WO2024246358A1

WO2024246358A1 - Thermostable compositions comprising mrna lipid nanoparticles

Info

Publication number: WO2024246358A1
Application number: PCT/EP2024/065175
Authority: WO
Inventors: Zhen Hu; Frank Derosa; Shrirang KARVE; Ashish Sarode; Fethi Bensaid; Federica COSTAMAGNA; Fanny MONTOUX; Rebecca Lee GOLDMAN; Neha KAUSHAL; Trent Northen; Dipen PARANDE
Original assignee: Sanofi SA
Current assignee: Sanofi SA
Priority date: 2023-06-01
Filing date: 2024-06-03
Publication date: 2024-12-05
Anticipated expiration: 2025-12-01

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 LIPID

NANOPARTICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] 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

[02] 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

[03] 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.

[04] 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.

[05] The rapid approval and remarkable success of COVID-19 vaccines, Comirnaty® (BNT162b2) and Spikevax (mRNA-1273), further demonstrated the clinical validation of LNP -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

[06] 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.

[07] 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 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.

[08] 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.

[09] 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 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. 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 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 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. [010] 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 one thermoreversible 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).

[012] 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% 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. 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.

[013] 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 about 4% 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.

[014] 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.

[015] 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.

[016] 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.

[017] 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 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. 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.

[018] 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. [019] 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 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.

BRIEF DESCRIPTION OF THE DRAWING

[020] 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.

[021] 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).

[022] 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.

[023] 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).

[024] 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-based formulation 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.

[025] 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).

[026] 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).

[027] 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. [028] 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. [029] 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.

[030] 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.

[031] 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.

[032] 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. [033] 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.

[034] 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.

[035] 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).

[036] 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.

[037] 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).

[038] 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).

[039] 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).

[040] 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.

[041] 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).

[042] 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.

[043] 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).

[044] 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.

[045] 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).

[046] 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.

[047] 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).

[048] 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).

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

[050] 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.

[051] 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.

[052] 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.

[053] 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.

[054] 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. [055] 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.

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

[057] 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

[058] 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.

[059] 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

[060] 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.

[061] 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 amount and 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.

[062] 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.

[063] 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. [064] 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.

[065] 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. [066] 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.

[067] 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.

[068] 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. [069] 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.

[070] 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.

[071] 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.

[072] 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.

[073] 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.”

[074] 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.

[075] 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.

[076] 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 that an 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.

[077] 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.

[078] 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).

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

[080] 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.

[081] 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

[082] 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.

[083] 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.

[084] 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.

[085] 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 1 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.

[086] 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

[087] 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. A representative 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.

[088] 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.

[089] 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. [090] 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.

[091] 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.

[092] 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.

[093] 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.

[094] 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.

[095] 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.

[096] 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.

[097] 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.

[098] 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

[099] 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 the disclosure 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 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 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 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 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 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.

[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 gelling agent. 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 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.

[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 combinations thereof. 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 one thermostabilizing 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 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 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 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 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 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 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 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 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 liquid compositions 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 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 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 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.

[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, and the 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 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 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 sucrose in 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 LNP aggregation. 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 by Dynamic 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 with other 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 the cholesterol-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 about 40: 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 virus proteins: 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 H3N2 influenza 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 some embodiments, 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 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), 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 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, 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 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 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, 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 40-110 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-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 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 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, 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 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, 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 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 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 compositions comprise 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 more LNP-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 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) 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 humoral response 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 its entirety. 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, 19^th ed., 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 reaches the 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 use comprises 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 or composition 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 to prevent 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 of age. 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, the encapsulation 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°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.

[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 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 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 subranges therebetween, 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 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 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 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 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 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 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.

[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 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 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 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 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 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 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 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 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 cKK-ElO-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 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.

[0274] In some embodiments, the method comprises adding, into a composition comprising 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, 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 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, 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 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, 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 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

I l l 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 GL-HEPES-E3-E12-DS-4-E10-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 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 GL-HEPES-E3-E12-DS-4-E10-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 GL-HEPES- E3-E12-DS-4-E10-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 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. [0275] In some embodiments, the at least one thermoreversible gelling agent is added into the composition in an amount sufficient so that (1) the mean particle size of the LNP does not increase more than about 40%, (2) the encapsulation efficiency of the LNP does not decrease more than about 10%, (3) the integrity of the one or more RNA molecules encapsulated in the LNP does not decrease more than about 10%, and/or (4) 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.

[0276] Any of the thermoreversible gelling agents or amounts thereof described herein can be used in any of the above methods. In some embodiments, the amount of the at least one thermoreversible gelling agent sufficient to practicing any of the above methods is 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. In some embodiments, the at least one thermoreversible gelling agent used in any of the above methods comprises gelatin and the amount used is about 1% by weight.

[0277] Also provided herein is a method of preventing thermal degradation of one or more RNA molecules encapsulated in a LNP, as described herein, 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 thermal degradation of the one or more RNA molecules following 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 is reduced as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the thermal degradation of the one or more RNA molecules following 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 is reduced as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the thermal degradation of the one or more RNA molecules following 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 is reduced as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0278] Further provided herein is a method of stabilizing a liquid composition comprising one or more RNA molecules encapsulated in a LNP, as described herein, the method comprising adding at least one thermostabilizing excipient selected from lipoic acid, L- theanine, vanillin, or combinations thereof in an amount sufficient to prevent the integrity of the one or more RNA molecules from decreasing as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the addition of the at least one thermostabilizing excipient prevents the integrity of the one or more RNA molecules in the liquid composition from decreasing by more than about 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 addition of the at least one thermostabilizing excipient prevents the integrity of the one or more RNA molecules in the liquid composition from decreasing by 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, or up to about 6 months as compared to a control liquid composition without the at least one thermostabilizing excipient. In some embodiments, the addition of the at least one thermostabilizing excipient prevents the integrity of the one or more RNA molecules in the liquid composition from decreasing by more than about 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 addition of the at least one thermostabilizing excipient prevents the integrity of the one or more RNA molecules in the liquid composition from decreasing by more than about 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 month as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0279] Any amount of the at least one thermostabilizing excipient selected from lipoic acid,

L-theanine, vanillin, or combinations thereof described herein can be used in any of the above methods. For instance, the at least one thermostabilizing excipient is used to practice any of the above methods at a concentration of from about 0.1 mM to about 20 mM in some embodiments, from about 0.5 mM to about 15 mM in other embodiments, or from about 1 mM to about 10 mM in some further embodiments, including all values and subranges therebetween. In some embodiments, the at least one thermostabilizing excipient is used to practice any of the above methods at a concentration of about 5 mM. In some embodiments, the at least one thermostabilizing excipient is used to practice any of the above methods at a concentration of about 10 mM. In other embodiments, the at least one thermostabilizing excipient is used to practice any of the above methods at a concentration of about 15 mM. In some embodiments, the at least one thermostabilizing excipient is used to practice any of the above methods at a concentration of about 20 mM.

[0280] The amount of the at least one thermostabilizing excipient sufficient to practice any of the above methods can also be expressed by a weight ratio between the at least one thermostabilizing excipient and the one or more RNA molecule as described herein. Accordingly, in some embodiments, the amount of the at least one thermostabilizing excipient used in any of the above methods is such that the at least one thermostabilizing excipient and the one or more RNA molecules are present in a weight ratio of from about 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 some embodiments, the at least one thermostabilizing excipient used in any of the above methods comprises lipoic acid and the amount used is such 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 at least one thermostabilizing excipient used in any of the above methods comprises L-theanine and the amount used is such 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 further embodiments, the at least one thermostabilizing excipient used in any of the above methods comprises vanillin and the amount used is such 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.

[0281] In any of the above methods, the one or more RNA molecules encapsulated in the LNP formulated in the presence of the at least one thermostabilizing excipient selected from lipoic acid, L-theanine, vanillin, or combinations thereof are thermally stable at an above-zero temperature (e.g., 4°C). “Thermally stable,” as used herein, means that the integrity of the one or more RNA molecules does not substantially decrease after storage of the liquid composition at an above-zero temperature (e.g., 4°C) for a certain period of time. In some embodiments, the one or more RNA molecules are thermally stable 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.

[0282] In certain embodiments, the integrity of the one or more RNA molecules in the liquid composition prepared by any of the above methods 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, up to about 6 months, up to about 7 months, up to about 8 months, including all values and subranges therebetween, or more than 8 months 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 does not decrease more than 30% after storage of the liquid composition at a temperature of 4°C for up to 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, including all values and subranges therebetween, or more than 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 RNA molecules does not decrease more than 45% after storage of the liquid composition at a temperature of 4°C for 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 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, up to about 5 weeks, up to about 6 weeks, including all values and subranges therebetween, or more than 6 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 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, up to about 5 weeks, up to about 6 weeks, including all values and subranges therebetween, or more than 6 weeks as compared to a control liquid composition without the at least one thermostabilizing excipient.

[0283] 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), and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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), and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0284] 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), and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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), and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0285] 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), and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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), and L- theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L- theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0286] 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0287] 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0288] 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L- theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0289] 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0290] 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0291] 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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0292] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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), and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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), and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0293] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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), and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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), and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0294] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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), and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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), and L- theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L- theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0295] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 20 mM of Tris, about 100 mM of NaCl, about 0.4-1.3% of trehalose, about 5% of sucrose, about 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0296] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 20 mM of Tris, about 100 mM of NaCl, about 0.4-1.3% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0297] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 20 mM of Tris, about 100 mM of NaCl, about 0.4- 1.3% of trehalose, about 5% of sucrose, about 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L- theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0298] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0299] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0300] In some embodiments, the method comprises adding, into a composition comprising CKK-ElO-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. 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 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0301] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 Pl 88, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8), and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, 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), and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0302] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 Pl 88, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8), and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, 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), and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0303] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 Pl 88, and about 10 pM of EDTA at a pH of 7.5 ± 0.3 (i.e., 7.2-7.8), and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, 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), and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0304] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, about 20 mM of Tris, about 100 mM of NaCl, about 0.4-1.3% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0305] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, about 20 mM of Tris, about 100 mM of NaCl, about 0.4- 1.3% of trehalose, about 5% of sucrose, about 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0306] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L- theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, about 20 mM of Tris, about 100 mM of NaCl, about 0.4-1.3% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0307] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, about 50 mM of Tris, about 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and lipoic acid as the thermostabilizing excipient in such amount that lipoic acid and the mRNA molecules are present in a weight ratio of from about 2.5: 1 to about 15.5: 1 and the amount of lipoic acid is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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

[0308] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, about 50 mM of Tris, about 50 mM of NaCl, about 2- 2.6% of trehalose, about 5% of sucrose, about 0.4% of Pl 88, and about 10 pM of EDTA, at a pH of about 7.7, and vanillin as the thermostabilizing excipient in such amount that vanillin and the mRNA molecules are present in a weight ratio of from about 12.5: 1 to about 50: 1 and the amount of vanillin is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0309] In some embodiments, the method comprises adding, into a composition comprising GL-HEPES-E3-E12-DS-4-E10-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 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L- theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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. In some embodiments, the method comprises adding, into a composition comprising 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, about 50 mM of Tris, about 50 mM of NaCl, about 2-2.6% of trehalose, about 5% of sucrose, about 0.4% of P188, and about 10 pM of EDTA, at a pH of about 7.7, and L-theanine as the thermostabilizing excipient in such amount that L-theanine and the mRNA molecules are present in a weight ratio of from about 10: 1 to about 30: 1 and the amount of L-theanine is such that the integrity of the RNA molecules in the liquid composition does not decrease more than about 50%, such as 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.

