Thermally stable composition containing MRNA lipid nanoparticles

Abstract

This application relates to a composition comprising lipid nanoparticles (LNPs) that encapsulate ribonucleic acid (RNA) molecules such as messenger RNA molecules, and stabilized with a heat-stable formulation comprising one or more thermoreversible gelling agents, one or more heat-stabilizing excipients, and / or buffers, pharmaceutically acceptable salts, disaccharides, surfactants, and chelating agents. Methods for preparing and using the stabilized composition, as well as methods for stabilizing a composition containing RNA-encapsulated LNPs, are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to European Patent Application No. 23305868.4, filed on 1 June 2023, and European Patent Application No. 23305942.7, filed on 14 June 2023, the entire contents of which are incorporated herein by reference.

[0002] This application relates to a composition comprising lipid nanoparticles (LNPs) encapsulating ribonucleic acid (RNA) molecules, a messenger RNA molecule, and a thermostable formulation comprising one or more thermoreversible gelling agents, one or more thermostabilizing excipients, and / or buffers, pharmaceutically acceptable salts, disaccharides, surfactants, and chelating agents. Methods for preparing and using the stabilized composition, as well as methods for stabilizing a composition containing RNA-encapsulated LNPs, are also provided. [Background technology]

[0003] The use of ribonucleic acid (RNA) molecules, such as messenger RNA (mRNA), as pharmaceuticals is attracting considerable interest in various applications, including therapeutic drugs, vaccines, and diagnostic agents. However, the effective intracellular delivery of formulations containing RNA molecules (e.g., mRNA) remains challenging due to the inherent instability of RNA, its potential to activate immune responses, and / or its susceptibility to degradation by nucleases. Any of these challenges can lead to a loss of translational efficacy of such RNA molecules (e.g., mRNA), thus hindering their effectiveness as pharmaceuticals.

[0004] Various delivery systems, particularly non-viral delivery systems, have been developed to overcome many challenges associated with the in vivo delivery of RNA molecules (e.g., mRNA). Among these delivery systems are lipid nanoparticle (LNP) delivery systems, which have attracted particular attention in recent years because various LNP formulations have proven promising for various pharmaceutical applications. See, for example, Kowalski et al., Molecular Therapy, 2019, 27(4):710-728; Gomez-Aguado et al., Nanomaterials (Basel), 2020, 10(2):364; and Wadhwa et al., Pharmaceutics, 2020, 12(2):102.

[0005] The rapid approval and remarkable success of the COVID-19 vaccines Comirnaty® (BNT162b2) and Spikevax (mRNA-1273) further demonstrated the clinical validity of LNP-formulated mRNA as a new class of highly effective nucleic acids in the vaccine field. However, both vaccines require ultra-low temperatures, sub-zero temperatures, for long-term storage, which is not patient- or pharmacy-friendly and not ideal for widespread use. Therefore, there is still a need for LNP formulations containing RNA molecules (e.g., mRNA) that can be stored for long periods at favorable temperatures, such as 4°C, without significant loss of RNA stability, thereby facilitating the transport and storage of RNA-LNP formulations and extending their shelf life. [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides for compositions and methods for stabilizing therapeutic drugs, which include ribonucleic acid (RNA) molecules, such as mRNA, encapsulated in lipid nanoparticles (LNPs), using a heat-stable formulation comprising one or more thermoreversible gelling agents (e.g., polypeptide-based or protein-based polymers, e.g., gelatin), one or more heat-stabilizing excipients (e.g., lipoic acid, L-theanine, vanillin, or a combination thereof), and / or a buffer, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant, and a chelating agent. In some embodiments, this disclosure encompasses the observation that formulation stability is substantially improved by a mixture of at least one thermoreversible gelling agent (polypeptide-based or protein-based polymer, e.g., gelatin) and / or at least one heat-stabilizing excipient (lipoic acid, L-theanine, vanillin, or a combination thereof) and RNA molecules, such as mRNA, encapsulated in LNPs, allowing the resulting formulations to be stored for relatively long periods at favorable temperatures, such as 2-8°C. This disclosure further encompasses the observation that, in other embodiments, formulation stability is substantially improved by formulating RNA molecules, such as mRNA encapsulated in LNPs, in formulations comprising buffers, pharmaceutically acceptable salts, one or more disaccharides, surfactants, and chelating agents, each present in prescribed amounts, thereby enabling the resulting formulations to be stored for relatively long periods at favorable temperatures, such as 2–8°C. Accordingly, in one embodiment, a composition is provided herein comprising one or more RNA molecules encapsulated in LNPs and at least one thermoreversible gelling agent, such as a thermoreversible gelling agent having an upper critical solution temperature (UCST) of about 12°C to about 50°C. In some embodiments, the composition has a liquid phase at temperatures above about 12°C and reversibly transitions to a gel state at temperatures of about 1–11°C. At least one thermoreversible gelling agent may be present in the composition in an amount of about 0.1% to about 30% by weight in some embodiments, about 0.25% to about 5% by weight in other embodiments, or about 0.5% to about 1.5% by weight in some further embodiments.At least one thermoreversible gelling agent may include a thermoreversible gelling polymer (e.g., polypeptide-based or protein-based polymer), such as gelatin, poly(N-acryloyl asparagamide), poly(ethylene glycol)-b-poly(N-acryloylglycinamide-co-acrylonitrile) (PEG-bP(NAGA-co-AN), poly(N-acryloylglycinamide-co-N-phenylacrylamide) (P(NAGA-co-NPhAm)), poly(N-(2-hydroxypropyl)methacrylamide-glycolamide) (P(HPMA-GA)), poly(acrylamide-co-acrylonitrile)-b-poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(acrylic acid-co-acrylonitrile) (P(AA-co- These include AN), imidazole-based poly(N-vinylimidazole-co-1-vinyl-2-(hydroxymethyl)imidazole), poly(sulfobetaine-co-sulfabetine)(P(SB-co-ZB), poly[2-(methacryloyloxy)ethylphosphocholine]-b-poly(2-ureidoethyl methacrylate)(PMPC20-b-PUEM165), or combinations thereof. In other embodiments, at least one thermoreversible gelling agent comprises a thermoreversible gelling polypeptide such as multi-L-arginyl-poly-L-aspartate (iMAPA)-PEG. In some further embodiments, at least one thermoreversible gelling agent comprises a thermoreversible gelling protein. In some embodiments, at least one thermoreversible gelling agent comprises gelatin and may be present in the composition in an amount of about 1% by weight.

[0007] In some embodiments, the composition is stable after storage at a temperature of about 4°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months, compared to a control composition that does not contain at least one thermoreversible gelling agent, and the stability of the composition is measured by the change in the average particle size of the LNPs, the encapsulation efficiency of the LNPs, and / or the integrity of one or more RNA molecules encapsulated in the LNPs. In some embodiments, the average particle size of the LNPs does not increase by more than about 40% after storage of the composition at a temperature of about 4°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months, compared to a control composition that does not contain at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of LNPs does not decrease by more than about 10% after storage of the composition at a temperature of about 4°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months, compared with a control composition that does not contain at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of LNPs is higher than that of a control composition that does not contain at least one thermoreversible gelling agent. In some embodiments, the integrity of one or more RNA molecules encapsulated in the LNPs does not decrease by more than about 10% after storage of the composition at a temperature of about 4°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months, compared with a control composition that does not contain at least one thermoreversible gelling agent.

[0008] In another embodiment, a liquid composition is provided herein comprising one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs) and at least one heat-stabilizing excipient, wherein the at least one heat-stabilizing excipient includes lipoic acid, L-theanine, vanillin, or a combination thereof. In some embodiments, the at least one heat-stabilizing excipient is present at a concentration of about 0.1 mM to about 20 mM, about 0.5 mM to about 15 mM, or about 1 mM to about 10 mM. In some embodiments, the at least one heat-stabilizing excipient is present at a concentration of about 5 mM, about 10 mM, or about 15 mM. In some embodiments, the at least one heat-stabilizing excipient and one or more RNA molecules are present in a weight ratio of about 5:1 to about 50:1. In some embodiments, at least one heat-stabilizing excipient contains or is lipoic acid, and optionally lipoic acid and one or more RNA molecules are present in a weight ratio of about 2.5:1 to about 15.5:1. In some embodiments, at least one heat-stabilizing excipient contains or is L-theanine, and optionally L-theanine and one or more RNA molecules are present in a weight ratio of about 10:1 to about 30:1. In some embodiments, at least one heat-stabilizing excipient contains or is vanillin, and optionally vanillin and one or more RNA molecules are present in a weight ratio of about 12.5:1 to about 50:1.

[0009] In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 20% after storage of the liquid composition at 37°C for at least 7 days, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 25% after storage of the liquid composition at 4°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 30% after storage of the liquid composition at 4°C for up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, or up to about 8 months, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 45% after storage of the liquid composition at a temperature of 4°C for up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, or up to about 12 months, compared to a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 50% after storage of the liquid composition at a temperature of 25°C for up to about 1 week, up to about 2 weeks, up to about 3 weeks, or up to about 4 weeks, compared to a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the average particle size of LNPs does not increase by more than 40% after storage of the liquid composition at a temperature of 4°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months, or a maximum of about 12 months. In some embodiments, the average particle size of LNPs does not increase by more than 20% after storage of the liquid composition at a temperature of 25°C for a maximum of about 1 week, a maximum of about 2 weeks, a maximum of about 3 weeks, a maximum of about 4 weeks, a maximum of about 5 weeks, a maximum of about 6 weeks, or a maximum of about 7 weeks.In some embodiments, the LNP encapsulation efficiency does not decrease by more than 20% after storage of the liquid composition at a temperature of 4°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months, or a maximum of about 12 months. In some embodiments, the LNP encapsulation efficiency does not decrease by more than 20% after storage of the liquid composition at a temperature of 25°C for a maximum of about 1 week, a maximum of about 2 weeks, a maximum of about 3 weeks, a maximum of about 4 weeks, a maximum of about 5 weeks, a maximum of about 6 weeks, or a maximum of about 7 weeks.

[0010] In yet another embodiment, compositions are provided herein comprising one or more RNA molecules encapsulated in LNPs, at least one thermoreversible gelling agent, and at least one heat-stabilizing excipient, wherein the at least one heat-stabilizing excipient contains or is lipoic acid. In some embodiments, the compositions are stable after storage at a temperature of about 2–8°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months, compared to a control composition that does not contain at least one thermoreversible gelling agent and at least one heat-stabilizing excipient, and the stability of the compositions is measured by the change in the average particle size of the LNPs, the encapsulation efficiency of the LNPs, and / or the integrity of the one or more RNA molecules encapsulated in the LNPs. In some embodiments, the at least one thermoreversible gelling agent contains or is gelatin. In some embodiments, the thermoreversible gelling agent contains or is gelatin in an amount of about 0.5% to about 1.5% by weight. In some embodiments, the gelatin is present in an amount of about 1% by weight. In some embodiments, lipoic acid is present at a concentration of about 1 mM to about 10 mM. In some embodiments, lipoic acid is present at a concentration of about 1 mM to about 5 mM. In some embodiments, lipoic acid and one or more RNA molecules are present in a weight ratio of about 2.5:1 to about 15.5:1.

[0011] In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid further comprises a buffer, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant and / or a chelating agent. In some embodiments, the buffer comprises or is tris(hydroxymethyl)aminomethane (tris). In some embodiments, the pharmaceutically acceptable salt comprises or is sodium chloride (NaCl). In some embodiments, one or more disaccharides comprises or is sucrose. In some embodiments, the surfactant comprises or is poloxamer 188 (P188). In some embodiments, the chelating agent comprises or is ethylenediaminetetraacetic acid (EDTA).

[0012] In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 10 mM to about 60 mM Tris, about 40 mM to about 150 mM NaCl, about 1% to about 10% by weight sucrose, about 0.2% to about 0.6% by volume P188, and about 5 μM to about 15 μM EDTA, wherein the composition has a pH of about 7.2 to about 7.8. In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 50 mM Tris, about 150 mM NaCl, about 5% by weight sucrose, about 0.4% by volume P188, and about 10 μM EDTA, and the composition has a pH of about 7.5 ± 0.3. In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 10 mM to about 60 mM tris, about 40 mM to about 110 mM NaCl, about 3% to about 6% by weight sucrose, about 0.2% to about 4% by weight trehalose, about 0.2% to about 0.6% by volume P188, and about 5 μM to about 15 μM EDTA, wherein the composition has a pH of about 7.5 to about 7.7. In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 50 mM Tris, about 50 mM NaCl, about 5% by weight sucrose, about 2-2.6% by weight trehalose, about 0.4% by volume P188, and about 10 μM EDTA, and the composition has a pH of about 7.7.In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 20 mM to about 50 mM Tris, about 50 mM to about 100 mM NaCl, about 2% to about 5% by weight sucrose, about 0.3% to about 3% by weight trehalose, about 0.2% to about 0.4% by volume P188, and about 10 μM to about 15 μM EDTA, wherein the composition has a pH of about 7.7. In some embodiments, a composition comprising at least one thermoreversible gelling agent, a liquid composition comprising at least one heat-stabilizing excipient, or a composition comprising at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 20 mM Tris, about 100 mM NaCl, about 5% by weight sucrose, about 0.4–1.3% by weight trehalose, about 0.4% by volume P188, and about 10 μM EDTA, wherein the composition has a pH of about 7.7.

[0013] In yet another embodiment, a liquid formulation is provided herein comprising one or more ribonucleic acids (RNAs) encapsulated in lipid nanoparticles (LNPs), about 10 mM to about 60 mM tris(hydroxymethyl)aminomethane (Tris), about 40 mM to about 150 mM sodium chloride (NaCl), about 1% to about 10% by weight sucrose, about 0.2% to about 0.6% by volume poloxamer 188 (P188), and about 5 μM to about 15 μM ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of about 7.2 to about 7.8. In some embodiments, the liquid formulation comprises about 50 mM Tris, about 150 mM NaCl, about 5% by weight sucrose, about 0.4% by volume P188, and about 10 μM EDTA, wherein the liquid formulation has a pH of 7.5 ± 0.3. Liquid formulations comprising one or more ribonucleic acids (RNA) encapsulated in lipid nanoparticles (LNPs), approximately 10 mM to approximately 60 mM tris(hydroxymethyl)aminomethane (Tris), approximately 40 mM to approximately 110 mM sodium chloride (NaCl), approximately 3% to approximately 6% by weight sucrose, approximately 0.2% to approximately 4% by weight trehalose, approximately 0.2% to approximately 0.6% by volume poloxamer 188 (P188), and approximately 5 μM to approximately 15 μM ethylenediaminetetraacetic acid (EDTA) are also provided herein, the liquid formulations having a pH of approximately 7.5 to approximately 7.7. In some embodiments, the liquid formulation contains about 50 mM Tris, about 50 mM NaCl, about 5% by weight sucrose, about 2-2.6% by weight trehalose, about 0.4% by volume P188, and about 10 μM EDTA, and the liquid formulation has a pH of about 7.7. Liquid formulations comprising one or more ribonucleic acids (RNA) encapsulated in lipid nanoparticles (LNPs), approximately 20 mM to approximately 50 mM tris(hydroxymethyl)aminomethane (Tris), approximately 50 mM to approximately 100 mM sodium chloride (NaCl), approximately 2% to approximately 5% by weight sucrose, approximately 0.3% to approximately 3% by weight trehalose, approximately 0.2% to approximately 0.4% by volume poloxamer 188 (P188), and approximately 10 μM to approximately 15 μM ethylenediaminetetraacetic acid (EDTA) are further provided herein, the liquid formulations having a pH of approximately 7.7.In some embodiments, the liquid formulation contains about 20 mM Tris, about 100 mM NaCl, about 5% by weight sucrose, about 0.4–1.3% by weight trehalose, about 0.4% by volume P188, and about 10 μM EDTA, and the liquid formulation has a pH of about 7.7.

[0014] In some embodiments, the one or more RNA molecules encapsulated in the LNP encode one or more viral proteins, such as influenza virus protein, respiratory syncytial virus (RSV) protein, coronavirus protein, or a combination thereof. In some embodiments, the one or more RNA molecules encapsulated in the LNP are messenger RNA (mRNA) molecules. The one or more RNA molecules encapsulated in the LNP may also, in some embodiments, include at least one chemically modified nucleotide, which may include pseudouridine (e.g., N1-methylpseudridine), 2'-fluororibonucleotide, or 2'-methoxyribonucleotide, and / or in other embodiments, a phosphorothioate bond. In some embodiments, each of the one or more RNA molecules encapsulated in the LNP is present in an amount ranging from about 0.1 μg to about 150 μg, for example, about 1 μg to about 60 μg or about 5 μg to about 45 μg.

[0015] In some embodiments, the LNPs contained in the compositions of the present disclosure include cationic lipids (e.g., cKK-E10), polyethylene glycol conjugate (PEGylated) lipids (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxy(DMG)-PEG2000), cholesterol-based lipids (e.g., cholesterol), and helper lipids (e.g., dioleoyl-SN-glycero-3-phosphoethanolamine). Cationic lipids (e.g., cKK-E10) can be present in a molar ratio of approximately 30% to 50% (e.g., approximately 40%), PEGylated lipids (e.g., 1,2-dimiristoyl-rac-glycero-3-methoxy(DMG)-PEG2000) can be present in a molar ratio of approximately 0.25% to 15% (e.g., approximately 1.5% to 5%), cholesterol-based lipids (e.g., cholesterol) can be present in a molar ratio of approximately 20% to 40% (e.g., approximately 25% or approximately 28.5%), and helper lipids (e.g., dioleoyl-SN-glycero-3-phosphoethanolamine) can be present in a molar ratio of approximately 20% to 40% (e.g., approximately 30%). In some embodiments, the cationic lipids include OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; the PEGylated lipids include 1,2-dimiristoyl-rac-glycero-3-methoxy(DMG)-PEG2000; the cholesterol-based lipids include cholesterol; and / or the helper lipids include dioleoyl-SN-glycero-3-phosphoethanolamine.Accordingly, in some embodiments, the LNPs contained in the compositions of the present disclosure include cationic lipids (e.g., OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14), PEGylated lipids (e.g., 1,2-dimiristoyl-rac-glycero-3-methoxy(DMG)-PEG2000), cholesterol-based lipids (e.g., cholesterol), and helper lipids (e.g., dioleoyl-SN-glycero-3-phosphoethanolamine) in molar ratios of about 40:1.5:28.5:30 or about 40:5:25:30. In other embodiments, the LNP contained in the composition of the present disclosure includes ALC-0315 as a cationic lipid, N,N-ditetradecylacetamido-polyethylene glycol as a PEGylated lipid, and distearoylphosphatidylcholine (DSPC) and cholesterol as helper lipids.

[0016] In some embodiments, the compositions and formulations of the Disclosure are formulated for sublingual, intramuscular, intradermal, subcutaneous, intravenous, intranasal, inhalation, or intraperitoneal administration. In some embodiments, the compositions of the Disclosure are immunogenic compositions.

[0017] In some embodiments, the compositions and formulations of the Disclosure are immunogenic compositions. Accordingly, in other embodiments, vaccines comprising the immunogenic compositions of the Disclosure and a pharmaceutically acceptable carrier, as well as methods of using them, such as methods for immunizing a subject or for reducing one or more symptoms of a viral infection in a subject, are provided herein. In some embodiments, methods of immunizing a subject with the vaccine of the Disclosure prevent viral infection in the subject, reduce the likelihood of the subject contracting a viral infection, or reduce the likelihood of the subject contracting a serious disease of viral infection origin. In other embodiments, methods of immunizing a subject with the vaccine of the Disclosure enhance the protective immune response in the subject. In some embodiments, the subject is a human, for example, a human being 6 months of age or older but under 18 years of age, at least 6 months of age and under 18 years of age, at least 18 years of age and under 65 years of age, at least 6 months of age and under 5 years of age, at least 5 years of age and under 65 years of age, at least 60 years of age, or at least 65 years of age. In some embodiments, the vaccines of the present disclosure may be administered to a subject intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally. In some embodiments, the vaccines of the present disclosure include one or more LNP-encapsulated RNA molecules encoding one or more viral proteins, such as influenza virus protein, respiratory syncytial virus protein, coronavirus protein, or a combination thereof.

[0018] In a further embodiment, a method for stabilizing a composition containing one or more RNA molecules encapsulated in LNPs is provided herein, the method comprising adding a sufficient amount of at least one thermoreversible gelling agent to the composition, maintaining the composition in the liquid phase at temperatures above about 12°C, and reversibly transitioning the composition to a gel state at temperatures of about 1 to 11°C (e.g., 2 to 8°C or 4°C). A method for preventing the degradation of one or more RNA molecules encapsulated in LNPs in a liquid composition is also provided herein, the method comprising adding a sufficient amount of at least one thermoreversible gelling agent to the liquid composition, maintaining the liquid composition in the liquid phase at temperatures above about 12°C, and reversibly transitioning the composition to a gel state at temperatures of about 1 to 11°C (e.g., 2 to 8°C or 4°C). In some embodiments, at least one thermoreversible gelling agent is present in an amount of about 0.1% to about 30% by weight, about 0.25% to about 5% by weight, or about 0.5% to about 1.5% by weight. In some embodiments, at least one thermoreversible gelling agent comprises gelatin in an amount of about 1% by weight. In some embodiments, one or more RNA molecules encode one or more viral proteins, such as influenza virus protein, respiratory syncytial virus protein, coronavirus protein, or a combination thereof. In some embodiments, the LNP comprises cationic lipids, polyethylene glycol (PEG) conjugated (PEGylated) lipids, cholesterol lipids, and helper lipids.

[0019] In yet another embodiment, a method is provided herein for preventing the thermal decomposition of one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs), the method comprising formulating a liquid composition containing LNPs and one or more RNA molecules in the presence of at least one thermal stabilizing excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof. In some embodiments, the at least one thermal stabilizing excipient is present at a concentration of about 0.1 mM to about 20 mM, about 0.5 mM to about 15 mM, or about 1 mM to about 10 mM. In some embodiments, the at least one thermal stabilizing excipient is present at a concentration of about 5 mM, about 10 mM, or about 15 mM. In some embodiments, the at least one thermal stabilizing excipient and one or more RNA molecules are present in a weight ratio of about 5:1 to about 50:1. In some embodiments, at least one heat-stabilizing excipient contains or is lipoic acid, and optionally lipoic acid and one or more RNA molecules are present in a weight ratio of about 2.5:1 to about 15.5:1. In some embodiments, at least one heat-stabilizing excipient contains or is L-theanine, and optionally L-theanine and one or more RNA molecules are present in a weight ratio of about 10:1 to about 30:1. In some embodiments, at least one heat-stabilizing excipient contains or is vanillin, and optionally vanillin and one or more RNA molecules are present in a weight ratio of about 12.5:1 to about 50:1.

