Stabilization of MRNA-LNPS with antioxidants

Abstract

Disclosed are solid compositions, comprising a lipid nanoparticle, a nucleic acid, and an antioxidant; wherein the nucleic acid and the antioxidant are encapsulated by the lipid nanoparticle.

Description

[0001] MTV-24725

[0002] STABILIZATION OF MRNA-LNPS WITH ANTIOXIDANTS

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application No.: 63 / 733,778, filed December 13, 2024. The contents of the aforementioned application are fully incorporated by reference herein.

[0005] BACKGROUND

[0006] Lipid-nanoparticle (LNP)-formulated RNA therapeutics have attracted increasing attention across diverse clinical areas, particularly following the success of SARS-CoV-2 mRNA vaccines. Current mRNA-LNP liquid formulations require cold storage at 4 °C for short-term or -20 °C for long-term, which poses a significant challenge for worldwide distribution, especially in resource-limited and rural areas. Solid-state mRNA-LNP formulations, such as those obtained through lyophilization or freeze-drying, could improve stability and eliminate cold-chain storage requirements. While excipients are commonly employed to improve the long-term stability of solid-state RNA-LNP formulations, the methods to identify optimal excipients in a high throughput manner remains inadequately explored. Accordingly, new compositions and methods for stabilizing lipid nanoparticles are required.

[0007] SUMMARY OF THE INVENTION

[0008] In certain aspects, the present disclosure provides solid compositions, comprising a lipid nanoparticle, a nucleic acid, and an antioxidant; wherein the nucleic acid and the antioxidant are encapsulated by the lipid nanoparticle.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1A is a schematic showing the selection process of various antioxidants.

[0011] FIG. IB is a schematic showing how exemplary LNP comprising antioxidants can be formulated.

[0012] FIG. 2 shows exemplary LNP formulations with antioxidants.

[0013] FIGs. 3A-3C show the properties of exemplary LNP formulations.

[0014] FIG. 4 is a schematic showing how exemplary LNP formulations can be incorporated into vaccine blocks. MTV-24725

[0015] FIG. 5 shows further exemplary LNP formulations with antioxidants.

[0016] FIGs. 6A and 6B show the properties of further exemplary LNP formulations.

[0017] FIG. 7 is a schematic showing the delivery of a COVID RBD vaccine using exemplary LNP formulations with antioxidants.

[0018] FIG. 8 shows the results of the delivery of a CO VID RBD vaccine using exemplary LNP formulations with antioxidants.

[0019] FIG. 9 shows the formulations of further exemplary LNP formulations with antioxidants.

[0020] FIG. 10A-10C show the stability of exemplary LNP formulations with antioxidants.

[0021] FIG. 11A and 11B show the encapsulation efficiency of exemplary LNP formulations with antioxidants.

[0022] FIG. 12A-12F show the properties of exemplary LNP formulations.

[0023] FIG. 13A-13C show the properties of exemplary LNP formulations.

[0024] FIG. 14A and 14B show the properties of exemplary LNP formulations.

[0025] FIG. 15A-15C show the properties of exemplary LNP formulations.

[0026] FIG. 16A-16E show the properties of exemplary LNP formulations.

[0027] FIG. 17A-17C show the properties of exemplary LNP formulations.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Oxidation is a key factor contributing to the degradation of both mRNA and LNP components, which can affect the integrity and effectiveness of mRNA-based vaccines. By integrating antioxidants into the LNP formulation, the stability of mRNA can be improved, thereby, enhancing vaccine performance and longevity.