[0310] In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the composition does not decrease more than 25% after storage of the 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 composition without the at least one thermostabilizing excipient. In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the composition does not decrease more than about 30% after storage of the 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 composition without the at least one thermostabilizing excipient. In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the composition does not decrease more than about 35% after storage of the 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 composition without the at least one thermostabilizing excipient. In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the composition does not decrease more than about 40% after storage of the 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 composition without the at least one thermostabilizing excipient. In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the composition does not decrease more than about 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 composition without the at least one thermostabilizing excipient.

[0311] In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the liquid composition 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 composition without the at least one thermostabilizing excipient.

[0312] In some embodiments, the at least one thermostabilizing excipient is added into the composition in an amount sufficient so that the integrity of the RNA molecules in the composition 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 composition without the at least one thermostabilizing excipient.

[0313] As describe herein, the integrity of the RNA molecules can be determined using any method known in the art, such as fragmentation analysis using capillary electrophoresis (CE). For instance, capillary gel electrophoresis (CGE) 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 is measured by capillary electrophoresis.

[0314] Any RNA molecule, LNP, or combination thereof described elsewhere in this application can be used in any of the above methods. 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 proteins, coronavirus proteins, or combinations thereof as described herein in some embodiments. The LNP of any of the above methods, in some embodiments, comprises any of the cationic lipids (e.g., OF-02, cKK-ElO, cKK-E12, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12- DS-3-E14), any of the PEGylated lipids (e.g., dimyristoyl-PEG2000 or N,N- ditetradecylacetamide-polyethylene glycol), any of the cholesterol-based lipids (e.g., cholesterol), and any of the helper lipids (e.g., DOPE or DSPC) described herein in any of the molar ratios described herein. First Set of Representative Embodiments of the Disclosure

[0315] Embodiment 1 : A composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) and at least one thermoreversible gelling agent.

[0316] Embodiment 2: The composition of Embodiment 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.

[0317] Embodiment 3: The composition of Embodiment 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.

[0318] Embodiment 4: The composition of any of Embodiments 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.

[0319] Embodiment 5: The composition of Embodiment 4, wherein the at least one thermoreversible gelling agent is present in an amount of from about 0.25% to about 5% by weight.

[0320] Embodiment 6: The composition of Embodiment 5, wherein the at least one thermoreversible gelling agent is present in an amount of from about 0.5% to about 1.5% by weight.

[0321] Embodiment 7: The composition of any one of Embodiments 1-6, wherein the at least one thermoreversible gelling agent comprises a thermoreversible gelling polymer, a thermoreversible gelling polypeptide, and/or a thermoreversible gelling protein.

[0322] Embodiment 8: The composition of Embodiment 7, wherein the thermoreversible gelling polymer comprises a polypeptide-based gelling polymer or a protein-based gelling polymer.

[0323] Embodiment 9: The composition of Embodiment 7, wherein the thermoreversible gelling polypeptide comprises multi-L-arginyl-poly-L-aspartate (iMAPA)-PEG.

[0324] Embodiment 10: The composition of Embodiment 7, 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.

[0325] Embodiment 11 : The composition of Embodiment 10, wherein the at least one thermoreversible gelling polymer comprises gelatin.

[0326] Embodiment 12: The composition of Embodiment 11, wherein the gelatin is present in an amount of about 1% by weight.

[0327] Embodiment 13: The composition of any one of Embodiments 1-12, further comprising one or more pharmaceutically acceptable excipients selected from a buffering agent, a pharmaceutically acceptable salt, a disaccharide, a surfactant, and a chelating agent.

[0328] Embodiment 14: The composition of Embodiment 13, wherein the composition comprises a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a surfactant, and a chelating agent.

[0329] Embodiment 15: The composition of Embodiment 13 or 14, wherein the composition comprises about 10-60 mM of a buffering agent, about 40-110 mM of a pharmaceutically acceptable salt, about 1-10% by weight of one or more disaccharides, about 5-15 pM of a chelating agent, and about 0.2-0.6% by volume of a surfactant.

[0330] Embodiment 16: The composition of any one of Embodiments 13-15, wherein the composition comprises about 20-50 mM of a buffering agent, about 50-100 mM of a pharmaceutically acceptable salt, about 3-8% by weight of one or more di saccharides, about 10-15 pM of a chelating agent, and about 0.2-0.4% by volume of a surfactant.

[0331] Embodiment 17: The composition of any one of Embodiments 13-16, wherein the composition comprises about 20 mM of a buffering agent, about 100 mM of a pharmaceutically acceptable salt, about 5-7% by weight of one or more disaccharides, about 10 pM of a chelating agent, and about 0.4% by volume of a surfactant.

[0332] Embodiment 18: The composition of any one of Embodiments 13-16, wherein the composition comprises about 50 mM of a buffering agent, about 50 mM of a pharmaceutically acceptable salt, about 7-9% by weight of one or more di saccharides, about 10 pM of a chelating agent, and about 0.4% by volume of a surfactant.

[0333] Embodiment 19: The composition of any one of Embodiments 13-18, wherein the buffering agent comprises Tris.

[0334] Embodiment 20: The composition of any one of Embodiments 13-19, wherein the pharmaceutically acceptable salt comprises NaCl. [0335] Embodiment 21: The composition of any one of Embodiments 13-20, wherein the disaccharide comprises trehalose and/or sucrose.

[0336] Embodiment 22: The composition of any one of Embodiments 13-21, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

[0337] Embodiment 23: The composition of any one of Embodiments 13-22, wherein the surfactant comprises a poloxamer.

[0338] Embodiment 24: The composition of Embodiment 23, wherein the poloxamer comprises Poloxamer 188 (Pl 88).

[0339] Embodiment 25: The composition of any one of Embodiments 1-24, wherein the one or more RNA molecules encode one or more virus proteins.

[0340] Embodiment 26: The composition of Embodiment 25, wherein the one or more virus proteins comprise influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof.

[0341] Embodiment 27: The composition of any one of Embodiments 1-26, wherein the one or more RNA molecules are messenger RNA (mRNA) molecules.

[0342] Embodiment 28: The composition of any one of Embodiments 1-27, wherein the one or more RNA molecules comprise at least one chemically modified nucleotide and/or a phosphorothioate bond.

[0343] Embodiment 29: The composition of Embodiment 28, wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'- methoxy ribonucleotide.

[0344] Embodiment 30: The composition of Embodiment 29, wherein the pseudouridine is a N1 -methylpseudouridine.

[0345] Embodiment 31 : The composition of any of Embodiments 1-30, wherein the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol- based lipid, and a helper lipid.

[0346] Embodiment 32: The composition of Embodiment 31, wherein: i) the cationic lipid is present at a molar ratio between about 30% and about 50%; ii) the PEGylated lipid is present at a molar ratio between about 0.25% and about 15%; iii) the cholesterol -based lipid is present at a molar ratio between about 20% and about 40%; and iv) the helper lipid is present at a molar ratio between about 20% and about 40%.

[0347] Embodiment 33: The composition of Embodiment 32, wherein: i) the cationic lipid is present at a molar ratio of about 40%; ii) the PEGylated lipid is present at a molar ratio of about 1.5%; iii) the cholesterol-based lipid is present at a molar ratio of about 28.5%; and iv) the helper lipid is present at a molar ratio of about 30%.

[0348] Embodiment 34: The composition of Embodiment 32, wherein: i) the cationic lipid is present at a molar ratio of about 40%; ii) the PEGylated lipid is present at a molar ratio of about 5%; iii) the cholesterol-based lipid is present at a molar ratio of about 25%; and iv) the helper lipid is present at a molar ratio of about 30%.

[0349] Embodiment 35: The composition of any one of Embodiments 31-34, wherein 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.

[0350] Embodiment 36: The composition of any one of Embodiments 31-35, wherein the PEGylated lipid comprises dimyristoyl-PEG2000.

[0351] Embodiment 37: The composition of any one of Embodiments 31-36, wherein the cholesterol-based lipid comprises cholesterol.

[0352] Embodiment 38: The composition of any one of Embodiments 31-37, wherein the helper lipid comprises dioleoyl-SN-glycero-3-phosphoethanolamine.

[0353] Embodiment 39: The composition of any one of Embodiments 31-38, wherein the LNP comprises: i) 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 at a molar ratio of about 40%; ii) dimyristoyl-

PEG2000 at a molar ratio of about 1.5%; iii) cholesterol at a molar ratio of about 28.5%; and iv) di oleoyl-SN-glycero-3 -phosphoethanolamine at a molar ratio of about 30%.

[0354] Embodiment 40: The composition of any one of Embodiments 31-38, wherein the LNP comprises: i) 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 at a molar ratio of about 40%; ii) dimyristoyl-

PEG2000 at a molar ratio of about 5%; iii) cholesterol at a molar ratio of about 25%; and iv) dioleoyl-SN-glycero-3-phosphoethanolamine at a molar ratio of about 30%.

[0355] Embodiment 41 : The composition of Embodiment 39 or 40, wherein the LNP comprises OF-02.

[0356] Embodiment 42: The composition of Embodiment 39 or 40, wherein the LNP comprises cKK-ElO.

[0357] Embodiment 43: The composition of Embodiment 39 or 40, wherein the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1.

[0358] Embodiment 44: The composition of Embodiment 39 or 40, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10. [0359] Embodiment 45: The composition of Embodiment 39 or 40, wherein the LNP comprises GL-HEPES-E3-E12-DS-3-E14.

[0360] Embodiment 46: The composition of any one of Embodiments 31-38, wherein the LNP comprises: i) ALC-0315 as the cationic lipid; ii) N,N-ditetradecylacetamide-polyethylene glycol as the PEGylated lipid; iii) distearoylphosphatidylcholine (DSPC) as the helper lipid; and iv) cholesterol.

[0361] Embodiment 47: The composition of any one of Embodiments 1-46, 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.

