to 1.
  • T in Z 1 is optionally an integer of 0 to 2.
  • U in Z 1 is optionally an integer of 0 to 2.
  • R 4 in Z 1 is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the process of synthesizing the ionizable lipid.
  • the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, and an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, fluorine atom, chlorine atom, bromine atom, iodine atom, alkylsulfanyl group having 1 to 4 carbon atoms, alkylsulfonyl group having 1 to 4 carbon atoms, 6 to 10 carbon atoms arylsulfonyl group, nitro group, trifluoromethyl
  • Z a may be different even identical to the Z b .
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or glutaric acid.
  • a C 12-22 aliphatic hydrocarbon group and optionally each independently an aliphatic hydrocarbon group having 12-22 carbon atoms.
  • Examples of the fat-soluble vitamin having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotaxolol, tocopherol, and tocotrienol.
  • the fat-soluble vitamin having a hydroxyl group is optionally tocopherol.
  • Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, 0-sitosterol, lanosterol, ergosterol and the like, optionally cholesterol or cholestanol.
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched.
  • the aliphatic hydrocarbon group may be saturated or unsaturated.
  • the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, optionally 1 to 3, or 1 to 2.
  • Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds.
  • the number of carbon atoms contained in the aliphatic hydrocarbon group is optionally 13 to 19, or 13 to 17.
  • the aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group and the like, and optionally includes an alkyl group or an alkenyl group.
  • aliphatic hydrocarbon group having 12 to 22 carbon atoms include dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, heicosyl, docosyl, dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icocenyl group, henicocenyl group, dococenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group,
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms is optionally a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1 -hexylnonyl group, or a tridecyl group, a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group.
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by R 3a and R 3b is derived from a fatty acid.
  • the carbonyl carbon derived from the fatty acid is contained in — CO — O — in the formula (1).
  • Specific examples of the aliphatic hydrocarbon group include a heptadecenyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.
  • R 3a may be different be the same as R 3b .
  • R l a is the same as R lb
  • X a is the same as X b
  • R 2a is the same as R 2b
  • Y a is the same as Y b
  • Z a is identical to the Z b
  • R 3a is the same as R 3b .
  • Preferable examples of the ionizable lipid represented by the formula (1) include the following ionizable lipids: Ionizable lipid (1-1); R la and R lb are each independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene group, ethylene group); X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., — N (CH 3 ) — ), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidylene group); R 2a and R 2b are each independently an alkylene group having 8 or less carbon atoms (e.g., methylene group, ethylene group, propylene group); Y a and Y b are each independently an ester bond or an amide bond
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic group having 12 to 22 carbon atoms, a hydrocarbon group (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group).
  • a fat-soluble vitamin having a hydroxyl group e.g., tocopherol
  • succinic anhydride or glutaric anhydride e.g., glutaric anhydride
  • an aliphatic group having 12 to 22 carbon atoms
  • a hydrocarbon group e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group.
  • R la and R lb are each independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group);
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g., — N (CH 3 ) — ), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g., piperidylene group);
  • R 2a and R 2b are each independently an alkylene group having 6 or less carbon atoms (e.g., methylene group, ethylene group, propylene group);
  • Y a and Y b are each independently an ester bond or an amide bond;
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., Heptadecenyl group, heptadecadienyl group, 1 -hexylnonyl group).
  • ionizable lipid according to Formula 1 of the present disclosure include the following O-Ph-P3Cl, O-Ph-P4Cl, O-Ph-P4C2, O-Bn-P4C2, E-Ph- P4C2, L-Ph- P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, and O-Ph-C3M as seen in the following charts.
  • Lipids having the structure of Formula I are shown in the chart below. For example, SS-
  • OP is also named 0-Ph-P4C2.
  • SS-OP analog refers to a compound of
  • an LNP of the disclosure comprises a cationic lipid, e.g. DOTAP or variations thereof.
  • the cationic lipid may be a “permanent cationic lipid.”
  • the term cationic lipid may be cationic in pH ranges found in mammalian physiological environments such as blood or interstitial fluids.
  • Cationic lipids may be composed of a cationic amine moiety and a lipid moiety, and the cationic amine moiety and a polyanion nucleic acid may interact to form a positively charged liposome or lipid membrane structure. Thus, uptake into cells may be promoted and nucleic acids delivered into cells.
  • the cationic lipid may selected from one or more of 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylarnmonium bromide (DABB), or l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).
  • DOTAP 1,2- dioleoyl-3-trimethylammonium-propane
  • DABB N,N-distearyl-N,N-dimethylarnmonium bromide
  • EPC l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine
  • an LNP comprises a ionizable lipid wherein the ionizable lipid is one or more of N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5- carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3- Dimethylammonium- Propane (DODAP), 11 ,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), l,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), l,2-dilinoleyloxy-N,N- dimethyl-3-amin
  • a cationic lipid refers to a cationic cholesterol lipid.
  • an LNP comprises imidazole cholesterol ester (ICE).
  • ICE structure is substantially similar to:
  • an LNP comprises 25-Hydroxycholesterol (25 OH Choi).
  • 25 OH Choi structure is substantially similar to:
  • an LNP comprises 20a-hydroxycholesterol 5-cholestene-3a.
  • the 20a-hydroxycholesterol 5-cholestene-3a (also known as 20a-diol or 20a chol structure) is substantially similar to:
  • a cationic lipid refers to dimethyldioctadecylammonium bromide (DDAB).
  • an LNP comprises dimethyldioctadecylammonium bromide (DDAB).
  • DDAB dimethyldioctadecylammonium bromide
  • the LNP comprises a structural lipid.
  • structural lipids are lipids that contribute a physical or chemical property to the LNP that is in addition to, or independent of, electrical charge.
  • structural lipids may tend to have a shape, size, rigidity, hydrophobicity, or other property that increases the diagnostic and/or therapeutic utility of the LNP, such as, for example, by increasing its stability, half-life, deformability, transfection efficiency, tropism, thermostability, resistance to aggregation, membrane fluidity, or other parameter.
  • structural lipids are neutral in charge, either due to lacking charged moieties, or due to being zwitterionic with balanced charges summing to zero net charge.
  • an LNP may comprise a structural lipid selected from one more of: l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerolmonooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC) , palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclo
  • an LNP may include one or more phosphatidyl lipids, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine).
  • an LNP may comprise sphingolipids, for example but not limited to, sphingosine, ceramide, sphingomyelin, cerebroside and ganglioside.
  • the aforementioned “structural” lipids contribute to the stability and/or specificity of the LNP composition. Cholesterol-based Lipids
  • an LNP may comprise one or more cholesterol-based lipids.
  • a cholesterol-based lipid may include but is not limited to: PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine.
  • an LNP may comprise one or more PEGylated lipids.
  • PEG polyethylene glycol
  • PEG-CER derivatized ceramides
  • C8 PEG-2000 ceramide N-Octanoyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000]
  • PEGylated lipids comprise PEG-ceramides having shorter acyl chains (e.g., C14 or Cl 8).
  • the PEGylated lipid DSPE-PEG- Maleimide-Lectin may be used.
  • PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C2o length.
  • the addition of PEGylated lipids may prevent complex aggregation and increase circulation lifetime to facilitate the delivery of the liposome encapsulated mRNA to the target cell.
  • Methods of diagnosis treatment, and/prevention as described herein in certain embodiments refer to transfection of various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject in need thereof by administration of an LNP of the present disclosure comprising one or more mRNA sequences.
  • compositions and methods of the disclosure may be used for the diagnosis, prevention and/or treatment of conditions in or involving various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment of fibrotic conditions, including fibrosis.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment of fibrotic conditions, including fibrosis induce encapsulated mRNA expression in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment do not induce cellular, tissue or systemic toxicity.
  • Compositions may be administered systemically (e.g.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment of conditions in or involving various tissues and cell types throughout the mammalian body including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells, do not induce cellular or systemic toxicity.
  • compositions comprising an exemplary LNP described herein and a nucleic acid encoding an active agent such as TERT mRNA for use in preventing, treating or ameliorating various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells conditions or diseases may be administered intravenously, transcutaneously, intracutaneous injection via microneedle array, topical delivery (e.g., in conjunction with a skin barrier- permeabilizing step such as microneedling or application of a permeabilizing agent such as a mixture containing proteases, lipases, or others known in the field).
  • an active agent such as TERT mRNA
  • lipid nanoparticle comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between 0.6 and 1, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell
  • lipid nanoparticle comprising: (i) DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (ii) a cationic lipid at a molar percentage of between about 50% and about 70%, with a cationic lipid: ionizable lipid (C/I) ratio between 1 and 2, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types in the subject.
  • LNP lipid nanoparticle
  • the PEGylated lipid is DMG-PEG2000.
  • the cationic lipid is DOTAP.
  • the LNP of the methods described above and herein is comprised of the lipids set forth in Table 7.
  • the target cell types are located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 4 to 6 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 5 to 8 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 7 to 10 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 9 to 12 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 10 to 15 distinct organs of the subject.
  • the delivery of the polynucleotide is for the diagnosis, prevention and/or treatment of conditions or disease of various tissues and cell types throughout the mammalian body.
  • conditions or diseases include influenza, asthma, diabetes mellitus type 1, diabetes mellitus type 2, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer’s disease, Parkinson’s disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn’s disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testi
  • COPD chronic obstructive
  • the cell types may include, for example, stem cells (e.g., hematopoietic stem cells, progenitor cells), differentiated cells, terminally-differentiated cell, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells, cancer cells, and/or bone cells in a subject.
  • stem cells e.g., hematopoietic stem cells, progenitor cells
  • differentiated cells e.g., differentiated cells, terminally-differentiated cell, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells, cancer cells, and/or bone cells in a subject.
  • the method of delivering the polynucleotide is for the modulation of a condition or disease of various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • the method of delivering the polynucleotide is for increasing or initiating expression of a protein (e.g., a therapeutic or supplemental protein) in a target cell in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • a protein e.g., a therapeutic or supplemental protein
  • the presently described LNP composition is administered at a time before the subject has or is suspected of having the condition or disease.
  • the presently contemplated LNPs are capable of targeting the cell type and tissue where the condition or disease manifests.
  • the presently described LNPs are shown to provide for the ability to transfect cells with the encapsulated polynucleotide throughout the whole body of the subject, including cell types that exist throughout the body.
  • the use of targeting ligands or moieties provides for more specific targeted cell type and tissue transfection, which is often desired based on the objective of diagnosis, prevention and/or treatment and the specific type or types of condition or disease that is the subject of the administration to a subject.
  • the polynucleotide encapsulated in the LNP will vary based on the condition or disease and the objective of the delivery (diagnosis, prevention or treatment), the presently described LNPs are provided as an enabling delivery vehicle for such polynucleotide such that it can be delivered to areas that have been difficult or not possible to deliver a polynucleotide in vivo in a subject (e.g., mammal) previously.
  • a subject e.g., mammal
  • an LNP pharmaceutical composition contemplated herein is administered in a dose of about 0.001 mg/kg per the subject’s body weight to about 2.0 mg/kg per the subject’s body weight to a subject in need thereof.
  • a Targeting LNP is administered to a subject in need thereof in a dose of about 0.01 mg/kg; in some embodiments in a dose of about 0.025 mg/kg; in some embodiments in a dose of about 0.05 mg/kg; in some embodiments in a dose of about 0.075 mg/kg; in some embodiments in a dose of about 0.1 mg/kg; in some embodiments in a dose of about 0.125 mg/kg; in some embodiments in a dose of about 0.150 mg/kg; in some embodiments in a dose of about 0.175 mg/kg; in some embodiments in a dose of about 0.2 mg/kg; in some embodiments in a dose of about 0.5 mg/kg; in some embodiments in a dose of about 0.75 mg/kg; in some embodiments in a dose of about 1.0 mg/kg; in some embodiments, in a dose of about 1.25 mg/kg; in some embodiment in a dose of about 1.5 mg/kg; or in some embodiment in a dose of about 2.0 mg/
  • LNP pharmaceutical compositions contemplated herein are administered to a subject in need thereof in a single dose.
  • the LNP is administered to a subject in need thereof two, three, four, or five or more times.
  • the LNP is administered twice a week, every week, every two weeks, every four weeks, every six weeks, every twelve weeks, or every fifteen weeks.
  • the LNP is administered every month, every two months, every three months, every six months, once a year, on an ongoing basis, or as determined by their physician.
  • the contemplated LNP pharmaceutical compositions are delivered in an aerosolized inhaled form, orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly.
  • targeting LNPs are administered intravenously.
  • an exemplary LNP pharmaceutical composition includes an excipient, or carrier, e.g., an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline.
  • compositions may contain pharmaceutically acceptable auxiliary substances as those required to approximate physiological conditions such as pH and buffering agents, toxicity countering agents, e.g., sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride, and sodium lactate.
  • toxicity countering agents e.g., sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride, and sodium lactate.
  • the pharmaceutical composition comprises 10 mM sodium citrate buffered to pH 6.4.
  • the composition may contain a cryoprotectant, e.g., glycerol, ethylene glycol, sucrose, propylene glycol, or dimethylsulfoxide (DMSO).
  • a cryoprotectant e.g., glycerol, ethylene glycol, sucrose, propylene glycol, or dimethylsulfoxide (DMSO).
  • the concentration of active agent in these formulations can vary and are selected based on fluid volumes, viscosities, and body weight in accordance with the particular mode of administration selected and the patient’s needs e.g., Remington’s Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).
  • the compounds or compositions are administered without isolating the cell or cells, the tissue, or the organ from the subject (i.e., the administration is in vivo).
  • the compound or composition is delivered to all, or almost all, cells in the subject’s body.
  • the compound or composition is delivered to a specific cell, cell type, tissue, or organ in the subject’s body.
  • telomerase repeat amplification protocol (TRAP) assay.
  • TRAP telomerase repeat amplification protocol
  • Commercial versions of the TRAP assay are available, for example the Trapeze® telomerase detection kit (Millipore), which provides a sensitive detection and quantitation of telomerase activity, although other measurement techniques are also possible.
  • telomerase reverse transcriptase gene persists in an episomal DNA moiety, or is inserted into the genomic sequence of the cell or otherwise permanently modifies the genetic make-up of the targeted cell and results in constitutive activity of the nucleic acid sequence.
  • the transient expression is independent of cell cycle.
  • Diagnostic and/or therapeutic kits comprising a pharmaceutical composition of an LNP contemplated herein, or lipid components thereof provided in separate containers, and instructions for making and /or using are also contemplated herein.
  • the diagnostic and/or therapeutic kit comprises devices for administration, including but not limited to syringes, microneedles, inhalers, nebulizers, and vials or containers.
  • kits for use in diagnosis, prevention and/or treatment of conditions or diseases of various tissues and cell types throughout the mammalian body including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells
  • the kit may include means for transcutaneous delivery, means for intracutaneous injection via microneedle array, means for skin barrier-permeabilizing step such as microneedles and/or one or more permeabilizing agent such as a mixture containing proteases, lipases, or others known in the field.
  • components of an exemplary diagnostic and/or therapeutic kit are provided such that they can be mixed with commercially available microfluidic or vortex mixers. Often such kits are provided with or without nucleic acid (e.g., mRNA) that is to be encapsulated in the LNPs made with the components of the diagnostic and/or therapeutic kit.
  • nucleic acid e.g., mRNA
  • kits for diagnosis, prevention and/or treatment of a condition or disease, e.g., for example for use in extending telomeres in a mammalian cell.
  • the kits comprise any of the above-described compounds or compositions, together with instructions for their use.
  • Such instructions include the packaging label approved by a regulatory agency in the country where the kit is sold, which instructions guide dosing schedules and include contraindications.
  • steps for combining agents in the kit are required, such instructions are included in the instructions for their use, including steps set forth in certain examples for preparing a final product herein.
  • kits further comprise packaging materials.
  • packaging materials are air tight.
  • the packaging materials may optionally be filled with an inert gas, such as, for example, nitrogen, argon, or the like.
  • the packaging materials comprise a metal foil container, such as, for example, a sealed aluminum pouch or the like.
  • a delivery vehicle such as a lipid as described herein.
  • one or more components of the formulation are provided frozen with a cryoprotectant, or lyophilized.
  • the kit may further comprise a desiccant, a culture medium, an RNase inhibitor, or other such components. In some embodiments, the kit may further comprise a combination of more than one of these additional components. In some kit embodiments, the composition of the kit is sterile.
  • FIGS. 1 and 48 depict exemplary pharmacokinetics of the 5 lipid LNPs of Table 1.
  • Timeline of plasma, lung, and liver levels of the 5 lipid LNPs as determined by measuring one of the lipids, DOTAP, are presented.
  • LNPs were formulated with TERT mRNA (Human TERT opti - SEQ ID NO: 1) and the lipids in the ratios of Table 1.
  • Male C57B1/6 mice were dosed at Img/kg and plasma and tissues were collected at the time points indicated above.
  • DOTAP was measured in the plasma, lung, and liver using LC-MS/MS.
  • the nitrogemphosphorus N/P charge ratio of the composition of Table 1 is 12.
  • FIG. 2 depicts the ratio of bioluminescence signal from the indicated organs, produced from luciferase protein translated from mRNA injected intravenously, at the indicated time points post intravenous injection of the 5 lipid LNP of Table 2.
  • LNPs were formulated with firefly luciferase mRNA and the lipids in the ratios of Table 2.