[0020] The accompanying drawings incorporated herein and constituting part of this specification illustrate specific embodiments and, together with the written description, help to illustrate the specific principles of the methods and compositions disclosed herein. [Brief explanation of the drawing]

[0021] [Figure 1A-1B] Figure 1A shows a typical phase of a thermoreversible gelling agent that reversibly transitions to a gel (or hydrogel) form at temperatures below its upper critical solution temperature (UCST) and has a liquid phase at temperatures above its upper critical solution temperature (UCST). Figure 1B shows aggregation or fusion of lipid nanoparticles (LNPs) during storage (top) and stabilization of LNPs using a hydrogel (bottom). [Figure 2A-2B] Exemplary thermoreversible gel-forming formulations according to the present disclosure filled in sealed vials (Figure 2A) and pre-filled syringes (Figure 2B) at 4 °C and room temperature (RT) are shown. Figure 2B shows that the thermoreversible gel-forming formulation reversibly transitioned from the gel form at 4 °C to the liquid phase within 15 minutes at room temperature. [Figure 3] Stability of a representative thermoreversible gel-forming formulation containing 1% gelatin according to the present disclosure after storage at 4 °C for 1, 2, 3 or 4 months, measured by the degree of decrease in mRNA integrity (top), change in particle size (center) and change in encapsulation efficiency (EE, bottom) of lipid nanoparticles. In each example, the control formulation contains the same mRNA-LNP formulation without the thermoreversible gelling agent (i.e., gelatin). [Figure 4A-4B] Stability of a representative thermoreversible gel-forming formulation containing 1% gelatin according to the present disclosure after storage at 4 °C for up to 4 months, measured by the increase in degraded mRNA products using capillary electrophoresis (CE) (RFU: relative fluorescence unit). Figure 4A: control at T0; Figure 4B: gelatin-based control at T0; Figure 4C: control after storage at 4 °C for 4 months; Figure 4D: gelatin-based formulation after storage at 4 °C for up to 4 months. [Figure 4C-4D] Same as above. [Figure 5A-5B] Stability of a representative thermoreversible gel-forming formulation containing 1% gelatin according to the present disclosure after storage at 4 °C for up to 9 months, measured by the degree of decrease in mRNA integrity (Figure 5A), change in encapsulation efficiency of lipid nanoparticles (Figure 5B) and change in particle size (Figure 5C). In each example, the control formulation contains the same mRNA-LNP formulation without the thermoreversible gelling agent (i.e., gelatin). [Figure 5C] Same as above. [Figure 6]This paper demonstrates the effect of gelatin on protein expression in mRNA-LNP formulations containing CKK-E10 as a cationic lipid and mRNA encoding human erythropoietin (hEPO). "Gelatin-buffered DP": A representative thermoreversible gel-forming formulation containing 1% gelatin according to this disclosure; "Bulk DP": A control formulation excluding the same mRNA-LNP formulation containing a thermoreversible gelling agent (i.e., gelatin); DP: Pharmaceutical product. The difference in protein expression between the two formulations is not significant (ns). [Figure 7A] This figure shows how the addition of various excipients to mRNA-LNP formulations affects mRNA integrity after storage at 37°C for at least one week. Figure 7A shows mRNA integrity data for all excipients screened after formulation with human erythropoietin (hEPO) encoding mRNA and the cationic lipid cKK-E12 (also known as ML2). The y-axis represents the change in RNA integrity (%mRNA integrity), and the x-axis represents the time in days. [Figure 7B] This paper demonstrates how the addition of various excipients to mRNA-LNP formulations affects mRNA integrity after storage at 37°C for at least one week. Figure 7B shows the tested excipients, which exhibit higher mRNA integrity after 7 days at 37°C compared to the naked mRNA control and the control formulation. The control formulation contains the same mRNA-LNP formulation but without the excipients. The naked mRNA control contains mRNA in water that is free of RNase. [Figures 8A-8C]Figure 8A shows that adding L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to the mRNA-LNP preparation reduced the amount of mRNA degradation after 7 days of storage at 37°C compared to the control preparation. Figure 8B shows that no significant change was observed in the LNP particle size (nm) after storing the liquid at 37°C for 7 days with L-theanine (10 mM) or vanillin (5 mM) added. Figure 8C shows that the mRNA encapsulation efficiency of LNPs remained unchanged for all preparations (L-theanine, lipoic acid, or vanillin) after 7 days of storage at 37°C. In each example, the control preparation contains the same mRNA-LNP preparation, but without the excipients. [Figure 9A-9B] The addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to mRNA-LNP preparations reduced the amount of mRNA degradation compared to the control preparation when stored at 4°C (Figures 9A, 9C, and 9D) and 25°C (Figures 9B, 9D, and 9F). The mRNA-LNP preparations were prepared using modified tetravalent influenza mRNA ("4Flu mRNA") and cationic lipids such as cKK-E10 (Figures 9A-9B), OF-02 (Figures 9C-9D), or GL-HEPES-E3-E12-DS-4-E10 (Figures 9E-9F). In each example, the control preparation contains the same mRNA-LNP preparation but without the excipients. [Figure 9C-9D] Same as above. [Figures 9E-9F] Same as above. [Figure 10A-10B]This study shows how the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to mRNA-LNP preparations affects the LNP particle size (nm) of mRNA-LNP preparations over time after storage at 4°C (Figures 10A, 10C, and 10D) and 25°C (Figures 10B, 10D, and 10F). The mRNA-LNP preparations were prepared using modified tetravalent influenza mRNA ("4Flu mRNA") and cationic lipids such as cKK-E10 (Figures 10A-10B), OF-02 (Figures 10C-10D), or GL-HEPES-E3-E12-DS-4-E10 (Figures 10E-10F). In each example, the control preparation contains the same mRNA-LNP preparation but without the excipients. [Figure 10C-10D] Same as above. [Figures 10E-10F] Same as above. [Figure 11A-11B] This section shows whether the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to mRNA-LNP preparations improved or had no effect compared to a control preparation after storage at 4°C (Figures 11A, 11C, and 11D) and 25°C (Figures 11B, 11D, and 11F). The mRNA-LNP preparations were prepared using modified tetravalent influenza mRNA ("4Flu mRNA") and cationic lipids such as cKK-E10 (Figures 11A-11B), OF-02 (Figures 11C-11D), or GL-HEPES-E3-E12-DS-4-E10 (Figures 11E-11F). In each example, the control preparation contains the same mRNA-LNP preparation but without the excipients. [Figure 11C-11D] Same as above. [Figure 11E-11F] Same as above. [Figures 12A-12B]The liquid stability of mRNA-LNP preparations after storage at 25°C and 30°C, induced by the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM), is shown. The mRNA-LNP preparations were prepared using modified monovalent influenza mRNA encoding influenza hemagglutinin from the Tasmanian strain and cationic lipids such as cKK-E10 (Figures 12A, 12C, and 12E), OF-02, or GL-HEPES-E3-E12-DS-4-E10 (Figures 12B, 12D, and 12F). Figures 12A and 12B show the decrease in mRNA integrity over 7 weeks. Figures 12C and 12D show the LNP particle size over 7 weeks. Figures 12E and 12F show the encapsulation efficiency over 7 weeks. In each example, the control preparation contains the same mRNA-LNP preparation but without the excipients. [Figures 12C-12D] Same as above. [Figures 12E-12F] Same as above. [Figure 13] The addition of L-theanine (10 mM), lipoic acid (5 mM), and vanillin (10 mM) as excipients to mRNA-LNP preparations containing OF-02, cKK-E10, or GL-HEPES-E3-E12-DS-4-E10 as cationic lipids did not affect mRNA delivery and protein production (hEPO protein (ng / mL)) in mice compared to the control preparation (n=4). In each example, the control preparation contained the same mRNA-LNP preparation but without the excipients. [Figure 14] The addition of L-theanine (10 mM), lipoic acid (5 mM), and vanillin (10 mM) as excipients to mRNA-LNP preparations containing influenza hemagglutinin-encoding mRNA and cationic lipids OF-02, cKK-E10, or GL-HEPES-E3-E12-DS-4-E10 did not reduce the HAI titer produced in mice (n=8 per group) compared to the control preparation. In each example, the control preparation contained the same mRNA-LNP preparation but without the excipients. Each point on the graph represents the titer of an individual mouse animal. Bars and error bars represent the geometric mean with a 95% confidence interval, respectively. [Figures 15A-15B] After storage at 2-8°C for 12 months, the mRNA-LNP preparations exhibit the stability conferred by the addition of gelatin, lipoic acid, or a combination of gelatin and lipoic acid ("gelatin + lipoic acid"). The mRNA-LNP preparations were prepared using tetravalent influenza mRNA (1 mg / mL) in 10% trehalose and GL-HEPES-E3-E12-DS-4-E10 as a cationic lipid, and diluted to a concentration of 0.2 mg / mL in 50 mM Tris, pH 7.5, 50 mM NaCl, 2% trehalose, 0.5% P188, and 10 μM EDTA. Figure 15A shows the decrease in mRNA integrity over 12 months. Figure 15B shows the inclusion efficiency over 12 months. Figure 15C shows the LNP particle size over 12 months. In each example, the control preparation contains the same mRNA-LNP preparation but without gelatin and lipoic acid. Figure 15D shows that after 12 months of storage at 2–8°C, no visible aggregates were observed in sealed vials containing mRNA-LNP preparations with gelatin or a combination of gelatin and lipoic acid (right), but visible aggregates were observed in sealed vials containing mRNA-LNP preparations with lipoic acid or a control preparation after 9 months of storage at 2–8°C (left). [Figures 15C-15D] Same as above. [Figure 16A-16C]After storage at 2-8°C for 6 months, the mRNA-LNP preparation exhibits the stability imparted by the addition of gelatin, lipoic acid, or a combination of gelatin and lipoic acid ("gelatin + lipoic acid"). The mRNA-LNP preparation was prepared using tetravalent influenza mRNA (1 mg / mL) in 100 mM Tris, pH 7.5, 50 mM NaCl, and 5% trehalose, and GL-HEPES-E3-E12-DS-4-E10 as a cationic lipid. It was then diluted to a concentration of 0.2 mg / mL in 50 mM Tris, pH 7.5, 50 mM NaCl, 2% trehalose, 0.5% P188, and 10 μM EDTA. Figure 16A shows the decrease in mRNA integrity over 6 months. Figure 16B shows the inclusion efficiency over 6 months. Figure 16C shows the LNP particle size over 6 months. In each example, the control formulation contains an mRNA-LNP formulation that is identical but does not contain gelatin or lipoic acid. [Figure 17] The average particle size of LNPs loaded with mRNA encoding influenza antigen ("monoFlu-LNP") is shown at pH 6-8 under different buffer conditions, as determined by dynamic light scattering. CL: Cationic lipid (CL-1: OF-02, CL-2: cKK-E10, CL-3: GL-HEPES-E3-E12-DS-4-E10). [Figure 18] The mRNA expression of monoFlu-LNP under different buffer conditions (pH 6-8), analyzed by flow cytometry (for CL-1 formulation) and Western blotting (for CL-2 and CL-3 formulations), is shown. CL: Cationic lipid (CL-1: OF-02, CL-2: cKK-E10, CL-3: GL-HEPES-E3-E12-DS-4-E10). [Figure 19] This shows the decrease in average particle size and mRNA integrity due to dynamic light scattering in monoFlu-LNP under different buffer conditions at pH 7.5 to 8.5. CL: Cationic lipid (CL2: cKK-E10, CL3: GL-HEPES-E3-E12-DS-4-E10). [Figures 20A-20B]Figure 20A shows the pH changes and %mRNA inclusion after 6 days under different buffer conditions in the presence of 0.26 mg / mL mRNA-LNP. Figure 20B shows the decrease in %mRNA integrity under different buffer conditions in the presence of 0.1 mg / mL mRNA-LNP. CL-3-containing QIV-LNP: LNP loaded with tetravalent influenza mRNA ("QIV-LNP") using GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid. [Figure 21] The average particle size of monoFlu-LNPs is shown by dynamic light scattering after freeze / thaw cycles at -70°C / room temperature (RT) (top) and -20°C / RT (bottom) in the presence of different concentrations of trehalose. CL: Cationic lipid (CL2: cKK-E10, CL3: GL-HEPES-E3-E12-DS-4-E10). [Figures 22A-22D] The visual aspects (Figure 22A), average particle size by dynamic light scattering (Figure 22B), visible particles (Figure 22C), and turbidity (Figure 22D) of monoFlu-LNP containing cationic lipid cKK-E10 after freeze / thaw cycles at -20°C / RT in the presence of different concentrations of trehalose or sucrose are shown. [Figure 23] This shows the mRNA inclusion efficiency (% mRNA inclusion) after freeze / thaw cycles at -20°C / RT and storage at 25°C for 2 weeks in the presence of sucrose. CL: Cationic lipid (CL2: cKK-E10, CL3: GL-HEPES-E3-E12-DS-4-E10). [Figures 24A-24D] Visual aspects of mRNA-LNPs containing the cationic lipid cKK-E10 (Figure 24A), average particle size by dynamic light scattering (Figure 24B), subvisible particles (Figure 24C), and % mRNA inclusion (Figure 24D) are shown after 3 days of orbital shaking stress in RT. [Figure 25] The changes in subvisible particles and turbidity in the presence of P188 or PS80 are shown after 3 days of orbital shaking stress at RT and 3 freeze / thaw (F / T) cycles at -20°C / RT. CL cationic lipids (CL-1: OF-02, CL-2: cKK-E10, CL-3: GL-HEPES-E3-E12-DS-4-E10). [Figure 26] This shows the change in the average particle size of mRNA-LNPs containing the cationic lipid cKK-E10 at 25°C in the presence of different concentrations of EDTA. [Figure 27] QIV-LNPs containing three different cationic lipids exhibit a decrease in %mRNA integrity at 25°C in the presence of EDTA. CL cationic lipids (CL-1: OF-02, CL-2: cKK-E10, CL-3: GL-HEPES-E3-E12-DS-4-E10). [Figure 28] Exemplary long-term stability data (LNP particle size by dynamic light scattering, % mRNA inclusion by RiboGreen assay, and % decrease in mRNA integrity by capillary electrophoresis) for QIV-LNP formulations containing three different cationic lipids are shown. CL cationic lipids (CL-1: OF-02, CL-2: cKK-E10, CL-3: GL-HEPES-E3-E12-DS-4-E10). [Figure 29] The main results of the optimization and stability studies described in Example 10 are shown below. [Figure 30] The stability profiles show the optimal settings for pH, buffer volume, and salt volume to maximize mRNA integrity for each storage temperature and time point tested. [Figure 30-1] Same as above. [Figure 30-2] Same as above. [Figure 31] This shows a stability profile (after 1 month of storage at 5°C) illustrating the effects of pH, buffer volume, and salt content on mRNA fragments, mRNA integrity, and mRNA-lipid adducts. [Figure 32] This shows the mRNA encapsulation efficiency, as evaluated by RiboGreen, after storage at 30°C for 2 weeks ("T2W 30°C") or 1 month ("T1M 30°C") with different buffer amounts, salt amounts, and pH levels. [Figure 33] mRNA expression, assessed by flow cytometry after storage for 1 week ("T1W") or 2 weeks ("T2W") at different buffer amounts, salt levels, and pH levels, is shown. The results are consistent for each strain used, and only iLogMFI for one representative strain is shown. [Figures 34A-34B] The effect of surfactant P188 on stability after storage for 1 day ("T1d"), 2 days ("T2d"), or 5 days ("T5d") at 5°C or 25°C, and after freeze / thaw cycles is shown. Figure 34A: LNP size by DLS; Figure 34B: Subvisible particles by FlowCam analysis. [Figures 35A-35B] The effect of sucrose on stability after storage at 5°C or 25°C for 1 day ("T1d"), 2 days ("T2d"), or 5 days ("T5d"), and after freeze / thaw cycles. Figure 35A: LNP size by DLS; Figure 35B: Subvisible particles by FlowCam analysis. [Figure 36] This shows the effect of EDTA on mRNA integrity as determined by reverse-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC). [Figures 37A-37C] The stability of 16:0-18:1 PE-based LNP preparations is demonstrated after storage at 2-8°C for 7 months, with the addition of gelatin. The mRNA-LNP preparations were prepared using tetravalent influenza mRNA in 10% trehalose, GL-HEPES-E3-E12-DS-4-E10 as a cationic lipid, and 16:0-18:1 PE as a helper lipid. Figure 37A shows the LNP particle size over 7 months. Figure 37B shows the encapsulation efficiency over 7 months. Figure 37C shows the decrease in mRNA integrity over 7 months. In each example, the control preparation contains the same but gelatin-free mRNA-LNP preparation. [Modes for carrying out the invention]

[0022] Herein, various exemplary embodiments are given in detail with reference to the accompanying drawings and described in the following detailed description. It should be understood that the following detailed description is provided to allow the reader to fully understand the details of the specific embodiments, features, and aspects of this disclosure and should not be construed as limiting the scope of this disclosure.

[0023] To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of the terms below and other terms may be provided throughout this specification. If any definition of a term provided below conflicts with a definition in an application or patent incorporated by reference, the meaning of that term should be understood using the definition provided in this application.

[0024] definition As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural nouns unless the context clearly indicates otherwise. For example, a reference to “method” includes one or more methods and / or processes of the type described herein, and / or it would be apparent to those skilled in the art by reading this disclosure, etc.

[0025] The term “approximately” is used herein to mean within a typical tolerance range in the art. For example, “approximately” can be understood as a standard deviation of about 2 from the mean. According to certain embodiments, when referring to measurable values ​​such as quantities, “approximately” means to include a variation of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, or ±0.1% from a specified value, such variation being appropriate for carrying out the methods of the Disclosure and / or for preparing and using the compositions of the Disclosure. When “approximately” precedes a set of numbers or ranges, it is understood that “approximately” can modify each of the numbers in that set of numbers or range.

[0026] The term "and / or," as used herein and in the claims, means "either or both" of the elements thus combined, that is, elements that exist in some cases conjugated and in other cases separately. Other elements other than those specifically identified by the phrase "and / or" may exist at their discretion, whether related to or unrelated to those identified elements, unless otherwise explicitly stated. As a non-restrictive example, with respect to "A and / or B," when used in combination with open-ended language such as "including," for example, in one embodiment, it may refer to A without B (optionally including elements other than B); in another embodiment, it may refer to B without A (optionally including elements other than A); and in yet another embodiment, it may refer to both A and B (optionally including other elements).

[0027] As used herein, the term “antigen” refers to an agent that, when exposed to or administered to an organism, elicits an immune response, and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including the production of antigen-specific antibodies); alternatively or additionally, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). Those skilled in the art will understand that a particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, ferret, rabbit, primate, human), but not in all members of the target species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of members of the target species, including all values ​​and partial ranges between them. In some embodiments, the antigen may or may not bind to antibodies and / or T cell receptors and induce specific physiological responses in the organism. In some embodiments, for example, the antigen may bind to antibodies and / or T cell receptors in vitro, regardless of whether such interactions occur in vivo. In some embodiments, the antigen reacts with specific humoral or cellular immunity products, including those induced by heterologous immunogens. The antigen includes the NA and HA forms described herein.

[0028] The terms “at least,” “less than,” “greater than,” or “at most” preceding a number or set of numbers (e.g., “at least 2”) are understood, as is clear from the context, to include the number adjacent to the term “at least,” “less than,” or “greater than,” and all subsequent numbers or integers. When the terms “at least,” “less than,” “greater than,” or “at most” precede a set of digits or a range, it is understood that “at least,” “less than,” “greater than,” or “at most” can modify each digit in the set of digits or within the range.

[0029] As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle administered with the composition. In some exemplary embodiments, the carrier may include, for example, water and oils, such as petroleum, animal oils, vegetable oils, or oils of synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil, as sterile liquids. In some embodiments, the carrier is or comprises one or more solid components.

[0030] As used herein, “a control composition that does not contain at least one thermoreversible gelling agent” means a composition that is identical to the composition being compared, except that it does not contain at least one thermoreversible gelling agent. As used herein, the term “compared to a control composition that does not contain at least one thermoreversible gelling agent” means the control composition that does not contain at least one thermoreversible gelling agent at time zero (T0), i.e., before storage, that is, the composition being compared.

[0031] As used herein, “a control liquid composition free of at least one heat-stabilizing excipient” refers to a composition that is identical to the composition being compared, except that it does not contain at least one heat-stabilizing excipient. As used herein, the term “compared to a control liquid composition free of at least one heat-stabilizing excipient” means the composition being compared to the control liquid composition free of at least one heat-stabilizing excipient at time zero (T0), i.e., before storage.

[0032] As used herein, “Inclusion Efficiency” or “EE” refers to the amount of therapeutic and / or prophylactic agent, such as the RNA molecule of this disclosure, that becomes part of the lipid nanoparticles (LNPs), compared to the initial total amount of the therapeutic and / or prophylactic agent used in the preparation of the LNPs. For example, if 97 mg of the therapeutic and / or prophylactic agent is encapsulated in the LNPs out of a total of 100 mg of the therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency may be given as 97%. The encapsulation efficiency may be determined, for example, by a RiboGreen assay or any method known in the art. As used herein, “Inclusion” may refer to complete, substantial or partial enclosure, containment, siege, or accommodation.

[0033] As used herein, "H1" refers to influenza virus subtype 1 hemagglutinin (HA). Influenza A viruses are divided into Group 1 and Group 2. Groups 1 and 2 are further divided into subtypes, which refer to the classification of viruses based on the sequences of two proteins on the viral surface: HA and neuraminidase (NA). Currently, 18 HA subtypes (H1-H18) are recognized. Therefore, H1 is distinct from the other HA subtypes, including H2-H18.

[0034] As used herein, "H3" refers to influenza virus subtype 3HA. Therefore, H3 is distinct from the other HA subtypes, including H1, H2, and H4-H18.

[0035] Where used herein, terms such as “in some embodiments,” “in certain embodiments,” “in other embodiments,” and “in some other embodiments” refer to embodiments of all aspects of the present disclosure unless the context clearly indicates otherwise.

[0036] As used herein, “particle size” and “average particle size” in relation to a lipid nanoparticle composition refer to the average diameter of the nanoparticle composition. Particle size can be determined using any method known in the art, such as dynamic light scattering (DLS). DLS typically measures particle size based on intensity, and the intensity-based particle size and size distribution can then be recalculated to convert it to a volume-based particle size and size distribution. Therefore, in some embodiments, the particle size as defined herein, when measured by DLS, relates to the volume-average diameter.

[0037] As used herein, the terms “RNA-LNP composition” or “RNA-LNP preparation” refer to a composition or preparation containing one or more RNA molecules, such as mRNA molecules encapsulated in LNPs. Therefore, a composition or preparation containing one or more mRNA molecules encapsulated in LNPs is called an “mRNA-LNP composition” or “mRNA-LNP preparation.”

[0038] As used herein, "N1" refers to influenza virus subtype 1 neuraminidase (NA). Influenza A viruses are divided into Group 1 and Group 2. Group 1 and Group 2 are further divided into subtypes, which refer to the classification of viruses based on the sequences of two proteins on the viral surface: HA and neuraminidase (NA). Currently, 11 NA subtypes (N1-N11) are recognized. Therefore, N1 is distinct from the other NA subtypes, including N2-N11.

[0039] As used herein, "N2" refers to influenza virus subtype 2 neuraminidase (NA). Therefore, N2 is distinct from other NA subtypes, including N1 and N3-N11.

[0040] The terms “preventing,” “preventing,” or “prevention,” as used herein, refer to the prevention, avoidance of the onset, delay of the onset, and / or reduction of the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection by a virus such as influenza virus, respiratory syncytial virus (RSV), or coronavirus). In some embodiments, prevention is evaluated on a population basis, and if a statistically significant reduction in the incidence, frequency and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition, then the drug is considered to “prevent” a particular disease, disorder, or condition.

[0041] As used herein, the terms “preventing” or “preventing” in relation to the thermal degradation of RNA molecules refer to the delay of RNA degradation and / or a reduction in the amount or rate of RNA degradation. RNA degradation can be assessed based on the loss of RNA integrity, which can be measured, for example, by extracting RNA and analyzing it with a fragment analyzer using capillary electrophoresis.

[0042] As used herein, the term “preventive dose” means an amount sufficient to prevent, avoid, delay, and / or reduce the frequency and / or severity of one or more symptoms of a particular disease, disorder or condition (for example, infection by a virus such as influenza virus, respiratory syncytial virus (RSV), or coronavirus).

[0043] As used herein, the term "room temperature" refers to a temperature of approximately 18–25°C.

[0044] The World Health Organization (WHO) annually selects influenza strains to be included in seasonal vaccine formulations based on intensive surveillance efforts. As used herein, the terms “standard therapeutic strain” or “SOC strain” refer to influenza strains selected by the World Health Organization (WHO) for inclusion in seasonal vaccine formulations. Standard therapeutic strains may include past, current, or future standard therapeutic strains.

[0045] As used herein, the term “subject” means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, non-human subjects are mammals (e.g., rodents, mice, rats, rabbits, ferrets, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, subjects may be transgenic animals, genetically modified animals, and / or clones. In some embodiments, subjects are adults, adolescents, or infants. In some embodiments, the terms “individual” or “patient” are used and are intended to be interchangeable with the term “subject.”

[0046] Thermoreversible gel-forming RNA-LNP compositions: Ribonucleic acid (RNA), such as messenger RNA (mRNA) molecules, has recently been used as a drug in various applications, including therapeutic agents, vaccines, and diagnostic agents. However, due to their inherent instability and susceptibility to degradation by nucleases, the storage and effective in vivo delivery of formulations containing RNA molecules (e.g., mRNA) remain challenges.

[0047] Lipid nanoparticle (LNP) formulations offer an opportunity to deliver various RNA molecules, such as mRNA, in vivo for applications where unencapsulated RNA molecules are ineffective; however, their broad utility has been hindered by insufficient RNA stability over the relevant timeframe. Because the loss of RNA stability makes long-term storage at refrigerated or room temperature conditions unusable, the use of such formulations is limited to applications where the degradation of RNA molecules within the LNP formulation results in a freeze-contained composition. Whether LNP formulations containing RNA (e.g., mRNA) are suitable for long-term storage at refrigerated conditions, such as around 1–11°C (e.g., 2–8°C or 4°C), remains unclear.

[0048] This disclosure is based, at least in part, on the surprising finding that including a thermoreversible gelling agent in RNA molecule-containing LNP formulations substantially improves stability, including RNA stability under refrigerated conditions such as temperatures of approximately 1–11°C (e.g., 2–8°C or 4°C), which is useful for the preparation, storage, and use of RNA molecules as therapeutic agents. For example, the inventors of this disclosure have surprisingly found that, for mRNA-LNP compositions, when combined with the thermoreversible gelling agent according to this disclosure, the rate of decline in the integrity of mRNA encapsulated within the LNP is dramatically suppressed after long-term storage at 4°C, for example, for up to 6 months. mRNA instability, specifically the decline in RNA integrity, is considered one of the biggest challenges to the fundamental therapeutic and commercial potential of mRNA. Including at least one thermoreversible gelling agent in the mRNA-LNP formulation according to this disclosure thereby provides an important solution to these problems.

[0049] The discovery that ribonucleic acid can be stabilized within lipid carriers, such as LNPs, using a thermoreversible gelling agent is both surprising and unexpected. This finding enables several important applications, including the long refrigerated storage life of complete liquid formulations. Achieving stable formulations also enables commercially available and therapeutically desirable packaging and delivery options, including prefilled syringes (PFS) and cartridges for patient-friendly autoinjectors and infusion pumps. Therefore, the ability to stabilize RNA solutions and pharmaceutical formulations, such as mRNA, and other therapeutic agents, represents a valuable technology that will facilitate the broad use of therapeutic compositions, such as mRNA compositions.

[0050] Accordingly, thermoreversible gel-forming compositions comprising a therapeutic agent and at least one thermoreversible gelling agent are provided herein. Typically, the therapeutic agent comprises one or more RNA molecules. In certain embodiments, one or more RNA molecules are encapsulated in LNPs. The thermoreversible gel-forming compositions of this disclosure generally have a liquid phase at temperatures above about 12°C, for example at room temperature, and, in the presence of at least one thermoreversible gelling agent, reversibly transition to a gel (or hydrogel) form at temperatures of about 1 to 11°C, for example at refrigeration temperatures (e.g., 2 to 8°C or 4°C). The thermoreversible gel-forming properties of the compositions disclosed herein offer a surprising and unexpected advantage in maintaining the thermal stability of the compositions without requiring ultra-low temperature or sub-zero conditions for long-term storage, thus extending their shelf life and facilitating their widespread use.

[0051] In this specification, the term "thermally reversible" refers to a material, such as a polymer, that exhibits a reversible change in its physical properties (e.g., physical state) in response to a change in temperature. A "thermally reversible gelling agent," also called a "thermally responsive gelling agent" or "thermal gelling agent," refers to an agent that may contain water-soluble units and units having an upper critical solution temperature ("UCST"). The "upper critical solution temperature" or "UCST" generally refers to the critical temperature at which the components of a mixture are miscible in any proportion. The term "upper limit" means that UCST is an upper limit to the temperature range of partial miscibility, or miscibility only for a particular composition. Above UCST, the active substance, such as a polymer, is substantially completely soluble in water, while below this temperature, the UCST portions aggregate, losing their solubility in water and thereby forming crosslinks between polymer chains. The active substance, such as a polymer, then forms a three-dimensional network-like structure, creating a gel or hydrogel. Therefore, changes in physical state are observed along with temperature changes, for example, when a composition incorporating an active substance such as a polymer is subjected to temperature changes. Since the transformation to a gel is physical and temperature-dependent, this thermal gelation phenomenon is completely reversible. A typical phase diagram of a thermally reversible gelling agent according to this disclosure is provided in Figure 1A. As shown in Figure 1B, the formation of a gel or hydrogel can stabilize LNPs and prevent aggregation or fusion of LNPs.