[0362] Embodiment 48: The composition of any one of Embodiments 1-47, wherein the composition is formulated for sublingual administration, intramuscular administration, intradermal administration, subcutaneous administration, intravenous administration, intranasal administration, administration by inhalation, or intraperitoneal administration.

[0363] Embodiment 49: The composition of any one of Embodiments 1-48, 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.

[0364] Embodiment 50: The composition of Embodiment 49, wherein 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.

[0365] Embodiment 51 : The composition of Embodiment 49 or 50, wherein 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.

[0366] Embodiment 52: The composition of any one of Embodiments 49-51, wherein the encapsulation efficiency of the LNP is higher than the encapsulation efficiency of a control composition without the at least one thermoreversible gelling agent. [0367] Embodiment 53: The composition of any one of Embodiments 49-52, wherein 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.

[0368] Embodiment 54: The composition of any one of Embodiments 1-53, wherein the composition is an immunogenic composition.

[0369] Embodiment 55: A vaccine comprising the composition of Embodiment 54 and a pharmaceutically acceptable carrier.

[0370] Embodiment 56: A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of Embodiment 55.

[0371] Embodiment 57: The method of Embodiment 56, wherein the method 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.

[0372] Embodiment 58: The method of Embodiment 56 or 57, wherein the method raises a protective immune response in the subject.

[0373] Embodiment 59: The method of any one of Embodiments 56-58, wherein the subject is a human.

[0374] Embodiment 60: The method of Embodiment 59, 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.

[0375] Embodiment 61 : The method of any one of Embodiments 56-60, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0376] Embodiment 62: 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 Embodiment 55.

[0377] Embodiment 63: The method of any one of Embodiments 56-62, 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. [0378] Embodiment 64: 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.

[0379] Embodiment 65: The method of Embodiment 64, 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, and 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 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 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/or c) 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 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.

[0380] Embodiment 66: 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.

[0381] Embodiment 67: The method of Embodiment 66, wherein 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 4°C for up to about 1 month, up to 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. [0382] Embodiment 68: A method of formulating a composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP), wherein the composition is stable at 4°C for up to about 1 month, up to 2 months, up to about 3 months, up to 4 months, up to 5 months, or up to about 6 months, 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 liquid composition to a gel form at a temperature of about 1-11°C.

[0383] Embodiment 69: The method of any one of Embodiments 64-68, wherein the at least one thermoreversible gelling agent has an upper critical solution temperature (UCST) between about 12°C and about 50°C.

[0384] Embodiment 70: The method of any one of Embodiments 64-69, wherein the at least one thermoreversible gelling agent comprises a thermoreversible gelling polymer, a thermoreversible gelling polypeptide, and/or a thermoreversible gelling protein.

[0385] Embodiment 71 : The method of Embodiment 70, wherein the thermoreversible gelling polymer comprises a polypeptide-based gelling polymer or a protein-based gelling polymer.

[0386] Embodiment 72: The method of Embodiment 70, wherein the thermoreversible gelling polypeptide comprises multi-L-arginyl-poly-L-aspartate (iMAPA)-PEG.

[0387] Embodiment 73: The method of Embodiment 70, wherein the thermoreversible gelling agent 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 -

[0388] Embodiment 74: The method of any one of Embodiments 64-73, 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.

[0389] Embodiment 75: The method of any one of Embodiments 64-74, wherein the at least one thermoreversible gelling agent comprises gelatin in an amount of about 1% by weight. [0390] Embodiment 76: The method of any one of Embodiments 64-75, wherein 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.

[0391] Embodiment 77: The method of any one of Embodiments 64-76, wherein the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol- based lipid, and a helper lipid.

Second Set of Representative Embodiments of the Disclosure

[0392] Embodiment 1 : A composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) and at least one thermoreversible gelling agent.

[0393] Embodiment 2: The composition of Embodiment 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.

[0394] Embodiment 3 : The composition of Embodiment 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.

[0395] Embodiment 4: The composition of any one of Embodiments 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.

[0396] Embodiment 5: The composition of any one of Embodiments 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.

[0397] Embodiment 6: The composition of Embodiment 5, wherein the thermoreversible gelling polymer comprises a polypeptide-based gelling polymer or a protein-based gelling polymer.

[0398] Embodiment 7: The composition of Embodiment 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), 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)), poly(N-vinylimidazole-co-l -vinyl-2 -

[0399] Embodiment 8: The composition of Embodiment 7, wherein the at least one thermoreversible gelling polymer comprises gelatin, for example wherein the gelatin is present in an amount of about 1% by weight.

[0400] Embodiment 9: The composition of any one of Embodiments 1-8, further comprising one or more pharmaceutically acceptable excipients selected from a buffering agent, a pharmaceutically acceptable salt, a disaccharide, a chelating agent, and a surfactant.

[0401] Embodiment 10: The composition of Embodiment 9, wherein the composition comprises a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, a chelating agent, and a surfactant.

[0402] Embodiment 11 : The composition of Embodiment 9 or 10, wherein the composition comprises about 10-60 mM of a buffering agent, about 40-110 mM of a pharmaceutically acceptable salt, about 1-10% by weight of one or more disaccharides, about 5-15 pM of a chelating agent, and about 0.2-0.6% by volume of a surfactant, or wherein the composition comprises about 20-50 mM of a buffering agent, about 50-100 mM of a pharmaceutically acceptable salt, about 3-8% by weight of one or more disaccharides, about 10-15 pM of a chelating agent, and about 0.2-0.4% by volume of a surfactant, or wherein the composition comprises about 20 mM of a buffering agent, about 100 mM of a pharmaceutically acceptable salt, about 5-7% by weight of one or more disaccharides, about 10 pM of a chelating agent, and about 0.4% by volume of a surfactant, or wherein the composition comprises about 50 mM of a buffering agent, about 50 mM of a pharmaceutically acceptable salt, about 7-9% by weight of one or more disaccharides, about 10 pM of a chelating agent, and about 0.4% by volume of a surfactant.

[0403] Embodiment 12: The composition of any one of Embodiments 9-11, wherein the buffering agent comprises Tris; and/or wherein the pharmaceutically acceptable salt comprises NaCl; and/or wherein the disaccharide comprises trehalose and/or sucrose; and/or wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA); and/or wherein the surfactant comprises a pol oxamer, for example Pol oxamer 188 (Pl 88).

[0404] Embodiment 13: The composition of any one of Embodiments 1-12, wherein the one or more RNA molecules encode one or more virus proteins. [0405] Embodiment 14: The composition of any of Embodiments 1-13, wherein the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol- based lipid, and a helper lipid.

[0406] Embodiment 15: 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.

Third Set of Representative Embodiments of the Disclosure

[0407] Embodiment 1 : A liquid composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) and at least one excipient, wherein the at least one excipient comprises lipoic acid, L-theanine, vanillin, or combinations thereof. [0408] Embodiment 2: The liquid composition of Embodiment 1, 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 excipient.

[0409] Embodiment 3 : The liquid composition of Embodiment 1 or 2, wherein 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 excipient.

[0410] Embodiment 4: The liquid composition of any one of Embodiments 1-3, wherein 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 excipient.

[0411] Embodiment 5: The liquid composition of any one of Embodiments 1-4, wherein 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 excipient. [0412] Embodiment 6: The liquid composition of any one of Embodiments 1-5, wherein 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 excipient.

[0413] Embodiment 7: The liquid composition of any one of Embodiments 2-6, wherein the integrity of the one or more RNA molecules is measured by capillary electrophoresis.

[0414] Embodiment 8: The liquid composition of any one of Embodiments 1-7, wherein 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.

[0415] Embodiment 9: The liquid composition of any one of Embodiments 1-8, wherein 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. [0416] Embodiment 10: The liquid composition of any one of Embodiments 1-9, wherein 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.

[0417] Embodiment 11 : The liquid composition of any one of Embodiments 1-10, wherein 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.

[0418] Embodiment 12: The liquid composition of any one of Embodiments 1-11, wherein the at least one 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.

[0419] Embodiment 13: The liquid composition of Embodiment 12, wherein the at least one excipient is present in a concentration of about 5 mM, about 10 mM, or about 15 mM. [0420] Embodiment 14: The liquid composition of any one of Embodiments 1-13, wherein the at least one excipient and the one or more RNA molecules are present in a weight ratio of from about 5 : 1 to about 50: 1.

[0421] Embodiment 15: The liquid composition of any one of Embodiments 1-14, further comprising one or more pharmaceutically acceptable excipients selected from a buffering agent, a pharmaceutically acceptable salt, a disaccharide, and a surfactant.

[0422] Embodiment 16: The liquid composition of Embodiment 15, wherein the liquid composition comprises a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, and a surfactant.

[0423] Embodiment 17: The liquid composition of Embodiment 15 or 16, wherein the liquid composition comprises about 10-60 mM of a buffering agent, about 40-110 mM of a pharmaceutically acceptable salt, about 1-10% by weight of one or more disaccharides, and about 0.2-0.6% by volume of a surfactant.

[0424] Embodiment 18: The liquid composition of any one of Embodiments 15-17, wherein the liquid composition comprises about 20-50 mM of a buffering agent, about 50-100 mM of a pharmaceutically acceptable salt, about 3-8% by weight of one or more disaccharides, and about 0.2-0.4% by volume of a surfactant.

[0425] Embodiment 19: The liquid composition of any one of Embodiments 15-18, wherein the liquid composition comprises about 20 mM of a buffering agent, about 100 mM of a pharmaceutically acceptable salt, about 5-7% by weight of one or more disaccharides, and about 0.4% by volume of a surfactant.

[0426] Embodiment 20: The liquid composition of any one of Embodiments 15-19, wherein the liquid composition comprises about 50 mM of a buffering agent, about 50 mM of a pharmaceutically acceptable salt, about 7-9% by weight of one or more disaccharides, and about 0.4% by volume of a surfactant.

[0427] Embodiment 21 : The liquid composition of any one of Embodiments 15-20, wherein the buffering agent is or comprises Tris.

[0428] Embodiment 22: The liquid composition of any one of Embodiments 15-21, wherein the pharmaceutically acceptable salt is or comprises NaCl.

[0429] Embodiment 23: The liquid composition of any one of Embodiments 15-22, wherein the disaccharide is or comprises trehalose and/or sucrose.

[0430] Embodiment 24: The liquid composition of any one of Embodiments 15-23, wherein the surfactant is or comprises a poloxamer. [0431] Embodiment 25 : The liquid composition of Embodiment 24, wherein the pol oxamer is or comprises Pol oxamer 188 (Pl 88).

[0432] Embodiment 26: The liquid composition of any one of Embodiments 15-25, wherein the liquid composition further comprises a chelating agent, such as ethylenediaminetetraacetic acid (EDTA), at a concentration of, for example, about 5-15 pM or 10-15 pM, such as 10 pM.