[0030] Disclosed here are methods to incorporate antioxidants into the vaccine design: (1) in the lipid phase with antioxidants like Vitamin A (VA), Vitamin E (VE), and butylated hydroxytoluene (BHT), (2) in the aqueous phase with Vitamin C (VC) and TPGS, and (3) in the ink phase. Preliminary screening demonstrated that antioxidants, particularly VE, VC, and TPGS, improved the encapsulation efficiency and maintained a uniform particle size distribution within the LNPs. Long-term stability was monitored using RiboGreen and Femto Pulse assays, which measured LNP stability and mRNA integrity, respectively. Early results indicate that formulations with antioxidants preserve higher mRNA integrity and yield greater deliverable mRNA mass, suggesting a promising approach for improving mRNA vaccine stability. MTV-24725

[0031] In certain aspects, the present disclosure provides solid compositions, comprising a lipid nanoparticle, a nucleic acid, and an antioxidant; wherein the nucleic acid and the antioxidant are encapsulated by the lipid nanoparticle.

[0032] In certain embodiments, the lipid nanoparticle comprises an ionizable lipid, cholesterol, a polyethyleneglcyol lipid, a phospholipid, or a combination of any of them. In certain preferred embodiments, the lipid nanoparticle comprises 1 -octylnonyl 8-[(2 -hydroxy ethyl)[8- (nonyloxy)-8-oxooctyl]amino]octanoate (i.e., lipid 5, CAS Ref. No.: 2089251-33-0), 3,6-bis(4- (bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (i.e., cKK-E12), l,2-di-(9Z- octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol, or a combination of any of them. In certain embodiments, the lipid nanoparticle consists essentially of 1 -octylnonyl 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]octanoate i.e., lipid 5, CAS Ref. No.: 2089251-33-0), 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (i.e., cKK-E12), l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol, or a combination of any of them.

[0033] In certain embodiments, the concentration of the nucleic acid is about 50 pg / mL to about 1,000 pg / mL; or about 50 pg / mL, about 100 pg / mL, about 150 pg / mL, about 200 pg / mL, about 250 pg / mL, about 300 pg / mL, about 350 pg / mL, about 400 pg / mL, about 450 pg / mL, about 500 pg / mL, about 550 pg / mL, about 600 pg / mL, about 650 pg / mL about 700 pg / mL, about 750 pg / mL, about 800 pg / mL, about 850 pg / mL, about 900 pg / mL, about 950 pg / mL or about 1,000 pg / mL. In preferred embodiments, the concentration of the nucleic acid is about 650 pg / mL, about 700 pg / mL, or about 750 pg / mL. In certain preferred embodiments, the concentration of the nucleic acid is about 700 pg / mL.

[0034] In certain embodiments, the nucleic acids are mRNA, siRNA, RNA, or DNA. In certain preferred embodiments, the nucleic acids are RNA (e.g., mRNA).

[0035] In certain embodiments, the antioxidant is hydrophobic. In other embodiments, the antioxidant is hydrophilic. In certain embodiments, the antioxidant is selected from the group consisting of vitamin A, vitamin E, butylated hydroxytoluene, ascorbic acid, and D-u- tocopheryl polyethylene glycol succinate (TPGS). In certain preferred embodiments, the antioxidant is TPGS.

[0036] In certain embodiments, the composition further comprises a polymer. In certain embodiments, the polymer is PVP. In certain preferred embodiments, the polymer is PVP10. In other preferred embodiments, the polymer is PVP60. In certain embodiments, the polymer is polyvinylalcohol. In certain embodiments the polymer comprises 50 - 500 repeat units.

[0037] In certain embodiments, the solid composition further comprises a copolymer. MTV-24725

[0038] In certain embodiments, the copolymer comprises a plurality of repeat units of vinylalcohol and a plurality of repeat units of vinylpyrrolidinone. In certain embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone is about 1:1 to about 6:1; or about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 6:1. In certain preferred embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone is about 1:1, about 2:1, or about 3:1.

[0039] In certain embodiments, the copolymer comprises a plurality of repeat units of vinylalcohol and a plurality of repeat units of sucrose. In certain embodiments, the mass ratio of polyvinylalcohol to sucrose is about 1:1 to about 6:1; or about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 6:1. In certain embodiments, the mass ratio of polyvinylalcohol to sucrose is about 1 : 1 or about 2:1.