[0052] In some embodiments, the thermoreversible gelling agents contained in the compositions disclosed herein have a UCST of about 12°C to about 100°C, for example, about 12°C to about 80°C, about 12°C to about 60°C, about 12°C to about 50°C, about 12°C to about 40°C, about 12°C to about 30°C, or about 12°C to about 20°C. In some embodiments, the thermoreversible gelling agents of the disclosure have a UCST of about 12°C. In some embodiments, the thermoreversible gelling agents of the disclosure have a UCST of about 15°C. In some embodiments, the thermoreversible gelling agents of the disclosure have a UCST of about 20°C. In some embodiments, the thermoreversible gelling agents of the disclosure have a UCST of about 25°C. In some embodiments, the thermoreversible gelling agents of the disclosure have a UCST of about 30°C. In some embodiments, the thermoreversible gelling agents of the disclosure have a UCST of about 35°C. In some embodiments, the thermoreversible gelling agent of the Disclosure has a UCST of about 40°C. In some embodiments, the thermoreversible gelling agent of the Disclosure has a UCST of about 45°C. In some embodiments, the thermoreversible gelling agent of the Disclosure has a UCST of about 50°C. In some embodiments, the thermoreversible gelling agent of the Disclosure has a UCST suitable for compositions and / or vaccines disclosed herein.

[0053] Thermoreversible gelling agents preferred to the present disclosure may include thermoreversible gelling polymers, thermoreversible gelling polypeptides, and / or thermoreversible gelling proteins. Therefore, in some embodiments, the thermoreversible gelling agent includes a thermoreversible gelling polymer. In other embodiments, the thermoreversible gelling agent includes a thermoreversible gelling polypeptide. In some other embodiments, the thermoreversible gelling agent includes a thermoreversible gelling protein. In some embodiments, the thermoreversible gelling agent of the present disclosure, whether a polymer, polypeptide, or protein, is a hydrogel at low temperatures, such as about 4°C, while being soluble in water at high temperatures, such as about 12°C, in which case the hydrogel is a three-dimensional network comprising a crosslinked polymer, polypeptide, or protein. In certain embodiments, the polymer, polypeptide, or protein is crosslinked by non-covalent bonds.

[0054] In some embodiments, any thermoreversible gelling agent having UCST phase separation behavior, preferably about 12°C to about 100°C, for example, about 12°C to about 80°C, about 12°C to about 60°C, about 12°C to about 50°C, about 12°C to about 40°C, about 12°C to about 30°C, about 12°C to about 20°C, or about 12°C, can be used.

[0055] In some embodiments, the thermoreversible gelling agent of this disclosure is gelatin. Gelatin is a bulk-acting substance and acceptable material for medical use. Gelatin is commonly used as a stabilizer due to its high biocompatibility, biodegradability, low immunogenicity and low material cost, although we do not wish to be bound by any theory.

[0056] Gelatin is generally derived from collagen extracted from animal body parts, primarily skin, bone, and connective tissue. Gelatin may be of porcine or bovine origin, including porcine and bovine bone gelatin produced by acid or alkali extraction methods, or it may be made from fish by-products. Examples of porcine or bovine gelatin include, but are not limited to, the beMatrix® gelatin series (Nitta Gelatin Inc., Osaka, Japan), hydrolyzed porcine gelatin (SOL-U-PRO; Dynagel Inc., IL), and X-Pure Gelatin® (Rousselot Inc., WI). Gelatin may also be of non-animal origin, such as recombinant human gelatin (FG-5001; FibroGen, Inc., CA). Therefore, in some embodiments, the thermoreversible gelling agents of this disclosure include porcine gelatin, such as hydrolyzed porcine gelatin. In some embodiments, the thermoreversible gelling agent of the Disclosure comprises bovine-derived gelatin. In some embodiments, the thermoreversible gelling agent of the Disclosure comprises non-animal-derived gelatin. In some embodiments, the thermoreversible gelling agent of the Disclosure comprises recombinant gelatin, such as recombinant human gelatin. In some embodiments, the thermoreversible gelling agent of the Disclosure comprises food-grade gelatin. In some embodiments, the thermoreversible gelling agent of the Disclosure comprises pharmaceutical-grade gelatin.

[0057] Gelatin is generally derived from collagen. This is the irreversibly hydrolyzed form of collagen, where hydrolysis reduces protein fibrils to smaller peptides. Collagen is a triple helix-forming protein. Common motifs in the amino acid sequence of collagen are glycine-proline-X and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline, or hydroxyproline.

[0058] Other non-limiting examples of the thermoreversible gelling polymers of this disclosure include poly(N-acryloyl asparagineamide), poly(ethylene glycol)-b-poly(N-acryloylglycinamide-co-acrylonitrile) (PEG-bP(NAGA-co-AN), poly(N-acryloylglycinamide-co-N-phenylacrylamide) (P(NAGA-co-NPhAm)), poly(N-(2-hydroxypropyl)methacrylamide-glycolamide) (P(HPMA-GA)), and poly(acrylamide-co-acrylonitrile)-b-poly(Ori Examples include, but are not limited to, poly(ethylene glycol)methyl ether methacrylate (POEGMA), poly(acrylonitrile acrylate) (P(AA-co-AN)), imidazole-based poly(N-vinylimidazole-co-1-vinyl-2-(hydroxymethyl)imidazole), poly(sulfobetaine-co-sulfabetine) (P(SB-co-ZB) and poly[2-(methacryloyloxy)ethylphosphocholine]-b-poly(2-ureidoethyl methacrylate) (PMPC20-b-PUEM165). Thermoresponsive polymers exhibiting UCST as described by et al. (eXPRESS Polymer Letters, 2019, 13(11):974-992, incorporated herein by reference) can also be used. In some embodiments, the thermoreversible gelling polymers of the Disclosure thus include gelatin, poly(N-acryloyl asparagine amide), PEG-bP(NAGA-co-AN), P(NAGA-co-NPhAm), P(HPMA-GA), POEGMA, P(AA-co-AN), poly(N-vinylimidazole-co-1-vinyl-2-(hydroxymethyl)imidazole), P(SB-co-ZB), PMPC20-b-PUEM165, or combinations thereof. In some embodiments, the thermoreversible gelling polymers of the Disclosure may be any polymer having the phase separation behavior disclosed herein.

[0059] In some embodiments, any polypeptide having UCST phase separation behavior, preferably around 12°C to 100°C, for example, around 12°C to 80°C, around 12°C to 60°C, around 12°C to 50°C, around 12°C to 40°C, around 12°C to 30°C, around 12°C to 20°C, or around 12°C, can be used. One non-limiting example of a thermoreversible gelling polypeptide of this disclosure is iMAPA-PEG, which is insoluble multi-L-arginyl-poly-L-aspartic acid (iMAPA) conjugated with polyethylene glycol (PEG). See, for example, Tseng et al., Biomacromolecules, 2018, 19(12):4585-4592, incorporated herein by reference. Multi-L-arginyl-poly-L-aspartate (MAPA), also known as cyanophycin or CGP (cyanophycin granule polypeptide), is a non-protein, non-ribosomal-producing amino acid polymer composed of an aspartic acid backbone and arginine side groups. Other non-limiting examples of the thermoreversible gelling polypeptides of this disclosure include those described by Kuroyanagi et al., J.Am.Chem.Soc., 2019, 141:1261-1268, which are incorporated herein by reference. In some embodiments, the thermoreversible gelling polypeptides of this disclosure include iMAPA-PEG.

[0060] For example, the thermoreversible gelling agents of the present disclosure, such as gelatin, are present in sufficient amounts in the composition of the present disclosure for compositions having a liquid phase at temperatures above about 12°C, e.g., room temperature, and a gel phase at refrigeration temperatures, e.g., about 1 to 11°C (e.g., 2 to 8°C or 4°C). In some embodiments, the thermoreversible gelling agent is present in the composition of the present disclosure in amounts including any of the values ​​and partial ranges between them, such as about 0.1% to about 30% by weight, e.g., about 0.1% to about 20% by weight, about 0.1% to about 10% by weight, about 0.1% to about 5% by weight, about 0.2% to about 6% by weight, about 0.25% to about 5% by weight, about 0.3% to about 4% by weight, about 0.4% to about 3% by weight, or about 0.5% to about 1.5% by weight.

[0061] In some embodiments, the thermoreversible gelling agent is present in an amount of about 0.1% by weight in the composition of the Disclosure. In some embodiments, the thermoreversible gelling agent is present in an amount of about 0.5% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 1% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 1.5% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 2% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 2.5% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 3% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 3.5% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 4% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 5% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 6% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 7% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 7.5% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 8% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 9% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 10% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 15% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 20% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 25% by weight. In some embodiments, the thermoreversible gelling agent is present in an amount of about 30% by weight.

[0062] In some embodiments, the thermoreversible gelling agent included in the composition of the Disclosure is gelatin, and in certain embodiments, the gelatin is present in an amount of about 1% by weight.

[0063] Stability: Surprisingly, the thermoreversible gel-forming compositions of this disclosure were found to remain stable in gel form even when stored at refrigerated temperatures, e.g., about 1–11°C (e.g., 2–8°C or 4°C), for relatively long periods. As demonstrated in the following examples, the thermoreversible gel-forming compositions of this disclosure remained stable, at least with respect to the particle size and encapsulation efficiency of the LNPs contained therein, and the integrity of the RNA encapsulated within the LNPs, after storage at 4°C for up to about 6 months. Such stability can be maintained for even longer periods, including all values ​​and partial ranges in between, e.g., up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, up to about 1 year, up to about 18 months, or up to about 2 years. In some embodiments, such stability can be maintained for more than 2 years.

[0064] Accordingly, in some embodiments, the thermoreversible gel-forming compositions of the present disclosure are stable at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C) for up to about 1 month. In some embodiments, the thermoreversible gel-forming compositions of the present disclosure are stable at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C) for up to about 1 month or longer, including all values ​​and partial ranges in between, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, and up to about 2 years. In some embodiments, the thermoreversible gel-forming compositions of the present disclosure are stable at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C) for more than 2 years. The stability of a thermoreversible gel-forming composition can be measured by various methods known in the art, such as measuring changes in LNP particle size, LNP encapsulation efficiency, or the integrity of RNA encapsulated in the LNPs. In some embodiments, the stability of the thermoreversible gel-forming composition of the Disclosure is compared to the same composition before storage. In some embodiments, the stability of the thermoreversible gel-forming composition of the Disclosure is compared to a control composition that is identical to the thermoreversible gel-forming composition of the Disclosure except that it does not contain at least one thermoreversible gel-forming agent, which is referred to herein as a "control composition that does not contain at least one thermoreversible gel-forming agent."

[0065] In some embodiments, the stability of a thermoreversible gel-forming composition is measured with respect to changes in the particle size of LNPs, and the compositions of the present disclosure are stable when the average particle size of LNPs does not increase by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, with respect to all values ​​and partial ranges in between, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), with respect to all values ​​and partial ranges in between, for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or after storage of the liquid composition for more than 2 years, with respect to all values ​​and partial ranges in between. In some embodiments, the average particle size of LNPs does not increase by more than 40% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the average particle size of LNPs does not increase by more than about 30% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.In some embodiments, the average particle size of LNPs does not increase by more than about 20% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the average particle size of LNPs does not increase by more than about 10% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0066] The particle size can be determined using any method known in the art, such as dynamic light scattering (DLS). Generally, before storage, the LNPs contained in the thermoreversible gel-forming composition of this disclosure have an average particle size in the range of about 10 nm to about 1000 nm, for example, about 15 nm to about 750 nm, about 30 nm to about 500 nm, about 50 nm to about 250 nm, about 75 nm to about 200 nm, or about 80 nm to about 150 nm.

[0067] In some embodiments, the stability of the composition is measured in relation to the change in LNP encapsulation efficiency, and the compositions of the present disclosure are stable when the LNP encapsulation efficiency does not decrease by more than about 20%, for example, about 15%, 10%, or 5%, including all values ​​and partial ranges in between, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, for a maximum of about 1 month or more, for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or more than 2 years after storage of the liquid composition, including all values ​​and partial ranges in between. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 15% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 10% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 5% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0068] In some embodiments, the encapsulation efficiency of LNPs is higher than that of a control composition that does not contain at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of LNPs is at least 5% higher than that of a control composition that does not contain at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of LNPs is at least 7.5% higher than that of a control composition that does not contain at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of LNPs is at least 10% higher than that of a control composition that does not contain at least one thermoreversible gelling agent.

[0069] The encapsulation efficiency of LNPs can be determined using any method known in the art, such as a fluorescence plate-based assay using RiboGreen reagent (Invitrogen).

[0070] In some embodiments, the stability of the composition is measured in terms of changes in the integrity of the RNA encapsulated in the LNP, and the composition of the present disclosure is stable when the integrity of the RNA encapsulated in the LNP does not decrease by more than about 30%, including all values ​​and partial ranges, after storage of the composition for more than 2 years, or after storage of the composition for more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C) for a maximum of about 1 month or more, including all values ​​and partial ranges in between, for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or after storage of the composition for more than 2 years, including all values ​​and partial ranges in between. In some embodiments, the integrity of the RNA molecule does not decrease by more than about 15% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the integrity of the RNA molecule does not decrease by more than 10% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.In some embodiments, the integrity of the RNA molecule does not decrease by more than about 5% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0071] The integrity of ribonucleic acid molecules can be determined using any method known in the art, such as fragmentation analysis using capillary electrophoresis (CE) and / or capillary gel electrophoresis (CGE).

[0072] In some embodiments, the RNA molecules encapsulated in the LNPs of the Disclosure encode one or more influenza virus proteins, e.g., HA and / or NA proteins, and the compositions of the Disclosure inhibit hemagglutination. The inhibitory (HAI) titer is stable when, compared to a control composition that does not contain at least one thermoreversible gelling agent, it does not decrease by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C) for a maximum of about 1 month or more, including all values ​​and partial ranges in between, for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or more than 2 years, including all values ​​and partial ranges in between, after storage of the liquid composition. In some embodiments, the HAI titer of the composition does not decrease by more than about 25% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the HAI titer of the composition does not decrease by more than about 20% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.In some embodiments, the HAI titer of the composition does not decrease by more than about 15% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the HAI titer of the composition does not decrease by more than 10% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the HAI titer of the composition does not decrease by more than about 5% after storage of the composition for up to about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0073] HAI titer can be measured using any method known in the art, such as using the influenza virus HAI test (Denka Seiken Co., Tokyo, Japan).

[0074] Thermostable RNA-LNP liquid composition This disclosure also builds, at least in part, on the surprising finding that including certain excipients in RNA molecule-containing LNP formulations substantially improves stability, including RNA stability, when stored as a liquid at temperatures above 0°C under refrigerated conditions (e.g., 4°C). For example, the inventors of this disclosure have surprisingly found that, with respect to mRNA-LNP compositions, including certain excipients according to this disclosure, the rate of loss in the integrity of mRNA encapsulated within the LNP is dramatically suppressed after long-term storage as a liquid at 4°C, including, for example, up to 12 months. RNA instability, such as that measured by the loss in mRNA integrity, is considered a major challenge to the fundamental therapeutic and commercial potential of mRNA. Including at least one of these excipients in mRNA-LNP formulations according to this disclosure thereby provides an important solution to these problems. Due to their ability to confer thermal stability, such excipients will also be referred to below as “thermal stabilizing excipients.”

[0075] The discovery that the use of such heat-stabilizing excipients makes it possible to stabilize RNA within lipid carriers, such as LNPs, when stored as a liquid formulation was surprising and unexpected. This finding enables several applications for these liquid formulations, including long refrigerated storage life, long shelf life at room temperature, and long-term stability in use at physiological temperatures up to 37°C. Achieving stable liquid formulations also enables commercially available and therapeutically desirable packaging and delivery options, including pre-filled syringes (PFS) and cartridges for patient-friendly autoinjectors and infusion pumps. Therefore, the ability to stabilize RNA, such as mRNA, and other therapeutic solutions and pharmaceutical formulations represents a fundamental technology that will facilitate the broad use of therapeutic compositions, such as mRNA compositions.

[0076] Heat-stabilizing excipients Liquid compositions comprising one or more RNA molecules encapsulated in LNPs and at least one heat-stabilizing excipient are provided herein, wherein the at least one heat-stabilizing excipient includes or is lipoic acid, L-theanine, vanillin, or a combination thereof. Other suitable heat-stabilizing excipients that may be used in the heat-stable liquid compositions of this disclosure include, but are not limited to, quercetin, glutathione, gallic acid, naringin, acetylsalicylic acid, ascorbic acid, and eugenol. The liquid compositions of this disclosure are generally heat-stable, and as a result, the integrity of the one or more RNA molecules encapsulated in LNPs is not substantially reduced after storage of the liquid composition at temperatures above 0°C for a period of time.

[0077] Accordingly, the present disclosure relates, in particular, to a thermally stable liquid composition comprising one or more RNA molecules encapsulated in an LNP and at least one thermally stabilizing excipient having one or more of the following characteristics: long refrigerated storage life, long shelf life at room temperature, and long-term stability in use at physiological temperatures up to 37°C.

[0078] The thermal stabilizing excipients of the present disclosure, such as lipoic acid, L-theanine, vanillin, or combinations thereof, are present in the liquid composition of the present disclosure in an amount sufficient to maintain the liquid stability of the composition, including stabilizing the integrity of RNA molecules and maintaining the average particle size and LNP encapsulation efficiency. In some embodiments, the heat-stabilizing excipients of the Disclosure, e.g., lipoic acid, L-theanine, vanillin, or combinations thereof, are present in the liquid composition of the Disclosure at concentrations ranging from about 0.1 mM to about 30 mM, including all values ​​and partial ranges between them, for example, about 0.1 mM to about 25 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 15 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 15 mM, about 0.5 mM to about 10 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, or about 1 mM to about 5 mM. In some embodiments, the heat-stabilizing excipients are present at concentrations ranging from about 0.1 mM to about 20 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 0.5 mM to about 15 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 5 mM to about 15 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 5 mM to about 10 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 10 mM to about 15 mM.

[0079] In some embodiments, the heat-stabilizing excipients of the Disclosure, such as lipoic acid, L-theanine, vanillin, or combinations thereof, are present in the liquid composition of the Disclosure at concentrations of about 0.1 mM, about 0.5 mM, about 1 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 7.5 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 12.5 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 17.5 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, or about 30 mM, encompassing all values ​​and partial ranges between them. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 5 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 10 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 15 mM. In some embodiments, the heat-stabilizing excipient is present at a concentration of about 20 mM.

[0080] The amount of at least one heat-stabilizing excipient present in the liquid composition of this disclosure can also be expressed by the weight ratio of at least one heat-stabilizing excipient to one or more RNA molecules. Accordingly, in certain embodiments, the heat-stabilizing excipients of the Disclosure, such as lipoic acid, L-theanine, vanillin, or combinations thereof, are present in the liquid composition of the Disclosure in such amounts that the heat-stabilizing excipient and one or more RNA molecules are present in a weight ratio of about 1:1 to about 100:1, for example, about 2:1 to about 50:1, about 2:1 to about 30:1, about 2:1 to about 15:1, about 3:1 to about 60:1, about 3:1 to about 30:1, about 5:1 to about 50:1, about 5:1 to about 30:1, about 10:1 to about 50:1, about 10:1 to about 30:1, about 12:1 to about 50:1, about 12:1 to about 30:1, about 12:1 to about 20:1, or about 15:1 to about 50:1, encompassing all values ​​and partial ranges between them. In some embodiments, one heat-stabilizing excipient and one or more RNA molecules are present in a weight ratio of about 5:1 to about 50:1. In certain embodiments, the liquid composition of the Disclosure contains lipoic acid as a heat-stabilizing excipient in an amount such that lipoic acid and one or more RNA molecules are present in a weight ratio of about 2.5:1 to about 15.5:1. In other embodiments, the liquid composition of the Disclosure contains L-theanine as a heat-stabilizing excipient in an amount such that L-theanine and one or more RNA molecules are present in a weight ratio of about 10:1 to about 30:1. In some embodiments, the liquid composition of the Disclosure contains vanillin as a heat-stabilizing excipient in an amount such that vanillin and one or more RNA molecules are present in a weight ratio of about 12.5:1 to about 50:1.

[0081] thermal stability The liquid compositions of this disclosure are remarkably thermally stable at a temperature of 37°C. Therefore, in some embodiments, the integrity of one or more RNA molecules encapsulated in the LNP does not decrease by more than about 20%, e.g., about 15%, 10%, or 5%, including all values ​​and partial ranges during storage, after the liquid composition has been stored at 37°C for at least 7 days, compared to a control liquid composition without at least one heat-stabilizing excipient. In other embodiments, the integrity of one or more RNA molecules encapsulated in the LNP does not decrease by more than about 15%, after the liquid composition has been stored at 37°C for at least 7 days, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules encapsulated in the LNP does not decrease by more than about 10%, after the liquid composition has been stored at 37°C for at least 7 days, compared to a control liquid composition without at least one heat-stabilizing excipient.

[0082] The inventors have demonstrated that, when stored at a temperature of 4°C, corresponding to standard refrigeration conditions, the liquid compositions of this disclosure can remain stable for at least several months. Therefore, in some embodiments, the integrity of one or more RNA molecules encapsulated in the LNPs of the liquid compositions disclosed herein does not decrease by more than about 50%, including all values ​​and partial ranges during storage of the liquid composition for up to about 1 month or more, including all values ​​and partial ranges during storage, at a temperature of 4°C for up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, including all values ​​and partial ranges during storage, by more than about 50%, including all values ​​and partial ranges during storage.

[0083] In some embodiments, the integrity of one or more RNA molecules does not decrease by more than about 25% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 30% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 35% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 40% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between.In some embodiments, the integrity of one or more RNA molecules does not decrease by more than 45% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between.

[0084] The inventors have also demonstrated that when stored at a temperature of 25-30°C, the liquid compositions of this disclosure can maintain a stable state for at least several weeks. Therefore, in some embodiments, the integrity of one or more RNA molecules encapsulated in the LNPs of the liquid compositions disclosed herein, compared to a control liquid composition without at least one heat-stabilizing excipient, does not decrease by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, after storage of the liquid composition at a temperature of 25°C for at least about one week, including all values ​​and partial ranges in between, for example, at least about two weeks, at least about three weeks, at least about four weeks, at least about six weeks, at least about seven weeks, at least about eight weeks, or at least eight weeks, including all values ​​and partial ranges in between.

[0085] In some embodiments, the integrity of one or more RNA molecules does not decrease by more than about 40% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, or more than 4 weeks, at a temperature of about 25°C, including all values ​​and partial ranges in between, compared with a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than about 45% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, or more than 4 weeks, at a temperature of about 25°C, including all values ​​and partial ranges in between, compared with a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than about 50% after storage of the liquid composition at a temperature of 25°C for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, or more than 4 weeks, including all values ​​and partial ranges in between, compared to a control liquid composition that does not contain at least one heat-stabilizing excipient.

[0086] In other embodiments, the integrity of one or more RNA molecules encapsulated in LNPs of the liquid compositions disclosed herein, compared to a control liquid composition without at least one heat-stabilizing excipient, does not decrease by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges during storage of the liquid composition at a temperature of 30°C for a maximum of about one week or more, including all values ​​and partial ranges during that period, for a maximum of about two weeks, a maximum of about three weeks, a maximum of about four weeks, a maximum of about five weeks, a maximum of about six weeks, a maximum of about seven weeks, a maximum of about eight weeks, or more than eight weeks, including all values ​​and partial ranges during that period.

[0087] In some embodiments, the integrity of one or more RNA molecules does not decrease by more than about 40% after storage of the liquid composition at a temperature of 30°C for a maximum of about 1 week, a maximum of about 2 weeks, a maximum of about 3 weeks, a maximum of about 4 weeks, or more than 4 weeks, including all values ​​and partial ranges in between, compared to a control liquid composition without at least one heat-stabilizing excipient. In certain embodiments, the integrity of one or more RNA molecules does not decrease by more than about 45% after storage of the liquid composition at a temperature of 30°C for a maximum of about 1 week, a maximum of about 2 weeks, a maximum of about 3 weeks, a maximum of about 4 weeks, or more than 4 weeks, including all values ​​and partial ranges in between, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the integrity of one or more RNA molecules does not decrease by more than about 50% after storage of the liquid composition at a temperature of 30°C for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, or more than 4 weeks, including all values ​​and partial ranges in between, compared to a control liquid composition that does not contain at least one heat-stabilizing excipient.

[0088] The integrity of RNA molecules can be determined using any method known in the art, such as fragmentation analysis using capillary electrophoresis (CE) and / or capillary gel electrophoresis (CGE). For example, the integrity of RNA molecules can be determined using capillary gel electrophoresis or a fragment analyzer system. In some embodiments, the integrity of one or more RNA molecules encapsulated in LNPs of the liquid compositions disclosed herein is measured by capillary electrophoresis. In some embodiments, the integrity of one or more RNA molecules is measured by capillary gel electrophoresis. In other embodiments, the integrity of one or more RNA molecules is measured by a fragment analyzer system.

[0089] The thermal stability of the liquid compositions of the present disclosure is further extended to maintain such that the average particle size of LNPs does not substantially increase after storage of the liquid composition at temperatures above 0°C for a certain period of time. Accordingly, in some embodiments, the average particle size of LNPs in the liquid compositions of the present disclosure does not increase by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, at a temperature of 4°C, after storage of the liquid composition for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months, or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or more than 2 years, including all values ​​and partial ranges in between.

[0090] In some embodiments, the average particle size of LNPs does not increase by more than 35% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the average particle size of LNPs does not increase by more than 40% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the average particle size of LNPs does not increase by more than 45% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between.

[0091] In certain embodiments, the average particle size of LNPs in the liquid composition of the Disclosure, compared to a control liquid composition without at least one heat-stabilizing excipient, does not increase by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, after storage of the liquid composition for more than about one week at a temperature of 25°C, including all values ​​and partial ranges in between, for example, up to about two weeks, up to about three weeks, up to about four weeks, up to about five weeks, up to about six weeks, up to about seven weeks, up to about eight weeks, or more.

[0092] In some embodiments, the average particle size of LNPs does not increase by more than about 15% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 25°C, including all values ​​and partial ranges in between, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the average particle size of LNPs does not increase by more than about 20% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 25°C, including all values ​​and partial ranges in between, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the average particle size of LNPs does not increase by more than about 25% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 25°C, including all values ​​and partial ranges in between, compared to a control liquid composition that does not contain at least one heat-stabilizing excipient.