[0433] Embodiment 27: The liquid composition of any one of Embodiments 1-26, wherein the one or more RNA molecules encode one or more virus proteins.

[0434] Embodiment 28: The liquid composition of Embodiment 27, wherein the one or more virus proteins comprise influenza virus proteins, respiratory syncytial virus proteins, coronavirus proteins, or combinations thereof.

[0435] Embodiment 29: The liquid composition of any one of Embodiments 1-28, wherein the one or more RNA molecules are messenger RNA (mRNA) molecules.

[0436] Embodiment 30: The liquid composition of any one of Embodiments 1-29, wherein the one or more RNA molecules comprise at least one chemically modified nucleotide and/or a phosphorothioate bond.

[0437] Embodiment 31 : The liquid composition of Embodiment 30, wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'-methoxy ribonucleotide.

[0438] Embodiment 32: The liquid composition of Embodiment 31, wherein the pseudouridine is a N1 -methylpseudouridine.

[0439] Embodiment 33: The liquid composition of any of Embodiments 1-32, wherein the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0440] Embodiment 34: The liquid composition of Embodiment 33, wherein: i) the cationic lipid is present at a molar ratio between about 30% and about 50%; ii) the PEGylated lipid is present at a molar ratio between about 0.25% and about 15%; iii) the cholesterol -based lipid is present at a molar ratio between about 20% and about 40%; and iv) the helper lipid is present at a molar ratio between about 20% and about 40%.

[0441] Embodiment 35: The liquid composition of Embodiment 34, wherein: i) the cationic lipid is present at a molar ratio of about 40%; ii) the PEGylated lipid is present at a molar ratio of about 1.5%; iii) the cholesterol-based lipid is present at a molar ratio of about 28.5%; and iv) the helper lipid is present at a molar ratio of about 30%. [0442] Embodiment 36: The liquid composition of claim 34, wherein: i) the cationic lipid is present at a molar ratio of about 40%; ii) the PEGylated lipid is present at a molar ratio of about 5%; iii) the cholesterol-based lipid is present at a molar ratio of about 25%; and iv) the helper lipid is present at a molar ratio of about 30%.

[0443] Embodiment 37: The liquid composition of any one of Embodiments 33-36, wherein the cationic lipid comprises OF-02, cKK-ElO, and/or GL-HEPES-E3-E12-DS-4-E10. [0444] Embodiment 38: The liquid composition of any one of Embodiments 33-37, wherein the PEGylated lipid comprises l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)- PEG2000.

[0445] Embodiment 39: The liquid composition of any one of Embodiments 33-38, wherein the cholesterol-based lipid comprises cholesterol.

[0446] Embodiment 40: The liquid composition of any one of Embodiments 33-39, wherein the helper lipid comprises dioleoyl-SN-glycero-3-phosphoethanolamine.

[0447] Embodiment 41 : The liquid composition of any one of Embodiments 33-40, wherein the LNP comprises: i) OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%; ii) l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000 at a molar ratio of about 1.5%; iii) cholesterol at a molar ratio of about 28.5%; and iv) dioleoyl-SN- glycero-3 -phosphoethanolamine at a molar ratio of about 30%.

[0448] Embodiment 42: The liquid composition of any one of Embodiments 33-40, wherein the LNP comprises: i) OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%; ii) l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000 at a molar ratio of about 5%; iii) cholesterol at a molar ratio of about 25%; and iv) dioleoyl-SN-glycero- 3 -phosphoethanolamine at a molar ratio of about 30%.

[0449] Embodiment 43 : The liquid composition of Embodiment 41 or 42, wherein the LNP comprises OF-02.

[0450] Embodiment 44: The liquid composition of Embodiment 41 or 42, wherein the LNP comprises cKK-ElO.

[0451] Embodiment 45: The liquid composition of Embodiment 41 or 42, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10.

[0452] Embodiment 46: The liquid composition of any one of Embodiments 1-45, wherein the at least one excipient comprises lipoic acid.

[0453] Embodiment 47: The liquid composition of Embodiment 46, 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. [0454] Embodiment 48: The liquid composition of any one of Embodiments 1-45, wherein the at least one excipient comprises L-theanine.

[0455] Embodiment 49: The liquid composition of Embodiment 48, 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.

[0456] Embodiment 50: The liquid composition of any one of Embodiments 1-48, wherein the at least one excipient comprises vanillin.

[0457] Embodiment 51 : The liquid composition of Embodiment 50, 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.

[0458] Embodiment 52: The liquid composition of any one of Embodiments 1-51, wherein the liquid composition has a N/P ratio of from about 1 to about 10, or from about 3 to about 6. [0459] Embodiment 53: The liquid composition of any one of Embodiments 1-52, wherein the liquid composition has a N/P ratio of about 4.

[0460] Embodiment 54: The liquid composition of any one of Embodiments 1-53, 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.

[0461] Embodiment 55: The liquid composition of any one of Embodiments 1-54, wherein the liquid composition is formulated for sublingual administration, intramuscular administration, intradermal administration, subcutaneous administration, intravenous administration, intranasal administration, administration by inhalation, or intraperitoneal administration.

[0462] Embodiment 56: The liquid composition of any one of Embodiments 1-55, wherein the liquid composition is an immunogenic composition.

[0463] Embodiment 57: A vaccine comprising the liquid composition of Embodiment 56 and a pharmaceutically acceptable carrier.

[0464] Embodiment 58: A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of Embodiment 57.

[0465] Embodiment 59: The method of Embodiment 58, wherein 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.

[0466] Embodiment 60: The method of Embodiment 58 or 59, wherein the method raises a protective immune response in the subject. [0467] Embodiment 61 : The method of any one of Embodiments 58-60, wherein the subject is a human.

[0468] Embodiment 62: The method of Embodiment 61, 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.

[0469] Embodiment 63: The method of any one of Embodiments 58-62, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0470] Embodiment 64: 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 Embodiment 57.

[0471] Embodiment 65: The method of any one of Embodiments 58-64, 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.

[0472] Embodiment 66: 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 excipient selected from lipoic acid, L-theanine, vanillin, or combinations thereof.

[0473] Embodiment 67: The method of Embodiment 66, wherein the thermal degradation of the one or more RNA molecules following 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 is reduced as compared to a control liquid composition without the at least one excipient.

[0474] Embodiment 68: A method of stabilizing a liquid composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP), the method comprising adding at least one excipient selected from lipoic acid, L-theanine, vanillin, or combinations thereof in an amount sufficient to prevent the integrity of the one or more RNA molecules from decreasing by: a) 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 excipient; b) 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 excipient; c) 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 excipient; or d) 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 excipient.

[0475] Embodiment 69: The method of any one of Embodiments 66-68, wherein the at least one excipient is present in the liquid composition at 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.

[0476] Embodiment 70: The method of any one of Embodiments 66-69, wherein the at least one excipient is present in the liquid composition at a concentration of about 5 mM, about 10 mM, or about 15 mM.

[0477] Embodiment 71 : The method of any one of Embodiments 66-70, wherein the at least one excipient and the one or more RNA molecules are present in a weight ratio of from about 5 : 1 to about 50: 1.

[0478] Embodiment 72: The method of any one of Embodiments 66-71, wherein the one or more RNA molecules are messenger RNA (mRNA) molecules.

[0479] Embodiment 73 : The method of any one of Embodiments 66-72, wherein the one or more RNA molecules encapsulated in the LNP formulated in the presence of the at least one excipient are thermally stable 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.

[0480] Embodiment 74: The method of Embodiment 73, wherein thermal stability of the one or more RNA molecules is measured by a decrease in the integrity of the one or more RNA molecules. [0481] Embodiment 75. The method of Embodiment 74, wherein 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 excipient.

[0482] Embodiment 76: The method of Embodiment 74 or 75, wherein 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 excipient. [0483] Embodiment 77: The method of any one of Embodiments 74-76, wherein 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 excipient.

[0484] Embodiment 78: The method of any one of Embodiments 74-77, wherein 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 excipient.

[0485] Embodiment 79: The method of any one of Embodiments 74-78, wherein the integrity of the one or more RNA molecules is measured by capillary electrophoresis.

[0486] Embodiment 80: The method of any one of Embodiments 66-79, wherein the at least one excipient comprises lipoic acid.

[0487] Embodiment 81 : The method of Embodiment 80, 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. [0488] Embodiment 82: The method of any one of Embodiments 66-79, wherein the at least one excipient comprises L-theanine.

[0489] Embodiment 83 : The method of Embodiment 82, 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.

[0490] Embodiment 84: The method of any one of Embodiments 66-79, wherein the at least one excipient comprises vanillin. [0491] Embodiment 85. The method of Embodiment 84, 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.

Fourth Set of Representative Embodiments of the Disclosure

[0492] Embodiment 1 : A thermostable liquid composition comprising one or more ribonucleic acid (RNA) molecules encapsulated in a lipid nanoparticle (LNP) and at least one excipient, wherein the at least one excipient is selected from lipoic acid, L-theanine, vanillin, or combinations thereof.

[0493] Embodiment 2: The liquid composition of Embodiment 1, wherein the at least one 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, optionally about 5 mM, about 10 mM, or about 15 mM.

[0494] Embodiment 3: The liquid composition of Embodiment 1 or 2, wherein the at least one excipient and the one or more RNA molecules are present in a weight ratio of from about 5: 1 to about 50: 1.

[0495] Embodiment 4: The liquid composition of any one of the preceding Embodiments, further comprising one or more pharmaceutically acceptable excipients selected from a buffering agent, a pharmaceutically acceptable salt, a disaccharide, and a surfactant.

[0496] Embodiment 5: The liquid composition of Embodiment 4, wherein the liquid composition comprises a buffering agent, a pharmaceutically acceptable salt, one or more disaccharides, and a surfactant.

[0497] Embodiment 6: The liquid composition of Embodiment 4 or 5, wherein the liquid composition comprises about 10-60 mM of a buffering agent, about 40-110 mM of a pharmaceutically acceptable salt, about 1-10% by weight of one or more disaccharides, and about 0.2-0.6% by volume of a surfactant.

[0498] Embodiment 7: The liquid composition of any one of Embodiments 4-6, wherein (a) the buffering agent comprises, or is, Tris; (b) the pharmaceutically acceptable salt comprises, or is, NaCl; (c) the disaccharide comprises, or is, trehalose and/or sucrose; and/or (d) the surfactant comprises, or is, a pol oxamer, e.g., Pol oxamer 188 (Pl 88).

[0499] Embodiment 8: The liquid composition of any one of the preceding Embodiments, wherein the one or more RNA molecules (a) 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 (b) 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 Nl- methylpseudouridine.