[0040] In certain embodiments, the copolymer comprises a plurality of repeat units of vinylalcohol, a plurality of repeat units of polyvinylpyrrolidone, and a plurality of repeat units of sucrose. In certain embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone to sucrose is about 1:1:1 to about 1:1:3; or about 1:1:1, about 1:1:2, or about 1:1:3. In certain preferred embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone to sucrose is about 1:1:2.

[0041] In certain embodiments, the copolymer comprises 250 - 1,500 repeat units.

[0042] In certain embodiments the copolymer is a block copolymer. In other embodiments, the copolymer is a random copolymer.

[0043] In certain embodiments, the w / w ratio of the polymer or the copolymer to the lipid nanoparticle is at least 100:1 (e.g., 100:1 to 10,000:1). In certain embodiments, the w / w ratio of the polymer or the copolymer to the lipid nanoparticle is at least 1000:1 (e.g., 1000:1 to 10,000:1). In certain embodiments, the w / w ratio of the polymer or the copolymer to the lipid nanoparticle is about 100:1 to about 500:1; or about 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400: 1, 450: 1, or 500: 1. In certain embodiments, the w / w ratio of the polymer or the copolymer to the nucleic acids is about 600:1 to about 10,000:1; or about 600:1, about 700:1, about 800:1, 900:1, or about 1000:1, about 1,250:1, about 1500:1, about 1750:1, about 2000:1, about 2,250:1, about 2,500:1, about 2,750:1, about 3000:1, about 4,000:1, about 5,000:1, about 6,000:1, about 7000:1, about 8,000:1, about 9,000:1, or about 10,000:1. In certain embodiments, the w / w ratio of polymer or copolymer to the nucleic acids is about 20: 1, about 40:1, about 60:1, about 80:1, about 100:1, 120:1, 140:1, 160:1, 180:1, 200:1, 220:1, 240:1, 260:1, 280:1, or about 300:1, about 320:1, about 340:1, about 360:1, about 380:1, or about 400:1. MTV-24725

[0044] In certain embodiments the solid composition further comprises an amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is arginine.

[0045] In certain embodiments, the nitrogen to phosphate ratio of the lipid nanoparticle is about 1.5. In certain embodiments, the nitrogen to phosphate ratio of the lipid nanoparticle is about 3.1. In certain embodiments, the nitrogen to phosphate ratio of the lipid nanoparticle is about 5.4

[0046] In certain embodiments, the diameter of the lipid nanoparticle is about 50 nm to about 300 nm; or about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250, about 275 nm, or about 300 nm.

[0047] In certain embodiments, the solid composition further comprises pharmaceutically acceptable carrier.

[0048] In certain embodiments, the solid composition further comprises an adjuvant.

[0049] In certain embodiments, the encapsulation efficiency of the lipid nanoparticle is greater than about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the encapsulation efficiency of the lipid nanoparticle is greater than about 60%.

[0050] In certain embodiments, the nucleic acid has improved stability (e.g., improved thermostability) as compared to the nucleic acid alone or the nucleic acid in a composition not disclosed herein.

[0051] In further aspects, the present disclosure provides methods of delivering a therapy to a subject in need thereof, comprising contacting the subject with the compositions disclosed herein. In certain embodiments, the therapy is a vaccine. In certain preferred embodiments, the therapy is an mRNA vaccine.

[0052] In further aspects, the present disclosure provides injectable needles comprising the compositions disclosed herein.

[0053] In further aspects, the present disclosure provides methods of delivering a therapy to a subject in need thereof, comprising contacting the subject with the injectable needles disclosed herein.

[0054] Definitions

[0055] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, MTV-24725 immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

[0056] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0057] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0058] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

[0059] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents e.g., mice and rats).

[0060] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0061] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, MTV-24725 prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0062] “Administering” or “administration of’ a substance, a composition or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a composition or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A composition or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the composition or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0063] Appropriate methods of administering a substance, a composition or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the composition or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a composition or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered composition or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0064] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compositions can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0065] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, MTV-24725 a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subj ect will depend upon, for example, the subj ect’ s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0066] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not.