[0093] In some embodiments, the average particle size of LNPs in the liquid compositions of the Disclosure, compared to a control liquid composition without at least one heat-stabilizing excipient, does not increase by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, after storage of the liquid composition for more than about one week at a temperature of 30°C, including all values ​​and partial ranges in between, for example, up to about two weeks, up to about three weeks, up to about four weeks, up to about five weeks, up to about six weeks, up to about seven weeks, up to about eight weeks, or more than eight weeks. In some embodiments, the average particle size of LNPs does not increase by more than about 15% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 30°C, including all values ​​and partial ranges in between, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the average particle size of LNPs does not increase by more than about 20% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 30°C, including all values ​​and partial ranges in between, compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the average particle size of LNPs does not increase by more than about 25% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 30°C, including all values ​​and partial ranges in between, compared to a control liquid composition that does not contain at least one heat-stabilizing excipient.

[0094] The particle size can be determined using any method known in the art, such as dynamic light scattering (DLS). Generally, before storage, the LNPs contained in the liquid composition of this disclosure have an average particle size in the range of about 10 nm to about 1000 nm, for example, about 15 nm to about 750 nm, about 30 nm to about 500 nm, about 50 nm to about 250 nm, about 75 nm to about 200 nm, or about 80 nm to about 150 nm.

[0095] The thermal stability of the liquid compositions of the present disclosure is also further extended to maintain LNP encapsulation efficiency such that it does not substantially decrease after storage of the liquid compositions at temperatures above 0°C for a certain period of time. Accordingly, in some embodiments, the LNP encapsulation efficiency in the liquid compositions of the present disclosure does not decrease by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, at a temperature of 4°C, including all values ​​and partial ranges in between, for a maximum of about 1 month or more, including all values ​​and partial ranges in between, for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months, or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or more than 2 years, including all values ​​and partial ranges in between, after storage of the liquid compositions.

[0096] In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 15% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 20% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 25% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 30% after storage of the liquid composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between.

[0097] In certain embodiments, the LNP encapsulation efficiency in the liquid compositions of the Disclosure, compared to a control liquid composition without at least one thermal stabilizing excipient, does not decrease by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, after storage of the liquid composition at a temperature of 25°C for a maximum of about one week or more, including all values ​​and partial ranges in between, for a maximum of about two weeks, a maximum of about three weeks, a maximum of about four weeks, a maximum of about five weeks, a maximum of about six weeks, a maximum of about seven weeks, a maximum of about eight weeks, or more than eight weeks, including all values ​​and partial ranges in between.

[0098] In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 15% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 25°C, including all values ​​and partial ranges in between, compared with a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 20% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 25°C, including all values ​​and partial ranges in between, compared with a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 25% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 25°C, including all values ​​and partial ranges in between, compared to a control liquid composition that does not contain at least one thermally stabilizing excipient.

[0099] In some embodiments, the LNP encapsulation efficiency in the liquid compositions of the Disclosure, compared to a control liquid composition without at least one thermal stabilizing excipient, does not decrease by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, after storage of the liquid composition for more than about 1 week at a temperature of about 30°C, including all values ​​and partial ranges in between, for example, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, or more, including all values ​​and partial ranges in between.

[0100] In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 15% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 30°C, including all values ​​and partial ranges in between, compared with a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 20% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 30°C, including all values ​​and partial ranges in between, compared with a control liquid composition that does not contain at least one heat-stabilizing excipient. In some embodiments, the LNP encapsulation efficiency does not decrease by more than about 25% after storage of the liquid composition for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to 8 weeks, or more than 8 weeks, at a temperature of 30°C, including all values ​​and partial ranges in between, compared to a control liquid composition that does not contain at least one thermally stabilizing excipient.

[0101] The encapsulation efficiency of LNPs can be determined using any method known in the art, such as a fluorescence plate-based assay using RiboGreen reagent (Invitrogen). The encapsulation efficiency (EE%) is calculated by dividing (total RNA added - free uncaptured RNA) by the total RNA added.

[0102] In some embodiments, the RNA molecules encapsulated in the LNPs of the Disclosure encode one or more influenza virus proteins, such as HA and / or NA proteins, and the liquid compositions of the Disclosure induce a robust hemagglutination inhibition (HAI) titer after storage. In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than about 30%, such as about 25%, 20%, 15%, 10%, 5%, including all values ​​and partial ranges in between, at a temperature of 4°C for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, including all values ​​and partial ranges in between, after storage of the liquid composition.

[0103] In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than about 25% after storage of the liquid composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than 20% after storage of the liquid composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than 15% after storage of the liquid composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between.In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than 10% after storage of the liquid composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between. In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than about 5% after storage of the liquid composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of 4°C, including all values ​​and partial ranges in between.

[0104] In some embodiments, the RNA molecules encapsulated in the LNPs of this disclosure encode one or more influenza virus proteins, such as HA and / or NA proteins, and the HAI titer induced by the liquid composition does not decrease by more than about 30%, such as about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the liquid composition at a temperature of 25°C for a maximum of about one week or more, for example, a maximum of about two weeks, a maximum of about three weeks, a maximum of about four weeks, a maximum of about five weeks, a maximum of about six weeks, a maximum of about seven weeks, a maximum of about eight weeks, or more than eight weeks, including all values ​​and partial ranges in between, when compared to a control liquid composition without at least one heat-stabilizing excipient. In some embodiments, the HAI titer induced by the liquid composition does not decrease by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the liquid composition for more than about 1 week at a temperature of about 30°C, including all values ​​and partial ranges in between, for example, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, or more than 8 weeks, including all values ​​and partial ranges in between.

[0105] HAI titer can be measured using any method known in the art, such as using the influenza virus HAI test (Denka Seiken Co., Tokyo, Japan).

[0106] Thermostable RNA-LNP composition Given the remarkable finding that including a thermoreversible gelling agent or a specific heat-stabilizing excipient in an RNA molecule-containing LNP formulation substantially improves stability, including RNA stability, when stored as a liquid at temperatures above 0°C under refrigerated conditions (e.g., 2-8°C, e.g., 4°C), a heat-stable RNA-LNP composition comprising at least one thermoreversible gelling agent and at least one heat-stabilizing excipient described herein is also provided herein. In some embodiments, the at least one thermoreversible gelling agent comprises or is gelatin. In some embodiments, the at least one heat-stabilizing excipient comprises or is lipoic acid. In some embodiments, a heat-stable RNA-LNP composition comprising gelatin and lipoic acid is provided herein.

[0107] In some embodiments, at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 0.5% to about 1.5% by weight. In some embodiments, at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 0.5% by weight. In some embodiments, at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 1% by weight. In some embodiments, at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 1.5% by weight.

[0108] In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 1 mM to about 10 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 1 mM to about 5 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 1 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of 2 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 2.5 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 3 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 4 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, is present at a concentration of about 5 mM. In some embodiments, at least one heat-stabilizing excipient, such as lipoic acid, and one or more RNA molecules are present in a weight ratio of approximately 2.5:1 to approximately 15.5:1.

[0109] In some embodiments, at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 0.5% to about 1.5% by weight or 1% by weight, and at least one heat-stabilizing excipient, such as lipoic acid, is present in a concentration of about 1 mM to about 10 mM or about 1 mM to about 5 mM, or 1 mM, 2 mM, 3 mM, 4 mM or 5 mM.

[0110] In some embodiments, the heat-stable RNA-LNP composition comprises about 1% by weight of gelatin and about 1 mM of lipoic acid. In some embodiments, the composition is stable after storage at a temperature of about 2–8°C for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 106 months, up to about 11 months, or up to about 1 year, or more than 1 year, including all values ​​and partial ranges in between. As described anywhere in this specification, the stability of the composition can be measured by changes in the average particle size of the LNPs, the encapsulation efficiency of the LNPs, and / or the integrity of one or more RNA molecules encapsulated in the LNPs, using any method known in the art, such as those exemplified in this disclosure.

[0111] Thermally stable RNA-LNP liquid formulation This disclosure is further based, at least in part, on the surprising finding that the stability of the resulting formulation, including mRNA stability, is substantially improved when certain formulations containing LNP-encapsulated RNA molecules are stored as liquids at temperatures above 0°C under refrigerated conditions (e.g., 2–8°C). For example, the inventors of this disclosure have surprisingly found that the stability of the resulting formulation, including mRNA stability, is substantially improved by formulating LNP-encapsulated mRNA molecules using combinations of buffers (e.g., tris-hydroxymethyl-aminomethane or tris), pharmaceutically acceptable salts (e.g., sodium chloride or NaCl), disaccharides (e.g., sucrose), surfactants (e.g., poloxamers such as P188), and chelating agents (e.g., disodium ethylenediaminetetraacetate or EDTA), each present in the prescribed amounts disclosed herein. For example, mRNA instability, as measured by a decrease in mRNA integrity, is considered a major challenge to the fundamental therapeutic and commercial potential of mRNA. Therefore, the improved mRNA stability conferred by the thermally stable RNA-LNP liquid formulation of this disclosure provides an important solution to these problems.

[0112] This finding enables several important applications of the formulations disclosed herein, including long-term refrigerated storage life, long-term use life at room temperature, and long-term use stability at physiological temperatures. Achieving thermally stable RNA-LNP liquid formulations also enables commercially available and therapeutically desirable packaging and delivery options, including pre-filled syringes (PFS) and cartridges for patient-friendly autoinjectors and infusion pumps. Thus, the ability to stabilize liquid formulations containing LNP-encapsulated RNA molecules such as mRNA represents a valuable technology that will facilitate the broad use of RNA-LNP formulations, such as mRNA vaccines.

[0113] Accordingly, as described elsewhere in this specification, a heat-stable liquid formulation is provided comprising one or more RNA molecules encapsulated in an LNP, and a buffer (e.g., Tris), a pharmaceutically acceptable salt (e.g., NaCl), a disaccharide (e.g., sucrose), a surfactant (e.g., a poloxamer such as P188), and a chelating agent (e.g., EDTA), each present in the formulated amounts disclosed below, at a physiological pH (e.g., 7.5 ± 0.3) to facilitate administration, as described elsewhere in this specification. In some embodiments, the heat-stable liquid formulation of this disclosure further comprises trehalose.

[0114] cushioning agent The thermally stable liquid formulations of this disclosure include, for example, buffers such as Tris. While we do not wish to be bound by any theory, buffers can be used to stabilize the pH of a solution. Commonly used buffers include, but are not limited to, Tris, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), monosodium phosphate, and saline sodium citrate (SSC).

[0115] In some embodiments, the buffer contained in the thermally stable liquid formulation of the Disclosure is or contains Tris in an amount of about 10 mM to about 100 mM, for example, about 15 mM to about 80 mM, or about 20 mM to about 50 mM, encompassing all values ​​and partial ranges in between. In some embodiments, the buffer is or contains Tris in an amount of about 10 mM. In some embodiments, the buffer is or contains Tris in an amount of about 20 mM. In some embodiments, the buffer is or contains Tris in an amount of about 30 mM. In some embodiments, the buffer is or contains Tris in an amount of about 40 mM. In some embodiments, the buffer is or contains Tris in an amount of about 50 mM. In some embodiments, the buffer is or contains Tris in an amount of about 100 mM.

[0116] Pharmaceutically acceptable salts The heat-stable liquid formulations of this disclosure contain, for example, pharmaceutically acceptable salts such as NaCl. As used herein, the term “pharmaceutically acceptable” refers to substances described throughout this disclosure, when mixed with the active ingredient of this disclosure (e.g., mRNA) that is suitable for administration to humans. While not intending to be bound by any theory, pharmaceutically acceptable salts such as NaCl can be used to increase stability. Commonly used pharmaceutically acceptable salts include, but are not limited to, NaCl and calcium chloride (CaCl2).

[0117] In some embodiments, the pharmaceutically acceptable salt contained in the heat-stable liquid formulation of the present disclosure is or contains NaCl in amounts of about 10 mM to about 150 mM, e.g., about 20 mM to about 130 mM, about 30 mM to about 120 mM, or about 50 mM to about 100 mM, encompassing all values ​​and partial ranges in between. In some embodiments, the pharmaceutically acceptable salt is or contains NaCl in amounts of about 10 mM. In some embodiments, the pharmaceutically acceptable salt is or contains NaCl in amounts of about 30 mM. In some embodiments, the pharmaceutically acceptable salt is or contains NaCl in amounts of about 50 mM. In some embodiments, the pharmaceutically acceptable salt is or contains NaCl in amounts of about 80 mM. In some embodiments, the pharmaceutically acceptable salt is or contains NaCl in amounts of about 100 mM. In some embodiments, the pharmaceutically acceptable salt is about 120 mM of NaCl or contains NaCl. In some embodiments, the pharmaceutically acceptable salt is about 150 mM of NaCl or contains NaCl.

[0118] disaccharide The heat-stable liquid formulations of this disclosure contain one or more disaccharides. Certain disaccharides, such as sucrose and trehalose, are commonly used as cryoprotective agents to protect biological tissues from freeze damage.

[0119] In some embodiments, the disaccharide contained in the heat-stable liquid formulations of the Disclosure is sucrose or contains sucrose in an amount of about 1% to about 10% by weight, for example, about 2% to about 8% by weight, about 3% to about 6% by weight, or about 4% to about 5% by weight, including all values ​​and partial ranges between those amounts. In some embodiments, the disaccharide is sucrose or contains sucrose in an amount of about 1% by weight. In some embodiments, the disaccharide is sucrose or contains sucrose in an amount of about 3% by weight. In some embodiments, the disaccharide is sucrose or contains sucrose in an amount of about 5% by weight. In some embodiments, the disaccharide is sucrose or contains sucrose in an amount of about 10% by weight. In some embodiments, sucrose is the only disaccharide contained in the heat-stable liquid formulations of the Disclosure.

[0120] In other embodiments, the heat-stable liquid formulations of the Disclosure also include, in addition to sucrose, trehalose in amounts of about 0.1% to about 5% by weight, for example, about 0.2% to about 4% by weight, about 0.3% to about 3% by weight, about 0.4% to about 2% by weight, about 0.4% to about 1.5% by weight, about 0.4% to about 1.3% by weight, about 0.5% to about 4% by weight, about 1% to about 4% by weight, about 1.5% to about 3% by weight, about 2% to about 2.8% by weight, about 2% to about 2.6% by weight, about 2.5% to about 5% by weight, or about 2.5% to about 3.5% by weight, including all values ​​and partial ranges between them. In some embodiments, the heat-stable liquid formulations include trehalose in amounts of about 0.4% to about 1.3% by weight. In some embodiments, the heat-stable liquid formulation contains trehalose in an amount of about 2% to about 2.6% by weight.

[0121] In some embodiments, the heat-stable liquid formulation contains about 0.1% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 0.4% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 1% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 1.3% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 1.5% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 2% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 2.4% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 2.6% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 3% by weight of trehalose. In some embodiments, the heat-stable liquid formulation contains about 5% by weight of trehalose.

[0122] surfactant The heat-stable liquid formulations of this disclosure include surfactants such as nonionic surfactants (e.g., poloxamers such as poloxamer 188 (P188)). While we do not wish to be bound by any theory, surfactants may be used to prevent LNP aggregation. In some embodiments, the surfactants included in the heat-stable liquid formulations of this disclosure are nonionic surfactants. Commonly used nonionic surfactants include, but are not limited to, P188, polysorbate 20, and polysorbate 80.

[0123] In some embodiments, the surfactant contained in the heat-stable liquid formulations of the present disclosure is or contains a poloxamer such as P188 in an amount of about 0.1% to about 1% by volume, for example, about 0.2% to about 0.8% by volume, about 0.3% to about 0.7% by volume, or about 0.4% to about 0.6% by volume, encompassing all values ​​and partial ranges in between. In some embodiments, the surfactant is or contains a poloxamer such as P188 in an amount of about 0.1% by volume. In some embodiments, the surfactant is or contains a poloxamer such as P188 in an amount of about 0.2% by volume. In some embodiments, the surfactant is or contains a poloxamer such as P188 in an amount of about 0.4% by volume. In some embodiments, the surfactant is or contains a poloxamer such as P188 in an amount of about 0.6% by volume. In some embodiments, the surfactant is a poloxamer such as P188 in an amount of about 0.8 volume percent, or comprises a poloxamer. In some embodiments, the surfactant is a poloxamer such as P188 in an amount of about 1 volume percent, or comprises a poloxamer.

[0124] Chelating agents The thermally stable liquid formulations of this disclosure include chelating agents. While we do not wish to be bound by any theory, chelating agents may be used as stabilizers to complex heavy metals that may promote instability. Commonly used chelating agents include, but are not limited to, EDTA.

[0125] In some embodiments, the chelating agent contained in the heat-stable liquid formulation of the Disclosure is or contains EDTA in an amount of about 1 μM to about 50 μM, for example, about 5 μM to about 30 μM, or about 10 μM to about 25 μM, encompassing all values ​​and partial ranges in between. In some embodiments, the chelating agent is or contains EDTA in an amount of about 1 μM. In some embodiments, the chelating agent is or contains EDTA in an amount of about 5 μM. In some embodiments, the chelating agent is or contains EDTA in an amount of about 10 μM. In some embodiments, the chelating agent is or contains EDTA in an amount of about 15 μM. In some embodiments, the chelating agent is or contains EDTA in an amount of about 20 μM. In some embodiments, the chelating agent is or contains EDTA in an amount of about 30 μM. In some embodiments, the chelating agent is or contains EDTA in an amount of about 50 μM.

[0126] pH The heat-stable liquid formulations of this disclosure are at a physiological pH for ease of administration. In some embodiments, the pH of the heat-stable liquid formulations of this disclosure is about 7 to about 8, e.g., about 7.2 to about 7.8, or about 7.4 to about 7.7, including all values ​​and partial ranges in between. In some embodiments, the pH of the heat-stable liquid formulation is about 7.0, e.g., 7.0 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is about 7.1, e.g., 7.1 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is about 7.2, e.g., 7.2 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is about 7.3, e.g., 7.3 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is about 7.4, e.g., 7.4 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is about 7.5, e.g., 7.5 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is approximately 7.6, for example, 7.6 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is approximately 7.7, for example, 7.7 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is approximately 7.8, for example, 7.8 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is approximately 7.9, for example, 7.9 ± 0.3. In some embodiments, the pH of the heat-stable liquid formulation is approximately 8.0, for example, 8.0 ± 0.3.

[0127] Exemplary thermally stable liquid formulations In some embodiments, the heat-stable liquid formulations of the present disclosure contain, in addition to the RNA molecule (e.g., mRNA) encapsulated in the LNP, about 10–60 mM Tris, about 40–150 mM NaCl, about 1–10% by weight sucrose, about 0.2–0.6% by volume P188, and about 5–15 μM EDTA with a pH of about 7.2–7.8. In some embodiments, the heat-stable liquid formulations of the present disclosure contain, in addition to the RNA molecule (e.g., mRNA) encapsulated in the LNP, about 10–60 mM Tris, about 40–110 mM NaCl, about 3–6% by weight sucrose, about 0.2–4% by weight trehalose, about 0.2–0.6% by volume P188, and about 5–15 μM EDTA with a pH of about 7.5–7.7. In some embodiments, the heat-stable liquid formulations of the present disclosure contain, in addition to RNA molecules (e.g., mRNA) encapsulated in LNPs, about 20–50 mM Tris, about 50–100 mM NaCl, about 2–5% by weight sucrose, about 0.3–3% by weight trehalose, about 0.2–0.4% by volume P188, and about 10–15 μM EDTA with a pH of about 7.7.

[0128] In some embodiments, the heat-stable liquid formulations of the present disclosure contain, in addition to the RNA molecule (e.g., mRNA) encapsulated in the LNP, about 50 mM Tris, about 150 mM NaCl, about 5% by weight sucrose, about 0.4% by volume P188, and about 10 μM EDTA with a pH of about 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable liquid formulations of the present disclosure contain, in addition to the RNA molecule (e.g., mRNA) encapsulated in the LNP, about 50 mM Tris, about 50 mM NaCl, about 5% by weight sucrose, about 2 to 2.6% by weight trehalose, about 0.4% by volume P188, and about 10 μM EDTA with a pH of about 7.7. In some embodiments, the heat-stable liquid formulations of the present disclosure contain, in addition to RNA molecules (e.g., mRNA) encapsulated in LNPs, about 20 mM Tris, about 100 mM NaCl, about 5% by weight sucrose, about 0.4–1.3% by weight trehalose, about 0.4% by volume P188, and about 10 μM EDTA with a pH of about 7.7.

[0129] thermal stability The inventors have demonstrated that the heat-stable liquid formulations of this disclosure can remain stable in liquid form after storage for several months at a temperature of approximately 2–8°C (e.g., 4°C), corresponding to standard refrigeration conditions. Therefore, in some embodiments, the heat-stable liquid formulations of this disclosure remain stable in liquid form after storage for up to approximately 1 month, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, up to approximately 9 months, up to approximately 10 months, up to approximately 11 months, or up to approximately 1 year, or more than 1 year, at a temperature of approximately 2–8°C (e.g., 4°C), including all values ​​and partial ranges in between. The stability of the heat-stable liquid formulations can be measured by changes in the average particle size of the LNPs, the encapsulation efficiency of the LNPs, and / or the integrity of one or more mRNA molecules encapsulated within the LNPs.

[0130] In some embodiments, the stability of a thermally stable liquid formulation is measured by the change in the average particle size of LNPs, where the average particle size of LNPs does not increase by more than about 20%, e.g., about 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the thermally stable liquid formulation for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months, or a maximum of about 1 year, or more than 1 year, including all values ​​and partial ranges in between. The particle size can be determined using any method known in the art, such as dynamic light scattering (DLS). Generally, before storage, the LNPs contained in the thermally stable liquid formulations of this disclosure have an average particle size in the range of about 10 nm to about 1000 nm, for example, about 15 nm to about 750 nm, about 30 nm to about 500 nm, about 50 nm to about 250 nm, about 75 nm to about 200 nm, or about 80 nm to about 150 nm.

[0131] In some embodiments, the stability of a heat-stable liquid formulation is measured by the change in LNP encapsulation efficiency, which does not decrease by more than about 20%, e.g., about 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the heat-stable liquid formulation for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, or up to about 1 year, or more than 1 year, including all values ​​and partial ranges in between. The LNP encapsulation efficiency can be determined using any method known in the art, such as a fluorescence plate-based assay using RiboGreen reagent (Invitrogen). The encapsulation efficiency (EE%) is calculated by dividing (total RNA added - free uncaptured RNA) by the total RNA added.

[0132] In some embodiments, the stability of a heat-stable liquid formulation is measured by the change in the integrity of one or more mRNA molecules encapsulated in the LNP, and the integrity of the mRNA molecules does not decrease by more than about 20%, e.g., about 15%, 10%, or 5%, including all values ​​and partial ranges during storage of the immunogenic composition for up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, or up to about 1 year, or after storage of the immunogenic composition for more than 1 year, including all values ​​and partial ranges during that period. The integrity of the mRNA molecules can be determined using any method known in the art, such as fragmentation analysis using capillary electrophoresis (CE) and / or capillary gel electrophoresis (CGE). For example, the integrity of mRNA molecules can be determined using capillary gel electrophoresis or a fragment analyzer system. In some embodiments, the integrity of one or more mRNA molecules encapsulated in LNPs of the thermally stable liquid formulations disclosed herein is measured by capillary electrophoresis. In some embodiments, the integrity of one or more mRNA molecules is measured by capillary gel electrophoresis. In other embodiments, the integrity of one or more mRNA molecules is measured by a fragment analyzer system.

[0133] Transport vehicle In certain embodiments, the heat-stable compositions of this disclosure comprise one or more RNA molecules, such as mRNA molecules, and a transport vehicle. As used herein, the term “transport vehicle” includes any of the following standard pharmaceutical carriers, diluents, excipients, etc., which are generally intended for use in connection with the administration of biologically active agents containing RNA (e.g., mRNA). The compositions, and in particular the transport vehicles described herein, are capable of delivering RNA (e.g., mRNA) of various sizes to their target cells or tissues. In some embodiments, the transport vehicles of this disclosure are capable of delivering large RNA molecules (e.g., RNA of at least 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5 kDa, 10 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa or larger, e.g., mRNA). The RNA (e.g., mRNA) can be formulated using one or more acceptable reagents that provide a vehicle for delivering such RNA (e.g., mRNA) to target cells. Appropriate reagents are generally selected with respect to several factors, including the biological or chemical properties of the RNA (e.g., charge), the intended administration route, the biological environment to which such RNA (e.g., mRNA) will be exposed, and the specific characteristics of the intended target cells. In some embodiments, a transport vehicle, such as a liposome, encapsulates the RNA (e.g., mRNA) without impairing its biological activity. In some embodiments, the transport vehicle demonstrates preferential and / or substantial binding to the target cell over non-target cells. In certain embodiments, the transport vehicle delivers its contents to the target cell so that the RNA (e.g., mRNA) reaches a suitable intracellular compartment, such as the cytoplasm.

[0134] In some embodiments, the transport vehicle is a liposomal vesicle or other means for facilitating the transport of one or more RNA (e.g., mRNA) molecules to target cells and tissues. Suitable transport vehicles include, but are not limited to, liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, nanoparticles, calcium phosphate-silicate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, nanocrystalline particles, semiconductor nanoparticles, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other vector-like tags. The use of bionanopapellet and other viral capsid protein assemblies as suitable transport vehicles is also envisioned. See, for example, Kasuya et al., Hum. Gene Ther., 2008, 19(9):887-895. The use of polymers as transport vehicles, either alone or in combination with other transport vehicles, is also envisioned. Suitable polymers may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumins, alginates, collagens, chitosans, cyclodextrins, and polyethyleneimines. In some embodiments, the transport vehicle is selected based on its ability to facilitate the transfection of one or more RNA (e.g., mRNA) molecules into target cells.

