Improved lnp formulations and uses thereof

Abstract

The invention relates to lipid nanoparticles, immunogenic compositions and methods for use thereof.

Description

[0001]PC073229A IMPROVED LNP FORMULATIONS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and U.S. Provisional Application No.63 / 762,775, filed on February 25, 2025, U.S. Provisional Application No.63 / 568,754, filed March 22, 2024, U.S. Provisional Application No.63 / 562,257, filed March 6, 2024 and U.S. Provisional Application No.63 / 562,255, filed March 6, 2024. The entire content of each of the foregoing applications is hereby incorporated herein by reference. REFERENCE TO SEQUENCE LISTING This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled "PC073229A Sequence Listing.xml" created on February 27, 2025 and having a size of 12 KB. The sequence listing contained in this .xml file is part of the specification and is hereby incorporated herein by reference in its entirety. BACKGROUND There are many challenges associated with the delivery of nucleic acids to affect a desired response in a biological system. Therapeutic nucleic acids include, but are not limited to, e.g. messenger RNA (mRNA), self-amplifying RNA (saRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme, or as a vaccine. The therapeutic applications of translatable nucleotide delivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether or not indigenous to the system. The expression products of the nucleic acid can augment existing levels of protein, replace missing or non-functional versions of a protein, or introduce new protein and associated functionality in a cell or organism. Preferably, these lipid nanoparticles would provide optimal drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. In addition, these lipid-nucleic acid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. mRNA-based therapeutics have shown their enormous benefit, for example, with the development of mRNA-LNP vaccines against SARS-CoV-2 during the 2020 pandemic (e.g. COMIRNATY®). However, RNA-LNPs are still faced with some challenges. Currently mRNA-LNP vaccine formulations must be stored at ultralow temperatures before administration, e.g. at -80ºC (Comirnaty®), and have a shelf life of less than 3 months once the frozen drug product is thawed and stored at 5ºC (Crommelin et al. (2021) “Addressing the Cold Reality of mRNA Vaccine Stability”, J. Pharm Sci 10 (3): 997-1001). It has also been shown that one of the causes of reduced protein expression in refrigerated mRNA-LNP vaccines is the formation of adducts over time during refrigerated storage of the thawed drug product. These adducts are a class of impurities which render the mRNA untranslatable, leading to loss of protein expression (Packer, et al. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat. Commun.12, 6777 (2021)). Combination vaccines are a major way to reduce the number of vaccine administrations, and thereby improve compliance with immunization schedules. However, it is not always possible to combine vaccines especially in cases where the combination is not fully stable due to negative physical interactions among vaccine components and excipients, preservatives, buffer components and / or adjuvants. A combination vaccine is a mixture of individual vaccines with the result that multiple vaccines are administered together. There are two general categories of combination vaccines: multi-disease combination vaccines (i.e. include individual vaccines for different diseases) and multivalent combination vaccines (i.e. directed to several types (serotypes or serogroups) of the same viral or bacterial pathogen). While multivalent combinations are customarily mixed during the manufacturing process, multi-disease combinations may be mixed at one of three stages: (i) during the manufacturing process such that the combination is filled into vials or syringes; (ii) at the time of administration by mixing different vaccines in a vial then taking the mixture up into a syringe for injection or (iii) at the moment of administration from the 2 chambers of a dual-chambered syringe. The goal is to achieve a formulation with a long enough shelf-life for marketing, usually ≥1 year. Therefore, any formulation must be optimized for its component antigens and their excipients (Ronald W Ellis, (1999) “Development of combination vaccines” Vaccine 17:1635-1642). In addition, there is a need to identify formulations of lipid-containing nanoparticle compositions which promote increased RNA expression and protein yield and have improved colloidal stability which maintains RNA expression and protein yield for long-term refrigerated storage. Accordingly, there is a need for immunogenic compositions comprising RNA-LNPs with improved colloidal stability and reduced adduct formation in order to maintain RNA expression and protein yield. In addition, there remains a need for immunogenic compositions comprising RNA-LNPs that are compatible with other vaccine products (e.g. protein subunits, adjuvants, conjugates, etc.) when combined, without a loss of performance of the immunogenic composition comprising the RNA-LNP or the other vaccine products combined therewith. SUMMARY The invention described herein provides improved immunogenic compositions comprising lipid nanoparticles (LNP) which have improved colloidal stability for storage at a variety of temperatures with reduced adduct formation in order to promote increased mRNA expression and protein yield, as well as the flexibility to ensure compatibility with other products, including but not limited to vaccine drug products such as other RNA-LNP drug products, protein subunits, and conjugates, and other excipients such as adjuvants, when combined. Described below are embodiments of the invention, where for convenience Embodiment 1 (E1) is identical to the embodiment of claim 1 provided herein. Exemplary embodiments (E) of the invention provided herein include: E1. An immunogenic composition comprising a) a fatty acid, including a derivative or salt thereof; b) one or more lipid nanoparticles (LNP), wherein the LNP comprises: i. an ionizable cationic lipid; ii. cholesterol, a cholesterol analog or a mixture of cholesterol and a cholesterol analog; iii. a neutral lipid; and iv. a polymer-conjugated lipid: and c) one or more ribonucleic acids (RNA) comprising at least one open reading frame (ORF) encoding a polypeptide of a gene of interest or an immunogenic fragment thereof, wherein the RNA is encapsulated in the LNP of item (b). E2. The immunogenic composition of embodiment E1, wherein the fatty acid is an unsaturated fatty acid. E3. The immunogenic composition of embodiment E2, wherein the unsaturated fatty acid is a monounsaturated fatty acid, a di-unsaturated fatty acid or a polyunsaturated fatty acid. E4. The immunogenic composition of embodiment E3, wherein the fatty acid is selected from oleic acid, arachidonic acid, eruric acid, linolenic acid, ricinoleic acid, palmitoleic acid, linoleic acid, or a derivative or salt thereof. E5. The immunogenic composition of embodiment E4, wherein the salt is selected from sodium, potassium, magnesium or calcium. E6. The immunogenic composition of embodiment E4, wherein the fatty acid is oleic acid. E7. The immunogenic composition of embodiment E5, wherein the fatty acid salt is sodium oleate. E8. The immunogenic composition according to any one of embodiments E1-E7, wherein the immunogenic composition comprises a fatty acid to RNA weight ratio (O:R) of from about 1.5:1 to about 24:1. E9. The immunogenic composition of embodiment E8, wherein the immunogenic composition has an O:R of at least 1.5:1. E10. The immunogenic composition of embodiment E9, wherein the immunogenic composition has an O:R selected from about 1.5:1, about 2:1, about 3:1, about 4:1, about 6:1, about 6.5:1, about 8:1, about 10:1, about 12:1, about 13:1 or about 24:1. E11. The immunogenic composition of embodiment E10, wherein the immunogenic composition has an O:R of 8:1. E12. A combination formulation comprising the immunogenic composition of any one of embodiments E1-E11 and one or more additional compositions. E13. The combination formulation of embodiment E12, wherein the one or more additional compositions comprise one or more LNPs. E14. The combination formulation of embodiment E13, wherein the one or more additional compositions do not comprise a LNP. E15. The combination formulation of embodiment E14, wherein the one or more additional compositions comprise proteins or protein subunits, conjugated antigens, inactivated viral or bacterial strains or fragments thereof, adjuvants, or any combination thereof. E16. The combination formulation of embodiments E12-E15, wherein the immunogenic composition of any one of embodiments E1-E11 and the additional composition are pre- mixed, post-mixed or co-lyophilized to form the combination formulation. E17. The immunogenic composition according to any one of embodiments E1-E11 and the combination formulation of embodiments E12-E16, wherein the immunogenic composition or combination formulation is liquid, frozen, or lyophilized. E18. The immunogenic composition according to any one of embodiments E1-E11 and the combination formulation of embodiments E12-E16, wherein the immunogenic composition or combination formulation has never been frozen. This invention also provides methods for delivering the immunogenic composition of any one of embodiments E1 to E11 or the combination formulation of embodiments E12-E16. This invention also provides methods for treating or preventing a disease or disorder in a subject comprising administering to the subject the immunogenic composition of any one of embodiments E1 to E11 or the combination formulation of embodiments E12-E16. This invention further provides methods of making the immunogenic composition and combination formulations of any one of embodiments E1-E16. DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG.1A-1B. FIG.1A depicts % of encapsulation and LNP size (d. nm) of LNPs before dialysis and after filtration. FIG.1B depicts % of positive expression in HEK293T cells when comparing LNP formulations in the presence of egg sphingomyelin (ESM) and cholesterol against a benchmark LNP formulation in the absence of ESM and cholesterol. FIG.2A-2C. FIG.2A depicts testing of various LNP formulations and measuring LNP size (d. nm), wherein the samples tested are described in Table 3. Successful combination of sitosterol and SM can be achieved using PBS as buffer. FIG.2B depicts fraction of positive expression in HEK293T, testing samples as described respectively in Table 3. FIG.2C depicts mean fluorescence intensity (MFI) of samples as described respectively in Table 3. FIG.3A-3B. FIG.3A depicts LNP size change of samples with various cationic lipid and ESM ratios. Samples tested are respectively described in Table 5. FIG.3B depicts % encapsulation efficiency (EE) change of samples with various cationic lipid and ESM ratios. Samples tested are respectively described in Table 5. FIG.4A-4B. FIG.4A depicts % of positive expression in HEK293T cells of samples with various cationic lipid and ESM ratios. Samples tested are respectively described in Table 6. FIG.4B depicts MFI of positive expression cells from samples respectively described in Table 6. FIG.5A-5B show the results of an in vivo immunogenicity study for Flu HA / Cali using ESM formulations at 3 weeks post dose 1 (3wks PD1; FIG.5A) and 2 weeks post dose 2 (2wks PD2; FIG.5B). FIG.6A-6B show the results of an in vivo immunogenicity study for RSV preF modRNA using ESM formulations at 3 weeks post dose 1 (3wks PD1; FIG.6A) and 2 weeks post dose 2 (2wks PD2; FIG.6B). FIG.7A-7B show the results of an in vivo immunogenicity study for H1N1 A / California modRNA using ESM formulations at 3 weeks post dose 1 (3wks PD1; FIG.7A) and 2 weeks post dose 2 (2wks PD2; FIG.7B). FIG.8 shows the post prime neutralization results (3wks post dose 1) from an in vivo study comparing the immunogenicity of 6 month aged Flu B / Austria modRNA-LNP drug product that was never frozen (NF), freeze / thawed (F / T) and lyophilized with and without sodium oleate. FIG.9 shows the post boost neutralization results (2wks post dose 2) from an in vivo study comparing the immunogenicity of 6 month aged Flu B / Austria modRNA-LNP drug product that was never frozen (NF), freeze / thawed (F / T) and lyophilized with and without sodium oleate. FIG.10A-10B depicts 50% neutralization titer results 3 weeks post dose 1 (3wks PD1; FIG. 10A) and 2 weeks post dose 2 (2wks PD2; FIG.10B) against H1N1 A / California strain from drug product formulations (see Table 21 and Table 22). FIG.11A-11B depicts 50% neutralization titer results 3 weeks post dose 1 (3wks PD1; FIG. 11A) and 2 weeks post dose 2 (2wks PD2; FIG.11B) against RSV M37 strain from drug product formulations (see Table 21 and Table 22). FIG.12A-12B depicts 50% neutralization titer results 3 weeks post dose 1 (3wks PD1; FIG. 12A) and 2 weeks post dose 2 (2wks PD2; FIG.12B) against RSV B18537 strain from drug product formulations (see Table 21 and Table 22). FIG.13A-13F show liquid stability for quadrivalent modRNA (QuadMod) LNP Flu formulations with sodium oleate post 1X freeze / thaw (F / T) followed by up to 12 months storage at 5ºC by measuring: %EE (FIG.13A); mRNA integrity by Fragment Analyzer (FIG.13B); LNP size (diameter in nm) (FIG.13C); PDI (FIG.13D); IVE (%Pos) (FIG.13E); and IVE (EC50) (FIG. 13F) wherein * denotes >top dose of curve. FIG.14A-14B show colloidal stability results for QuadMod Flu formulations with sodium oleate up to 12 months at 5ºC: percent late migrating species (LMS) (FIG 14A); percent lipid-RNA adducts (FIG.14B). FIG.15A-15B show various methods for addition of a fatty acid or a derivative or salt thereof, to a RNA-LNP drug product: FIG.15A shows Methods 1-3; and FIG.15B shows Methods 4A, 4B and 5. FIG.16A-F show the results of stability evaluation of a low concentration (0.01 mg / mL) Flu B modRNA drug product + / - sodium oleate after freeze / thaw and storage at 5ºC over a 6 month period of time: particle size (nm) (FIG.16A); PDI (FIG.16B); encapsulation efficiency (%EE) (FIG.16C); mRNA Integrity (%Main) (FIG.16D); % late migrating species (LMS) (FIG.16E); in vitro expression in adherent cells (IVE EC50 (pg / well)) (FIG.16F). DETAILED DESCRIPTION The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. It is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. It is to be also understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Described below are embodiments of the invention, where for convenience Embodiment 1 (E1) is identical to the embodiment of claim 1 provided herein. Exemplary embodiments (E) of the invention provided herein include: E1. An immunogenic composition comprising a) a fatty acid, including a derivative or salt thereof; b) one or more lipid nanoparticles (LNP), wherein the LNP comprises: i. an ionizable cationic lipid; ii. cholesterol, a cholesterol analog or a mixture of cholesterol and a cholesterol analog; iii. a neutral lipid; and iv. a polymer-conjugated lipid: and c) one or more ribonucleic acids (RNA) comprising at least one open reading frame (ORF) encoding a polypeptide of a gene of interest or an immunogenic fragment thereof, wherein the RNA is encapsulated in the LNP of item (b). E2. The immunogenic composition of embodiment E1, wherein the fatty acid is an unsaturated fatty acid. E3. The immunogenic composition of embodiment E2, wherein the unsaturated fatty acid is a monounsaturated fatty acid, a di-unsaturated fatty acid or a polyunsaturated fatty acid. E4. The immunogenic composition of embodiment E3, wherein the fatty acid is selected from oleic acid, arachidonic acid, eruric acid, linolenic acid, ricinoleic acid, palmitoleic acid, linoleic acid, or a derivative or salt thereof. E5. The immunogenic composition of embodiment E4, wherein the salt is selected from sodium, potassium, magnesium or calcium. E6. The immunogenic composition of embodiment E4, wherein the fatty acid is oleic acid. E7. The immunogenic composition of embodiment E5, wherein the fatty acid salt is sodium oleate. E8. The immunogenic composition of embodiment E4, wherein the fatty acid derivative is a derivative of ricinoleic acid. As used herein, the term “fatty acid” shall mean an aliphatic acid, consisting of a carboxylic acid functional group at the polar end and a hydrocarbon chain at the non-polar end of the fatty acid. The general structure for a fatty acid in mono form is: wherein R is a hydrogen atom or an ammonium, sodium, potassium, magnesium, orcalcium cation. The chain lengths for fatty acids are 4 to 40 carbons in length (i.e. n is 2 to 38).Examples of fatty acids include, but are not limited to, oleic acid, arachidonic acid, eruric acid, linolenic acid, ricinoleic acid, palmitoleic acid, linoleic acid. A “fatty acid salt” e.g. sodium salt of oleic acid, also known as sodium oleate, is a compound formed when the carboxylic acid group of fatty acid is neutralized by sodium hydroxide (NaOH) or other neutralizing agent, essentially creating a salt with the chemical formula "CH3(CH2)7CH=CH(CH2)7COONa" where "COONa" represents the sodium carboxylate group. Salts include, but are not limited to, sodium, potassium, magnesium or calcium. E9. The immunogenic composition of embodiment E1, wherein the fatty acid derivative is Kolliphor EL® or Cremaphor EL®. E10. The immunogenic composition according to any one of embodiments E1-E9, wherein the immunogenic composition comprises a fatty acid to RNA weight ratio (O:R) of from about 1.5:1 to about 24:1. E11. The immunogenic composition of embodiment E10, wherein the immunogenic composition has an O:R of at least 1.5:1. E12. The immunogenic composition of embodiment E11, wherein the immunogenic composition has an O:R selected from about 1.5:1, about 2:1, about 3:1, about 4:1, about 6:1, about 6.5:1, about 8:1, about 10:1, about 12:1, about 13:1 or about 24:1. E13. The immunogenic composition of embodiment E12, wherein the immunogenic composition has an O:R of 8:1. E14. The immunogenic composition according to any one of embodiments E1-E13, wherein the cholesterol analog is selected from sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, β-sitosterol, stigmastanol, β-sitostanol, ergosterol, fecosterol, lupeol, cycloartenol, Δ5-avenasterol, Δ7-avenasterol or a Δ7-stigmasterol, tomatidine, ursolic acid or alpha-tocopherol, including analogs, salts or esters thereof. E15. The immunogenic composition of embodiment E14, wherein the cholesterol analog is β- sitosterol, stigmasterol or campesterol. E16. The immunogenic composition of embodiment E15, wherein the cholesterol analog is β- sitosterol. E17. The immunogenic composition according to any one of embodiments E1-E16, wherein the molar ratio of the cholesterol analog to cholesterol in the mixture of the cholesterol analog and cholesterol is 6:4, 1:1 or 4:6. E18. The immunogenic composition of embodiment E17, wherein the mixture of the cholesterol analog and cholesterol has a molar ratio of 6:4 of cholesterol analog to cholesterol. E19. The immunogenic composition of embodiment E18, wherein the mixture comprises β- sitosterol and cholesterol. E20. The immunogenic composition