  • Male C57B16 mice were dosed at 1.3 mg/kg and tissues were collected and imaged ex vivo at the time points indicated above.
  • Mean radiance values were baseline normalized by subtracting the mean radiance from the negative control (no LNP) treated animal.
  • the relative radiance between lung, liver, and spleen are plotted in FIG. 2.
  • the tissues were also homogenized and the bioluminescence was measured from equal amounts by weight of homogenate.
  • the nitrogemphosphorus N/P charge ratio of the composition of Table 2 is 12.
  • FIGS. 3-17 and FIGS. 20-21 depict experiments involving the use of a different 5 lipid LNP composition where lung fibrosis was induced in third generation (G3) TERT knock-out (KO) mice, which have telomere lengths that are similar to those of humans.
  • Bleomycin was administered via oropharyngeal aspiration at 2.0 U/kg.
  • an initial dose of bleomycin was administered via oropharyngeal aspiration at 0.5 U/kg one week prior to the 2.0 U/kg dose.
  • LNPs were formulated with firefly luciferase mRNA (Ctrl) or TERT mRNA (mouse SEQ ID NO: 2, including synthetic 5’ UTR (SEQ ID NO: 3), wt mTert coding sequence (SEQ ID NO: 4) and mouse alpha-globin 3’ UTR (SEQ ID NO: 5)) (referred to as “TERT mRNA LNP” herein or “TERT” or “TERT mRNA” in the figures) and the lipids in the ratios of Table 3.
  • the DOTAP molar ratio is 2.27 times higher in the 5 lipid LNP of Table 3. Mice were dosed at 1.5mg/kg twice a week for two weeks beginning on day 7 post bleomycin.
  • mice received bleomycin, but not LNP treatment (No LNP), and control mice received saline instead of bleomycin and LNP treatment (No Bleo).
  • Tissues were harvested on day 24 post bleomycin. Lungs were paraffin fixed, formalin embedded. To measure telomere length, sections were stained using the Q-FISH method with the telomere probe and antibody against pro- surfactant protein C (SPC) to label the alveolar type II (AT2) cells. Telomerase activity was measured in lung tissue lysate using the telomeric repeat amplification protocol (TRAP) assay.
  • SPC pro- surfactant protein C
  • N/P ratio nitrogen:phosphorus charge ratio
  • FIG. 3 depicts a representative immunofluorescence micrograph of a lung section used to measure telomere length by the Q-FISH method known in the field, in which the intensity of a telomere probe is proportional to telomere length, and the cells were costained with an antibody to the AT2 cell marker pro-SPC.
  • FIG. 4 depicts measurements of telomere length by Q-FISH in AT2 cells in mice in which fibrosis had been induced using bleomycin (“Bleo”) as described above.
  • Treatment with TERT mRNA LNPs (“TERT” in the figure) of Table 3 increased the median telomere length and 10 th percentile telomere length in AT2 cells (cells that were scored as pro- SPC+ by immunofluorescence staining) as measured by Q-FISH, relative to mice receiving luciferase mRNA LNPs (referred to as “Ctrl” in the figure), almost to the length found in normal mice that had not received bleomycin (“No Bleo” in the figure).
  • FIG. 5 depicts measurements of telomere length by Q-FISH in AT2 cells in mice in which fibrosis had been induced using bleomycin (“Bleo”) as described above.
  • Treatment with TERT mRNA LNPs (“TERT” in the figure) of Table 3 increased the median telomere length and 10 th percentile telomere length in lung alveolar cells as measured by Q- FISH, relative to mice receiving luciferase mRNA LNPs (referred to as “Ctrl” in the figure), almost to the length found in normal mice that had not received bleomycin (“No Bleo” in the figure).
  • alveolar tissue includes epithelial, endothelial, fibroblasts, immune cells, and other cell types.
  • FIG. 6A depicts the timeline of the bleomycin mouse experiment described above. “CT” indicates x-ray computed tomography scanning of the mice to quantify normo- aerative lung volume.
  • FIG. 6B depicts the timeline of the bleomycin mouse experiment for FIGS. 13- 16.
  • FIG. 7 depicts false color images of lung sections stained with Sirius Red for quantification of fibrotic foci and fibrosis by area of tissue covered with collagen deposition. IV infusion of TERT mRNA LNPs of Table 3 reduced fibrosis by 62%.
  • FIG. 8 depicts reduction of fibrotic foci by TERT mRNA LNP of Table 3 treatment in bleomycin-treated mice as measured by Sirius Red staining.
  • FIG. 9 depicts increased normo-aerative lung ratio, or amount of useful lung volume, by TERT mRNA LNP of Table 3 treatment in bleomycin-treated mice as measured by x-ray computed tomography imaging followed by quantification of fibrotic tissue based on x- ray intensity of the voxels.
  • FIG. 10 depicts improvement of lung elastance by TERT mRNA LNP of Table 3 treatment in bleomycin-treated mice as measured by the Flexivent system.
  • FIGS. 11 and 12 depict improvement of lung forced expiratory volume (FEV0.1) and forced vital capacity (FVC), respectively, by TERT mRNA LNP of Table 3 treatment in bleomycin-treated mice as measured by the Flexivent system.
  • FEV0.1 lung forced expiratory volume
  • FVC forced vital capacity
  • FIG. 13, 14, and 15 depicts improvement in alveolar density, alveolar diameter, and alveolar circularity, respectively, by TERT mRNA LNP of Table 3 treatment in bleomycin-treated mice as measured by machine vision analysis of micrographs of lung sections by the company Biocellvia.
  • FIG. 16 depicts examples of false color images produced by Biocellvia of lung tissue sections during their analysis of alveolar architecture quantified in FIGS 13-15.
  • FIG. 17 depicts measurements of telomerase activity in isolated primary human alveolar epithelial cells from a 50 year-old donor at the indicated time points after treatment with TERT mRNA LNPs of Table 3 at a concentration of 500ng/ml as measured using the telomeric repeat amplification protocol (TRAP) assay.
  • TERT mRNA LNPs of Table 3 at a concentration of 500ng/ml as measured using the telomeric repeat amplification protocol (TRAP) assay.
  • FIG. 18 depicts the timeline of an experiment to measure the effect of TERT mRNA LNP of Table 3 treatment on the colony formation capacity of isolated primary human alveolar epithelial cells from a 50 year-old donor.
  • the cells were plated in medium that supports colony formation, treated with TERT mRNA LNPs at a concentration of 500 ng/ml of TERT mRNA, and incubated for 7 days after which colonies were counted, as reported in FIG. 19.
  • FIG. 19 depicts an increase in colony formation capacity of human alveolar epithelial cells from a 50 year-old donor by TERT mRNA LNP of Table 3 treatment.
  • Example 4 Reduction in senescence after telomerase mRNA LNP treatment in mice
  • Methods The experiment was performed as described in connection with FIGS. 3-12.
  • Results As shown in FIGS. 20-21, a 40% reduction in senescence marker P21 was shown in the lung alveolar cells of mice treated with telomerase mRNA LNP compared to mice treated with luciferase mRNA LNP (Ctrl). In FIG. 21, dark cells are positive. Increase in senescence following bleomycin treatment.
  • Example 6 Titrating lipid : mRNA ratio in 2 lipid LNP
  • DOTAP and PEG were formulated with firefly luciferase mRNA (Luc).
  • the lipid ratios were 99% DOTAP and 1% PEG.
  • the lipid : mRNA ratios is presented as NP; the ratio of nitrogen to phosphate molecules.
  • Each DOTAP molecule has a single nitrogen and each nucleoside on the mRNA has one phosphate.
  • C57B1/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and lungs were removed for ex vivo imaging 23hr later. Shown in the FIG. 23 is the mean radiance (photons/s/cm 2 /sr).
  • Example 7 Time course of protein expression after delivery of lung-targeting mRNA-LNP encoding firefly luciferase
  • LNPs was formulated with firefly luciferase mRNA (Luc) per ratios in the Table 3 of provisional.
  • C57B1/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 16hr (“Day 1”), 33hr (“Day 2”), 73hr (“Day 3”), 91hr (“Day 4”). Shown in the FIG. 24 is the mean radiance (photons/s/cm 2 /sr).
  • Example 8 Titrating SS-QP lipid into the ⁇ DOTAP + PEG’ LNP
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 25D.
  • C57B1/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 22hr later. Shown in the FIG. 25A is the mean radiance (photons/ s/cm 2 /sr) .
  • Liver- targeting LNP was formulated per lipid ratios in Table 4, and lung-targeting LNP was formulated per lipid ratios in Table 3. Both were formulated with firefly luciferase mRNA (Luc).
  • CD1 male mice were dosed at l.Omg/kg via intravenous injection (IV) and organs were imaged ex vivo 19hr later.
  • IV intravenous injection
  • FIGS. 26A and 26B provide mean radiance (photons/s/cm 2 /sr).
  • Liver BL1 signal was variable, however it was very low in the condition where the lung LNP was dosed first.
  • FIG. 26B The Liver BL1 signal was variable, however it was very low in the condition where the lung LNP was dosed first.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in the table on FIGS. 27A.
  • C57B1/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 23hr later.
  • Shown in the FIG. 27C is the mean radiance (photons/s/cm 2 /sr).
  • Body weights were measured and clinical signs assessed using a scoring system consisting of visual assessment of mouse appearance and behavior, specifically focusing on locomotion, posture, and eye (abbreviated “LPE”) (Nunamaker 2013, PMID: 24209966).
  • LPE locomotion, posture, and eye
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in Table 3. They were either formulated and dosed on the same day ‘Fresh’ or cryopreserved at -20C, -80C, or liquid nitrogen and thawed 70 days later. Sucrose was added to 7% prior to freezing as a cryoprotectant. CD1 male mice were dosed at 1.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 23hr later. Shown in the FIG. 28A is the mean radiance (photons/s/cm 2 /sr) +/- S.E.M.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in Table 3. They were either formulated and dosed on the same day ‘Fresh’ or cryopreserved at 4C, -20C, -80C, or liquid nitrogen and thawed 3 days later. Sucrose was added to 15% prior to freezing as a cryoprotectant, except in the “4C w/o sucrose” condition. CD1 female mice were dosed at 0.4 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 23hr later. Shown in FIG. 29A is the mean radiance (photons/s/cm 2 /sr).
  • Example 13 Titrating N/P ratios and comparing 2, 3, and 5 lipid formulations
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in the FIG. 30A.
  • C57B1/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 23hr later. Shown in FIG. 30B is the mean radiance (photons/ s/cm 2 /sr) .
  • Example 14 Titrating N/P ratio (mRNA to lipid ratio) with 3 and 5 lipid formulations
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 31A.
  • Bl/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 21hr later.
  • Shown in FIG. 31C is the mean radiance (photons/s/cm 2 /sr) .
  • Example 15 Testing vortex vs microfluidic mixing for mRNA-LNP formulation
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in Table 3.
  • Balb c/J male mice were dosed at 0. 1 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 21hr later. Shown in FIG. 32A is the mean radiance (photons/ s/cm 2 /sr) .
  • Example 16 Titrating NP ratio for 5 lipid mix, comparing concentrations of inputs [00315]
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in the table on FIG. 33A.
  • C57B1/6 male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 21hr later. Shown in FIG. 33B is the mean radiance (photons/s/cm 2 /sr).
  • the mRNA and lipid are diluted in buffer to 30% of original prior to mixing, but the ratios of the components is kept the same.
  • the mRNA concentration for the input went from 0.147mg/ml to 0.44mg/ml.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 35A.
  • C57B1/6 male mice were dosed at 2.0mg/kg via intravenous injection (IV) and organs were imaged ex vivo 17hr later.
  • Shown in the FIG. 35C is the mean radiance (photons/s/cm 2 /sr).
  • the formulations were the same, the variable changed was the flow rate through the Precision Nanosystems Nanoassemblr microfluidic mixing platform.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 36A.
  • BALB/cJ male mice were dosed at 1.5mg/kg via intravenous injection (IV) and organs were imaged ex vivo 26hr later.
  • Shown in FIG. 36B is the mean radiance (photons/s/cm 2 /sr).
  • Total flow-rate was varied, but the ratio of the mRNA (aqueous) to lipid (organic) streams was maintained at 3 : 1.
  • the flow rate for the mRNA stream was total flow rate multiplied by 0.75 and the flow rate of the lipid stream was the total flow rate multiplied by 0.25.
  • Example 20 Substituting SS-QP in the 3-lipid LNP with a different ionizable lipid: DLin- MC3-DMA (“MC3”). Titrating MC3 vs DOTAP ratios from 9 to 50% MC3.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 37A. Encapsulation percentage for the LNPS is provided in FIG 37B. BALB/cJ female mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 24hr later. Shown in FIG. 37C is the mean radiance (photons/s/cm 2 /sr).
  • Example 21 Swapping out ionizable lipids SS-OP for DLin-MC3-DMA in 3 lipid LNPs.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 38A.
  • MC3 is short for DLin-MC3-DMA.
  • CD1 female mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 26hr later. Shown in FIG. 38C is the mean radiance (photons/s/cm 2 /sr).
  • Example 22 Swapping out ionizable lipids SS-QP for DLin-MC3-DMA in 5 lipid LNPs.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 39A.
  • MC3 is short for DLin-MC3-DMA.
  • CD1 female mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 26hr later.
  • Shown in FIG. 39C is the mean radiance (photons/s/cm 2 /sr).
  • FIG. 39B Mean lung radiance was at least 7.2X greater in the 5-lipid condition with SS-OP than in any of the LNPs containing DLin-MC3-DMA.
  • FIG. 39C Mean lung radiance was at least 7.2X greater in the 5-lipid condition with SS-OP than in any of the LNPs containing DLin-MC3-DMA.
  • Example 23 Titrating time that mRNA is adsorbed to formed LNP in 2-lipid formulation
  • Methods DOTAP and DMG-PEG2000 at a 100 to 1 molar ratio underwent microfluidic mixing with malic acid followed by buffer exchange to 0. IM sodium acetate and concentration. The lipid concentration was measured by HPLC with UV detector and then firefly luciferase mRNA (Luc) was added to the lipid to a final N/P ratio of 15. The mRNA was allowed to adsorb for the time noted in FIG. 40A before the addition of PBS.
  • CD1 female mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 24hr later. Shown in FIG. 40A is the mean radiance (photons/s/cm 2 /sr).
  • Encapsulation efficiency was greater than 98% for all conditions.
  • Example 24 - Titrating mRNA Lipid ratio for 2-lipid LNP
  • DOTAP and DMG-PEG2000 at a 100 to 1 molar ratio underwent microfluidic mixing with malic acid followed by buffer exchange to 0. IM sodium acetate and concentration.
  • the lipid concentration was measured by HPLC and then firefly luciferase mRNA (Luc) was added to the lipid to an N/P ratio as denoted in FIG. 41A.
  • Encapsulation percentage for each composition is provided in FIG. 41B.
  • the mRNA was allowed to adsorb for 30 minutes before the addition of PBS.
  • C57BL/6 female mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 27hr later. Shown in FIG. 41A is the mean radiance (photons/s/cm 2 /sr).
  • LNP with NP 2 did not yield enough mRNA to dose.
  • LNP “PDS” was formulated with firefly luciferase mRNA (Luc) per ratios in Table 5. Encapsulation percentage for each composition is provided in FIG. 42B. 3- MC3 LNPs were formulated with a molar percentage of 39.6% MC3, 59.4% DOTAP, and 1% DMG-PEG2000 at the N/P ratios listed in the graph C57BL/6 male mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 20hr later. Shown in FIG. 42A is the mean radiance (photons/s/cm 2 /sr).
  • NP 1.5 and NP 2 had low yields, so only a single animal was dosed.
  • DOTAP and DMG-PEG2000 at a molar percentage as indicated in FIGS. 43A and 43B for the PEGylated lipid underwent microfluidic mixing with malic acid followed by buffer exchange to 0. IM sodium acetate and concentration. The lipid concentration was measured by HPLC and then firefly luciferase mRNA (Luc) was added to a N/P ratio of 5.0. The mRNA was allowed to adsorb for 30 minutes before the addition of PBS. C57BL/6 female mice were dosed at 2.0 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 20hr later. Shown in FIG. 43A is the mean radiance (photons/s/cm 2 /sr).
  • Single cationic lipid - DOTAP - is sufficient for tolerable lung-targeting.
  • Example 27 Microfluidic vs manual mixing of lipids in ethanol with malic acid buffer
  • Methods DOTAP and DMG-PEG2000 at a molar ratio of 100 to 1 underwent microfluidic or manual vortex mixing with malic acid followed by buffer exchange to 0. IM sodium acetate and concentration. The lipid concentration was measured by HPLC and then firefly luciferase mRNA (Luc) was added to a N/P ratio of 5.0. The mRNA was allowed to adsorb for 30 minutes before the addition of PBS.
  • mice C57BL/6 female mice were dosed at 0.5 mg/kg via intravenous injection (IV) and organs were imaged ex vivo 20hr later. Shown in FIG. 44 is the mean radiance (photons/s/cm 2 /sr).
  • Example 28 Comparing published MC3 formulation to 5-lipid LNP with SS-OP
  • Methods 1 LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in FIG. 45 A. C57BL/6J female mice were dosed at the indicated mg/kg via intravenous injection (IV) and organs were imaged ex vivo 20hr later. Shown in FIG. 45B is the mean radiance (photons/s/cm 2 /sr).
  • the MC3 formulation was based on Dilliard et al 2021 PNAS (https://doi.org/10.1073/pnas.2109256118).