[0135] Lipid nanoparticles In some embodiments, the transport vehicle is formulated as lipid nanoparticles (LNPs). The terms “lipid nanoparticles” or “LNPs” refer to particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm), comprising one or more lipids, e.g., cationic and / or non-cationic lipids, and one or more excipients selected from neutral lipids, anionic lipids, zwitterionic lipids, ionizable lipids, steroids, and polymer-conjugated lipids (e.g., PEGylated lipids). Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). In certain embodiments, the compositions of this disclosure comprise one or more RNA (e.g., mRNA) molecules encapsulated in the LNPs. RNA-encapsulating LNP compositions are known in the art, for example, as described in PCT publications WO2021 (International Publication No. / 237084) and WO2022 (International Publication No. / 099003), the contents of which are all incorporated herein by reference. Any known LNP formulation may be used in the embodiments disclosed herein. In some embodiments, the LNP comprises a mixture of four types of lipids: ionized (e.g., cationic) lipids, polyethylene glycol (PEG) complexed lipids, cholesterol-based lipids, and helper lipids such as phospholipids. The LNP is used to encapsulate RNA molecules (e.g., mRNA molecules). The encapsulated RNA molecules (e.g., mRNA molecules) may consist of native ribonucleotides, chemically modified nucleotides, or combinations thereof, and may encode one or more proteins individually or collectively.

[0136] Ionizable or cationic lipids Ionizable lipids can be cationic lipids that promote the encapsulation of RNA molecules (e.g., mRNA molecules). Cationic lipids provide a positively charged environment at low pH, making it easier to efficiently encapsulate, for example, negatively charged RNA molecules (e.g., mRNA molecules). Suitable cationic lipids for LNP formulations include, but are not limited to, ALC-0315, OF-02, cKK-E10, cKK-E12, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14.

[0137] ALC-0315 ([(4-hydroxybutyl)azanejyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)) has the following chemical structure: [ka] It is a synthetic lipid containing [a specific compound / component]. ALC-0315 is a colorless oily substance and is attracting attention as a component of the SARS-CoV-2 vaccine Comirnaty® (BNT162b2) developed by Pfizer-BioNTech. Below physiological pH, ALC-0315 is protonated by a nitrogen atom, producing an ammonium cation that is attracted to anionic messenger RNA (mRNA).

[0138] OF-02 (3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadieno-1-yl]amino]butyl]-2,5-piperazinedione, CAS number 1883431-67-1) is an alkenyl amino alcohol (AAA) ionizing lipid for highly potent in vivo mRNA delivery, and has the following chemical structure: [ka] OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains a degradable ester bond connecting a diketopiperazine core and a double unsaturated tail, while OF-02 contains a non-degradable 1,2-amino-alcohol bond connecting the same diketopiperazine core and double unsaturated tail. See Fenton et al., Adv. Mater., 2016, 28(15):2939-2943; U.S. Patent No. 10,201,618, incorporated herein by reference.

[0139] cKK-E10 and cKK-E12 are two cationic lipids that can be used in lipid nanoparticles for delivering nucleic acids to various cell types (Dong et al., PNAS, 2014, 111(11):3955-3960; U.S. Patent No. 9,512,073, both of which are incorporated herein by reference). cKK-E12 has been used to deliver siRNA to mice, rats, and primates (ED50 = 0.002, 0.01 & 0.3 mg / kg, respectively). See Dong et al. above. It has been shown to have low toxicity and is selective to hepatic parenchymal cells more than to liver, heart, lung, and kidney endothelial cells.

[0140] cKK-E10 has the following chemical structure: [ka] It holds.

[0141] cKK-E12 (3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, catalog number 1432494-65-9) has the following chemical structure: [ka] It holds.

[0142] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14, described in PCT Publication No. 2022 / 221688A1 (incorporated herein by reference), are HEPES-based disulfide cationic lipids having a piperazine core. GL-HEPES-E3-E10-DS-3-E18-1 ((2-(4-(2-((3-(bis((Z)-2-hydroxyoctadeca-9-en-1-yl)amino)propyl)disulfanyl)ethyl)piperazine-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate) has the following chemical structure: [ka] It holds.

[0143] GL-HEPES-E3-E12-DS-4-E10(2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate) has the following chemical structure: [ka] It holds.

[0144] GL-HEPES-E3-E12-DS-3-E14(2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate) has the following chemical structure: [ka] It holds.

[0145] Other cationic lipids that may be used include those described above in Dong et al.; U.S. Patent No. 10,201,618, and PCT Publication No. International Publication No. / 221688A1 (all of which are incorporated herein by reference).

[0146] Accordingly, in some embodiments, the cationic lipids used to form LNPs according to the Disclosure include ALC-0315, OF-02, cKK-E10, cKK-E12, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10 and / or GL-HEPES-E3-E12-DS-3-E14. In some embodiments, the cationic lipid includes ALC-0315. In some embodiments, the cationic lipid includes OF-02. In some embodiments, the cationic lipid includes cKK-E10. In some embodiments, the cationic lipid includes cKK-E12. In some embodiments, the cationic lipid includes GL-HEPES-E3-E10-DS-3-E18-1. In some embodiments, the cationic lipid includes GL-HEPES-E3-E12-DS-4-E10. In some embodiments, the cationic lipid includes GL-HEPES-E3-E12-DS-3-E14.

[0147] PEGylated lipids PEGylated lipid components provide control over the particle size and stability of nanoparticles. Adding such components can prevent complex aggregation, extend circulating life, and provide a means to increase the delivery of lipid-nucleic acid drug compositions to target tissues. See Klibanov et al., FEBS Letters, 1990, 268(1):235-237. These components may be selected to be rapidly replaced from the drug composition in vivo. See, for example, U.S. Patent No. 5,885,613.

[0148] The intended PEGylated lipids include derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)]) (C8PEG-ceramide), such as C6-C 20 (For example, C8, C 10 , C 12 , C 14 , C 16 or C 18 Examples include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length, covalently bonded to a lipid having an alkyl chain of ) length. In some embodiments, the PEGylated lipids include 1,2-dimiristoyl-rac-glycero-3-methoxy-polyethylene glycol (DMG-PEG, also known as DMG-PEG2000); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG) and / or N,N-ditetradecylacetamide-polyethylene glycol (e.g., ALC-0159). In some embodiments, the PEGylated lipids used in the LNPs of this disclosure include 1,2-dimiristoyl-rac-glycero-3-methoxy-polyethylene glycol (DSG-PEG2000). In some embodiments, the PEGylated lipid comprises N,N-ditetradecylacetamide-polyethylene glycol.

[0149] PEG preferably has a high molecular weight, for example, 2000 to 2400 g / mol. In some embodiments, PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipids as used herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, and / or C8PEG2000. In some embodiments, the PEGylated lipid is dimyristoyl-PEG2000.

[0150] Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao et al., Biochem. Biophys. Res. Comm., 1991, 179:280; Wolf et al., BioTechniques, 1997, 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol ester ("ICE"; International Publication No. 2011 / 068810), β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP of this disclosure is cholesterol.

[0151] Helper lipids Helper lipids enhance the structural stability of LNPs and assist in LNP extrusion into endosomes. This improves the uptake and release of ribonucleic acid molecules (e.g., mRNA) drug payloads. In some embodiments, the helper lipid is a zwitterionic lipid having fusion properties to improve drug payload uptake and release. In some embodiments, the helper lipid is a phospholipid. Examples of helper lipids include 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dieridoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE). In some embodiments, the helper lipids used in the LNPs of this disclosure include DOPE. In some embodiments, the helper lipid includes DSPC.

[0152] Other exemplary helper lipids include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and dipalmitoylphosphatidyl These include ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof.

[0153] Exemplary lipid nanoparticle composition Accordingly, in some embodiments, the LNP according to the present disclosure comprises (i) cationic lipids, e.g., OF-02, cKK-E10, cKK-E12, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14 and / or ALC-0315; (ii) PEGylated lipids, e.g., DMG-PEG2000 or N,N-ditetradecylacetamido-polyethylene glycol; (iii) cholesterol-based lipids, e.g., cholesterol and (iv) helper lipids, e.g., DOPE or DSPC. In some embodiments, the LNP comprises OF-02 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol and DOPE as a helper lipid. In some embodiments, LNP comprises cKK-E10 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid. In some embodiments, LNP comprises cKK-E12 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid. In some embodiments, LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid. In some embodiments, LNP comprises GL-HEPES-E3-E12-DS-4-E10 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid. In some embodiments, LNP comprises GL-HEPES-E3-E12-DS-3-E14 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid. In some embodiments, LNP comprises ALC-0315 as a cationic lipid, N,N-ditetradecylacetamide-polyethylene glycol as a PEGylated lipid, cholesterol, and DSPC as a helper lipid.

[0154] Molar ratio of lipid components The molar ratios of the LNP components described above can enhance the effectiveness of the LNP in the delivery of the encapsulated RNA molecule (e.g., mRNA). The molar ratio of cationic lipids, PEGylated lipids, cholesterol lipids, and helper lipids is A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipids in the LNP to total lipids (i.e., A) is about 30-60%, for example, about 30-50%, 30-45%, 30-40%, 35-55%, 35-50%, 35-45%, 30-50%, or 30-40%, encompassing all values ​​and sub-ranges in between. In some embodiments, the molar ratio of PEGylated lipid components to total lipids (i.e., B) is about 0.25–15%, for example, about 0.25–10%, 0.25–7.5%, 0.25–5%, 0.5–15%, 0.5–10%, 0.5–7.5%, 0.5–5%, 1–15%, 1–10%, 1–7.5%, or 1–5%, encompassing all values ​​and partial ranges in between. In some embodiments, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is about 20–40%, for example, about 20–35%, 20–30%, 25–40%, 25–35%, or 25–30%, encompassing all values ​​and partial ranges in between. In some embodiments, the molar ratio of helper lipids to total lipids (i.e., D) is about 20–40%, for example, about 20–35%, 20–30%, 25–40%, 25–35%, or 25–30%, encompassing all values ​​and partial ranges in between. In some embodiments, the molar ratio of cationic lipids in LNP to total lipids (i.e., A) is about 30–50%, the molar ratio of PEGylated lipid components to total lipids (i.e., B) is about 0.25–15%, the molar ratio of cholesterol lipids to total lipids (i.e., C) is about 20–40%, and the molar ratio of helper lipids to total lipids (i.e., D) is about 20–40%. In some embodiments, the molar ratio of cationic lipids in LNP to total lipids (i.e., A) is approximately 35-45%, the molar ratio of PEGylated lipid components to total lipids (i.e., B) is approximately 0.25-7.5%, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is approximately 25-35%, and the molar ratio of helper lipids to total lipids (i.e., D) is approximately 25-35%.In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the helper lipid. In some embodiments, the molar ratio of cationic lipid to helper lipid in the LNP is greater than 1.

[0155] To calculate the actual amount of each lipid contained in an LNP formulation, first determine the molar amount of the cationic lipid based on the desired N / P ratio (where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the ribonucleic acid molecule (e.g., mRNA) transported by the LNP). Next, calculate the molar amount of each of the other lipids based on the molar amount of the cationic lipid and the selected molar ratio. Then, convert these molar amounts to weight using the molecular weight of each lipid.

[0156] In some embodiments, LNP contains cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids in a molar ratio of approximately 40:1.5:28.5:30, i.e., cationic lipids are present in a molar ratio of approximately 40%, PEGylated lipids in a molar ratio of approximately 1.5%, cholesterol-based lipids in a molar ratio of approximately 28.5%, and helper lipids in a molar ratio of approximately 30%. In some embodiments, LNP contains OF-02 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, and DOPE as cholesterol and helper lipids in a molar ratio of approximately 40:1.5:28.5:30. In some embodiments, LNP contains cKK-E10 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, and DOPE as cholesterol and helper lipids in a molar ratio of approximately 40:1.5:28.5:30. In some embodiments, LNP contains cKK-E12 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:1.5:28.5:30. In some embodiments, LNP contains GL-HEPES-E3-E10-DS-3-E18-1 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:1.5:28.5:30. In some embodiments, LNP contains GL-HEPES-E3-E12-DS-4-E10 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:1.5:28.5:30. In some embodiments, LNP contains GL-HEPES-E3-E12-DS-3-E14 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:1.5:28.5:30.

[0157] In some embodiments, LNP contains cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids in a molar ratio of approximately 40:5:25:30, i.e., cationic lipids are present in a molar ratio of approximately 40%, PEGylated lipids in a molar ratio of approximately 5%, cholesterol-based lipids in a molar ratio of approximately 25%, and helper lipids in a molar ratio of approximately 30%. In some embodiments, LNP contains OF-02 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, and DOPE as cholesterol and helper lipids in a molar ratio of approximately 40:5:25:30. In some embodiments, LNP contains cKK-E10 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, and DOPE as cholesterol and helper lipids in a molar ratio of approximately 40:5:25:30. In some embodiments, LNP contains cKK-E12 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:5:25:30. In some embodiments, LNP contains GL-HEPES-E3-E10-DS-3-E18-1 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:5:25:30. In some embodiments, LNP contains GL-HEPES-E3-E12-DS-4-E10 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:5:25:30. In some embodiments, LNP contains GL-HEPES-E3-E12-DS-3-E14 as a cationic lipid, DMG-PEG2000 as a PEGylated lipid, cholesterol, and DOPE as a helper lipid in a molar ratio of approximately 40:5:25:30.

[0158] In some embodiments, the LNP is (i) ALC-0315 as a cationic lipid in a molar ratio of about 25% to about 65%, for example, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%; (ii) N,N-ditetradecyl as a PEGylated lipid in a molar ratio of about 0.5% to about 3%, for example, about 0.5%, 1%, 1.5%, 2%, 2.5%, or 3%. (iii) DSPC as a helper lipid in molar ratios of approximately 5% to approximately 15%, for example, approximately 5%, 7.5%, 10%, 12.5%, or 15%, and (iv) cholesterol in molar ratios of approximately 20% to approximately 60%, for example, approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0159] RNA molecule Any RNA molecule may be encapsulated in the LNP formulations of this disclosure, but are not limited to, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small activating RNAs (saRNAs), microRNAs (miRNAs), aptamers, long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs). The remarkable success of the COVID-19 vaccines Comirnaty® (BNT162b2) and Spikevax (mRNA-1273) demonstrated the clinical validation of mRNA formulated in lipid nanoparticles as a new class of highly effective nucleic acids in the vaccine field. Therefore, in some embodiments, the RNA molecule encapsulated in the LNPs according to this disclosure is an mRNA molecule.

[0160] In some embodiments, the LNP or LNP formulation according to the Disclosure may be monovalent, meaning that the LNP encapsulates an RNA molecule (e.g., mRNA) encoding the same protein, such as an antigen in some embodiments. In some embodiments, the LNP or LNP formulation according to the Disclosure may be polyvalent, meaning that the LNP encapsulates at least two different proteins, such as RNA molecules (e.g., mRNA) encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different proteins. In some embodiments, if the LNP or LNP formulation according to the Disclosure is polyvalent, the RNA molecule (e.g., mRNA) encapsulated in the LNP may encode at least two different antigens of the same or different pathogens (e.g., viruses), such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different antigens. For example, an LNP may contain multiple RNA molecules (e.g., mRNA) each encoding a different antigen; or it may contain polycistronic mRNA that can be translated into two or more antigens (e.g., each antigen-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). An LNP containing different RNA molecules (e.g., mRNA) typically contains (encapsulates) multiple copies of each mRNA molecule. For example, an LNP containing or encapsulating two different RNA molecules (e.g., mRNA) typically contains multiple copies of each of the two different RNA molecules (e.g., mRNA).

[0161] In some embodiments, a single LNP formulation may contain multiple types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of LNPs, each carrying a different RNA molecule (e.g., mRNA).

[0162] mRNA molecule In some embodiments, the RNA molecule (e.g., mRNA) encapsulated in the LNP or LNP formulation according to this disclosure may encode one or more viral polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) of the same or different viruses. For example, in some embodiments, the RNA molecule (e.g., mRNA) encapsulated in the LNP or LNP formulation may encode one or more influenza virus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10), such as influenza hemagglutinin (HA) and / or neuraminidase (NA) proteins of the same or different types of influenza virus. In some embodiments, the RNA molecule (e.g., mRNA) may encode one or more influenza virus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) selected from H1, H3, B / Victoria lineage HA and / or B / Yamagata lineage HA. In some embodiments, the RNA molecule (e.g., mRNA) encodes three different influenza virus proteins (e.g., trivalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, and HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage. In some embodiments, the RNA molecule (e.g., mRNA) encodes four different influenza virus proteins (e.g., tetravalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain derived from the B / Yamagata lineage. In some embodiments, the LNP or LNP formulation according to this disclosure is trivalent if the RNA molecule (e.g., mRNA) encodes three different influenza virus proteins, such as H1, H3, and HA from the B / Victoria lineage. In some embodiments, the LNP or LNP formulation according to the present disclosure is tetravalent when the RNA molecule (e.g., mRNA) encodes four different influenza virus proteins, such as H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage.In some embodiments, H1 is derived from an H1N1 influenza virus strain. In some embodiments, H3 is derived from an H3N2 influenza virus strain. In some embodiments, H1 is derived from an H1N1 influenza virus strain, and H3 is derived from an H3N2 influenza virus strain.

[0163] The World Health Organization (WHO) annually selects influenza strains to be included in seasonal vaccine formulations based on intensive surveillance efforts. Therefore, as used herein, the term “standard therapeutic strain” refers to the influenza strain selected by the World Health Organization (WHO) for inclusion in seasonal vaccine formulations. Standard therapeutic strains may include past, current, or future standard therapeutic strains.

[0164] In other embodiments, the RNA molecule (e.g., mRNA) encodes one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) selected from N1, N2, NA from the B / Victoria lineage and / or NA from the B / Yamagata lineage. In some embodiments, the RNA molecule (e.g., mRNA) encodes three different influenza virus proteins: N1 from a first standard treatment influenza virus strain, N2 from a second standard treatment influenza virus strain, and NA from a third standard treatment influenza virus strain of the B / Victoria lineage. In some embodiments, the RNA molecule (e.g., mRNA) encodes four different influenza virus proteins: N1 from a first standard treatment influenza virus strain, N2 from a second standard treatment influenza virus strain, NA from a third standard treatment influenza virus strain of the B / Victoria lineage, and NA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage. In some embodiments, the LNP or LNP formulation according to the Disclosure is trivalent if the RNA molecule (e.g., mRNA) encodes three different influenza virus proteins, such as N1, N2, and NA from the B / Victoria lineage. In some embodiments, the LNP or LNP formulation according to the Disclosure is tetravalent if the RNA molecule (e.g., mRNA) encodes four different influenza virus proteins, such as N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage. In some embodiments, N1 is derived from the H1N1 influenza virus strain. In some embodiments, N3 is derived from the H3N2 influenza virus strain. In some embodiments, N1 is derived from the H1N1 influenza virus strain and N3 is derived from the H3N2 influenza virus strain.

[0165] In other embodiments, the RNA molecule (e.g., mRNA) encodes one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) selected from HAs derived from H1, H3, and B / Victoria lineages, and / or HAs derived from B / Yamagata lineages, and one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) selected from N1, N2, and NAs derived from B / Victoria lineages, and / or NAs derived from B / Yamagata lineages. In some embodiments, an RNA molecule (e.g., mRNA) encodes eight different influenza virus proteins (e.g., octavalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, N1 from a fifth standard treatment influenza virus strain, N2 from a sixth standard treatment influenza virus strain, NA from a seventh standard treatment influenza virus strain of the B / Victoria lineage, and NA from an eighth standard treatment influenza virus strain of the B / Yamagata lineage. In some embodiments, H1 is derived from an H1N1 influenza virus strain. In some embodiments, H3 is derived from an H3N2 influenza virus strain. In some embodiments, H1 is derived from an H1N1 influenza virus strain and H3 is derived from an H3N2 influenza virus strain. In some embodiments, N1 is derived from an H1N1 influenza virus strain. In some embodiments, N2 is derived from an H3N2 influenza virus strain. In some embodiments, N1 is derived from an H1N1 influenza virus strain and N2 is derived from an H3N2 influenza virus strain. In some embodiments, H1 is derived from an H1N1 influenza virus strain and H3 is derived from an H3N2 influenza virus strain, and N1 is derived from an H1N1 influenza virus strain and N2 is derived from an H3N2 influenza virus strain. In some embodiments, H1 and N1 are derived from the same H1N1 influenza virus strain. In some embodiments, H1 and N1 are derived from different H1N1 influenza virus strains.In some embodiments, H3 and N2 are derived from the same H3N2 influenza virus strain. In some embodiments, H3 and N2 are derived from different H3N2 influenza virus strains. In some embodiments, HA and NA of the B / Yamagata lineage are derived from the same influenza virus strain. In some embodiments, HA and NA of the B / Yamagata lineage are derived from different influenza virus strains. In some embodiments, HA and NA of the B / Victoria lineage are derived from the same influenza virus strain. In some embodiments, HA and NA of the B / Victoria lineage are derived from different influenza virus strains.

[0166] In some embodiments, the RNA molecule (e.g., mRNA) encodes one or more respiratory syncytial virus (RSV) polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10), such as receptor-binding glycoproteins (G), fusion proteins (F), and / or short hydrophobic (SH) proteins derived from the same or different subtypes of respiratory syncytial virus (RSV). In other embodiments, the RNA molecule (e.g., mRNA) encodes one or more coronavirus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10), particularly the spike protein (S).

[0167] The RNA molecules (e.g., mRNA) encapsulated in the LNP or LNP formulations of this disclosure can also encode one or more viral polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) of different viruses. Therefore, in some embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) and one or more (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) coronavirus proteins. In some embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) and one or more (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) respiratory syncytial virus (RSV) proteins. In some embodiments, the RNA molecule (e.g., mRNA) encodes one or more coronavirus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) and one or more (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) respiratory syncytial virus (RSV) proteins. In some embodiments, the RNA molecule (e.g., mRNA) encodes one or more influenza virus polypeptides (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) coronavirus proteins, and one or more (e.g., types 2, 3, 4, 5, 6, 7, 8, 9, 10) respiratory syncytial virus (RSV) proteins. In some embodiments, the RNA molecule (e.g., mRNA) encodes a polypeptide derived from any combination of viral proteins.

[0168] The RNA molecules (e.g., mRNA) according to this disclosure may be self-amplified mRNA. Conventional antigen expression from mRNA is proportional to the number of mRNA molecules successfully delivered to the target from the immunogenic composition or vaccine. However, self-amplified mRNA comprises genetically engineered replicons derived from self-replicating viruses and may therefore be added to immunogenic compositions or vaccines at lower doses than conventional mRNA while achieving equivalent results.

[0169] The self-replicating mRNA may encode any of the viral proteins disclosed herein, including, for example, influenza virus HA (e.g., HA from H1, H3, B / Victoria lineages and / or HA from B / Yamagata lineages), influenza virus NA (e.g., N1, N2, NA from B / Victoria lineages and / or NA from B / Yamagata lineages), respiratory syncytial virus (RSV) proteins (e.g., G protein, F protein and / or SH protein), and coronavirus proteins (e.g., spike protein).

[0170] The RNA molecule (e.g., mRNA) may be unmodified (i.e., containing only natural ribonucleotides A, U, C and / or G linked by phosphodiester bonds) or chemically modified (e.g., pseudouridine (e.g., N1--methylpseudridine), nucleotide analogs including 2'-fluororibonucleotide or 2-methoxyribonucleotide, and / or phosphorothioate bonds). The RNA molecule (e.g., mRNA) may contain a 5' cap and a poly-A tail. In some embodiments, one or more RNA molecules contain one or more modified nucleotides, and in some embodiments, one or more modified nucleotides are selected from pseudouridine, methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, all uridines in the ribonucleic acid molecule are replaced by pseudouridines, such as methylpseudridine, such as 1N-methylpseudridine. In some embodiments, one or more RNA molecules contain one or more phosphorothioate bonds.

[0171] When used as an immunogenic composition or vaccine, each RNA molecule is present in the composition disclosed herein in an amount effective to induce an immune response in the subject to which the composition or vaccine is administered. In some embodiments, each RNA molecule may be present in the composition disclosed herein in amounts ranging from, for example, about 0.1 μg to about 150 μg, for example, about 5 μg to about 120 μg, about 10 μg to about 60 μg, about 1 μg to about 60 μg, about 5 μg to about 45 μg, or about 15 μg to about 45 μg. In some embodiments, each RNA molecule is present in the composition in an amount sufficient to encode viral proteins, for example, about 5 μg to about 120 μg, for example, about 10 μg to about 60 μg, or about 15 μg to about 45 μg, such as influenza virus HA or NA protein, respiratory syncytial virus (RSV) protein (e.g., G protein, F protein and / or SH protein), and / or coronavirus protein (e.g., spike protein).

[0172] The molar ratio of nitrogen (N) in ionizable lipids to phosphate (P) in RNA molecules has an effect on the in vitro and in vivo interactions of RNA-LNP complexes. See, for example, Gary et al., Macromol. Biosci., 2013, 13(8):1059-1071. Accordingly, in some embodiments, the liquid compositions of the present disclosure have an N / P ratio of about 1 to about 10, e.g., about 1 to about 8, about 1 to about 6, about 1 to about 4, about 2 to about 8, about 2 to about 6, about 3 to about 6, or about 4 to about 6, encompassing all values ​​and partial ranges in between. In some embodiments, the liquid compositions of the present disclosure have an N / P ratio of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0173] Exemplary Thermostable RNA-LNP Compositions The thermoreversible gelling agents, thermostable excipients, and thermostable formulations disclosed herein can be used alone or in combination to stabilize compositions containing LNPs that encapsulate RNA molecules (including mRNA molecules). For example, one or more thermoreversible gelling agents disclosed herein can be used together with one or more thermostable excipients disclosed herein, and / or one of the thermostable formulations disclosed herein to stabilize a composition containing LNPs that encapsulate RNA (such as mRNA) encoding one or more viral polypeptides (e.g., influenza HA and / or the same or different types of influenza viruses). Exemplary thermostable RNA-LNP compositions according to this disclosure are provided herein below.