[0500] Embodiment 9: The liquid composition of any of any one of the preceding Embodiments, wherein the LNP comprises a cationic lipid, a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0501] Embodiment 10: The liquid composition of claim 9, 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%.

[0502] Embodiment 11 : The liquid composition of Embodiment 10, 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.

[0503] Embodiment 12: The liquid composition of any one of claims 9-11, wherein (a) the cationic lipid comprises, oris, OF-02, cKK-ElO, and/or GL-HEPES-E3-E12-DS-4-E10; and/or (b) 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.

[0504] Embodiment 13: The liquid composition of any one of the preceding claims, wherein the liquid composition has an N/P ratio of from about 1 to about 10, or from about 3 to about 6, optionally of about 4.

[0505] Embodiment 14: The liquid composition of any one of the preceding Embodiments, 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.

[0506] Embodiment 15: 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 excipient selected from lipoic acid, L-theanine, vanillin, or combinations thereof. EXAMPLES

[0507] The following examples are to be considered illustrative and not limiting on the scope of the disclosure described above.

Example 1. Evaluation of Gelatin as a Thermoreversible Gelling Agent in mRNA-LNP Formulations.

[0508] Gelatin was used to study its potential as a thermoreversible gelling agent in mRNA-LNP formulations. The LNP formulation used in this example contained a cationic lipid (cKK-ElO), a PEGylated lipid (l,2-dimyristoyl-rac-glycero-3 -methoxy (DMG)- PEG2000), a cholesterol-based lipid (cholesterol), and a helper lipid (DOPE). In this example, the LNPs were loaded with a monovalent influenza mRNA encoding an influenza antigen from a Tasmania strain. A liquid flowable buffered drug product containing the resultant mRNA- LNP formulation with or without 1% gelatin was manufactured and kept at 4°C. The buffered drug product was formulated to contain either (1) 20 mM Tris, 100 mM NaCl, 0.4-1.3% trehalose, 5% sucrose, 10 pM EDTA, 0.4% P188, pH 7.7, or (2) 50 mM Tris, 50 mM NaCl, 2- 2.6% trehalose, 5% sucrose, 10 pM EDTA, 0.4% P188, pH 7.7.

[0509] FIGs. 2A-2B show the buffered drug product formulated with 1% gelatin filled into a sealed vial (FIG. 2A) and a pre-filled syringe (FIG. 2B) at 4°C and at room temperature (RT). As shown in FIGs. 2A-2B, the buffered drug product formulated with 1% gelatin had a liquid phase at room temperature (RT, right) which is reversibly transitioned to a gel form at 4°C (left). FIG. 2B further shows that the buffered drug product formulated with 1% gelatin was reversibly transitioned to the liquid phase at RT from the gel form at 4°C within 15 minutes.

[0510] The buffered drug product formulated with 1% gelatin was stored at 4°C and evaluated for improvement in stability as compared to a control containing only the buffered drug product without gelatin at specific timepoints for up to 6 months by measuring the change in mean particle size and encapsulation efficiency of the LNP and the change in mRNA integrity. The particle size was measured using Dynamic Light Scattering, the mRNA encapsulation efficiency was measured using a fluorescence plate-based assay, and the mRNA integrity was measured by extracting the mRNA from the LNP and analyzing on a fragment analyzer using capillary electrophoresis.

[0511] As shown in FIG. 3, similar to the control, the mean particle size (FIG. 3, middle) and the encapsulation efficiency (FIG. 3, bottom) of the mRNA-LNPs tested remained relatively constant after storage at 4°C for at least 4 months, with the mRNA-LNP formulated with gelatin having slightly improved encapsulation efficiency as compared to the control without gelatin. The buffered drug product formulated with 1% gelatin also showed less decrease in mRNA integrity after storage at 4°C for 4 months as compared to the control (FIG. 3, top), indicating an improvement in stability with the gelatin formulation. FIGs. 4A-4D are gel electrophoresis graphs showing that mRNA-LNPs formulated with 1% gelatin generated less degraded mRNA following 4 months of storage at 4°C, as compared to the control without gelatin, indicating that the gelatin formulation improves stability.

[0512] FIGs. 5A-5C show that the improvement in stability with 1% gelatin was extended to up to 9 months. As shown in FIG. 5A, the mRNA-LNP formulated with 1% gelatin had less decrease in mRNA integrity after storage at 4°C for up to 9 months as compared to the control. The mRNA-LNP formulated with 1% gelatin also had slightly improved encapsulation efficiency as compared to the control after storage at 4°C for up to 9 months (FIG. 5B). The control exhibited physical instability after 6 months of storage at 4°C as evidenced by increased particle size and visible aggregates, neither of which was observed in the mRNA-LNP formulated with 1% gelatin.

Example 2. In vivo Study on the Effect of Adding Gelatin to the mRNA-LNP Formulations on Protein Production.

[0513] The mRNA LNPs formulated with gelatin were analyzed for protein production. The LNP formulation used in this example contained a cationic lipid (CKK-E10), a PEGylated lipid (l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000), a cholesterol-based lipid (cholesterol), and a helper lipid (DOPE). In this example, the LNPs were loaded with human erythropoietin (hEPO) mRNA. A buffered drug product containing the resultant mRNA-LNP formulation with or without 1% gelatin was manufactured and kept at 4°C. Mice were then dosed with a single intramuscular injection of the resultant mRNA-LNP formulation. Serum was collected at 6 hours and 24 hours post administration to detect the amount of EPO protein in the blood using ELISA.

[0514] As shown in FIG. 6, the addition of gelatin to the mRNA-LNP formulations did not affect protein production in mice as compared to the control, which contains the same buffered drug product but without gelatin. Thus, using gelatin in mRNA-LNPs does not interfere with in vivo protein production. Example 3. Excipient Screening for Improved mRNA Stability in a Liquid LNP Formulation.

[0515] Over 80 different excipients were evaluated for their ability to prevent mRNA degradation overtime in a liquid LNP formulation.

[0516] Various excipients were incorporated into an mRNA-containing LNP formulation and screened for their ability to affect mRNA stability. The LNP formulation contained a cationic lipid (cKK-E12; also known as ML2), aPEGylated lipid (1,2-dimyristoyl-rac-glycero- 3 -methoxy (DMG)-PEG2000), a cholesterol -based lipid (cholesterol), and a helper lipid (DOPE). In this example, the LNP contained a mole composition of 5% PEGylated lipid, 40% cationic lipid, 25% cholesterol -based lipid and 30% helper lipid and the mRNA encoded human erythropoietin (hEPO). To make the formulation, the excipients were added to the acidification buffer (1 mM citrate/150 mM NaCl, pH 3.5) or ethanol side with the dissolved lipids before a T-tube mixing processes. The concentration of the excipients ranged from 0.5 mM to 10 mM depending on their solubility in water or ethanol. The formulation was then filtered, diafiltrated with 10% trehalose, concentrated and stored at -80°C.

[0517] Once the formulations were made with excipients, the resultant liquid formulations were placed at 37°C for 7 days to screen them under accelerated mRNA degradation conditions. The particle size and mRNA encapsulation efficiency of the LNP, as well as the decrease in mRNA integrity, were measured at specific timepoints for up to 7 days. The particle size was measured using Dynamic Light Scattering, the mRNA encapsulation efficiency was measured using a fluorescence plate-based assay, and the mRNA integrity was measured by extracting the mRNA from the LNP and analyzing on a fragment analyzer using capillary electrophoresis. The excipients tested in this screening are listed in Table 1.

Table 1. List of excipients screened for improved mRNA stability in a liquid LNP formulation.

[0518] As shown in FIGs. 7A-7B, several of the excipients tested prevented mRNA degradation and showed less of a decrease in the mRNA integrity as compared to the formulation control containing the same mRNA-LNP formulation but without the excipient. The mRNA integrity data of some of the excipients screened are shown in FIG. 7A with the y- axis representing the change in percentage of mRNA integrity (% mRNA integrity) and the x- axis representing the time in days. The data from FIG. 7A are summarized in Table 2. FIG. 7B shows that the top excipients, having higher mRNA integrity values after 7 days at 37°C compared to the naked mRNA control and the formulation control, include quercetin, glutathione, salicylic acid, vanillin, L-theanine, and lipoic acid. The data from FIG. 7B are summarized in Table 3. The formulation control contains the same mRNA-LNP formulation but without the excipient.

Table 2. Change in percentage of mRNA integrity (% mRNA integrity) of the screened excipients shown in FIG. 7 after 7 days at 37°C.

Table 3. Change in percentage of mRNA integrity (% mRNA integrity) of the top excipients after 7 days at 37°C.

[0519] Three of the top excipients were selected for further study, namely L-theanine, lipoic acid, and vanillin. Each of these three excipients was incorporated into the mRNA-LNP formulation as described above. As shown in FIG. 8A, the addition of L-theanine (10 mM), lipoic acid (5 mM), or vanillin (10 mM) in the mRNA-LNP formulation decreased the amount of mRNA degradation under the accelerated degradation condition at 37°C as compared to the formulation control. No significant change in the particle size of the LNP was observed (FIG. 8B) and the mRNA encapsulation efficiency of the LNP remained unchanged (FIG. 8C) for all the formulations after storage at 37°C for 7 days. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

Example 4. Long-Term Liquid Stability Studies with mRNA-LNP Formulations Containing L-Theanine, Lipoic Acid, or Vanillin as Excipient at 4°C and 25°C. [0520] The three top excipients selected in Example 3, namely L-theanine, lipoic acid, and vanillin, were used to study their potential in conferring long-term liquid stability in mRNA- LNP formulations at 4°C and 25°C. The LNP formulation contained a cationic lipid (OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10), a PEGylated lipid (1,2-dimyristoyl-rac-glycero- 3 -methoxy (DMG)-PEG2000), a cholesterol -based lipid (cholesterol), and a helper lipid (DOPE). In this example, the LNP contained a mole composition of 1.5% PEGylated lipid, 40% cationic lipid, 28.5% cholesterol -based lipid and 30% helper lipid and were loaded with four modified mRNAs encoding influenza hemagglutinins from Tasmania, Washington, Wisconsin and Phuket strains, respectively. The mRNAs were modified with 1 -methyl peusdouridine. The excipients were added to the mRNA-LNP formulations during the T-tube mixing processes as described above in the amount of 10 mM L-theanine (mRNA side), 5 mM lipoic acid (lipid side), or 10 mM vanillin (lipid side). The N/P ratio of the formulations was 4 and the final buffer contains 10% trehalose. The bulk drug products were finally concentrated to 1 mg/mL of total mRNA and then diluted to 0.2 mg/mL concentration of total mRNA using two different buffers that contain Tris, NaCl, P 188 and 10% trehalose. These final quadrivalent influenza formulations containing either (1) 40 mM Tris, 75 mM NaCl, 2% trehalose, 0.5% P188, pH 7.5 (Buffer 1) or (2) 50 mM Tris, 50 mM NaCl, 2% trehalose, 0.5% P188, pH 7.5 (Buffer 2) were then stored at 4°C or 25°C as a liquid, and the liquid formulation stability was then analyzed at specific timepoints for up to 12 months by measuring the particle size and mRNA encapsulation efficiency of the LNP, as well as the decrease in mRNA integrity. The particle size was measured using Dynamic Light Scattering, the mRNA encapsulation efficiency was measured using a fluorescence plate-based assay, and the mRNA integrity was measured by extracting the mRNA from the LNP and analyzing on a fragment analyzer using capillary electrophoresis.