  • the 5-lipid SS-OP containing LNP had 6.3X and 5.2 higher mean lung radiance compared to the published DLin-MC3-DMA containing lung-targeted LNP in the 0.5 and 2.0 mg/kg dose group respectively.
  • FIG. 45B
  • Encapsulation was greater than >98% in both LNPs, and overall yields were comparable.
  • Modal particle size by dynamic light scattering was between 35-55 nm.
  • Methods 2 LNPs were also formulated with Cre mRNA per ratios in FIG. 45 A.
  • the MC3 formulation was based on Dilliard et al 2021 PNAS
  • Ai 14 Tomato fl/fl female mice were dosed with Cre mRNA intravenously at 2.0 mg/kg. Lungs were harvested 3 days later and processed via formalin fixation and paraffin embedding. Positive cells were labeled with anti-tdTomato antibody. Shown in FIG. 45C are percent positive cells in the lung alveolar cells.
  • the 5-lipid SS-OP containing LNP had 4.9X higher rate of transfection of lung alveolar cells. 2. Shown is a representative image of the lung alveolar region
  • LNPs were formulated using lipid molar ratios and lipid:mRNA ratios as shown in FIG. 46A.
  • the PD(*) formulation was prepared as described above (in Example 26).
  • Ail4 Tomato fl/fl female mice were dosed with Cre mRNA intravenously at 1.5 mg/kg.
  • FIG. 46E Dark cells are Cre mRNA transfected tdTomato positive cells.
  • the 5 lipid LNP (5Lip) and 3-lipid LNP (PDS) had comparable transfection in the lung and spleen, but the 5 -lipid LNP had 8X higher transfection of hepatocytes in the liver.
  • FIG. 45D Shown in FIG. 45D is a representative image of the lung alveolar region. Dark cells are transfected (tdTomato positive) and include both endothelial and epithelial cells.
  • PD formulation was prepared as described above (in Example 26) with mRNA encoding human telomerase.
  • Fig. l C57B1/6 male mice were dosed at 1.0 mg/kg, and plasma and tissue were collected at the time points indicated.
  • DOTAP was measured using LC-MS/MS.
  • LNPs were formulated using lipid molar ratios in Table 1 with mRNA encoding firefly Luciferase. New Zealand white rabbits were dosed intravenously (IV) at the level indicated in the graph for mg mRNA/kg of body weight. Organs were imaged ex vivo 6hr later. Shown in the graph is the mean radiance (photons/s/cm 2 /sr).
  • Results are provided in FIGS. 49A and 49B.
  • FIGS. 50A is the mean radiance of each organ imaged (photons/s/cm 2 /sr).
  • FIGS 50B and 50C shows the relative radiance amongst the organs for the low and high dose.
  • FIGS. 50D-50F show representative bioluminescent images of organs from a high dose animal. Shown in Table 6
  • LNPs were formulated using lipid molar ratios in Table 3 with mRNA encoding human telomerase or mCherry as a control.
  • LNP was added to human small airway epithelial cells (SAECs) obtained from Lonza in culture at 0.5 mg/ml. SAECs were grown in Lonza SAGM until 70% confluent One day prior, drugs representing standard-of-care (SOC) for patients with pulmonary fibrosis, pirfenidone (luM) and nintedanib (0.5 uM), were added to the culture, and replaced at every media change.
  • SOC standard-of-care
  • telomere activity For measurement of telomerase activity, cells were harvested and lysed in CHAPS buffer for the TRAP assay 24 hours post treatment with LNP. The lysate was exposed to an artificial telomerase single- stranded DNA template, and PCR amplification was used to detect telomerase activity.
  • FIG. 5 IB For measurement of telomerase activity, cells were harvested and lysed in CHAPS buffer for the TRAP assay 24 hours post treatment with LNP. The lysate was exposed to an artificial telomerase single- stranded DNA template, and PCR amplification was used to detect telomerase activity.
  • FIG. 5 IB For measurement of telomerase activity, cells were harvested and lysed in CHAPS buffer for the TRAP assay 24 hours post treatment with LNP. The lysate was exposed to an artificial telomerase single- stranded DNA template, and PCR amplification was used to detect telomerase activity.
  • FIG. 5 IB For measurement of telome
  • telomere length measurement the cells were harvested and fixed five days post addition of telomerase mRNA LNPs.
  • FIG. 51 A The Q-FISH protocol was performed as follows: cells were spun down on glass slides and were permeabilized and had the telomeres labeled with a fluorescent probe.
  • FIG. 5 IB Microscopy and quantitative image analysis were performed to determine lengths of individual telomeres. All of the hTERT LNP samples (+/- SOC) were pooled in the analysis to test for differences in telomere length.
  • LNPs were formulated using lipid molar ratios in Table 3 with mRNA encoding human telomerase or mCherry as a control.
  • LNP was added to fetal human lung fibroblast MRC-5 cells (passage 5) were grown in DMEM+10% FBS until 70-80% confluency.
  • 24 hrs prior to the LNP treatment MRC-5 cells were pre-treated with the standard- of-care drugs pirfenidone (luM) and nintedanib (luM), following by treatment with 500 ng/ml hTERT LNPs or 500 ng/ml of mCherry LNPs (control).
  • Standard-of-care drugs were added fresh with every media change.
  • telomere activity For measurement of telomerase activity, cells were harvested and lysed in CHAPS buffer for the TRAP assay 24 hours post treatment with LNP. The lysate was exposed to an artificial telomerase single-stranded DNA template, and PCR amplification was used to detect telomerase activity. FIG. 52.
  • LNPs are formulated using lipid molar ratios described herein with mRNA encoding firefly luciferase.
  • the LNP ratios used are described in Table 7.
  • LNPs were mixed with 15% sucrose and frozen at -80°C. Next, they were placed into a pre-frozen shelf of the L-200 Pro Lyovapor freeze dryer. The vacuum was set to Imbar, the condenser was set to - 55°C, and the total run-time was 22 hours.
  • the LNPs were stored at 4°C for 2 days prior to resuspension with water.
  • As a control “PDS” LNP was frozen with 15% sucrose and thawed without lyophilization.
  • 2 nd generation Tert -/- female mice on a C57B1/6 background are dosed at 0.3 mg mRNA/kg of body weight via intravenous injection (IV) and organs are imaged ex vivo 18hr later.
  • Table 7 provide the molar percentage composition, N/P ratio, molar ratio of cationic lipid : ionizable lipid , and lipid:mRNA ratio (wt/wt), of exemplary LNPs of the present disclosure named, respectively, “SSOP-DOTAP”, “5 lipid”, “PDS”, “3-MC3”, “PD”. Table 7
  • C/I Ratio Optimal cationic lipid to ionizable lipid ratios
  • N/P ratios identified by the present inventors for LNPs containing either the SS-OP family or DLin-MC3- DMA family of ionizable lipids are modeled in FIG. 56.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in Table 7.
  • Luc firefly luciferase mRNA
  • Table 7 For LNPs with an asterisk (*), the microfluidic mixing was performed with buffer only, no mRNA. This was followed by buffer exchange to 0.1M sodium acetate and concentration. The firefly luciferase mRNA (Luc) was added to the lipid to an N/P ratio as denoted in the graph. The mRNA was allowed to adsorb for 2 hours before the addition of PBS, 0.2pM filtration, and dosing. mRNA LNPs were delivered intravenously to C57BL/6 male mice and organs were imaged ex vivo 17 hr post.
  • LNPs were formulated with firefly luciferase mRNA (Luc) per ratios in Table 7.
  • Luc firefly luciferase mRNA
  • Table 7 For PD(*), the microfluidic mixing was performed with buffer only, no mRNA. This was followed by buffer exchange to 0.1M sodium acetate and concentration. The firefly luciferase mRNA (Luc) was added to the lipid to an N/P ratio as denoted in the graph. The mRNA was allowed to adsorb for 2 hours before the addition of PBS, 0.2pM filtration, and dosing.
  • mRNA LNPs were delivered intravenously to BALB/cJ male mice at 1.5mg/kg and organs were imaged ex vivo 26 hr post.
  • FIG. 54A 1. Adding the mRNA at the end for the PD formulation resulted in a 31.8X increase in bioluminescence signal.
  • FIG. 54A 1. Adding the mRNA at the end for the PD formulation resulted in a 31.8X increase in bioluminescence signal.
  • LNPs were formulated with mRNA encoding Cre recombinase using lipid molar ratios as shown in Table 7.
  • Ail4 Tomato fl/fl mice were intravenously dosed with either 3-lipid MC3 (3-MC3) or 3-lipid SS-OP (PDS) LNPs operably containing Cre mRNA at 1.2 mg/kg or PBS as a control.
  • 3-lipid MC3 3-lipid MC3
  • PDS-OP 3-lipid SS-OP
  • Bone marrow cells were collected 6 days after the above-mentioned administration and analyzed by flow cytometry using the gating scheme of FIGS. 57, 58A, 58B and 58C, including DAPI to gate for live cells.
  • the PDS and 3-MC3 LNP formulations were found to transfect bone marrow cells (hematopoietic stem cells) at 5.9% and 12.2% respectively as show in FIG. 58D.
  • mice were imaged using a Lago whole body fluorescence imager (Spectral Instruments Imaging) 120 days later.
  • the signal was captured in the Cy3 channel (FIG. 59A) (535nm excitation, 590nm emission, 60 sec exposure), and as a control the mice were imaged in the Cy7 channel (FIG. 59B) (745 excitation, 790 emission, 20 sec exposure).
  • the Cy3 signal depicts the locations where Cre mRNA LNPs transfected cells are located, and where the mRNA was translated into Cre recombinase, which performs a recombination event to remove the stop codon in the tdTomato gene allowing for robust tdTomato expression. It was concluded that both of the 3-MC3 and PDS LNPs provide for broad transfection distribution, including in the limbs, tail, and throughout the abdominal area.
  • FIG. 61 A Quantification of the limbs, tail and pelvis Cy3 images from the mouse dorsal side (500nm excitation, 590nm emission, 60 sec exposure time) is shows in FIG. 6 IB. Shown is mean radiance (photons/s/cm A 2/sr), which is area normalized. Signal is distributed along the spinal column, limbs, tail, and pelvis in both the 3-MC3 and PDS images.
  • Cre mRNA half-life is less than one day and Cre protein half-life is only a few days, and therefore the transfection appears to have occurred within the first few days, thereby permanently activating tdTomato in the transfected cells.
  • Previous experiments have shown that the tdTomato signal is detectable within 1-3 days of Cre mRNA injection.
  • the formulations were well-tolerated as assessed by cage-side observations of locomotion, eye appearance, and posture.
  • Bone marrow cells were isolated from these mice at 126 days post-dosing and subject to flow cytometry.
  • the gating structure was as shown in FIGS. 57 and 58A-58C.
  • the percentage of tdTomato-i- cells in the bone marrow for PBS control, 3-MC3, and PDS is shown in FIG. 62A.
  • Tissues were fixed in formalin and embedded in paraffin. Sections were stained with anti-tdTomato antibody and digitally scanned. QuPath was used to identify nuclei and quantify % of positive cells in each tissue with quantitative results shown in FIG. 63A. The results showed that the Ail 4 tdTomato fl/fl mice receiving Cre mRNA formulated with either 3-MC3 or PDS and injected intravenously at a dose of 1.2 mg/kg resulted in broad transfection of various tissues of the mice, including those specifically listed. Tissues additional to those listed in FIG. 63A appear to have been transfected as well and studies are planned for evaluating these additional cells and tissues.
  • FIG. 63B Immunohistochemistry tissue section images shown in liver (FIG. 63B), spleen (FIG. 63C), kidney tissue (FIG. 63D), leg (FIG. 63E) and tail (FIG. 63F).
  • sections of tissue were harvested from the mice and immunostained with antibody to tdTomato conjugated to horseradish peroxidase. The darkened (brown in the original) stain indicates transfection with Cre mRNA permanently activating tdTomato expression in the transfected cells (tdTomato positive).
  • Example 41 Transfection employing LNPs bearing an antibody targeting ligand
  • LNPs were formulated with 100% N1 -methylpseudouridine uridinesubstituted mRNA encoding Cre recombinase in an LNP formulated using standard microfluidic mixing methods (Ignite, PNI) using a 1:3 mixing ratio of lipid to mRNA solutions, and comprising MC3 and a maleimide-PEG-lipid conjugated to a monoclonal antibody specific to the stem and progenitor cell marker c-Kit.
  • Ignite, PNI microfluidic mixing methods
  • the antibody was conjugated using methods known in the field, comprising reduction of the antibody with TCEP followed by reaction with maleimide-PEG-lipid on the LNP at room temperature for Ih with slow rotation, followed by size exclusion column purification (Izon) to remove unconjugated antibody.
  • the LNPs were dosed at 0.2 mg/kg mRNA to tdTomato mice and the bone marrow cells were harvested on day-6 post-dosing, immunostained with a hematopoietic lineage antibody panel, Seal, and C-Kit antibodies, and analyzed by flow cytometry according to the gating scheme depicted in Fig.
  • Example 42 - PDS LNPs were formulated with Cre mRNA with lipid ratios and procedures as in Example 37, Table 7.
  • Ail4 tdTomato fl/fl male mice were dosed intravenously at 1.5 mg (high dose) or 0.5 mg (lose dose) of mRNA per kg of body weight.
  • Bone marrow cells were harvested 8 days post-dose. Cells that were transfected were LdTomato-i-.
  • HSCs hematopoietic stem cells
  • the Lineage- FITC is a cocktail of antibodies including anti-mouse CD3, CD45R (B220), CDl lb, TER-119, and Ly-G6.
  • FIG. 65 shows transfection of hematopoietic stem cells (Lineage-, Scal+, cKit-i- bone marrow cells) using PDS LNP at multiple doses.
  • a method of delivering a polynucleotide to one or more of a wide range of different cell types of a subject comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types the subject.
  • LNP lipid nanoparticle
  • PEGylated lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-Maleimide.
  • a method of delivering a polynucleotide to one or more of a wide range of different cell types of a subject comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (ii) a cationic lipid at a molar percentage of between about 50% and about 70%, with a cationic lipid: ionizable lipid (C/I) ratio between 1 and 2, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types in the subject.
  • LNP lipid nanoparticle
  • the PEGylated lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-Maleimide.
  • the one or more different cell types are selected from the group consisting of a hematopoietic stem cell, a progenitor cell, a spleen cell, a hepatocyte, a kidney cell, an endothelial cell, an epithelial cell, and cell of tissues of limbs of the subject.
  • a method of delivering a polynucleotide to a bone marrow cell comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising:
  • a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1; and/or
  • DLin-MC3-DMA at a molar percentage of between about 30% and about 50%
  • DMG-PEG2000 at a molar percentage of between about 0.5% and about 2.5%
  • a cationic lipid at a molar percentage of between about 50% and about 70%
  • C/I ionizable lipid ratio between 1 and 2
  • the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in the bone marrow cell of a subject.
  • the PEGylated lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-Maleimide.
  • a method of delivering a polynucleotide to one or more of a bone marrow cell, spleen cell, a hepatocyte, and/or a kidney cell comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising:
  • a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1; and/or
  • DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and a cationic lipid at a molar percentage of between about 50% and about 70%, with a cationic lipid: ionizable lipid (C/I) ratio between 1 and 2, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in the one or more of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell of a subject.
  • C/I ionizable lipid
  • the PEGylated lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-Maleimide.
  • cationic lipid is DOTAP.
  • PEGylated lipid is DMG-PEG2000 and wherein the cationic lipid is DOTAP.
  • the bone marrow cell is a bone marrow stem cell and/or a progenitor cell.
  • administration is intravenous, transcutaneous, intracutaneous injection via microneedle array, or topical delivery involving a step of permeabilizing the skin barrier using a mechanical means such as microneedling and/or application of a skin permeabilizing agent.
  • telomerase reverse transcriptase telomerase reverse transcriptase
  • polynucleotide comprises a nucleic acid sequence at least at least 90% identical to SEQ ID NO: 1 or a fragment thereof.
  • polynucleotide comprises a sequence encoding a diagnostic or therapeutic protein that can be expressed in the bone marrow stem cell and/or progenitor cell.
  • telomerase reverse transcriptase telomerase reverse transcriptase
  • polynucleotide comprises a nucleic acid sequence at least at least 90% identical to SEQ ID NO: 1 or a fragment thereof.
  • the targeting ligand includes a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody adapted to bind the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer- antibody conjugate.
  • a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody adapted to bind the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer- antibody conjugate.
  • condition or disease is or includes influenza, asthma, diabetes mellitus type 1, diabetes mellitus type 2, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer’s disease, Parkinson’s disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn’s disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, stomach cancer, brain cancer, endometrial cancer, bone cancer, sarcoma, multiple myeloma,
  • COPD chronic obstructive
  • RNA- and DNA-based biologies have expansive capacities to modulate cellular activities for treating inherited and acquired diseases.
  • the clinical success of LNPs has gained recent widespread attention.
  • Most lipid-based nucleic acid delivery platforms that are undergoing clinical studies or on the market consist of four or five lipid components. Recent studies have reported that not only the choice of lipid components, but also the relative proportions of the lipid ingredients in the formulation, greatly influence in vivo transfection efficiency and tissue-specific delivery.
  • lipid nanoparticle comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different
  • lipid nanoparticle comprising: (i) DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (ii) a cationic lipid at a molar percentage of between about 50% and about 70%, with a cationic lipid: ionizable lipid (C/I) ratio between 1 and 2, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types in the subject.