[0174] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 to 10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 10 to 60 mM of a buffer (e.g., Tris), about 40 to 150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 1 to 10% by weight of a disaccharide (e.g., sucrose), about 0.2 to 0.6% by volume of a surfactant (e.g., poloxamer such as P188), and about 5 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.2 to 7.8. In some embodiments, the thermally stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 to 10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 10 to 60 mM of a buffer (e.g., Tris), about 40 to 110 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 1 to 10% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2 to 0.6 volume of a surfactant (e.g., poloxamer such as P188), and about 5 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.5 to 5% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 20 to 50 mM of a buffer (e.g., Tris), about 50 to 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 3 to 8% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2 to 0.4 volume of a surfactant (e.g., poloxamer such as P188), and about 10 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0175] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 50 mM of a buffer (e.g., Tris), about 150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5% by weight of one or more disaccharides (e.g., sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the thermally stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 20 mM of a buffer (e.g., Tris), about 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5-7% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), 50 mM of a buffer (e.g., Tris), about 50 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 7-9% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0176] In some embodiments, the thermally stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (e.g., mRNA) and LNPs, comprise about 0.1 to 10% by weight of gelatin, about 10 to 60 mM of Tris, about 40 to 150 mM of NaCl, about 1 to 10% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.2 to 7.8. In some embodiments, the thermally stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (e.g., mRNA) and LNPs, about 0.1 to 10% by weight of gelatin, about 10 to 60 mM of Tris, about 40 to 110 mM of NaCl, about 0.2 to 3% by weight of trehalose, about 2 to 7% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (e.g., mRNA) and LNP, about 0.5–5 wt% gelatin, about 20–50 mM Tris, about 50–100 mM NaCl, about 0.4–2.6 wt% trehalose, about 3–5 wt% sucrose, about 0.2–0.4 vol% P188, and about 10–15 μM EDTA with a pH of about 7.7. In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (e.g., mRNA) and LNP, about 1 wt% gelatin, about 50 mM Tris, about 150 mM NaCl, about 5 wt% sucrose, about 0.4 vol% P188, and about 10 μM EDTA with a pH of about 7.5 ± 0.3 (i.e., 7.2–7.8). In some embodiments, the heat-stable RNA-LNP composition of the Disclosure comprises, in addition to RNA molecules (e.g., mRNA) and LNP, about 1% by weight of gelatin, about 20 mM of Tris, about 100 mM of NaCl, about 0.4–1.3% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP composition of the Disclosure comprises, in addition to RNA molecules (e.g., mRNA) and LNP, about 1% by weight of gelatin, about 50 mM of Tris, about 50 mM of NaCl, about 2–2.6% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7.

[0177] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof; about 10 to 60 mM of a buffer (e.g., Tris); about 40 to 150 mM of a pharmaceutically acceptable salt (e.g., NaCl); about 1 to 10% by weight of a disaccharide (e.g., sucrose); about 0.2 to 0.6% by volume of a surfactant (e.g., poloxamer such as P188); and about 5 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof; about 10 to 60 mM of a buffer (e.g., Tris); about 40 to 110 mM of a pharmaceutically acceptable salt (e.g., NaCl); about 1 to 10% by weight of one or more disaccharides (e.g., trehalose and / or sucrose); about 0.2 to 0.6 volume of a surfactant (e.g., poloxamer such as P188); and about 5 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof; about 20 to 50 mM of a buffer (e.g., Tris); about 50 to 100 mM of a pharmaceutically acceptable salt (e.g., NaCl); about 3 to 8% by weight of one or more disaccharides (e.g., trehalose and / or sucrose); about 0.2 to 0.4% by volume of a surfactant (e.g., poloxamer such as P188); and about 10 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0178] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 50 mM of a buffer (e.g., Tris), about 150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5% by weight of one or more disaccharides (e.g., sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 20 mM of a buffer (e.g., Tris), about 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5 to 7% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof; about 50 mM of a buffer (e.g., Tris); about 50 mM of a pharmaceutically acceptable salt (e.g., NaCl); about 7 to 9% by weight of one or more disaccharides (e.g., trehalose and / or sucrose); about 0.4% by volume of a surfactant (e.g., poloxamer such as P188); and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0179] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or a combination thereof, about 10 to 60 mM of Tris, about 40 to 150 mM of NaCl, about 1 to 10% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 10 to 60 mM of Tris, about 40 to 110 mM of NaCl, about 0.2 to 3% by weight of trehalose, about 2 to 7% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or a combination thereof, about 20 to 50 mM of Tris, about 50 to 100 mM of NaCl, about 0.4 to 2.6% by weight of trehalose, about 3 to 5% by weight of sucrose, about 0.2 to 0.4% by volume of P188, and about 10 to 15 μM of EDTA with a pH of about 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 mM to about 20 mM of at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, about 50 mM Tris, about 150 mM NaCl, about 5% by weight sucrose, about 0.4% by volume P188, and about 10 μM EDTA with a pH of about 7.5 ± 0.3 (i.e., 7.2 to 7.8).In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in a concentration of about 0.1 mM to about 20 mM, about 20 mM Tris, about 100 mM NaCl, about 0.4 to 1.3 wt% trehalose, about 5 wt% sucrose, about 0.4 vol% P188, and about 10 μM EDTA with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in a concentration of about 0.1 mM to about 20 mM, 50 mM Tris, about 50 mM NaCl, about 2 to 2.6% by weight trehalose, about 5% by weight sucrose, about 0.4% by volume P188, and about 10 μM EDTA with a pH of about 7.7.

[0180] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 10 to 60 mM buffering agent (e.g., Tris), about 40 to 150 mM pharmaceutically acceptable salt (e.g., NaCl), about 1 to 10% by weight disaccharide (e.g., sucrose), about 0.2 to 0.6% by volume surfactant (e.g., poloxamer such as P188), and about 5 to 15 μM chelating agent (e.g., EDTA) with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 10 to 60 mM buffering agent (e.g., Tris), about 40 to 110 mM pharmaceutically acceptable salt (e.g., NaCl), about 1 to 10% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2 to 0.6 volume% surfactant (e.g., poloxamer such as P188), and about 5 to 15 μM chelating agent (e.g., EDTA) with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 20 to 50 mM of a buffer (e.g., Tris), about 50 to 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 3 to 8% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2 to 0.4 volume% of a surfactant (e.g., poloxamer such as P188), and about 10 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0181] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 50 mM buffering agent (e.g., Tris), about 150 mM pharmaceutically acceptable salt (e.g., NaCl), about 5% by weight of one or more disaccharides (e.g., sucrose), about 0.4% by volume surfactant (e.g., poloxamer such as P188), and about 10 μM chelating agent (e.g., EDTA) with a pH of 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 20 mM buffering agent (e.g., Tris), about 100 mM pharmaceutically acceptable salt (e.g., NaCl), about 5 to 7% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume surfactant (e.g., poloxamer such as P188), and about 10 μM chelating agent (e.g., EDTA) with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 50 mM buffering agent (e.g., Tris), about 50 mM pharmaceutically acceptable salt (e.g., NaCl), about 7 to 9% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume surfactant (e.g., poloxamer such as P188), and about 10 μM chelating agent (e.g., EDTA) with a pH of about 7.7.

[0182] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 10 to 60 mM Tris, about 40 to 150 mM NaCl, about 1 to 10% by weight sucrose, about 0.2 to 0.6% by volume P188, and about 5 to 15 μM EDTA with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 10 to 60 mM Tris, about 40 to 110 mM NaCl, about 0.2 to 3% by weight trehalose, about 2 to 7% by weight sucrose, about 0.2 to 0.6% by volume P188, and about 5 to 15 μM EDTA with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 20 to 50 mM Tris, about 50 to 100 mM NaCl, about 0.4 to 2.6% by weight trehalose, about 3 to 5% by weight sucrose, about 0.2 to 0.4% by volume P188 and about 10 to 15 μM EDTA with a pH of about 7.7.In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, at least one heat-stable excipient and at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 50 mM Tris, about 150 mM NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA at a pH of about 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, at least one heat-stable excipient and at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 20 mM Tris, about 100 mM NaCl, about 0.4 to 1.3% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNPs, at least one heat-stable excipient and at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, 50 mM Tris, about 50 mM NaCl, about 2 to 2.6% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7.

[0183] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 to 10% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 0.1 mM to about 20 mM of at least one heat-stabilizing excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 10 to 60 mM of a buffer (e.g., Tris), about 40 to 150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 1 to 10% by weight of a disaccharide (e.g., sucrose), about 0.2 to 0.6% by volume of a surfactant (e.g., poloxamer such as P188), and about 5 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 0.1 to 10% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 0.1 mM to about 20 mM of at least one heat-stabilizing excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 10 to 60 mM of a buffer (e.g., Tris), about 40 to 110 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 1 to 10% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2 to 0.6 volume of a surfactant (e.g., poloxamer such as P188), and about 5 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.5 to 7.7.In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNPs, comprise about 0.5 to 5% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 0.1 mM to about 20 mM of at least one heat-stabilizing excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 20 to 50 mM of a buffer (e.g., Tris), about 50 to 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 3 to 8% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2 to 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 to 15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0184] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 0.1 mM to about 20 mM of at least one heat-stabilizing excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 50 mM of a buffer (e.g., Tris), about 150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5% by weight of one or more disaccharides (e.g., sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) at a pH of about 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 0.1 mM to about 20 mM of at least one heat-stabilizing excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 20 mM of a buffer (e.g., Tris), about 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5 to 7% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.In other embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), about 0.1 mM to about 20 mM of at least one heat-stabilizing excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin, or a combination thereof, about 50 mM of a buffer (e.g., Tris), about 50 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 7 to 9% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0185] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNPs, include about 0.1 to 10% by weight of gelatin, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or a combination thereof, about 10 to 60 mM of Tris, about 40 to 150 mM of NaCl, about 1 to 10% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNPs, include about 0.1 to 10% by weight of gelatin, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or a combination thereof, about 10 to 60 mM of Tris, about 40 to 110 mM of NaCl, about 0.2 to 3% by weight of trehalose, about 2 to 7% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNPs, include about 0.5 to 5% by weight of gelatin, about 0.1 mM to about 20 mM of at least one heat-stable excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or a combination thereof, about 20 to 50 mM of Tris, about 50 to 100 mM of NaCl, about 0.4 to 2.6% by weight of trehalose, about 3 to 5% by weight of sucrose, about 0.2 to 0.4% by volume of P188, and about 10 to 15 μM of EDTA with a pH of about 7.7. In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one heat-stable excipient selected from gelatin, lipoic acid, L-theanine, vanillin or a combination thereof in a concentration of about 0.1 mM to about 20 mM, about 50 mM Tris, about 150 mM NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA at a pH of about 7.5 ± 0.3 (i.e., 7.2 to 7.8).In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one heat-stable excipient selected from gelatin, lipoic acid, L-theanine, vanillin or a combination thereof in a concentration of about 0.1 mM to about 20 mM, about 20 mM Tris, about 100 mM NaCl, about 0.4 to 1.3% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNPs, about 1% by weight of at least one heat-stable excipient selected from gelatin, lipoic acid, L-theanine, vanillin or a combination thereof in a concentration of about 0.1 mM to about 20 mM, 50 mM Tris, about 50 mM NaCl, about 2 to 2.6% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7.

[0186] In some embodiments, the heat-stable RNA-LNP composition of the present disclosure contains, in addition to RNA molecules (mRNA) and LNP, at least 0.1 to 10% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), at least one heat-stabilizing excipient, and RNA molecules (e.g., mRNA) in an amount such that they are present in a weight ratio of about 5:1 to about 50:1. It comprises one type of heat-stabilizing excipient (such as lipoic acid, L-theanine, vanillin, or a combination thereof), about 10–60 mM of a buffer (e.g., Tris), about 40–150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 1–10% by weight of a disaccharide (e.g., sucrose), about 0.2–0.6% by volume of a surfactant (e.g., poloxamer such as P188), and about 5–15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.2–7.8. In some embodiments, the heat-stable RNA-LNP composition of the present disclosure includes, in addition to RNA molecules (mRNA) and LNP, about 0.1 to 10% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), and at least one heat-stable excipient in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1 of at least one heat-stable excipient disclosed herein. The solution comprises an agent (such as lipoic acid, L-theanine, vanillin, or a combination thereof), about 10–60 mM of a buffer (e.g., Tris), about 40–110 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 1–10% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2–0.6 volume% of a surfactant (e.g., poloxamer such as P188), and about 5–15 μM of a chelating agent (e.g., EDTA) with a pH of about 7.5–7.7.In some embodiments, the heat-stable RNA-LNP composition of the present disclosure includes, in addition to RNA molecules (mRNA) and LNP, about 0.5 to 5% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), at least one heat-stabilizing excipient, and RNA molecules (e.g., mRNA) in an amount such that they are present in a weight ratio of about 5:1 to about 50:1 of at least one heat-stabilizing excipient disclosed herein. It comprises excipients (such as lipoic acid, L-theanine, vanillin, or a combination thereof), about 20-50 mM buffering agent (e.g., Tris), about 50-100 mM pharmaceutically acceptable salt (e.g., NaCl), about 3-8% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.2-0.4% by volume surfactant (e.g., poloxamer such as P188), and about 10-15 μM chelating agent (e.g., EDTA) with a pH of about 7.7.

[0187] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNP, about 1 wt% of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), at least one heat-stable excipient (such as lipoic acid, L-theanine, vanillin, or a combination thereof) in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, about 50 mM of a buffer (e.g., Tris), about 150 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5 wt% of one or more disaccharides (e.g., sucrose), about 0.4 volume% of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure include, in addition to RNA molecules (mRNA) and LNP, about 1% by weight of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), at least one heat-stable excipient (such as lipoic acid, L-theanine, vanillin, or a combination thereof) in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, about 20 mM of a buffer (e.g., Tris), about 100 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 5–7% by weight of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4% by volume of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.In other embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, about 1 wt% of at least one heat-reversible gelling agent disclosed herein (e.g., polypeptide-based or protein-based polymer, e.g., gelatin), at least one heat-stable excipient (such as lipoic acid, L-theanine, vanillin, or a combination thereof) in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, about 50 mM of a buffer (e.g., Tris), about 50 mM of a pharmaceutically acceptable salt (e.g., NaCl), about 7 to 9 wt% of one or more disaccharides (e.g., trehalose and / or sucrose), about 0.4 volume% of a surfactant (e.g., poloxamer such as P188), and about 10 μM of a chelating agent (e.g., EDTA) with a pH of about 7.7.

[0188] In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNPs, include about 0.1 to 10% by weight of gelatin, at least one heat-stable excipient (such as lipoic acid, L-theanine, vanillin, or a combination thereof) in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, 10 to 60 mM Tris, about 40 to 150 mM NaCl, about 1 to 10% by weight of sucrose, about 0.2 to 0.6% by volume of P188, and about 5 to 15 μM of EDTA with a pH of about 7.2 to 7.8. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNP, include about 0.1 to 10% by weight of gelatin, at least one heat-stable excipient and at least one heat-stable excipient disclosed herein, e.g., lipoic acid, L-theanine, vanillin or a combination thereof, in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, about 10 to 60 mM Tris, about 40 to 110 mM NaCl, about 0.2 to 3% by weight of trehalose, about 2 to 7% by weight of sucrose, about 0.2 to 0.6% by volume of P188 and about 5 to 15 μM of EDTA with a pH of about 7.5 to 7.7. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure, in addition to RNA molecules (mRNA) and LNPs, include about 0.5 to 5% by weight of gelatin, at least one heat-stable excipient (such as lipoic acid, L-theanine, vanillin, or a combination thereof) in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, about 20 to 50 mM of Tris, about 50 to 100 mM of NaCl, about 0.4 to 2.6% by weight of trehalose, about 3 to 5% by weight of sucrose, about 0.2 to 0.4% by volume of P188, and about 10 to 15 μM of EDTA with a pH of about 7.7.In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, about 1% by weight of gelatin, at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in an amount such that the RNA molecules (e.g., mRNA) are present in a weight ratio of about 5:1 to about 50:1, about 50 mM Tris, about 150 mM NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA at a pH of about 7.5 ± 0.3 (i.e., 7.2 to 7.8). In some embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, about 1% by weight of gelatin, at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, about 20 mM Tris, about 100 mM NaCl, about 0.4 to 1.3% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7. In other embodiments, the heat-stable RNA-LNP composition of the present disclosure comprises, in addition to RNA molecules (mRNA) and LNP, about 1% by weight of gelatin, at least one heat-stable excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof, in an amount such that the RNA molecule (e.g., mRNA) is present in a weight ratio of about 5:1 to about 50:1, 50 mM Tris, about 50 mM NaCl, about 2 to 2.6% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 μM of EDTA with a pH of about 7.7.

[0189] In some embodiments, the heat-stable RNA-LNP composition exemplified above comprises OF-02 LNPs encapsulating one or more mRNA molecules. In some embodiments, the heat-stable RNA-LNP composition exemplified above comprises cKK-E10 LNPs encapsulating one or more mRNA molecules. In some embodiments, the heat-stable RNA-LNP composition exemplified above comprises GL-HEPES-E3-E12-DS-4-E10 LNPs encapsulating one or more mRNA molecules.

[0190] In some embodiments, the heat-stable RNA-LNP composition illustrated above comprises OF-02 LNPs encapsulating mRNA molecules encoding three different influenza virus proteins (e.g., trivalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, and HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage. In some embodiments, the heat-stable RNA-LNP composition illustrated above comprises OF-02 LNPs encapsulating four different influenza virus proteins (e.g., tetravalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain derived from the B / Yamagata lineage. In some embodiments, the heat-stable RNA-LNP compositions illustrated above include OF-02 system LNPs that encapsulate mRNA molecules encoding eight different influenza virus proteins (e.g., octavalent), such as H1, H3, HA from the B / Victoria lineage, HA from the B / Yamagata lineage, N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage.

[0191] In some embodiments, the heat-stable RNA-LNP composition illustrated above comprises cKK-E10 LNPs encapsulating mRNA molecules encoding three different influenza virus proteins (e.g., trivalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, and HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage. In some embodiments, the heat-stable RNA-LNP composition illustrated above comprises cKK-E10 LNPs encapsulating four different influenza virus proteins (e.g., tetravalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain derived from the B / Yamagata lineage. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure illustrated above include cKK-E10 LNPs that encapsulate mRNA molecules encoding eight different influenza virus proteins (e.g., octavalent), such as H1, H3, HA from the B / Victoria lineage, HA from the B / Yamagata lineage, N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage.

[0192] In some embodiments, the heat-stable RNA-LNP composition illustrated above comprises GL-HEPES-E3-E12-DS-4-E10 LNPs encapsulating mRNA molecules encoding three different influenza virus proteins (e.g., trivalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, and HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage. In some embodiments, the heat-stable RNA-LNP composition illustrated above comprises GL-HEPES-E3-E12-DS-4-E10 LNPs encapsulating four different influenza virus proteins (e.g., tetravalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain derived from the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain derived from the B / Yamagata lineage. In some embodiments, the heat-stable RNA-LNP compositions of the present disclosure illustrated above include GL-HEPES-E3-E12-DS-4-E10 LNPs that encapsulate mRNA molecules encoding eight different influenza virus proteins (e.g., octavalent), such as H1, H3, HA from the B / Victoria lineage, HA from the B / Yamagata lineage, N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage.

[0193] Administration The heat-stable RNA-LNP compositions of this disclosure can be formulated for administration by any method known in the art of drug delivery, such as oral, parenteral, intravenous, intramuscular, subcutaneous, intradermal, transdermal, intrathecal, submucosal, sublingual, rectal, or vaginal. In some embodiments, the compositions are formulated for sublingual, intramuscular, intradermal, subcutaneous, intravenous, intranasal, inhalation, or intraperitoneal administration. In some embodiments, the compositions are formulated for sublingual administration. In some embodiments, the compositions are formulated for intramuscular injection.

[0194] The heat-stable RNA-LNP compositions of this disclosure may be packaged in containers such as pre-filled syringes, vials, or auto-injectors. In some embodiments, the compositions of this disclosure are packaged in pre-filled syringes. In some embodiments, the compositions of this disclosure are packaged in vials. In some embodiments, the compositions of this disclosure are packaged in auto-injectors. In other embodiments, the compositions of this disclosure are cartridges packaged for patient-friendly auto-injectors and infusion pump devices.

[0195] Prefilled syringes offer several advantages over other types of packaging, such as convenience, affordability, accuracy, sterility, and safety. Accordingly, in some embodiments, prefilled syringes containing any of the heat-stable RNA-LNP compositions disclosed herein are provided in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL. In some embodiments, the prefilled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more influenza virus polypeptides, e.g., influenza HA and / or NA proteins derived from the same or different types of influenza viruses, and one or more LNP-encapsulated RNA molecules (e.g., mRNA). In some embodiments, the pre-filled syringes of the Disclosure contain a composition in volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides selected from H1, H3, HA from the B / Victoria lineage and / or HA from the B / Yamagata lineage. In some embodiments, the pre-filled syringes of the Disclosure contain a composition in volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding three different influenza virus proteins (e.g., trivalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, and HA from a third standard treatment influenza virus strain of the B / Victoria lineage.In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of approximately 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins (e.g., tetravalent): H1 of a first standard treatment influenza virus strain, H3 of a second standard treatment influenza virus strain, HA of a third standard treatment influenza virus strain of the B / Victoria lineage, and HA of a fourth standard treatment influenza virus strain of the B / Yamagata lineage. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins, such as H1, H3, HA from the B / Victoria lineage and / or HA from the B / Yamagata lineage.

[0196] In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) selected from N1, N2, NA from the B / Victoria lineage and / or NA from the B / Yamagata lineage. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising three different influenza virus proteins (e.g., trivalent): N1 of a first standard treatment influenza virus strain, N2 of a second standard treatment influenza virus strain, and one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding NA from a third standard treatment influenza virus strain of the B / Victoria lineage. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of approximately 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins (e.g., tetravalent): N1 of a first standard treatment influenza virus strain, N2 of a second standard treatment influenza virus strain, NA of a third standard treatment influenza virus strain of the B / Victoria lineage, and NA of a fourth standard treatment influenza virus strain of the B / Yamagata lineage. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins, such as N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage.

[0197] In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding eight different influenza virus proteins (e.g., octavalent), such as H1, H3, HA from the B / Victoria lineage, HA from the B / Yamagata lineage, N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage.

[0198] In some embodiments, the pre-filled syringes of the present disclosure contain a composition in volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more respiratory syncytial virus (RSV) polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), such as receptor-binding glycoprotein (G), fusion protein (F), and / or short hydrophobic (SH) proteins derived from the same or different subtypes of respiratory syncytial virus (RSV). In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more coronavirus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), and in particular one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding a spike protein (S).

[0199] In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more viral polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) derived from different viruses. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) and one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) coronavirus proteins. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) and one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more respiratory syncytial virus (RSV) proteins. In some embodiments, the pre-filled syringes of the present disclosure contain a composition in volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more coronavirus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) and one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more respiratory syncytial virus (RSV) proteins.In some embodiments, the pre-filled syringes of the present disclosure contain a composition in volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more influenza virus polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), one or more coronavirus proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), and one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more respiratory syncytial virus (RSV) proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the pre-filled syringe of the present disclosure contains a composition in a volume of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL, comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding polypeptides from any combination of viral proteins.

[0200] Immunogenic compositions and vaccines In some embodiments, the heat-stable RNA-LNP compositions of this disclosure are immunogenic compositions. As used herein, the term “immunogenic composition” means a composition that elicits a protective immune response or an immune response that may or may not be protective. The term “immune response” means the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells, to a stimulus such as an antigen, immunogen, or vaccine. An immune response may include any cells of the body involved in a host defense response, including, for example, epithelial cells that secrete interferon or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses. Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activity of lymphocytes (such as B cells or T cells), measuring the secretion of cytokines or chemokines, measuring inflammation, and measuring antibody production. An antibody response or humoral response is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and / or other leukocytes.

[0201] Vaccines comprising immunogenic compositions and pharmaceutically acceptable carriers of the present disclosure are also provided herein. As used herein, the term “vaccine” means a composition that induces a protective immune response or protective immunity in a subject. A “protective immune response” or “protective immunity” means an immune response that protects a subject from infection (prevents infection or the development of an infection-related disease) or reduces the symptoms of an infection (e.g., infection by the influenza virus). Vaccines can induce both prophylactic (preventive) and therapeutic responses. Methods of administration vary by vaccine and may include inoculation, ingestion, inhalation or other forms of administration. Inoculation can be delivered by any of many routes, including parenteral administration such as intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, inhalation or intramuscular.

[0202] Adjuvant In some embodiments, the immunogenic compositions of this disclosure include an adjuvant. In other embodiments, the immunogenic compositions of this disclosure do not contain an adjuvant. Similarly, in some embodiments, the vaccines of this disclosure may be administered with an adjuvant to enhance the immune response. In other embodiments, the vaccines may be administered without an adjuvant. As used herein, the term “adjuvant” means a substance or combination of substances that may be used to enhance the immune response to the antigenic component of a vaccine or immunogenic composition. Examples of adjuvants include suspensions of inorganic substances to which the antigen is adsorbed (alum, aluminum salts (e.g., aluminum hydroxide / aluminum oxyhydroxide (AIOOH), aluminum phosphate (AIPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or potassium aluminum sulfate)); or water-in-oil emulsions in which the antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), which may also include dead mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides (e.g., those containing CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Other adjuvants include biomolecules such as lipids and co-stimulatory molecules. Examples of biological adjuvants include AS04 (Didierlaurent et al., J.Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41 BBL.

[0203] In certain embodiments, the adjuvant is a squalene-based adjuvant comprising a water-in-oil adjuvant emulsion containing at least squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant. In certain embodiments, the emulsion is thermoreversible and optionally, about 90% of the population, on a volume basis, have a size of less than about 200 nm.

[0204] In certain embodiments, the polyoxyethylene alkyl ether is CH3-(CH2) x -(O-CH2-CH2) n It is a -OH compound, where n is an integer from 10 to 60 and x is an integer from 11 to 17. In a particular embodiment, the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12)cetostearyl ether.

[0205] In certain embodiments, approximately 90% of the oil droplets have a size of less than approximately 160 nm based on volume. In certain embodiments, approximately 90% of the oil droplets have a size of less than approximately 150 nm based on volume. In certain embodiments, approximately 50% of the oil droplets have a size of less than approximately 100 nm based on volume. In certain embodiments, approximately 50% of the oil droplets have a size of less than approximately 90 nm based on volume.

[0206] In certain embodiments, the adjuvant further comprises at least one algitol, including but not limited to glycerol, erythritol, xylitol, sorbitol, and mannitol.

[0207] In some embodiments, the hydrophilic-lipophilic balance (HLB) of the hydrophilic nonionic surfactant is about 10 or more. In certain embodiments, the HLB of the hydrophobic nonionic surfactant is less than about 9. In certain embodiments, the HLB of the hydrophilic nonionic surfactant is about 10 or more, and the HLB of the hydrophobic nonionic surfactant is less than about 9.

