Thermostable compositions comprising mRNA lipid nanoparticles

Abstract

This application relates to compositions comprising lipid nanoparticles (LNPs) which encapsulate ribonucleic acid (RNA) molecules, including messenger RNA molecules, stabilized with one or more thermoreversible gelling agents, one or more thermostabilizing excipients, and / or a thermostable formulation comprising a buffering agent, a pharmaceutically acceptable salt, a disaccharide, a surfactant, and a chelating agent. Methods of making and of use of the stabilized compositions, as well as methods of stabilizing compositions comprising RNA-encapsulated LNPs, are also provided.

Description

THERMOSTABLE COMPOSITIONS COMPRISING MRNA LIPIDNANOPARTICLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to European Application No. 23305868.4 filed 1 June 2023 and European Application No. 23305942.7 filed on 14 June 2023, the contents of which are incorporated herein by reference in their entireties.FIELD

[0002] This application relates to compositions comprising lipid nanoparticles (LNPs) which encapsulate ribonucleic acid (RNA) molecules, including messenger RNA molecules, stabilized with one or more thermoreversible gelling agents, one or more thermostabilizing excipients, and / or a thermostable formulation comprising a buffering agent, a pharmaceutically acceptable salt, a disaccharide, a surfactant, and a chelating agent. Methods of making and of use of the stabilized compositions, as well as methods of stabilizing compositions comprising RNA-encapsulated LNPs, are also provided.BACKGROUND

[0003] The use of ribonucleic acid (RNA) molecules, such as messenger RNAs (mRNAs), as pharmaceutical agents is of great interest for a variety of applications, including in therapeutics, vaccines, and diagnostics. Effective in vivo delivery of formulations containing RNA molecules (e.g., mRNAs) represents a continuing challenge because RNA is inherently unstable, can activate an immune response, and / or is susceptible to degradation by nucleases. Any of these challenges can lead to loss of translational potency of such RNA molecules (e.g., mRNAs) and thus hinders their efficacy as pharmaceutical agents.

[0004] Various delivery systems, particularly non-viral delivery systems, have been developed to overcome many challenges associated with in vivo delivery of RNA molecules (e.g., mRNAs). Amongst those delivery systems are lipid nanoparticle (LNPs) based delivery systems, which have drawn particular attention in recent years as various LNP formulations have shown promise in a variety of pharmaceutical applications. See e.g., Kowalski et al., Molecular Therapy, 2019, 27(4):710-728; Gomez-Aguado et al., Nanomaterials (Basel), 2020, 10(2):364; Wadhwa et al., Pharmaceutics, 2020, 12(2): 102.

[0005] The rapid approval and remarkable success of COVID-19 vaccines, Comirnaty® (BNT162b2) and Spikevax (mRNA-1273), further demonstrated the clinical validation ofLNP -formulated mRNA as a new class of highly efficacious nucleic acids in the field of vaccines. However, both vaccines require ultra-cold, sub-zero temperatures for long-term storage, which is not patient- and pharmacy-friendly and is not ideal for widespread use. Thus, there remains a need for LNP formulations containing RNA molecules (e.g., mRNAs) that can be stored at convenient temperatures, such as 4°C, for an extended period of time without significant loss of RNA stability, thus facilitating transportation and storage of RNA-LNP formulations and prolonging their shelf life.SUMMARY

[0006] Disclosed herein are compositions and methods for the stabilization of therapeutic agents, including ribonucleic acid (RNA) molecules, such as mRNAs, encapsulated in lipid nanoparticles (LNPs) using one or more thermoreversible gelling agents (such as polypeptide- or protein-based polymers, e.g., gelatin), one or more thermostabilizing excipients (such as lipoic acid, L-theanine, vanillin, or combinations thereof), and / or a thermostable formulation comprising a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant, and a chelating agent. The present disclosure encompasses, in some aspects, the observation that a mixture of at least one thermoreversible gelling agent (such as a polypeptide- or protein-based polymer, e.g., gelatin) and / or at least one thermostabilizing excipient (such as lipoic acid, L-theanine, vanillin, or combinations thereof) and RNA molecules, such as mRNAs, encapsulated in a LNP resulted in substantially improved formulation stability, which allows for the resultant formulations to be stored at a convenient temperature, such as 2-8°C, for a relatively long period of time. The present disclosure further encompasses, in other aspects, the observation that formulating RNA molecules, such as mRNAs, encapsulated in a LNP in a formulation comprising a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant, and a chelating agent, each present at a prescribed amount, resulted in substantially improved formulation stability, which allows for the resultant formulation to be stored at a convenient temperature, such as 2-8°C, for a relatively long period of time. Accordingly, in one aspect, provided herein is a composition comprising one or more RNA molecules encapsulated in a LNP and at least one thermoreversible gelling agent, such as a thermoreversible gelling agent having an upper critical solution temperature (UCST) between about 12°C and about 50°C. In some embodiments, the composition has a liquid phase at a temperature above about 12°C and is reversibly transitioned to a gel form at a temperature of about 1-11°C. The at least one thermoreversible gelling agent can be present in the composition in an amount of from about 0.1% to about 30% by weight in some embodiments,from about 0.25% to about 5% by weight in other embodiments, or from about 0.5% to about 1.5% by weight in some further embodiments. The at least one thermoreversible gelling agent can comprise a thermoreversible gelling polymer (e.g., a polypeptide- or protein-based polymer), such as gelatin, poly(N-acryloylasparaginamide), poly(ethylene glycol)-b-poly(N- acryloylglycine amide-co-acrylonitrile) (PEG-b-P(NAGA-co-AN), poly(N- acryloylglycineamide-co-N-phenylacrylamide) (P(NAGA-co-NPhAm)), poly(N-(2- hydroxypropyl) methacrylamide)-glycolamide) (P(HPMA-GA)), P(AAm-co-AN)-b- poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly(acrylic acid-co- acrylonitrile) (P(AA-co-AN)), imidazole-based poly(N-vinylimidazole-co-l-vinyl-2- (hydroxymethyl)imidazole), poly(sulfobetaine-co-sulfabetaine) (P(SB-co-ZB), poly(2- (methacryloyloxy)ethylphosphocholine)-b-poly(2-ureidoethyl methacrylate) (PMPC20-b- PUEM165), or combinations thereof. In other embodiments, the at least one thermoreversible gelling agent comprises a thermoreversible gelling polypeptide, such as polypeptide-based multi-L-arginyl-poly-L-aspartate (iMAPA)-PEG. In some further embodiments, the at least one thermoreversible gelling agent comprises a thermoreversible gelling protein. In some embodiments, the at least one thermoreversible gelling agent comprises gelatin, which can 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 as compared to a control composition without the at least one thermoreversible gelling agent, wherein stability of the composition is measured by a change in mean particle size of the LNP, encapsulation efficiency of the LNP, and / or integrity of the one or more RNA molecules encapsulated in the LNP. In some embodiments, the mean particle size of the LNP does not increase 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 as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP does not decrease 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 as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP is higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent. In some embodiments, the integrity of the one or more RNAmolecules encapsulated in the LNP does not decrease 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 as compared to a control composition without the at least one thermoreversible gelling agent.

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

[0009] In some embodiments, the integrity of the one or more RNA molecules does not decrease more than 20% after storage of the liquid composition at a temperature of 37°C for at least 7 days as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than 25% after storage of the liquid composition at a temperature of 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 as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than 30% after storage of the liquid composition 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, or up to about 8 months as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNAmolecules does not decrease 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 as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease 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 as compared to a control liquid composition without the at least one thermostabilizing excipient, In some embodiments, the mean particle size of the LNP does not increase more than 40% after storage of the liquid composition at a temperature of 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, 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. In some embodiments, the mean particle size of the LNP does not increase more than 20% 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, up to about 5 weeks, up to about 6 weeks, or up to about 7 weeks. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than 20% after storage of the liquid composition at a temperature of 4°C for up to about 1 month, up to about2 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. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than 20% 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 about3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, or up to about 7 weeks.

[0010] In yet another aspect, provided herein is a composition comprising one or more RNA molecules encapsulated in a LNP, at least one thermoreversible gelling agent, and at least one thermostabilizing excipient, wherein the at least one thermostabilizing excipient comprises or is 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, or up to about 6 months as compared to a control composition without the at least one thermoreversible gelling agent and the at least one thermostabilizing excipient, wherein stability of the composition is measured by a change in mean particle size of the LNP, encapsulation efficiency of the LNP, and / or integrity of the one or more RNA molecules encapsulated in the LNP. In some embodiments, the at least onethermoreversible gelling agent comprises or is gelatin. In some embodiments, the at least one thermoreversible gelling agent comprises or is gelatin in an amount of from 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, the lipoic acid is present in a concentration of from about 1 mM to about 10 mM. In some embodiments, the lipoic acid is present in a concentration of from about 1 mM to about 5 mM. In some embodiments, the lipoic acid and the one or more RNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1.[OH] In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid further comprises a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant, and / or a chelating agent. In some embodiments, the buffering agent comprises or is tris(hydroxymethyl)aminomethane) (Tris). In some embodiments, the pharmaceutically acceptable salt comprises or is sodium chloride (NaCl). In some embodiments, the one or more disaccharides comprise or are sucrose. In some embodiments, the surfactant comprises or is Poloxamer 188 (Pl 88). In some embodiments, the chelating agent comprises or is ethylenediaminetetraacetic acid (EDTA).

[0012] In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises from about 10 mM to about 60 mM of Tris, from about 40 mM to about 150 mM of NaCl, from about 1% to about 10% by weight of sucrose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.2 to about 7.8. In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the composition has a pH of 7.5 ± 0.3. In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises from about 10 mM to about 60 mM of Tris, from about 40 mM to about 110 mM of NaCl, from about 3% to about 6% by weight of sucrose, from about 0.2% to about 4% byweight of trehalose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.5 to about 7.7. In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7. In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises from about 20 mM to about 50 mM of Tris, from about 50 mM to about 100 mM of NaCl, from about 2% to about 5% by weight of sucrose, from about 0.3% to about 3% by weight of trehalose, from about 0.2% to about 0.4% by volume of Pl 88, and from about 10 pM to about 15 pM of EDTA, wherein the composition has a pH of about 7.7. In some embodiments, the composition comprising the at least one thermoreversible gelling agent, the liquid composition comprising the at least one thermostabilizing excipient, or the composition comprising the at least one thermoreversible gelling agent (e.g., gelatin) and lipoic acid comprises about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4-1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7.

[0013] In yet another aspect, provided herein is a liquid formulation comprising one or more ribonucleic acid (RNA) encapsulated in a lipid nanoparticle (LNP), from about 10 mM to about 60 mM of tris(hydroxymethyl)aminomethane) (Tris), from about 40 mM to about 150 mM of sodium chloride (NaCl), from about 1% to about 10% by weight of sucrose, from about 0.2% to about 0.6% by volume of Poloxamer 188 (P188), and from about 5 pM to about 15 pM of ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of from about 7.2 to about 7.8. In some embodiments, the liquid formulation comprises about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the liquid formulation has a pH of 7.5 ± 0.3. Also provided herein is a liquid formulation comprising one or more ribonucleic acid (RNA) encapsulated within a lipid nanoparticle (LNP), from about 10 mM to about 60 mM of tris(hydroxymethyl)aminomethane) (Tris), from about 40 mM to about 110 mM of sodium chloride (NaCl), from about 3% to about 6% by weight of sucrose, from about 0.2% to about4% by weight of trehalose, from about 0.2% to about 0.6% by volume of Pol oxamer 188 (Pl 88), and from about 5 pM to about 15 pM of ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of from about 7.5 to about 7.7. In some embodiments, the liquid formulation comprises about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the liquid formulation has a pH of about 7.7. Further provided herein is a liquid formulation comprising one or more ribonucleic acid (RNA) encapsulated within a lipid nanoparticle (LNP), from about 20 mM to about 50 mM of tris(hydroxymethyl)aminomethane) (Tris), from about 50 mM to about 100 mM of sodium chloride (NaCl), from about 2% to about 5% by weight of sucrose, from about 0.3% to about 3% by weight of trehalose, from about 0.2% to about 0.4% by volume of Pol oxamer 188 (P188), and from about 10 pM to about 15 pM of ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of about 7.7. In some embodiments, the liquid formulation comprises about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4-1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein 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 virus proteins, such as influenza virus proteins, respiratory syncytial virus (RSV) proteins, coronavirus proteins, or combinations 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 can also comprise at least one chemically modified nucleotide in some embodiments, which can comprise a pseudouridine (such as a N1 -methylpseudouridine), a 2'-fluoro ribonucleotide, or a 2'-m ethoxy ribonucleotide, and / or a phosphorothioate bond in other embodiments. 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 pg to about 150 pg, such as from about 1 pg to about 60 pg or from about 5 pg to about 45 pg.

[0015] In some embodiments, the LNP comprised in the composition of the disclosure comprises a cationic lipid (e.g., cKK-ElO), a polyethylene glycol conjugated (PEGylated) lipid (e.g., l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000), a cholesterol-based lipid (e.g., cholesterol), and a helper lipid (e.g., dioleoyl-SN-glycero-3-phosphoethanolamine). The cationic lipid (e.g., cKK-ElO) can be present at a molar ratio between about 30% and about 50% (e.g., about 40%), the PEGylated lipid (e.g., l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000) can be present at a molar ratio between about 0.25% and about 15%, (e.g.,about 1.5% or about 5%), the cholesterol-based lipid (e.g., cholesterol) can be present at a molar ratio between about 20% and about 40% (e.g., about 25% or about 28.5%), and the helper lipid (e.g., dioleoyl-SN-glycero-3-phosphoethanolamine) can be present at a molar ratio between about 20% and about 40% (e.g., about 30%). In some embodiments, the cationic lipid comprises OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4- E10, and / or GL-HEPES-E3-E12-DS-3-E14, the PEGylated lipid comprises 1,2-dimyristoyl- rac-glycero-3 -methoxy (DMG)-PEG2000, the cholesterol-based lipid comprises cholesterol, and / or the helper lipid comprises di oleoyl-SN-glycero-3 -phosphoethanolamine. Accordingly, in some embodiments, the LNP comprised in the composition of the disclosure comprises a cationic lipid (e.g, OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12- DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14), a PEGylated lipid (e.g., 1,2-dimyristoyl-rac- glycero-3 -methoxy (DMG)-PEG2000), a cholesterol-based lipid (e.g., cholesterol), and a helper lipid (e.g., di oleoyl-SN-glycero-3 -phosphoethanolamine) at a molar ratio of about 40: 1.5:28.5:30, or about 40:5:25:30. In other embodiments, the LNP comprised in the composition of the disclosure comprises ALC-0315 as the cationic lipid, N,N- ditetradecylacetamide-polyethylene glycol as the PEGylated lipid, distearoylphosphatidylcholine (DSPC) as the helper lipid, and cholesterol.

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

[0017] In some embodiments, the compositions and formulations of the disclosure are immunogenic compositions. In another aspect, accordingly, provided herein is a vaccine comprising the immunogenic composition of the disclosure and a pharmaceutically acceptable carrier as well as methods of using the same, such as a method of immunizing a subject or a method of reducing one or more symptoms of a virus infection in a subject. In some embodiments, the method of immunizing a subject with the vaccine of the disclosure prevents a virus infection in the subject, decreases the subject’s likelihood of getting a virus infection, or reduces the subject’s likelihood of getting serious illness from a virus infection. In other embodiments, the method of immunizing a subject with the vaccine of the disclosure raises a protective immune response in the subject. In some embodiments, the subject is a human, such as a human of 6 months of age or older, less than 18 years of age, at least 6 months of age and less than 18 years of age, at least 18 years of age and less than 65 years of age, at least 6 monthsof age and less than 5 years of age, at least 5 years of age and less than 65 years of age, at least 60 years of age, or at least 65 years of age. The vaccine of the disclosure can be administered to the subject, in some embodiments, intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally. In some embodiments, the vaccine of the disclosure comprises one or more LNP-encapsulated RNA molecules which encode one or more virus proteins, such as influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof.

[0018] In a further aspect, provided herein is a method of stabilizing a composition comprising one or more RNA molecules encapsulated in aLNP, the method comprising adding at least one thermoreversible gelling agent to the composition in an amount sufficient to maintain the composition in a liquid phase at a temperature above about 12°C and reversibly transition the composition to a gel form at a temperature of about 1-11°C (e.g., 2-8°C or 4°C). Also provided herein is a method of preventing degradation of one or more RNA molecules encapsulated in a LNP 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 a liquid phase at a temperature above about 12°C and reversibly transition the liquid composition to a gel form at a temperature of about 1-11°C (e.g., 2-8°C or 4°C). In some embodiments, the at least one thermoreversible gelling agent is present in an amount of from about 0.1% to about 30% by weight, from about 0.25% to about 5% by weight, or from about 0.5% to about 1.5% by weight. In some embodiments, the at least one thermoreversible gelling agent comprises gelatin in an amount of about 1% by weight. In some embodiments, the one or more RNA molecules encode one or more virus proteins, such as influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof. In some embodiments, the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0019] In yet another aspect, provided herein is a method of preventing thermal degradation of one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP), the method comprising formulating a liquid composition comprising the LNP and the one or more RNA molecules in the presence of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combinations thereof. In some embodiments, the at least one thermostabilizing excipient is present in a concentration of from about 0.1 mM to about 20 mM, from about 0.5 mM to about 15 mM, or from about 1 mM to about 10 mM. In some embodiments, the at least one thermostabilizing excipient is present in a concentration of about 5 mM, about 10 mM, or about 15 mM. In some embodiments, the at least one thermostabilizingexcipient and the one or more RNA molecules are present in a weight ratio of from about 5: 1 to about 50: 1. In some embodiments, the at least one thermostabilizing excipient comprises or is lipoic acid, optionally wherein the lipoic acid and the one or more RNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5:1. In some embodiments, the at least one thermostabilizing excipient comprises or is L-theanine, optionally wherein the L-theanine and the one or more RNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1. In some embodiments, the at least one thermostabilizing excipient comprises or is vanillin, optionally wherein the vanillin and the one or more RNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1.BRIEF DESCRIPTION OF THE DRAWING

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and compositions disclosed herein.

[0021] FIG. 1A depicts a representative phase diagram of a thermoreversible gelling agent having a liquid phase at a temperature above its upper critical solution temperature (UCST) which is reversibly transitioned to a gel (or hydrogel) form at a temperature below its UCST. FIG. IB depicts aggregation or fusion of lipid nanoparticles (LNPs) during storage (top) and stabilization of LNPs with hydrogel (bottom).

[0022] FIGs. 2A-2B depict exemplary thermoreversible gel forming formulations according to the disclosure filled into a sealed vial (FIG. 2A) and a pre-filled syringe (FIG. 2B) at 4°C and at room temperature (RT). FIG. 2B shows that a thermoreversible gel forming formulation was reversibly transitioned to the liquid phase at RT from the gel form at 4°C within 15 minutes.

[0023] FIG. 3 depicts the stability of a representative thermoreversible gel forming formulation containing 1% gelatin according to the disclosure after storage at 4°C for 1, 2, 3, or 4 months measured by the degree of decrease in mRNA integrity (top), the change in particle size (middle), and the change in encapsulation efficiency (EE; bottom) of the lipid nanoparticles. In each instance, the control formulation contains the same mRNA-LNP formulation without the thermoreversible gelling agent (i.e., gelatin).