  • LNP lipid nanoparticle
  • the PEGylated lipid is DMG-PEG2000.
  • the cationic lipid is DOTAP.
  • the LNP of the methods described above and herein is comprised of the lipids set forth in Table 7.
  • the target cell types are located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 4 to 6 distinct organs
  • the target cell types are located in between 5 to 8 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 7 to 10 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 9 to 12 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 10 to 15 distinct organs of the subject.
  • the delivery is to a target cell.
  • target cell is often in any of a variety of tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, hematopoietic stem cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • the administration is intravenous, transcutaneous, intracutaneous injection via microneedle array, or topical delivery involving a step of permeabilizing the skin barrier using a mechanical means such as microneedling and/or application of a skin permeabilizing agent.
  • exemplary skin permeabilizing agents include solutions and mixtures containing one or more protease, one or more lipase, or other skin permeabilization agents known in the art.
  • the corresponding protein encoded by the synthetic RNA is expressed in two or more of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell. According to certain embodiments, the corresponding protein encoded by the synthetic RNA is expressed in each of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell. According to related embodiments, the bone marrow cell is a bone marrow stem cell and/or a progenitor cell.
  • the corresponding protein encoded by the synthetic RNA is expressed in two or more of a stem cell, progenitor cell, germ cell, differentiated cell, or terminally differentiated cell, a cancer cell, endothelial cell, and/or bone cell.
  • the corresponding protein encoded by the synthetic RNA is expressed in each of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell.
  • the bone marrow cell is a bone marrow stem cell and/or a progenitor cell and/or stem cell such as a hematopoietic stem cell.
  • the polynucleotide in the LNP will generally comprise a sequence encoding any therapeutic protein or protein useful in a diagnostic that can be expressed in a target cell, such as, for example, telomerase reverse transcriptase (TERT) protein or part thereof, selected from
  • the polynucleotide e.g., mRNA
  • the polynucleotide often comprises SEQ ID NO: 1 or a fragment thereof.
  • the synthetic ribonucleic acid encodes telomerase reverse transcriptase (TERT), wherein optionally the TERT mRNA comprises a nucleic acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOS: 1-5, or SEQ ID NOS: 38-40 of PCT/US22/22642.
  • TERT telomerase reverse transcriptase
  • the TERT synthetic mRNA comprises a 5' cap structure, wherein the 5' cap structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, CapO, Capl, ARCA, inosine, Nl- methylguanosine, 2'fluoro-guanosine, 7-deaza-guanosine, CleanCap®, 8-oxo-guanosine, 2- aminoguanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.
  • the 5' cap structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, CapO, Capl, ARCA, inosine, Nl- methylguanosine, 2'fluoro-guanosine, 7-deaza-guanosine, CleanCap®, 8-oxo-gu
  • methods of delivering a polynucleotide to a bone marrow cell comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2%, and a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between .6 and 1; and/or (ii) DLin-MC3- DMA at a molar percentage of between about 30% and about 50%, DMG-PEG2000 at a molar percentage of between about 0.5% and about 2%, and a cationic lipid at a molar percentage of between about 50% and about 70%, with
  • the PEGylated lipid is DMG-PEG2000.
  • the cationic lipid is DOTAP.
  • the PEGylated lipid is DMG-PEG2000 and wherein the cationic lipid is DOTAP.
  • methods of delivering a polynucleotide to one or more of a bone marrow cell, spleen cell, a hepatocyte, and/or a kidney cell comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, a PEGylated lipid at a molar percentage of between about 0.5% and about 2%, and a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic
  • LNP lipid nanoparticle
  • the PEGylated lipid is DMG-PEG2000.
  • the cationic lipid is DOTAP.
  • the PEGylated lipid is DMG-PEG2000 and wherein the cationic lipid is DOTAP.
  • the corresponding protein encoded by the polynucleotide is expressed in two or more of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell. Often, the corresponding protein encoded by the polynucleotide is expressed in each of the bone marrow cell, spleen cell, hepatocyte, and/or kidney cell.
  • the bone marrow cell is a bone marrow stem cell and/or a progenitor cell.
  • the polynucleotide often comprises a sequence encoding a diagnostic or therapeutic protein that can be expressed in a target cell.
  • the LNP further comprises a targeting ligand adapted to specifically target the cell.
  • the adaptation to specifically target the cell is often a binding or other interaction of the LNP with the cell.
  • the targeting ligand includes a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody adapted to bind the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer- antibody conjugate.
  • a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody adapted to bind the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer- antibody conjugate.
  • the method is adapted for diagnosis, prevention or treatment of a condition or disease involving the cell.
  • the delivery of the polynucleotide is for the diagnosis, prevention and/or treatment of conditions or disease of various tissues and cell types throughout the mammalian body.
  • conditions or diseases include influenza, asthma, diabetes mellitus type 1, diabetes mellitus type 2, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer’s disease, Parkinson’s disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn’s
  • - 5 - disease ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, stomach cancer, brain cancer, endometrial cancer, bone cancer, sarcoma, multiple myeloma, skin cancer, basal cell carcinoma, squamous cell carcinoma, tuberculosis, pneumonia, bronchitis, sinusitis, otitis media, urinary tract infection (UTI), hepatitis A, hepatitis B, hepatitis C, HIV/AIDS, syphilis, gonorrhea, chlamydi
  • - 6 - disorder delirium, dementia, fetal alcohol syndrome, neonatal abstinence syndrome, Pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), liver cirrhosis, hepatic fibrosis, nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), chronic hepatitis B-related fibrosis, chronic hepatitis C-related fibrosis, systemic sclerosis (scleroderma), cystic fibrosis, cardiac fibrosis, hypertrophic cardiomyopathy, restrictive cardiomyopathy, dilated cardiomyopathy, chronic kidney disease (CKD), glomerulonephritis, diabetic nephropathy, interstitial nephritis, retroperitoneal fibrosis, peritoneal fibrosis, Dupuytren’s contracture, Peyronie’s disease, my
  • the cell types may include, for example, stem cells (e.g., hematopoietic stem cells, progenitor cells), differentiated cells, terminally-differentiated cell, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells, cancer cells, and/or bone cells in a subject.
  • stem cells e.g., hematopoietic stem cells, progenitor cells
  • differentiated cells e.g., differentiated cells, terminally-differentiated cell, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells, cancer cells, and/or bone cells in a subject.
  • the method of delivering the polynucleotide is for the modulation of a condition or disease of various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • the method of delivering the polynucleotide is for increasing or initiating expression of a protein (e.g., a therapeutic or supplemental protein) in a target cell in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • a protein e.g., a therapeutic or supplemental protein
  • the presently described LNP composition is administered at a time before the subject has or is suspected of having the condition or disease.
  • the presently contemplated LNPs are capable of targeting the cell type and tissue where the condition or disease manifests.
  • the presently described LNPs are shown to provide for the ability to transfect cells with the encapsulated polynucleotide throughout the whole body of the subject, including cell types that exist throughout the body.
  • the use of targeting ligands or moieties provides for more specific
  • LNPs are provided as an enabling delivery vehicle for such polynucleotide such that it can be delivered to areas that have been difficult or not possible to deliver a polynucleotide in vivo in a subject (e.g., mammal) previously.
  • the LNP is selected from an LNP of Table 7.
  • the LNP has only a single charged lipid, for example DOTAP.
  • LNPs e.g., 3-MC3 and/or PDS
  • the LNPs are manufactured in a manner that forms the LNP prior to addition of nucleic acid.
  • such LNPs are 2, 3 or 5 lipids LNPs described herein in Table 7 and the method employs vortex mixing during the addition of the nucleic acid.
  • the LNP is composed of 2 or 3 lipid LNPs described herein.
  • FIGS. 1 A-1D depict the pharmacokinetics of specific lipid nanoparticles (LNPs).
  • Timeline of serum (FIG. 1A), lung (FIG. IB), and liver (FIG. 1C) levels of the LNPs as determined by measuring one of the lipids, DOTAP.
  • FIG. ID provides tabular data related to FIGS. 1A-1C.
  • FIG. 2 depicts the ratio of bioluminescence signal from the indicated organs, produced from luciferase protein translated from a formulation of mRNA in LNPs (“mRNA LNPs”) injected intravenously, at the indicated time points post intravenous injection.
  • mRNA LNPs mRNA in LNPs
  • FIG. 3 shows that LNPs formulated with TERT mRNA (“TERT mRNA LNPs”) extend telomeres in lung cells in vivo, where telomere length was measured in lung tissue sections using Q-FISH, and the telomere signal intensity (green/lighter) is proportional to signal length.
  • FIG. 4 shows that TERT mRNA LNPs extend telomeres in lung cells in vivo, where TERT mRNA LNPs extend median and average telomere length in AT2 cells. At least 50 pro- SPC-positive cell nuclei were quantified per mouse. The error bars are standard error of the mean (SEM) for the telomere length between mice.
  • SEM standard error of the mean
  • FIG. 5 shows that TERT mRNA LNPs extend telomeres in lung cells in vivo, where TERT mRNA LNPs extend median and average telomere length in lung alveolar cells. Telomeres of at least 100 alveolar cells were quantified per mouse. The error bars are standard error of the mean (SEM) for the telomere length between mice.
  • FIG. 6A shows experimental design and timeline of dosing for data of the present disclosure presented in FIGS 3-12 and FIGS 20-21.
  • FIG. 6B shows experimental design and timeline of dosing for data of the present disclosure presented in FIGS 13-16.
  • FIG. 7 shows that TERT mRNA LNPs reduce fibrosis in lung in vivo, where high- density fibrotic foci (yellow/darker) are mapped using picrosirius red staining in lungs in bleomycin-treated mice treated with either control mRNA LNPs (carrying luciferase mRNA) or TERT mRNA LNPs. “No bleo” indicates a lung section from a normal healthy mouse, not treated with bleomycin.
  • FIG. 8 shows that TERT mRNA LNPs reduce fibrosis in lung in vivo, as measured by quantification of high-density fibrotic foci in Picrosirius Red stained tissue sections.
  • FIG. 9 shows that TERT mRNA LNPs increase the amount of useful lung volume in vivo, as determined by quantification of the change in normo-aerative lung ratio from baseline using 3D quantification of fibrosis detected by signal intensity in x-ray computed tomography scan data.
  • FIG. 10 shows that TERT mRNA LNPs improve tissue elastance in vivo, as determined by quantification of measurement of tissue elastance in mice at the end of the study using the Flexivent system.
  • FIG. 11 shows that TERT mRNA LNPs improve lung function in vivo, as determined by measurement of forced expiratory volume in mice at the end of the study using the Flexivent system.
  • FIG. 12 shows that TERT mRNA LNPs improve lung function in vivo as determined by measurement of forced vital capacity in mice at the end of the study using the Flexivent system.
  • FIG. 13 shows that TERT mRNA ENPs improve lung architecture in vivo, where Morpho-Quant Fung software was used to analyze digitized images of tissue sections to quantify alveolar number per unit area.
  • FIG. 14 shows that TERT mRNA LNPs improve lung architecture in vivo, where Morpho-Quant Lung software was used to analyze digitized images of tissue sections to quantify alveolar diameter.
  • FIG. 15 shows that TERT mRNA LNPs improve lung architecture in vivo, where Morpho-Quant Lung software was used to analyze digitized images of tissue sections to quantify alveolar circularity.
  • FIG. 16 shows that TERT mRNA LNPs improve lung architecture in vivo, where representative images of alveoli false colored by Morpho-Quant Lung software during the digitized analysis of alveolar architecture are shown.
  • FIG. 17 shows pharmacodynamic data of TERT mRNA in vivo following intravenous infusion in a formulation of TERT mRNA LNPs, where in the level of telomerase activity in lung tissue is quantified at the indicated time points following infusion.
  • FIG. 18 shows an experimental schema for quantifying the effect of TERT mRNA LNPs on human primary epithelial cell colony formation capacity.
  • FIG. 19 shows that TERT mRNA LNPs increase the colony formation capacity of human primary epithelial cells in the experimental model shown in FIG. 18.
  • FIG. 20 depicts levels of P21+ senescent lung alveolar cells involving the LNP of Table 3, based on the experimental model shown in FIG. 6A.
  • FIG. 21 is a representative immunohistochemistry image of anti-P21 staining of lung sections, as quantified in FIG. 20.
  • FIGS. 22A, 22B, 22C and 22D provide lung delivery data obtained using 2 lipid LNP compositions.
  • FIG. 23 depicts the results of titrating lipid : mRNA ratio in 2 lipid LNP.
  • FIG. 24 depicts the time course of protein expression after delivery of lung-targeting mRNA-LNP encoding firefly luciferase using the LNPs of Table 3.
  • FIGS. 25 A, 25B and 25C provide lung radiance, encapsulation efficiency, and body weight variance in titrating SS-OP lipid into the ‘DOTAP + PEG’ LNP using LNPs of FIG. 25D.
  • FIGS. 26A and 26B lung and liver delivery data using the LNP of Table 4.
  • FIGS. 27A and 27B provide exemplary LNPs and encapsulation efficiency thereof.
  • FIGS. 27C and 27D provide lung delivery data and body weight variance data in titrating SS-OP lipid into the ‘DOTAP + PEG’ LNP using LNPs of FIGS. 27A.
  • FIGS. 28A and 28B provide comparative fresh versus long term cryopreserved lung transfection and weight data for LNPs of Table 3.
  • FIGS. 29A and 29B provide comparative fresh versus short term cryopreserved lung transfection and particle size data for LNPs of Table 3.
  • FIG. 30A provides exemplary 2, 3 and 5 lipid LNPs.
  • FIGS. 30B and 30C provide lung delivery data and body weight variance data in titrating N/P ratios and comparing 2, 3, and 5 lipid formulations of FIG. 30A.
  • FIGS. 31A and 3 IB provide exemplary 3 and 5 lipid LNPs and encapsulation efficiencies thereof.
  • FIGS. 31C provides lung delivery data in titrating N/P ratio (mRNA to lipid ratio) with 3 and 5 lipid formulations 30A.
  • FIGS. 32A and 32B provide lung transfection data and LNP size and encapsulation efficiency data for LNPS having lipid ratios of Table 3 prepared using vortex or microfluidic mixing.
  • FIG. 33A provides exemplary 3 and 5 lipid LNPs.
  • FIG. 33B provides lung transfection data using the LNPs of FIG. 33A.
  • FIG. 34A provides exemplary 2, 3 and 5 lipid LNPs.
  • FIG. 34B and 34C provide lung transfection data using, and particle size and encapsulation efficiency of the LNPs of FIG. 34A.
  • FIG. 35A provides exemplary 5 lipid LNPs prepared using different flow rates.
  • FIG. 35B and 35C provide encapsulation percentage and lung transfection data using the LNPs of FIG. 35 A.
  • FIG. 36A provides exemplary 3 lipid LNPs prepared using different flow rates.
  • FIG. 36B and 36C provide lung transfection and body weight change data using the LNPs of FIG. 36A.
  • FIGS. 37A and 37B provide exemplary LNPs prepared by substituting SS-OP with a different ionizable lipid (DLin-MC3-DMA), including encapsulation percentages.
  • DLin-MC3-DMA ionizable lipid
  • FIG. 37C and 37D provide lung transfection and body weight change data using the LNPs of FIG. 37A.
  • FIG. 38A provides exemplary LNPs prepared by swapping out SS-OP for DLin-MC3- DMA in 3 lipid LNPs.
  • FIGS. 38B and 38C provide encapsulation percentage and lung transfection data using the LNPs of FIG. 38 A.
  • FIG. 39A provides exemplary LNPs prepared by swapping out SS-OP for DLin-MC3- DMA in 5 lipid LNPs.
  • FIGS. 39B and 39C provide encapsulation percentage and lung transfection data using the LNPs of FIG. 39A.
  • FIG. 40A provides a depiction of titrating time that mRNA is adsorbed to form LNP in a 2-lipid formulation.
  • FIG. 40B provides body weight change data using the LNPs of FIG. 40A.
  • FIGS. 41 A and 4 IB provide lung transfection and encapsulation percentage data of 2- lipid LNPs made while titrating mRNAdipid ratio.
  • FIGS. 42A and 42B provide lung transfection and encapsulation percentage data of 3- lipid LNPs made while titrating N/P ratio.
  • FIGS. 43 A and 43B provide lung transfection and body weight change data using the listed 2-lipid LNPS titrated with DMG-PEG2000.
  • FIG. 44 provides lung mean radiance in a comparison of microfluidic vs manual mixing of lipids in ethanol with malic acid buffer.
  • FIGS. 45A-45D provide formulation, transfection and IHC data comparing an MC3 formulation with a 5 lipid LNP composition of the present specification, demonstrating transfection of endothelial and epithelial cells of the lung alveolar region.
  • FIG. 46A provides exemplary 2, 3 and 5 lipid LNPs.
  • FIGS. 46B, 46C and 46D provide lung, liver and spleen cell transfection data using the LNPs of FIG. 46A.
  • FIG. 46E provides IHC data related to use of exemplary 2, 3 and 5 lipid LNPs in lung, liver and spleen tissue.
  • FIGS. 47A, 47B and 47C depict the pharmacokinetics of specific 2-lipid LNPs in plasma, lung and liver.
  • FIG. 48 depicts the pharmacokinetics of specific 5-lipid LNPs from Table 1.
  • FIGS. 49A and 49B depict biodistribution of exemplary 5-lipid LNPs from Table 1 when administered to a mammal.