[0208] In certain embodiments, the hydrophobic nonionic surfactant is a sorbitan ester such as sorbitan monooleate, or a mannide ester surfactant. In certain embodiments, the amount of squalene is about 5% to about 45%. In certain embodiments, the amount of polyoxyethylene alkyl ether surfactant is about 0.9% to about 9%. In certain embodiments, the amount of hydrophobic nonionic surfactant is about 0.7% to about 7%. In certain embodiments, the adjuvant contains i) about 32.5% squalene, ii) about 6.18% polyoxyethylene(12) cetostearyl ether, iii) about 4.82% sorbitan monooleate, and iv) about 6% mannitol.

[0209] In certain embodiments, the adjuvant further comprises an alkyl polyglycoside and / or an antifreeze agent, such as a sugar, particularly dodecyl maltoside and / or sucrose.

[0210] In certain embodiments, the adjuvant includes AF03 as described in Klucker et al., J.Pharm.Sci., 2012, 101(12):4490-4500 (this document is incorporated herein by reference in its entirety). In certain embodiments, the adjuvant includes a liposomal adjuvant such as SPA14. SPA14 is a liposome-based adjuvant (AS01-like) containing a Toll-like receptor 4 (TLR4) agonist (E6020) and a saponin (QS21).

[0211] In some embodiments, including those in which one or more nucleic acids are encapsulated in LNPs, the vaccine composition does not contain adjuvants. In certain embodiments, one or more RNA molecules, such as one or more mRNA molecules, are encapsulated in LNPs that may help support one or more recombinant proteins (e.g., viral proteins) in the composition. See, for example, Shirai et al., Vaccines, 2020, 8(433):1-18.

[0212] Administration In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal or intramuscular administration. The immunogenic composition or vaccine of the present disclosure can also be formulated for nasal or inhalation administration. The immunogenic composition or vaccine of the present disclosure can also be formulated for any other intended route of administration.

[0213] In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for intradermal injection, nasal administration or intramuscular injection. General considerations in the formulation and manufacture of pharmaceuticals for administration by these routes are, for example, described in Remington’s Pharmaceutical Sciences, 19 thThis can be found in ed., Mack Publishing Co., Easton, PA, 1995 (incorporated herein by reference). Currently, oral, nasal spray, or aerosol routes (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, the immunogenic compositions or vaccines of this disclosure are administered using devices for delivering quantitative vaccine compositions. Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include, for example, those described in U.S. Patents No. 4,886,499, No. 5,190,521, No. 5,328,483, No. 5,527,288, No. 4,270,537, No. 5,015,235, No. 5,141,496, and No. 5,417,662, all of which are incorporated herein by reference. Intradermal compositions may also be administered by devices that limit the effective penetration length of a needle into the skin, such as those described in International Publication No. 1999 / 34850, incorporated herein by reference, and by functional equivalents thereof. Equally preferred are jet injectors that deliver liquid vaccines to the dermis via liquid jet syringes or via needles that puncture the stratum corneum and generate a jet that reaches the dermis.Jet injection devices are, for example, those described herein by reference in U.S. Patents No. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, and 5,466,220. This is described in U.S. Patent Nos. 5,339,163, 5,312,335, 5,503,627, U.S. Patent Nos. 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, and in International Publication No. 1997 / 37705 and International Publication No. 1997 / 13537. In addition, conventional syringes may be used in the classic Mantot method of intradermal administration.

[0214] Typical parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions containing saline and buffer media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose-sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous vehicles include fluids and nutritional supplements, and electrolyte supplements (e.g., ringer's dextrose-based). Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0215] How to use Methods for administering the vaccines described herein to a subject are also provided herein. A subject may be vaccinated using these methods to prevent an infectious disease in the subject (e.g., a viral infection such as influenza, coronavirus, or respiratory syncytial virus (RSV) infection), to reduce the subject's likelihood of contracting an infectious disease (e.g., a viral infection), or to reduce the subject's likelihood of contracting a serious disease of infectious origin (e.g., a viral infection such as influenza virus, coronavirus, or RSV infection). Similarly, this disclosure provides any of the vaccine compositions described herein for use in vaccinating a subject against an infectious disease (e.g., a viral infection such as influenza virus, coronavirus, or RSV infection). Any of the vaccine compositions described herein are also disclosed for manufacturing a vaccine for use in vaccinating a subject against an infectious disease (e.g., a viral infection such as influenza virus, coronavirus, or RSV infection). In some embodiments, the vaccination method and use involve administering an immunologically effective dose of any of the vaccines described herein to a subject in need.

[0216] As used herein, the terms “immunologically effective dose” or “therapeutic dose” mean an amount sufficient to immunize a subject. In some embodiments, an immunologically effective dose or therapeutic dose is capable of inducing protective immunity against an infection, including but not limited to an increase in antibody titers and / or T-cell immunity against the infection. In some embodiments, an immunologically effective dose or therapeutic dose of the vaccine or composition disclosed herein increases the protective immunity of a subject by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and about 100%, including values ​​and partial ranges between them, compared to a subject that has not been administered the vaccine or composition disclosed herein.

[0217] Accordingly, in some embodiments, the Disclosure provides a method for immunizing a subject, comprising administering an immunologically effective amount of any of the vaccines described herein to the subject in need. In certain embodiments, the Disclosure provides a method for immunizing a subject, comprising administering an immunologically effective amount of any of the vaccines described herein to the subject in need. As used herein, “immunize” or “immunize” means to induce a protective immune response in a subject to an infectious disease (e.g., a viral infection such as influenza, coronavirus, or RSV infection). Similarly, the Disclosure provides any of the vaccine compositions described herein for use in immunizing a subject to an infectious disease (e.g., a viral infection such as influenza, coronavirus, or RSV infection). Any of the vaccine compositions described herein are also disclosed for manufacturing a vaccine for use in immunizing a subject to an infectious disease (e.g., a viral infection such as influenza virus, coronavirus, or RSV infection).

[0218] In some embodiments, the method or use prevents a viral infection or disease caused by a viral infection in a subject. In some embodiments, the method or use reduces the likelihood of a subject becoming infected with a viral infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and about 100%, including values ​​and partial ranges between them. In some embodiments, the method or use reduces the likelihood of a subject contracting a serious illness of an infectious disease (e.g., a viral infection, e.g., influenza, coronavirus, or RSV infection) by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and about 100%, including values ​​and partial ranges between them, compared to a control that has not been administered the vaccine or composition disclosed herein. In some embodiments, the method or use induces a protective immune response in the subject. In some embodiments, this protective immune response is an antibody response.

[0219] In some embodiments, methods are also provided for alleviating one or more symptoms of an infectious disease (e.g., a viral infection such as influenza virus infection, coronavirus infection, or RSV infection), comprising administering one of the vaccines described herein to a subject in need.

[0220] This disclosure provides any of the vaccine compositions described herein for use in reducing one or more symptoms of an infectious disease (e.g., viral infections such as influenza virus infection, coronavirus infection, or RSV infection). Also disclosed herein are any of the immunogenic compositions described herein for manufacturing a vaccine for use in reducing one or more symptoms of an infectious disease (e.g., viral infections such as influenza virus infection, coronavirus infection, or RSV infection) in a subject.

[0221] In some embodiments, the methods or uses of the present disclosure reduce one or more symptoms of an infectious disease (e.g., a viral infection, e.g., influenza, coronavirus, or RSV infection) by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and about 100%, including all values ​​and partial ranges in between.

[0222] In some embodiments, the vaccine and any adjuvant may be administered before or after the onset of one or more symptoms of an infection (e.g., a viral infection such as influenza virus infection, coronavirus infection, or RSV infection). That is, in some embodiments, the vaccine described herein may be administered prophylactically to prevent an infection (e.g., a viral infection such as influenza virus infection, coronavirus infection, or RSV infection) or to alleviate symptoms of a potential infection (e.g., a viral infection such as influenza virus infection, coronavirus infection, or RSV infection).

[0223] In some embodiments, a subject is at risk of infection if the subject is likely to come into contact with other individuals or livestock (e.g., pigs) that are known to be infected with or suspected to be infected with a particular infectious agent (e.g., a virus such as influenza, coronavirus, or RSV), and / or if the subject is likely to be located in an area where an infectious disease (e.g., a viral infection) is known to be or is likely to be endemic. In some embodiments, the vaccine is administered to a subject who has an infectious disease (e.g., a viral infection such as influenza virus, coronavirus, or RSV infection), or the subject is exhibiting one or more symptoms commonly associated with an infectious disease (e.g., a viral infection such as influenza virus, coronavirus, or RSV infection). In some embodiments, the subject is known to or is likely to have been exposed to an infectious agent (e.g., a virus such as influenza virus, coronavirus, or RSV).

[0224] The vaccines according to this disclosure may be administered in any amount or dose appropriate to achieve the desired outcome. In some embodiments, the desired outcome is the induction of a sustained adaptive immune response to the virus. In some embodiments, the desired outcome is a reduction in the intensity, severity, and / or frequency of one or more symptoms associated with the viral infection and / or a delay in their onset. The required dose may vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the infection being treated, the specific composition used, and the method of administration thereof.

[0225] In some embodiments, the vaccines described herein are administered to a subject, which may be any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human subject is a bird (e.g., chicken or bird), reptile, amphibian, fish, insect, and / or worm. In some embodiments, the non-human subject is a mammal (e.g., ferret, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, and / or pig).

[0226] In some embodiments, the vaccines described herein are administered to human subjects. In some embodiments, human subjects are 6 months of age or older, 6 to 35 months of age, at least 2 years of age, at least 3 years of age, 36 months to 8 years of age, 9 years of age or older, at least 6 months of age or older and under 5 years of age, at least 6 months of age or older and under 18 years of age, or at least 3 years of age or older and under 18 years of age. In some embodiments, human subjects are infants (under 36 months of age). In some embodiments, human subjects are children or adolescents (under 18 years of age). In some embodiments, human subjects are children at least 6 months of age or older and under 5 years of age. In some embodiments, human subjects are at least 5 years of age or older and under 60 years of age. In some embodiments, human subjects are at least 5 years of age or older and under 65 years of age. In some embodiments, human subjects are elderly (at least 60 years of age or at least 65 years of age). In some embodiments, human subjects are non-elderly adults (at least 18 years of age or older and under 65 years of age, or at least 18 years of age or older and under 60 years of age).

[0227] The methods and uses of vaccines described herein involve administering a single dose (i.e., not a booster dose) to a subject. In some embodiments, the methods and uses of vaccines described herein include a prime-boost vaccination strategy. Prime-boost vaccination involves administering a prime vaccine, and then, after a certain period of time, administering a boost vaccine to the subject. The immune response is “pre-stimulated” at the time of prime vaccine administration and “enhanced” at the time of boost vaccine administration. The prime vaccine may include the vaccines described herein and any adjuvants. Similarly, the boost vaccine may include the vaccines described herein and any adjuvants. The prime vaccine may be, but does not have to be, the same as the boost vaccine. The administration of the boost vaccine is generally several weeks or months after the administration of the prime composition, preferably about 2-3 weeks, or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks. In certain embodiments, the recipients of the Prime Boost vaccine are naive subjects, typically naive infants or children.

[0228] The vaccine can be administered using any preferred route of administration, including, for example, parenteral delivery, as described above. In some embodiments, the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0229] Other methods A method for stabilizing a composition comprising one or more RNA molecules encapsulated in LNPs described herein is also provided herein, the method comprising adding a sufficient amount of at least one thermoreversible gelling agent described herein to the composition, maintaining the composition in the liquid phase at temperatures above about 12°C (e.g., room temperature), and reversibly transitioning the composition to a gel state at temperatures of about 1 to 11°C (e.g., 2 to 8°C or 4°C). As described herein, the stability of the composition may be measured by the average particle size of the LNPs in some embodiments, the encapsulation efficiency of the LNPs in other embodiments, and / or the integrity of the one or more RNA molecules encapsulated in the LNPs in some further embodiments.

[0230] Accordingly, in certain embodiments, the method includes adding at least one thermoreversible gelling agent in a sufficient amount so that the average particle size of the LNPs does not increase by more than about 50%, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, after storage of the composition for more than two years or more at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the method includes adding at least one thermoreversible gelling agent in sufficient quantity so that the average particle size of the LNPs does not increase by more than about 40% after storage of the composition for a maximum of about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0231] In other embodiments, the method includes adding at least one thermoreversible gelling agent in a sufficient amount so that the LNP encapsulation efficiency does not decrease by more than about 20%, for example, about 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the composition for a maximum of about 1 month or more, including all values ​​and partial ranges in between, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, for a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months or a maximum of about 12 months, a maximum of about 18 months, a maximum of about 2 years, or more than 2 years, including all values ​​and partial ranges in between. In some embodiments, the method includes adding at least one thermoreversible gelling agent in a sufficient amount so that the LNP encapsulation efficiency does not decrease by more than about 10% after storage of the composition for up to about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0232] In some embodiments, the method includes adding at least one thermoreversible gelling agent in a sufficient amount so that the integrity of one or more RNA molecules encapsulated in the LNP does not decrease by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the composition for more than two years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, for a maximum of about one month or more, for a maximum of about two months, a maximum of about three months, a maximum of about four months, a maximum of about five months, a maximum of about six months, a maximum of about seven months, a maximum of about eight months, a maximum of about nine months, a maximum of about ten months, a maximum of about eleven months or a maximum of about twelve months, a maximum of about eighteen months, a maximum of about two years, or more than two years, including all values ​​and partial ranges in between. In some embodiments, the method includes adding at least one thermoreversible gelling agent in sufficient quantity to ensure that the integrity of one or more RNA molecules encapsulated in the LNP does not decrease by more than about 10% after storage of the composition for a maximum of about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0233] In some embodiments in which one or more RNA molecules encode one or more influenza virus proteins, the method includes adding at least one thermoreversible gelling agent in a sufficient amount so that the HAI titer of the composition does not decrease by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, after storage of the composition for a maximum of about one month or longer, for example, up to about two months, up to about three months, up to about four months, up to about five months, up to about six months, up to about seven months, up to about eight months, up to about nine months, up to about ten months, up to about eleven months, or up to about twelve months, up to about eighteen months, up to about two years, or more than two years, including all values ​​and partial ranges in between, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. In some embodiments, the method includes adding at least one thermoreversible gelling agent in a sufficient amount such that the HAI titer of the composition does not decrease by more than about 25% after storage of the composition for a maximum of about 1 month or longer, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about 2 years, or more than 2 years, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between.

[0234] Methods for preventing the degradation of one or more RNA molecules encapsulated in LNPs in a liquid composition are also provided herein, the method comprising adding at least one thermoreversible gelling agent described herein to the liquid composition in a sufficient amount to maintain the composition in the liquid phase at a temperature higher than about 12 °C (e.g., room temperature), and reversibly transitioning the liquid composition into a gel form at a temperature of about 1-11 °C (e.g., 2-8 °C or 4 °C). In some embodiments, the integrity of one or more RNA molecules encapsulated in the LNP, when compared to a control composition that does not contain at least one thermoreversible gelling agent, is at a temperature of about 1-11 °C (e.g., 2-8 °C or 4 °C), including all values and subranges therebetween, at most about 1 month or more, e.g., at most about 2 months, at most about 3 months, at most about 4 months, at most about 5 months, at most about 6 months, at most about 7 months, at most about 8 months, at most about 9 months, at most about 10 months, at most about 11 months or at most about 12 months, at most about 18 months, at most about 2 years, or after storage of the liquid composition for more than 2 years, does not decrease by more than about 30%, e.g., about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween. In some embodiments, the integrity of one or more RNA molecules encapsulated in the LNP, when compared to a control composition that does not contain at least one thermoreversible gelling agent, is at a temperature of about 1-11 °C (e.g., 2-8 °C or 4 °C), including all values and subranges therebetween, at most about 1 month or more, e.g., at most about 2 months, at most about 3 months, at most about 4 months, at most about 5 months, at most about 6 months, at most about 7 months, at most about 8 months, at most about 9 months, at most about 10 months, at most about 11 months or at most about 12 months, at most about 18 months, at most about 2 years, or after storage of the liquid composition for more than 2 years, does not decrease by more than about 10%.

[0235] In other embodiments, a method is provided herein for formulating a composition comprising one or more RNA molecules encapsulated in an LNP, wherein the composition is stable at a temperature of 4°C for up to about one month or more, for example, up to about two months, up to about three months, up to about four months, up to about five months, up to about six months, up to about seven months, up to about eight months, up to about nine months, up to about ten months, up to about eleven months or up to about twelve months, up to about eighteen months, up to about two years or more, including all values ​​and partial ranges in between, and the method comprises adding a sufficient amount of at least one thermoreversible gelling agent described herein to the composition, maintaining the composition in a liquid phase at temperatures above about 12°C (e.g., room temperature), and reversibly transitioning the liquid composition to a gel form at temperatures of about 1 to 11°C (e.g., 2 to 8°C or 4°C).

[0236] In some embodiments, the method involves a composition containing OF-02 system LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins (e.g., tetravalent): H1 of a first standard treatment influenza virus strain, H3 of a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, to which about 50 mM Tris and about 150 mM NaC are added. Compared to a control composition containing approximately 5% by weight of sucrose, approximately 0.4% by volume of P188, and approximately 10 μM of EDTA with a pH of 7.5 ± 0.3 (i.e., 7.2 to 7.8), and at a temperature of approximately 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, it can withstand for up to approximately 1 month or longer, for example, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, up to approximately 9 months. After storage of the liquid composition for months, up to approximately 10 months, up to approximately 11 months, up to approximately 12 months, up to approximately 18 months, up to approximately 2 years, or more than 2 years, (1) the average particle size of LNPs shall not increase by more than approximately 50%, including all values ​​and partial ranges in between, for example, approximately 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, and (2) the encapsulation efficiency of LNPs shall not decrease by more than approximately 20%, including all values ​​and partial ranges in between, for example, approximately 15%, 10%, or 5%. (3) The integrity of one or more RNA molecules encapsulated in the LNP is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, and / or (4) The HAI titer of the composition is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between. This includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin).In some embodiments, the method involves a composition containing OF-02 system LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins (e.g., tetravalent): H1 of a first standard treatment influenza virus strain, H3 of a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, to which about 20 mM Tris and about 100 mM NaC are added. Compared to a control composition containing approximately 0.4-1.3% trehalose, approximately 5% sucrose, approximately 10 μM EDTA, and approximately 0.4% P188 with a pH of approximately 7.7, and no thermoreversible gelling agent, the composition can be frozen for up to approximately 1 month or longer, for example, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, and up to approximately 1 month at a temperature of approximately 1-11°C (e.g., 2-8°C or 4°C), including all values ​​and partial ranges in between. After storage of the liquid composition for 9 months, up to approximately 10 months, up to approximately 11 months, or up to approximately 12 months, up to approximately 18 months, up to approximately 2 years, or more than 2 years, (1) the average particle size of the LNPs shall not increase by more than approximately 50%, for example, approximately 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, and (2) the encapsulation efficiency of the LNPs shall not decrease by more than approximately 20%, for example, approximately 15%, 10%, or 5%, including all values ​​and partial ranges in between. To that end, (3) the integrity of one or more RNA molecules encapsulated in the LNP is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, and / or (4) the HAI titer of the composition is not reduced by more than about 30%, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, by more than about 30%, about 25%, 20%, 15%, 10%, or 5%. This includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin).In some embodiments, the method involves a composition containing OF-02 system LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins (e.g., tetravalent): H1 of a first standard treatment influenza virus strain, H3 of a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, to which about 50 mM Tris and about 50 mM NaCl are added. Compared to a control composition containing approximately 2-2.6% trehalose, approximately 5% sucrose, approximately 10 μM EDTA, and approximately 0.4% P188 with a pH of approximately 7.7, and no thermoreversible gelling agent, the gelling time is approximately 1 month or longer, for example, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, and approximately 9 months, at a temperature of approximately 1-11°C (e.g., 2-8°C or 4°C), including all values ​​and partial ranges in between. After storage of the liquid composition for a maximum of approximately 10 months, a maximum of approximately 11 months or a maximum of approximately 12 months, a maximum of approximately 18 months, a maximum of approximately 2 years, or more than 2 years, (1) the average particle size of the LNPs shall not increase by more than approximately 50%, for example, approximately 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values ​​and partial ranges in between, and (2) the encapsulation efficiency of the LNPs shall not decrease by more than approximately 20%, for example, approximately 15%, 10%, or 5%, including all values ​​and partial ranges in between. (3) The integrity of one or more RNA molecules encapsulated in the LNP is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, and / or (4) The HAI titer of the composition is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between. This includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin).In some embodiments, the method involves a composition comprising OF-02 system LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins, e.g., H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage, along with approximately 50 mM Tris, approximately 150 mM NaCl, approximately 5% by weight sucrose, approximately 0.4% by volume P188, and approximately 10 μM EDTA at pH 7.5 ± 0.3 (i.e., 7.2 to 7.8), and at least Compared to a control composition that does not contain one type of thermoreversible gelling agent, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, it lasts for a maximum of about 1 month or more, for example, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, a maximum of about 6 months, a maximum of about 7 months, a maximum of about 8 months, a maximum of about 9 months, a maximum of about 10 months, a maximum of about 11 months or a maximum of about 12 months, a maximum of about 18 months, and a maximum of about 2 months. The method includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin) so that after storage of the liquid composition for one year or more than two years, (1) the average particle size of the LNPs does not increase by more than about 50%, including all values ​​and partial ranges in between, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, (2) the encapsulation efficiency of the LNPs does not decrease by more than about 20%, including all values ​​and partial ranges in between, for example, about 15%, 10%, or 5%, (3) the integrity of one or more RNA molecules encapsulated in the LNPs does not decrease by more than about 30%, including all values ​​and partial ranges in between, for example, about 25%, 20%, 15%, 10%, or 5%, and / or (4) the HAI titer of the composition does not decrease by more than about 30%, including all values ​​and partial ranges in between, for example, about 25%, 20%, 15%, 10%, or 5%,In some embodiments, the method involves a composition containing OF-02 LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins, e.g., H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage, to which about 20 mM Tris, about 100 mM NaCl, about 0.4–1.3% trehalose, about 5% sucrose, about 10 μM EDTA, and about 0.4% P188 with a pH of about 7.7, at least Compared to a control composition that does not contain one type of thermoreversible gelling agent, at a temperature of about 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, it lasts for a maximum of about 1 month or more, for example, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months or up to about 12 months, up to about 18 months, up to about The method includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin) so that after storage of the liquid composition for two years or more than two years, (1) the average particle size of the LNPs does not increase by more than about 50%, including all values ​​and partial ranges in between, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, (2) the encapsulation efficiency of the LNPs does not decrease by more than about 20%, including all values ​​and partial ranges in between, for example, about 15%, 10%, or 5%, (3) the integrity of one or more RNA molecules encapsulated in the LNPs does not decrease by more than about 30%, including all values ​​and partial ranges in between, for example, about 25%, 20%, 15%, 10%, or 5%, and / or (4) the HAI titer of the composition does not decrease by more than about 30%, including all values ​​and partial ranges in between, for example, about 25%, 20%, 15%, 10%, or 5%,In some embodiments, the method involves a composition comprising OF-02 LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins, e.g., H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage, to which about 50 mM Tris, about 50 mM NaCl, about 2-2.6% trehalose, about 5% sucrose, about 10 μM EDTA, and about 0.4% P188 with a pH of about 7.7, at least one of these. Compared to a control composition that does not contain a thermoreversible gelling agent, at a temperature of approximately 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between, it lasts for up to approximately 1 month or longer, for example, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, up to approximately 9 months, up to approximately 10 months, up to approximately 11 months or up to approximately 12 months, up to approximately 18 months, and up to approximately 2 years. The method includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin) so that after storage of the liquid composition for a period of time or more than two years, (1) the average particle size of the LNPs does not increase by more than about 50%, including all values ​​and partial ranges in between, for example, about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, (2) the encapsulation efficiency of the LNPs does not decrease by more than about 20%, including all values ​​and partial ranges in between, for example, about 15%, 10%, or 5%, (3) the integrity of one or more RNA molecules encapsulated in the LNPs does not decrease by more than about 30%, including all values ​​and partial ranges in between, for example, about 25%, 20%, 15%, 10%, or 5%, and / or (4) the HAI titer of the composition does not decrease by more than about 30%, including all values ​​and partial ranges in between, for example, about 25%, 20%, 15%, 10%, or 5%,

[0237] In some embodiments, the method involves a composition containing cKK-E10 LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins (e.g., tetravalent): H1 from a first standard treatment influenza virus strain, H3 from a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, to which about 50 mM Tris and about 150 mM Na are added. Compared to a control composition that does not contain Cl, approximately 5% by weight sucrose, approximately 0.4% by volume P188, and approximately 10 μM EDTA with a pH of 7.5 ± 0.3 (i.e., 7.2 to 7.8), and at least one thermoreversible gelling agent, the composition can be frozen for up to approximately 1 month or longer, for example, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, up to approximately 9 months, at a temperature of approximately 1 to 11°C (e.g., 2 to 8°C or 4°C), including all values ​​and partial ranges in between. After storage of the liquid composition for months, up to approximately 10 months, up to approximately 11 months, or up to approximately 12 months, up to approximately 18 months, up to approximately 2 years, or more than 2 years, (1) the average particle size of the LNPs shall not increase by more than approximately 50%, including all values ​​and partial ranges in between, for example, approximately 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, and (2) the encapsulation efficiency of the LNPs shall not decrease by more than approximately 20%, including all values ​​and partial ranges in between, for example, approximately 15%, 10%, or 5%. (3) The integrity of one or more RNA molecules encapsulated in the LNP is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, and / or (4) The HAI titer of the composition is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between. This includes adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin).In some embodiments, the method involves a composition containing cKK-E10 LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins (e.g., tetravalent): H1 of a first standard treatment influenza virus strain, H3 of a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, to which about 20 mM Tris and about 100 mM N are added. Compared to a control composition containing aCl, approximately 0.4-1.3% trehalose, approximately 5% sucrose, approximately 10 μM EDTA, and approximately 0.4% P188 with a pH of approximately 7.7, and at least one thermoreversible gelling agent, the composition can be frozen for up to approximately 1 month or longer, for example, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, and up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, and up to approximately 5 months. After storage of the liquid composition for approximately 9 months, up to approximately 10 months, up to approximately 11 months, or up to approximately 12 months, up to approximately 18 months, up to approximately 2 years, or more than 2 years, (1) the average particle size of the LNPs shall not increase by more than approximately 50%, including all values ​​and partial ranges in between, for example, approximately 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, and (2) the encapsulation efficiency of the LNPs shall not decrease by more than approximately 20%, including all values ​​and partial ranges in between, for example, approximately 15%, 10%, or 5%. (3) The integrity of one or more RNA molecules encapsulated in the LNP is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, and / or (4) The HAI titer of the composition is not reduced by more than about 30%, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, by more than about 30%, about 25%, 20%, 15%, 10%, or 5%, including all values ​​and partial ranges in between, by adding a sufficient amount of at least one thermoreversible gelling agent (e.g., about 1% gelatin).In some embodiments, the method involves a composition containing cKK-E10 LNPs that encapsulate mRNA molecules encoding four different influenza virus proteins (e.g., tetravalent): H1 of a first standard treatment influenza virus strain, H3 of a second standard treatment influenza virus strain, HA from a third standard treatment influenza virus strain of the B / Victoria lineage, and HA from a fourth standard treatment influenza virus strain of the B / Yamagata lineage, to which about 50 mM Tris and about 50 mM Na are added. Compared to a control composition containing Cl, approximately 2-2.6% trehalose, approximately 5% sucrose, approximately 10 μM EDTA, and approximately 0.4% P188 with a pH of approximately 7.7, and no thermoreversible gelling agent, the composition showed a maximum shelf life of approximately 1 month or more, for example, up to approximately 2 months, up to approximately 3 months, up to approximately 4 months, up to approximately 5 months, up to approximately 6 months, up to approximately 7 months, up to approximately 8 months, and up to approximately 9 months, at a temperature of approximately 1-11°C (e.g., 2-8°C or 4°C), including all values ​​and partial ranges in between. After storage of the liquid composition for months, up to approximately 10 months, up to approximately 11 months, up to approximately 12 months, up to approximately 18 months, up to approximately 2 years, or more than 2 years, (1) the average particle size of LNPs shall not increase by more than approximately 50%, including all values ​​and partial ranges in between, for example, approximately 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, and (2) the encapsulation efficiency of LNPs shall not decrease by more than approximately 20%, including all values ​​and partial ranges in between, for example, approximately 15%, 10%, or 5%. (3) The integrity of one or more RNA molecules encapsulated in the LNP is not reduced by more than about 30%, for example, about 25%, 20%, 15%, 10%, or 5%, inclu...