[0024] FIGs. 4A-4D depict the stability of a representative thermoreversible gel forming formulation containing 1% gelatin according to the disclosure after storage at 4°C for up to 4 months measured by the increase of degraded mRNA products using capillary electrophoresis (CE) (RFU: relative fluorescence unit). FIG. 4A: control at To; FIG. 4B: gelatin-basedformulation at To; FIG. 4C: control after storage at 4°C for 4 months; FIG. 4D: gelatin-based formulation after storage at 4°C for up to 4 months.

[0025] FIGs. 5A-5C depict the stability of a representative thermoreversible gel forming formulation containing 1% gelatin according to the disclosure after storage at 4°C for up to 9 months measured by the degree of decrease in mRNA integrity (FIG. 5A), the change in encapsulation efficiency of the lipid nanoparticles (FIG. 5B), and the change in particle size (FIG. 5C). In each instance, the control formulation contains the same mRNA-LNP formulation without the thermoreversible gelling agent (i.e., gelatin).

[0026] FIG. 6 depicts the effect of gelatin on protein expression in an mRNA-LNP formulation containing CKK-E10 as the cationic lipid and mRNA encoding human erythropoietin (hEPO). “Gelatin Buffered DP”: a representative thermoreversible gel forming formulation containing 1% gelatin according to the disclosure; “Bulk DP”: a control formulation containing the same mRNA-LNP formulation without the thermoreversible gelling agent (i.e., gelatin); DP: drug product. The difference in protein expression between two formulations is non-significant (ns).

[0027] FIGs. 7A-7B show how the addition of various excipients to an mRNA-LNP formulation affects mRNA integrity following storage for at least one week at 37°C. FIG. 7A shows the mRNA integrity data of all the excipients screened after being formulated with a mRNA encoding human erythropoietin (hEPO) and the cationic lipid cKK-E12 (also known as ML2). The j’-axis represents the change in percentage of RNA integrity (% mRNA integrity) and the x-axis shows the time in days. FIG. 7B shows the tested excipients that had higher mRNA integrity values after 7 days at 37°C as compared to the naked mRNA control and the formulation control. The formulation control contains the same mRNA-LNP formulation but without the excipient. The naked mRNA control contains mRNA that is in RNase-free water.

[0028] FIG. 8A shows that the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) into the mRNA-LNP formulation decreased the amount of mRNA degradation following storage at 37°C for 7 days as compared to the formulation control. FIG. 8B shows that no significant change in the LNP particle size (nm) of the LNP was observed with the addition of L-theanine (10 mM) or vanillin (5 mM) after liquid storage at 37°C for 7 days. FIG. 8C shows that the mRNA encapsulation efficiency of the LNP remained unchanged for all formulations (L-theanine, lipoic acid, or vanillin) after storage at 37°C for 7 days. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0029] FIGs. 9A-9F show that the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to an mRNA-LNP formulation decreased the amount of mRNA degradation when stored at 4°C (FIG. 9A, FIG. 9C, and FIG. 9D) and 25 °C (FIG. 9B, FIG. 9D, and FIG. 9F) as compared to the formulation control. The mRNA-LNP formulations were made with a modified quadrivalent influenza mRNA (“4 Flu mRNAs”) and cKK-ElO (FIGs. 9A-9B), OF- 02 (FIGs. 9C-9D) or GL-HEPES-E3-E12-DS-4-E10 (FIGs. 9E-9F) as the cationic lipid. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0030] FIGs. 10A-10F show how the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to an mRNA-LNP formulation affects LNP particle size (nm) in the mRNA-LNP formulations following storage at 4°C (FIG. 10A, FIG. 10C, and FIG. 10D) and 25°C (FIG. 10B, FIG. 10D, and FIG. 10F) overtime. The mRNA-LNP formulations were made with a modified quadrivalent influenza mRNA (“4 Flu mRNAs”) and cKK-ElO (FIGs. 10A-10B), OF-02 (FIGs. 10C-10D) or GL-HEPES-E3-E12-DS-4-E10 (FIGs. 10E-10F) as the cationic lipid. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0031] FIGs. 11A-11F show that the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) to mRNA-LNP formulations improved or had no effect on the encapsulation efficiency following storage at 4°C (FIG. 11 A, FIG. 11C, and FIG. 11D) or 25°C (FIG. 11B, FIG. 11D, and FIG. 11F) as compared to the control formulation. The mRNA-LNP formulations were made with a modified quadrivalent influenza mRNA (“4 Flu mRNAs”) and cKK-ElO (FIGs. 11A-11B), OF-02 (FIGs. 11C-11D) or GL-HEPES-E3-E12- DS-4-E10 (FIGs. 11E-11F) as the cationic lipid. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0032] FIGs. 12A-12F show the liquid stability conferred by the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) in the mRNA-LNP formulations following storage at 25°C and 30°C. The mRNA-LNP formulations were made with a modified monovalent influenza mRNA encoding an influenza hemagglutinin from a Tasmania strain and cKK-ElO (FIG. 12A, FIG. 12C, and FIG. 12E), OF-02 or GL-HEPES-E3-E12-DS-4-E10 (FIG. 12B, FIG. 12D, and FIG. 12F) as the cationic lipid. FIG. 12A and FIG. 12B show the reduction in mRNA integrity over 7 weeks. FIG. 12C and FIG. 12D show the LNP particle size over 7 weeks. FIG. 12E and FIG. 12F show the encapsulation efficiency over 7 weeks. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0033] FIG. 13 shows that the addition of L-theanine (10 mM), lipoic acid (5 mM), and vanillin (10 mM) as an excipient to mRNA-LNP formulations containing OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid did not disrupt mRNA delivery and protein production (hEPO protein (ng / mL) in mice as compared to the formulation control (n=4). In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0034] FIG. 14 shows that the addition of L-theanine (10 mM), lipoic acid (5 mM), and vanillin (10 mM) as an excipient to mRNA-LNP formulations containing an mRNA encoding influenza hemagglutinin and OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid did not lower HAI titers produced in mice (n=8 per group) as compared to the formulation control. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient. Each dot on the graph represents individual mouse animal titer values. The bars and error bars represent the geometric mean with 95% confidence intervals, respectively.

[0035] FIGs. 15A-15D show the stability conferred by the addition of gelatin, lipoic acid, or combination of gelatin and lipoic acid (“Gelatin + Lipoic Acid”) in the mRNA-LNP formulations following storage at 2-8°C for 12 months. The mRNA-LNP formulations were prepared with a quadrivalent influenza mRNA in 10% trehalose (1 mg / mL) and GL-HEPES- E3-E12-DS-4-E10 as the 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 pM EDTA. FIG. 15A shows the reduction in mRNA integrity over 12 months. FIG. 15B shows the encapsulation efficiency over 12 months. FIG. 15C shows the LNP particle size over 12 months. In each instance, the formulation control contains the same mRNA-LNP formulation but without gelatin and lipoic acid. FIG. 15D shows no visible aggregates in the sealed vial containing the mRNA-LNP formulation with the addition of gelatin or combination of gelatin and lipoic acid after storage at 2-8°C for 12 months (right), while visible aggregates can be seen in the sealed vial containing the mRNA-LNP formulation with the addition of lipoic acid or the formulation control after storage at 2-8°C for 9 months (left).

[0036] FIGs. 16A-16C show the stability conferred by the addition of gelatin, lipoic acid, or combination of gelatin and lipoic acid (“Gelatin + Lipoic Acid”) in the mRNA-LNP formulations following storage at 2-8°C for 6 months. The mRNA-LNP formulations were made with a quadrivalent 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 the 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% P-188,and 10 pM EDTA. FIG. 16A shows the reduction in mRNA integrity over 6 months. FIG. 16B shows the encapsulation efficiency over 6 months. FIG. 16C shows the LNP particle size over 6 months. In each instance, the formulation control contains the same mRNA-LNP formulation but without gelatin and lipoic acid.

[0037] FIG. 17 depicts the mean particle size of LNPs loaded with an mRNA encoding an influenza antigen (“monoFlu-LNPs”) by Dynamic Light Scattering in different buffer conditions, in the pH range 6-8. CL: cationic lipid (CL 1 : OF-02; CL 2: cKK-ElO; CL 3: GL- HEPES-E3-E12-DS-4-E10).

[0038] FIG. 18 depicts mRNA expression of monoFlu-LNPs in different buffer conditions, in the pH range 6-8, analyzed by Flow Cytometry (for CL-1 formulations) and by Western blot (for CL-2 and CL-3 formulations). CL: cationic lipid (CL-1 : OF-02; CL-2: cKK-ElO; CL-3: GL-HEPES-E3-E12-DS-4-E10).

[0039] FIG. 19 depicts the mean particle size by Dynamic Light Scattering and %mRNA integrity drop on monoFlu-LNPs in different buffer conditions, in the pH range 7.5-8.5. CL: cationic lipid (CL 2: cKK-ElO; CL 3: GL-HEPES-E3-E12-DS-4-E10).

[0040] FIG. 20A depicts pH evolution and %mRNA encapsulation after 6 days in different buffer conditions in the presence of 0.26 mg / mL mRNA-LNPs. FIG. 20B depicts %mRNA integrity drop in different buffer conditions in the presence of 0.1 mg / mL mRNA-LNPs. QIV- LNPs with CL-3 : LNPs loaded with a quadrivalent influenza mRNA (“QIV-LNPs”) using GL- HEPES-E3-E12-DS-4-E10 as the cationic lipid.

[0041] FIG. 21 depicts the mean particle size of monoFlu-LNPs 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 trehalose concentrations. CL: cationic lipid (CL 2: cKK- E10; CL 3: GL-HEPES-E3-E12-DS-4-E10).

[0042] FIGs. 22A-22D depict the visual aspect (FIG. 22A), mean particle size by Dynamic Light Scattering (FIG. 22B), visible particles (FIG. 22C), and turbidity (FIG. 22D) of monoFlu-LNPs with cationic lipid cKK-ElO after Freeze / Thaw cycles at -20°C / RT in the presence of different concentrations of trehalose or sucrose.

[0043] FIG. 23 depicts the mRNA encapsulation rate (%mRNA encapsulation) after Freeze / Thaw cycles at -20°C / RT and after storage at 25°C for 2 weeks in the presence of sucrose. CL: cationic lipid (CL 2: cKK-ElO; CL 3: GL-HEPES-E3-E12-DS-4-E10).

[0044] FIGs. 24A-24D depict the visual aspect (FIG. 24A), mean particle size by Dynamic Light Scattering (FIG. 24B), subvisible particles (FIG. 24C), and %mRNA encapsulation(FIG. 24D) of mRNA-LNPs with cationic lipid cKK-ElO after 3 days of orbital shaking stress at RT.

[0045] FIG. 25 depicts the subvisible particles evolution and turbidity evolution in the presence of Pl 88 or PS80 after 3 days of orbital shaking stress at RT and 3 Freeze / Thaw (F / T) cycles at -20°C / RT. CL: cationic lipid (CL 1 : OF-02; CL 2: cKK-ElO; CL 3: GL-HEPES-E3- E12-DS-4-E10).

[0046] FIG. 26 depicts mean particle size evolution of mRNA-LNPs with cationic lipid cKK-ElO at 25°C in the presence of different concentrations of EDTA.

[0047] FIG. 27 depicts %mRNA integrity decrease at 25°C in the presence of EDTA on QIV-LNPs with three different cationic lipids. CL: cationic lipid (CL 1 : OF-02; CL 2: cKK- E10; CL 3: GL-HEPES-E3-E12-DS-4-E10).

[0048] FIG. 28 depicts exemplary long term stability data of QIV-LNP formulations with the three different cationic lipids (LNPs particle size by Dynamic Light Scattering, %mRNA encapsulation by RiboGreen assay, %decrease of mRNA integrity by capillary electrophoresis). CL: cationic lipid (CL-1 : OF-02; CL-2: cKK-ElO; CL-3: GL-HEPES-E3- E12-DS-4-E10).

[0049] FIG. 29 depicts the main outcomes from the optimization and stability studies described in Example 10.

[0050] FIG. 30 depicts stability profilers showing the optimal settings in terms of pH, buffer amount, and salt amount to maximize mRNA integrity for each storage temperature and timepoint tested.

[0051] FIG. 31 depicts stability profilers (after storage at 5°C for 1 month) showing the impact of pH, buffer amount, and salt amount on mRNA fragments, mRNA integrity, and mRNA-lipid adducts.

[0052] FIG. 32 depicts mRNA encapsulation rate evaluated by RiboGreen after storage at 30°C for 2 weeks (“T2W 30°C”) or 1 month (“TIM 30°C”) in different buffer amount, salt amount, and pH.

[0053] FIG. 33 depicts mRNA expression evaluated by Flow Cytometry after storage for 1 week (“T1W”) or 2 weeks (“T2W”) in different buffer amount, salt amount, and pH. The results are consistent for each strain used and only iLogMFI for one representative strain is shown.

[0054] FIGs. 34A-34B depict impact of surfactant Pl 88 on stability after storage at 5°C or 25°C for 1 day (“Tld”), 2 days “(“T2d”), or 5 days (“T5d”), and after Freeze / Thaw cycles. FIG. 34A: LNP size by DLS; FIG. 34B: Subvisible particles by FlowCam analysis.

[0055] FIGs. 35A-35B depict impact of sucrose on stability after storage at 5°C or 25°C for 1 day (“Tld”), 2 days “(“T2d”), or 5 days (“T5d”), and after Freeze / Thaw cycles. FIG. 35A: LNP size by DLS; FIG. 35B: Subvisible particles by FlowCam analysis.

[0056] FIG. 36 depicts the impact of EDTA on mRNA integrity by reverse-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC).

[0057] FIGs. 37A-37C shows the stability conferred by the addition of gelatin in the 16:0- 18: 1 PE based LNP formulations following storage at 2-8°C for 7 months. The mRNA-LNP formulations were made with a quadrivalent influenza mRNA in 10% trehalose, GL-HEPES- E3-E12-DS-4-E10 as the cationic lipid, and 16:0-18: 1 PE as helper lipid. FIG. 37A shows the LNP particle size over 7 months. FIG. 37B shows the encapsulation efficiency over 7 months. FIG. 37C shows the reduction in mRNA integrity over 7 months. In each instance, the formulation control contains the same mRNA-LNP formulation but without gelatin.DETAILED DESCRIPTION

[0058] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as limiting the scope of the disclosure.

[0059] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.Definitions

[0060] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0061] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amountand the like, “about” is meant to encompass variations 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 the specified value as such variations are appropriate to perform the disclosed methods and / or to make and use the disclosed compositions. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[0062] The term “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0063] As used herein, the term “antigen” refers to an agent that elicits an immune response; and / or (ii) an agent that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody (e.g., produced by a B cell) when exposed or administered to an organism. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism. It will be appreciated by those skilled in the art that a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, ferrets, rabbits, primates, humans), but not in all members of the target organism species. In some embodiments, an 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%, 99%, including all values and subranges therebetween, of the members of a target organism species. In some embodiments, an antigen binds to an antibody and / or T cell receptor and may or may not induce a particular physiological response in an organism. In some embodiments, for example, an antigen may bind to an antibody and / or to a T cell receptor in vitro, whether or not such an interaction occurs in vivo. In some embodiments, an antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Antigens include the NA and HA forms as described herein.

[0064] The term “at least,” “less than,” “more than,” or “up to” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “less than” or “more than,” and all subsequent numbers or integers that could logically be included, as clear from context. When the term “at least,” “less than,” “more than,” or “up to” is present before a series of numbers or a range, it is understood that “at least,” “less than,” “more than,” or “up to” can modify each of the numbers in the series or range.

[0065] The term “carrier,” as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are, or include, one or more solid components.

[0066] A “control composition without the at least one thermoreversible gelling agent,” as used herein, refers to a composition that is identical to the composition being compared to except that it does not contain the at least one thermoreversible gelling agent. As used herein, the term “as compared to a control composition without the at least one thermoreversible gelling agent” means that it is the control composition without the at least one thermoreversible gelling agent at time-zero (To), i.e., prior to storage, being compared to.

[0067] A “control liquid composition without the at least one thermostabilizing excipient,” as used herein, refers to a liquid composition that is identical to the liquid composition being compared to except that it does not contain the at least one thermostabilizing excipient. As used herein, the term “as compared to a control liquid composition without the at least one thermostabilizing excipient” means that the liquid composition is compared to the control liquid composition without the at least one thermostabilizing excipient at time-zero (To), i.e., prior to storage.

[0068] As used herein, “encapsulation efficiency” or “EE” refers to the amount of a therapeutic and / or prophylactic, such as a RNA molecule of the disclosure, that becomes part of a lipid nanoparticle (LNP), relative to the initial total amount of therapeutic and / or prophylactic used in the preparation of a LNP. For example, if 97 mg of therapeutic and / or prophylactic are encapsulated in a LNP out of a total 100 mg of therapeutic and / or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. Encapsulation efficiency can be determined by, for instance, the RiboGreen assay or any method known in the art. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0069] As used herein, “Hl” refers to an influenza virus subtype 1 hemagglutinin (HA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and neuraminidase (NA). Currently, there are 18 recognized HA subtypes (Hl -Hl 8). Hl is thus distinct from the other HA subtypes, including H2-H18.

[0070] As used herein, “H3” refers to an influenza virus subtype 3 HA. H3 is thus distinct from the other HA subtypes, including Hl, H2 and H4-H18.

[0071] As used herein, the term “in some embodiments,” “in certain embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0072] As used herein, “particle size” or “mean particle size” in the context of lipid nanoparticle compositions refers to the mean diameter of a nanoparticle composition. Particle size can be determined using any method known in the art, such as by 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 and transformed into volume-based particle size and size distribution. Accordingly, in some embodiments, the defined particle sizes in the present disclosure, when measured by DLS, relate to volume mean diameter.

[0073] As used herein, the term “RNA-LNP composition” or “RNA-LNP formulation” refers to a composition or formulation comprising one or more RNA molecules, such as mRNA molecules, encapsulated in LNPs. Thus, a composition or formulation comprising one or more mRNA molecules encapsulated in LNPs is referred to as “mRNA-LNP composition” or “mRNA-LNP formulation.”

[0074] As used herein, “Nl” refers to an influenza virus subtype 1 neuraminidase (NA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and neuraminidase (NA). Currently, there are 11 recognized NA subtypes (Nl-Nl l). Nl is thus distinct from the other NA subtypes, including N2-N11.

[0075] As used herein, “N2” refers to an influenza virus subtype 2 neuraminidase (NA). N2 is thus distinct from the other NA subtypes, including N 1 and N3-N11.

[0076] The term “prevent,” “preventing,” or “prevention,” as used herein, refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and / or reduction in frequency and / or severity of one or more symptoms of a particular disease, disorder or condition (e.g., infection with, for example, a virus, such as influenza virus, respiratory syncytial virus (RSV), or coronavirus). In some embodiments, prevention is assessed on a population basis such thatan agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, 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.

[0077] The term “prevent” or “preventing,” as used herein in the context of thermal degradation of RNA molecules, refers to a delay of RNA degradation and / or a decrease in amount or percentage of RNA degradation. RNA degradation can be assessed based on the loss in RNA integrity, which can be measured by, for instance, extracting the RNA and analyzed on a fragment analyzer using capillary electrophoresis.