  • FIGS. 50A, 50B and 50C depict biodistribution of 3-lipid LNP as described in FIG. 46A when administered to a mammal.
  • FIGS. 50D, 50E and 50F depict organ level bioluminescence related to the use of 3- lipid LNPs in mammals as described in Example 33.
  • FIG. 50G depicts biodistribution of exemplary 3-lipid LNPs as described in FIG. 46A when administered to a mammal.
  • FIGS. 51 A and 5 IB provide data related to telomere extension and telomerase activity in lung epithelial cells using the LNP of Table 3.
  • FIG. 52 provides data related to telomerase activity in lung fibroblasts using the LNP of Table 3.
  • FIGS. 53A and 53B provide lung transfection and encapsulation efficiency data using the LNPs of Example 38.
  • FIGS. 54A and 54B provide lung transfection and encapsulation efficiency data using the LNPs of Example 39.
  • FIG. 55 demonstrates lung mean radiance of the freeze-dried (lyophilized) LNPs of Example 36.
  • FIG. 56 models optimal cationic lipid to ionizable lipid ratio (“C/I Ratio”) and optimal lipid nitrogen : polynucleotide phosphate ratio (“N/P ratio”) for LNPs containing either the SS-OP family or DLin-MC3-DMA family of ionizable lipids.
  • the grey boxes indicate the optimal ranges of C/I ratio and N/P ratio.
  • the horizontal dashed lines indicate thresholds of relative lung radiance selected so that the convex peak(s) of relative lung radiance in each graph is/are substantially above the dashed lines.
  • the horizonal dotted line is the threshold of relative yield such that the convex curve of relative yield is substantially above the dashed line.
  • the limits of the optimal ranges are determined by the intersections of the curves of relative lung radiance and relative yield with the horizontal dashed or dotted threshold lines, respectively.
  • FIG. 57 demonstrates an exemplary flow cytometry gating strategy including DAPI to gate for live bone marrow cells.
  • FIGS. 58A, 58B and 58C provide flow cytometric results of bone marrow cells collected six days after administration of mRNA-LNPs formulated with mRNA encoding Cre recombinase and lipids in the exemplary PDS and 3-MC3 LNPs.
  • FIG. 58D refers to the results of FIGS. 58A, 58B and 58C and provides results of the bone marrow transfection efficiency for the exemplary PDS and 3-MC3 LNPs.
  • FIGS. 59A and 59B show fluorescent imaging data in Ail4 LdTomato flox/flox mice that have received an injection of exemplary 3-MC3 and PDS LNPs containing Cre
  • FIG. 59A shows the test Cy3 channel and FIG. 59B shows the control Cy7 channel.
  • FIG. 60A shows fluorescent imaging data, with whole body gating, of the dorsal side of Ail4 tdTomato flox/flox mice that have received an injection of exemplary 3-MC3 and PDS LNPs containing Cre mRNA (compared with control).
  • FIG. 60B shows the quantification of the Cy3 image in FIG. 60A.
  • FIG. 61 A shows fluorescent imaging data, with gating around limbs, tail and pelvis, of the dorsal side of Ail4 tdTomato flox/flox mice that have received an injection of exemplary 3-MC3 and PDS LNPs containing Cre mRNA (compared with control).
  • FIG. 61B shows the quantification of the Cy3 image in FIG. 61A.
  • FIG. 62A shows the results of an assay where bone marrow cells were isolated from mice in the previous slide at 126 days post-dosing.
  • the gating structure is shown in FIGS. 57 and 58A-58C.
  • the percentage of tdTomato-i- cells in the bone marrow for PBS control, 3- MC3, and PDS is shown.
  • FIG. 62B is representative of the same data of FIG. 62A, but the percent tdTomato+ cells in the PBS control was subtracted from all samples at each time point to allow direct comparison.
  • FIG. 63A shows quantification of the percentage of transfected cells in liver hepatocytes, spleen white pulp cells, spleen red pulp cells, kidney cells, brain tissue cells, and cells obtained from the foot and leg tissues of the same Ail4 mice discussed in connection with the data of FIGS. 58-62, taken on day 126 post-delivery.
  • FIGS. 63B-63F show IHC data related to use of exemplary 3-MC3 and PDS LNPs in liver (FIG. 63B), spleen (FIG. 63C), kidney tissue (FIG. 63D), leg (FIG. 63E) and tail (FIG. 63F) of the same Ail4 mice discussed in connection with the data of FIGS. 58-63.
  • FIGS. 64A-64B show a flow cytometric experiment demonstrating hematopoietic stem cell targeting in a mammal (Lineage-, Scal+, cKit-i- bone marrow cells) using a lipid nanoparticle targeted with an anti-cKit antibody conjugated to a pegylated lipid.
  • FIG. 65 shows transfection of hematopoietic stem cells (Lineage-, Scal+, cKit+ bone marrow cells) using PDS LNP at multiple doses.
  • the term “approximately” or “about” may refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the term “and/or” may mean “and,” it may mean “or,” it may mean “exclusive-or,” it may mean “one,” it may mean “some, but not all,” it may mean “neither,” and/or it may mean “both.”
  • the terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, prevention, or treatment is desired.
  • the subject may be a mammalian subject.
  • Mammalian subjects include, e. g., humans, non-human primates, rodents, (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc.
  • the subject is a human.
  • the subject is a non-human primate, for example a cynomolgus monkey.
  • the subject is a companion or service animal (e.g. cats or dogs).
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating nucleic acid sequences which, upon being translated (if RNA) or transcribed and translated (if DNA), produce the same sequence of amino acid residues as the original nucleic acid sequence; or, in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • nucleotide sequence refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • ribonucleic acid means a polymer of ribonucleotides.
  • mRNA species means mRNA molecules with identical ribonucleotide lengths and sequences.
  • compositions capable of delivery of a polynucleotide to traditionally hard to reach places (tissues and cells) in a subject. This includes, for example, transfection of stem cells (e.g., hematopoietic stem cells, progenitor cells), tumor cells or areas receiving low level of circulation.
  • stem cells e.g., hematopoietic stem cells, progenitor cells
  • the broad transfection ability demonstrated by the present compositions provides for the ability to deliver a nucleic acid payload to any of a variety of body tissues and cells with or without specific targeting ligands (which are also contemplated for inclusion in the present LNPs (e.g., 3-MC3 and/or PDS)), including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNPs e.g., 3-MC3 and/or PDS
  • Such LNPs have very diverse use opportunities since they have been demonstrated as capable of transfecting a broad variety of cells and tissue types.
  • LNPS low-density polypeptides
  • stem cells progenitor cells, germ cells, differentiated cells, or terminally differentiated cells
  • cancer cells endothelial cells
  • the presently described LNPs can further include attachment of ligands to the liposome surface.
  • the lipid particle comprises a targeting agent, such as a targeting lipid described herein.
  • the targeting is often moiety specific to a cell type or tissue.
  • the targeting of LNPs (e.g., 3-MC3 and/or PDS) using various targeting moieties such as ligands, cell surface receptors, glycoproteins, vitamins (eg riboflavin) and monoclonal antibodies is a known concept.
  • the targeting moiety can include the entire protein or a fragment thereof.
  • the targeting mechanism usually requires that the targeting agent be positioned on the surface of the lipid particle in such a way that the targeting moiety is available for interaction with the target (e.g., a cell surface receptor).
  • the targeting agent e.g., a cell surface receptor
  • a ligand that targets lipid particles is bound to the polar head group of the lipid that forms the lipid particle.
  • Standard methods for binding the target agent can be used.
  • targeting moieties may include other proteins specific for cellular components, including antigens associated with stem cells, progenitor cells, germ cells, and differentiated and terminally-differentiated cells, neoplasms, and tumors. Proteins used as targeting moieties can be covalently attached to liposomes (see Heath, Covalent Attachment of Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)).
  • Other targeting methods include the biotin-avidin system, maleimide-thiol reactions, and succinimidyl ester-amine reactions.
  • moieties are ligands that are bound either directly or indirectly via an intervening tether, preferably covalently.
  • the ligand alters the distribution, targeting or lifetime of the molecule in which it is incorporated.
  • the ligand is selected from a target (e.g., molecule, cell or cell type, compartment (e.g., cell compartment or organ compartment), tissue, Enhance affinity for organs or body regions).
  • a ligand that enhances affinity for a selected target is also referred to as a targeting ligand.
  • a preferred ligand for conjugation to the lipids of the present invention is a targeting ligand.
  • Some ligands can have endosomal lytic properties. Endosomal lytic ligands promote endosomal lysis and / or transport of the compositions of the invention, or components thereof, from the endosome to the cytoplasm of the cell.
  • the endosomal lytic ligand can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane
  • the endosomal lytic ligand assumes its active conformation at endosomal pH.
  • An “active” conformation is a conformation in which the endosomal lytic ligand facilitates endosomal lysis and / or transport of the composition of the invention, or components thereof, from the endosome to the cytoplasm of the cell.
  • Exemplary endosomal lytic ligands include GALA peptides, and derivatives thereof.
  • the endosomal lytic component can include chemical groups (e.g., amino acids) that change charge or protonation in response to changes in pH.
  • the endosomal lytic component may be linear or branched.
  • Preferred ligands can improve transport properties, hybridization properties, and specificity properties, and the resulting natural or modified oligoribonucleotides, or monomers and / or natural ribonucleotides described herein. It may also improve the nuclease resistance of polymer molecules containing any combination of nucleotides or modified ribonucleotides.
  • Ligands generally include, for example, therapeutic modifiers to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; crosslinkers; and moieties that confer nuclease resistance, it can. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.
  • Ligands include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids.
  • the ligand may be a recombinant molecule or a synthetic molecule, such as a synthetic polymer (e.g., a synthetic polyamino acid), an oligonucleotide (e.g., an aptamer).
  • polyamino acids examples include polylysine (PLL), poly L-aspartic acid, poly L- glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer, polyamino acids that are N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphadine.
  • PLL polylysine
  • poly L-aspartic acid examples include poly L-aspartic acid, poly L- glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer,
  • polyamines examples include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine Grade salts, or alpha helix peptides.
  • the ligand can also include a targeting group, such as a cell targeting agent or tissue targeting agent (e.g., a lectin, glycoprotein, lipid or protein (e.g., an antibody that binds to a particular cell type such as a stem cell, progenitor cell, germ cell, differentiated cell, terminally-differentiated cell, or a specific cell type, for example a kidney cell)).
  • a cell targeting agent or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein (e.g., an antibody that binds to a particular cell type such as a stem cell, progenitor cell, germ cell, differentiated cell, terminally-differentiated cell, or a specific cell type, for example a kidney cell).
  • a targeting group such as a cell targeting agent or tissue targeting agent (e.g., a lectin, glycoprotein, lipid or protein (e.g., an antibody that binds to a particular cell type such as a stem cell,
  • targeting groups include antibodies, peptides, aptamers, small molecules, carbohydrates, lipids, nanobodies, and aptamer- antibody conjugates.
  • targeting groups include thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, Glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B 12, biotin, RSG peptide, RSG peptide mimic, or can be an aptamer.
  • antibodies, nanobodies, aptamers targeting clusters of differentiation on cell surfaces for example, CDla, CDlb, CDlc, CDld, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CDlla, CDllb, CDllc, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49d, CD49e, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62L, CD63, CD64, CD65, CD66, CD67,
  • ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, suffirin), polycyclic aromatic hydrocarbons (e.g., phenazine), Dihydrophenazine), artificial endonucleases (eg EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1 -pyrenebutyric acid, dihydrotestosterone, l,3-bis-0 (hexadecyl) glycerol, geranyloxy Hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoy
  • a ligand can be a protein (e.g., a glycoprotein), or a peptide (e.g., a molecule with specific affinity for a co-ligand), or an antibody (e.g., with specificity to specific types of cells, for example a stem cell, progenitor cell, germ cell, differentiated cell, or terminally differentiated cell, a cancer cell, endothelial cell, or bone cell).
  • Ligands can also include hormones and hormone receptors.
  • Ligand includes non-peptide such as lipid, lectin, carbohydrate, vitamin, cofactor, polyvalent lactose, polyvalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, or aptamer Species can also be included.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
  • a ligand is a substance that increases the uptake of a polynucleotide of the LNP into a cell, for example, by increasing the affinity of the LNP for a molecule on the surface of the cell, or increasing the affinity of the LNP for a molecule that in turn binds to a molecule on the surface of the cell, or by disrupting the cytoskeleton of the cell (e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell, e.g., a drug).
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanosine, or myoserbin.
  • the ligand can increase the uptake of the polynucleotide of the LNP into the cell, for example, via endocytosis, or by activating an inflammatory response.
  • exemplary ligands having such effects include tumor necrosis factor a (TNFa), interleukin- 113, or y interferon.
  • the ligand is a lipid or lipid-based molecule.
  • lipids or lipid-based molecules preferably bind to serum proteins such as human serum albumin (HSA).
  • HSA binding ligands allow the conjugate to be distributed to target tissues (e.g., target tissues other than the body's kidneys).
  • target tissues e.g., target tissues other than the body's kidneys.
  • the target tissue can be bone marrow containing hematopoietic stem cells.
  • Other molecules that can bind to a target tissue can also be used as ligands.
  • Lipid based ligands can be used, for example, to control the binding of the conjugate to the target tissue.
  • the inclusion of one or more targeting ligand with the LNPs of the present disclosure is provided for diagnosis, prevention and/or treatment of the suspected or actual underlying disease or condition (including those listed above in the Summary) in the specifically targeted cell type and/or tissue.
  • the presently contemplated LNPs are capable of targeting the cell type and tissue where the condition or disease manifests.
  • the presently described LNPs are shown to provide for the ability to transfect cells with the encapsulated polynucleotide throughout the whole body of the subject, including cell types that exist throughout the body.
  • the polynucleotide encapsulated in the LNP will vary based on the condition or disease and the objective of the delivery (diagnosis, prevention or treatment), the presently described LNPs are provided as an enabling delivery vehicle for such polynucleotide such that it can be delivered to areas that have been difficult or not possible to deliver a polynucleotide in vivo in a subject (e.g., mammal) previously.
  • a subject e.g., mammal
  • telomere extension offers potential benefits for mitigating some age- related changes, it is crucial to consider the associated risks such as cellular immortalization, which can occur with delivery of telomerase DNA to cells.
  • delivery of mRNA or circular RNA encoding telomerase reverse transcriptase (TERT) enables safe telomere extension in a wide range of different cell populations in vitro and in vivo without risk of immortalization.
  • telomere extension in various tissues and cell types throughout the mammalian body including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells using TERT mRNA or TERT circular RNA delivered in vivo using the delivery vehicles disclosed herein provides a way to provide a variety of benefits.
  • One such benefit includes boosting replicative and regenerative capacity of a wide range of different cell populations, where increased telomerase activity can delay replicative senescence in a specifically targeted cell population, promoting tissue renewal and homeostasis.
  • Another benefit is to help preserve or enhance tissue structure in a variety of organs and tissues, wherein improved structural integrity through telomere extension can protect against or slow the rate of degradation of that tissue. Still another such benefit includes the ability to accelerate wound healing in tissues, where promoting cellular proliferation and migration can enhance the tissue’s regenerative capacity. Also, another benefit includes reducing the risk of cancer in a wide range of different cell populations, whereby maintaining telomere length and preventing chromosomal instability, telomere extension strategies might offer a potential approach to reduce the risk of a variety of cancers associated with aging.
  • the delivery of diagnostic and/or therapeutic agents such as mRNAs (including TERT mRNA) using LNPs described herein to cells can be provided in a method to treat, ameliorate or prevent a condition or disease that can be modulated by an engineered polynucleotide such as a modified mRNA.
  • an engineered polynucleotide such as a modified mRNA.
  • contemplated a variety of conditions or diseases where the condition or disease itself or its symptoms that may be modulated (e.g., treated, ameliorated or prevented) by the presently described and encompassed LNP compositions and formulations are contemplated.
  • These include preventing, treating or ameliorating various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells conditions or diseases.
  • the data provided herein demonstrate or support, for example, pharmacokinetics of the exemplified LNPs in vivo, and particularly information and data related to the time course of concentration levels of a lipid from the LNPs in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • L P compositions and formulations are provided that are rapidly cleared from the circulatory system of the subject but have a longer half-life in the body tissues.
  • the data provided herein also demonstrate or support, for example, the use of LNPs in which the mRNA encodes telomerase.
  • LNPs for use in introducing nucleic acids to body tissues including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • the LNPs are manufactured in a manner that forms the LNP prior to addition of nucleic acid.
  • such LNPs are 2, 3 or 5 lipids LNPs described herein and the method often employs vortex mixing during the addition of the nucleic acid.
  • the present inventors have found that in LNPs adapted to work according to the presently contemplated methods and uses, the ratios of cationic lipid to ionizable lipid (e.g. SSOP : DOTAP or MC3 : DOTAP) are unpredictable based on the specific constituents of the LNP. Specifically, while not intending to be bound by any specific theory, experimentally it appears that the particular ionizable lipid in the LNP affects the cationic lipid to ionizable lipid ratio in a manner that requires experimentation to confirm.
  • the experimental evidence provided herein provides support for a specific cationic lipid to ionizable lipid ratio, or specific ratio range, based on the identified ionizable lipid in the LNP.
  • the tables and Figures provided herein, for example Table 7, provide additional examples of specific ratios of cationic lipid to ionizable lipid of 3- and 5- lipid LNPs contemplated herein.