[0521] As shown in FIGs. 9A-9D, the addition of L-theanine (10 mM), lipoic acid (5 mM), and vanillin (10 mM) in the mRNA-LNP formulations containing cKK-ElO or OF-02 as the cationic lipid decreased the amount of mRNA degradation when stored at 4°C and 25 °C as compared to the formulation control. More specifically, in the mRNA-LNP formulations containing cKK-ElO as the cationic lipid, all three excipients decreased the amount of quadrivalent flu mRNA degradation in a liquid formulation when stored at 25°C for up to 2 months (FIG. 9B) and 4°C for up to 12 months (FIG. 9A) as compared to the formulation control. In the mRNA-LNP formulations containing OF-02 as the cationic lipid, all three excipients decreased the amount of mRNA degradation in a liquid formulation when stored at 25°C for up to 2 months (FIG. 9D) and 4°C for up to 9 months (FIG. 9C) as compared to the formulation control. In the mRNA-LNP formulations containing GL-HEPES-E3-E12-DS-4- E10 as the cationic lipid, no mRNA stability benefit was observed from the addition of any of the three excipients at 4°C (FIG. 9E) or 25 °C (FIG. 9F) as compared to the formulation control. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0522] As shown in FIGs. 10A-10B and FIGs. 10E-10F, an increase in particle size was observed in the mRNA-LNP formulations containing cKK-ElO or GL-HEPES-E3-E12-DS-4- E10 as the cationic lipid when stored at 4°C and 25°C. More specifically, in the mRNA-LNP formulations containing cKK-ElO as the cationic lipid, all the formulations increased in particle size when stored as a liquid at 4°C (FIG. 10A) and 25 °C (FIG. 10B). In the mRNA-LNP formulations containing OF-02 as the cationic lipid, no significant change in particle size was observed for all the formulations after storage as a liquid at 25°C (FIG. 10D) for up to 3 months and at 4°C for up to 9 months (FIG. 10C). In the mRNA-LNP formulations containing GL- HEPES-E3-E12-DS-4-E10 as the cationic lipid, the addition of L-theanine, lipoic acid, or vanillin to the formulation generally resulted in smaller particle size as compared to the formulation control and an increase in particle size was observed when stored as a liquid at 4°C (FIG. 10E) and 25°C (FIG. 10F). In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient.

[0523] As shown in FIGs. 11A-11B and FIGs. 11E-11F, the addition of L-theanine or vanillin to the mRNA-LNP formulations containing cKK-ElO or GL-HEPES-E3-E12-DS-4- E10 as the cationic lipid improved the encapsulation efficiency for liquid formulations stored at 4°C as compared to the control formulation. More specifically, the encapsulation efficiency was improved in the mRNA-LNP formulations containing cKK-ElO as the cationic lipid with the addition of L-theanine or vanillin when stored as a liquid at 4°C for up to 12 months (FIG. 11 A) and 25 °C for up to 2 months (FIG. 11B) as compared to the control formulation. For the mRNA-LNP formulations containing OF-02 as the cationic lipid, the encapsulation efficiency remained stable for all the formulations when stored as a liquid at 4°C for up to 9 months (FIG. 11C) and 25°C for up to 3 months (FIG. 11D). In the mRNA-LNP formulations containing GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid, the encapsulation efficiency was improved with the addition of L-theanine or vanillin when stored as a liquid at 4°C for up to 6 months (FIG. HE) and comparable or better with the addition of lipoic acid, L-theanine, or vanillin when stored as a liquid at 25°C for up to 3 months (FIG. HF) as compared to the control formulation. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient. Example 5. Liquid Stability Studies with mRNA-LNP Formulations Containing L- Theanine, Lipoic Acid, or Vanillin as Excipient at 25°C and 30°C.

[0524] The three top excipients selected in Example 3, namely L-theanine, lipoic acid, and vanillin, were used to study their potential in conferring liquid stability in mRNA-LNP formulations at 25°C and 30°C. The LNP formulation contained a cationic lipid (OF-02, cKK- E10, or GL-HEPES-E3-E12-DS-4-E10), a PEGylated lipid (l,2-dimyristoyl-rac-glycero-3- methoxy (DMG)-PEG2000), a cholesterol-based lipid (cholesterol), and a helper lipid (DOPE). In this example, the LNPs were formulated using a mole composition of 1.5% PEGylated lipid, 40% cationic lipid, 28.5% cholesterol -based lipid and 30% helper lipid and were loaded with a modified monovalent influenza mRNA encoding an influenza hemagglutinin from a Tasmania strain. The mRNA modification is the 1 -methyl peusdouri dine. The excipients were added to the mRNA-LNP formulations during the T-tube mixing processes as described above in the amount of 10 mM L-theanine, 5 mM lipoic acid, or 10 mM vanillin. The resultant formulations were then stored at 25 °C and 30°C as a liquid, and the liquid formulation stability was then analyzed at specific timepoints for up to 7 weeks by measuring the particle size and mRNA encapsulation efficiency of the LNP, as well as the decrease in mRNA integrity. The particle size was measured using Dynamic Light Scattering, the mRNA encapsulation efficiency was measured using a fluorescence plate-based assay, and the mRNA integrity was measured by extracting the mRNA from the LNP and analyzing on a fragment analyzer using capillary electrophoresis.

[0525] As shown in FIGs. 12A-12F, the addition of L-theanine, lipoic acid, and vanillin to the mRNA-LNP formulations containing the monovalent influenza mRNA and OF-02, cKK- E10, or GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid decreased the amount of mRNA degradation when stored as a liquid at 25°C as compared to the formulation control. More specifically, in the mRNA-LNP formulations containing cKK-ElO as the cationic lipid, all three excipients decreased the amount of mRNA degradation when stored as a liquid at 25 °C for up to 7 weeks and 30°C for up to 6 weeks as compared to the formulation control (FIG. 12A). In the mRNA-LNP formulations containing OF-02 or GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid, all three excipients decreased the amount of mRNA degradation when stored as a liquid at 25°C as compared to the formulation control (FIG. 12B). In the mRNA- LNP formulations containing cKK-ElO as the cationic lipid, all the formulations increased in particle size overtime when stored as a liquid at 25°C and 30°C (FIG. 12C). In the mRNA- LNP formulations containing OF-02 or GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid, the addition of L-theanine, lipoic acid, or vanillin to the formulation generally resulted in smaller particle size as compared to the formulation control and all the formulations maintained the particle size when stored as a liquid at 25°C overtime (FIG. 12D). In the mRNA-LNP formulations containing cKK-ElO as the cationic lipid, the encapsulation efficiency of the LNP remained constant for all the formulations stored as a liquid at 25°C and 30°C for up to 7 weeks (FIG. 12E). In the mRNA-LNP formulations containing OF-02 or GL-HEPES-E3-E12-DS- 4-E10 as the cationic lipid, the encapsulation efficiency of the LNP remained constant for all the formulations stored as a liquid at 25°C for up to 7 weeks (FIG. 12F). In each instance, the formulation control contains the same mRNA-LNP formulation without the excipient.

Example 6. In vivo Study on the Effect of Adding L-Theanine, Lipoic Acid, and Vanillin as an Excipient to the mRNA-LNP Formulations on mRNA Delivery and Protein Production in Mice.

[0526] LNP formulations containing mRNA and L-theanine, lipoic acid, or vanillin were analyzed for in vivo mRNA delivery and protein production. The LNP formulation contained a cationic lipid (OF-02, cKK-ElO, or GL-HEPES-E3-E12-DS-4-E10), a PEGylated lipid (1,2- dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000), a cholesterol-based lipid (cholesterol), and a helper lipid (DOPE). In this example, the LNPs were formulated using OF-02, cKK- E10, or GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid at a mole composition of 1.5% PEGylated lipid, 40% cationic lipid, 28.5% cholesterol -based lipid, and 30% helper lipid, with an N/P ratio of 4, and were loaded with human EPO mRNA. The excipients were added to the mRNA-LNP formulations during the T-tube mixing processes as described above in the amount of 10 mM L-theanine, 5 mM lipoic acid, or 10 mM vanillin. Mice (n=4 per group) were then dosed with a single intramuscular injection of 0.1 pg/30pL of the resultant mRNA- LNP formulation. Serum was collected at 6 hours and 24 hours post administration to detect the amount of EPO protein in the blood using ELISA.

[0527] As shown in FIG. 13, the addition of L-theanine, lipoic acid, and vanillin as an excipient to the mRNA-LNP formulations containing human EPO mRNA and OF-02 (A), cKK-ElO (B), or GL-HEPES-E3-E12-DS-4-E10 (C) as the cationic lipid did not affect mRNA delivery and protein production in mice as compared to the formulation control. In each instance, the formulation control contains the same mRNA-LNP formulation but without the excipient. Thus, using the excipients L-theanine, lipoic acid, and vanillin in mRNA-LNPs does not interfere with in vivo mRNA delivery and protein production. Example 7. In vivo Study on the Effect of Adding L-Theanine, Lipoic Acid, and Vanillin as an Excipient to the mRNA-LNP Formulations on HAI Titers Produced in Mice with mRNA Encoding an Influenza Virus Antigen.

[0528] LNP formulations containing mRNA and L-theanine, lipoic acid, or vanillin were analyzed for in vivo antibody titer measured by the hemagglutination inhibition (HAI) assay (i.e., HAI titers). The LNP formulation contained a cationic lipid (OF-02, cKK-ElO, or GL- HEPES-E3-E12-DS-4-E10), a PEGylated lipid (l,2-dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000), a cholesterol-based lipid (cholesterol), and a helper lipid (DOPE). In this example, LNPs were formulated at a mole composition of 1.5% PEGylated lipid, 40% cationic lipid, 28.5% cholesterol -based lipid, and 30% helper lipid, with an N/P ratio of 4, and were loaded with a mRNA that encodes an influenza virus hemagglutinin. The excipients were added to the mRNA-LNP formulations during the T-tube mixing processes as described above in the amount of 10 mM L-theanine, 5 mM lipoic acid, or 10 mM vanillin. Mice (n=8 per group) were immunized at day 0 and day 21 with a dose of 0.4 pg mRNA. Serum was collected at day 35 to measure functional antibody titers by the hemagglutination inhibition (HAI) assay. [0529] As shown in FIG. 14, the addition of L-theanine, lipoic acid, and vanillin as an excipient to the mRNA-LNP formulations containing OF-02, cKK-ElO, or GL-HEPES-E3- E12-DS-4-E10 as the cationic lipid did not lower HAI titers produced in mice 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.