[0078] As used herein, the term “prophylactically effective amount” means an amount sufficient to avoid disease manifestation, delay onset of and / or reduce in frequency and / or severity one or more symptoms of a particular disease, disorder or condition (e.g., infection with, for example, a virus, such as influenza virus, respiratory syncytial virus (RSV), or coronavirus).

[0079] As used herein, the term “room temperature” refers to a temperature of about 18- 25°C.

[0080] Each year, based on intensive surveillance efforts, the World Health Organization (WHO) selects influenza strains to be included in the seasonal vaccine preparations. As used herein, the term “standard of care strain” or “SOC strain” refers to an influenza strain that is selected by the WHO to be included in the seasonal vaccine preparations. A standard of care strain can include a historical standard of care strain, a current standard of care strain or a future standard of care strain.

[0081] 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 nonhuman animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, and / or a clone. In some embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, the term “individual” or “patient” is used and is intended to be interchangeable with the term “subject.”Thermoreversible Gel Forming RNA-LNP Compositions

[0082] Ribonucleic acid (RNA) molecules, such as messenger RNAs (mRNAs), have been used as pharmaceutical agents in a variety of applications, such as therapeutics, vaccines and diagnostics, in recent years. However, due to their inherent instability and susceptibility to degradation by nucleases, storage and effective in vivo delivery of formulations containing RNA molecules (e.g., mRNAs) continue to be a challenge.

[0083] Lipid nanoparticle (LNP) formulations offer an opportunity to deliver various RNA molecules, such as mRNAs, in vivo for applications in which unencapsulated RNA molecules would be ineffective, but their broad utility has been hindered by insufficient RNA stability over relevant timeframes. Degradation of RNA molecules within LNP formulations limits the use of such formulations to applications in which frozen compositions are acceptable, as longterm storage in refrigerated or room temperature conditions is not possible due to loss of stability of the RNAs. Whether LNP formulations comprising RNAs (such as mRNAs) could be amenable to long-term storage in refrigerated conditions, such as at a temperature of about 1-11°C (e.g., 2-8°C or 4°C), remains unclear.

[0084] The present disclosure is based, at least in part, on the surprising finding that the inclusion of a thermoreversible gelling agent in LNP formulations containing RNA molecules resulted in substantially improved stability, including RNA stability, in refrigerated conditions, such as at a temperature of about 1-11°C (e.g., 2-8°C or 4°C), which is useful for preparation, storage and use of RNA molecules as therapeutic agents. For instance, the inventors of the present disclosure surprisingly found that, for mRNA-LNP compositions, combination with a thermoreversible gelling agent according to the present disclosure dramatically inhibits the rate of decrease in integrity of mRNA encapsulated within the LNP after storage at 4°C for extended periods, including at least up to 6 months. The instability of mRNA, specifically decrease in RNA integrity, is considered one of the greatest challenges to its fundamental therapeutic and commercial viability. The inclusion of at least one thermoreversible gelling agent in the mRNA-LNP formulations, according to the present disclosure, thus provides a significant solution to such problems.

[0085] The discovery that using a thermoreversible gelling agent is able to stabilize ribonucleic acids within a lipid carrier, such as an LNP, is surprising and unexpected. This finding enables several significant applications, including extended refrigerated shelf-life of fully liquid formulation. Achieving a stable formulation also enables commercially and therapeutically desirable packaging and delivery options including prefilled syringes (PFS) and 1cartridges for patient-friendly autoinjector and infusion pump devices. The ability to stabilize solutions and pharmaceutical preparations of RNAs, such as mRNAs, and other therapeutics therefore represents a valuable technology facilitating broader use of therapeutic compositions such as mRNA compositions.

[0086] Accordingly, provided herein is a thermoreversible gel forming composition comprising a therapeutic agent and at least one thermoreversible gelling agent. Typically, the therapeutic agent comprises one or more RNA molecules. In certain embodiments, the one or more RNA molecules are encapsulated in a LNP. The thermoreversible gel forming composition of the disclosure generally has a liquid phase at a temperature above about 12°C, such as at room temperature, and is reversibly transitioned to a gel (or hydrogel) form at a temperature of about 1-11°C, such as at refrigerated temperatures (e.g., 2-8°C or 4°C) due to the presence of the at least one thermoreversible gelling agent. The thermoreversible gel forming property of the compositions disclosed herein provides surprising and unexpected advantages in maintaining the thermostability of the compositions without a need of ultra-cold, sub-zero conditions for long-term storage, thus prolongs their shelf life and facilitates widespread use.Thermoreversible gelling agents

[0087] As used herein, the term “thermoreversible” refers to a material, such as a polymer, that exhibits a reversible change in a physical property (e.g., physical state) in response to a change in temperature. “Thermoreversible gelling agent,” also referred to as “thermoresponsive gelling agent” or “thermogelling agent,” refers to an agent which may comprise water-soluble units and units having an upper critical solution temperature (“UCST”). “Upper critical solution temperature” or “UCST” generally refers to the critical temperature above which the components of a mixture are miscible in all proportions. The word “upper” indicates that the UCST is an upper bound to a temperature range of partial miscibility, or miscibility for certain compositions only. Above the UCST, the agent, such as a polymer, is substantially completely soluble in water, whereas below this temperature, the UCST portions aggregate and lose their solubility in water, thus forming crosslinks between the polymer chains. The agent, such as a polymer, then becomes like a three-dimensional network, leading to formation of a gel or hydrogel. Thus, a change in physical state is seen with a change in temperature, for example when a composition in which the agents, such as polymers, are incorporated is subjected to a change in temperature. Because the change to a gel is physical and temperature-dependent, this phenomenon of thermal gelling is completely reversible. Arepresentative phase diagram of a thermoreversible gelling agent according to the disclosure is provided in FIG. 1A. As shown in FIG. IB, the formation of a gel or hydrogel can stabilize LNPs and prevent aggregation or fusion of LNPs.

[0088] In some embodiments, the thermoreversible gelling agents comprised in the compositions disclosed herein have an UCST between about 12°C and about 100°C, such as between about 12°C and about 80°C, between about 12°C and about 60°C, between about 12°C and about 50°C, between about 12°C and about 40°C, between about 12°C and about 30°C, or between about 12°C and about 20°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 12°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 15°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 20°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 25°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 30°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 35°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 40°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 45°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST of about 50°C. In some embodiments, the thermoreversible gelling agents of the disclosure have an UCST that is suitable for the composition and / or vaccine disclosed in the present disclosure.

[0089] Thermoreversible gelling agents suitable for the present disclosure may comprise thermoreversible gelling polymers, thermoreversible gelling polypeptides, and / or thermoreversible gelling proteins. Accordingly, in some embodiments, the thermoreversible gelling agent comprises a thermoreversible gelling polymer. In other embodiments, the thermoreversible gelling agent comprises a thermoreversible gelling polypeptide. In some other embodiments, the thermoreversible gelling agent comprises a thermoreversible gelling protein. In some embodiments, the thermoreversible gelling agent of the disclosure, whether a polymer, polypeptide, or protein, is a hydrogel at a lower temperature, such as about 4°C, while soluble in water at a higher temperature, such as about 12°C, where the hydrogel is a three-dimensional network comprising cross-linked, polymers, polypeptides, or proteins. In certain embodiments, the polymers, polypeptides, or proteins are cross linked through non- covalent bonds.

[0090] In some embodiments, any thermoreversible gelling agent with UCST phase separation behavior, preferably an UCST between about 12°C and about 100°C, such as between about 12°C and about 80°C, between about 12°C and about 60°C, between about 12°C and about 50°C, between about 12°C and about 40°C, between about 12°C and about 30°C, between about 12°C and about 20°C, or about 12°C can be used.

[0091] In some embodiments, the thermoreversible gelling agent of the disclosure is gelatin. Gelatin is a bulking agent and acceptable material for medical use. Without wishing to be bound by any theory, gelatin is generally used as a stabilizer due to its high biocompatibility, biodegradability, low immunogenicity, and low material cost.

[0092] Gelatin is commonly derived from collagen taken from animal body parts, mainly pieces of skin, bones, and connective tissue. Gelatin can be of porcine or bovine origin, which includes pigskin and bovine bone gelatin resulting from acid or alkaline extraction methods or made from fish by-products. Examples of gelatin of porcine or bovine origin include, but are not limited to, beMatrix™ Gelatin series (Nitta Gelatin Inc., Osaka, Japan), hydrolyzed porcine gelatin (SOL-U-PRO; Dynagel Inc., IL), and X-Pure® gelatins (Rousselot Inc., WI). Gelatin can also be of non-animal origin, such as recombinant origin, such as recombinant human gelatin (FG-5001; FibroGen, Inc., CA). Accordingly, in some embodiments, the thermoreversible gelling agents of the disclosure comprise gelatin of porcine origin, such as hydrolyzed porcine gelatin. In some embodiments, the thermoreversible gelling agents of the disclosure comprise gelatin of bovine origin. In some embodiments, the thermoreversible gelling agents of the disclosure comprise gelatin of non-animal origin. In some embodiments, the thermoreversible gelling agents of the disclosure comprise recombinant gelatin, such as recombinant human gelatin. In some embodiments, the thermoreversible gelling agents of the disclosure comprise a food grade gelatin. In some embodiments, the thermoreversible gelling agents of the disclosure comprise a pharmaceutical grade gelatin.

[0093] Gelatin is commonly derived from collagen. It is an irreversibly hydrolyzed form of collagen, wherein the hydrolysis reduces protein fibrils into 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, wherein X is any amino acid other than glycine, proline or hydroxyproline.

[0094] Other non-limiting examples of the thermoreversible gelling polymers of the disclosure include, but are not limited to, poly(N-acryloylasparaginamide), polyethylene glycol)-b-poly(N-acryloylglycine amide-co-acrylonitrile) (PEG-b-P(NAGA-co-AN), poly(N- acryloylglycineamide-co-N-phenylacrylamide) (P(NAGA-co-NPhAm)), poly(N-(2-hydroxypropyl) methacrylamide)-glycolamide) (P(HPMA-GA)), P(AAm-co-AN)-b- poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly(acrylic acid-co- acrylonitrile) (P(AA-co-AN)), imidazole-based poly(N-vinylimidazole-co-l-vinyl-2- (hydroxymethyl)imidazole), poly(sulfobetaine-co-sulfabetaine) (P(SB-co-ZB), and poly(2- (methacryloyloxy)ethylphosphocholine)-b-poly(2-ureidoethyl methacrylate) (PMPC20-b- PUEM165). The thermoresponsive polymers exhibiting UCST described in Kuldeep et al. (eXPRESS Polymer Letters, 2019, 13(11):974-992, incorporated herein by reference) can also be used. In some embodiments, accordingly, the thermoreversible gelling polymers of the disclosure comprises gelatin, poly(N-acryloylasparaginamide), PEG-b-P(NAGA-co-AN), P(NAGA-co-NPhAm), P(HPMA-GA), POEGMA, P(AA-co-AN), poly(N-vinylimidazole-co- l-vinyl-2-(hydroxymethyl)imidazole), P(SB-co-ZB), PMPC20-b-PUEM165, or combinations thereof. In some embodiments, the thermoreversible gelling polymers of the disclosure can be any polymer with phase separation behavior as disclosed herein.

[0095] In some embodiments, any polypeptide with UCST phase separation behavior, preferably an UCST between about 12°C and about 100°C, such as between about 12°C and about 80°C, between about 12°C and about 60°C, between about 12°C and about 50°C, between about 12°C and about 40°C, between about 12°C and about 30°C, between about 12°C and about 20°C, or about 12°C can be used. One non -limiting example of the thermoreversible gelling polypeptides of the disclosure is iMAPA-PEG, which is insoluble multi-L-arginyl-poly- L-aspartate (iMAPA) conjugated with polyethylene glycol (PEG). See e.g., Tseng et al., Biomacromolecules, 2018, 19(12):4585-4592, incorporated herein by reference. Multi-L- arginyl-poly-L-aspartate (MAP A), also known as cyanophycin or CGP (cyanophycin granule polypeptide), is a non-protein, non-ribosomally produced amino acid polymer composed of an aspartic acid backbone and arginine side groups. Other non-limiting examples of the thermoreversible gelling polypeptides of the disclosure include those described in Kuroyanagi et al., J. Am. Chem. Soc., 2019, 141 : 1261-1268, incorporated herein by reference. In some embodiments, the thermoreversible gelling polypeptides of the disclosure comprise iMAPA- PEG.

[0096] The thermoreversible gelling agent of the disclosure, such as gelatin, is present in the composition of the disclosure in an amount sufficient for the composition to have a liquid phase at a temperature above about 12°C, such as room temperature, and a gel phase at refrigerated temperature, such as at about 1-11°C (e.g., 2-8°C or 4°C). In some embodiments, the thermoreversible gelling agent is present in the composition of the disclosure in an amount of from about 0.1% to about 30% by weight, such as from about 0.1% to about 20% by weight,from about 0.1% to about 10% by weight, from about 0.1% to about 5% by weight, from about 0.2% to about 6% by weight, from about 0.25% to about 5% by weight, from about 0.3% to about 4% by weight, from about 0.4% to about 3% by weight, or from about 0.5% to about 1.5% by weight, including any values and subranges therebetween.

[0097] In some embodiments, the thermoreversible gelling agent is present in the composition of the disclosure in an amount of about 0.1% by weight. 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.

[0098] In some embodiments, the thermoreversible gelling agent comprised in the composition of the disclosure comprises gelatin and in certain embodiments, the gelatin is present in an amount of about 1% by weight.Stability

[0099] It has been surprisingly discovered that the thermoreversible gel forming composition of the disclosure was stable in a gel form, even when stored at refrigerated temperature, such as at about 1-11°C (e.g., 2-8°C or 4°C), for a relatively long period of time. As demonstrated in the examples below, the thermoreversible gel forming composition of the disclosure was stable at least in terms of the particle size and encapsulation efficiency of the LNP comprised therein, as well as the integrity of the RNA molecule encapsulated in the LNP, after storage at 4°C for up to about 6 months. It is expected that such stability can be maintained even longer, such as 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, including all values and subranges therebetween. In some embodiments, such stability can be maintained for more than 2 years.

[0100] Accordingly, in some embodiments, the thermoreversible gel forming composition of the disclosure is stable at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month. In some embodiments, the thermoreversible gel forming composition of the disclosure is stable at a temperature of about 1 -11 °C (e.g., 2-8°C or 4°C) for more than 1 month, such as 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, up to about 12 months, up to about 18 months, or up to about 2 years, including all values and subranges therebetween. In some embodiments, the thermoreversible gel forming composition of the disclosure is stable at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for more than 2 years. The stability of the thermoreversible gel forming composition can be measured in various ways as known in the art, such as measuring the change in the particle size of the LNP, the encapsulation efficiency of the LNP, or the integrity of the RNA molecule encapsulated in the LNP. 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 the at least one thermoreversible gelling agent, referring to herein “a control composition without the at least one thermoreversible gelling agent.”

[0101] In some embodiments, the stability of the thermoreversible gel forming composition is measured in terms of the change in the particle size of the LNP, and the composition of thedisclosure is stable when the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the mean particle size of the LNP does not increase more than about 40% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the mean particle size of the LNP does not increase more than about 30% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the mean particle size of the LNP does not increase more than about 20% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the mean particle size of the LNP does not increase more than about 10% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0102] Particle size can be determined using any method known in the art, such as by Dynamic Light Scattering (DLS). In general, prior to storage, the LNPs comprised in the thermoreversible gel forming compositions of the disclosure have a mean particle size ranging from about 10 nm to about 1000 nm, such as from about 15 nm to about 750 nm, from about 30 nm to about 500 nm, from about 50 nm to about 250 nm, from about 75 nm to about 200 nm, or from about 80 nm to about 150 nm.

[0103] In some embodiments, the stability of the composition is measured in terms of the change in the encapsulation efficiency of the LNP, and the composition of the disclosure is stable when the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 15% after storage of the composition at a temperature of about 1-11 °C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 10% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 5% after storage of the composition at a temperature of about 1-11 °C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0104] In some embodiments, the encapsulation efficiency of the LNP is higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP is at least 5% higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP is at least 7.5% higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent. In some embodiments, the encapsulation efficiency of the LNP is at least 10% higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent.

[0105] Encapsulation efficiency of the LNP can be determined using any method known in the art, such as a fluorescence plate-based assay using the RiboGreen reagent (Invitrogen).

[0106] In some embodiments, the stability of the composition is measured in terms of the change in the integrity of the RNA molecule encapsulated in the LNP, and the composition of the disclosure is stable when the integrity of the RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the integrity of the RNA molecules does not decrease more than about 15% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the integrity of the RNA molecules does not decrease more than about 10% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the integrity of the RNA molecules does not decrease more than about 5% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0107] Integrity of the 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).

[0108] In some embodiments, the RNA molecules encapsulated in the LNP of the disclosure encode one or more influenza virus proteins, such as HA and / or NA proteins, and the composition of the disclosure is stable when the hemagglutination inhibition (HAI) titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10%, 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gellingagent. In some embodiments, the HAI titers of the composition does not decrease more than about 25% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the HAI titers of the composition does not decrease more than about 20% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the HAI titers of the composition does not decrease more than about 15% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the HAI titers of the composition does not decrease more than about 10% after storage of the composition at a temperature of about 1-11 °C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the HAI titers of the composition does not decrease more than about 5% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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 10months, up to about 11 months, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0109] HAI titers can be measured using any method known in the art, such as using an influenza virus HAI test (Denka Seiken Co., Tokyo, Japan).Thermostable RNA-LNP Liquid Compositions

[0110] The present disclosure is also based, at least in part, on the surprising finding that the inclusion of certain excipients in LNP formulations containing RNA molecules resulted in substantially improved stability, including RNA stability, when stored as a liquid at an abovezero temperature, such as in refrigerated conditions (e.g., 4°C). For instance, the inventors of the present disclosure surprisingly found that, for mRNA-LNP compositions, inclusion of certain excipients according to the present disclosure dramatically inhibits the rate of decrease in the integrity of mRNA encapsulated within the LNP after storage as a liquid at 4°C for extended periods, including up to 12 months. The instability of mRNA, as measured, for example, by a decrease in RNA integrity, is considered a major challenge to mRNA’s fundamental therapeutic and commercial viability. The inclusion of at least one of those excipients in the mRNA-LNP formulations, according to the present disclosure, thus provides a significant solution to such problems. Because of their ability to confer thermostability, such excipients are also called hereinafter “thermostabilizing excipients.”[oni] The discovery that using such thermostabilizing excipients is able to stabilize RNA within a lipid carrier, such as an LNP, when stored as a liquid formulation is surprising and unexpected. This finding enables several significant applications of these liquid formulations, including extended refrigerated shelf-life, extended in-use periods at room temperature, and extended in-use stability at physiological temperatures up to 37°C. Achieving a stable liquid formulation also enables commercially and therapeutically desirable packaging and delivery options including prefilled syringes (PFS) and cartridges for patient-friendly autoinjector and infusion pump devices. The ability to stabilize solutions and pharmaceutical preparations of RNAs, such as mRNAs, and other therapeutics therefore represents a valuable technology, facilitating broader use of therapeutic compositions, such as mRNA compositions.Thermostabilizing excipients

[0112] Provided herein is a liquid composition comprising one or more RNA molecules encapsulated in a LNP and at least one thermostabilizing excipient, wherein the at least one thermostabilizing excipient comprises or is lipoic acid, L-theanine, vanillin, or combinationsthereof. Other suitable thermostabilizing excipients that may be used in the thermostable liquid compositions of the present disclosure include, but are not limited to, quercetin, glutathione, gallic acid, naringin, acetyl salicylic acid, ascorbic acid, and eugenol. The liquid composition of the present disclosure is generally thermostable, such that the integrity of the one or more RNA molecules encapsulated in the LNP does not substantially decrease after storage of the liquid composition at an above-zero temperature for a period of time.