  • the cationic lipid to ionizable lipid ratio is at or about 0.8 and the ionizable lipid is SS-OP.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 0.8 and is composed of 3 or 5 lipids.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 0.8 and is composed of 3 or 5 lipids, including DOTAP.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 0.8 and is composed of 3 or 5 lipids, including a pegylated lipid (e.g., DMG-PEG2000) and DOTAP.
  • a pegylated lipid e.g., DMG-PEG2000
  • DOTAP DOTAP
  • LNPs are used in the presently described methods to introduce a genetic pay load (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic pay load e.g., mRNA, including TERT mRNA
  • the cationic lipid to ionizable lipid ratio is at or about 1.8 and the ionizable lipid is SS-OP.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.8 and is composed of 5 lipids.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.8 and is composed of 5 lipids, including DOTAP and DOPC.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.8 and is composed of 5 lipids, including a pegylated lipid (e.g., DMG-PEG2000), DOPC and DOTAP.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • mRNA including TERT mRNA
  • the LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • mRNA including TERT mRNA
  • the cationic lipid to ionizable lipid ratio is at or about 1.5 and the ionizable lipid is DLin-MC3-DMA.
  • the LNP has a cationic lipid to ionizable lipid ratio is at or about 1.5 and is composed of 3 lipids.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.5 and is composed of 3 lipids, including DOTAP.
  • the LNP has a cationic lipid to ionizable lipid ratio at or about 1.5 and is composed of 3 lipids, including a pegylated lipid (e.g., DMG-PEG2000) and DOTAP.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • mRNA including TERT mRNA
  • the LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • mRNA including TERT mRNA
  • LNP compositions of the present disclosure include and encompass a variety of lipid ratios and lipid : mRNA ratios (lipid : mRNA ratios are also referred to herein as “N/P ratio”). When mentioned herein LNP compositions refers to either or both the final LNP
  • nucleic acid containing LNP compositions composed of 5 different lipids, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 10 and 30 are provided.
  • Table 7 provides some such exemplary compositions.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of between 0.8 to 1.8.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of at or about 0.8.
  • such LNP compositions include SS-OP as the ionizable lipid in the composition.
  • LNPs are used in the presently described methods to introduce a genetic pay load (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic pay load e.g., mRNA, including TERT mRNA
  • such LNP compositions are used in vivo in a mammal subject and target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a manner that provides for transfection of the nucleic acid payload in the various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions are contemplated herein as adapted to various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNP compositions preferentially target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • such LNP compositions have an increased
  • LNP compositions have a half-life in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNP compositions have a half-life in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells or over 30 hours, or between 30 hours and 40 hours, or about 37 hours.
  • nucleic acid containing LNP compositions composed of 3 different lipids, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 4 and 24 are provided, or an N/P ratio of between 4.5 and 12.
  • Table 7 provides some such exemplary compositions.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of between 0.8 to 1.5.
  • such LNP compositions are further characterized as having a cationic lipid to ionizable lipid ratio of at or about 0.8.
  • such LNP compositions include SS-OP as the ionizable lipid in the composition and a cationic lipid to ionizable lipid ratio of at or about 0.8. Also according to such embodiments such LNP compositions include DLin-MC3-DMA as the ionizable lipid in the composition and a cationic lipid to ionizable lipid ratio of at or about 1.5, and optionally an N/P ratio of at or about 4.5.
  • such LNP compositions are used in vivo in a mammal subject and target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a manner that provides for transfection of the nucleic acid payload in the various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions are contemplated herein as adapted to target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • LNP compositions preferentially target various tissues and cell types throughout the mammalian body, including stem cells, progenitor
  • - 26 - cells germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • nucleic acid containing LNP compositions composed of 3 or 5 different lipids including DOTAP, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 4 and 24 are provided, or an N/P ratio of at or about 12. Also according to preferred embodiments herein, nucleic acid containing LNP compositions composed of 3 different lipids including DLin-MC3-DMA, including an ionizable lipid and a cationic lipid, and having a N/P ratio of between 4 and 24 are provided, or an N/P ratio of at or about 4.5.
  • LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • nucleic acid containing LNP compositions composed of 2 different lipids having a N/P ratio of at or about 4 are provided.
  • one of the two lipids is or comprises DOTAP.
  • such LNP compositions are used in vivo in a mammal subject and target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a manner that provides for transfection of the nucleic acid payload in the various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions are contemplated herein as adapted to target various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • sucrose is a surprisingly good cryoprotectant for the presently contemplated LNP compositions. Specifically, it has been found that sucrose included at a range of between 5% to 25% of the final composition provides for preferred levels of cryoprotection. In specific embodiments the cryoprotectant (e.g.,
  • LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in operable form such that it is expressed in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • a genetic payload e.g., mRNA, including TERT mRNA
  • telomere delivery of synthetic nucleoside-modified mRNA encoding TERT enables transient elevation of telomerase activity in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells which extends telomeres in hours sufficiently to reverse years of telomere shortening.
  • LNP lipid nanoparticle
  • IV intravenous
  • Administration may also be provided transcutaneously, intracutaneous injection via microneedle array, topical delivery (e.g., in conjunction with a skin barrier-permeabilizing step such as microneedling or application of a permeabilizing agent such as a mixture containing proteases, lipases, or others known in the field).
  • RNA ribonucleic acid
  • RNA may refer to any RNA sequence comprising a mutation (point or deletion) or additional nucleotides not found in the wild type sequence.
  • a messenger RNA mRNA
  • mRNA messenger RNA
  • the nucleotides themselves may encode amino acids distinct from the wild type, or be modified to reduce immunogenicity in the cell or tissue.
  • An mRNA sequence in some embodiments may comprise any of the following modifications, including but not limited to an untranslated region (UTR), a 5' cap, and a poly-adenosine tail.
  • the RNA may be circular and/or self-replicating.
  • a mRNA may comprise a codon-optimized sequence. In some embodiments, a mRNA may comprise a uridine depleted sequence.
  • the 5' cap of the ribonucleic acid is a non-immunogenic cap.
  • the 5' cap may increase the translation of the ribonucleic acid.
  • the 5' cap may be treated with phosphatase to modulate the innate immunogenicity of the ribonucleic acid.
  • the 5' cap is an anti-reverse cap analog (“ARCA”), such as a 3'-O-Me-m7G(5')ppp(5')G RNA cap structure analog.
  • the 5' cap is m7G(5')ppp(5')(2'OmeA)pG (also known as CleanCap® AG).
  • the 5' cap is m7(3'OmeG)(5')ppp(5')(2'OmeA)pG (also known as CleanCap® AG (3' OMe)).
  • the above features, or others may increase translation of the protein encoded by the ribonucleic acid, may increase or decrease the stability of the ribonucleic acid itself in a cell type-specific or cell type-independent manner, or may do both.
  • the 5' UTR and/or the 3' UTR are from a gene that has a very stable mRNA and/or an mRNA that is rapidly translated, for example, a-globin or P-globin, c-fos, or tobacco etch virus.
  • the 5' UTR and 3' UTR are from different genes or are from different species than the species into which the compositions are being delivered.
  • the UTRs may also be assemblies of parts of UTRs from the mRNAs of different genes, where the parts are selected to achieve a certain combination of stability and efficiency of translation.
  • the UTRs may also comprise designed sequences that confer properties to the RNA such as cell type-specific stability or cell type-independent stability.
  • the ribonucleic acids of the present disclosure may comprise one or more modified nucleosides, and/or comprise primary sequences of nucleosides, that modulate translation, stability, or immunogenicity of the RNA.
  • Most mature RNA molecules in eukaryotic cells contain nucleosides that are modified versions of the canonical unmodified RNA nucleosides, adenine, cytidine, guanosine, and uridine. For example, the 5' cap of mature RNA nucleosides, adenine, cytidine, guanosine, and uridine. For example, the 5' cap of mature RNA nucleosides, adenine, cytidine, guanosine, and uridine. For example, the 5' cap of mature RNA nucleosides, adenine, cytidine, guanosine, and uridine. For example, the 5' cap of mature RNA nucleosides, adenine
  • RNA - 29 - RNA comprises a modified nucleoside, and other modified nucleosides often occur elsewhere in the RNA. Those modifications may prevent the RNA from being recognized as a foreign RNA. Synthetic RNA molecules made using certain nucleosides are much less immunogenic than unmodified RNA. The immunogenicity can be reduced even further by purifying the synthetic mRNA, for example by using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the modified nucleosides may be, for example, chosen from the nucleosides listed below.
  • the nucleosides are, in some embodiments, pseudouridine, 1 -methylpseudouridine, 2- thiouridine, 5-methoxyuridine, or 5 -methylcytidine.
  • the primary sequence may be modified in ways that increase or decrease immunogenicity. Under some circumstances, it may be desirable for the modified RNA to retain some immunogenicity.
  • the ribonucleic acids of the instant compositions comprise a 1 -methylpseudouridine, pseudouridine, a 5-methoxyuridine (5-moU), a 2-thiouridine, a 5 -methylcytidine, or another modified nucleoside.
  • Modified nucleosides found in eukaryotic cells include mlA 1 -methyladenosine, m6A N6-methyladenosine, Am 2'- O-methyladenosine, i6A N6-isopentenyladenosine, io6A N6-(cis- hydroxyisopentenyl)adenosine, ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, g6A N6-glycinylcarbamoyladenosine, t6A N6-threonylcarbamoyladenosine, ms2t6A 2-methylthio-N6-threonyl carbamoyladenosine, Ar(p) 2'-O-ribosyladenosine (phosphate), mb 2A N6,N6-dimethyladenosine, m6Am N6,2
  • the presence of the modified nucleosides, and/or sequences of nucleosides that alter secondary structure of the RNA and/or binding of RNA to RNA binding proteins or microRNA, may enable mRNA to avoid activation of an immune response mediated by various receptors, including the Toll-like receptors and RIG-1.
  • Non-immunogenic mRNA has been used as a therapeutic agent in mice via topical delivery. Kormann et al. (2011) Nature Biotechnology 29:154-157. In some
  • the ribonucleic acids comprise more than one of the above nucleosides or combination of the above nucleosides. In some embodiments, the ribonucleic acids comprise 1- methylpseudouridine, 5-methoxyuridine, or pseudouridine and 5 -methylcytidine.
  • an immune response to the mRNA may be desired, and the RNA may be modified to induce an optimal level of innate immunity. In other embodiments, an immune response to the mRNA may not be desired, and the RNA may be modified in order to minimize such a reaction.
  • the RNA can be modified for either situation.
  • the ribonucleic acid molecules can be synthetic ribonucleic acids.
  • the term “synthetic”, as used herein, can mean that the ribonucleic acids are in some embodiments prepared using the tools of molecular biology under the direction of a human, for example as described below.
  • the synthetic ribonucleic acids may, for example, be prepared by in vitro synthesis using cellular extracts or purified enzymes and nucleic acid templates.
  • the synthetic ribonucleic acids may in some embodiments be prepared by chemical synthesis, either partially or completely.
  • the synthetic ribonucleic acids may in some embodiments be prepared by engineered expression in a cell, followed by disruption of the cell and at least partial purification of the ribonucleic acid.
  • the ribonucleic acids of the present disclosure may be prepared using a variety of techniques, as would be understood by one of ordinary skill in the art.
  • the ribonucleic acids may be prepared by in vitro synthesis.
  • the ribonucleic acids may be prepared by chemical synthesis.
  • the ribonucleic acids may be prepared by a combination of in vitro synthesis and chemical synthesis.
  • synthetic should be understood to include ribonucleic acids that are prepared either by chemical synthesis, by in vitro synthesis, by expression in vivo and at least partial purification, or by a combination of such, or other, chemical or molecular biological methods.
  • the ribonucleic acids may, in some embodiments, be purified. As noted above, purification may reduce immunogenicity of the ribonucleic acids and may be advantageous in some circumstances. In some embodiments, the ribonucleic acids are purified by one or more of HPLC, DNAse treatment, protease treatment, or by affinity capture and elution.
  • an mRNA sequence may be synthesized as an unmodified or modified mRNA.
  • An mRNA may be modified to enhance stability and/or evade immune detection and degradation.
  • a modified mRNA may include, for example, one or more of a nucleotide modification, a nucleoside modification, a backbone modification, a sugar modification, and/or a base modification.
  • the modified nucleoside is
  • the modified nucleoside is 5- methoxyuridine.
  • a modified nucleoside as used herein may comprise any of the moieties listed in Table A.
  • an RNA e.g. an mRNA
  • an RNA may be synthesized from naturally occurring nucleosides and/or nucleoside analogs (modified nucleosides) including, but not limited to, nucleosides comprising adenosine (A), guanosine (G)) or pyrimidines (thymine (T), cytidine (C), uridine (U)), and nucleoside comprising analogues and derivatives thereof, e.g., 3 '-deoxy adenosine (cordycepin), 3 '-deoxyuridine, 3'-deoxycytosine, 3'- deoxyguanosine, 3 '-deoxy thymine, 2',3'-dideoxynucleosides, 2', 3'- dideoxyadenosine, 2', 3'- dideoxyuridine, 2', 3 '-dideoxycytosine
  • uracil nucleosides of the mRNA are about 80%, about 90%, 95%, 99%, or 100% depleted and replaced with a uracil nucleoside analog, e.g., pseudouridine, 5-methoxyuridine, or N-l-methyl-pseudouridine.
  • a uracil nucleoside analog e.g., pseudouridine, 5-methoxyuridine, or N-l-methyl-pseudouridine.
  • an RNA may contain an RNA backbone modification.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are chemically modified.
  • Exemplary backbone modifications may include, but are not limited to, modifications in which the phosphodiester linkage is replaced with a member from the group consisting of peptides, methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-0-(l-
  • an RNA may contain sugar modifications.
  • a sugar modification may include but is not limited to, 2' O-methyl sugar modifications, 2' fluoro sugar modifications (e.g. 2'-fluororibose), 3' amino sugar modifications, 2' thio sugar modifications, 2'-O-alkyl sugar modifications, 5 -methylthioribose, and sugar modifications of 2'-deoxy-2'- fluoro-ribonucleotide (2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine), 2'-deoxy-2'- deamine-ribonucleotide (2'-amino-2'-deoxycytidine, 2,-amino-2'-deoxyuridine), 2'-O- alkylribonucleotide, 2'-deoxy-2'-C-alkylribonucleotide (2'-O-methylcytidine, 2'- methyluridine), 2'-C-
  • an RNA may be synthesized from one or more of the nucleotide triphosphates comprising any of the nucleosides and nucleotides disclosed herein, or any of the following nucleoside triphosphates: 2'-Deoxyadenosine-5'-O-(l-Thiotriphosphate), 2'-Deoxycytidine-5'-O-(l -Thiotriphosphate), 2'-Deoxyguanosine-5'-O-(l-Thiotriphosphate), 2'-Deoxythymidine-5'-O-(l -Thiotriphosphate), Adenosine-5'-O-(l-Thiotriphosphate), Cytidine-5 '-O-( 1 -Thiotriphosphate) , Gu nosine-5 '-O-( 1 -Thiotriphosphate) , Uridine-5 '-O-( 1 - Thio triphosphate), 2',3'-Dide
  • an mRNA may include the addition of a “cap” on the N- terminal (5') end, and a “tail” on the C-terminal (3') end.
  • the presence of the cap may provide resistance to nucleases found in eukaryotic cells.
  • the presence of a “tail” may protect the mRNA from exonuclease degradation.
  • an mRNA may include a 5' cap structure.
  • a 5' cap may comprise for example, a triphosphate linkage and a guanine nucleotide in which the 7-nitrogen is methylated.
  • Examples of cap structures include, but are not limited to, m7G(5')ppp (5') A, G(5')ppp(5')A, and G(5')ppp(5')G.
  • Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5 '-end of the first transcribed
  • - 42 - nucleotide resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside.
  • the cap is added in the nucleus by the enzyme guanylyl transferase immediately after initiation of transcription.
  • a 5 'cap may comprise an m7(3'OmeG)(5')ppp(5')(2'OmeA)pG or (CleanCap® 3' OMe) structure.
  • a 5' cap may comprise a m7G(5')ppp(5')G.
  • the AntiReverse Cap Analog (“ARCA”) or modified ARCA is a 5' cap in which the 2' or 3' OH group is replaced with -OCH3.
  • the ARCA comprises an 3'-0-Me- m7G(5')ppp(5')G structure.
  • the 5' cap comprises m7G(5')ppp(5')(2'OmeA)pG.
  • Additional mRNA caps may include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated caps e.g., GpppG); a memethylated cap (e.g., m2'7GpppG), a trimethylated cap analog, or anti reverse cap analogs (e.g., ARCA; m7,2'0meGpppG, m72'dGpppG, m7’3'0meGpppG, m7,3 dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al, ‘Wove anti-reverse cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).
  • a suitable cap is a 7-methyl guanylate (“m7G”) linked via a triphosphate bridge to the 5 ‘-end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleoside.
  • An embodiment of an m7G cap utilized in embodiments of the disclosure is m7G(5')ppp(5')G.
  • the cap is a CapO structure. CapO structures lack a 2'-0- methyl residue of the ribose attached to bases 1 and 2.
  • the cap is a Capl structure. Capl structures have a 2'-0-methyl residue at base 2.
  • the cap is a Cap2 structure. Cap2 structures have a 2'-0-methyl residue attached to both bases 2 and 3.