[0067] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0068] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0069] As used herein, the phrase “encapsulation efficiency” refers to the percentage of a compound (e.g., drug) or other material (c.g, adjuvant) that is successfully trapped inside a nanoparticle (e.g., lipid nanoparticle) or micelle following a process (e.g., a chemical reaction) design to trap the compound or other material inside the nanoparticle or micelle.

[0070] EXAMPLES

[0071] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[0072] Example 1 : Synthesis of Exemplary Nanoparticles of the Disclosure

[0073] We formulated lipid nanoparticles (LNPs) using a combination of heptadecane-9-yl 8- ((2-hydroxyethyl)(8-nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), cholesterol, and l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14- MTV-24725

[0074] PEG2000), along with antioxidants as specified in the tables. Lipids were combined at specific molar ratios, and Luciferase mRNA was incorporated into these formulations.

[0075] High-purity lipids were utilized for the synthesis of LNPs, including heptadecane-9-yl 8-((2-hydroxyethyl)(8-nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), cholesterol, and l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14- PEG2000). The lipids were dissolved in ethanol to create the organic phase, with molar ratios specified in accompanying tables, and included antioxidants, if applicable.

[0076] Purified mRNAs featuring a CleanCap AG cap, complete Nl-methyl-pseudouridine substitution, and a polyadenylated tail of 120 adenosines were prepared. These mRNA solutions were buffered with citrate at pH 3, forming the aqueous phase, and included antioxidants, if applicable.

[0077] For LNP synthesis, the organic phase was rapidly combined with the aqueous phase at a 3 : 1 volume ratio (aqueous:organic) using a flow rate of 3 mL / min. In cases where LNPs were directly employed in solution form, the resulting nanoparticles were dialyzed in PBS at 4°C for 2 hours using a dialysis cassette with a 20,000 molecular weight cutoff. For applications requiring solid materials incorporating polymers, the LNPs were dialyzed under the same conditions but using water instead of PBS.

[0078] Example 2: Properties of Exemplary Nanoparticles of the Disclosure

[0079] To assess the long-term stability of antioxidant-stabilized LNPs under physiologically relevant conditions, we conducted two in vitro studies associated with SEAL microparticles (MPs). Initially, we prepared LNPs in a solid formulation by embedding them in a polymer blend of polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) (PVA), forming blocks that were then incorporated into SEAL MPs.

[0080] UnSEALed study: Each block was placed in 5 pL of water and stored in a 37°C incubator. During incubation, samples were characterized at designated time points using RiboGreen and Femto Pulse assays to measure LNP encapsulation efficiency (EE) and mRNA integrity.

[0081] SEALed study: Each MP containing one block was placed in 50 pL of water and stored in a 37°C incubator. For analysis, MPs were manually broken to release LNPs, followed by RiboGreen and Femto Pulse characterization to assess LNP EE and mRNA integrity.

[0082] For both studies, the polymer formulation ratio was PVP:PVA at 1 :2 (w / w), and the mRNA:polymer ratio was 1 :320 (w / w). MTV-24725

[0083] Encapsulation efficiency (EE) was calculated based on the equation below. TX is TE buffer mixed with Triton X-100.

[0084] Total mRNA (TX) — Free mRNA (TE)

[0085] EE% = -7- - — - x 100%

[0086] Total mRNA (TX) mRNAtotai= TX Concentration x Dilution x Volume mRN AencapsuiatedmRN Atotaix EE° / o mRNA integrity was calculated based on the equation below.

[0087] The deliverable mRNA was calculated based on the equation below: mRN AencapsuiatedX mRNAintact% mRN Adeliverable

[0088] Example 3: Vaccination using Exemplary Nanoparticles of the Disclosure

[0089] We conducted an in vivo study using the CO VID receptor-binding domain (RBD) as a model antigen to assess the long-term stabilization and controlled release of an mRNA-LNP vaccine. Based on findings from our in vitro studies, we selected TPGS1 as the optimal formulation among the antioxidant-enhanced options for the booster dose, delivered via the SEAL microparticle (MP). For the prime dose, administered via the CRIS needle, we used the BARDA formulation, which does not include antioxidants, as long-term stabilization was not required for this initial dose.