[0113] Accordingly, the present disclosure relates, among other things, to a thermostable liquid composition comprising one or more RNA molecules encapsulated in a LNP and at least one thermostabilizing excipient comprising lipoic acid, L-theanine, vanillin, or combinations thereof, and having one or more of the following characteristics: extended refrigerated shelflife, extended in-use periods at room temperature, and extended in-use stability at physiological temperatures up to 37°C.

[0114] The thermostabilizing excipient of the present disclosure, such as lipoic acid, L- theanine, vanillin, or combinations thereof, is 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 the RNA molecules and maintaining the mean particle size and encapsulation efficiency of the LNP. In some embodiments, the thermostabilizing excipient of the present disclosure, such as lipoic acid, L-theanine, vanillin, or combinations thereof, is present in the liquid composition of the present disclosure in a concentration of from about 0.1 mM to about 30 mM, such as from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 15 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 15 mM, from about 0.5 mM to about 10 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 15 mM, from about 1 mM to about 10 mM, or from about 1 mM to about 5 mM, including all values and subranges therebetween. In some embodiments, the thermostabilizing excipient is present in a concentration of from about 0.1 mM to about 20 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of from about 0.5 mM to about 15 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of from about 1 mM to about 10 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of from about 5 mM to about 15 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of from about 5 mM to about 10 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of from about 10 mM to about 15 mM.

[0115] In some embodiments, the thermostabilizing excipient of the present disclosure, such as lipoic acid, L-theanine, vanillin, or combinations thereof, is present in the liquid composition of the present disclosure in a concentration 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 9mM, 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 17m M, about 17.5 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, or about 30 mM, including all values and subranges therebetween. In some embodiments, the thermostabilizing excipient is present in a concentration of about 5 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of 10 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of 15 mM. In some embodiments, the thermostabilizing excipient is present in a concentration of 20 mM.

[0116] The amount of the at least one thermostabilizing excipient present in the liquid composition of the present disclosure can also be expressed by a weight ratio between the at least one thermostabilizing excipient and the one or more RNA molecule. Accordingly, in certain embodiments, the thermostabilizing excipient of the present disclosure, such as lipoic acid, L-theanine, vanillin, or combinations thereof, is present in the liquid composition of the present disclosure in an amount so that the thermostabilizing excipient and the one or more RNA molecules are present in a weight ratio of from about 1 : 1 to about 100: 1, such as from about 2: 1 to about 50: 1, from about 2: 1 to about 30: 1, from about 2: 1 to about 15: 1, from about 3: 1 to about 60: 1, from about 3: 1 to about 30: 1, from about 5: 1 to about 50: 1, from about 5: 1 to about 30: 1, from about 10: 1 to about 50: 1, from about 10: 1 to about 30: 1, from about 12: 1 to about 50: 1, from about 12: 1 to about 30: 1, from about 12: 1 to about 20: 1, or from about 15: 1 to about 50: 1, including all values and subranges therebetween,. In some embodiments, the thermostabilizing excipient and the one or more RNA molecules are present in a weight ratio of from about 5 : 1 to about 50: 1. In certain embodiments, the liquid composition of the present disclosure comprises lipoic acid as the thermostabilizing excipient in an amount so that the lipoic acid and the one or more RNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1. In other embodiments, the liquid composition of the present disclosure comprises L-theanine as the thermostabilizing excipient in an amount so that the L-theanine and the one or more RNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1. In some embodiments, the liquid composition of the present disclosure comprises vanillin as the thermostabilizing excipient in an amount so that the vanillin and the one or more RNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1.Thermostability

[0117] The liquid compositions of the present disclosure are surprisingly thermostable at a temperature of 37°C. Thus, in some embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 37°C for at least 7 days as compared to a control liquid composition without the at least one thermostabilizing excipient. In other embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 15% after storage of the liquid composition at a temperature of 37°C for at least 7 days as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 10% after storage of the liquid composition at a temperature of 37°C for at least 7 days as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0118] It has been demonstrated by the inventors that, when stored at a temperature of 4°C, which corresponds to standard refrigerated conditions, the liquid composition of the present disclosure can remain stable for months. Thus, in some embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP of the liquid composition disclosed herein does not decrease more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0119] In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 25% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least onethermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 30% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 35% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 40% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 45% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0120] It has also been demonstrated by the inventors that, when stored at a temperature of 25°C to 30°C, the liquid composition of the present disclosure can remain stable for weeks. Thus, in some embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP of the liquid composition disclosed herein does not decrease more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all valuesand subranges therebetween, after storage of the liquid composition at a temperature of 25°C for up to about 1 week or longer, such as up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0121] In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 40% 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, including all values and subranges therebetween, or more than 4 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 45% 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, including all values and subranges therebetween, or more than 4 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease 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, including all values and subranges therebetween, or more than 4 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0122] In other embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP of the liquid composition disclosed herein does not decrease more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 30°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0123] In some embodiments, the integrity of the one or more RNA molecules does not decrease more than about 40% 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, including all values and subranges therebetween, or more than 4 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In certain embodiments, the integrity of the one or more RNA molecules does not decrease more than about 45% after storage of the liquid composition at a temperature of 30°C for up to about 1 week, up to about2 weeks, up to about 3 weeks, up to about 4 weeks, including all values and subranges therebetween, or more than 4 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the integrity of the one or more RNA molecules does not decrease 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, including all values and subranges therebetween, or more than 4 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0124] Integrity of the 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 instance, capillary gel electrophoresis or a fragment analyzer system can be used to determine the integrity of the RNA molecules. In some embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP of the liquid composition disclosed herein is measured by capillary electrophoresis. In some embodiments, the integrity of the one or more RNA molecules is measured by capillary gel electrophoresis. In other embodiments, the integrity of the one or more RNA molecules is measured by a fragment analyzer system.

[0125] The thermostability of the liquid composition of the present disclosure is further extended to maintain the mean particle size of the LNP so that it does not substantially increase after storage of the liquid composition at an above-zero temperature for a period of time. Accordingly, in some embodiments, the mean particle size of the LNP in the liquid composition of the present disclosure does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0126] In some embodiments, the mean particle size of the LNP does not increase more than about 35% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 40% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 45% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0127] In certain embodiments, the mean particle size of the LNP in the liquid composition of the present disclosure does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 25°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient

[0128] In some embodiments, the mean particle size of the LNP does not increase more than about 15% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 20% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 25% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0129] In some embodiments, the mean particle size of the LNP in the liquid composition of the present disclosure does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 30°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 15% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 20% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the mean particle size of the LNP does not increase more than about 25% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0130] Particle size can be determined using any method known in the art, such as by Dynamic Light Scattering (DLS). In general, prior to storage, the LNPs comprised in the liquidcompositions of the present disclosure have a mean particle size ranging from about 10 nm to about 1000 nm, such as from about 15 nm to about 750 nm, from about 30 nm to about 500 nm, from about 50 nm to about 250 nm, from about 75 nm to about 200 nm, or from about 80 nm to about 150 nm.

[0131] The thermostability of the liquid composition of the present disclosure is also extended to maintain the encapsulation efficiency of the LNP so that it does not substantially decrease after storage of the liquid composition at an above-zero temperature for a period of time. Accordingly, in some embodiments, the encapsulation efficiency of the LNP in the liquid composition of the present disclosure does not decrease more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0132] In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 15% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 20% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 25% after storage of the liquid composition at a temperature of 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 about5 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 30% after storage of the liquid composition at a temperature of 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, 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0133] In certain embodiments, the encapsulation efficiency of the LNP in the liquid composition of the present disclosure does not decrease more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 25°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0134] In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 15% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 20% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 25% 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, up to about 5weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0135] In some embodiments, the encapsulation efficiency of the LNP in the liquid composition of the present disclosure does not decrease more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 30°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0136] In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 15% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 20% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the encapsulation efficiency of the LNP does not decrease more than about 25% 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, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0137] Encapsulation efficiency of the LNP can be determined using any method known in the art, such as a fluorescence plate-based assay using the RiboGreen reagent (Invitrogen). Encapsulation efficiency (EE%) is calculated by (total RNA added - free non-entrapped RNA) divided by the total RNA added.

[0138] In some embodiments, the RNA molecules encapsulated in the LNP of the present disclosure encode one or more influenza virus proteins, such as HA and / or NA proteins, andthe liquid composition of the present disclosure induces a robust hemagglutination inhibition (HAI) titer following storage. In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10%, 5%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0139] In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 25% after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 20% after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 15% after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 10% after storage of the liquid composition ata temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 5% after storage of the liquid composition at a temperature of 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0140] In some embodiments, the RNA molecules encapsulated in the LNP of the present 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 more than about 30%, such as more than about 25%, 20%, 15%, 10%, 5%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 25°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the HAI titer induced by the liquid composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10%, 5%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of 30°C for up to about 1 week or longer, such as 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, including all values and subranges therebetween, or more than 8 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0141] HAI titers can be measured using any method known in the art, such as using an influenza virus HAI test (Denka Seiken Co., Tokyo, Japan).Thermostable RNA-LNP Compositions

[0142] In view of the surprising findings that the inclusion of a thermoreversible gelling agent or certain thermostabilizing excipients in LNP formulations containing RNA molecules resulted in substantially improved stability, including RNA stability, when stored at an abovezero temperature, such as in refrigerated conditions (e.g., 2-8°C, such as 4°C), also provided herein is a thermostable RNA-LNP composition comprising at least one thermoreversible gelling agent as described herein and at least one thermostabilizing excipient as described herein. In some embodiments, the at least one thermoreversible gelling agent comprises or is gelatin. In some embodiments, the at least one thermostabilizing excipient comprises or is lipoic acid. In some embodiments, provided herein is a thermostable RNA-LNP composition comprising gelatin and lipoic acid.

[0143] In some embodiments, the at least one thermoreversible gelling agent, such as gelatin, is present in an amount of from about 0.5% to about 1.5% by weight. In some embodiments, the at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 0.5% by weight. In some embodiments, the at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 1% by weight. In some embodiments, the at least one thermoreversible gelling agent, such as gelatin, is present in an amount of about 1.5% by weight.

[0144] In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of from about 1 mM to about 10 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of from about 1 mM to about 5 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of about 1 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of 2 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of about 2.5 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of about 3 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of about 4 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of about 5 mM. In some embodiments, the at least one thermostabilizing excipient, such as lipoic acid, and the one or more RNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1.

[0145] In some embodiments, the at least one thermoreversible gelling agent, such as gelatin, is present in an amount of from about 0.5% to about 1.5% by weight or 1% by weight, and the at least one thermostabilizing excipient, such as lipoic acid, is present in a concentration of from about 1 mM to about 10 mM or from about 1 mM to about 5 mM, or about 1 mM, 2mM, 3mM, 4mM or 5mM.

[0146] In some embodiments, the thermostable 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, up to about 1 year, including all values and subranges therebetween, or more than 1 year as compared to a control composition without gelatin and lipoic acid. As described herein elsewhere, stability of the composition can be measured by a change in mean particle size of the LNP, encapsulation efficiency of the LNP, and / or integrity of the one or more RNA molecules encapsulated in the LNP using any methods known in the art, such as those exemplified in the present disclosure.Thermostable RNA-LNP Liquid Formulations

[0147] The present disclosure is further based, at least in part, on the surprising finding that certain formulations containing LNP-encapsulated RNA molecules exhibited substantially improved formulation stability, including mRNA stability, when stored as a liquid at an abovezero temperature, such as in refrigerated conditions (e.g., 2-8°C). For instance, the inventors of the present disclosure surprisingly found that, by formulating LNP-encapsulated mRNA molecules with a combination of a buffering agent (e.g., Tris-hydroxymethyl-aminomethane or Tris), a pharmaceutically acceptable salt (e.g., sodium chloride or NaCl), a disaccharide (e.g., sucrose), a surfactant (e.g., a poloxamer, such as P188), and a chelating agent (e.g., ethylenediaminetetraacetic acid disodium salt or EDTA), each present at a prescribed amount as disclosed herein, the stability of the resultant formulation, including mRNA stability, was substantially improved. The instability of mRNA, as measured, for example, by a decrease in mRNA integrity, is considered a major challenge to mRNA’s fundamental therapeutic and commercial viability. The improved mRNA stability conferred by the thermostable RNA-LNP liquid formulations of the present disclosure thus provides a significant solution to such problems.

[0148] This finding enables several significant applications of the formulations of the disclosure, including extended refrigerated shelf-life, extended in-use periods at room temperature, and extended in-use stability at physiological temperatures. Achieving a thermostable RNA-LNP liquid formulation also enables commercially and therapeutically desirable packaging and delivery options including prefilled syringes (PFS) and cartridges for patient-friendly autoinjector and infusion pump devices. The ability to stabilize liquid formulations containing LNP-encapsulated RNA molecules, such as mRNAs, therefore represents a valuable technology, facilitating broader use of RNA-LNP formulations, such as mRNA vaccines.

[0149] Accordingly, provided herein are thermostable liquid formulations comprising one or more RNA molecules encapsulated in a LNP, as described herein elsewhere, and a buffering agent (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 at a prescribed amount as disclosed herein below, at a physiological pH for ease of administration (e.g., 7.5 ± 0.3). In some embodiments, the thermostable liquid formulations of the present disclosure further comprise trehalose.Buffering agent

[0150] The thermostable liquid formulations of the present disclosure comprise a buffering agent, such as Tris. Without wishing to be bound by any theory, buffering agents can be used to stabilize the pH of solutions. Commonly used buffering agents include, but are not limited to, Tris, 4-(2-hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 2-(N- morpholino)ethanesulfonic acid (MES), monosodium phosphate, and saline sodium citrate (SSC).

[0151] In some embodiments, the buffering agent comprised in the thermostable liquid formulations of the present disclosure is or comprises Tris in the amount of from about 10 mM to about 100 mM, such as from about 15 mM to about 80 mM, or from about 20 mM to about 50 mM, including all values and subranges therebetween. In some embodiments, the buffering agent is or comprises Tris in the amount of about 10 mM. In some embodiments, the buffering agent is or comprises Tris in the amount of about 20 mM. In some embodiments, the buffering agent is or comprises Tris in the amount of about 30 mM. In some embodiments, the buffering agent is or comprises Tris in the amount of about 40 mM. In some embodiments, the buffering agent is or comprises Tris in the amount of about 50 mM. In some embodiments, the buffering agent is or comprises Tris in the amount of about 100 mM.Pharmaceutically acceptable salt

[0152] The thermostable liquid formulations of the present disclosure comprise a pharmaceutically acceptable salt, such as NaCl. As used herein, the term “pharmaceutically acceptable” refers to a substance, as described throughout the present disclosure, which is admixed with an active ingredient (e.g., a mRNA) of the disclosure that is suitable for administration to humans. Without wishing to be bound by any theory, a pharmaceutically acceptable salt, 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).

[0153] In some embodiments, the pharmaceutically acceptable salt comprised in the thermostable liquid formulations of the present disclosure is or comprises NaCl in the amount of from about 10 mM to about 150 mM, such as from about 20 mM to about 130 mM, from about 30 mM to about 120 mM, or from about 50 mM to about 100 mM, including all values and subranges therebetween. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 10 mM. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 30 mM. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 50 mM. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 80 mM. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 100 mM. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 120 mM. In some embodiments, the pharmaceutically acceptable salt is or comprises NaCl in the amount of about 150 mM.Disaccharide(s)

[0154] The thermostable liquid formulations of the present disclosure comprise one or more disaccharides. Certain disaccharides, such as sucrose and trehalose, are commonly used as cryoprotectants to protect biological tissue from freezing damage.

[0155] In some embodiments, the disaccharide comprised in the thermostable liquid formulations of the present disclosure is or comprises sucrose in the amount of from about 1% to about 10% by weight, such as from about 2% to about 8% by weight, from 3% to about 6% by weight, or from about 4% to about 5% by weight, including all values and subranges therebetween. In some embodiments, the disaccharide is or comprises sucrose in the amount of about 1% by weight. In some embodiments, the disaccharide is or comprises sucrose in the amount of about 3% by weight. In some embodiments, the disaccharide is or comprises sucrosein the amount of about 5% by weight. In some embodiments, the disaccharide is or comprises sucrose in the amount of about 10% by weight. In some embodiments, sucrose is the only disaccharide comprised in the thermostable liquid formulations of the present disclosure.

[0156] In other embodiments, the thermostable liquid formulations of the present disclosure, in addition to sucrose, also comprise trehalose in the amount of from about 0.1% to about 5% by weight, such as from about 0.2% to about 4% by weight, from about 0.3% to about 3% by weight, from about 0.4% to about 2% by weight, from about 0.4% to about 1.5% by weight, from about 0.4% to about 1.3% by weight, from about 0.5% to about 4% by weight, from about 1% to about 4% by weight, from about 1.5% to about 3% by weight, from about 2% to about 2.8% by weight, from about 2% to about 2.6% by weight, from about 2.5% to about 5% by weight, or from about 2.5% to about 3.5% by weight, including all values and subranges therebetween. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of from about 0.4% to about 1.3% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of from about 2% to about 2.6% by weight.

[0157] In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 0.1% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 0.4% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 1% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 1.3% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 1.5% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 2% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 2.4% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 2.6% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 3% by weight. In some embodiments, the thermostable liquid formulations comprise trehalose in the amount of about 5% by weight.Surfactant

[0158] The thermostable liquid formulations of the present disclosure comprise a surfactant, such as a non-ionic surfactant (e.g., a poloxamer, such as poloxamer 188 (P188)). Without wishing to be bound by any theory, surfactants can be used to prevent LNPaggregation. In some embodiments, the surfactant comprised in the thermostable liquid formulations of the present disclosure is a non-ionic surfactant. Commonly used non-ionic surfactant include, but are not limited to, Pl 88, polysorbate 20, and polysorbate 80.

[0159] In some embodiments, the surfactant comprised in the thermostable liquid formulations of the present disclosure is or comprises a poloxamer, such as P188, in the amount of from about 0.1% to about 1% by volume, such as from about 0.2% to about 0.8% by volume, from about 0.3% to about 0.7% by volume, or from about 0.4% to about 0.6% by volume, including all values and subranges therebetween. In some embodiments, the surfactant is or comprises a poloxamer, such as P188, in the amount of about 0.1% by volume. In some embodiments, the surfactant is or comprises a poloxamer, such as Pl 88, in the amount of about 0.2% by volume. In some embodiments, the surfactant is or comprises a poloxamer, such as Pl 88, in the amount of about 0.4% by volume. In some embodiments, the surfactant is or comprises a poloxamer, such as P188, in the amount of about 0.6% by volume. In some embodiments, the surfactant is or comprises a poloxamer, such as Pl 88, in the amount of about 0.8% by volume. In some embodiments, the surfactant is or comprises a poloxamer, such as P188, in the amount of about 1% by volume.Chelating agent

[0160] The thermostable liquid formulations of the present disclosure comprise a chelating agent. Without wishing to be bound by any theory, chelating agents can be used as stabilizers to complex heavy metals that might promote instability. Commonly used chelating agents include, but are not limited to, EDTA.