  • m7G cap analogs are known in the art, many of which are commercially available. These include the m7 GpppG described above, as well as the ARCA 3'- OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the disclosure include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E.
  • RNA, 10: 1479-1487 (2004) phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al, RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Patent Nos. 8,093,367 and 8,304,529, incorporated by reference herein.
  • the 5' cap is inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, CleanCap®,
  • the 5' cap comprises or consists of an internal ribosome entry site (IRES).
  • IRES is within the 5' UTR.
  • the 5' cap comprises or consists of a 2A self-cleavage peptide, e.g, one or more of P2A, T2A, E2A and F2A.
  • a “tail” may serve to protect an mRNA from exonuclease degradation.
  • the poly-A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly-A tail can be added to an mRNA molecule thus rendering the RNA more stable.
  • Poly-A tails can be added using a variety of art- recognized techniques. For example, long poly-A tails can be added to synthetic or in vitro transcribed RNA using poly-A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly-A tails. In addition, poly-A tails can be added by transcription directly from PCR products.
  • Poly-A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2 nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
  • an mRNA may include a 3' poly(A) tail structure.
  • the length of the poly-A tail may be at least about 10, 50, 100, 200, 300, 400 or at least about 500 nucleotides.
  • a poly-A tail on the 3' terminus of an mRNA may include about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • the poly A tail is 120 adenosine nucleotides.
  • an mRNA may include a 3' poly-C tail structure.
  • a poly- C tail on the 3' terminus of mRNA may include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • the length of the poly-A or poly C tail is associated with the stability of a modified sense mRNA and, therefore, the transcription of the protein.
  • the length of the poly-A tail may influence the half-life of a sense mRNA molecule, the length
  • poly-A tail may be adjusted to modify the level of resistance of the mRNA to nucleases, thereby providing more control over the time course of polynucleotide expression and/or polypeptide production.
  • an mRNA may include 5' untranslated region (UTR) and/or a 3' UTR.
  • a 5' UTR may include one or more elements that affect the stability or translation of an mRNA.
  • the 5'UTR for example, may include an iron responsive element.
  • 5' UTR may be between about 50 to about 100, or from about 50 to about 500 nucleotides in length.
  • 3' UTR includes one or more of a poly-A signal, a binding site for proteins that may affect mRNA stability or localization, or one or more binding sites for miRNAs.
  • 3' UTR may be between about 0 and about 50 nucleotides, or about 50 to about 100 nucleotides in length
  • Example 3' an’ 5' UTR sequences may be derived from mRNAs with relatively long half-lives (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule.
  • 5' UTR sequence may include a partial sequence of a cytomegalovirus (CMV) immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide.
  • CMV cytomegalovirus
  • IE1 immediate-early 1
  • the 5 ’ UTR could include the sequence of the tobacco etch virus (TEV).
  • these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides resistance to in vivo nuclease digestion.
  • a UTR may improve tissue specific expression, e.g., in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • the UTR may be any of, or functional variants of, those described in any of PCT Application No. WO2017053297A1 and Patent No. US10519189B2, both of which are incorporated herein in their entirety. Ionizable lipids
  • an LNP may comprise an ionizable lipid, e.g. SS-OP or analogs thereof, or MC3 or analogs thereof.
  • the charge of the lipid may depend on pH of the surrounding solution, making it an ionizable lipid.
  • the ionizable lipid may also be cleavable.
  • the ionizable lipid may be cationic at ranges of pH found in endosomes or lysosomes in mammalian cells.
  • An ionizable lipid may refer to any of a number of lipid species that have a net positive charge at a selected pH, such as a physiological pH.
  • an LNP may comprise an ionizable lipid as disclosed in either of WO 2010/053572 or WO 2012/170930, or variations thereof, both of which are incorporated herein by reference in their entirety.
  • an LNP may comprise one or more of MC3 (((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate), DLin- MC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yL 10,13-nonadecadien-l-yl ester), or analogs thereof including but not limited to LenMC3, y- LenMC3, MC3MC, MC2C, MC2MC, MC3 Thioester, MC3 Ether, MC4 Ether, MC3 Alkyne, MC3 Amide, Pan-MC3, Pan-MC4, Pan-MC5, CP-LenMC3, CP-y-LenMC3,
  • an LNP may comprise one or more of cKK-E12 (3,6- Bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione), DLinDAP (l,2-dilineoyl-3- dimethylammonium-propane), DLin-DMA, DLin-D-DMA, DLin-KC2-DMA (2,2-dilinoleyL 4-dimethylaminoethyl-[l,3]-dioxolane), and DODMA.
  • cKK-E12 3,6- Bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione
  • DLinDAP l,2-dilineoyl-3- dimethylammonium-propane
  • DLin-DMA DLin-D-DMA
  • DLin-KC2-DMA 2,2-dilinoleyL 4-dimethyl
  • the ionizable lipid may comprise SS-OP or analogs thereof. In some embodiments, the ionizable lipid is a compound of Formula (1):
  • R la and R lb each independently represents an alkylene group having 1 to 6 carbon atoms, and may be linear or branched.
  • the alkylene group may have 1 to 4 carbon atoms, or may have 1 to 2.
  • Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group,
  • R la and R lb may be each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group, or a tetramethylene group, and may be an ethylene group.
  • R la may be different or be the same as R lb .
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups, and/or each independently a cyclic alkylene having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups and an alkylene tertiary amino group.
  • the alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is branched even if it is linear.
  • the alkyl group may be annular.
  • the alkyl group may have 1 to 3 carbon atoms.
  • Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • Neopentyl group, t-pentyl group, 1 ,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, A cyclohexyl group etc. can be mentioned.
  • a specific structure of an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is represented by X 1 .
  • R 5 of X 1 represents an alkyl group having 1 to 6 carbon atoms and may be linear, branched or cyclic.
  • the alkyl group may have 1 to 3 carbon atoms.
  • Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec -butyl group, isobutyl group, tert-butyl group, pentyl group, and isopentyl group.
  • the number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups may be 4 to 5.
  • Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include aziridylene group, azetidylene group, pyrrolidylene group, piperidylene group,
  • - 47 - imidazolidylene group a piperazylene group, optionally a pyrrolidylene group, a piperidylene group or a piperazylene group.
  • Number is 2 to 5 carbon atoms, and specific structure of alkylene tertiary amino groups containing 1 annular tertiary amino group represented by X 2 .
  • P of X 2 is 1 or 2.
  • X 2 is a pyrrolidylene group, and when p is 2,
  • X 2 is a piperidylene group.
  • a specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is represented by X 3 .
  • W of X 3 is 1 or 2.
  • X 3 is an imidazolidylene group, and when w is
  • X 3 is a piperazylene group.
  • X a may be different be identical to X b .
  • R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having 8 or less carbon atoms, optionally each independently an alkylene group having 8 or less carbon atoms.
  • the alkylene group having 8 or less carbon atoms may be linear or branched but is optionally linear.
  • the number of carbon atoms contained in the alkylene group is optionally
  • alkylene group having 8 or less carbon atoms include methylene group, ethylene group, propylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, and the like.
  • a methylene group, an ethylene group, a propylene group, and a tetramethylene group are included.
  • the oxydialkylene group having 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) via an ether bond, and the total number of carbon atoms of two alkylene groups is 8 or less.
  • the two alkylenes may be the same or different, but are optionally the same.
  • Specific examples of the oxydialkylene group having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydipropylene group, and an oxydibutylene group.
  • R 2a may be the same or different from R 2b .
  • Y a and Y b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, optionally each independently an ester bond, an amide bond or a carbamate bond. While Y binding orientation of Y a and Y b are not limited, if Y a and Y b is an ester bond, optionally, -Z a -CO — R 2a - and -Z b -CO-O-R 2b -structure. [00219] Y a may be different or identical to Y b .
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom.
  • the number of carbon atoms contained in the aromatic compound is optionally 6 to 12, or 6 to 7.
  • the number of aromatic rings contained in the aromatic compound is optionally one.
  • aromatic rings contained in the aromatic compound having 3 to 16 carbon atoms as for aromatic hydrocarbon rings, benzene ring, naphthalene ring, anthracene ring, and aromatic heterocycles as imidazole ring, pyrazole ring, oxazole ring, Isoxazole ring, thiazole ring, isothiazole ring, triazine ring, pyrrole ring, furanthiophene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, purine ring, pteridine ring, benzimidazole ring, indole ring, benzofuran ring, quinazoline ring, phthalazine ring, quinoline ring, isoquinoline ring, coumarin ring, chromone ring, benzodiazepine ring, phenoxazine
  • the aromatic ring may have a substituent.
  • substituents include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, and 2 to 2 carbon atoms.
  • acyloxy groups acylamino groups having 2 to 4 carbon atoms, alkoxycarbonylamino groups having 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylsulfanyl groups having 1 to 4 carbon atoms, 1 carbon atom Alkylsulfonyl group having 4 to 4, arylsulfonyl group having 6 to 10 carbon atoms, nitro group, trifluoromethyl group, cyano group, alkyl group having 1 to 4 carbon atoms, ureido group having 1 to 4 carbon atoms, 1 to carbon atoms 4 alkoxy groups, aryl groups having 6 to 10 carbon atoms, aryloxy groups having 6 to 10 carbon atoms, and the like.
  • Some examples include acetyl groups, methoxycarbonyl groups, methyl carbonate groups, and the like, moyl group, acetoxy group, acetamide group, methoxycarbonylamino group, fluorine atom, chlorine atom, bromine atom, iodine atom, methylsulfanyl group, phenylsulfonyl group, nitro group, trifluoromethyl group, cyano group,
  • a specific structure of Z a and Z b includes Z 1 .
  • s represents an integer of 0 to 3
  • t represents an integer of 0 to 3
  • u represents an integer of 0 to 4
  • S in Z 1 is optionally an integer of 0 to 1.
  • T in Z 1 is optionally an integer of 0 to 2.
  • U in Z 1 is optionally an integer of 0 to 2.
  • R 4 in Z 1 is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the process of synthesizing the ionizable lipid.
  • the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, and an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, fluorine atom, chlorine atom, bromine atom, iodine atom, alkylsulfanyl group having 1 to 4 carbon atoms, alkylsulfonyl group having 1 to 4 carbon atoms, 6 to 10 carbon atoms arylsulfonyl group, nitro group, trifluoromethyl
  • Z a may be different even identical to the Z b .
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or
  • sterol derivative having a hydroxyl group and succinic anhydride or glutaric acid.
  • a C 12-22 aliphatic hydrocarbon group and optionally each independently an aliphatic hydrocarbon group having 12-22 carbon atoms.
  • Examples of the fat-soluble vitamin having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotaxolol, tocopherol, and tocotrienol.
  • the fat-soluble vitamin having a hydroxyl group is optionally tocopherol.
  • Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, 0-sitosterol, lanosterol, ergosterol and the like, optionally cholesterol or cholestanol.
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched.
  • the aliphatic hydrocarbon group may be saturated or unsaturated.
  • the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, optionally 1 to 3, or 1 to 2.
  • Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds.
  • the number of carbon atoms contained in the aliphatic hydrocarbon group is optionally 13 to 19, or 13 to 17.
  • the aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group and the like, and optionally includes an alkyl group or an alkenyl group.
  • aliphatic hydrocarbon group having 12 to 22 carbon atoms include dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, heicosyl, docosyl, dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icocenyl group, henicocenyl group, dococenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group,
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms is optionally a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group,
  • - 51 - or a 1 -hexylnonyl group or a tridecyl group, a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group.
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by R 3a and R 3b is derived from a fatty acid.
  • the carbonyl carbon derived from the fatty acid is contained in — CO — O — in the formula (1).
  • Specific examples of the aliphatic hydrocarbon group include a heptadecenyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.
  • R 3a may be different be the same as R 3b .
  • R l a is the same as R lb
  • X a is the same as X b
  • R 2a is the same as R 2b
  • Y a is the same as Y b
  • Z a is identical to the Z b
  • R 3a is the same as R 3b .
  • Preferable examples of the ionizable lipid represented by the formula (1) include the following ionizable lipids: Ionizable lipid (1-1); R la and R lb are each independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene group, ethylene group); X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., — N (CH 3 ) — ), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidylene group); R 2a and R 2b are each independently an alkylene group having 8 or less carbon atoms (e.g., methylene group, ethylene group, propylene group); Y a and Y b are each independently an ester bond or an amide bond
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic group having 12 to 22 carbon atoms, a hydrocarbon group (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group).
  • a fat-soluble vitamin having a hydroxyl group e.g., tocopherol
  • succinic anhydride or glutaric anhydride e.g., glutaric anhydride
  • an aliphatic group having 12 to 22 carbon atoms
  • a hydrocarbon group e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group.
  • R la and R lb are each independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group);
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g., — N (CH 3 ) — ), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g., piperidylene group);
  • R 2a and R 2b are each independently an alkylene group having 6 or less carbon atoms (e.g., methylene group, ethylene group, propylene group);
  • Y a and Y b are each independently an ester bond or an amide
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a hetero atom (e.g., — C 6 H 4 —CH 2 — , —CH 2 — C 6 H 4 —CH 2 — );
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., , Heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group).
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., Heptadecenyl group, heptadecadienyl group, 1 -hexylnonyl group).
  • ionizable lipid include the following O-Ph-P3Cl, O-Ph-P4Cl, O-Ph-P4C2, O-Bn-P4C2, E-Ph-
  • Lipids having the structure of Formula I are shown in the chart below. For example, SS-
  • OP is also named 0-Ph-P4C2.
  • SS-OP analog refers to a compound of
  • an LNP of the disclosure comprises a cationic lipid, e.g. DOTAP or variations thereof.
  • the cationic lipid may be a “permanent cationic lipid.”
  • the term cationic lipid may be cationic in pH ranges found in mammalian physiological environments such as blood or interstitial fluids.
  • Cationic lipids may be composed of a cationic amine moiety and a lipid moiety, and the cationic amine moiety and a polyanion nucleic acid may interact to form a positively charged liposome or lipid membrane structure. Thus, uptake into cells may be promoted and nucleic acids delivered into cells.
  • the cationic lipid may selected from one or more of 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylarnmonium bromide (DABB), or l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).
  • DOTAP 1,2- dioleoyl-3-trimethylammonium-propane
  • DABB N,N-distearyl-N,N-dimethylarnmonium bromide
  • EPC l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine
  • an LNP comprises a ionizable lipid wherein the ionizable lipid is one or more of N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5- carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3- Dimethylammonium- Propane (DODAP), 11 ,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), l,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), l,2-dilinoleyloxy-N,N- dimethyl-3-amin
  • DLin-K-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DLin-K- XTC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • a cationic lipid refers to a cationic cholesterol lipid.
  • an LNP comprises imidazole cholesterol ester (ICE).
  • ICE structure is substantially similar to:
  • an LNP comprises 25-Hydroxycholesterol (25 OH Choi).
  • 25 OH Choi structure is substantially similar to:
  • an LNP comprises 20a-hydroxycholesterol 5-cholestene-3a.
  • the 20a-hydroxycholesterol 5-cholestene-3a (also known as 20a-diol or 20a chol structure) is substantially similar to:
  • a cationic lipid refers to dimethyldioctadecylammonium bromide (DDAB).
  • an LNP comprises dimethyldioctadecylammonium bromide (DDAB).
  • DDAB dimethyldioctadecylammonium bromide
  • the LNP comprises a structural lipid.
  • structural lipids are lipids that contribute a physical or chemical property to the LNP that is in addition to, or independent of, electrical charge.
  • structural lipids may tend to have a shape, size, rigidity, hydrophobicity, or other property that increases the diagnostic and/or therapeutic utility of the LNP, such as, for example, by increasing its stability, half-life, deformability, transfection efficiency, tropism, thermostability, resistance to aggregation, membrane fluidity, or other parameter.
  • structural lipids are neutral in charge, either due to lacking charged moieties, or due to being zwitterionic with balanced charges summing to zero net charge.
  • an LNP may comprise a structural lipid selected from one more of: l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerolmonooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC) , palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclo
  • an LNP may include one or more phosphatidyl lipids, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine).
  • an LNP may comprise sphingolipids, for example but not limited to, sphingosine, ceramide, sphingomyelin, cerebroside and ganglioside.
  • the aforementioned “structural” lipids contribute to the stability and/or specificity of the LNP composition.
  • an LNP may comprise one or more cholesterol-based lipids.
  • a cholesterol-based lipid may include but is not limited to: PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine.
  • an LNP may comprise one or more PEGylated lipids.
  • PEG polyethylene glycol
  • PEG-CER derivatized ceramides
  • C8 PEG-2000 ceramide N-Octanoyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000]
  • PEGylated lipids comprise PEG-ceramides having shorter acyl chains (e.g., C14 or Cl 8).
  • the PEGylated lipid DSPE-PEG- Maleimide-Lectin may be used.
  • PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C2o length.
  • the addition of PEGylated lipids may prevent complex aggregation and increase circulation lifetime to facilitate the delivery of the liposome encapsulated mRNA to the target cell.
  • Methods of diagnosis treatment, and/prevention as described herein in certain embodiments refer to transfection of various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject in need thereof by administration of an LNP of the present disclosure comprising one or more mRNA sequences.
  • compositions and methods of the disclosure may be used for the diagnosis, prevention and/or treatment of conditions in or involving various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment of fibrotic conditions, including fibrosis.