Example 8. Thermostability Improvement Using Combination of Gelatin and Lipoic Acid.

[0530] As shown in the above examples, adding gelatin or certain excipients, such as lipoic acid, L-theanine, and vanillin, to mRNA-LNP formulations resulted in substantially improved stability, including RNA stability, when stored at an above-zero temperature, such as in refrigerated conditions (e.g., 4°C). This example describes a thermostability improvement using a combination of gelatin and one of the aforementioned excipients, lipoic acid.

[0531] In this example, the mRNA-LNPs were first prepared with a quadrivalent influenza mRNA in 10% trehalose (1 mg/mL). The LNP formulation used in this example contained a cationic lipid (GL-HEPES-E3-E12-DS-4-E10, 40% by mol), a PEGylated lipid (1,2- dimyristoyl-rac-glycero-3-methoxy (DMG)-PEG2000, 1.5% by mol), a cholesterol-based lipid (cholesterol, 28.5% by mol), and a helper lipid (DOPE, 30% by mol). The mRNA-LNPs were diluted to a concentration of 0.2 mg/mL in 50 mM Tris, pH 7.5, 50 mM NaCl, 2% trehalose, 0.5% Pl 88, and 10 pM EDTA to form the mRNA-LNP formulation. The resultant mRNA- LNP formulation was stored at 2-8°C in the presence of 1% gelatin, 5 mM lipoic acid, or 1% gelatin and 5 mM lipoic acid, or with no addition of gelatin and lipoic acid as the formulation control. The formulation stability was then analyzed at specific timepoints for up to 12 months by measuring the particle size and mRNA encapsulation efficiency of the LNPs, as well as the decrease in mRNA integrity. The particle size was measured using Dynamic Light Scattering, the mRNA encapsulation efficiency was measured using a fluorescence plate-based assay, and the mRNA integrity was measured by extracting the mRNA from the LNPs and analyzing on a fragment analyzer using capillary electrophoresis.

[0532] As shown in FIGs. 15A-15C, while the addition of gelatin or lipoic acid to the mRNA-LNP formulation decreased the amount of mRNA degradation as compared to the formulation control (FIG. 15A), the combination of gelatin and lipoic acid (“Gelatin + Lipoic Acid”) conferred a better overall thermostability profile for the mRNA-LNP formulation stored at 2-8°C for 12 months. However, as shown in FIG. 15D, visible aggregates were observed in the sealed vial containing the mRNA-LNP formulation with the addition of 5 mM lipoic acid after storage at 2-8°C for 9 months, similar to the sealed vial containing the formulation control (FIG. 15D, left), while no visible aggregates were observed 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 (FIG. 15D, right), In view of this observation, a slightly modified mRNA-LNP formulation was prepared and tested with a reduced concentration of lipoic acid.

[0533] The modified mRNA-LNP formulation was prepared with the same quadrivalent influenza mRNA in 100 mM Tris, pH 7.5, 50 mM NaCl, and 5% trehalose (1 mg/mL). The LNP formulation was the same as described above, which contained a cationic lipid (GL- HEPES-E3-E12-DS-4-E10, 40% by mol), a PEGylated lipid (l,2-dimyristoyl-rac-glycero-3- methoxy (DMG)-PEG2000, 1.5% by mol), a cholesterol-based lipid (cholesterol, 28.5% by mol), and a helper lipid (DOPE, 30% by mol). The resultant mRNA-LNPs were 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 to form the modified mRNA-LNP formulation. This modified mRNA-LNP formulation was stored at 2-8°C in the presence of 1% gelatin, 1 mM lipoic acid, or 1% gelatin and ImM lipoic acid, or with no addition of gelatin and lipoic acid as the formulation control. The formulation stability was then analyzed at specific timepoints for up to 6 months by measuring the particle size and mRNA encapsulation efficiency of the LNPs, as well as the decrease in mRNA integrity, as described above.

[0534] Similar to the results observed previously, the combination of gelatin and lipoic acid (“Gelatin + Lipoic Acid”), even with a reduced concentration of lipoic acid, again conferred a better overall thermostability profile for this modified mRNA-LNP formulation stored at 2-8°C for 5 months as shown in FIGs. 16A-16C.

Example 9. mRNA-LNP Formulations Stability Studies.

[0535] One of the main challenges inherent to the development of mRNA-LNP drug products is the achievement of maintaining stability at different temperatures. Various quality attributes have to be monitored during the mRNA-LNP formulation development, linked on one side to the chemical stability of mRNA and excipients and on the other to the physical stability of the LNPs. As described above, addition of gelatin and/or certain excipients, such as lipoic acid, L-theanine, and vanillin, to mRNA-LNP formulations resulted in substantially improved stability, including RNA stability, when stored at an above-zero temperature, such as in refrigerated conditions (e.g., 2-8°C). This example describes a yet another approach by developing thermostable mRNA-LNP liquid formulations.

[0536] The starting materials were mRNA-LNPs prepared with a quadrivalent influenza mRNA in 10% trehalose (1 mg/mL) and three different cationic lipids, namely OF-02 (CL-1), cKK-ElO (CL-2), and GL-HEPES-E3-E12-DS-4-E10 (CL-3). The stabilized mRNA-LNP liquid formulations were obtained by diluting the mRNA-LNPs in concentrated excipients solutions containing a buffering agent (Tris-hydroxymethyl-aminomethane or Tris), an osmotic agent (sodium chloride orNaCl), a disaccharide (sucrose), a surfactant (P188), and a chelating agent (ethylenediaminetetraacetic acid disodium salt or EDTA) and contained a residue amount of trehalose from the mRNA solution used to prepare the mRNA-LNPs. The final mRNA concentration was between 0.04 mg/mL and 0.26 mg/mL and the final pH is 7.7 ± 0.3.

[0537] Two formulations were identified: Formulation A and Formulation B. Formulation A is suitable for mRNA-LNP concentration in the range of 0.04-0.13 mg/mL and Formulation B is suitable for mRNA-LNP concentration in the range of 0.2-0.26 mg/mL. Formulation A contains 20 mM Tris, 100 mM NaCl, 5% sucrose, 0.4% P188, and 10 pM EDTA. Formulation B contains 50 mM Tris, 50 mM NaCl, 5% sucrose, 0.4% Pl 88, and 10 pM EDTA. The final pH for both formulations is 7.7 ± 0.3.

[0538] To identify these two formulations, stability studies upon various stress conditions were performed to compare the stability of several mRNA-LNP formulations. The physical and chemical stability of mRNA-LNP formulations were assessed. The final composition of the two formulations, i.e., Formulation A and Formulation B, was defined according to the outcomes summarized below. i. Buffer

[0539] The buffer was selected based on preformulation studies on LNPs loaded with a monovalent influenza mRNA (“monoFlu-LNPs”) and LNPs loaded with a quadrivalent influenza mRNA (“QIV-LNPs”) using the three different cationic lipids (CL-1 : OF-02; CL-2: cKK-ElO; CL-3: GL-HEPES-E3-E12-DS-4-E10). Physical and chemical stability and mRNA expression of LNPs in different buffers at different pH was tested and the results are shown in FIG. 17, FIG. 18, and FIG. 19. The effect of NaCl to modulate the ionic strength was investigated as well.

[0540] FIG. 17 shows the particle size of monoFlu-LNPs by Dynamic Light Scattering in different buffer conditions, in the pH range 6-8. For each buffer, a condition without NaCl and a condition with 150 mM NaCl were tested. Tris buffer at pH 8 maintained the particle size of monoFlu-LNPs with all the three cationic lipids.

[0541] FIG. 18 shows 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). Tris buffer at pH 8 maintained good mRNA expression with all the three cationic lipids.

[0542] FIG. 19 shows the particle size by Dynamic Light Scattering and decrease of mRNA integrity (“%mRNA integrity vs TO”) on monoFlu-LNPs in different buffer conditions, in the pH range 7.5-8.5. For each buffer, a condition without NaCl and different NaCl concentrations (50mM, lOOmM, 150mM) were tested. Tris buffer at a pH range between 7.5 and 8 with NaCl maintained the particle size of monoFlu-LNPs and reduced the decrease of mRNA integrity.

[0543] Overall, Tris buffer with NaCl at a pH 7.5-8 ensured higher physical stability of mRNA-LNPs and lower mRNA integrity decrease, compared to other conditions tested. Based on these results, Tris 20 mM/NaCl 100 mM (Formulation A) is the composition selected for low mRNA-LNP concentrations and Tris 50mM/NaCl 50mM (Formulation B) is the composition selected for higher mRNA-LNP concentrations. In fact, as shown in FIGs. 20A- 20B, at high mRNA-LNP concentrations, Tris 20 mM/NaCl 100 mM had not enough buffering capacity to maintain pH stability and mRNA encapsulation drops, while Tris 50 mM/NaCl 50 mM ensured good stability in this case. On the contrary, Tris 20 mM/NaCl 100 mM ensured good physical stability and lower mRNA integrity drop at low mRNA-LNP concentrations. ii. Cryoprotectant (Disaccharide(s))

[0544] The cryoprotectant was selected based on Freeze/Thaw studies conducted on monoFlu-LNPs with CL-2 and CL-3. Physical and chemical stability of LNPs in the presence of different cryoprotectants was tested and the results are shown in FIG. 21, FIGs. 22A-22D, and FIG. 23.

[0545] FIG. 21 shows the particle size of monoFlu-LNPs by Dynamic Light Scattering after Freeze/Thaw cycles at -70°C/room temperature (RT) (using CL-2 as cationic lipid) and at -20°C/RT (using CL-3 as cationic lipid) in the presence of different trehalose concentrations. The particle size and poly dispersity index (pDI) remained acceptable after 3 Freeze/Thaw cycles at -70°C/RT in the presence of 10% trehalose, while aggregation was faster in the presence of lower trehalose concentrations (e.g., 1% or 5%) (FIG. 21, top). The particle size and pDI remained acceptable after 3 Freeze/Thaw cycles at -20°C/RT only with lower trehalose concentrations (e.g., 1%), while aggregation was faster in the presence of higher trehalose concentrations (e.g., 10% or 15%) (FIG. 21, bottom).