[0161] In some embodiments, the chelating agent comprised in the thermostable liquid formulations of the present disclosure is or comprises EDTA in the amount of from about 1 pM to about 50 pM, such as from about 5 pM to about 30 pM, or from about 10 pM to about 25 pM, including all values and subranges therebetween. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 1 pM. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 5 pM. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 10 pM. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 15 pM. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 20 pM. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 30 pM. In some embodiments, the chelating agent is or comprises EDTA in the amount of about 50 pM.pH

[0162] The thermostable liquid formulations of the present disclosure are at a physiological pH for ease of administration. In some embodiments, the pH of the thermostable liquid formulations of the present disclosure are at a pH of from about 7 to about 8, such as from about 7.2 to about 7.8, or from about 7.4 to about 7.7, including all values and subranges therebetween. In some embodiments, the pH of the thermostable liquid formulations is about 7.0, such as 7.0 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.1, such as 7.1 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.2, such as 7.2 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.3, such as 7.3 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.4, such as 7.4 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.5, such as 7.5 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.6, such as 7.6 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.7, such as 7.7 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.8, such as 7.8 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 7.9, such as 7.9 ± 0.3. In some embodiments, the pH of the thermostable liquid formulations is about 8.0, such as 8.0 ± 0.3.Exemplary thermostable liquid formulations

[0163] In some embodiments, the thermostable liquid formulations of the present disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) encapsulated in a LNP, about 10-60 mM of Tris, about 40-150 mM of NaCl, about 1-10% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.2-7.8. In some embodiments, the thermostable liquid formulations of the present disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) encapsulated in a LNP, about 10-60 mM of Tris, about 40-110 mM of NaCl, about 3-6% by weight of sucrose, about 0.2-4% by weight of trehalose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.5-7.7. In some embodiments, the thermostable liquid formulations of the present disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) encapsulated in a LNP, about 20- 50 mM of Tris, about 50-100 mM of NaCl, about 2-5% by weight of sucrose, about 0.3-3% by weight of trehalose, about 0.2-0.4% by volume of P188, and about 10-15 pM of EDTA at a pH of about 7.7.

[0164] In some embodiments, the thermostable liquid formulations of the present disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) encapsulated in a LNP, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable liquid formulations of the present disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) encapsulated in a LNP, about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of about 7.7. In some embodiments, the thermostable liquid formulations of the present disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) encapsulated in a LNP, about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4-1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of about 7.7.Thermostability

[0165] It has been demonstrated by the inventors that the thermostable liquid formulations of the present disclosure can be stable in liquid form after storage for several months at a temperature of about 2-8°C (e.g., 4°C), which corresponds to standard refrigerated conditions. Thus, in some embodiments, the thermostable liquid formulations of the present disclosure are stable in liquid form after storage at a temperature of about 2-8°C (e.g., 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, 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, up to about 1 year, or more than 1 year, including all values and subranges therebetween. The stability of the thermostable liquid formulations can be measured by a change in mean particle size of the LNP, encapsulation efficiency of the LNP, and / or integrity of the one or more mRNA molecules encapsulated in the LNP.

[0166] In some embodiments, the stability of the thermostable liquid formulations is measured by a change in mean particle size of the LNP and the mean particle size of the LNP does not increase more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, after storage of the thermostable liquid formulations at a temperature of about 2-8°C (e.g., 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, 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, up to about 1 year, or more than 1 year, including all values and subranges therebetween. Particle size can be determined using any method known in the art, such as byDynamic Light Scattering (DLS). In general, prior to storage, the LNPs comprised in the thermostable liquid formulations of the present disclosure have a mean particle size ranging from about 10 nm to about 1000 nm, such as from about 15 nm to about 750 nm, from about 30 nm to about 500 nm, from about 50 nm to about 250 nm, from about 75 nm to about 200 nm, or from about 80 nm to about 150 nm.

[0167] In some embodiments, the stability of the thermostable liquid formulations is measured by a change in encapsulation efficiency of the LNP and the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, after storage of the thermostable liquid formulations at a temperature of about 2-8°C (e.g., 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, 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, up to about 1 year, or more than 1 year, including all values and subranges therebetween. Encapsulation efficiency of the LNP can be determined using any method known in the art, such as a fluorescence plate-based assay using the RiboGreen reagent (Invitrogen). Encapsulation efficiency (EE%) is calculated by (total RNA added - free non-entrapped RNA) divided by the total RNA added.

[0168] In some embodiments, the stability of the thermostable liquid formulations is measured by a change in the integrity of the one or more mRNA molecules encapsulated in the LNP and the integrity of the mRNA molecules does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, after storage of the immunogenic composition at a temperature of about 2-8°C (e.g., 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, 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, up to about 1 year, or more than 1 year, including all values and subranges therebetween. 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 instance, capillary gel electrophoresis or a fragment analyzer system can be used to determine the integrity of the mRNA molecules. In some embodiments, the integrity of the one or more mRNA molecules encapsulated in the LNP of the thermostable liquid formulations disclosed herein is measured by capillary electrophoresis. In some embodiments, the integrity of the one or more mRNA molecules is measured by capillary gel electrophoresis. In other embodiments, the integrity of the one or more mRNA molecules is measured by a fragment analyzer system.Transfer Vehicle

[0169] In certain embodiments, the thermostable compositions of the disclosure comprise one or more RNA molecules, such as mRNA molecules, and a transfer vehicle. As used herein, the term “transfer vehicle” includes any of the standard pharmaceutical carriers, diluents, excipients, and the like which are generally intended for use in connection with the administration of biologically active agents, including RNAs (e.g., mRNAs). The compositions and in particular the transfer vehicles described herein are capable of delivering RNAs (e.g., mRNAs) of varying sizes to their target cells or tissues. In some embodiments, the transfer vehicles of the present disclosure are capable of delivering large RNA molecules (e.g., RNAs, such as mRNAs, of at least 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5kDa, 10 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or more). The RNAs (e.g., mRNAs) can be formulated with one or more acceptable reagents, which provide a vehicle for delivering such RNAs (e.g., mRNAs) to target cells. Appropriate reagents are generally selected with regards to a number of factors, which include, among other things, the biological or chemical properties of the RNAs (e.g., charge), the intended route of administration, the anticipated biological environment to which such RNAs (e.g., mRNAs) will be exposed and the specific properties of the intended target cells. In some embodiments, transfer vehicles, such as liposomes, encapsulate the RNAs (e.g., mRNAs) without compromising biological activity. In some embodiments, the transfer vehicle demonstrates preferential and / or substantial binding to a target cell relative to non-target cells. In certain embodiments, the transfer vehicle delivers its contents to the target cell such that the RNAs (e.g., mRNAs) are delivered to the appropriate subcellular compartment, such as the cytoplasm.

[0170] In some embodiments, the transfer vehicle is a liposomal vesicle, or other means to facilitate the transfer of the one or more RNA (e.g., mRNA) molecules to target cells and tissues. Suitable transfer vehicles include, but are not limited to, liposomes, nano liposomes, ceramide-containing nanoliposomes, proteoliposomes, nanoparticulates, calcium phosphorsilicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags. Also contemplated is the use of bionanocapsules and other viral capsid proteins assemblies as a suitable transfer vehicle. See e.g., Kasuya et al., Hum. Gene Ther., 2008, 19(9):887-895. Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination withother transfer vehicles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, alginate, collagen, chitosan, cyclodextrins and polyethylenimine. In some embodiments, the transfer vehicle is selected based upon its ability to facilitate the transfection of one or more RNA (e.g., mRNA) molecules to a target cell.Lipid Nanoparticles

[0171] In some embodiments, the transfer vehicle is formulated as a lipid nanoparticle (LNP). The term “lipid nanoparticle” or “LNP” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids, for example a cationic and / or non-cationic lipid, and one or more excipients selected from neutral lipids, anionic lipids, zwitterionic lipids, ionizable lipids, steroids, and polymer conjugated lipids (e.g., a pegylated lipid). Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). In certain embodiments, the compositions of the present disclosure comprise one or more RNA (e.g., mRNA) molecules encapsulated in a LNP. RNA-encapsulated LNP compositions are known in the art, such as those described in PCT Publication Nos. WO 2021 / 237084 and WO 2022 / 099003, the entire contents of which are incorporated by reference herein. Any known LNP formulations may be used in the embodiments disclosed herein. In some embodiments, the LNPs comprise a mixture of four lipids: an ionizable (e.g., cationic) lipid, a polyethylene glycol (PEG)-conjugated lipid, a cholesterol-based lipid, and a helper lipid, such as a phospholipid. The LNPs are used to encapsulate RNA molecules (e.g., mRNA molecules). The encapsulated RNA molecules (e.g., mRNA molecules) can be comprised of naturally- occurring ribonucleotides, chemically-modified nucleotides, or a combination thereof, and can each or collectively code for one or more proteins.Ionizable or cationic lipids

[0172] The ionizable lipid facilitates encapsulation of the RNA molecules (e.g., mRNA molecules) and may be a cationic lipid. A cationic lipid affords a positively charged environment at low pH to facilitate efficient encapsulation of, for instance, the negatively charged RNA molecules (e.g., mRNA molecules). Suitable cationic lipids for LNP formulation include, but are not limited to, ALC-0315, OF-02, cKK-ElO, 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.

[0173] ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl)bis(2- hexyl decanoate)) is a synthetic lipid having the following chemical structure:ALC-0315 is a colorless oily material and has attracted attention as a component of the SARS- CoV-2 vaccine Comirnaty® (BNT162b2) by Pfizer-BioNTech. Below physiological pH, ALC-0315 becomes protonated at the nitrogen atom, yielding an ammonium cation that is attracted to the messenger RNA (mRNA), which is anionic.

[0174] OF-02 (3,6-Z>A[4-[Z>A[(9Z,12Z)-2-hydroxy-9,12-octadecadien-l-yl]amino] butyl]-2,5-piperazinedione, CAS No. 1883431-67-1) is an alkenyl amino alcohol (AAA) ionizable lipid for highly potent in vivo mRNA delivery and has the following chemical structure:OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains degradable ester linkages to attach the diketopiperazine core and the doubly-unsaturated tails, whereas OF- 02 contains non-degradable 1,2-amino-alcohol linkages to attach the same diketopiperazine core and the doubly-unsaturated tails. See, Fenton et al., Adv. Mater., 2016, 28(15):2939-2943; U.S. Pat. No. 10,201,618, both incorporated herein by reference.

[0175] cKK-ElO and cKK-E12 are two cationic lipids that can be used in lipid nanoparticles for delivery of nucleic acids to various cell types (Dong et al., PNAS, 2014, 111(11):3955-3960; U.S. Pat. No. 9,512,073, both of which are incorporated herein by reference). cKK-E12 has been used to deliver siRNA in mice, rats, and primates (ED50 =0.002, 0.01, & 0.3 mg / kg respectively). See, Dong et al., supra. It shows low toxicity and is selective for liver parenchymal cells over liver, heart, lung, and kidney endothelial cells.

[0176] cKK-ElO has the following chemical structure:

[0177] cKK-E12 (3,6-Z>75[4-[Z>75(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione,CAS No. 1432494-65-9) has the following chemical structure:

[0178] 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. WO 2022 / 221688 Al (incorporated herein by reference), are HEPES-based disulfide cationic lipids with a piperazine core. GL-HEPES-E3-E10-DS-3-E18-1 (2-(4-(2-((3-(Bis((Z)-2- hydroxyoctadec-9-en- 1 -yl)amino) propyl)disulfaneyl)ethyl)piperazin- 1 -yl)ethyl 4-(bis(2- hydroxydecyl)amino)butanoate) has the following chemical structure:

[0179] GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl) disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate) has the following chemical structure:

[0180] GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(Bis(2-hydroxytetradecyl)amino) propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl4-(bis(2-hydroxydodecyl)amino)butanoate) has the following chemical structure:

[0181] Other cationic lipids that can be used include those described in Dong et al., supra, U.S. Pat. No. 10,201,618, and PCT publication No. WO 2022 / 221688A1, all of which are incorporated herein by reference.

[0182] Accordingly, in some embodiments, the cationic lipid used to form the LNP according to the disclosure comprises ALC-0315, OF-02, cKK-ElO, 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 comprises ALC-0315. In some embodiments, the cationic lipid comprises OF-02. In some embodiments, the cationic lipid comprises cKK-ElO. In some embodiments, the cationic lipid comprises cKK-E12. In some embodiments, the cationic lipid comprises GL-HEPES-E3-E10-DS-3-E18-1. In some embodiments, the cationic lipid comprises GL-HEPES-E3-E12-DS-4-E10. In some embodiments, the cationic lipid comprises GL-HEPES-E3-E12-DS-3-E14.PEGylated lipids

[0183] The PEGylated lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and provide a means for increasing circulation lifetime and increasing the delivery of the lipidnucleic acid pharmaceutical composition to target tissues. See, Klibanov et al., FEBS Letters, 1990, 268(l):235-237. These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo. See e.g., U.S. Pat. No. 5,885,613.

[0184] Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 (e.g., Cs, C10, C12, C14, Ci6, or Cis) length, such as a derivatized ceramide (e.g., N- octanoyl-sphingosine-l-[succinyl(m ethoxypoly ethylene glycol)] (Cs PEG ceramide)). In some embodiments, the PEGylated lipid comprises l,2-dimyristoyl-rac-glycero-3-methoxy- poly ethylene glycol (DMG-PEG, also known as DMG-PEG 2000); 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); l,2-dilauroyl-sn-glycero-3- phosphoethanolamine-poly ethylene glycol (DLPE-PEG); 1,2-distearoyl-rac-glycero- polyethelene glycol (DSG-PEG); and / or N,N ditetradecylacetamide-polyethylene glycol (e.g., ALC-0159). In some embodiments, the PEGylated lipid used in the LNPs of the disclosure comprises l,2-dimyristoyl-rac-glycero-3-methoxy-polyethylene glycol (DMG-PEG 2000). In some embodiments, the PEGylated lipid comprises N,N-ditetradecylacetamide-polyethylene glycol.

[0185] The PEG preferably has a high molecular weight, e.g., 2000-2400 g / mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipid herein comprises DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, and / or Cs PEG2000. In some embodiments, the PEGylated lipid comprises dimyristoyl-PEG2000.Cholesterol-based lipids

[0186] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNPs comprise one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, but are not limited to, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem. Biophys. Res. Comm., 1991, 179:280; Wolf et al., BioTechniques, 1997, 23: 139; U.S. Pat. No. 5,744,335), imidazole cholesterol ester (“ICE”; WO 2011 / 068810), 0- sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNPs of the disclosure comprises cholesterol.Helper lipids

[0187] A helper lipid enhances the structural stability of the LNP and helps the LNP in endosome escape. It improves uptake and release of the ribonucleic acid molecules (e.g., mRNA) drug payload. In some embodiments, the helper lipid is a zwitterionic lipid, which has fusogenic properties for enhancing uptake and release of the drug payload. In some embodiments, the helper lipid is a phospholipid. Examples of helper lipids are 1,2-dioleoyl- SN-glycero-3-phosphoethanolamine (DOPE); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE); and l,2-dioleoyl-sn-glycero-3 -phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), and l,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE). In some embodiments, the helper lipid used in the LNPs of the disclosure comprises DOPE. In some embodiments, the helper lipid comprises DSPC.

[0188] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides,gangliosides, 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), or a combination thereof.Exemplary lipid nanoparticle compositions

[0189] Accordingly, in some embodiments, the LNP according to the disclosure comprises (i) a cationic lipid, such as OF-02, cKK-ElO, 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) a PEGylated lipid, such as DMG-PEG2000 or N,N-ditetradecylacetamide-polyethylene glycol; (iii) a cholesterol-based lipid, such as cholesterol; and (iv) a helper lipid, such as DOPE or DSPC. In some embodiments, the LNP comprises OF-02 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid. In some embodiments, the LNP comprises cKK-ElO as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid. In some embodiments, the LNP comprises cKK- E12 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid. In some embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid. In some embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid. In some embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid. In some embodiments, the LNP comprises ALC-0315 as the cationic lipid, N,N- ditetradecylacetamide-polyethylene glycol as the PEGylated lipid, cholesterol, and DSPC as the helper lipid.Molar ratios of lipid components

[0190] The molar ratios of the above LNP components may assist in the LNPs’ effectiveness in delivering the RNA molecules (e.g., mRNA) encapsulated therein. The molar ratio of the cationic lipid, the PEGylated lipid, the cholesterol-based lipid, and the helper lipid is A:B:C:D, wherein A+B+C+D = 100%. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is about 30-60%, such as about 30-50%, 30-45%, 30-40%, 35-55%, 35-50%, 35-45%, 30-50%, or 30-40%, including all values and subranges therebetween. In some embodiments, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is about 0.25-15%, such as 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%, including all values and subranges therebetween. In some embodiments, the molar ratio of thecholesterol-based lipid relative to the total lipids (i.e., C) is about 20-40%, such as 20-35%, 20- 30%, 25-40%, 25-35%, or 25-30%, including all values and subranges therebetween. In some embodiments, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is about 20- 40%, such as 20-35%, 20-30%, 25-40%, 25-35%, or 25-30%, including all values and subranges therebetween. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is about 30-50%, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is about 0.25-15%, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is about 20-40%, and the molar ratio of the helper lipid relative to the total lipids (i.e., D) is about 20-40%. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is about 35- 45%, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is about 0.25-7.5%, the molar ratio of the cholesterol-based lipid relative to the total lipids (i.e., C) is about 25-35%, and the molar ratio of the helper lipid relative to the total lipids (i.e., D) is about 25-35%. In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of the cationic lipid to the helper lipid that is more than 1.

[0191] To calculate the actual amount of each lipid to be put into an LNP formulation, the molar amount of the cationic lipid is first determined based on a 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 molecules (e.g., mRNA) to be transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.

[0192] In some embodiments, the LNPs contain a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid at a molar ratio of about 40: 1.5:28.5:30, that is the cationic lipid is present at a molar ratio of about 40%, the PEGylated lipid is present at a molar ratio of about 1.5%, the cholesterol-based lipid is present at a molar ratio of about 28.5%, and the helper lipid is present at a molar ratio of about 30%. In some embodiments, the LNPs contain OF-02 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40: 1.5:28.5:30. In some embodiments, the LNPs contain cKK-ElO as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40: 1.5:28.5:30. In some embodiments, the LNPs contain cKK-E12 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about40: 1.5:28.5:30. In some embodiments, the LNPs contain GL-HEPES-E3-E10-DS-3-E18-1 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40: 1.5:28.5:30. In some embodiments, the LNPs contain GL- HEPES-E3-E12-DS-4-E10 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40: 1.5:28.5:30. In some embodiments, the LNPs contain GL-HEPES-E3-E12-DS-3-E14 as the cationic lipid, DMG- PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40: 1.5:28.5:30.