  • compositions and/or methods of use of compositions of the disclosure may be used for the diagnosis, prevention and/or treatment of conditions in or involving various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment do not induce cellular, tissue or systemic toxicity.
  • Compositions may be administered systemically (e.g.
  • compositions and/or methods of use of compositions of the disclosure intended for diagnosis, prevention and/or treatment of conditions in or involving various tissues and cell types throughout the mammalian body including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells, do not induce cellular or systemic toxicity.
  • compositions comprising an exemplary LNP described herein and a nucleic acid encoding an active agent such as TERT mRNA for use in preventing, treating or ameliorating various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells conditions or diseases may be administered intravenously, transcutaneously, intracutaneous injection via microneedle array, topical delivery (e.g., in conjunction with a skin barrier- permeabilizing step such as microneedling or application of a permeabilizing agent such as a mixture containing proteases, lipases, or others known in the field).
  • an active agent such as TERT mRNA
  • lipid nanoparticle comprising: (i) a SS-OP or an SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, with a cationic lipid: ionizable lipid (C/I) ratio between 0.6 and 1, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the
  • RNA - 61 - synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types in the subject.
  • lipid nanoparticle comprising: (i) DLin-MC3-DMA at a molar percentage of between about 30% and about 50%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (ii) a cationic lipid at a molar percentage of between about 50% and about 70%, with a cationic lipid: ionizable lipid (C/I) ratio between 1 and 2, wherein the polynucleotide comprises a synthetic RNA, which upon or after administration of the LNP, the synthetic RNA is translated into a corresponding protein encoded by the synthetic RNA in vivo in one or more different cell types in the subject.
  • LNP lipid nanoparticle
  • the PEGylated lipid is DMG-PEG2000.
  • the cationic lipid is DOTAP.
  • the LNP of the methods described above and herein is comprised of the lipids set forth in Table 7.
  • the target cell types are located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 4 to 6 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 5 to 8 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 7 to 10 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 9 to 12 distinct organs of the subject. According to related embodiments described herein the target cell types are located in between 10 to 15 distinct organs of the subject.
  • the delivery of the polynucleotide is for the diagnosis, prevention and/or treatment of conditions or disease of various tissues and cell types throughout the mammalian body.
  • conditions or diseases include influenza, asthma, diabetes mellitus type 1, diabetes mellitus type 2, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer’s disease, Parkinson’s disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn’s disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease,
  • COPD chronic obstructive pulmonary disease
  • - 62 - hemophilia leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, stomach cancer, brain cancer, endometrial cancer, bone cancer, sarcoma, multiple myeloma, skin cancer, basal cell carcinoma, squamous cell carcinoma, tuberculosis, pneumonia, bronchitis, sinusitis, otitis media, urinary tract infection (UTI), hepatitis A, hepatitis B, hepatitis C, HIV/AIDS, syphilis, gonorrhea, chlamydia, herpes simplex virus (HS V), human papillomavirus (HPV), scabies, athlete’s foot, ringworm, lice infestation, measles, mumps, rubella, chickenpox
  • NASH - 63 - alcoholic steatohepatitis
  • PBC primary biliary cholangitis
  • PSC primary sclerosing cholangitis
  • chronic hepatitis B-related fibrosis chronic hepatitis C-related fibrosis
  • systemic sclerosis systemic sclerosis (scleroderma)
  • cystic fibrosis cardiac fibrosis
  • hypertrophic cardiomyopathy restrictive cardiomyopathy
  • dilated cardiomyopathy chronic kidney disease (CKD)
  • CKD chronic kidney disease
  • CKD chronic kidney disease
  • glomerulonephritis diabetic nephropathy, interstitial nephritis, retroperitoneal fibrosis, peritoneal fibrosis
  • Dupuytren’s contracture Peyronie’s disease, myelofibrosis, bone marrow fibrosis, keloid formation, scar tissue formation, adhesive capsulitis (frozen shoulder), chronic pan
  • the cell types may include, for example, stem cells (e.g., hematopoietic stem cells, progenitor cells), differentiated cells, terminally-differentiated cell, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells, cancer cells, and/or bone cells in a subject.
  • stem cells e.g., hematopoietic stem cells, progenitor cells
  • differentiated cells e.g., differentiated cells, terminally-differentiated cell, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells, cancer cells, and/or bone cells in a subject.
  • the method of delivering the polynucleotide is for the modulation of a condition or disease of various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • the method of delivering the polynucleotide is for increasing or initiating expression of a protein (e.g., a therapeutic or supplemental protein) in a target cell in various tissues and cell types throughout the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells in a subject.
  • a protein e.g., a therapeutic or supplemental protein
  • the presently described LNP composition is administered at a time before the subject has or is suspected of having the condition or disease.
  • the presently contemplated LNPs are capable of targeting the cell type and tissue where the condition or disease manifests.
  • the presently described LNPs are shown to provide for the ability to transfect cells with the encapsulated polynucleotide throughout the whole body of the subject, including cell types that exist throughout the body.
  • the use of targeting ligands or moieties provides for more specific targeted cell type and tissue transfection, which is often desired based on the objective of diagnosis, prevention and/or treatment and the specific type or types of condition or disease
  • the polynucleotide encapsulated in the LNP will vary based on the condition or disease and the objective of the delivery (diagnosis, prevention or treatment), the presently described LNPs are provided as an enabling delivery vehicle for such polynucleotide such that it can be delivered to areas that have been difficult or not possible to deliver a polynucleotide in vivo in a subject (e.g., mammal) previously.
  • an LNP pharmaceutical composition contemplated herein is administered in a dose of about 0.001 mg/kg per the subject’s body weight to about 2.0 mg/kg per the subject’s body weight to a subject in need thereof.
  • a Targeting LNP is administered to a subject in need thereof in a dose of about 0.01 mg/kg; in some embodiments in a dose of about 0.025 mg/kg; in some embodiments in a dose of about 0.05 mg/kg; in some embodiments in a dose of about 0.075 mg/kg; in some embodiments in a dose of about 0.1 mg/kg; in some embodiments in a dose of about 0.125 mg/kg; in some embodiments in a dose of about 0.150 mg/kg; in some embodiments in a dose of about 0.175 mg/kg; in some embodiments in a dose of about 0.2 mg/kg; in some embodiments in a dose of about 0.5 mg/kg; in some embodiments in a dose of about 0.75 mg/kg; in some embodiments in a dose of about 1.0 mg/kg; in some embodiments, in a dose of about 1.25 mg/kg; in some embodiment in a dose of about 1.5 mg/kg; or in some embodiment in a dose of about 2.0 mg/
  • LNP pharmaceutical compositions contemplated herein are administered to a subject in need thereof in a single dose.
  • the LNP is administered to a subject in need thereof two, three, four, or five or more times.
  • the LNP is administered twice a week, every week, every two weeks, every four weeks, every six weeks, every twelve weeks, or every fifteen weeks.
  • the LNP is administered every month, every two months, every three months, every six months, once a year, on an ongoing basis, or as determined by their physician.
  • the contemplated LNP pharmaceutical compositions are delivered in an aerosolized inhaled form, orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly.
  • targeting LNPs are administered intravenously.
  • an exemplary LNP pharmaceutical composition includes an excipient, or carrier, e.g., an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as those required to approximate physiological conditions such as pH and buffering agents, toxicity countering agents, e.g., sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride, and sodium lactate.
  • the pharmaceutical composition comprises 10 mM sodium citrate buffered to pH 6.4.
  • the composition may contain a cryoprotectant, e.g., glycerol, ethylene glycol, sucrose, propylene glycol, or dimethylsulfoxide (DMSO).
  • a cryoprotectant e.g., glycerol, ethylene glycol, sucrose, propylene glycol, or dimethylsulfoxide (DMSO).
  • concentration of active agent in these formulations can vary and are selected based on fluid volumes, viscosities, and body weight in accordance with the particular mode of administration selected and the patient’s needs e.g., Remington’s Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).
  • the compounds or compositions are administered without isolating the cell or cells, the tissue, or the organ from the subject (i.e., the administration is in vivo).
  • the compound or composition is delivered to all, or almost all, cells in the subject’s body.
  • the compound or composition is delivered to a specific cell, cell type, tissue, or organ in the subject’s body.
  • telomerase repeat amplification protocol (TRAP) assay.
  • TRAP telomerase repeat amplification protocol
  • Commercial versions of the TRAP assay are available, for example the Trapeze® telomerase detection kit (Millipore), which provides a sensitive detection and quantitation of telomerase activity, although other measurement techniques are also possible.
  • telomerase reverse transcriptase gene persists in an episomal DNA moiety, or is inserted into the genomic sequence of the cell or otherwise permanently modifies the genetic make-up of the targeted cell and results in constitutive activity of the nucleic acid sequence.
  • the transient expression is independent of cell cycle.
  • Diagnostic and/or therapeutic kits comprising a pharmaceutical composition of an LNP contemplated herein, or lipid components thereof provided in separate containers, and instructions for making and /or using are also contemplated herein.
  • the diagnostic and/or therapeutic kit comprises devices for administration, including but not limited to syringes, microneedles, inhalers, nebulizers, and vials or containers.
  • kits for use in diagnosis, prevention and/or treatment of conditions or diseases of various tissues and cell types throughout the mammalian body including stem cells, progenitor cells, germ cells, differentiated cells, or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, hepatocytes, kidney cells and/or bone cells
  • the kit may include means for transcutaneous delivery, means for intracutaneous injection via microneedle array, means for skin barrier-permeabilizing step such as microneedles and/or one or more permeabilizing agent such as a mixture containing proteases, lipases, or others known in the field.
  • components of an exemplary diagnostic and/or therapeutic kit are provided such that they can be mixed with commercially available microfluidic or vortex mixers. Often such kits are provided with or without nucleic acid (e.g., mRNA) that is to be encapsulated in the LNPs made with the components of the diagnostic and/or therapeutic kit.
  • nucleic acid e.g., mRNA
  • kits for diagnosis, prevention and/or treatment of a condition or disease, e.g., for example for use in extending telomeres in a mammalian cell.
  • the kits comprise any of the above-described compounds or compositions, together with instructions for their use.
  • Such instructions include the packaging label approved by a regulatory agency in the country where the kit is sold, which instructions guide dosing schedules and include contraindications.
  • steps for combining agents in the kit are required, such instructions are included in the instructions for their use, including steps set forth in certain examples for preparing a final product herein.
  • kits further comprise packaging materials.
  • packaging materials are air tight.
  • the packaging materials may optionally be filled with an inert gas, such as, for example, nitrogen, argon, or the like.
  • the packaging materials comprise a metal foil container, such as, for example, a sealed aluminum pouch or the like. Such packaging materials are well known by those of ordinary skill in the art.
  • the kit may also comprise a delivery vehicle, such as a lipid as
  • one or more components of the formulation are provided frozen with a cryoprotectant, or lyophilized.
  • the kit may further comprise a desiccant, a culture medium, an RNase inhibitor, or other such components. In some embodiments, the kit may further comprise a combination of more than one of these additional components. In some kit embodiments, the composition of the kit is sterile.
  • FIGS. 1 and 48 depict exemplary pharmacokinetics of the 5 lipid LNPs of Table 1.
  • Timeline of plasma, lung, and liver levels of the 5 lipid LNPs as determined by measuring one of the lipids, DOTAP, are presented.
  • LNPs were formulated with TERT mRNA (Human TERT opti - SEQ ID NO: 1) and the lipids in the ratios of Table 1.
  • Male C57B1/6 mice were dosed at Img/kg and plasma and tissues were collected at the time points indicated above.
  • DOTAP was measured in the plasma, lung, and liver using LC-MS/MS.
  • the nitrogemphosphorus N/P charge ratio of the composition of Table 1 is 12.
  • FIG. 2 depicts the ratio of bioluminescence signal from the indicated organs, produced from luciferase protein translated from mRNA injected intravenously, at the indicated time points post intravenous injection of the 5 lipid LNP of Table 2.
  • LNPs were formulated with firefly luciferase mRNA and the lipids in the ratios of Table 2.
  • Male C57B16 mice were dosed at 1.3 mg/kg and tissues were collected and imaged ex vivo at the time points indicated above.
  • Mean radiance values were baseline normalized by subtracting the mean radiance from the negative control (no LNP) treated animal.
  • the relative radiance between lung, liver, and spleen are plotted in FIG. 2.
  • the tissues were also homogenized and the bioluminescence was measured from equal amounts by weight of homogenate.
  • the nitrogemphosphorus N/P charge ratio of the composition of Table 2 is 12.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Nanotechnology (AREA)
  • Dermatology (AREA)
  • Biophysics (AREA)
  • Optics & Photonics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des compositions de nanoparticules lipidiques (NPL), et des polynucléotides diagnostiques ou thérapeutiques, par exemple, de l'ARNm de TERT, qui peuvent être administrés avec les compositions de NPL dans des formulations au niveau de divers tissus et types de cellules dans tout le corps d'un mammifère, y compris des cellules souches, des cellules progénitrices, des cellules germinales, des cellules différenciées, ou des cellules à différenciation terminale, des cellules cancéreuses, des cellules endothéliales, des cellules épithéliales, des cellules spléniques, des hépatocytes, des cellules rénales et/ou des cellules osseuses, par exemple, pour le diagnostic, la prévention et/ou le traitement d'affections ou d'une maladie.<i />
PCT/US2024/044269 2023-08-29 2024-08-28 Compositions et méthodes pour l'administration large d'arn dans un tissu Pending WO2025049632A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363579345P 2023-08-29 2023-08-29
US63/579,345 2023-08-29

Publications (1)

Publication Number Publication Date
WO2025049632A1 true WO2025049632A1 (fr) 2025-03-06

Family

ID=94820316

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/044269 Pending WO2025049632A1 (fr) 2023-08-29 2024-08-28 Compositions et méthodes pour l'administration large d'arn dans un tissu

Country Status (1)

Country Link
WO (1) WO2025049632A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210059953A1 (en) * 2017-09-08 2021-03-04 Generation Bio Co. Lipid nanoparticle formulations of non-viral, capsid-free dna vectors
WO2022147039A1 (fr) * 2020-12-29 2022-07-07 Rejuvenation Technologies Inc. Compositions et méthodes pour l'administration d'arn
WO2022232540A1 (fr) * 2021-04-30 2022-11-03 The Trustees Of The University Of Pennsylvania Nanoparticules lipidiques ciblées de cd-90
US20220347112A1 (en) * 2021-03-31 2022-11-03 Rejuvenation Technologies Inc. Compositions and methods for delivery of rna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210059953A1 (en) * 2017-09-08 2021-03-04 Generation Bio Co. Lipid nanoparticle formulations of non-viral, capsid-free dna vectors
WO2022147039A1 (fr) * 2020-12-29 2022-07-07 Rejuvenation Technologies Inc. Compositions et méthodes pour l'administration d'arn
US20220347112A1 (en) * 2021-03-31 2022-11-03 Rejuvenation Technologies Inc. Compositions and methods for delivery of rna
WO2022232540A1 (fr) * 2021-04-30 2022-11-03 The Trustees Of The University Of Pennsylvania Nanoparticules lipidiques ciblées de cd-90

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAO KEWA, LI JIE, SONG HENGYUE, HAN HESONG, WANG YONGHENG, YIN BOYAN, FARMER DIANA L., MURTHY NIREN, WANG AIJUN: "In utero delivery of mRNA to the heart, diaphragm and muscle with lipid nanoparticles", BIOACTIVE MATERIALS, vol. 25, 1 July 2023 (2023-07-01), pages 387 - 398, XP093290477, ISSN: 2452-199X, DOI: 10.1016/j.bioactmat.2023.02.011 *

Similar Documents

Publication Publication Date Title
US20230165973A1 (en) Compositions and methods for delivering messenger rna
CN116322788A (zh) 环状rna组合物和方法
JP2022173314A (ja) メッセンジャーｒｎａの皮下送達
WO2020061457A1 (fr) Préparation de nanoparticules lipidiques et leurs méthodes d&#39;administration
KR20220078557A (ko) 개선된 mrna 로딩된 지질 나노입자 및 이의 제조 방법
KR20230078723A (ko) 향상된 약동학을 나타내는 조작된 세포외 소포
CN120129532A (zh) 稳定的脂质或类脂质纳米颗粒悬浮液
WO2024023504A1 (fr) Vésicule extracellulaire chargée
JP2024501388A (ja) Ｒｎａの送達のための組成物および方法
EP3866825A1 (fr) Encapsulation sans pompe d&#39;arn messager
WO2025049632A1 (fr) Compositions et méthodes pour l&#39;administration large d&#39;arn dans un tissu
CN117396193A (zh) 用于递送rna的组合物和方法
AU2023334154A1 (en) Compositions and methods for delivery of rna
US20220287966A1 (en) Stable Liquid Lipid Nanoparticle Formulations
EP4633673A1 (fr) Arnm codant pour une particule de type virus de la grippe
JP2025517105A (ja) 改変された細胞外小胞のための新規の足場タンパク質
WO2024200823A1 (fr) Nanoparticule à base de lipide ciblant des cellules immunitaires activées pour l&#39;expression d&#39;une molécule d&#39;amélioration de cellule immunitaire et son utilisation
WO2024112882A1 (fr) Administration ciblée de constructions d&#39;édition génique et leurs méthodes d&#39;utilisation
HK40023090A (en) Compositions and methods for delivering messenger rna
HK1219719B (en) Compositions and methods for delivering messenger rna

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24860968

Country of ref document: EP

Kind code of ref document: A1