[0090] We conducted an in vivo study using the SARS-CoV-2 receptor-binding domain (RBD) as a model antigen to evaluate the long-term stabilization and controlled release of an mRNA- LNP vaccine. Based on findings from in vitro studies, we identified TPGS-1 as the optimal formulation among antioxidant-enhanced options for the booster dose, which was delivered via the SEAL microparticle (MP) platform. For the prime dose, administered using the CRIS needle, we utilized the BARDA formulation, which lacks antioxidants, as long-term stabilization was not required for this initial administration.

[0091] Study Design

[0092] Formulations:

[0093] • CRIS needle: PVP10:PVA (1 : 1) / 1 :320 / no antioxidant

[0094] • Block: PVP10:PVA (1 :2) / 1 :50 / TPGS-1

[0095] • MP base: 858s

[0096] • MP cap: 502 MTV-24725

[0097] Study Groups:

[0098] 1. Group 1: Soluble + Soluble

[0099] 2. Group 2: CRIS needle + Soluble

[0100] 3. Group 3: Soluble + Block (3 blocks)

[0101] 4. Group 4: Soluble + SEAL (3 MPs)

[0102] 5. Group 5: CRIS-SEAL (1 CRIS MP + 2 MPs)

[0103] Dosing Details:

[0104] • Target dose: 5 pg for CRIS needle, 5 pg for soluble formulations, 15 pg for block or MPs.

[0105] • N = 5 mice per group.

[0106] • Administration route: Subcutaneous.

[0107] Characterization Methods:

[0108] • Antigen-specific IgG responses were quantified using an ELISA assay.

[0109] • Antigen cross-reactivity and broad immunity were analyzed with an MSD assay.

[0110] ELISA Assay Method

[0111] 96-well plates were coated with purified recombinant SARS-CoV-2 Spike Sl-RBD antigen. Heat-inactivated sera were serially diluted and incubated at 37°C. A rabbit anti-mouse IgG-horseradish peroxidase conjugate (1 : 10,000 dilution) was used as the secondary antibody, and 3,5,3',5'-tetramethylbenzidine (TMB) served as the substrate. Interpolated endpoint titers were calculated as the dilution at which the optical density exceeded 3* the background, determined from sera of naive mice.

[0112] MSD Assay Method

[0113] ECLA plates were predesigned with four antigen spots per well: WA1 / 2020, B.1.1.7, P. l, and B.1.351 Sl-RBD. Plates were blocked with 50 pL of 1% BSA solution for at least 30 minutes at room temperature with shaking (700 rpm). During blocking, serum samples were diluted 1 :5,000 in Diluent 100. Plates were washed with 150 pL of wash buffer, blotted dry, and loaded with 50 pL of diluted samples in duplicate. After a 2-hour incubation at room temperature with shaking (700 rpm), secondary detection was performed using rabbit antimouse IgG conjugated to MSD GOLD SULFO-TAG. Plates were washed three times, and 50 pL of SULFO-tagged detection antibody (diluted lx in Diluent 100) was added and incubated for 1 hour. After final washes, 150 pL of MSD GOLD Read Buffer B was added to each well, and the plates were read using a MESO Quick-Plex SQ 120. MSD titers were reported as relative light units (RLU), calculated as the sample signal minus blank for each antigen spot. The limit of detection was set at 500 RLU for all assays. MTV-24725

[0114] INCORPORATION BY REFERENCE

[0115] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0116] EQUIVALENTS

[0117] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

MTV-24725We claim:

1. A solid composition, comprising a lipid nanoparticle, a nucleic acid, and an antioxidant; wherein the nucleic acid and the antioxidant are encapsulated by the lipid nanoparticle.

2. The solid composition of claim 1, wherein the lipid nanoparticle comprises an ionizable lipid, cholesterol, a polyethyleneglcyol lipid, a phospholipid, or a combination of any of them.