[0193] In some embodiments, the LNPs contain a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid at a molar ratio of about 40:5:25:30, that is the cationic lipid is present at a molar ratio of about 40%, the PEGylated lipid is present at a molar ratio of about 5%, the cholesterol-based lipid is present at a molar ratio of about 25%, and the helper lipid is present at a molar ratio of about 30%. In some embodiments, the LNPs contain OF-02 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40:5:25:30. In some embodiments, the LNPs contain cKK-ElO as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40:5:25:30. In some embodiments, the LNPs contain cKK-E12 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40:5:25:30. In some embodiments, the LNPs contain GL-HEPES-E3-E10-DS-3-E18-1 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40:5:25:30. In some embodiments, the LNPs contain GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid, DMG- PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40:5:25:30. In some embodiments, the LNPs contain GL-HEPES-E3-E12-DS-3-E14 as the cationic lipid, DMG-PEG2000 as the PEGylated lipid, cholesterol, and DOPE as the helper lipid at a molar ratio of about 40:5:25:30.

[0194] In some embodiments, the LNP comprises (i) ALC-0315 as the cationic lipid at a molar ratio of about 25% to about 65%, such as about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%; (ii) N,N-ditetradecylacetamide-polyethylene glycol (e.g., ALC-0159) as the PEGylated lipid at a molar ratio of about 0.5% to about 3%, such as about 0.5%, 1%, 1.5%, 2%, 2.5% or 3%; (iii) DSPC as the helper lipid at a molar ratio of about 5% to about 15%, such as about 5%, 7.5%, 10%, 12.5%, or 15%, and (iv) cholesterol at a molar ratio of about 20% to about 60%, such as about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.RNA Molecules

[0195] Any RNA molecules may be encapsulated in the LNP formulation of the disclosure, which include, but are not limited to, antisense oligonucleotides (ASO), small interfering RNA (siRNA), small activating RNAs (saRNA), microRNAs (miRNAs), aptamers, long non-coding RNAs (IncRNAs), and messenger RNA (mRNA). The remarkable success of COVID-19 vaccines Comimaty® (BNT162b2) and Spikevax (mRNA-1273) have demonstrated the clinical validation of lipid nanoparticle-formulated mRNA as a new class of highly efficacious nucleic acids in the field of vaccines. Accordingly, in some embodiments, the RNA molecules encapsulated in the LNP according to the disclosure are mRNA molecules.

[0196] In some embodiments, the LNP or the LNP formulation according to the disclosure may be mono-valent, which means that the LNP encapsulates RNA molecules (e.g., mRNA) that encode the same protein, such as an antigen in some embodiments. In some embodiments, the LNP or the LNP formulation according to the disclosure may be multi-valent, which means that the LNP encapsulates RNA molecules (e.g., mRNA) that encode at least two different proteins, such as two, three, four, five, six, seven, eight, nine, ten, or more different proteins. In some embodiments, when the LNP or the LNP formulation according to the disclosure is multi-valent, the RNA molecules (e.g., mRNA) encapsulated in the LNP may encode at least two different antigens, such as two, three, four, five, six, seven, eight, nine, ten, or more different antigens, from the same or different pathogens (e.g., virus). For example, the LNP may carry multiple RNA molecules (e.g., mRNA), each encoding a different antigen; or carry a polycistronic mRNA that can be translated into more than one antigen (e.g., each antigencoding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). An LNP carrying different RNA molecules (e.g., mRNA) typically comprises (encapsulate) multiple copies of each mRNA molecule. For example, an LNP carrying or encapsulating two different RNA molecules (e.g., mRNA) typically carries multiple copies of each of the two different RNA molecules (e.g., mRNA).

[0197] In some embodiments, a single LNP formulation may comprise multiple kinds (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of LNPs, each kind carrying a different RNA molecule (e.g., mRNA). mRNA Molecules

[0198] In some embodiments, the RNA molecules (e.g., mRNA) encapsulated in the LNP or the LNP formulation according to the disclosure may encode one or more virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) from the same or different viruses.For instance, in some embodiments, the RNA molecules (e.g., mRNA) encapsulated in the LNP or the LNP formulation encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), such as influenza hemagglutinin (HA) and / or neuraminidase (NA) proteins from the same or different type of influenza viruses. In some embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) selected from Hl, H3, HA from a B / Victoria lineage, and / or HA from a B / Yamagata lineage. In some embodiments, the RNA molecules (e.g., mRNA) encode three different influenza virus proteins (e.g., trivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, and an HA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the RNA molecules (e.g., mRNA) encode four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the LNP or the LNP formulation according to the disclosure are trivalent when the RNA molecules (e.g., mRNA) encode three different influenza virus proteins such as Hl, H3, and HA from a B / Victoria lineage. In some embodiments, the LNP or the LNP formulation according to the disclosure are quadrivalent when the RNA molecules (e.g., mRNA) encode four different influenza virus proteins such as Hl, H3, HA from a B / Victoria lineage, and HA from a B / Yamagata lineage. In some embodiments, the Hl is from an H1N1 influenza virus strain. In some embodiments, the H3 is from an H3N2 influenza virus strain. In some embodiments, the Hl is from an H1N1 influenza virus strain and the H3 is from an H3N2 influenza virus strain.

[0199] Each year, based on intensive surveillance efforts, the World Health Organization (WHO) selects influenza strains to be included in the seasonal vaccine preparations. Accordingly, as used herein, the term “standard of care strain” refers to an influenza strain that is selected by the WHO to be included in the seasonal vaccine preparations. A standard of care strain can include a historical standard of care strain, a current standard of care strain or a future standard of care strain.

[0200] In other embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) selected from Nl, N2, NA from a B / Victoria lineage, and / or NA from a B / Yamagata lineage. In some embodiments, the RNA molecules (e.g., mRNA) encode three different influenza virusproteins: a N1 from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, and an NA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the RNA molecules (e.g., mRNA) encode four different influenza virus proteins: a N1 from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, an NA from a third standard of care influenza virus strain from the B / Victoria lineage, and an NA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the LNP or the LNP formulation according to the disclosure are trivalent when the RNA molecules (e.g., mRNA) encode three different influenza virus proteins, such as Nl, N2, and NA from a B / Victoria lineage. In some embodiments, the LNP or the LNP formulation according to the disclosure are quadrivalent when the RNA molecules (e.g., mRNA) encode four different influenza virus proteins, such as Nl, N2, NA from a B / Victoria lineage, and NA from a B / Yamagata lineage. In some embodiments, the Nl is from an H1N1 influenza virus strain. In some embodiments, the N3 is from an H3N2 influenza virus strain. In some embodiments, the Nl is from an H1N1 influenza virus strain and the N3 is from an H3N2 influenza virus strain.

[0201] In other embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) selected from Hl, H3, HA from a B / Victoria lineage, and / or HA from a B / Yamagata lineage and one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) selected from Nl, N2, NA from a B / Victoria lineage, and / or NA from a B / Yamagata lineage. In some embodiments, the RNA molecules (e.g., mRNA) encode eight different influenza virus proteins (e.g., octavalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage, an Nl from a fifth standard of care influenza virus strain, an N2 from a sixth standard of care influenza virus strain, an NA from a seventh standard of care influenza virus strain from a B / Victoria lineage, and an NA from a eighth standard of care influenza virus strain from a B / Yamagata lineage. In some embodiments, the Hl is from an H1N1 influenza virus strain. In some embodiments, the H3 is from an H3N2 influenza virus strain. In some embodiments, the Hl is from an H1N1 influenza virus strain and the H3 is from an H3N2 influenza virus strain. In some embodiments, the Nl is from an H1N1 influenza virus strain. In some embodiments, the N2 is from an H3N2 influenza virus strain. In some embodiments, the Nl is from an H1N1 influenza virus strain and the N2 is from an H3N2influenza virus strain. In some embodiments, the Hl is from an H1N1 influenza virus strain, the H3 is from an H3N2 influenza virus strain, the N1 is from an H1N1 influenza virus strain, and the N2 is from an H3N2 influenza virus strain. In some embodiments, the Hl and the N1 are from the same H1N1 influenza virus strain. In some embodiments, the Hl and the N1 are from different H1N1 influenza virus strains. In some embodiments, the H3 and the N2 are from the same H3N2 influenza virus strain. In some embodiments, the H3 and the N2 are from different H3N2 influenza virus strains. In some embodiments, the HA of a B / Yamagata lineage and the NA of a B / Yamagata lineage are from the same influenza virus strain. In some embodiments, the HA of a B / Yamagata lineage and the NA of a B / Yamagata lineage are from different influenza virus strains. In some embodiments, the HA of a B / Victoria lineage and the NA of a B / Victoria lineage are from the same influenza virus strain. In some embodiments, the HA of a B / Victoria lineage and the NA of a B / Victoria lineage are from different influenza virus strains.

[0202] In some embodiments, the RNA molecules (e.g., mRNA) encode one or more respiratory syncytial virus (RSV) polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), such as the receptor attachment glycoprotein (G), the fusion protein (F), and / or a short hydrophobic (SH) protein from the same or different subtypes of respiratory syncytial virus (RSV). In other embodiments, the RNA molecules (e.g., mRNA) encode one or more coronavirus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), particularly the Spike protein (S).

[0203] The RNA molecules (e.g., mRNA) encapsulated in the LNP or LNP formulation of the disclosure can also encode one or more virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) from different viruses. In some embodiments therefore, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) coronavirus proteins. In some embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) respiratory syncytial virus (RSV) proteins. In some embodiments, the RNA molecules (e.g., mRNA) encode one or more coronavirus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) respiratory syncytial virus (RSV) proteins. In some embodiments, the RNA molecules (e.g., mRNA) encode one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten)coronavirus proteins, and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) respiratory syncytial virus (RSV) proteins. In some embodiments, the RNA molecules (e.g., mRNA) encode polypeptides from any combinations of virus proteins.

[0204] The RNA molecules (e.g., mRNA) according to the disclosure can be selfamplifying RNAs. Antigen expression from traditional mRNA is proportional to the number of mRNA molecules successfully delivered to a subject from an immunogenic composition or a vaccine. Self-amplifying mRNA, however, comprise genetically-engineered replicons derived from self-replicating viruses and therefore, may be added to an immunogenic composition or a vaccine in lower dosages than traditional mRNA while achieving comparable results.

[0205] The self-amplifying mRNA may encode any of the virus proteins disclosed herein, including, for example, influenza virus HAs (e.g., Hl, H3, HA from the B / Victoria lineage, and / or HA from the B / Yamagata lineage), influenza virus NAs (e.g., Nl, N2, NA from the B / Victoria lineage, and / or NA from the B / Yamagata lineage), respiratory syncytial virus (RSV) proteins (e.g., G protein, F protein, and / or SH protein), and coronavirus proteins (e.g., Spike protein).

[0206] The RNA molecules (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., including nucleotide analogs, such as pseudouridines (e.g., N-l-methyl pseudouridine), 2'-fluoro ribonucleotides, and 2'-methoxy ribonucleotides, and / or phosphorothioate bonds). The RNA molecules (e.g., mRNA) may comprise a 5' cap and a polyA tail. In some embodiments, the one or more RNA molecules comprise one or more modified nucleotides, and in some embodiments, the one or more modified nucleotides are selected from pseudouridine, methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l- methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyl uridine. In some embodiments, every uridine in the ribonucleic acid molecule is replaced by a pseudouridine, e.g., a methylpseudouridine, such as IN-methylpseudouridine. In some embodiments, the one or more RNA molecules comprise one or more phosphorothioate bonds.

[0207] When used as an immunogenic composition or a vaccine, each RNA molecule is present in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition or vaccine is administered. In someembodiments, each RNA molecule may be present in the compositions disclosed herein in an amount ranging from, for example, about 0.1 pg to about 150 pg, such as from about 5 pg to about 120 pg, from about 10 pg to about 60 pg, from about 1 pg to about 60 pg, from about 5 pg to about 45 pg, or from about 15 pg to about 45 pg. In some embodiments, each RNA molecule is present in the composition in an amount sufficient to encode, for example, from about 5 pg to about 120 pg, such as from about 10 pg to about 60 pg, or about 15 pg to about 45 pg of virus proteins, such as influenza virus HA or NA proteins, respiratory syncytial virus (RSV) proteins (e.g., G protein, F protein, and / or SH protein), and / or coronavirus proteins (e.g., Spike protein).

[0208] The molar ratio between nitrogen (N) on the ionizable lipid to phosphate (P) on RNA molecules has effects on the in vitro and in vivo interaction properties of the RNA-LNP complexes. See e.g., Gary et al., Macromol. Biosci., 2013, 13(8): 1059-1071. Thus, in some embodiments, the liquid composition of the disclosure has a N / P ratio of from about 1 to about 10, such as from about 1 to about 8, from about 1 to about 6, from about 1 to about 4, from about 2 to about 8, from about 2 to about 6, from about 3 to about 6, or from about 4 to about 6, including all values and subranges therebetween. In some embodiments, the liquid composition of the disclosure has a 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.Exemplary Thermostable RNA-LNP Compositions

[0209] The thermoreversible gelling agents, thermostabilizing excipients, and thermostable formulations disclosed herein can be used alone or in combination to stabilize compositions comprising LNPs which encapsulate RNA molecules, including mRNA molecules. For instance, one or more of the thermoreversible gelling agents disclosed herein can be used together with one or more of the thermostabilizing excipients disclosed herein and / or one of the thermostable formulations disclosed herein to stabilize compositions comprising LNPs encapsulating RNAs, such as mRNAs encoding one or more virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), such as influenza HA and / or NA proteins from the same or different type of influenza viruses. Exemplary thermostable RNA-LNP compositions according to the present disclosure are provided herein below.

[0210] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), about 10-60 mM of a buffering agent (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., a poloxamer, such as P188), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), about 10-60 mM of a buffering agent (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% by volume of a surfactant (e.g., a poloxamer, such as P188), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.5-5% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), about 20-50 mM of a buffering agent (e.g., Tris), about 50-100 mM of a 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 of a surfactant (e.g., a poloxamer, such as P188), and about 10-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0211] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), about 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), about 20 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosurecomprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0212] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of gelatin, about 10-60 mM of Tris, about 40-150 mM of NaCl, about 1-10% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of gelatin, about 10-60 mM of Tris, about 40-110 mM of NaCl, about 0.2-3% by weight of trehalose, about 2-7% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5- 15 pM of EDTA at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.5-5% by weight of gelatin, about 20-50 mM of Tris, about 50-100 mM of NaCl, about 0.4-2.6% by weight of trehalose, about 3.-5% by weight of sucrose, about 0.2-0.4% by volume of P188, and about 10-15 pM of EDTA at a pH of about 7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) 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 Pl 88, and about 10 pM of EDTA at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, 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 pM of EDTA at a pH of about 7.7.

[0213] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM toabout 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of a buffering agent (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., a poloxamer, such as P188), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of a buffering agent (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% by volume of a surfactant (e.g., a poloxamer, such as Pl 88), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 20-50 mM of a buffering agent (e.g., Tris), about 50-100 mM of a 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 of a surfactant (e.g., a poloxamer, such as P188), and about 10-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0214] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 20 mM of a buffering agent (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., a poloxamer, such as Pl 88), and about 10 pM of a chelating agent (e.g., EDTA) at a pHof about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0215] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of Tris, about 40-150 mM of NaCl, about 1-10% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of Tris, about 40-110 mM of NaCl, about 0.2-3% by weight of trehalose, about 2-7% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 20-50 mM of Tris, about 50-100 mM of NaCl, about 0.4-2.6% by weight of trehalose, about 3.-5% by weight of sucrose, about 0.2- 0.4% by volume of P188, and about 10-15 pM of EDTA at a pH of about 7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, 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 Pl 88, and about 10 pM of EDTA at a pH of about 7.7. In other embodiments, the thermostable RNA- LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, 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 pM of EDTA at a pH of about 7.7.

[0216] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of a buffering agent (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., a pol oxamer, such as Pl 88), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of a buffering agent (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% by volume of a surfactant (e.g., a pol oxamer, such as Pl 88), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20-50 mM of a buffering agent (e.g., Tris), about 50-100 mM of a 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 of a surfactant (e.g., a pol oxamer, such as Pl 88), and about 10-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0217] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 50 mM of a buffering agent (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., a pol oxamer, such as Pl 88), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, 50 mM of a buffering agent (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., a poloxamer, such as Pl 88), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0218] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of Tris, about 40-150 mM of NaCl, about 1-10% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, inaddition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of Tris, about 40-110 mM of NaCl, about 0.2-3% by weight of trehalose, about 2-7% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20-50 mM of Tris, about 50-100 mM of NaCl, about 0.4-2.6% by weight of trehalose, about 3.-5% by weight of sucrose, about 0.2-0.4% by volume of P188, and about 10-15 pM of EDTA at a pH of about 7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20 mM of Tris, about 100 mM ofNaCl, about 0.4-1.3% by weight of trehalose, about 5% by weight of sucrose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA at a pH of about 7.7. In other embodiments, the thermostable RNA- LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, 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 Pl 88, and about 10 pM of EDTA at a pH of about 7.7.

[0219] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of a buffering agent (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., a poloxamer, such as P188), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of a buffering agent (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% by volume of a surfactant (e.g., a poloxamer, such as Pl 88), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA- LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.5-5% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 20-50 mM of a buffering agent (e.g., Tris), about 50-100 mM of a 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 of a surfactant (e.g., a poloxamer, such as P188), and about 10-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0220] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 50 mM of a buffering agent (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., a pol oxamer, such as Pl 88), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 20 mM of a buffering agent (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., a pol oxamer, such as Pl 88), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0221] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of gelatin, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of Tris, about 40-150 mM of NaCl, about 1-10% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of gelatin, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 10-60 mM of Tris, about 40-110 mM of NaCl, about 0.2-3% by weight of trehalose, about 2-7% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.5-5% by weight of gelatin,from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, about 20-50 mM of Tris, about 50-100 mM of NaCl, about 0.4-2.6% by weight of trehalose, about 3.-5% by weight of sucrose, about 0.2-0.4% by volume of P188, and about 10-15 pM of EDTA at a pH of about 7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, 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 Pl 88, and about 10 pM of EDTA at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, from about 0.1 mM to about 20 mM of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, 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 Pl 88, and about 10 pM of EDTA at a pH of about 7.7.

[0222] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of a buffering agent (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., a poloxamer, such as P188), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-basedpolymer, such as gelatin), at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of a buffering agent (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% by volume of a surfactant (e.g., a pol oxamer, such as Pl 88), and about 5-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.5-5% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20-50 mM of a buffering agent (e.g., Tris), about 50-100 mM of a 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 of a surfactant (e.g., a poloxamer, such as P188), and about 10-15 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0223] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or protein-based polymer, such as gelatin), at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratioof from about 5: 1 to about 50: 1, about 20 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of at least one thermoreversible gelling agent disclosed herein (e.g., a polypeptide- or proteinbased polymer, such as gelatin), at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, 50 mM of a buffering agent (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., a poloxamer, such as P188), and about 10 pM of a chelating agent (e.g., EDTA) at a pH of about 7.7.