Claims

1. A composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs) and at least one thermoreversible gelling agent.

2. The composition according to claim 1, wherein the composition has a liquid phase at temperatures above about 12°C and reversibly transitions to a gel state at temperatures of about 1 to 11°C.

3. The composition according to claim 1 or 2, wherein the at least one thermoreversible gelling agent has an upper critical solution temperature (UCST) of about 12°C to about 50°C.

4. The composition according to any one of claims 1 to 3, wherein the at least one thermoreversible gelling agent is present in an amount of about 0.1% to about 30% by weight, about 0.25% to about 5% by weight, or about 0.5% to about 1.5% by weight.

5. The composition according to any one of claims 1 to 4, wherein the at least one thermoreversible gelling agent comprises a thermoreversible gelling polymer, a thermoreversible gelling polypeptide, and / or a thermoreversible gelling protein.

6. The composition according to claim 5, wherein the thermoreversible gelling polymer comprises a polypeptide-based gel-forming polymer or a protein-based gel-forming polymer.

7. The thermoreversible gelling polypeptide comprises multi-L-arginyl-poly-L-aspartate (iMAPA)-PEG, or the thermoreversible gelling polymer comprises gelatin, poly(N-acryloyl asparagine amide), poly(ethylene glycol)-b-poly(N-acryloylglycinamide-co-acrylonitrile)(PEG-b-P(NAGA-co-AN), poly(N-acryloylglycinamide-co-N-phenylacrylamide)(P(NAGA-co-NPhAm)), poly(N-(2-hydroxypropyl)methacrylamide-glycolamide)(P(HPMA-GA)), poly The composition according to claim 5, comprising (acrylamide-co-acrylonitrile)-b-poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(acrylic acid-co-acrylonitrile) (P(AA-co-AN)), poly(N-vinylimidazole-co-1-vinyl-2-(hydroxymethyl)imidazole), poly(sulfobetaine-co-sulfabetine) (P(SB-co-ZB), poly[2-(methacryloyloxy)ethylphosphocholine]-b-poly(2-ureidoethyl methacrylate) (PMPC20-b-PUEM165) or a combination thereof.

8. The composition according to claim 7, wherein the at least one thermoreversible gelling polymer comprises gelatin, and optionally the gelatin is present in an amount of about 1% by weight.

9. The composition according to any one of claims 1 to 8, further comprising a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant and / or a chelating agent.

10. a) The buffer contains or is tris(hydroxymethyl)aminomethane (tris), b) The pharmaceutically acceptable salt contains sodium chloride (NaCl) or is sodium chloride (NaCl), c) The one or more disaccharides include sucrose or are sucrose, d) The surfactant contains or is poloxamer 188 (P188), and / or e) The composition according to claim 9, wherein the chelating agent contains ethylenediaminetetraacetic acid (EDTA) or is ethylenediaminetetraacetic acid (EDTA).

11. The composition is a) comprising approximately 10 mM to approximately 60 mM Tris, approximately 40 mM to approximately 150 mM NaCl, approximately 1% to approximately 10% by weight Sucrose, approximately 0.2% to approximately 0.6% by volume P188, and approximately 5 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.2 to approximately 7.

8. b) A composition comprising approximately 50 mM Tris, approximately 150 mM NaCl, approximately 5% by weight sucrose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the composition has a pH of approximately 7.5 ± 0.

3. c) A composition comprising approximately 10 mM to approximately 60 mM tris, approximately 40 mM to approximately 110 mM NaCl, approximately 3% to approximately 6% by weight sucrose, approximately 0.2% to approximately 4% by weight trehalose, approximately 0.2% to approximately 0.6% by volume P188, and approximately 5 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.5 to approximately 7.

7. d) A composition comprising approximately 50 mM Tris, approximately 50 mM NaCl, approximately 5% by weight Sucrose, approximately 2-2.6% by weight Trehalose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the composition has a pH of approximately 7.

7. e) A composition comprising approximately 20 mM to approximately 50 mM Tris, approximately 50 mM to approximately 100 mM NaCl, approximately 2% to approximately 5% by weight Sucrose, approximately 0.3% to approximately 3% by weight Trehalose, approximately 0.2% to approximately 0.4% by volume P188, and approximately 10 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.7, or f) The composition according to any one of claims 1 to 10, comprising about 20 mM Tris, about 100 mM NaCl, about 5% by weight Sucrose, about 0.4 to 1.3% by weight Trehalose, about 0.4% by volume P188, and about 10 μM EDTA, wherein the composition has a pH of about 7.

7.

12. The composition according to any one of claims 1 to 11, wherein the composition is stable after storage at a temperature of about 4°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, or a maximum of about 6 months, compared to a control composition that does not contain the at least one thermoreversible gelling agent, and the stability of the composition is measured by changes in the average particle size of the LNPs, the encapsulation efficiency of the LNPs, and / or the integrity of the one or more RNA molecules encapsulated in the LNPs.

13. a) The average particle size of the LNP does not increase by more than 40% after storage of the composition at a temperature of about 4°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, or a maximum of about 6 months, compared to a control composition that does not contain at least one thermoreversible gelling agent. b) The encapsulation efficiency of the LNP does not decrease by more than 10% after storage of the composition at a temperature of about 4°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, or a maximum of about 6 months, compared to a control composition that does not contain at least one thermoreversible gelling agent. c) The encapsulation efficiency of the LNP is higher than that of the control composition which does not contain at least one thermoreversible gelling agent, and / or d) The composition according to claim 12, wherein the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease by more than 10% after storage of the composition at a temperature of about 4°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, or a maximum of about 6 months, compared to a control composition that does not contain the at least one thermoreversible gelling agent.

14. A liquid composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs) and at least one heat-stabilizing excipient, wherein the at least one heat-stabilizing excipient comprises lipoic acid, L-theanine, vanillin, or a combination thereof.

15. The liquid composition according to claim 14, wherein the integrity of the one or more RNA molecules does not decrease by more than 20% after storage of the liquid composition at a temperature of 37°C for at least 7 days, compared to a control liquid composition that does not contain the at least one heat-stabilizing excipient.

16. a) The integrity of the one or more RNA molecules does not decrease by more than 25% after storage of the liquid composition at a temperature of 4°C for a maximum of approximately 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, compared to a control liquid composition that does not contain the at least one heat-stabilizing excipient. b) The integrity of the one or more RNA molecules does not decrease by more than 30% after storage of the liquid composition at a temperature of 4°C for a maximum of approximately 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, or 8 months, compared to a control liquid composition that does not contain the at least one heat-stabilizing excipient. c) The integrity of the one or more RNA molecules does not decrease by more than 45% after storage of the liquid composition at a temperature of 4°C for a maximum of approximately 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, compared to a control liquid composition that does not contain the at least one heat-stabilizing excipient. d) The integrity of the one or more RNA molecules does not decrease by more than 50% after storage of the liquid composition at a temperature of 25°C for a maximum of approximately 1 week, a maximum of approximately 2 weeks, a maximum of approximately 3 weeks, or a maximum of approximately 4 weeks, compared to a control liquid composition that does not contain the at least one heat-stabilizing excipient. e) The average particle size of the LNP does not decrease by more than 40% after storage of the liquid composition at a temperature of 4°C for a maximum of approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. f) The average particle size of the LNP does not increase by more than 20% after storage of the liquid composition at a temperature of 25°C for a maximum of approximately 1 week, a maximum of approximately 2 weeks, a maximum of approximately 3 weeks, a maximum of approximately 4 weeks, a maximum of approximately 5 weeks, a maximum of approximately 6 weeks, or a maximum of approximately 7 weeks. g) The encapsulation efficiency of the LNP does not decrease by more than 20% after storage of the liquid composition at a temperature of 4°C for a maximum of approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, and / or h) The liquid composition according to claim 14 or 15, wherein the encapsulation efficiency of the LNP does not decrease by more than 20% after storage of the liquid composition at a temperature of 25°C for a maximum of about 1 week, a maximum of about 2 weeks, a maximum of about 3 weeks, a maximum of about 4 weeks, a maximum of about 5 weeks, a maximum of about 6 weeks, or a maximum of about 7 weeks.

17. a) The at least one heat-stabilizing excipient is present at a concentration of about 0.1 mM to about 20 mM, about 0.5 mM to about 15 mM, or about 1 mM to about 10 mM. b) The at least one of the heat-stabilizing excipients is present at a concentration of about 5 mM, about 10 mM, or about 15 mM, and / or c) The liquid composition according to any one of claims 14 to 16, wherein the at least one heat-stabilizing excipient and the one or more RNA molecules are present in a weight ratio of about 5:1 to about 50:

1.

18. a) The at least one heat-stabilizing excipient contains lipoic acid or is lipoic acid, and optionally the lipoic acid and the one or more RNA molecules are present in a weight ratio of about 2.5:1 to about 15.5:

1. b) The at least one heat-stabilizing excipient contains L-theanine or is L-theanine, and optionally the L-theanine and the one or more RNA molecules are present in a weight ratio of about 10:1 to about 30:

1. c) The liquid composition according to claims 14 to 17, wherein the at least one heat-stabilizing excipient contains vanillin or is vanillin, and optionally the vanillin and the one or more RNA molecules are present in a weight ratio of about 12.5:1 to about 50:

1.

19. A liquid composition according to any one of claims 14 to 18, further comprising a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant and / or a chelating agent.

20. a) The buffer contains or is tris(hydroxymethyl)aminomethane (tris), b) The pharmaceutically acceptable salt contains sodium chloride (NaCl) or is sodium chloride (NaCl), c) The one or more disaccharides include sucrose or are sucrose, d) The surfactant contains or is poloxamer 188 (P188), and / or e) The liquid composition according to claim 19, wherein the chelating agent contains or is ethylenediaminetetraacetic acid (EDTA).

21. The liquid composition is a) comprising approximately 10 mM to approximately 60 mM Tris, approximately 40 mM to approximately 150 mM NaCl, approximately 1% to approximately 10% by weight Sucrose, approximately 0.2% to approximately 0.6% by volume P188, and approximately 5 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.2 to approximately 7.

8. b) A composition comprising approximately 50 mM Tris, approximately 150 mM NaCl, approximately 5% by weight sucrose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the composition has a pH of approximately 7.5 ± 0.

3. c) A composition comprising approximately 10 mM to approximately 60 mM tris, approximately 40 mM to approximately 110 mM NaCl, approximately 3% to approximately 6% by weight sucrose, approximately 0.2% to approximately 4% by weight trehalose, approximately 0.2% to approximately 0.6% by volume P188, and approximately 5 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.5 to approximately 7.

7. d) A composition comprising approximately 50 mM Tris, approximately 50 mM NaCl, approximately 5% by weight Sucrose, approximately 2-2.6% by weight Trehalose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the composition has a pH of approximately 7.

7. e) A composition comprising approximately 20 mM to approximately 50 mM Tris, approximately 50 mM to approximately 100 mM NaCl, approximately 2% to approximately 5% by weight Sucrose, approximately 0.3% to approximately 3% by weight Trehalose, approximately 0.2% to approximately 0.4% by volume P188, and approximately 10 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.7, or f) The liquid composition according to any one of claims 14 to 20, comprising about 20 mM Tris, about 100 mM NaCl, about 5% by weight Sucrose, about 0.4 to 1.3% by weight Trehalose, about 0.4% by volume P188, and about 10 μM EDTA, wherein the composition has a pH of about 7.

7.

22. A liquid formulation comprising one or more ribonucleic acids (RNA) encapsulated in lipid nanoparticles (LNPs), about 10 mM to about 60 mM tris(hydroxymethyl)aminomethane (Tris), about 40 mM to about 150 mM sodium chloride (NaCl), about 1% to about 10% by weight sucrose, about 0.2% to about 0.6% by volume poloxamer 188 (P188), and about 5 μM to about 15 μM ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of about 7.2 to about 7.

8.

23. The liquid formulation according to claim 22, comprising approximately 50 mM Tris, approximately 150 mM NaCl, approximately 5% by weight sucrose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the liquid formulation has a pH of 7.5 ± 0.

3.

24. A liquid formulation comprising one or more ribonucleic acids (RNA) encapsulated in lipid nanoparticles (LNPs), about 10 mM to about 60 mM tris(hydroxymethyl)aminomethane (Tris), about 40 mM to about 110 mM sodium chloride (NaCl), about 3% to about 6% by weight sucrose, about 0.2% to about 4% by weight trehalose, about 0.2% to about 0.6% by volume poloxamer 188 (P188), and about 5 μM to about 15 μM ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of about 7.5 to about 7.

7.

25. The liquid formulation according to claim 24, comprising approximately 50 mM Tris, approximately 50 mM NaCl, approximately 5% by weight Sucrose, approximately 2-2.6% by weight Trehalose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the liquid formulation has a pH of approximately 7.

7.

26. A liquid formulation comprising one or more ribonucleic acids (RNA) encapsulated in lipid nanoparticles (LNPs), about 20 mM to about 50 mM tris(hydroxymethyl)aminomethane (Tris), about 50 mM to about 100 mM sodium chloride (NaCl), about 2% to about 5% by weight sucrose, about 0.3% to about 3% by weight trehalose, about 0.2% to about 0.4% by volume poloxamer 188 (P188), and about 10 μM to about 15 μM ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of about 7.

7.

27. The liquid formulation according to claim 26, comprising approximately 20 mM Tris, approximately 100 mM NaCl, approximately 5% by weight Sucrose, approximately 0.4 to 1.3% by weight Trehalose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the liquid formulation has a pH of approximately 7.

7.

28. A composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs), at least one thermoreversible gelling agent, and at least one heat-stabilizing excipient, wherein the at least one heat-stabilizing excipient contains or is lipoic acid.

29. The composition according to claim 28, wherein the at least one thermoreversible gelling agent contains or is gelatin, and optionally the gelatin is present in an amount of about 0.5% to about 1.5% by weight, for example, about 1% by weight.

30. The composition according to claim 28 or 29, wherein the lipoic acid is present at a concentration of about 1 mM to about 10 mM, for example, about 1 mM to about 5 mM, or the lipoic acid and the one or more RNA molecules are present in a weight ratio of about 2.5:1 to about 15.5:

1.

31. The composition according to any one of claims 28 to 30, further comprising a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant and / or a chelating agent.

32. a) The buffer contains or is tris(hydroxymethyl)aminomethane (tris), b) The pharmaceutically acceptable salt contains sodium chloride (NaCl) or is sodium chloride (NaCl), c) The one or more disaccharides include sucrose or are sucrose, d) The surfactant contains or is poloxamer 188 (P188), and / or e) The composition according to claim 31, wherein the chelating agent contains ethylenediaminetetraacetic acid (EDTA) or is ethylenediaminetetraacetic acid (EDTA).

33. The composition is a) comprising approximately 10 mM to approximately 60 mM Tris, approximately 40 mM to approximately 150 mM NaCl, approximately 1% to approximately 10% by weight Sucrose, approximately 0.2% to approximately 0.6% by volume P188, and approximately 5 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.2 to approximately 7.

8. b) A composition comprising approximately 50 mM Tris, approximately 150 mM NaCl, approximately 5% by weight sucrose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the composition has a pH of approximately 7.5 ± 0.

3. c) A composition comprising approximately 10 mM to approximately 60 mM tris, approximately 40 mM to approximately 110 mM NaCl, approximately 3% to approximately 6% by weight sucrose, approximately 0.2% to approximately 4% by weight trehalose, approximately 0.2% to approximately 0.6% by volume P188, and approximately 5 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.5 to approximately 7.

7. d) A composition comprising approximately 50 mM Tris, approximately 50 mM NaCl, approximately 5% by weight Sucrose, approximately 2-2.6% by weight Trehalose, approximately 0.4% by volume P188, and approximately 10 μM EDTA, wherein the composition has a pH of approximately 7.

7. e) A composition comprising approximately 20 mM to approximately 50 mM Tris, approximately 50 mM to approximately 100 mM NaCl, approximately 2% to approximately 5% by weight Sucrose, approximately 0.3% to approximately 3% by weight Trehalose, approximately 0.2% to approximately 0.4% by volume P188, and approximately 10 μM to approximately 15 μM EDTA, wherein the composition has a pH of approximately 7.7, or f) The composition according to any one of claims 28 to 32, comprising about 20 mM Tris, about 100 mM NaCl, about 5% by weight Sucrose, about 0.4 to 1.3% by weight Trehalose, about 0.4% by volume P188, and about 10 μM EDTA, wherein the composition has a pH of about 7.

7.

34. The composition according to any one of claims 28 to 33, wherein, compared to a control composition that does not contain the at least one thermoreversible gelling agent and the at least one heat-stabilizing excipient, the composition is stable after storage at a temperature of about 2 to 8°C for a maximum of about 1 month, a maximum of about 2 months, a maximum of about 3 months, a maximum of about 4 months, a maximum of about 5 months, or a maximum of about 6 months, and the stability of the composition is measured by the change in the average particle size of the LNPs, the encapsulation efficiency of the LNPs, and / or the integrity of the one or more RNA molecules encapsulated in the LNPs.

35. The aforementioned one or more RNA molecules: a) Messenger RNA (mRNA) molecule, b) Encoding one or more viral proteins, wherein the one or more viral proteins optionally include influenza virus protein, respiratory syncytial virus protein, coronavirus protein, or a combination thereof, and / or c) A composition according to any one of claims 1 to 13 and 28 to 34, comprising at least one chemically modified nucleotide and / or a phosphorothioate bond, wherein the at least one chemically modified nucleotide comprises pseudouridine, 2'-fluororibonucleotide, or 2'-methoxyribonucleotide, and wherein the pseudouridine is N1-methylpseudridine; a liquid composition according to any one of claims 14 to 20; or a liquid formulation according to any one of claims 21 to 27.

36. The composition, liquid composition, or liquid formulation according to any one of claims 1 to 35, wherein the LNP comprises a cationic lipid, a polyethylene glycol conjugate (PEG-conjugated) lipid, a cholesterol-based lipid, and a helper lipid.

37. a) The cationic lipid is present in a molar ratio of approximately 30% to approximately 50%, a) The PEGylated lipid is present in a molar ratio of approximately 0.25% to approximately 15%. a) The cholesterol-based lipids are present in a molar ratio of approximately 20% to approximately 40%, a) The composition, liquid composition, or liquid formulation according to claim 36, wherein the helper lipid is present in a molar ratio of about 20% to about 40%.

38. The cationic lipid, the PEG-modified lipid, the cholesterol-based lipid, and the helper lipid are a) Approximately 40%, 1.5%, 28.5%, and 30%, respectively, b) Approximately 40%, 5%, 25%, and 30%, respectively The composition, liquid composition, or liquid formulation according to claim 37, which is present in a molar ratio of .

39. a) The cationic lipid includes OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10 and / or GL-HEPES-E3-E12-DS-3-E14 and / or b) The PEG-modified lipid contains or is 1,2-dimiristoyl-rac-glycero-3-methoxy(DMG)-PEG2000, and / or c) The cholesterol-based lipid contains cholesterol, or is cholesterol, and / or d) The composition, liquid composition, or liquid formulation according to any one of claims 36 to 38, wherein the helper lipid comprises or is dioleoyl-SN-glycero-3-phosphoethanolamine.

40. The composition, liquid composition, or liquid formulation according to any one of claims 1 to 39, wherein the composition, liquid composition, or liquid formulation has an N / P ratio of about 1 to about 10 or about 3 to about 6, optionally about 4.

41. The composition, liquid composition, or liquid formulation according to any one of claims 1 to 40, wherein each of the one or more RNA molecules is present in an amount ranging from about 0.1 μg to about 150 μg, about 1 μg to about 60 μg, or about 5 μg to about 45 μg.

42. The composition, liquid composition, or liquid formulation according to any one of claims 1 to 41, wherein the composition, liquid composition, or liquid formulation is formulated for sublingual, intramuscular, intradermal, subcutaneous, intravenous, intranasal, inhalation, or intraperitoneal administration.

43. The composition, liquid composition, or liquid formulation according to any one of claims 1 to 42, wherein the composition, liquid composition, or liquid formulation is an immunogenic composition.

44. A vaccine comprising the immunogenic composition and a pharmaceutically acceptable carrier according to claim 43.

45. A method for immunizing a target, wherein the method comprises administering the vaccine described in claim 44 to the target in need, wherein the vaccine is optionally administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally, and optionally, a) The method prevents viral infection in the subject, reduces the likelihood of the subject contracting a viral infection, and / or reduces the likelihood of the subject contracting a serious disease resulting from a viral infection, and / or b) A method wherein the method elicits a protective immune response in the subject.

46. The method according to claim 45, wherein the subject is a human, and optionally the human is, for example, 6 months old or older and under 18 years old, at least 6 months old and under 18 years old, at least 18 years old and under 65 years old, at least 6 months old and under 5 years old, at least 5 years old and under 65 years old, at least 60 years old, or at least 65 years old.

47. A method for alleviating one or more symptoms of a viral infection, wherein the method comprises administering the vaccine described in claim 44 to a subject in need.

48. The method according to any one of claims 45 to 47, wherein the vaccine comprises one or more LNP-encapsulated RNA molecules encoding one or more viral proteins, and the one or more viral proteins comprise influenza virus protein, respiratory syncytial virus, coronavirus protein, or a combination thereof.

49. A method for stabilizing a composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs), the method comprising adding at least one thermoreversible gelling agent to the composition in an amount sufficient to maintain the composition in the liquid phase at temperatures above about 12°C and to reversibly transition the composition to a gel form at temperatures between about 1 and 11°C.

50. A method for preventing the degradation of one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs) in a liquid composition, the method comprising adding at least one thermoreversible gelling agent to the liquid composition in an amount sufficient to maintain the liquid composition in the liquid phase at temperatures above about 12°C and to reversibly transition the liquid composition to a gel form at temperatures between about 1 and 11°C.

51. The method according to claim 49 or 50, wherein the at least one thermoreversible gelling agent is present in an amount of about 0.1% to about 30% by weight, about 0.25% to about 5% by weight, or about 0.5% to about 1.5% by weight, and optionally the at least one thermoreversible gelling agent contains or is gelatin in an amount of about 1% by weight.

52. a) The one or more RNA molecules encode one or more viral proteins, such as influenza virus protein, respiratory syncytial virus protein, coronavirus protein, or a combination thereof. b) The method according to any one of claims 49 to 51, wherein the LNP comprises a cationic lipid, a polyethylene glycol (PEG) conjugate (PEG-modified) lipid, a cholesterol-based lipid, and a helper lipid.

53. A method for preventing thermal decomposition of one or more ribonucleic acid (RNA) molecules encapsulated in lipid nanoparticles (LNPs), the method comprising formulating a liquid composition containing the LNPs and the one or more RNA molecules in the presence of at least one thermally stabilizing excipient selected from lipoic acid, L-theanine, vanillin, or a combination thereof.

54. a) The at least one heat-stabilizing excipient is present at a concentration of about 0.1 mM to about 20 mM, about 0.5 mM to about 15 mM, or about 1 mM to about 10 mM. b) The at least one of the heat-stabilizing excipients is present at a concentration of about 5 mM, about 10 mM, or about 15 mM, and / or c) The method according to claim 53, wherein the at least one heat-stabilizing excipient and the one or more RNA molecules are present in a weight ratio of about 5:1 to about 50:

1.

55. a) The at least one heat-stabilizing excipient contains lipoic acid or is lipoic acid, and optionally the lipoic acid and the one or more RNA molecules are present in a weight ratio of approximately 2.5:1 to approximately 15.5:

1. b) The at least one heat-stabilizing excipient contains L-theanine or is L-theanine, and optionally the L-theanine and the one or more RNA molecules are present in a weight ratio of about 10:1 to about 30:1, or c) The method according to claim 53 or 54, wherein the at least one heat-stabilizing excipient contains vanillin or is vanillin, and optionally the vanillin and the one or more RNA molecules are present in a weight ratio of about 12.5:1 to about 50:1.

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