3. The solid composition of claim 1 or 2, wherein the lipid nanoparticle comprises 1- octylnonyl 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]octanoate (i.e., lipid 5, CAS Ref. No.: 2089251-33-0), 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (z.e., cKK-E12), l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol, or a combination of any of them.

4. The solid composition of claim 1 or 2, wherein the lipid nanoparticle consists essentially of 1 -octylnonyl 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]octanoate (i.e., lipid 5, CAS Ref. No.: 2089251-33-0), 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (z.e., cKK-E12), l,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, or a combination of any of them.

5. The solid composition of any one of claims 1-4, wherein the concentration of the nucleic acid is about 50 pg / mL to about 1,000 pg / mL; or about 50 pg / mL, about 100 pg / mL, about 150 pg / mL, about 200 pg / mL, about 250 pg / mL, about 300 pg / mL, about 350 pg / mL, about 400 pg / mL, about 450 pg / mL, about 500 pg / mL, about 550 pg / mL, about 600 pg / mL, about 650 pg / mL about 700 pg / mL, about 750 pg / mL, about 800 pg / mL, about 850 pg / mL, about 900 pg / mL, about 950 pg / mL or about 1,000 pg / mL.

6. The solid composition of any one of claims 1-4, wherein the concentration of the nucleic acid is about 650 pg / mL, about 700 pg / mL, or about 750 pg / mL.

7. The solid composition of any one of claims 1-4, wherein the concentration of the nucleic acid is about 700 pg / mL.MTV-247258. The solid composition of any one of claims 1-7, wherein the nucleic acids are mRNA, siRNA, RNA, or DNA.

9. The solid composition of any one of claims 1-7, wherein the nucleic acids are RNA (e.g., mRNA).

10. The solid composition of any one of claims 1-9, wherein the antioxidant is hydrophobic.

11. The solid composition of any one of claims 1-9, wherein the antioxidant is hydrophilic.

12. The solid composition of any one of claims 1-9, wherein the antioxidant is selected from the group consisting of vitamin A, vitamin E, butylated hydroxytoluene, ascorbic acid, and D-u-tocopheryl polyethylene glycol succinate (TPGS).

13. The solid composition of any one of claims 1-9, wherein the antioxidant is TPGS.

14. The solid composition of any one of claims 1-13, further comprising a polymer.

15. The solid composition of claim 14, wherein the polymer is PVP.

16. The solid composition of claim 14, wherein the polymer is PVP10.

17. The solid composition of claim 14, wherein the polymer is PVP60.

18. The solid composition of claim 14, wherein the polymer is polyvinylalcohol.

19. The solid composition of any one of claims 14-18, wherein the polymer comprises 50 - 500 repeat units.

20. The solid composition of any one of claims 1-13, further comprising a copolymer.

21. The solid composition of claim 20, wherein the copolymer comprises a plurality of repeat units of vinylalcohol and a plurality of repeat units of vinylpyrrolidinone.MTV-2472522. The solid composition of claim 21, wherein the mass ratio of polyvinylalcohol to polyvinylpyrrolidone is about 1:1 to about 6:1; or about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 6:1.

23. The solid composition of claim 21, wherein the mass ratio of polyvinylalcohol to polyvinylpyrrolidone is about 1 : 1, about 2: 1, or about 3:1.

24. The solid composition of claim 20, wherein the copolymer comprises a plurality of repeat units of vinylalcohol and a plurality of repeat units of sucrose.

25. The solid composition of claims 24, wherein the mass ratio of polyvinylalcohol to sucrose is about 1:1 to about 6:1; or about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 6:1.

26. The solid composition of claims 24, wherein the mass ratio of polyvinylalcohol to sucrose is about 1 : 1 or about 2:1.

27. The solid composition of claim 25, wherein the copolymer comprises a plurality of repeat units of vinylalcohol, a plurality of repeat units of polyvinylpyrrolidone, and a plurality of repeat units of sucrose.

28. The solid composition of claims 27, wherein the mass ratio of polyvinylalcohol to polyvinylpyrrolidone to sucrose is about 1:1:1 to about 1:1:3; or about 1:1:1, about 1:1:2, or about 1:1:3.