[0224] In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1-10% by weight of gelatin, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L- theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of Tris, about 40-150 mM of NaCl, about 1- 10% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.2-7.8. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.1- 10% by weight of gelatin, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 10-60 mM of Tris, about 40-110 mM of NaCl, about 0.2-3% by weight of trehalose, about 2-7% by weight of sucrose, about 0.2-0.6% by volume of P188, and about 5-15 pM of EDTA at a pH of about 7.5-7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 0.5-5% by weight of gelatin, at least one thermostabilizing excipient disclosed herein, such as lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and theRNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20-50 mM of Tris, about 50-100 mM of NaCl, about 0.4-2.6% by weight of trehalose, about 3.-5% by weight of sucrose, about 0.2-0.4% by volume of P188, and about 10-15 pM of EDTA at a pH of about 7.7. In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, at least one thermostabilizing excipient selected from lipoic acid, L- theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8). In some embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, about 20 mM of Tris, about 100 mM ofNaCl, 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 pM of EDTA at a pH of about 7.7. In other embodiments, the thermostable RNA-LNP compositions of the disclosure comprise, in addition to the RNA molecules (e.g., mRNAs) and LNP, about 1% by weight of gelatin, at least one thermostabilizing excipient selected from lipoic acid, L- theanine, vanillin, or combination thereof, in an amount so that the at least one thermostabilizing excipient and the RNA molecules (e.g., mRNAs) are present in a weight ratio of from about 5: 1 to about 50: 1, 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 pM of EDTA at a pH of about 7.7.

[0225] In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise OF-02-based LNPs encapsulating one or more mRNA molecules. In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise cKK-ElO-based LNPs encapsulating one or more mRNA molecules. In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise GL-HEPES-E3-E12- DS-4-E10-based LNPs encapsulating one or more mRNA molecules.

[0226] In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise OF-02-based LNPs encapsulating mRNA molecules encoding three different influenza virus proteins (e.g., trivalent): an Hl from a first standard of care influenzavirus strain, an H3 from a second standard of care influenza virus strain, and an HA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise OF-02-based LNPs encapsulating mRNA molecules encoding eight different influenza virus proteins (e.g., octavalent), such as an Hl, an H3, an HA from a B / Victoria lineage, an HA from a B / Yamagata lineage, an Nl, an N2, an NA from a B / Victoria lineage, and an NA from a B / Yamagata lineage.

[0227] In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise cKK-El 0-based LNPs encapsulating mRNA molecules encoding three different influenza virus proteins (e.g., trivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, and an HA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise cKK-ElO-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the above exemplified thermostable RNA-LNP compositions of the disclosure comprise cKK-ElO-based LNPs encapsulating mRNA molecules encoding eight different influenza virus proteins (e.g., octavalent), such as an Hl, an H3, an HA from a B / Victoria lineage, an HA from a B / Yamagata lineage, an Nl, an N2, an NA from a B / Victoria lineage, and an NA from a B / Yamagata lineage. .

[0228] In some embodiments, the above exemplified thermostable RNA-LNP compositions comprise GL-HEPES-E3-E12-DS-4-E10-based LNPs encapsulating mRNA molecules encoding three different influenza virus proteins (e.g., trivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, and an HA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the above exemplified thermostable RNA-LNP compositionscomprise GL-HEPES-E3-E12-DS-4-E10-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the above exemplified thermostable RNA-LNP compositions of the disclosure comprise GL-HEPES-E3-E12-DS-4-E10-based LNPs encapsulating mRNA molecules encoding eight different influenza virus proteins (e.g., octavalent), such as an Hl, an H3, an HA from a B / Victoria lineage, an HA from a B / Yamagata lineage, an Nl, an N2, an NA from a B / Victoria lineage, and an NA from a B / Yamagata lineage.A dministration

[0229] The thermostable RNA-LNP compositions of the present disclosure can be formulated for administration in any way known in the art of drug delivery, for example, orally, parenterally, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intrathecally, submucosally, sublingually, rectally, vaginally, etc. In some embodiments, the composition is formulated for sublingual administration, intramuscular administration, intradermal administration, subcutaneous administration, intravenous administration, intranasal administration, administration by inhalation, or intraperitoneal administration. In some embodiments, the composition is formulated for sublingual administration. In some embodiments, the composition is formulated for intramuscular injection.

[0230] The thermostable RNA-LNP compositions of the disclosure may be packaged in a container, such as a prefilled syringe, a vial, or an autoinjector. In some embodiments, the compositions of the disclosure are packaged in a prefilled syringe. In some embodiments, the compositions of the disclosure are packaged in a vial. In some embodiments, the compositions of the disclosure are packaged in an autoinjector. In other embodiments, the compositions of the disclosure are packaged cartridges for patient-friendly autoinjector and infusion pump devices.

[0231] Prefilled syringes provide several advantages over other types of packages, such as convenience, affordability, accuracy, sterility, and safety. Accordingly, in some embodiments, provided herein is a pre-filled syringe comprising about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of any of the thermostable RNA-LNP compositions disclosed herein. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides, such as influenza HA and / or NA proteins from the same or different type of influenza viruses. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides selected from Hl, H3, HA from a B / Victoria lineage, and / or HA from a B / Yamagata lineage. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding three different influenza virus proteins (e.g., trivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, and an HA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins such as Hl, H3, HA from a B / Victoria lineage, and HA from a B / Yamagata lineage.

[0232] In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) selected from Nl, N2, NA from a B / Victoria lineage, and / or NA from a B / Yamagata lineage. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding three different influenza virus proteins (e.g., trivalent): a Nl from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, and an NA from a third standard of care influenza virus strain from the B / Victoria lineage. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or moreLNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins (e.g., quadrivalent): a N1 from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, an NA from a third standard of care influenza virus strain from the B / Victoria lineage, and an NA from a fourth standard of care influenza virus strain from the B / Yamagata lineage. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding four different influenza virus proteins, such as Nl, N2, NA from a B / Victoria lineage, and NA from a B / Yamagata lineage.

[0233] In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding eight different influenza virus proteins (e.g., octavalent), such as an Hl, an H3, an HA from a B / Victoria lineage, an HA from a B / Yamagata lineage, an Nl, an N2, an NA from a B / Victoria lineage, and an NA from a B / Yamagata lineage.

[0234] In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more respiratory syncytial virus (RSV) polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), such as the receptor attachment glycoprotein (G), the fusion protein (F), and / or a short hydrophobic (SH) protein from the same or different subtypes of respiratory syncytial virus (RSV). In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more coronavirus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), particularly the Spike protein (S).

[0235] In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) from different viruses. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) coronavirus proteins. In some embodiments, the pre-filled syringeof the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) respiratory syncytial virus (RSV) proteins. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more coronavirus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten) and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) respiratory syncytial virus (RSV) proteins. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding one or more influenza virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten), one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) coronavirus proteins, and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) respiratory syncytial virus (RSV) proteins. In some embodiments, the pre-filled syringe of the disclosure comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of a composition comprising one or more LNP-encapsulated RNA molecules (e.g., mRNA) encoding polypeptides from any combinations of virus proteins.Immunogenic Compositions and Vaccines

[0236] In some embodiments, the thermostable RNA-LNP compositions of the present disclosure are immunogenic compositions. As used herein, the term “immunogenic composition” refers to a composition that generates an immune response that may or may not be a protective immune response or protective immunity. The term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response. Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. An antibody response or humoralresponse is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and / or other white blood cells.

[0237] Also provided herein is a vaccine comprising the immunogenic composition of the disclosure and a pharmaceutically acceptable carrier. As used herein, the term “vaccine” refers to a composition that generates a protective immune response or a protective immunity in a subject. A “protective immune response” or “protective immunity” refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection) or reduces the symptoms of infection (for instance, an infection by an influenza virus). Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, by inhalation, or intramuscular.Adjuvants

[0238] In some embodiments, the immunogenic composition of the disclosure comprises an adjuvant. In other embodiments, the immunogenic composition of the disclosure does not contain an adjuvant. Similarly, in some embodiments, the vaccine of the disclosure can be administered with an adjuvant to boost the immune response. In other embodiments, the vaccines can be administered without an adjuvant. As used herein, the term “adjuvant” refers to a substance or combination of substances that may be used to enhance an immune response to an antigen component of a vaccine or immunogenic composition. Adjuvants can include a suspension of minerals (alum, aluminum salts, including, for example, aluminum hydroxide / oxyhydroxide (A100H), aluminum phosphate (AIPO4), aluminum hydroxyphosphate sulfate (AAHS) and / or potassium aluminum sulfate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see 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). Adjuvants also include biological molecules, such as lipids and costimulatory molecules. Exemplary biological adjuvants include, but are not limited to, AS04 (Didierlaurent et al., J. Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

[0239] In certain embodiments, the adjuvant is a squalene-based adjuvant comprising an oil- in-water adjuvant emulsion comprising 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, optionally wherein about 90% of the population by volume of the oil drops has a size less than about 200 nm.

[0240] In certain embodiments, the polyoxyethylene alkyl ether is of formula CH3-(CH2)X- (O-CH2-CH2)n-OH, in which n is an integer from 10 to 60, and x is an integer from 11 to 17. In certain embodiments, the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12) cetostearyl ether.

[0241] In certain embodiments, about 90% of the population by volume of the oil drops has a size less than about 160 nm. In certain embodiments, about 90% of the population by volume of the oil drops has a size less than about 150 nm. In certain embodiments, about 50% of the population by volume of the oil drops has a size less than about 100 nm. In certain embodiments, about 50% of the population by volume of the oil drops has a size less than about 90 nm.

[0242] In certain embodiments, the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol and mannitol.

[0243] In some embodiments the hydrophilic / lipophilic balance (HLB) of the hydrophilic nonionic surfactant is greater than or equal to about 10. 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 greater than or equal to about 10 and the HLB of the hydrophobic nonionic surfactant is less than about 9.

[0244] 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 between about 5% and about 45%. In certain embodiments, the amount of polyoxyethylene alkyl ether surfactant is between about 0.9% and about 9%. In certain embodiments, the amount of hydrophobic nonionic surfactant is between about 0.7% and about 7%. In certain embodiments, the adjuvant comprises: i) about 32.5% of squalene, ii) about 6.18% of polyoxyethylene(12) cetostearyl ether, iii) about 4.82% of sorbitan monooleate, and iv) about 6% of mannitol.

[0245] In certain embodiments, the adjuvant further comprises an alkylpolyglycoside and / or a cryoprotective agent, such as a sugar, in particular dodecylmaltoside and / or sucrose.

[0246] In certain embodiments, the adjuvant comprises AF03, as described in Klucker et al., J. Pharm. Sci., 2012, 101(12):4490-4500, which is hereby incorporated by reference in itsentirety. In certain embodiments, the adjuvant comprises a liposome-based adjuvant, such as SPAM. SPAM is a liposome-based adjuvant (ASOl-like) containing a toll-like receptor 4 (TLR4) agonist (E6020) and saponin (QS21).

[0247] In some embodiments, including embodiments where the one or more nucleic acids are encapsulated in a LNP, the vaccine composition does not comprise an adjuvant. In certain embodiments, the one or more RNA molecules, such as one or more mRNA molecules, are encapsulated in a LNP that may serve to adjuvate one or more of the recombinant proteins (e.g., viral proteins) in the composition. See e.g., Shirai et al., Vaccines, 2020, 8(433): 1-18.Administration

[0248] In some embodiments, the immunogenic composition or vaccine of the disclosure is formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. The immunogenic composition or vaccine of the disclosure may also be formulated for intranasal or inhalation administration. The immunogenic composition or vaccine of the disclosure can also be formulated for any other intended route of administration.

[0249] In some embodiments, the immunogenic composition or vaccine of the disclosure is formulated for intradermal injection, intranasal administration or intramuscular injection. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington’s Pharmaceutical Sciences, 19thed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. At present the oral or nasal spray or aerosol route (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, the immunogenic composition or vaccine of the disclosure is administered using a device that delivers a metered dosage of the vaccine composition. Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. PatentNo. 5,417,662, all of which are incorporated herein by reference. Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO 1999 / 34850, incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reachesthe dermis. Jet injection devices are described for example in U.S. Patent No. 5,480,381, U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No. 5,704,911, U.S. Patent No. 5,383,851, U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Patent No. 5,520,639, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 4,940,460, WO1997 / 37705, and WO1997 / 13537, all of which are incorporated herein by reference. Additionally, conventional syringes may be used in the classical Mantoux method of intradermal administration.

[0250] Preparations for parenteral administration typically include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are 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, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.Methods of Use

[0251] Also provided herein are methods of administering the vaccines described herein to a subject. The methods may be used to vaccinate a subject to prevent an infectious disease (e.g., virus infection, such as influenza, coronavirus, or a respiratory syncytial virus (RSV) infection) in the subject, to decrease the subject’s likelihood of getting an infectious disease (e.g., virus infection), or to reduce the subject’s likelihood of getting serious illness from an infectious disease (e.g., virus infection, such as an influenza virus, coronavirus, or RSV infection). Likewise, the present disclosure provides any of the vaccine compositions described herein for use in vaccinating a subject against an infectious disease (e.g., virus infection, such as an influenza virus, coronavirus, or RSV infection). Also disclosed is any of the vaccine compositions as described herein, for the manufacture of a vaccine for use in vaccinating a subject against an infectious disease (e.g., virus infection, such as an influenza virus, coronavirus, or RSV infection). In some embodiments, the vaccination method or usecomprises administering to a subject in need thereof an immunologically effective amount of any of the vaccines described herein.

[0252] As used herein, the term “immunologically effective amount” or “therapeutically effective amount” means an amount sufficient to immunize a subject. In some embodiments, the immunologically effective amount or therapeutically effective amount is capable of eliciting protective immunity against an infectious disease, which include, but are not limited to, an increase of antibody titers and / or T cell immunity against an infectious disease. In some embodiments, an immunologically effective amount or therapeutically effective amount of the vaccine or composition as disclosed herein increases protective immunity in 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%, about 100%, including values and subranges therebetween, when compared with a subject who is not administered with the vaccine or composition as disclosed herein.

[0253] Accordingly, in some embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof an immunologically effective amount of any of the vaccines described herein. In particular embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof an immunologically effective amount any of the vaccines described herein. As used herein, “immunize” or “immunizing” means to induce in a subject a protective immune response against an infectious disease (e.g., viral infection, such as influenza, coronavirus, or RSV infection). Likewise, the present disclosure provides any of the vaccine compositions described herein for use in immunizing a subject against an infectious disease (e.g., viral infection, such as influenza, coronavirus, or RSV infection). Also disclosed is any of the vaccine compositions as described herein, for the manufacture of a vaccine for use in immunizing a subject against an infectious disease (e.g., virus infection, such as an influenza virus, coronavirus, or RSV infection).

[0254] In some embodiments, the method or use prevents virus infection or disease caused by the virus infection in the subject. In some embodiments, the method or use decreases the subject’s likelihood of getting a virus 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%, about 100%, including values and subranges therebetween, when compared with a subject who is not administered with the vaccine orcomposition as disclosed herein. In some embodiments, the method or use reduces the subject’s likelihood of getting serious illness from the infectious disease (e.g., viral infection, such as 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%, about 100%, including values and subranges therebetween, when compared with a subject who is not administered with the vaccine or composition as disclosed herein. In some embodiments, the method or use raises a protective immune response in the subject. In some embodiments, the protective immune response is an antibody response.

[0255] Also provided, in some embodiments, is a method of reducing one or more symptoms of an infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection) comprising administering to a subject in need thereof any of the vaccines described herein. In some embodiments, provided herein is a method of reducing one or more symptoms of an infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection) comprising administering to a subject in need thereof a prophylactically effective amount of any of the vaccines described herein.

[0256] The present disclosure provides any of the vaccine compositions described herein for use in reducing one or more symptoms of an infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection). Also disclosed is any of the immunogenic compositions as described herein, for the manufacture of a vaccine for use in reducing one or more symptoms of an infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection) in a subject.

[0257] In some embodiments, the method or use of the present disclosure reduces one or more symptoms of an infectious disease (e.g., viral infection, such as 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%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or composition as disclosed herein.

[0258] In some embodiments, the vaccine, and an optional adjuvant, may be administered prior to or after development of one or more symptoms of the infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection). That is, in some embodiments, the vaccines described herein may be administered prophylactically toprevent the infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection) or ameliorate the symptoms of a potential infectious disease (e.g., virus infection, such as influenza virus infection, coronavirus infection, or RSV infection).

[0259] In some embodiments, the subject is at risk of infection if the subject will be in contact with other individuals or livestock (e.g., swine) known or suspected to have been infected with a particular infectious agent (e.g., virus, such as influenza, coronavirus, or RSV) and / or if the subject will be present in a location in which infectious disease (e.g., virus infection) is known or thought to be prevalent or endemic. In some embodiments, the vaccines are administered to a subject suffering from an infectious disease (e.g., virus infection, such as an influenza virus, coronavirus, or RSV infection), or the subject is displaying one or more symptoms commonly associated with an infectious disease (e.g., virus infection, such as an influenza virus, coronavirus, or RSV infection). In some embodiments, the subject is known or believed to have been exposed to an infectious agent (e.g., a virus, such as an influenzas virus, coronavirus, or RSV).

[0260] Vaccines in accordance with the disclosure may be administered in any amount or dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is induction of a lasting adaptive immune response against the virus. In some embodiments, the desired outcome is reduction in intensity, severity, and / or frequency, and / or delay of onset of one or more symptoms associated with virus infection. The dose required 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 particular composition being used, and its mode of administration.

[0261] In some embodiments, the vaccines described herein are administered to subjects, wherein the subjects can 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 an avian (e.g., a chicken or a bird), a reptile, an amphibian, a fish, an insect, and / or a worm. In some embodiments, the non-human subject is a mammal (e.g., a ferret, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig).

[0262] In some embodiments, the vaccines described herein are administered to a human subject. In some embodiments, a human subject is 6 months of age or older, 6 months through 35 months of age, at least two years of age, at least 3 years of age, 36 months through 8 years of age, 9 years of age or older, at least 6 months of age and less than 5 years of age, at least 6 months of age and less than 18 years of age, or at least 3 years of age and less than 18 years ofage. In some embodiments, the human subject is an infant (less than 36 months). In some embodiments, the human subject is a child or adolescent (less than 18 years of age). In some embodiments, the human subject is a child of at least 6 months of age and less than 5 years of age. In some embodiments, the human subject is at least 5 years of age and less than 60 years of age. In some embodiments, the human subject is at least 5 years of age and less than 65 years of age. In some embodiments, the human subject is elderly (at least 60 years of age or at least 65 years of age). In some embodiments, the human subject is a non-elderly adult (at least 18 years of age and less than 65 years of age or at least 18 years of age and less than 60 years of age).

[0263] The methods and uses of the vaccines described herein include administration of a single dose to a subject (i.e., no booster dose). In some embodiments, the methods and uses of the vaccines described herein include prime-boost vaccination strategies. Prime-boost vaccination comprises administering a priming vaccine and then, after a period of time has passed, administering to the subject a boosting vaccine. The immune response is “primed” upon administration of the priming vaccine and is “boosted” upon administration of the boosting vaccine. The priming vaccine can include a vaccine as described herein and an optional adjuvant. Likewise, the boosting vaccine can include a vaccine as described herein and an optional adjuvant. The priming vaccine can be, but need not be, the same as the boosting vaccine. Administration of the boosting vaccine is generally weeks or months after administration of the priming 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 recipient of the prime-boost vaccination is a naive subject, typically a naive infant or child.