[0546] FIGs. 22A-22D shows the visual aspect (FIG. 22A), 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. In the presence of high concentrations of trehalose, the particle size, pDI, and turbidity all increased after Freeze/Thaw cycles at - 20°C/RT. Conversely, the presence of sucrose maintained the stability of the formulation up to 3 Freeze/Thaw cycles at -20°C/RT.

[0547] FIG. 23 shows 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.

[0548] Overall, trehalose prevented aggregation upon storage at -70°C only at concentrations higher than 5% but did not prevent aggregation upon storage at -20°C. In addition, increasing trehalose concentration led to faster aggregation (FIG. 21). Sucrose prevented aggregation upon storage at -20°C, while did not negatively impact mRNA encapsulation rate and other attributes (FIGs. 22A-22D and FIG. 23).

Hi. Surfactant

[0549] The surfactant was selected based on preliminary stability studies on monoFlu- LNPs with CL-2 and on stability studies on QIV-LNPs with CL-1, CL-2, and CL-3.

[0550] As shown in FIGs. 24A-24D, the presence of surfactant (Pl 88) was needed to prevent visual aggregation (FIG. 24A), particle size increase (FIG. 24B), subvisible particles increase (FIG. 24C), and the %mRNA encapsulation decrease (FIG. 24D) after 3 days of orbital shaking stress at RT.

[0551] Two surfactants (Pl 88 and PS80) were compared on QIV-LNPs with CL-1, CL-2, and CL-3 in their ability to maintain physical stability upon different stresses. 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 cycles at -20°C/RT are shown in FIG. 25. As can be seen in FIG. 25, P188 was much more efficient in maintaining physical stability, i.e., in inhibiting turbidity evolution and subvisible particles evolution, upon shaking stress and Freeze/Thaw stress. iv. Chelate Agent

[0552] The addition of EDTA as a chelate agent was preliminary tested on monoFlu-LNPs with CL-2. To this end, different concentrations of EDTA were tested for their impact on physical stability of LNPs at 25°C. As shown in FIG. 26, the presence of different concentrations of EDTA in the buffer did not negatively impact the physical stability of LNPs. [0553] Using QIV-LNPs with CL-1, CL-2, and CL-3, EDTA was shown to be able to slow mRNA integrity decrease by chelating metals potentially present in the formulation (FIG. 27). [0554] Based on the stability studies upon various stress conditions, Formulation A (20 mM Tris, 100 mM NaCl, 5% sucrose, 0.4% Pl 88, and 10 pM EDTA) and Formulation B (50 mM Tris, 50 mM NaCl, 5% sucrose, 0.4% Pl 88, and 10 pM EDTA) were identified as being able to maintain colloidal stability and chemical stability of mRNA-LNPs. Stabilized mRNA- LNP Formulations A and B generally maintained a particle size of an average diameter lower than about 150 nm and an mRNA encapsulation rate more than about 80% following 9 months storage at 5°C or 6 months at a temperature ranging from -50°C to -15°C. They were physically resistant to Freeze/Thaw cycles as well. The decrease of mRNA integrity over time was also reduced, which could be attributed to the stabilizing effect of the formulations. FIG. 28 shows exemplary long term stability data of QIV-LNP formulations with the three cationic lipids (i.e., CL-1, CL-2, and CL-3) (LNPs particle size by Dynamic Light Scattering, %mRNA encapsulation by RiboGreen Assay, %decrease of mRNA integrity by capillary electrophoresis).

Example 10. Optimized Thermostable mRNA-LNP Formulation.

[0555] In searching for ways to improve stability of mRNA-LNP formulations, it was discovered that addition of gelatin and/or certain excipients, such as lipoic acid, L-theanine, and vanillin, to mRNA-LNP formulations led to substantially improved stability, including RNA stability, as described in Examples 1-8. It was also discovered that formulating mRNA- LNPs in solutions containing a defined amount of a buffering agent (e.g., Tris), an osmotic agent (e.g., NaCl), a disaccharide (e.g., sucrose), a surfactant (e.g., P188), and a chelating agent (e.g., EDTA) in the presence of a residue amount of trehalose also led to substantially improved stability, including RNA stability, as described in Example 9. This example describes another optimized thermostable mRNA-LNP formulation.

[0556] Similar to the stabilized formulations described in Example 9, the optimized thermostable mRNA-LNP formulation also contains a buffering agent (e.g., Tris), an osmotic agent (e.g., NaCl), a disaccharide (e.g., sucrose), a surfactant (e.g., P188), and a chelating agent (e.g., EDTA). However, unlike the stabilized formulations described in Example 9, the optimized thermostable mRNA-LNP formulation described in this example does not contain any amount of trehalose. The optimization and stability studies upon various stress conditions were performed on different formulation prototypes and the physical and chemical stability of mRNA-LNP formulations in storage conditions were assessed. The final optimized thermostable mRNA-LNP formulation was defined according to the outcomes summarized below. i. Buffer

[0557] The buffer and pH of the formulation were selected based on a design of experiments (DOE) study on mRNA-LNPs prepared with a quadrivalent influenza mRNA using GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid at a concentration of 0.26 mg/mL. The effects of Tris concentration, NaCl concentration, and pH on physical stability, chemical stability, and mRNA expression of mRNA-LNPs were evaluated. Tris concentration range evaluated was 20-50 mM, NaCl concentration range assessed was 50-150 mM, and pH screened was between 7.2 and 7.7. Main outcomes are summarized in FIG. 29.

[0558] Based on this study, the optimal settings to maximize mRNA integrity are 20 mM Tris, 150 mM NaCl, and pH 7.5. The buffer amount was weakly associated with mRNA integrity in the range tested, as shown in FIG. 30. Tris and NaCl were shown to be fundamental to maintain LNPs physical stability, especially at pH closed to 7.0, and beneficial in reducing mRNA-lipid adducts (FIG. 31). However, both could increase mRNA fragmentation rate at higher concentration. pH played a major role on both LNPs physical stability and chemical stability of mRNA. At pH close to 7.0, physical stability issues were observed without buffers with high ionic strength: at pH 7.2, only the formulation containing 50 mM Tris and 150 mM NaCl showed good stability (FIG. 32). Regarding mRNA integrity, pH close to 7.0 favorized mRNA-lipid adducts formation, while mRNA fragmentation increased at pH higher than 7.5 (FIG. 31)

[0559] mRNA expression evaluated by Flow Cytometry put in evidence the inferiority of formulations with lower pH and lower concentration of Tris/NaCl (FIG. 33).

[0560] In order to select a formulation optimized for mRNA integrity and physically stable in a range of pH ± 0.3 in the final formulation, the final buffer composition and pH were set as follows: 50 mM Tris, 150 mM NaCl, pH 7.5. ii. Surfactant

[0561] The surfactant was added in the formulation based on stability studies at 5°C and 25°C, and Freeze/Thaw studies on the same mRNA-LNPs but at a concentration of 1 mg/mL. Based on previous studies, only Pl 88 was tested.

[0562] The presence of P188 at 0.4% (w/v) prevented size increase at 5°C and 25°C, and after Freeze/Thaw cycles (FIG. 34A). Moreover, the presence of P 188 was shown to be critical to prevent subvisible particles increase, after freezing, when added in combination with sucrose (FIG. 34B)

Hi. Cryoprotectant (Disaccharide(s))

[0563] The cryoprotectant was selected based on Freeze/Thaw stress studies conducted on the same mRNA-LNPs at a concentration of 1 mg/mL.

[0564] Physical and chemical stability of LNPs in the presence of sucrose at different concentrations was evaluated. As shown in FIGs. 35A-35B, sucrose prevented aggregation upon Freeze/Thaw cycles (-70°C/RT) only at concentrations higher than or equal to 5%, and in combination with 0.4% (w/v) P188. iv. Chelate Agent

[0565] The addition of EDTA to the mRNA-LNP formulation was tested on the same mRNA-LNPs.

[0566] The presence of EDTA in the final buffer did not negatively impact the physical stability of LNPs. Based on literature, EDTA can slow mRNA integrity drop by chelating metals potentially present in the formulation. As shown in FIG. 36, the presence of EDTA in the final buffer did not negatively impact mRNA integrity in the conditions tested.

[0567] Based on these studies, the final optimized thermostable mRNA-LNP formulation contains 50 mM Tris, 150 mM NaCl, 5% sucrose, 0.4% P188, and 10 pM EDTA, pH 7.5 ± 0.3. The final optimized formulation maintained colloidal stability and chemical stability of mRNA-LNP in a broad range of mRNA-LNP concentration (0.04-1 mg/mL). The stabilized mRNA-LNP formulation maintained an average diameter lower than 150 nm and an mRNA encapsulation rate more than about 80% following 6 months storage at 5 °C or at a temperature ranging from -80°C to -15°C. Physical stability upon Freeze/Thaw cycles was documented as well. The decrease of mRNA integrity over time was reduced because of the stabilizing effect of the formulation.

Example 11. Thermostability Improvement Using Gelatin for 16:0-18:1 PE Helper Lipid Based LNPs.

[0568] This example describes another thermostability improvement study using gelatin for 16:0-18: 1 PE helper lipid based LNPs.

[0569] The mRNA-LNPs were prepared with a quadrivalent influenza mRNA in 10% trehalose (1 mg/mL). The LNP formulation used in this example contained a cationic lipid (GL-HEPES-E3-E12-DS-4-E10, 50% by mol), a PEGylated lipid (1,2-dimyristoyl-rac- glycero-3 -methoxy (DMG)-PEG2000, 1.5% by mol), a cholesterol-based lipid (cholesterol, 38.5% by mol), and a helper lipid (16:0-18: 1 PE, 10% by mol). The mRNA-LNPs were diluted to a concentration of 0.2 mg/mL in 50 mM Tris, pH 7.5, 50 mM NaCl, 2% trehalose, 0.5% Pl 88, and 10 pM EDTA to form the mRNA-LNP formulation. The resultant mRNA-LNP formulation was stored at 2-8°C in the presence of 1% gelatin or without gelatin as the formulation control. The formulation stability was then analyzed at specific timepoints for up to 7 months by measuring the particle size and mRNA encapsulation efficiency of the LNPs, as well as the decrease in mRNA integrity. The particle size was measured using Dynamic Light Scattering, the mRNA encapsulation efficiency was measured using a fluorescence platebased assay, and the mRNA integrity was measured by extracting the mRNA from the LNPs and analyzing on a fragment analyzer using capillary electrophoresis.

[0570] As shown in FIGs. 37A-37C, the addition of gelatin to the mRNA-LNP formations provided an overall better thermostability profile after storage at 2-8°C for at least 7 months as compared to the formulation control without gelatin.

[0571] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations. [0572] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. In general, where embodiments or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0573] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

1. 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 -

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 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; 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; 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.

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 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 (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/or b) 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.