29. The solid composition of claims 27, wherein the mass ratio of polyvinylalcohol to polyvinylpyrrolidone to sucrose is about 1:1:2.

30. The solid composition of any one of claims 20-29, wherein the copolymer comprises 250 - 1,500 repeat units.

31. The solid composition of any one of claims 20-30, wherein the copolymer is a block copolymer.MTV-2472532. The solid composition of any one of claims 20-30, wherein the copolymer is a random copolymer.

33. The solid composition of any one of claims 14-32, wherein the w / w ratio of the polymer or the copolymer to the lipid nanoparticle is at least 100: 1 (e.g., 100: 1 to 10,000: 1).

34. The solid composition of any one of claims 14-32, wherein the w / w ratio of the polymer or the copolymer to the lipid nanoparticle is at least 1000: 1 (e.g., 1000: 1 to 10,000:1).

35. The solid composition of any one of claims 14-32, wherein the w / w ratio of the polymer or the copolymer to the lipid nanoparticle is about 100: 1 to about 500: 1; or about 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, or 500:1.

36. The solid composition of any one of claims 14-35, wherein the w / w ratio of the polymer or the copolymer to the nucleic acids is about 600: 1 to about 10,000: 1; or about 600:1, about 700:1, about 800:1, 900:1, or about 1000:1, about 1,250:1, about 1500:1, about 1750:1, about 2000:1, about 2,250:1, about 2,500:1, about 2,750:1, about 3000:1, about 4,000:1, about 5,000:1, about 6,000:1, about 7000:1, about 8,000:1, about 9,000:1, or about 10,000:1.

37. The solid composition of any one of claims 13-35, wherein the w / w ratio of polymer or copolymer to the nucleic acids is about 20: 1, about 40: 1, about 60: 1, about 80: 1, about 100:1, 120:1, 140:1, 160:1, 180:1, 200:1, 220:1, 240:1, 260:1, 280:1, or about 300:1, about 320:1, about 340:1, about 360:1, about 380:1, or about 400:1.

38. The solid composition of any one of claims 1-37, further comprising an amino acid.

39. The solid composition of claim 38, wherein the amino acid is a naturally occurring amino acid.

40. The solid composition of claim 38, wherein the amino acid is arginine.

41. The solid composition of any one of claims 1-40, wherein the nitrogen to phosphate ratio of the lipid nanoparticle is about 1.5.MTV-2472542. The solid composition of any one of claims 1-40, wherein the nitrogen to phosphate ratio of the lipid nanoparticle is about 3.1.

43. The solid composition of any one of claims 1-40, wherein the nitrogen to phosphate ratio of the lipid nanoparticle is about 5.

444. The solid composition of any one of claims 1-43, wherein the diameter of the lipid nanoparticle is about 50 nm to about 300 nm; or about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250, about 275 nm, or about 300 nm.

45. The solid composition of any one of claims 1-44, further comprising a pharmaceutically acceptable carrier.

46. The solid composition of any one of claims 1-45, further comprising an adjuvant.

47. The solid composition of any one of claims 1-46, wherein the encapsulation efficiency of the lipid nanoparticle is greater than about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

48. The solid composition of any one of claims 1-46, wherein the encapsulation efficiency of the lipid nanoparticle is greater than about 60%.

49. The solid composition of any one of claims 1-48, wherein the nucleic acid has improved stability (e.g., improved thermostability) as compared to the nucleic acid alone or the nucleic acid in a composition not recited in any one of claims 1-48.

50. A method of delivering a therapy, comprising contacting a subject in need thereof with the composition of any one of claims 1-48.

51. The method of claim 50, wherein the therapy is a vaccine.

52. The method of claim 50, wherein the therapy is an mRNA vaccine.

53. An injectable needle, comprising the composition of any one of claims 1-49.MTV-2472554. A method of delivering a therapy, comprising contacting a subject in need thereof with the injectable needle of claim 53.

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