[0264] The vaccine can be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above. In some embodiments, the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.Other Methods

[0265] Also provided herein is a method of stabilizing a composition comprising one or more RNA molecules encapsulated in a LNP, as described herein, the method comprising adding at least one thermoreversible gelling agent, as described herein, to the composition in an amount sufficient to maintain the composition in a liquid phase at a temperature above about 12°C (e.g., room temperature) and reversibly transition the composition to a gel form at a temperature of about 1-11°C (e.g., 2-8°C or 4°C). As described herein, the stability of the composition can be measured by the mean particle size of the LNP in some embodiments, theencapsulation efficiency of the LNP in other embodiments, and / or the integrity of the one or more RNA molecules encapsulated in the LNP in some further embodiments.

[0266] Accordingly, in certain embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the mean particle size of the LNP does not increase more than about 40% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0267] In other embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the encapsulation efficiency of the LNP does not decrease more than about 10% after storage of the composition at a temperature of about 1-11 °C (e.g., 2-8°Cor 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0268] In some embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 10% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0269] In some embodiments where the one or more RNA molecules encode one or more influenza virus proteins, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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 11months, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding the at least one thermoreversible gelling agent in an amount sufficient so that the HAI titers of the composition does not decrease more than about 25% after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0270] Also provided herein is a method of preventing degradation of one or more RNA molecules encapsulated in a LNP in a liquid composition, the method comprising adding the at least one thermoreversible gelling agent, as described herein, to the liquid composition in an amount sufficient to maintain the liquid composition in a liquid phase at a temperature above about 12°C (e.g., room temperature) and reversibly transition the liquid composition to 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 the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the liquid composition at a temperature of about 1-11 °C (e.g., 2- 8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 10% after storage of the liquid composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subrangestherebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0271] In other embodiments, provided herein is a method of formulating a composition comprising one or more RNA molecules encapsulated in a LNP, wherein the composition is stable at 4°C for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years, the method comprising adding the at least one thermoreversible gelling agent, as described herein, to the composition in an amount sufficient to maintain the composition in a liquid phase at a temperature above about 12°C, such as room temperature, and reversibly transition the liquid composition to a gel form at a temperature of about 1-11°C (e.g., 2-8°C or 4°C).

[0272] In some embodiments, the method comprises adding, into a composition comprising OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8), the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up toabout 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding, into a composition comprising OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage, about 20 mM of Tris, about 100 mM of NaCl, about 0.4-1.3% of trehalose, about 5% of sucrose, about 10 pM of EDTA, and about 0.4% of P188 at a pH of about 7.7, the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding, into a composition comprising OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage, about 50 mM of Tris, about 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 10 pM of EDTA, and about 0.4% of P188 at a pH of about 7.7, the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increasemore than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding, into a composition comprising OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins, such as Hl, H3, HA from a B / Victoria lineage, and HA from a B / Yamagata lineage, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8), the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years ascompared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding, into a composition comprising OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins, such as Hl, H3, HA from a B / Victoria lineage, and HA from a B / Yamagata lineage, about 20 mM of Tris, about 100 mM of NaCl, about 0.4- 1.3% of trehalose, about 5% of sucrose, about 10 pM of EDTA, and about 0.4% of Pl 88 at a pH of about 7.7, the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding, into a composition comprising OF-02-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins, such as Hl, H3, HA from a B / Victoria lineage, and HA from a B / Yamagata lineage, about 50 mM of Tris, about 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 10 pM of EDTA, and about 0.4% of P188 at a pH of about 7.7, the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the compositiondoes not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent.

[0273] In some embodiments, the method comprises adding, into a composition comprising cKK-ElO-based LNPs encapsulating mRNA molecules encoding four different influenza virus proteins (e.g., quadrivalent): an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B / Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B / Yamagata lineage, about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8), the at least one thermoreversible gelling agent in an amount sufficient (e.g., about 1% gelatin) so that (1) the mean particle size of the LNP does not increase more than about 50%, such as more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, including all values and subranges therebetween, (2) the encapsulation efficiency of the LNP does not decrease more than about 20%, such as more than about 15%, 10%, or 5%, including all values and subranges therebetween, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, and / or (4) the HAI titers of the composition does not decrease more than about 30%, such as more than about 25%, 20%, 15%, 10% or 5%, including all values and subranges therebetween, after storage of the composition at a temperature of about 1-11°C (e.g., 2-8°C or 4°C) for up to about 1 month or longer, such as 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, up to about 12 months, up to about 18 months, up to about 2 years, including all values and subranges therebetween, or more than 2 years as compared to a control composition without the at least one thermoreversible gelling agent. In some embodiments, the method comprises adding, into a composition comprising cKK-ElO-based LN...

Claims

CLAIMS1. A composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) and at least one thermoreversible gelling agent.

2. The composition of claim 1, wherein the composition has a liquid phase at a temperature above about 12°C and is reversibly transitioned to a gel form at a temperature of about 1-11°C.

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

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

5. The composition of any one of claims 1-4, 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 of claim 5, wherein the thermoreversible gelling polymer comprises a polypeptide-based gelling polymer or a protein-based gelling polymer.

7. The composition of claim 5, wherein the thermoreversible gelling polypeptide comprises multi-L-arginyl-poly-L-aspartate (iMAPA)-PEG, or wherein the thermoreversible gelling polymer comprises gelatin, poly(N-acryloylasparaginamide), poly(ethylene glycol)-b- poly(N-acryloylglycine amide-co-acrylonitrile) (PEG-b-P(NAGA-co-AN), poly(N- acryloylglycineamide-co-N-phenylacrylamide) (P(NAGA-co-NPhAm)), poly(N-(2- hydroxypropyl) methacrylamide)-glycolamide) (P(HPMA-GA)), P(AAm-co-AN)-b- poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly(acrylic acid-co- acrylonitrile) (P(AA-co-AN)), poly(N-vinylimidazole-co-l -vinyl-2 -(hydroxymethyl)imidazole), poly(sulfobetaine-co-sulfabetaine) (P(SB-co-ZB), poly(2- (methacryloyloxy)ethylphosphocholine)-b-poly(2-ureidoethyl methacrylate) (PMPC20-b- PUEM165), or combinations thereof.

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

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

10. The composition of claim 9, wherein: a) the buffering agent comprises or is tris(hydroxymethyl)aminomethane) (Tris); b) the pharmaceutically acceptable salt comprises or is sodium chloride (NaCl); c) the one or more disaccharides comprise or are sucrose; d) the surfactant comprises or is Pol oxamer 188 (Pl 88); and / or e) the chelating agent comprises or is ethylenedi aminetetraacetic acid (EDTA).

11. The composition of any one of claims 1-10, wherein the composition comprises: a) from about 10 mM to about 60 mM of Tris, from about 40 mM to about 150 mM of NaCl, from about 1% to about 10% by weight of sucrose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.2 to about 7.8; b) about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of 7.5 ± 0.3; c) from about 10 mM to about 60 mM of Tris, from about 40 mM to about 110 mM of NaCl, from about 3% to about 6% by weight of sucrose, from about 0.2% to about 4% by weight of trehalose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.5 to about 7.7; d) about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7; e) from about 20 mM to about 50 mM of Tris, from about 50 mM to about 100 mM of NaCl, from about 2% to about 5% by weight of sucrose, from about 0.3% to about 3%by weight of trehalose, from about 0.2% to about 0.4% by volume of Pl 88, and from about 10 pM to about 15 pM of EDTA, wherein the composition has a pH of about 7.7; or f) about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4-1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7.

12. The composition of any one of claims 1-11, wherein 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 as compared to a control composition without the at least one thermoreversible gelling agent, wherein stability of the composition is measured by a change in mean particle size of the LNP, encapsulation efficiency of the LNP, and / or integrity of the one or more RNA molecules encapsulated in the LNP.

13. The composition of claim 12, wherein: a) the mean particle size of the LNP does not increase 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 as compared to a control composition without the at least one thermoreversible gelling agent; b) the encapsulation efficiency of the LNP does not decrease 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 as compared to a control composition without the at least one thermoreversible gelling agent; c) the encapsulation efficiency of the LNP is higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent; and / or d) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease 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 as compared to a control composition without the at least one thermoreversible gelling agent.

14. A liquid composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) and at least one thermostabilizing excipient, wherein the at least one thermostabilizing excipient comprises lipoic acid, L-theanine, vanillin, or combinations thereof.

15. The liquid composition of claim 14, wherein the integrity of the one or more RNA molecules does not decrease more than 20% after storage of the liquid composition at a temperature of 37°C for at least 7 days as compared to a control liquid composition without the at least one thermostabilizing excipient.

16. The liquid composition of claim 14 or 15, wherein: a) the integrity of the one or more RNA molecules does not decrease more than 25% after storage of the liquid composition at a temperature of 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 as compared to a control liquid composition without the at least one thermostabilizing excipient; b) the integrity of the one or more RNA molecules does not decrease more than 30% after storage of the liquid composition 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, or up to about 8 months as compared to a control liquid composition without the at least one thermostabilizing excipient; c) the integrity of the one or more RNA molecules does not decrease 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 as compared to a control liquid composition without the at least one thermostabilizing excipient; d) the integrity of the one or more RNA molecules does not decrease 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 as compared to a control liquid composition without the at least one thermostabilizing excipient; e) the mean particle size of the LNP does not increase more than 40% after storage of the liquid composition at a temperature of 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, up to about 6 months, up toabout 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; f) the mean particle size of the LNP does not increase more than 20% 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, up to about 5 weeks, up to about 6 weeks, or up to about 7 weeks; g) the encapsulation efficiency of the LNP does not decrease more than 20% after storage of the liquid composition at a temperature of 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, 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; and / or h) the encapsulation efficiency of the LNP does not decrease more than 20% 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, up to about 5 weeks, up to about 6 weeks, or up to about 7 weeks.

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

18. The liquid composition of any one of claims 14-17, wherein: a) the at least one thermostabilizing excipient comprises or is lipoic acid, optionally wherein the lipoic acid and the one or more RNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1; b) the at least one thermostabilizing excipient comprises or is L-theanine, optionally wherein the L-theanine and the one or more RNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1; orc) the at least one thermostabilizing excipient comprises or is vanillin, optionally wherein the vanillin and the one or more RNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1.

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

20. The liquid composition of claim 19, wherein: a) the buffering agent comprises or is tri s(hydroxymethyl)aminom ethane) (Tris); b) the pharmaceutically acceptable salt comprises or is sodium chloride (NaCl); c) the one or more disaccharides comprise or are sucrose; d) the surfactant comprises or is Pol oxamer 188 (Pl 88); and / or e) the chelating agent comprises or is ethylenediaminetetraacetic acid (EDTA).

21. The liquid composition of any one of claims 14-20, wherein the liquid composition comprises: a) from about 10 mM to about 60 mM of Tris, from about 40 mM to about 150 mM of NaCl, from about 1% to about 10% by weight of sucrose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.2 to about 7.8; b) about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of 7.5 ± 0.3; c) from about 10 mM to about 60 mM of Tris, from about 40 mM to about 110 mM of NaCl, from about 3% to about 6% by weight of sucrose, from about 0.2% to about 4% by weight of trehalose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.5 to about 7.7; d) about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7; e) from about 20 mM to about 50 mM of Tris, from about 50 mM to about 100 mM of NaCl, from about 2% to about 5% by weight of sucrose, from about 0.3% to about 3%by weight of trehalose, from about 0.2% to about 0.4% by volume of Pl 88, and from about 10 pM to about 15 pM of EDTA, wherein the composition has a pH of about 7.7; or f) about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4-1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7.

22. A liquid formulation comprising one or more ribonucleic acid (RNA) encapsulated in a lipid nanoparticle (LNP), from about 10 mM to about 60 mM of tris(hydroxymethyl)aminomethane) (Tris), from about 40 mM to about 150 mM of sodium chloride (NaCl), from about 1% to about 10% by weight of sucrose, from about 0.2% to about 0.6% by volume of Poloxamer 188 (P188), and from about 5 pM to about 15 pM of ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of from about 7.2 to about 7.8.

23. The liquid formulation of claim 22, comprising about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the liquid formulation has a pH of 7.5 ± 0.3.

24. A liquid formulation comprising one or more ribonucleic acid (RNA) encapsulated within a lipid nanoparticle (LNP), from about 10 mM to about 60 mM of tris(hydroxymethyl)aminomethane) (Tris), from about 40 mM to about 110 mM of sodium chloride (NaCl), from about 3% to about 6% by weight of sucrose, from about 0.2% to about 4% by weight of trehalose, from about 0.2% to about 0.6% by volume of Poloxamer 188 (Pl 88), and from about 5 pM to about 15 pM of ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of from about 7.5 to about 7.7.

25. The liquid formulation of claim 24, comprising about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the liquid formulation has a pH of about 7.7.

26. A liquid formulation comprising one or more ribonucleic acid (RNA) encapsulated within a lipid nanoparticle (LNP), from about 20 mM to about 50 mM of tris(hydroxymethyl)aminomethane) (Tris), from about 50 mM to about 100 mM of sodiumchloride (NaCl), from about 2% to about 5% by weight of sucrose, from about 0.3% to about 3% by weight of trehalose, from about 0.2% to about 0.4% by volume of Pol oxamer 188 (Pl 88), and from about 10 pM to about 15 pM of ethylenediaminetetraacetic acid (EDTA), wherein the liquid formulation has a pH of about 7.7.

27. The liquid formulation of claim 26, comprising about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4- 1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the liquid formulation has a pH of about 7.7.

28. A composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP), at least one thermoreversible gelling agent, and at least one thermostabilizing excipient, wherein the at least one thermostabilizing excipient comprises or is lipoic acid.

29. The composition of claim 28, wherein the at least one thermoreversible gelling agent comprises or is gelatin, wherein optionally the gelatin is present in an amount of from about 0.5% to about 1.5% by weight, such as about 1% by weight.

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

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

32. The composition of claim 31, wherein: a) the buffering agent comprises or is tris(hydroxymethyl)aminomethane) (Tris); b) the pharmaceutically acceptable salt comprises or is sodium chloride (NaCl); c) the one or more disaccharides comprise or is sucrose; d) the surfactant comprises or is Pol oxamer 188 (Pl 88); and / or e) the chelating agent comprises or is ethylenedi aminetetraacetic acid (EDTA).

33. The composition of any one of claims 28-32, wherein the composition comprises: a) from about 10 mM to about 60 mM of Tris, from about 40 mM to about 150 mM of NaCl, from about 1% to about 10% by weight of sucrose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.2 to about 7.8; b) about 50 mM of Tris, about 150 mM of NaCl, about 5% by weight of sucrose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of 7.5 ± 0.3; c) from about 10 mM to about 60 mM of Tris, from about 40 mM to about 110 mM of NaCl, from about 3% to about 6% by weight of sucrose, from about 0.2% to about 4% by weight of trehalose, from about 0.2% to about 0.6% by volume of P188, and from about 5 pM to about 15 pM of EDTA, wherein the composition has a pH of from about 7.5 to about 7.7; d) about 50 mM of Tris, about 50 mM of NaCl, about 5% by weight of sucrose, about 2-2.6% by weight of trehalose, about 0.4% by volume of Pl 88, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7; e) from about 20 mM to about 50 mM of Tris, from about 50 mM to about 100 mM of NaCl, from about 2% to about 5% by weight of sucrose, from about 0.3% to about 3% by weight of trehalose, from about 0.2% to about 0.4% by volume of Pl 88, and from about 10 pM to about 15 pM of EDTA, wherein the composition has a pH of about 7.7; or f) about 20 mM of Tris, about 100 mM of NaCl, about 5% by weight of sucrose, about 0.4-1.3% by weight of trehalose, about 0.4% by volume of P188, and about 10 pM of EDTA, wherein the composition has a pH of about 7.7.

34. The composition of any one of claims 28-33, wherein 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, or up to about 6 months as compared to a control composition without the at least one thermoreversible gelling agent and the at least one thermostabilizing excipient, wherein stability of the composition is measured by a change in mean particle size of the LNP, encapsulation efficiency of the LNP, and / or integrity of the one or more RNA molecules encapsulated in the LNP.

35. The composition of any one of claims 1-13 and 28-34, the liquid composition of any one of claims 14-20, or the liquid formulation of any one of claims 21-27, wherein the one or more RNA molecules: a) are messenger RNA (mRNA) molecules; b) encode one or more virus proteins, optionally wherein the one or more virus proteins comprise influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof; and / or c) comprise at least one chemically modified nucleotide and / or a phosphorothioate bond, optionally wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'-methoxy ribonucleotide, optionally wherein the pseudouridine is a N1 -methylpseudouridine.

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

37. The composition, liquid composition, or liquid formulation of claim 36, wherein: a) the cationic lipid is present at a molar ratio between about 30% and about 50%; b) the PEGylated lipid is present at a molar ratio between about 0.25% and about 15%; c) the cholesterol-based lipid is present at a molar ratio between about 20% and about 40%; and d) the helper lipid is present at a molar ratio between about 20% and about 40%.

38. The composition, liquid composition, or liquid formulation of claim 37, wherein the cationic lipid, the PEGylated lipid, the cholesterol-based lipid, and the helper lipid are present at a molar ratio of: a) about 40%, about 1.5%, about 28.5%, and about 30%, respectively; or b) about 40%, about 5%, about 25%, and about 30%, respectively.

39. The composition, liquid composition, or liquid formulation of any one of claims36-38, wherein: a) the cationic lipid comprises or is OF-02, cKK-ElO, 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 / orb) the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000; and / or c) the cholesterol-based lipid comprises or is cholesterol; and / or d) the helper lipid comprises, or is, dioleoyl-SN-glycero-3-phosphoethanolamine.

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

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

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

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

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

45. A method of immunizing a subj ect, the method comprising administering to the subj ect in need thereof the vaccine of claim 44, optionally the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally, optionally wherein: a) the method prevents a virus infection in the subject, decreases the subject’s likelihood of getting a virus infection, and / or reduces the subject’s likelihood of getting serious illness from a virus infection; and / or b) the method raises a protective immune response in the subject.

46. The method of claim 45, wherein the subject is a human, optionally wherein the human is 6 months of age or older, less than 18 years of age, at least 6 months of age and less than 18 years of age, at least 18 years of age and less than 65 years of age, at least 6 months of age and less than 5 years of age, at least 5 years of age and less than 65 years of age, at least 60 years of age, or at least 65 years of age.

47. A method of reducing one or more symptoms of a virus infection, the method comprising administering to a subject in need thereof the vaccine of claim 44.

48. The method of any one of claims 45-47, wherein the vaccine comprises one or more LNP-encapsulated RNA molecules which encode one or more virus proteins, and wherein the one or more virus proteins comprise influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof.

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

50. A method of preventing degradation of one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) 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 a liquid phase at a temperature above about 12°C and reversibly transition the liquid composition to a gel form at a temperature of about 1-11°C.

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

52. The method of any one of claims 49-51, wherein:a) the one or more RNA molecules encode one or more virus proteins, such as influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof, and / or b) the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

53. A method of preventing thermal degradation of one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP), the method comprising formulating a liquid composition comprising the LNP and the one or more RNA molecules in the presence of at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combinations thereof.

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

55. The method of claim 53 or 54, wherein: a) the at least one thermostabilizing excipient comprises or is lipoic acid, optionally wherein the lipoic acid and the one or more RNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1; b) the at least one thermostabilizing excipient comprises or is L-theanine, optionally wherein the L-theanine and the one or more RNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1; or c) the at least one thermostabilizing excipient comprises or is vanillin, optionally wherein the vanillin and the one or more RNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1.

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