of embodiment E19, wherein the mixture comprises a molar ratio of β-sitosterol:cholesterol of 6:4. E21. The immunogenic composition of any one of embodiments E1- E20, wherein the ionizable cationic lipid is selected from N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3]-dioxolane (DLin-K-DMA), (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315), 2-hexyldecyl 6-[(2-{[4-(heptylcarbonylamino)butyl]-N- methylamino}ethyl)[5-(2-hexyldecyloxycarbonyl)pentyl]amino]hexanoate (ALC-0515), 2-(7- (4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diyl bis(2-heptylnonanoate) (PF- 9032), heptadecane-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM-102), or a mixture thereof. E22. The immunogenic composition of embodiment E21, wherein the ionizable cationic lipid is ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC- 0315) having the structure: . E23. The immunogenic composition of embodiment E21, wherein the ionizable cationic lipid is 2-hexyldecyl 6-[(2-{[4-(heptylcarbonylamino)butyl]-N-methylamino}ethyl)[5-(2- hexyldecyloxycarbonyl)pentyl]amino]hexanoate (ALC-0515) having the structure: . E24. The immunogenic composition of embodiment E21, wherein the ionizable cationic lipid is 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diyl bis(2- heptylnonanoate) (PF-9032) having the structure: . E25. The immunogenic composition of any one of embodiments E1-E24, wherein the neutral lipid is a phospholipid. E26. The immunogenic composition of embodiment E25, wherein the phospholipid is a sphingolipid. E27. The immunogenic composition of embodiment E26, wherein the sphingolipid is a sphingomyelin. E28. The immunogenic composition of any one of embodiments E1-E27, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16- O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2- oleoylphosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidyl serine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diemcoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), l,2-dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE); l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, or a mixture thereof. E29. The immunogenic composition of embodiment E28, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl- oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18- 1-trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE), or a mixture thereof. E30. The immunogenic composition of embodiment E29, wherein the neutral lipid is 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC). E31. The immunogenic composition of embodiment E28, wherein the neutral lipid is egg sphingomyelin (ESM). E32. The immunogenic composition of any one of embodiments E1-E31, wherein the polymer-conjugated lipid is a pegylated lipid. E33. The immunogenic composition of embodiment E32, wherein the pegylated lipid is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) or a PEG dialkyoxypropylcarba PEGylated diacylglycerol (PEG-DAG), 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), 4-O- (2′,3′-di(tetradecanoyloxy)propyl-1-O-((O-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), PEG-ceramide, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159) or α-(3’-[(1,2-di[myristyloxy]propanoxy) carbonylamino]propyl)-ω-methoxy, polyoxyethylene (PEG-C-DMG). E34. The immunogenic composition of embodiment E33, wherein the pegylated lipid is ALC- 0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide) having the structure: . E35. The immunogenic composition of any one of embodiments E1-E31, wherein the polymer-conjugated lipid is a terminally activated polyoxazoline (POZ)-conjugated lipid. E36. The immunogenic composition of embodiment E35, wherein the POZ-conjugated lipid is polyethyloxazoline-dimyristoylamide (PEOZ-dma) having the structure: , wherein n is an integer from 18-22. E37. The immunogenic composition of any one of embodiments E1-E36, wherein the LNP comprises about 15-60 mol % ionizable cationic lipid, about 13-60 mol % cholesterol or mixture of cholesterol and a cholesterol analog, about 3-30 mol % neutral lipid, and about 0.5-10 mol % polymer-conjugated lipid. E38. The immunogenic composition according to any one of embodiments E1-E37, wherein the lipid nanoparticle comprises about 20 mol % to about 60 mol % ionizable cationic lipids, about 18.5 mol % to about 48.5 mol % mixture of cholesterol and a cholesterol analog, about 0 mol % to about 30 mol % neutral lipids, and about 0 mol % to about 10 mol % polymer-conjugated lipid. E39. The immunogenic composition of any of embodiments E37-E38, wherein the immunogenic composition comprises a mol% ratio of ionizable cationic lipid:cholesterol:neutral lipid:polymer-conjugated lipid:fatty acid or derivative or salt thereof selected from the group: a) 42.22:36.18:8.89:1.6:11.11; b) 40.71:34.88:8.57:1.54:14.29; c) 38:32.56:8:1.44:20; d) 35.62:30.52:7.5:1.35:25; e) 31.67:27.13:6.67:1.2:33.34; f) 30.81:26.4:6.49:1.17:35.14; g) 28.5:24.42:6:1.08:40; h) 23.75;20.35:5:0.90:50; or i) 15.83:13.57:3.33:0.6:66.67. E40. The immunogenic composition of any of embodiments E37-E38, wherein the immunogenic composition comprises a mol% ratio of ionizable cationic lipid:cholesterol analog:cholesterol:neutral lipid:polymer conjugated lipid:fatty acid or fatty acid salt selected from the group: a) 42.22:21.71:14.47:8.89:1.6:11.11; b) 40.71:20.93:13.95:8.57:1.54:14.29; c) 38:13.02:19.54:8:1.44:20; d) 35.62:18.31:12.21:7.5:1.35:25; e) 31.67:10.85:16.28:6.67:1.2:33.34; f) 30.81:10.56:15.84:6.49:1.17:35.14; g) 28.5:14.65:9.77:6:1.08:40; h) 25.91:13.32:8.88:5.45:0.98:45.46; or i) 22.8:11.72:7.81:4.8:0.86:52. E41. The immunogenic composition of any one of embodiments E37, E38 and E40, wherein the cholesterol analog is β-sitosterol. E42. The immunogenic composition of embodiment E41, wherein the LNP comprises a combination of lipids selected from: a) ALC-0315, beta-sitosterol, cholesterol, DSPC, and ALC-0159; b) ALC-0315, beta-sitosterol, cholesterol, ESM, and ALC-0159; c) ALC-0315, beta-sitosterol, cholesterol, DSPC, and PEOZ; d) ALC-0315, beta-sitosterol, cholesterol, ESM, and PEOZ; e) ALC-0515, beta-sitosterol, cholesterol, DSPC, and ALC-0159; f) ALC-0515, beta-sitosterol, cholesterol, ESM, and ALC-0159; g) ALC-0515, beta-sitosterol, cholesterol, DSPC, and PEOZ; h) ALC-0515, beta-sitosterol, cholesterol, ESM, and PEOZ; i) PF-9032, beta-sitosterol, cholesterol, DSPC, and ALC-0159; j) PF-9032, beta-sitosterol, cholesterol, ESM, and ALC-0159; k) PF-9032, beta-sitosterol, cholesterol, DSPC, and PEOZ; or l) PF-9032, beta-sitosterol, cholesterol, ESM, and PEOZ. E43. The immunogenic composition of embodiment E39, wherein the LNP comprises a combination of lipids selected from: a) ALC-0315, cholesterol, DSPC, and ALC-0159; b) ALC-0315, cholesterol, ESM, and ALC-0159; c) ALC-0315, cholesterol, DSPC, and PEOZ; d) ALC-0315, cholesterol, ESM, and PEOZ; e) ALC-0515, cholesterol, DSPC, and ALC-0159; f) ALC-0515, cholesterol, ESM, and ALC-0159; g) ALC-0515, cholesterol, DSPC, and PEOZ; h) ALC-0515, cholesterol, ESM, and PEOZ; i) PF-9032, cholesterol, DSPC, and ALC-0159; j) PF-9032, cholesterol, ESM, and ALC-0159; k) PF-9032, cholesterol, DSPC, and PEOZ; or l) PF-9032, cholesterol, ESM, and PEOZ. E44. The immunogenic composition according to any of embodiments E1-E43, wherein the LNP size is at least 40 nm. E45. The immunogenic composition according to any of embodiments E1-E44, wherein the LNP size is at most 180 nm. E46. The immunogenic composition according to any one of embodiments E1-E45, wherein the RNA is modRNA or saRNA. E47. The immunogenic composition of embodiment E46, wherein the RNA comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail. E48. The immunogenic composition of embodiment E47, wherein the 5’ cap is a 5’ cap analog. E49. The immunogenic composition according to any of embodiments 46 to 48, wherein the RNA further comprises one or more modified nucleotides. E50. The immunogenic composition of embodiment E49, wherein the modified nucleotide is selected from pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5- methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2′-O-methyl uridine or N1-Methylpseudourodine-5’-triphosphate (m1ΨTP). E51.The immunogenic composition of embodiment E50, wherein the modified nucleotide is N1- Methylpseudourodine-5’-triphosphate (m1ΨTP). E52. The immunogenic composition according to any of embodiments E46-E51, wherein the RNA open reading frame is codon-optimized. E53. The immunogenic composition of according to any one of embodiments E1-E52, wherein the RNA has an integrity greater than 85%. E54. The immunogenic composition of according to any one of embodiments E1-E53, wherein the RNA has a purity of greater than 85%. E55. The immunogenic composition according to any one of embodiments E1-54, wherein at least 80% of the total RNA in the immunogenic composition is encapsulated. E56. The immunogenic composition according to any one of embodiments E1-E55, wherein the percentage of intact mRNA encapsulated in the LNP is at least 80%. E57. The immunogenic composition according to any one of embodiments E1-E56, wherein the LNP comprises an N:P ratio of from about 2:1 to about 30:1. E58. The immunogenic composition of embodiment E57, wherein the LNP has an N:P ratio of at least 5. E59. The immunogenic composition of embodiment E57, wherein the LNP has an N:P ratio of at least 6. E60. The immunogenic composition of embodiment E57, wherein the LNP has an N:P ratio of 10. E61. The immunogenic composition of embodiment E57, wherein the LNP has an N:P ratio of 6. E62. The immunogenic composition according to any one of embodiments E1-E61, wherein the immunogenic composition further comprises a buffer. E63. The immunogenic composition of embodiment E62, wherein the buffer is Tris. E64. The immunogenic composition of embodiment E63, wherein the buffer is 10 mM Tris. E65. The immunogenic composition according to any one of embodiment E62, wherein the buffer is phosphate buffered saline (PBS). E66. The immunogenic composition according to any one of embodiments E1-E65, wherein the immunogenic composition further comprises sucrose. E67. The immunogenic composition of embodiment E66, wherein the immunogenic composition comprises 300 mM sucrose. E68. The immunogenic composition according to any one of embodiments E1-E67, wherein the immunogenic composition does not comprise sodium chloride. E69. The immunogenic composition according to any one of embodiments E1-E68, wherein the immunogenic composition has a pH from about 4 to about 11, from about 5 to about 10, from about 6 to about 9, from about 7 to about 8, or from about 7.3 to about 7.5. E70. The immunogenic composition according to embodiment E69, wherein the immunogenic composition has a pH of 7.4. E71. The immunogenic composition according to any one of embodiments E1-E70, wherein the immunogenic composition has less than or equal to 12.5 EU / mL of bacterial endotoxins. E72. A combination formulation comprising the immunogenic composition of any one of embodiments E1-E71 and one or more additional compositions. E73. The combination formulation of embodiment E72, wherein the one or more additional compositions comprise one or more LNPs. E74. The combination formulation of embodiment E73, wherein the one or more additional compositions do not comprise any LNP. E75. The combination formulation of embodiment E74, wherein the one or more additional compositions comprise proteins or protein subunits, conjugated antigens, inactivated viral or bacterial strains or fragments thereof, adjuvants, or any combination thereof. E76. The combination formulation of embodiments E72-E75, wherein the additional composition comprises TritonX. E77. The combination formulation of embodiments E72-E76, wherein the immunogenic composition of any one of embodiments E1-E71 and the additional composition are pre- mixed, post-mixed or co-lyophilized to form the combination formulation. E78. The immunogenic composition according to any one of embodiments E1-E71 and the combination formulation of embodiments E72-E77, wherein the immunogenic composition or combination formulation is liquid, frozen, or lyophilized. E79. The immunogenic composition according to any one of embodiments E1-E71 and the combination formulation of embodiments E72-E77, wherein the immunogenic composition or combination formulation has never been frozen. E80. The immunogenic composition or the combination formulation of any one of embodiments E1-E79, wherein the immunogenic composition or combination formulation has a particle size which is less than that of an immunogenic composition which does not comprise a fatty acid, derivative or salt thereof. E81. The immunogenic composition or the combination formulation of any one of embodiments E1-E79, wherein the immunogenic composition or combination formulation has a PDI which is less than that of an immunogenic composition which does not comprise a fatty acid, derivative or salt thereof. E82. The immunogenic composition or combination formulation of embodiments E80 or E81, wherein the particle size or PDI of the immunogenic composition is about 95% or less, about 90% or less, about 85% or less, about % 80 less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, as compared to that of an immunogenic composition which does not comprise a fatty acid, derivative or salt thereof. E83. The immunogenic composition or the combination formulation of any one of embodiments E1-E79, wherein the immunogenic composition or combination formulation has less adducts than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E84. The immunogenic composition or the combination formulation of any one of embodiments E1-E79, wherein the immunogenic composition or combination formulation is more stable at 5ºC over time than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E85. The immunogenic composition or combination formulation of embodiment E84 , wherein the immunogenic composition or combination formulation is stable up to about 5 ºC for at least 3 months. E86. The immunogenic composition or combination formulation of embodiment E84 , wherein the immunogenic composition or combination formulation is stable up to about 5 ºC for at least 3 months, at least 6 months, at least 9 months or at least 12 months or longer. E87. The immunogenic composition or the combination formulation of any one of embodiments E1-E79, wherein the immunogenic composition or combination formulation is more stable after one or more freeze / thaw cycles than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E88. The immunogenic composition or combination formulation of embodiment E79, wherein the immunogenic composition or combination formulation has more RNA integrity than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E89. The immunogenic composition or combination formulation of embodiment E79, wherein the immunogenic composition or combination formulation has higher RNA encapsulation than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E90. The immunogenic composition or combination formulation of embodiment E79, wherein the immunogenic composition or combination formulation has higher in vitro expression than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E91. The immunogenic composition or combination formulation of embodiment E79, wherein the immunogenic composition or combination formulation has higher immunogenicity than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof. E92. The immunogenic composition or combination formulation of embodiment E79, wherein the immunogenic composition or combination formulation is a vaccine. E93. A method of making the immunogenic composition of any one of embodiments E1-E71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with the lipids of an LNP (organic phase); ii) filtering the mixture of step (i) via Tangential Flow Filtration / BBR; iii) dissolving a fatty acid salt in a buffer in an amount that will achieve the desired O:R ratio of the composition; iv) compounding the buffer solution of step (iii) with the filtered mixture of step (ii); and v) the compounded mixture of step (iv) undergoes terminal filtration, thereby making the immunogenic composition of any one of embodiments E1-E71. E94. A method of producing a stable immunogenic composition of any one of embodiments E1- E71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with the lipids of an LNP (organic phase); ii) filtering the mixture of step (i) via Tangential Flow Filtration / BBR; iii) mixing a fatty acid with a compound in a buffer to make a fatty acid salt in an amount that will achieve the desired O:R ratio of the composition; iv) adding the buffer solution of step (iii) to the filtered mixture of step (ii) and compounding; and v) terminal filtration of the compounded mixture of step (iv), thereby making the immunogenic composition of any one of embodiments E1-E71. E95. A method of making the immunogenic composition of any one of embodiments E1-E71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with an organic phase comprising the lipids of an LNP and a fatty acid in an amount that will achieve the desired O:R ratio of the composition; ii) filtering the mixture of step (i) via Tangential Flow Filtration / BBR; iii) compounding the filtered mixture of step (ii); and iv) the compounded mixture of step (iii) undergoes terminal filtration, thereby making the immunogenic composition of any one of embodiments E1-E71. E96. A method of making the immunogenic composition of any one of embodiments E1-E71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with the lipids of an LNP (organic phase); ii) mixing a fatty acid salt or a fatty acid with a compound to make a fatty acid salt with a buffer in an amount that will achieve the desired O:R ratio of the composition (quench buffer); iii) mixing the mixture of step (i) with the quench buffer of step (ii) and filtering via Tangential Flow Filtration / BBR; iv) compounding the filtered mixture of step (iii); and v) the compounded mixture of step (iv) undergoes terminal filtration, thereby making the immunogenic composition of any one of embodiments E1-E71. E97. A method of making the immunogenic composition of any one of embodiments E1-E71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with an organic phase comprising the lipids of an LNP and a fatty acid salt in an amount that will achieve the desired O:R ratio of the composition; ii) mixing the mixture of step (i) with a quench buffer having pH 7.4; iii) filtering the mixture of step (ii) via Tangential Flow Filtration / BBR; iv) compounding the filtered mixture of step (iii); and v) the compounded mixture of step (iv) undergoes terminal filtration, thereby making the immunogenic composition of any one of embodiments E1-E71. E98. A method for delivering a ribonucleic acid to a subject in need thereof comprising administering to the subject the immunogenic composition or combination formulation of any one of embodiments E1-E79. E99. The immunogenic composition or combination formulation of any one of embodiments E1- E79 for use as a component of a medicament. E100. Use of the immunogenic composition or combination formulation of any one of embodiments E1-E79 for the manufacture of a medicament. “Stability,” “stabilized,” and “stable” as used herein refers to the resistance of LNPs to chemical or physical changes (e.g., chemical degradation, particle size change, phase separation, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and / or in-use conditions, e.g., when stress is applied such as shear force, freeze / thaw stress, etc. The “stable” formulations shall include the immunogenic compositions and combinations thereof of the disclosure. The “stable” formulations shall preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the chemical purity (e.g., chromatographic purity) of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions. The “stable” formulations of the disclosure also preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the physical properties (e.g., phase homogeneity or heterogeneity, particle size, turbidity, encapsulation efficiency, etc.) of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions. For example, the “stable” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the physical stability of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions. For example, the physical stability refers to little or no phase separation of LNP components, e.g., phase separation of a fraction of the structure lipid such as cholesterol or phase separation of a fraction of the ionizable lipid such as an ionizable amino lipid from the remainder of LNP. For example, the “stable” formulations of the disclosure also preferably have an increase of about 20%, 10%, 5%, 1%, 0.5% or less of a starting, standard, or reference LNP mean size under given manufacturing, preparation, transportation, storage and / or in-use conditions. For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at 5° C or lower for at least one month. For example, the formulation has an increase in LNP mean size of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after 1, 2, 3, or more, up to 30 freeze / thaw cycles. For example, the “stable” formulation has an increase in turbidity of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at 5°C or lower for at least one month, e.g., via nephelometric turbidity analysis. For example, the “stable” formulation has an increase in turbidity of about 20% or less (e.g., about 15%, about 10%, about 5% or less) after after 1, 2, 3, or more, up to 30 freeze / thaw cycles, e.g., via nephelometric turbidity analysis. For example, the physical stability of LNPs is determined by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), turbidity analysis, flow microscopy analysis, flow cytometry, FTIR microscopy, resonant mass measurement (RMM), Raman microscopy, filtration, laser diffraction, electron microscopy, atomic force microscopy (AFM), static light scattering (SLS), multi-angle static light scattering (MALS), field flow fractionation (FFF), analytical ultracentrifugation (AUC), or any combination thereof. For example, the “stable” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the encapsulation efficiency of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions. For example, the encapsulation efficiency is substantially the same after storage at about 5° C. or lower for at least one month. For example, the encapsulation efficiency may decrease for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about 5°C. or lower for at least one month. For example, the encapsulation efficiency is substantially the same after 1, 2, 3, or more, up to 30 freeze / thaw cycles. The “stable” formulations of the disclosure may also preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the biological activity of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions. For example, the formulation has little or no immunogenicity. For example, the immunogenicity is substantially the same after storage at about 5° C or lower for at least one month. For example, the immunogenicity may increase for about 20% or less (e.g., about 15%, about 10%, about 5% or less) after storage at about 5° C or lower for at least one month. For example, the immunogenicity is substantially the same after 1, 2, 3, or more, up to 30 freeze / thaw cycles. For example, the formulation has a lower immunogenicity as compared to a corresponding formulation which does not comprise the fatty acid, derivative or salt thereof. The “stable” formulations of the disclosure also preferably has an increase of about 20% 10%, 5%, 1%, 0.5% or less of a starting, standard, or reference or benchmark amount of impurities or adducts under given manufacturing, preparation, transportation, storage and / or in- use conditions. The “stable” formulations of the disclosure also preferably has an increase of about 20% 10%, 5%, 1%, 0.5% or less of a starting, standard, or reference or benchmark amount of sub- visible particles or adducts under given manufacturing, preparation, transportation, storage and / or in-use conditions. The purity, LNP mean size, encapsulation efficiency, biological activity, immunogenicity, amount of impurities (or adducts) can be determined using any art-recognized method. For example, the LNP mean size can be measured dynamic light scattering (DLS). For example, the concentration of a component of the formulation can be determined using routine methods such as UV-Vis spectrophotometry and high pressure liquid chromatography (HPLC). For example, amount of sub-visible particles can be determined by micro-flow imaging (MFI) and adducts by RP-HPLC. In certain embodiments, the present formulations are stable at temperatures of about 5° C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stable for at least 6-12 months at 5° C. In certain embodiments, the present formulations are stable at temperatures ranging from about 2 to 8° C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stable for at least 2 months at 2 to 8° C. In certain embodiments, the present formulations are stable at a temperature of about 5° C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In one embodiment, the formulation is stable for at least 2 months at about 5° C. In a particular embodiment, a formulation of the disclosure is stable at a temperature ranging between about −20° C. and 5° C at a nucleic acid concentration (e.g., an mRNA concentration) of up to 2 mg / mL for at least 2 weeks, for at least 4 weeks, for at least 8 weeks, for at least 12 weeks, for at least 16 weeks, for at least 32 weeks, for at least a year, or for at least two years. In a particular embodiment, a formulation of the disclosure is stable at a temperature ranging between about −20° C. and 5° C at a nucleic acid concentration (e.g., an mRNA concentration) of up to 1 mg / mL for at least 2 weeks, for at least 4 weeks, for at least 8 weeks, for at least 12 weeks, or for at least 16 weeks. The present disclosure relates to mRNA vaccines in general. Several mRNA vaccine platforms are available in the prior art. The basic structure of in vitro transcribed (IVT) mRNA closely resembles “mature” eukaryotic mRNA and includes (i) a protein-encoding open reading frame (ORF), flanked by (ii) 5′ and 3′ untranslated regions (UTRs), and at the end sides (iii) a 7- methyl guanosine 5′ cap structure and (iv) a 3′ poly(A) tail. The non-coding structural features play important roles in the pharmacology of mRNA and can be individually optimized to modulate the mRNA stability, translation efficiency, and immunogenicity. By incorporating modified nucleosides, mRNA transcripts referred to as “nucleoside-modified mRNA” or “modRNA” can be produced with reduced immunostimulatory activity, and therefore an improved safety profile can be obtained. In addition, modified nucleosides allow the design of mRNA vaccines with strongly enhanced stability and translation capacity, as they can avoid the direct antiviral pathways that are induced by type IFNs and are programmed to degrade and inhibit invading mRNA. For instance, the replacement of uridine with pseudouridine in IVT of mRNA reduces the activity of 2′-5′-oligoadenylate synthetase, which regulates the mRNA cleavage by RNase L. In addition, lower activities are measured for protein kinase R, an enzyme that is associated with the inhibition of the mRNA translation process. Besides the incorporation of modified nucleotides, other approaches have been validated to increase the translation capacity and stability of mRNA. One example is the development of “sequence-engineered mRNA”. Here, mRNA expression can be strongly increased by sequence optimizations in the ORF and UTRs of mRNA, for instance by enriching the GC content, or by selecting the UTRs of long-lived mRNA molecules. Also, several modifications have been implemented at the end structures of mRNA. Anti- reverse cap (ARCA) modifications can ensure the correct cap orientation at the 5′ end, which yields almost complete fractions of mRNA that can efficiently bind the ribosomes. Other cap modifications, such as phosphorothioate cap analogs, can further improve the affinity towards the eukaryotic translation initiation factor 4E, and increase the resistance against the RNA decapping complex. Conversely, by modifying its structure, the potency of mRNA to trigger innate immune responses can be further improved, but to the detriment of translation capacity. By stabilizing the mRNA with either a phosphorothioate backbone, or by its precipitation with the cationic protein protamine, antigen expression can be diminished, but stronger immune-stimulating capacities can be obtained. In one aspect the invention relates to an immunogenic composition comprising an mRNA molecule that encodes one or more polypeptides or fragments thereof of an antigen. In some embodiments, the mRNA molecule comprises a nucleoside-modified mRNA. mRNA useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 '-terminus of the first region (e.g., a 5’-UTR), a second flanking region located at the 3 '-terminus of the first region (e.g., a 3’-UTR), at least one 5 '-cap region, and a 3 '-stabilizing region. In some embodiments, the mRNA of the disclosure further includes a poly-A region or a Kozak sequence (e.g., in the 5 '-UTR). In some cases, mRNA of the disclosure may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, mRNA of the disclosure may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence, and / or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3 '-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-0-methyl nucleoside and / or the coding region, 5 '-UTR, 3 '-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5-methoxyuridine), a 1-substituted pseudouridine (e.g., 1-methyl-pseudouridine), and / or a 5- substituted cytidine (e.g., 5-methyl-cytidine). The compositions described herein comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame (ORF), and encodes a 3′ UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template. A “5′ untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (e.g., 5′) from the start codon (e.g., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide of an antigen of interest. A “3′ untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (e.g., 3′) from the stop codon (e.g., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide of an antigen of interest. An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide of an antigen of interest. A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (e.g., 3′), from the 3′ UTR which contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation. In preferred embodiments, the 3′ polyadenylation tail comprises about 80 adenosine monophosphates (e.g.80A). In one aspect, the 3′ polyadenylation tail comprises SEQ ID NO: 3. In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides. In some embodiments, a LNP includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the phosphate groups in an RNA. In general, a lower N:P ratio is preferred. In one embodiment, the lipid is an ionizable lipid. In another embodiment, the lipid is an ionizable cationic lipid. The one or more RNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1 or 30:1. In certain embodiments, the N:P ratio may be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1 , about 5.5 :1, about 5.67:1, about 6.0:1, about 6.5:1 , or about 7.0:1. For example, the N:P ratio may be about 5.67:1. In some embodiments, the composition comprises a second LNP, wherein the second LNP does not encapsulate a polynucleotide as described in WO2023057930, the entirety of which is hereby incorporated herein by reference. In some aspects, the compositions and methods described herein relate to frozen or lyophilized lipid nanoparticles encapsulating or associated with RNA in the presence of a cryoprotectant, preferably a carbohydrate cryoprotectant, and / or further in the presence of lipid nanoparticles that are devoid of nucleic acid, (e.g., not encapsulating and not associated with RNA (also referred herein as “blank” LNPs), or liposomes, or a higher cryoprotectant concentration) resulting in a composition comprising LNPs encapsulating RNA or associated with RNA that is characterized by, among other things, an improved integrity of the RNA after completion of the respective freezing or lyophilization process and which is further characterized by increased storage stability, such as, for example, with respect to storage for extended periods and / or under non-cooling conditions, as compared to a composition comprising lipid nanoparticles encapsulating or associated with RNA in the absence of the blank LNPs, or liposomes, or a higher cryoprotectant concentration when assessed under identical conditions. In other words, in some aspects, the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA, a second lipid nanoparticle that is devoid of nucleic acid, and a cryoprotectant that results in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. In some aspects, the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA and an increased cryoprotectant concentration that results in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. In some aspects, the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA and a second lipid nanoparticle that is devoid of nucleic acid that results in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. In some aspects, the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA and a liposome that results in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. In some aspects, the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA, a liposome, and an increased cryoprotectant concentration that result in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. In some aspects, the compositions and methods described herein relate to liquid (never frozen), frozen or lyophilized lipid nanoparticles encapsulating or associated with RNA in the presence of cholesterol or a cholesterol analog, and further in the presence of a fatty acid or derivative or salt thereof resulting in a composition comprising LNPs encapsulating RNA or associated with RNA that is characterized by, among other things, an improved integrity of the RNA and quality of the lipid nanoparticle after completion of the respective freezing and thawing process or lyophilization process, and which is also characterized by increased storage stability, such as, for example, with respect to storage for extended periods and / or under non-cooling conditions, and is further characterized by a decrease in lipid adduct formation, as compared to a composition comprising lipid nanoparticles encapsulating or associated with RNA in the absence of the cholesterol analog and the fatty acid or derivative or salt thereof when assessed under identical conditions, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. In addition, the composition and methods described herein result in a lyophilized, frozen, or liquid lipid nanoparticle drug product capable of combination with other product modalities, including but not limited to for example, subunit protein vaccines, conjugate vaccines or adjuvants, while maintaining the quality attributes of the original LNP particle, and which is also characterized by increased storage stability preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine. Advantageously, the compositions and methods thereof described herein are suitable for use at an industrial scale. The methods described herein may be used to produce, for example, a liquid (never frozen), frozen or lyophilized composition comprising LNPs encapsulating or associated with RNA having the above-mentioned properties in a reproducible and cost-effective manner. The composition comprising LNPs encapsulating or associated with RNA may advantageously be stored, shipped and applied, e.g., as a vaccine, without a cold chain, while the integrity and the biological activity of the RNA in the composition remain unexpectedly high. mRNA of the disclosure may include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the poly A tail, and any combination of (a, b, c, or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). mRNA of the disclosure may include one or more alternative components, as described herein, which impart useful properties including increased stability and / or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. For example, a modRNA may exhibit reduced degradation in a cell into which the modRNA is introduced, relative to a corresponding unaltered mRNA. These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and / or viability of contacted cells, as well as possess reduced immunogenicity. mRNA of the disclosure may include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof. The mRNA useful in a LNP can include any useful modification or alteration, such as to the nucleobase, the sugar, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). In certain embodiments, alterations (e.g., one or more alterations) are present in each of the nucleobase, the sugar, and the internucleoside linkage. Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs), e.g., the substitution of the 2'-OH of the ribofuranosyl ring to 2'-H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or hybrids thereof. Additional alterations are described herein. mRNA of the disclosure may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly altered in a mRNA, or in a given predetermined sequence region thereof. In some instances, all nucleotides X in a mRNA (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C. Different sugar alterations and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in a polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. An alteration may also be a 5'- or 3 '-terminal alteration. In some embodiments, the polynucleotide includes an alteration at the 3 '-terminus. The polynucleotide may contain from about 1% to about 100% alternative nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, e.g., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of a canonical nucleotide (e.g., A, G, U, or C). Polynucleotides may contain at a minimum zero and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least 5% alternative nucleotides, at least 10% alternative nucleotides, at least 25% alternative nucleotides, at least 50% alternative nucleotides, at least 80% alternative nucleotides, or at least 90% alternative nucleotides. For example, polynucleotides may contain an alternative pyrimidine such as an alternative uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in a polynucleotide is replaced with an alternative uracil (e.g., a 5-substituted uracil). The alternative uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with an alternative cytosine (e.g., a 5-substituted cytosine). The alternative cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some instances, nucleic acids do not substantially induce an innate immune response of a cell into which the polynucleotide (e.g., mRNA) is introduced. Features of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc., and / or 3) termination or reduction in protein translation. In some embodiments, the mRNA comprises one or more alternative nucleoside or nucleotides. The alternative nucleosides and nucleotides can include an alternative nucleobase. A nucleobase of a nucleic acid is an organic base such as a purine or pyrimidine or a derivative thereof. A nucleobase may be a canonical base (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties, e.g., increased stability such as resistance to nucleases. Non-canonical or modified bases may include, for example, one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction. In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (ψ), pyridin-4- one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio- uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uracil (ho5U), 5-aminoallyl- uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m U), 5-methoxy- uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1 -carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl- uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5- methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5- aminomethyl-2-thio-uracil (nmVu), 5-methylaminomethyl-uracil (mnm5U), 5- methylaminomethyl-2-thio-uracil (mnmVu), 5-methylaminomethyl-2-seleno-uracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uracil (cmnmVu), 5-propynyl-uracil, 1- propynyl-pseudouracil, 5-taurinomethyl-uracil (xm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio- uracil(xm5s2U), 1 -taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, e.g., having the nucleobase deoxythymine), 1-methyl-pseudouridine (mV), 5-methyl-2- thio-uracil (m5s2U), l- methyl-4-thio-pseudouridine (m xj / ), 4-thio- 1-methyl-pseudouridine, 3- methyl-pseudouridine (m \| / ), 2 -thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l -methyl- 1- deaza-pseudouri dine, dihydrouracil (D), dihydropseudouridine, 5,6- dihydrouracil, 5-methyl- dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2- methoxy-4-thio-uracil, 4-methoxy- pseudouridine, 4-methoxy -2-thio-pseudouridine, N1-methyl- pseudouridine, 3-(3-amino-3- carboxypropyl)uracil (acp U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp ψ), 5- (isopentenylaminomethyl)uracil (inm5U), 5- (isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2'-0-dimethyl-uridine (m5Um), 2-thio-2'- 0_methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-0-methyl-uridine (mem Um), 5- carbamoylmethyl-2'-0-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyl- uridine (cmnm5Um), 3,2'-0- dimethyl-uridine (m Um), and 5-(isopentenylaminomethyl)-2'-0- methyl-uridine (inm5Um), 1- thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5- (carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio- uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(l-E-propenylamino)]uracil. In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza-cytosine, 6-aza- cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl- cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5- iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo- cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio- pseudoisocy tidine, 4-thio- 1 -methy 1-pseudoisocy tidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5 - methy 1- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2- methoxy-5- methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl- pseudoisocytidine, lysidine (k2C), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acetyl-2'-0-methyl- cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formyl-2'-0-methyl-cytidine (f5Cm), N4,N4,2'-0- trimethyl-cytidine (m42Cm), 1 -thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)- cytosine, and 5-(2-azidoethyl)-cytosine. In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6- chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methy 1-adenine (ml A), 2-methyl-adenine (m2A), N6- methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl- adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl- adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl- adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2- methylthio-adenine, 2-methoxy -adenine, N6,2'-0-dimethyl-adenosine (m6Am), N6,N6,2'-0- trimethyl-adenosine (m62Am), l,2'-0-dimethyl-adenosine (ml Am), 2-amino-N6-methyl-purine, 1- thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl- adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine. In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl- inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl), archaeosine (G+), 7-deaza-8-aza-guanine, 6- thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6- thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1 -methyl-guanine (mlG), N2- methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2, N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1 - methyl-6-thio- guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl- 2'-0-methyl- guanosine (m2Gm), N2,N2-dimethyl-2'-0-methyl-guanosine (m22Gm), 1 -methyl-2'- 0-methyl- guanosine (mlGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'-0-methyl- inosine (Im), l,2'-0-dimethyl-inosine (mllm), 1 -thio-guanine, and O-6-methyl-guanine. The alternative nucleobase of a nucleotide can be independently a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7- deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a] l,3,5 triazinones, 9- deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4- triazine, pyridazine; or 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and / or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine). The mRNA may include a 5 '-cap structure. The 5 '-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5 '-proximal introns removal during mRNA splicing. Endogenous polynucleotide molecules may be 5 '-end capped generating a 5 '-ppp-5' - triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the polynucleotide. This 5 '-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5 ' end of the polynucleotide may optionally also be 2'-0-methylated.5 '-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. Alterations to polynucleotides may generate a non-hydrolyzable cap structure preventing decapping and thus increasing polynucleotide half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, alternative nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 '-ppp-5 ' cap. Additional alternative guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides. Additional alterations include, but are not limited to, 2'-0-methylation of the ribose sugars of 5'-terminal and / or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxy group of the sugar. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (e.g., endogenous, wild-type, or physiological) 5 '-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (e.g., non-enzymatically) or enzymatically synthesized and / linked to a polynucleotide. For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5 '-5 '-triphosphate group, wherein one guanosine contains an N7-methyl group as well as a 3'-0-methyl group (e.g., N7, '-0-dimethyl-guanosine-5 '- triphosphate-5 '-guanosine, m7G-3'mppp-G, which may equivalently be designated 3' 0-Me- m7G(5')ppp(5')G). The 3'-0 atom of the other, unaltered, guanosine becomes linked to the 5 '- terminal nucleotide of the capped polynucleotide (e.g., an mRNA). The N7- and 3'-0-methylated guanosine provides the terminal moiety of the capped polynucleotide (e.g., mRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (e.g., N7,2'-0-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine, m7Gm- ppp-G). A cap may be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. 8,519,110, the cap structures of which are herein incorporated by reference. Alternatively, a cap analog may be a N7-(4-chlorophenoxy ethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7- (4-chlorophenoxy ethyl) substituted dinucleotide cap analogs include a N7-(4- chlorophenoxyethyl)-G(5 )ppp(5 ')G and a N7-(4-chlorophenoxyethyl)-m3 '-OG(5 )ppp(5 ')G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321 :4570-4574; the cap structures of which are herein incorporated by reference). In other instances, a cap analog useful in the polynucleotides of the present disclosure is a 4-chloro / bromophenoxy ethyl analog. While cap analogs allow for the concomitant capping of a polynucleotide in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5 '-cap structures of polynucleotides produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability. Alternative polynucleotides may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function, and / or structure as compared to synthetic features or analogs of the prior art, or which outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more authentic 5 '-cap structures useful in the polynucleotides of the present disclosure are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5'-endonucleases, and / or reduced 5'- decapping, as compared to synthetic 5 '-cap structures known in the art (or to a wild-type, natural or physiological 5 '-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '- triphosphate linkage between the 5 '-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide wherein the cap guanosine contains an N7-methylation and the 5 '-terminal nucleotide of the polynucleotide contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency, cellular stability, and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 ' cap analog structures known in the art. Other exemplary cap structures include 7mG(5 ')ppp(5 ')N,pN2p (Cap 0), 7mG(5 ')ppp(5 ')NlmpNp (Cap 1), 7mG(5 ')-ppp(5')NlmpN2mp (Cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (Cap 4). Because the alternative polynucleotides may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the mRNA may be capped. This is in contrast to -80% when a cap analog is linked to a polynucleotide in the course of an in vitro transcription reaction. 5 '-terminal caps may include endogenous caps or cap analogs. A 5 '- terminal cap may include a guanosine analog. Useful guanosine analogs include inosine, N1- methyl- guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA- guanosine, and 2-azido-guanosine. In some cases, a polynucleotide contains a modified 5 '-cap. A modification on the 5 '-cap may increase the stability of polynucleotide, increase the half-life of the polynucleotide, and could increase the polynucleotide translational efficiency. The modified 5 '-cap may include, but is not limited to, one or more of the following modifications: modification at the 2'- and / or 3 '-position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety. A 5'-UTR may be provided as a flanking region to the mRNA. A 5’ -UTR may be homologous or heterologous to the coding region found in a polynucleotide. Multiple 5 '-UTRs may be included in the flanking region and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and / or after codon optimization. In one embodiment, an ORF encoding an antigen is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein or incorporated by reference herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art—non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.To alter one or more properties of an mRNA, 5 '-UTRs which are heterologous to the coding region of an mRNA may be engineered. The mRNA may then be administered to cells, tissue or organisms and outcomes such as protein level, localization, and / or half-life may be measured to evaluate the beneficial effects the heterologous 5 ' -UTR may have on the mRNA. Variants of the 5 '-UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.5 '-UTRs may also be codon-optimized, or altered in any manner described herein. mRNAs may include a stem loop such as, but not limited to, a histone stem loop. The stem loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length. The histone stem loop may be located 3 '-relative to the coding region (e.g., at the 3 '-terminus of the coding region). As a non-limiting example, the stem loop may be located at the 3 '-end of a polynucleotide described herein. In some cases, an mRNA includes more than one stem loop (e.g., two stem loops). A stem loop may be located in a second terminal region of a polynucleotide. As a non-limiting example, the stem loop may be located within an untranslated region (e.g., 3'-UTR) in a second terminal region. In some cases, a mRNA which includes the histone stem loop may be stabilized by the addition of a 3 '-stabilizing region (e.g., a 3'- stabilizing region including at least one chain terminating nucleoside). Not wishing to be bound by theory, the addition of at least one chain terminating nucleoside may slow the degradation of a polynucleotide and thus can increase the half-life of the polynucleotide. In other cases, a mRNA, which includes the histone stem loop may be stabilized by an alteration to the 3 '-region of the polynucleotide that can prevent and / or inhibit the addition of oligio(U). In yet other cases, a mRNA, which includes the histone stem loop may be stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2',3 '-dideoxynucleoside 3 '-0- methylnucleosides, 3 -0- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein. In some instances, the mRNA of the present disclosure may include a histone stem loop, a poly-A region, and / or a 5 '-cap structure. The histone stem loop may be before and / or after the poly-A region. The polynucleotides including the histone stem loop and a poly-A region sequence may include a chain terminating nucleoside described herein. In other instances, the polynucleotides of the present disclosure may include a histone stem loop and a 5 '-cap structure. The 5 '-cap structure may include, but is not limited to, those described herein and / or known in the art. In some cases, the conserved stem loop region may include a miR sequence described herein. As a non-limiting example, the stem loop region may include the seed sequence of a miR sequence described herein. In another non-limiting example, the stem loop region may include a miR- 122 seed sequence. mRNA may include at least one histone stem-loop and a poly-A region or polyadenylation signal. In certain cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for a pathogen antigen or fragment thereof. In other cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for a therapeutic protein. In some cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for a tumor antigen or fragment thereof. In other cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for an allergenic antigen or an autoimmune self-antigen. An mRNA may include a polyA sequence and / or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may be a tail located adjacent to a 3' untranslated region of a nucleic acid. During RNA processing, a long chain of adenosine nucleotides (poly-A region) is normally added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3'-end of the transcript is cleaved to free a 3'-hydroxy. Then poly-A polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A region that is between 100 and 250 residues long. Unique poly- A region lengths may provide certain advantages to the alternative polynucleotides of the present disclosure. Generally, the length of a poly-A region of the present disclosure is at least 30 nucleotides in length. In another embodiment, the poly-A region is at least 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 70 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1700 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 1900 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In some instances, the poly-A region may be 80 nucleotides, 120 nucleotides, 160 nucleotides in length on an alternative polynucleotide molecule described herein. In other instances, the poly-A region may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length on an alternative polynucleotide molecule described herein. In some cases, the poly-A region is designed relative to the length of the overall alternative polynucleotide. This design may be based on the length of the coding region of the alternative polynucleotide, the length of a particular feature or region of the alternative polynucleotide (such as mRNA) or based on the length of the ultimate product expressed from the alternative polynucleotide. When relative to any feature of the alternative polynucleotide (e.g., other than the mRNA portion which includes the poly-A region) the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature. The poly-A region may also be designed as a fraction of the alternative polynucleotide to which it belongs. In this context, the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A region. In certain cases, engineered binding sites and / or the conjugation of mRNA for poly-A binding protein may be used to enhance expression. The engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the mRNA. As a non-limiting example, the mRNA may include at least one engineered binding site to alter the binding affinity of poly-A binding protein (PABP) and analogs thereof. The incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof. Additionally, multiple distinct mRNA may be linked together to the PABP (poly-A binding protein) through the 3'-end using alternative nucleotides at the 3'- terminus of the poly-A region. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and day 7 post-transfection. As a non-limiting example, the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site. In certain cases, a poly-A region may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A region recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis. In some cases, a poly-A region may also be used in the present disclosure to protect against 3 '-5 '-exonuclease digestion. In some instances, an mRNA may include a polyA-G Quartet. The G- quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A region. The resultant mRNA may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A region of 120 nucleotides alone. In some cases, mRNA may include a poly-A region and may be stabilized by the addition of a 3 '-stabilizing region. The mRNA with a poly-A region may further include a 5 '-cap structure. In other cases, mRNA may include a poly- A-G Quartet. The mRNA with a poly-A-G Quartet may further include a 5 '-cap structure. In some cases, the 3 '-stabilizing region which may be used to stabilize mRNA includes a poly-A region or poly-A-G Quartet. In other cases, the 3 '-stabilizing region which may be used with the present disclosure include a chain termination nucleoside such as 3 '-deoxyadenosine (cordycepin), 3 '-deoxyuridine, 3 '- deoxycytosine, 3 '-deoxyguanosine, 3 '-deoxy thymine, 2',3'- dideoxynucleosides, such as 2',3 '- dideoxyadenosine, 2',3 '-dideoxyuridine, 2',3 '- dideoxycytosine, 2', 3 '- dideoxyguanosine, 2',3 '-dideoxythymine, a 2'-deoxynucleoside, or an O-methylnucleoside. In other cases, mRNA which includes a polyA region or a poly-A-G Quartet may be stabilized by an alteration to the 3 '-region of the polynucleotide that can prevent and / or inhibit the addition of oligio(U). In yet other instances, mRNA which includes a poly-A region or a poly-A-G Quartet may be stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2',3 '-dideoxynucleoside 3 -O- methylnucleosides, 3 '-O- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein. The modRNA immunogenic composition is based on a modRNA platform technology. The single stranded, 5′-capped modRNA contains an open reading frame encoding the vaccine antigen of interest and features structural elements optimized for high efficacy of the RNA. The modRNA also contains a substitution of 1-methyl-pseudouridine for each uridine to decrease recognition of the vaccine RNA by innate immune sensors, such as TLRs 7 and 8, resulting in decreased innate immune activation and increased protein translation. The modRNA is encapsulated in a LNP for delivery into target cells. In an embodiment, the mRNA vaccines of the disclosure comprise lipids. The lipids and mRNA can together form nanoparticles. The lipids can encapsulate the mRNA in the form of a lipid nanoparticle (LNP) to aid cell entry and stability of the RNA / lipid nanoparticles. Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic. A LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements. The efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation. Lipid nanoparticles may be designed for one or more specific applications or targets. For example, a LNP may be designed to deliver a therapeutic and / or prophylactic such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal's body. The LNP delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen and / or other antigens in the composition. Physiochemical properties of lipid nanoparticles may be altered to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The therapeutic and / or prophylactic included in a LNP may also be selected based on the desired delivery target or targets. For example, a therapeutic and / or prophylactic may be selected for a particular indication, condition, disease, or disorder and / or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery). In certain embodiments, a LNP may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a particular organ. In some embodiments, a composition may be designed to be specifically delivered to a mammalian liver. In some embodiments, a composition may be designed to be specifically delivered to a lymph node. In some embodiments, a composition may be designed to be specifically delivered to a mammalian spleen. A LNP may include one or more components described herein. In some embodiments, the LNP formulation of the disclosure includes at least one lipid nanoparticle component. Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic, such as a nucleic acid. A LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements. The efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation. In some embodiments, for example, a polymer may be included in and / or used to encapsulate or partially encapsulate or be conjugated to a lipid (polymer-conjugated lipid or polymer lipid) in a LNP. A polymer may be biodegradable and / or biocompatible. A polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L- lactic acid) (PLLA), poly(gly colic acid) (PGA), poly(lactic acid-co-gly colic acid) (PLGA), poly(L- lactic acid-co-gly colic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L- lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co- glycolide), poly(D,L- lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co- caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2- oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), poly(2-oxazoline)(POZ), which is a synthetic, water-soluble, and low-viscosity polymer, and polyglycerol. In some embodiments, the amount of polymer-lipid in the lipid composition of a pharmaceutical composition disclosed herein ranges from about 0.1 mol % to about 5 mol %, from about 0.5 mol % to about 5 mol %, from about 1 mol % to about 5 mol %, from about 1.5 mol % to about 5 mol %, from about 2 mol % to about 5 mol %, from about 0.1 mol % to about 4 mol %, from about 0.5 mol % to about 4 mol %, from about 1 mol % to about 4 mol %, from about 1.5 mol % to about 4 mol %, from about 2 mol % to about 4 mol %, from about 0.1 mol % to about 3 mol %, from about 0.5 mol % to about 3 mol %, from about 1 mol % to about 3 mol %, from about 1.5 mol % to about 3 mol %, from about 2 mol % to about 3 mol %, from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 1.5 mol % to about 2 mol %, from about 0.1 mol % to about 1.5 mol %, from about 0.5 mol % to about 1.5 mol %, or from about 1 mol % to about 1.5 mol %. In some embodiments, the amount of polymer-lipid in the lipid composition disclosed herein is about 2 mol %. In some embodiments, the amount of polymer-lipid in the lipid composition disclosed herein is about 1.5 mol %. In some embodiments, the amount of polymer-lipid in the lipid composition disclosed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol %. In a preferred embodiment, the amount of polymer-lipid (or polymer-conjugated lipid) in the lipid composition is 1.8 mol %. In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid. Surface altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl- ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surface altering agent may be disposed within a nanoparticle and / or on the surface of a LNP (e.g., by coating, adsorption, covalent linkage, or other process). A LNP may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging. The surface of a LNP may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art. In addition to these components, lipid nanoparticles may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surface active agents, buffering agents, preservatives, and other species. Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxy ethylene sorbitan monolaurate [TWEEN®20], polyoxy ethylene sorbitan [TWEEN® 60], polyoxy ethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof. Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®. An exemplary free radical scavenger includes butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine. Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, Tris buffer, and / or combinations thereof. In some embodiments, the formulation including a LNP may further include a salt, such as a chloride salt. In some embodiments, the formulation including a LNP may further includes a sugar such as a disaccharide. In some embodiments, the formulation further includes a sugar but not a salt, such as a chloride salt.In some embodiments, a LNP may further include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). The characteristics of a LNP may depend on the components thereof. For example, a LNP including cholesterol as a structural lipid may have different characteristics than a LNP that includes a different structural lipid. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. The lipid nanoparticle compositions may include a mixture of structural lipids. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol and cholesterol analogs such as fecosterol, sitosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, sitostanol, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In some preferred embodiments, the sterol comprises In some preferred embodiments, the sterol comprises In some preferred embodiments, the sterol comprises stigmasterol. In some preferred embodiments, the sterol comprises β-sitosterol . In some embodiments, the structural lipid is a sitosterol, a stigmasterol, a campesterol, a sitostanol, a campestanol, a brassicasterol, a fucosterol, beta-sitosterol, stigmastanol, beta- sitostanol, ergosterol, lupeol, cycloartenol, Δ5-avenasterol, Δ7-avenasterol or a Δ7- stigmasterol, including analogs, salts or esters thereof, alone or in combination. In some embodiments, the sterol component of a LNP of the disclosure is a single phytosterol. In some embodiments, the phytosterol component of a LNP of the disclosure is a mixture of different phytosterols (e.g.2, 3, 4, 5 or 6 different phytosterols). In some embodiments, the phytosterol component of an LNP of the disclosure is a blend of one or more phytosterols and one or more zoosterols, such as a blend of a phytosterol (e.g., a sitosterol, such as beta-sitosterol) and cholesterol. In some embodiments, the phytosterol is β-sitosterol, campesterol, sigmastanol, or any combination thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the cholesterol analog comprises β-sitosterol, campesterol, and stigmasterol. In some embodiments, the mixture of structural lipids comprises a mixture of β-sitosterol and cholesterol. In one embodiment, the mixture of structural lipids is a mixture of β-sitosterol and cholesterol. In some embodiments, the mixture of structural lipids comprises about 35% to about 85% of β-sitosterol, about 5% to about 35% stigmasterol, and about 5% to about 35% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 80% of β-sitosterol, about 10% to about 30% stigmasterol, and about 10% to about 30% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 70% of β-sitosterol, about 10% to about 25% stigmasterol, and about 10% to about 25% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 70% of β-sitosterol, about 15% to about 25% stigmasterol, and about 15% to about 25% of campesterol. In some embodiments, the mixture of structural lipids comprises about 35% to about 45% of β-sitosterol, about 20% to about 30% stigmasterol, and about 20% to about 30% of campesterol. In some embodiments, the the mixture of structural lipids comprises about 40% to about 50% of β-sitosterol, about 25% to about 35% stigmasterol, and about 25% to about 35% of campesterol. In some embodiments, the mixture of structural lipids comprises about 65% to about 75% of β-sitosterol, about 5% to about 15% stigmasterol, and about 5% to about 15% of campesterol. In some embodiments, the mixture of structural lipids comprises about 35% to about 85% of β-sitosterol, about 5% to about 35% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 80% of β- sitosterol, about 10% to about 30% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 70% of β-sitosterol, about 10% to about 25% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 70% of β-sitosterol, about 15% to about 25% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 35% to about 45% of β-sitosterol, about 20% to about 30% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 50% of β-sitosterol, about 25% to about 35% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 65% to about 75% of β-sitosterol, about 5% to about 15% stigmasterol, and 0% of campesterol. Accordingly, in some preferred embodiments, the composition does not comprise campesterol. In some embodiments, the composition comprises a mixture of structural lipids comprising about 10% to about 30% of cholesterol, about 10% to about 30% β-sitosterol, and about 10% to about 30% stigmasterol, and 0% campesterol. In some embodiments, the composition further comprises about 30-50% cationic lipid and about 5-25% phospholipid. In some embodiments, the mixture of structural lipids is between about 30 mol% and about 60 mol%. In some embodiments, the mixture of structural lipids is between about 40 mol% and about 60 mol%. In some embodiments, the mixture of structural lipids is between about 30 mol% and about 50 mol%. In some embodiments, the mixture of structural lipids is between about 40 mol% and about 50 mol%. In some embodiments, the mixture of structural lipids is about 30 mol%. In some embodiments, the mixture of structural lipids is about 40 mol%. In some embodiments, the mixture of structural lipids is about 50 mol%. In some embodiments, the mixture of structural lipids is about 60 mol%. In some embodiments, the mol % of the structural lipids is between about 1% and 50% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the mol % of the structural lipids is between about 10% and 40% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the mol % of the structural lipids is between about 20% and 30% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the mol % of the structural lipids is about 30% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the lipid nanoparticle compositions described herein can comprise about 20 mol% to about 60 mol% structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 30 mol% to about 50 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 35 mol% to about 45 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 37 mol% to about 42 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 35, about 36, about 37, about 38, about 39, or about 40 mol% of structural lipids. In some embodiments, the nanoparticle comprises about 39 to about 40 mol% structural lipids. In some aspects, the lipid nanoparticle compositions comprising a mixture of structural lipids does not comprise a mixture of cholesterol and β- sitosterol of about 38.5 mol%. In some embodiments, the amount of the structural lipid or mixture of structural lipids in the composition disclosed herein is at least about 30.0, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32.0, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33.0, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34.0, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35.0, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36.0, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38.0, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39.0, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, 50.0, 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, 51.0, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52.0, 52.1, 52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53.0, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54.0, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, 57.0, 57.1, 57.2, 57.3, 57.4, 57.5, 57.6, 57.7, 57.8, 57.9, 58.0, 58.1, 58.2, 58.3, 58.4, 58.5, 58.6, 58.7, 58.8, 58.9, 59.0, 59.1, 59.2, 59.3, 59.4, 59.5, 59.6, 59.7, 59.8, 59.9, or 60.0 mol %. In some aspects, the composition comprising a mixture of structural lipids does not comprise a mixture of cholesterol and β-sitosterol of about 38.5 mol%. In some embodiments, the composition comprises a mixture of structural lipids comprising cholesterol and a cholesterol analog, wherein the mixture is about 15 mol% to about 25 mol% of cholesterol and about 15 mol% to about 25 mol% cholesterol analog. In some embodiments, the amount of cholesterol in the mixture of structural lipids in the composition disclosed herein is at least about 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, or 25.9 mol % of cholesterol and at least about 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, or 25.9 mol % of cholesterol analog. In some aspects, the cholesterol analog is β-sitosterol, stigmasterol or campesterol. In a preferred embodiment, the cholesterol analog is β-sitosterol. In some embodiments, the amount of cholesterol in the mixture of structural lipids in the composition disclosed herein is at least about 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, or 25.9 mol % of cholesterol and at least about 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, or 25.9 mol % of β- sitosterol. In some aspects, the composition comprising a mixture of cholesterol and β-sitosterol is not about 38.5 mol%. In some embodiments, the composition comprises a mixture of structural lipids comprising cholesterol and a cholesterol analog, wherein the molar ratio between the cholesterol and the cholesterol analog in the mixture is about 6:4, 1:1 or 4:6. In some embodiments, the composition further comprises about 30-50% cationic lipid and about 5-25% phospholipid. In some embodiments, a LNP of the invention comprises an N:P ratio of from about 2:1 to about 30:1. In some embodiments, a LNP of the invention comprises an N:P ratio of about 10:1. In some embodiments, a LNP of the invention comprises an N:P ratio of about 6:1. In some embodiments, a LNP of the invention comprises an N:P ratio of about 3:1, 4:1, or 5:1. In some embodiments, the characteristics of a LNP may depend on the absolute or relative amounts of its components. For instance, a LNP including a higher molar fraction of a phospholipid may have different characteristics than a LNP including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the lipid nanoparticle. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, and 2-lysophosphatidyl choline. Further examples of a phospholipid moiety for the lipid nanoparticle include a lipid that is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1- trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidyl serine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diemcoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl- phosphatidylethanolamine (DEPE), l,2-dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE); l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. In some embodiments, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. In some embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidyl-ethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. In some embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. Lipid nanoparticles may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a LNP. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a LNP, such as particle size, polydispersity index, and zeta potential. The mean size of a LNP may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a LNP may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a LNP may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm. A LNP may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20. The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about - 10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about - 5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. The efficiency of encapsulation of a therapeutic and / or prophylactic describes the amount of therapeutic and / or prophylactic that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic (e.g., RNA) in a solution. For example, if 97 mg of mRNA are encapsulated in a composition out of a total 100 mg of mRNA initially provided to the composition, the encapsulation efficiency may be given as 97%. For the lipid nanoparticles described herein, the encapsulation efficiency of a therapeutic and / or prophylactic may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. A LNP may optionally comprise one or more coatings. For example, a LNP may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density. Formulations comprising amphiphilic polymers and lipid nanoparticles may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more amphiphilic polymers and one or more lipid nanoparticles. For example, a pharmaceutical composition may include one or more amphiphilic polymers and one or more lipid nanoparticles including one or more different therapeutics and / or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a LNP or the one or more amphiphilic polymers in the formulation of the disclosure. An excipient or accessory ingredient may be incompatible with a component of a LNP or the amphiphilic polymer of the formulation if its combination with the component or amphiphilic polymer may result in any undesirable biological effect or otherwise deleterious effect. In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a LNP. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia. Relative amounts of the one or more amphiphilic polymers, the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition may comprise between 0.1% and 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w / v). In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of about 5 °C or lower, such as a temperature between about -150 °C and about 5 °C or between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about 5 °C, 0 °C, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, - 80 °C, -90 °C, -130 °C or -150 °C). For example, the pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and / or shipment at, for example, about - 20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C. In certain embodiments, the disclosure also relates to a method of increasing stability of the lipid nanoparticles by adding an effective amount of an amphiphilic polymer and by storing the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 4 °C or lower, such as a temperature between about - 150 °C and about 0 °C or between about -80 °C and about -20 °C, e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C). The chemical properties of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure may be characterized by a variety of methods. In some embodiments, electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reverse phase liquid chromatography) may be used to examine the mRNA integrity. The efficacy of a product is dependent on expression of the delivered RNA, which requires a sufficiently intact RNA molecule. RNA integrity is a measure of RNA quality that quantitates intact RNA. The method is also capable of detecting potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. The initial specification is set to ensure sufficient RNA integrity in drug product preparations. In some embodiments, the RNA polynucleotide has an integrity of at least about 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 95%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 98%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 99%. In preferred embodiments, the RNA polynucleotide has a clinical grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%. In some embodiments, the purity of the RNA polynucleotide is between about 80% and 99%. In some embodiments, the purity of the RNA polynucleotide is between about 90% and about 99%. In some embodiments, wherein the purified mRNA has a clinical grade purity without further purification. In some embodiments, the clinical grade purity is achieved through a method including tangential flow filtration (TFF) purification. In some embodiments, the clinical grade purity is achieved without the further purification selected from high performance liquid chromatography (HPLC) purification, ligand or binding based purification, and / or ion exchange chromatography. In some embodiments, the method of producing the RNA polynucleotides removes long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA residual solvent and / or residual salt. In some embodiments, the short abortive transcript contaminants comprise less than 15 bases. In some embodiments, the short abortive transcript contaminants comprise about 8-12 bases. In some embodiments, the method of the invention also removes RNAse inhibitor. In some embodiments, the purified RNA polynucleotide comprises 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less or is substantially free of protein contaminants as determined by capillary electrophoresis. In some embodiments, the purified RNA polynucleotide comprises less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, or is substantially free of salt contaminants determined by high performance liquid chromatography (HPLC). In some embodiments, the purified RNA polynucleotide comprises 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less or is substantially free of short abortive transcript contaminants determined by known methods, such as, e.g., high performance liquid chromatography (HPLC). In some embodiments, the purified RNA polynucleotide has integrity of 60% or greater, 70% or greater, 80% or greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater as determined by a known method, such as, e.g., capillary electrophoresis. Modified nucleobases Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules include, for example, m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1-methyladenosine); m2A (2- methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2- methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0- ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (l,2'-0- dimethylinosine); m3C (3-methylcytidine); Cm (2T-0-methylcytidine); s2C (2- thiocytidine); ac4C (N4- acetylcytidine); £5C (5-fonnylcytidine); m5Cm (5,2-0- dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mlG (1- methylguanosine); m2G (N2- methylguanosine); m7G (7-methylguanosine); Gm (2'-0- methylguanosine); m22G (N2,N2- dimethylguanosine); m2Gm (N2,2'-0- dimethylguanosine); m22Gm (N2,N2,2'-0- trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7- aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0- dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'- O- methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5- hydroxyuridine); mo5U (5- methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl- 2-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl-2-thiouridine); nm5s2U (5- aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5- methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L- Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6- dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-0- dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); rn62Am (N6,N6,0-2- trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7- trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0- methylcytidine); mlGm (l,2'-0-dimethylguanosine); m'Am (1,2-0- dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG- 14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7- substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-C6)-alkyluracil, 5- methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)- alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5- hydroxycytosine, 5-(Ci-C6 )- alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2- C6)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2- C6)alkynylguanine, 7-deaza- 8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8- azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. In some aspects, one or more of the modified nucleosides in the list may be excluded. Additional exemplary modified nucleotides include any one of N-1-methylpseudouridine; pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some embodiments, the modified nucleotide is N-1-methylpseudouridine. In some embodiments, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. In some embodiments, the RNA molecule includes a modified nucleotide selected from any one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methoxyuridine and 2′-O-methyl uridine. In some embodiments, the modified or unnatural nucleotides are selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyl uridine. In some embodiments, the modified or unnatural nucleotides are selected from the group consisting of 5- methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine. In some embodiments, at least 10% of a total population of a particular nucleotide in the RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of the particular nucleotide population in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least a portion, or all of a total population of a particular nucleotide in the RNA molecule has been replaced with two modified or unnatural nucleotides. In some embodiments, the two modified or unnatural nucleotides are provided in a ratio equal to any one of, at least any one of, at most any one of, or between any two of 1:99 to 99:1, including 1:99; 2:98; 3:97; 4:96; 5:95; 6:94; 7:93; 8:92; 9:91; 10:90; 11:89; 12:88; 13:87; 14:86; 15:85; 16:84; 17:83; 18:82, 19:81; 20:80; 21:79; 22:78; 23:77; 24:76; 25:75; 26:74; 27:73; 28:72; 29:71; 30:70; 31:69; 32:68; 33:67; 34:66; 35:65; 36:64; 37:63; 38:62; 39:61; 40:60; 41:59; 42:58; 43:57; 44:56; 45:55; 46:54; 47:53; 48:52; 49:51; 50:50; 51:49; 52:48; 53:47; 54:46; 55:45; 56:44; 57:43; 58:42; 59:41; 60:40; 61:39; 62:38; 63:37; 64:36; 65:35; 66:34; 67:33; 68:32; 69:31; 70:30; 71:29; 72:28; 73:27; 74:26; 75:25; 76:24; 77:23; 78:22; 79:21; 80:20; 81:19; 82:18; 83:17; 84:16; 85:15; 86:14; 87:13; 88:12; 89:11; 90:10; 91:9; 92:8; 93:7; 94:6; 95:5; 96:4; 97:3; 98:2; and 99:1, or any range derivable therein. In some embodiments, at least 10% of a total population of a first particular nucleotide in a RNA molecule as disclosed herein has been replaced with one or more modified or unnatural nucleotides, and at least 10% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of uridine nucleotides in the RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1- methylpseudouridine. In some embodiments, at least 75% of a total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with N1- methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5-methoxyuridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the molecule has been replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 2-thiouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methoxyuridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with about 25% 5- methoxyuridine and about 75% N1-methylpseudouridine. UTRs The 5′ untranslated regions (UTR) is a regulatory region of DNA situated at the 5′ end of a protein coding sequence that is transcribed into mRNA but not translated into protein.5′ UTRs may contain various regulatory elements, e.g., 5′ cap structure, stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 3′ UTR, situated downstream of a protein coding sequence, may be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. In some embodiments, the UTR is derived from an mRNA that is naturally abundant in a specific tissue (e.g., lymphoid tissue), to which the mRNA expression is targeted. In some embodiments, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR may increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and / or the rate at which ribosomes initiate translation on the message (message translation efficiency). According, the UTR sequence may prolong protein synthesis in a tissue-specific manner. In some embodiments, the 5′ UTR and the 3′ UTR sequences are computationally derived. In some embodiments, the 5′ UTR and the 3′ UTRs are derived from a naturally abundant mRNA in a tissue. The tissue may be, for example, liver, a stem cell, or lymphoid tissue. The lymphoid tissue may include, for example, any one of a lymphocyte (e.g., a B-lymphocyte, a helper T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil and a reticulocyte. In some embodiments, the 5′ UTR and the 3′ UTR are derived from an alphavirus. In some embodiments, the 5′ UTR and the 3′ UTR are from a wild-type alphavirus. Examples of alphaviruses are described below. In some embodiments, the first RNA molecule includes a 5′ UTR and the 3′ UTR derived from a naturally abundant mRNA in a tissue. In some embodiments, the first RNA molecule includes a 5′ UTR and the 3′ UTR derived from an alphavirus. In some embodiments, the second mRNA or the saRNA molecule includes a 5′ UTR and the 3′ UTR derived from an alphavirus. In some embodiments, the second mRNA or the saRNA molecule includes a 5′ UTR and the 3′ UTR from a wild-type alphavirus. In some embodiments, the RNA molecule includes a 5’ cap. In some embodiments, the RNA molecule includes a 5’UTR or 3’UTR as described in WO2024 / 154061, which is hereby incorporated herein by reference in its entirety, including all sequences set forth therein. In some embodiments, the RNA molecule comprises a 5’ UTR having the sequence GAAΨAAAC ΨAGΨAΨΨCΨΨ CΨGGΨCCCCA CAGACΨCAGA GAGAACCCGC CACC (SEQ ID NO: 1) , wherein is 1-methyl-3'-pseudouridylyl. In some embodiments, the RNA molecule comprises a 5’ UTR having the sequence AATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC (5’ WHO UTR1) (SEQ ID NO: 4). In some embodiments, the RNA molecule comprises a 5’ UTR comprises the sequence GAGAAΨAAACΨAGΨAΨΨCΨΨ CΨGGΨCCCCA CAGACΨCAGA GAGAACCCGCCACC (SEQ ID NO: 5), wherein is 1-methyl-3'-pseudouridylyl. In some embodiments, the RNA molecule comprises a 5’ UTR comprises the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC (5’ WHO UTR1). (SEQ ID NO: 6). In some embodiments, the RNA molecule comprises a 3’ UTR comprises the sequence C ΨCGAGCΨGGΨ ACΨGCAΨGCA CGCAAΨGCΨA GCΨGCCCCΨΨ ΨCCCGΨCCΨG GGΨACCCCGA GΨCΨCCCCCG ACCΨCGGGΨC CCAGGΨAΨGC ΨCCCACCΨCC ACCΨGCCCCA CΨCACCACCΨ CΨGCΨAGΨΨC CAGACACCΨC CCAAGCACGC AGCAAΨGCAG CΨCAAAACGC ΨΨAGCCΨAGC CACACCCCCA CGGGAAACAG CAGΨGAΨΨAA CCΨΨΨAGCAA ΨAAACGAAAG ΨΨΨAACΨAAG CΨAΨACΨAAC CCCAGGGΨΨG GΨCAAΨΨΨCG ΨGCCAGCCAC ACCCΨGGAGC ΨAGC (SEQ ID NO: 2), wherein is 1-methyl-3'-pseudouridylyl. In some embodiments, the RNA molecule comprises a 3’ UTR comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCC CGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCAC CACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAG CCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUU AACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUA GC (3’ WHO UTR2). (SEQ ID NO: 7). In some embodiments, the RNA molecule comprises a 3’ UTR comprises the sequence CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCCΨΨΨCCCGΨCCΨGGGΨAC CCCGAGΨCΨCCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨCCACCΨGCCCCAC ΨCACCACCΨCΨGCΨAGΨΨCCAGACACCΨCCCAAGCACGCAGCAAΨGCAGCΨCAAAAC GCΨΨAGCCΨAGCCACACCCCCACGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAAC GAAAGΨΨΨAACΨAAGCΨAΨACΨAACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACA CCCΨGGAGCΨAGC (3’ WHO ΨTR2) (SEQ ID NO: 8), wherein is 1-methyl-3'-pseudouridylyl. Open reading frame (ORF) The 5′ and 3′ UTRs may be operably linked to an open reading frame (ORF), which may be a sequence of codons derived from a gene of interest that is capable of being translated into a polypeptide of interest. As stated above, the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs). In some embodiments, the ORF encodes a non-structural viral gene. In some embodiments, the ORF further includes one or more subgenomic promoters. In some embodiments, the RNA molecule includes a subgenomic promoter operably linked to the ORF. In some embodiments, the subgenomic promoter comprises a cis-acting regulatory element. In some embodiments, the cis-acting regulatory element is immediately downstream (5’-3’) of B2. In some embodiments, the cis-acting regulatory element is immediately downstream (5’-3’) of a guanine that is immediately downstream of B2. In some embodiments, the cis-acting regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauau, auauauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug. In some embodiments, the second mRNA or the saRNA molecule may include (i) an ORF encoding a replicase which may transcribe RNA from the second mRNA or the saRNA molecule and (ii) an ORF encoding at least one an antigen or polypeptide of interest. The polymerase may be an alphavirus replicase e.g., including any one of the non- structural alphavirus proteins nsP1, nsP2, nsP3 and nsP4, or a combination thereof. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP1. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP2. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP4. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4. In some embodiments, an open reading frame of an RNA (e.g., modified mRNA or saRNA) composition is codon-optimized. In some embodiments, the open reading frame from which the polypeptide or fragment thereof is encoded is codon-optimized. Genes of Interest The RNA molecules described herein include a gene of interest. The gene of interest encodes a polypeptide of interest selected from, e.g., biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. The sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GenBank. n some aspects, the RNA molecule includes a coding region for an antigen preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Escherichia coli Fimbrial antigen H, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), Parainfluenza virus (PIV), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis. 5’ cap In some embodiments, the mRNA or saRNA molecule described herein includes a 5’ cap. In some embodiments, the 5'-cap moiety is a natural 5'-cap. A “natural 5'-cap” is defined as a cap that includes 7-methylguanosine connected to the 5’ end of an mRNA molecule through a 5′ to 5′ triphosphate linkage. In some embodiments, the 5'-cap moiety is a 5'- cap analog. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping), wherein “N” is any ribonucleotide. In some embodiments, the 5’ end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule. An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyl- transferase, and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures. Cap 0 structure can help maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2 '-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '-Ο] N), which may further increase translation efficacy. In some embodiments, the RNA molecule may be enzymatically capped at the 5′ end using Vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to yield cap 0 structure. An inverted 7- methylguanosine cap is added via a 5′ to 5′ triphosphate bridge. Alternatively, use of a 2′O- methyltransferase with Vaccinia guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2′OH group is methylated on the penultimate nucleotide. S-adenosyl- L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. Non-limiting examples of 5′ cap structures are those which, among other things, have enhanced binding of cap binding polypeptides, increased half-life, reduced susceptibility to 5′ endonucleases and / or reduced 5′ decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme may create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5′-terminal nucleotide of the mRNA includes a 2′-O- methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational- competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5′)ppp(5′)N,pN2p (cap 0) and 7mG(5′)ppp(5′)N1mpNp (cap 1). Cap 0 is a N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, typically referred to as m7G cap or m7Gppp. In the cell, the cap 0 structure can help provide for efficient translation of the mRNA that carries the cap. An additional methylation on the 2′O position of the initiating nucleotide generates Cap 1, or refers to as m7GpppNm-, wherein Nm denotes any nucleotide with a 2′O methylation. In some embodiments, the 5′ terminal cap includes a cap analog, for example, a 5′ terminal cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the capping region may include a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent. In some embodiments, the first and second operational regions may be equal to any one of, at least any one of, at most any one of, or between any two of 3 to 40, e.g., 5-30, 10-20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and / or Stop codon, one or more signal and / or restriction sequences. In some embodiments, the 5’ Cap is represented by Formula I: where R1and R2are each independently H or Me, and B1and B2are each independently guanine, adenine, or uracil. In some embodiments, B1and B2are naturally-occurring bases. In some embodiments, R1is methyl and R2is hydrogen. In some embodiments, B1is guanine. In some embodiments, B1is adenine. In some embodiments, B2is adenine. In some embodiments, B2is uracil. In some embodiments, B2is uracil and at least 5% of a total population of uracil nucleotides in the molecule that are downstream of B2have been replaced with one or more modified or unnatural nucleotides. In some embodiments, the nucleotide immediately downstream (5’ to 3’ direction) of the 5’ Cap comprises guanine. In some embodiments, B1is adenine and B2is uracil. In some embodiments, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen. In some instances, the mRNA or saRNA does not comprise a 5’ Cap. In some instances, the 5’ Cap is not represented by Formula I. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen; this embodiment corresponds to CleanCap AU, and the inclusion of B2= uracil, while optionally substuting uracil nucleotides downstream of B2, has been shown to improve mRNA or saRNA functionality in some embodiments. In some embodiments, the RNA molecule further comprises: (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the RNA molecule encodes at least one antigen. In some embodiments, the RNA molecule comprises at least 7000 nucleotides. In some embodiments, the RNA molecule comprises at least 8000 nucleotides. In some embodiments, at least 80% of the total RNA molecules are full length. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus. In some embodiments, the alphavirus is Semliki Forest virus. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1- methylpseudouridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5- methoxyuridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5- methyluridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 25% 5-methoxyuridine and about 75% N1-methylpseudouridine. In some embodiments, a 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp. In some preferred . In some embodiments, the 5’ cap comprises CLEANCAP® Reagent AG (3' OMe) for co- transcriptional capping of mRNA, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, . In alternative embodiments, the 5’ cap comprises CLEANCAP® AU for Self-Amplifying mRNA, CLEANCAP® Reagent AU for co-transcriptional capping of mRNA, m7G(5')ppp(5')(2'OMeA)pU, . Poly-A tail As used herein, “poly A tail” refers to a stretch of consecutive adenine residues, which may be attached to the 3’ end of the RNA molecule. The poly-A tail may increase the half-life of the RNA molecule. Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation. Exemplary designs include a poly-A tails of about 40 adenine residues to about 80 adenine residues. In some embodiments, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly A tail sequence. In some embodiments, such as for the second mRNA or the saRNA molecule, the RNA molecule further includes a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. A “full length” RNA molecule is one that includes a 5’-cap and a poly A tail. In some embodiments, the poly A tail includes 5-400 adenine nucleotides in length. The poly A tail nucleotide length may be equal to any one of, at least any one of, at most any one of, or between any two of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400. In some embodiments, the RNA molecule includes a poly A tail that includes about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly A tail includes a sequence of greater than 30 adenosine nucleotides (“As”). In some embodiments, the RNA molecule includes a poly A tail that includes about 40 As. In some embodiments, the RNA molecule includes a poly A tail that includes about 80 As. As used herein, the term “about” refers to a deviation of ±10% of the value(s) to which it is attached. In some embodiments, the 3’ poly- A tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some embodiments, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 70 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 80 consecutive adenosine residues. Compositions In some instances, the compositions described herein include at least one RNA as described herein. In some embodiments, equal to any one of, at least any one of, at most any one of, or between any two of 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules (capped and uncapped) in the composition are capped. In some embodiments, equal to any one of, at least any one of, at most any one of, or between any two of 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition are full length RNA transcripts. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measure by, e.g., peak area of full- length RNA molecule relative to total peak. In some embodiments, a fragment analyzer (FA) may be used to quantify and purify the RNA. The fragment analyzer automates capillary electrophoresis and HPLC. In some embodiments, the composition is substantially free of one or more impurities or contaminants including the linear DNA template and / or reverse complement transcription products and, for instance, includes RNA molecules that are equal to any one of, at least any one of, at most any one of, or between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure, or at least 99% pure. In some embodiments, the composition comprises an amount of the first RNA molecule that is greater than the amount of the second RNA molecule. In some embodiments, the composition comprises an amount of the first RNA molecule that is at least about 1 to 2 times greater than the amount of the second RNA molecule. In some embodiments, the composition comprises an amount of the first RNA molecule that is at least about 1 to 100 times greater than the amount of the second RNA molecule. In some embodiments, the composition further includes a pharmaceutically acceptable carrier. In some embodiments, the composition further includes a pharmaceutically acceptable vehicle. In some embodiments, the composition further includes a lipid-based delivery system, which delivers an RNA molecule to the interior of a cell, where it can then replicate and / or express the encoded polypeptide of interest. The delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen. In some embodiments, the composition further includes one or more each of neutral lipids, cationic lipids, structural lipids (e.g. cholesterol or combinations thereof), and polymer conjugate lipids such as polyethylene glycol conjugated lipids (PEG-lipids), and forms nanoparticles that encompass the RNA molecules. In some embodiments, the composition further includes any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil- in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion. In some embodiments, the RNA molecule is encapsulated in, bound to or adsorbed on any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion, or a combination thereof. In some instances, the compositions described herein include at least two RNA molecules: a first RNA molecule and a second RNA molecule as described herein. To protect against more than one strain of a virus or different viruses, a combination vaccine composition may be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first virus or organism and further includes a second RNA molecule encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second virus or organism. RNA can be co-formulated, for example, in a single lipid nanoparticle (LNP) or can be formulated in separate LNPs for co-administration. In some embodiments, the second RNA molecule includes any one of a 5’ cap, a 5’ UTR, an open reading frame, a 3’ UTR, and a poly A sequence, or any combination thereof. In some embodiments, the second RNA molecule includes a 5’ cap moiety. In some embodiments, the second RNA molecule includes a 5’ UTR and a 3’UTR. In some embodiments, the second RNA molecule includes a 5’UTR, an open reading frame, a 3’UTR, and does not further include a 5’ cap. In some embodiments, the second RNA molecule includes a 5’ cap moiety, 5’ UTR, coding region, 3’ UTR, and a 3’ poly A sequence. In some embodiments, the second RNA molecule includes a 5’ cap moiety, 5’ UTR, noncoding region, 3’ UTR, and a 3’ poly A sequence. In some embodiments, the second RNA molecule includes a noncoding region and does not further comprise any one of a 5’ cap moiety, 5’ UTR, 3’ UTR, and a 3’ poly A sequence. In some embodiments, the second RNA molecule includes a 5’ cap moiety, a 5’ untranslated region (5’ UTR), a modified nucleotide, an open reading frame, a 3’ untranslated region (3’ UTR), and a 3’ poly A sequence. Some aspects of the disclosure are directed to a composition comprising (i) first RNA molecule encoding a gene of interest; and (ii) a second RNA molecule comprising a modified or unnatural nucleotide. In some instances, the first RNA molecule comprises a 5’ Cap, a 5’ untranslated region, a coding region for a nonstructural protein comprising a RNA replicase, a subgenomic promoter, an open reading frame encoding a gene of interest, a 3’ untranslated region, and a 3’ poly A sequence. In some instances, at least 5% of a total population of a particular nucleotide in the first RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some instances, the RNA molecule comprises natural, unmodified nucleotides and does not include a modified or unnatural nucleotide. In some instances, the 5’ Cap is represented by Formula I, where R1and R2are each independently H or Me, B1and B2are each independently guanine, adenine, or uracil, a 5’ untranslated region, a coding region for a nonstructural protein derived from an alphavirus, a subgenomic promoter, such as one derived from an alphavirus, an open reading frame encoding a gene of interest, a 3’ untranslated region, and a 3’ poly A sequence. In some embodiments, B1and B2are naturally-occurring bases. In some embodiments, R1is methyl and R2is hydrogen. In some embodiments, B1is guanine. In some embodiments, B1is adenine. In some embodiments, B2is adenine. In some embodiments, B2is uracil. In some embodiments, the nucleotide immediately downstream (5’ to 3’ direction) of the 5’ Cap comprises guanine. In some embodiments, B1is adenine and B2is uracil. In some embodiments, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen; this embodiment corresponds to CLEANCAP AU (Trilink), and the inclusion of B2= uracil, while optionally substituting uracil nucleotides downstream of B2, which has been shown to provide increased RNA functionality in some embodiments. In some embodiments, at least 10% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of a particular nucleotide population in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, the one or more modified or unnatural replacement nucleotides comprise two modified or unnatural nucleotides provided in a ratio ranging from 1:99 to 99:1, or any derivable range therein. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 10% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 75% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5- methoxyuridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with 5- methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the first RNA molecule has been replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the first RNA molecule have been replaced with 5- methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with 2-thiouridine. In some embodiments, at least 25% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 75% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1- methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 5-methoxyuridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the second RNA molecule has been replaced with 5- methylcytosine. In some embodiments, essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 2- thiouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 2-thiouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methyluridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with about 25% 5-methoxyuridine and about 75% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and essentially all uridine nucleotides in the second RNA molecule have been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5- methyluridine, and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and essentially all uridine nucleotides in the second RNA molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. saRNA In some embodiments, the RNA molecule, such as the first RNA molecule, is an saRNA. “saRNA,” “self-amplifying RNA,” and “replicon” refer to RNA with the ability to replicate itself. Self- amplifying RNA molecules may be produced by using replication elements derived from a virus or viruses, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein of interest, e.g., an antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g., a viral antigen, can become a major polypeptide product of the cells. In some embodiments, the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins. In some embodiments, the self-amplifying RNA may also include 5'- and 3 '-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES). In some embodiments, the self-amplifying RNA molecule is not encapsulated in a virus- like particle. Self-amplifying RNA molecules described herein may be designed so that the self- amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA. For example, when the self- amplifying RNA molecule is based on an alphavirus, such as Sinbis virus (SIN), Semliki forest virus and Venezuelan equine encephalitis virus (VEE), one or more genes encoding viral structural proteins, such as capsid and / or envelope glycoproteins, may be omitted. In some embodiments, a self-amplifying RNA molecule described herein encodes (i) an RNA-dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen. In some embodiments, the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus protein nsP1, nsP2, nsP3, nsP4, and any combination thereof. In some embodiments, the self-amplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine,N1-methylpseudouridine, N6-methyladenosine, 5-methylcytidine, 5- methyluridine). In some embodiments, the self-amplifying RNA molecules does not include a modified nucleotide (e.g., pseudouridine, N1-methylpseudouridine, N6-methyladenosine, 5- methylcytidine, 5-methyluridine). The saRNA construct may encode at least one non-structural protein (NSP), disposed 5’ or 3’ of the sequence encoding at least one peptide or polypeptide of interest. In some embodiments, the sequence encoding at least one NSP is disposed 5’ of the sequences encoding the peptide or polypeptide of interest. Thus, the sequence encoding at least one NSP may be disposed at the 5’ end of the RNA construct. In some embodiments, at least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4. In some embodiments, the saRNA construct encodes nsP1, nsP2, nsP3 and, nsP4. As is known in the art, nsP1 is the viral capping enzyme and membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing. nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP-ribosylation. nsP4 is the core viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Whereas natural alphavirus genomes encode structural virion proteins in addition to the non- structural replicase polypeptide, in some embodiments, the self-amplifying RNA molecules do not encode alphavirus structural proteins. In some embodiments, the self-amplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the self-amplifying RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses can be absent from self-amplifying RNAs of the present disclosure and their place can be taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins. In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame can encode a replicase; the second (3') open reading frame can encode a polypeptide comprising an antigen of interest. In some embodiments the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides. In some embodiments, the second RNA or the saRNA molecule further includes (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase. Optionally, self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection. In some embodiments, the saRNA molecule is alphavirus-based. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and virus subtypes within the alphavirus genus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. As such, the self-amplifying RNA described herein may incorporate an RNA replicase derived from any one of semliki forest virus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross-River virus (RRV), or other viruses belonging to the alphavirus family. In some embodiments, the self-amplifying RNA described herein may incorporate sequences derived from a mutant or wild-type virus sequence, e.g., the attenuated TC83 mutant of VEEV has been used in saRNAs. Alphavirus-based saRNAs are (+)-stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or replicase- transcriptase). The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA. These (-)-strand transcripts may themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the desired gene product. Translation of the subgenomic transcript thus leads to in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs may use a replicase from a sindbis virus, a semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, or mutant variants thereof. In some embodiments, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, such as a positive-stranded RNA virus, and in particular a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild- type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR- 1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR- 70, ATCC VR- 1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some aspects, one or more of the alphaviruses in the list may be excluded. In some embodiments, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., saRNA). Typically, the self-amplifying RNA molecules described herein include at least about 4 kb. For example, the self-amplifying RNA may be equal to any one of, at least any one of, at most any one of, or between any two of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb. In some instances the self-amplifying RNA may include at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In certain examples, the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences. In some embodiments, the saRNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. Alternatively, or in addition, one saRNA molecule may also encode more than one polypeptide of interest or more, such as an antigen, e.g., a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens. The term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" means not only a fusion of a first RNA molecule to a RNA molecule at the 5’-end or the 3’-end, but also includes insertion of the whole first RNA molecule into any two nucleotides in the second RNA molecule. The first second RNA molecule can be linked to a second RNA molecule by a phosphodiester bond or a linker. The linker can be, e.g., a polynucleotide. In some embodiments, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. In some embodiments, the self-amplifying RNA described herein may encode epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both. In some embodiments, the saRNA molecule is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration. Some embodiments of the disclosure are directed to a composition comprising a self- amplifying RNA molecule comprising a 5’ Cap, a 5’ untranslated region, a coding region comprising a sequence encoding an RNA-dependent RNA polymerase (also referred to as a “replicase”), a subgenomic promoter, such as one derived from an alphavirus, an open reading frame encoding a gene of interest (e.g., an antigen derived from a virus), a 3’ untranslated region, and a 3’ poly A sequence. In some embodiments, at least 5% of a total population of a particular nucleotide in the saRNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, the saRNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some embodiments, the saRNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some embodiments, the RNA may include a 5' cap comprising a 7'-methylguanosine, and the first 1, 2 or 35' ribonucleotides may be methylated at the 2' position of the ribose. In alternative embodiments, each RNA polynucleotide encoding a particular antigen is formulated in an individual LNP, such that each LNP encapsulates an RNA polynucleotide encoding identical antigens. Such embodiments may be referred herein as "post-mix". Accordingly, in some embodiments, the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; the fifth RNA polynucleotide is formulated in a fifth LNP; the sixth RNA polynucleotide is formulated in a sixth LNP; the seventh RNA polynucleotide is formulated in a seventh LNP; and the eighth RNA polynucleotide is formulated in an eighth LNP. In some embodiments, the molar ratio of the first LNP to the second LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the second LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the third LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the third LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the fourth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the fourth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the fifth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the sixth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the seventh LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the eighth LNP is greater than 1:1. Surprisingly, it has been discovered that regardless of the process, the resulting ratio of RNA polynucleotide is comparable whether the plurality of RNA polynucleotides are mixed prior to formulation in an LNP (pre-mixed) or whether the RNA polynucleotides encoding a particular antigen are formulated in an individual LNP and the plurality of LNPs for different antigens are mixed (post-mixed). As a result of this discovery, there may be an option for medical professionals to mix and administer different ratios of antigens depending on the need, e.g. influenza season, particularly when the individual LNPs encapsulate RNA for a single antigen. In some embodiments, the mRNAs encoding antigens are separately encapsulated (e.g., separate encapsulation of each mRNA encoding an antigen; encapsulation of mRNA(s) encoding a first antigen (e.g. influenza type A antigen) in a first population of nanoparticles (e.g., LNPs) and encapsulation of mRNA(s) encoding a second antigen (e.g. influenza type B antigen) in a second population of nanoparticles (e.g., LNPs); or encapsulation of mRNA(s) encoding a first antigen (e.g. influenza type A antigen) in a first population of nanoparticles (e.g., LNPs) and encapsulation of each mRNA encoding a second antigen (e.g. influenza type B antigen) in a separate population of nanoparticles (e.g., LNPs)). In some embodiments, the composition comprises co-encapsulated mRNAs encoding an antigen. In some embodiments, a composition comprising (i) two or more different RNAs, each encoding an antigenic polypeptide (e.g., an HA protein) of a first virus (e.g., influenza type A virus), e.g. so that the two or more different RNAs, together, encode two or more different polypeptides (e.g. two or more different influenza A HA polypeptides, and (ii) two or more different RNAs, each encoding an antigenic polypeptide (e.g., an HA protein) of a second virus (e.g., an influenza type B virus), e.g. so that the two or more different RNAs, together, encode two or more different polypeptides (e.g. two or more different influenza B HA polypeptides are formulated in nanoparticles (e.g., LNPs) such that: each RNA encoding an antigenic polypeptide of a first virus (e.g. influenza type A virus) is encapsulated in a first population of nanoparticles and each RNA encoding an antigenic polypeptide of a second virus (e.g. influenza type B virus) is encapsulated in a second population of nanoparticles; each RNA is encapsulated in a separate nanoparticle; or each RNA encoding a polypeptide of a first antigen (e.g. influenza type A antigenic polypeptide)is encapsulated in a first population of nanoparticles and the two RNAs encoding a polypeptide of second antigen (e.g. influenza type B antigenic polypeptide) are each encapsulated in separate populations of nanoparticles. Methods of use The RNA compositions may be utilized to treat and / or prevent a disease, disorder or infection comprising administering to a subject any of the RNA compositions described herein. In one embodiment, the RNA composition produces an antigen specific immune response. In some embodiments, the antigen specific immune response comprises a T cell response. In some embodiments, the antigen specific immune response comprises a B cell response. In some embodiments, the antigen specific immune response comprises both a T cell response and a B cell response. In some embodiments, the method of producing an antigen specific immune response involves a single administration of the RNA composition. In some embodiments, the RNA composition is administered to the subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration. In some embodiments, the nanoparticle has a net neutral charge at a neutral pH value. In some embodiments, the RNA (e.g., mRNA) vaccine is multivalent. In some embodiments, the RNA polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof as an antigen. Some aspects of the disclosure are directed to a method of inducing an immune response in a subject, comprising administering to the subject in need thereof an amount of a composition as disclosed herein that produces an immune response comprising an antibody response. In some embodiments, a composition as disclosed herein elicits an immune response comprising a T cell response. One aspect of the disclosure is directed to a method comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein, wherein the amount of the composition is an amount effective to produce an immune response, to treat or to prevent a disease, disorder or infection e.g. “an effective amount”. Some aspects of the disclosure are directed to a method of vaccinating a subject, comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein. The compounds of the invention may be useful for treating or preventing a disease, disorder, or condition or delivering an active agent to treat or prevent a disease, disorder, or condition. In particular, such compositions may be useful in treating or preventing a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. Diseases, disorders, and / or conditions characterized by dysfunctional or aberrant protein or polypeptide activity for which a composition may be administered include, but are not limited to rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases. Administration and Dosing The term "treating", "treat" or "treatment" as used herein embraces both preventative, e.g., prophylactic, and palliative treatment, e.g., relieve, alleviate, or slow the progression of the patient’s disease (or condition) or any tissue damage associated with the disease. As used herein, the terms, “subject, “individual” or “patient,” used interchangeably, refer to any animal, including mammals. Mammals according to the invention include canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, humans and the like, and encompass mammals in utero. In an embodiment, humans are suitable subjects. Human subjects may be of any gender and at any stage of development. As used herein, the phrase “therapeutically effective amount” or “effective amount” refers to the amount of active compound or pharmaceutical agent, such as a nucleic acid, that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include one or more of the following: (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (e.g., arresting (or slowing) further development of the pathology or symptomatology or both); and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (e.g., reversing the pathology or symptomatology or both). An “effective amount” or “therapeutically effective amount” of nucleic acid is an amount sufficient to produce the desired effect, e.g. an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid. In the case where the expression product is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art. The phrase “induce expression of a desired protein” refers to the ability of a nucleic acid to increase expression of the desired protein. To examine the extent of protein expression, a test sample (e.g., a sample of cells in culture expressing the desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., nucleic acid in combination with a lipid of the present invention). Expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g., a sample of cells in culture expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid. When the desired protein is present in a control sample or a control mammal, the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1.0. In particular embodiments, inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1, for example, about 1.1, 1.5, 2.0.5.0 or 10.0. When a desired protein is not present in a control sample or a control mammal, inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected. One of ordinary skill in the art will understand appropriate assays to determine the level of protein expression in a sample, for example dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions. The phrase “inhibiting expression of a target gene” refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid. The expression of the target gene in a control sample or a control mammal may be assigned a value of 100%. In particular embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Typically, a compound of the invention is administered in an amount effective to treat a condition as described herein or to deliver an active agent to treat a condition as described herein. The compounds of the invention may be administered as compound per se, or alternatively, as a pharmaceutically acceptable salt. For administration and dosing purposes, the compound per se or pharmaceutically acceptable salt thereof will simply be referred to as the compounds of the invention. The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compounds of the invention may be administered orally, rectally, vaginally, parenterally, topically, intranasally, or by inhalation. The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the bloodstream directly from the mouth. In another embodiment, the compounds of the invention may also be administered parenterally, for example directly into the bloodstream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques. In another embodiment, the compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. In another embodiment, the compounds of the invention may also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention may be administered rectally or vaginally. In another embodiment, the compounds of the invention may also be administered directly to the eye or ear. The dosage regimen for the compounds of the invention or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary widely. In one embodiment, the total daily dose of a compound of the invention is typically from about 0.01 to about 100 mg / kg (e.g., mg compound of the invention per kg body weight) for the treatment of the indicated conditions discussed herein or to deliver an active agent by using the compounds of the invention. In another embodiment, total daily dose of the compound of the invention is from about 0.00001 to about 50 mg / kg, and in another embodiment, from about 0.0001 to about 30 mg / kg. It is not uncommon that the administration of the compounds of the invention will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired. Nucleic Acids In certain embodiments, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, mRNA (e.g. modified RNA), saRNA, and complementary sequences of the foregoing described herein. Nucleic acids that encode an epitope to which antibodies may bind. Nucleic acids encoding fusion proteins that include these polypeptides are also provided. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids). The term “polynucleotide” refers to a nucleic acid molecule that can be recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide. In this respect, the term “gene” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post- translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar polypeptide. In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 95% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. The nucleic acid sequences, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, equal to any one of, at least any one of, at most any one of, or between any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide. RNA TRANSCRIPTION In some aspects, the RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein. According to the present disclosure, “transcription” comprises “in vitro transcription” or “IVT,” which refers to the process whereby transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for use in various applications, including, e.g., production of protein or polypeptides. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R.1972. In vitro transcription, Ann Rev Biochem, 41409-46; Kamakaka, R. T. and Kraus, W. L. 2001. In vitro Transcription, Current Protocols in Cell Biology, 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B. 2010. Synthesis of RNA by In vitro Transcription in RNA, Methods in Molecular Biology, 703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five – In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology 530:101-114; all of which are incorporated herein by reference). Cloning vectors may be applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term “vector.” According to specific aspects, the RNA used is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template, and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses, Methods 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012, each incorporated herein by reference). The promoter for controlling transcription may be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. Synthetic IVT RNA products may be translated in vitro or introduced directly into cells, where they may be translated. With respect to RNA, the term “expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein. Such synthetic RNA products include but are not limited to, e.g., mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, and the like. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing synthetic RNA products may be excluded. An IVT reaction typically utilizes a DNA template (e.g., a linear DNA template) as described and / or utilized herein, ribonucleotides (e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase. In some aspects, an mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, an RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription may be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. In some aspects, starting material for IVT may include linearized DNA template, nucleotides, Rnase inhibitor, pyrophosphatase, and / or a polymerase (e.g., a T7 RNA polymerase). The nucleotides may be manufactured in house, may be obtained from a supplier, or may be synthesized. The nucleotides may be, but are not limited to, those described herein including natural and unnatural (modified) nucleotides. Any number of RNA polymerases or variants may be used, including, but not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing RNA polymerases may be excluded from. Some embodiments exclude the use of Dnase. In some aspects, the IVT process is conducted in a bioreactor. The bioreactor may comprise a mixer. In some aspects, nucleotides may be added into the bioreactor throughout the IVT process. In some aspects, one or more post-IVT agents are added into the IVT mixture comprising RNA in the bioreactor after the IVT process. Exemplary post-IVT agents may include DNAse I configured to digest the linearized DNA template and / or proteinase K configured to digest DNAse I and T7 RNA polymerase. In some aspects, the post-IVT agents are incubated with the mixture in the bioreactor after IVT. In some aspects, the bioreactor may contain at least, at most, exactly, or between (inclusive or exclusive) any two of 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 ,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500 or more liters IVT mixture. The IVT mixture may have an RNA concentration that is or is not at least, at most, exactly, or between (inclusive or exclusive) any two of 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL or more RNA. In some aspects, the IVT mixture may include residual spermidine, residual DNA, residual proteins, peptides, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+, Na+, Ca2+), RNA fragments, residual nucleotides, free phosphates, or any combinations thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing can be excluded from the IVT mixture. Isolation and / or purification of the nucleic acids described herein may include, but is not limited to, phenol / chloroform extraction and / or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid clean-up, quality assurance and quality control. Additional, non-limiting examples of purification procedures include AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). In some aspects, 1, 2, 3, 4, 5, or more of the foregoing purification may be excluded. The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment and / or purification method. In some aspects, at least a portion of the IVT mixture is filtered. The IVT mixture may be filtered via ultrafiltration and / or diafiltration to remove at least some impurities from the IVT mixture and / or to change buffer solution for the at least a portion of IVT mixture to produce a concentrated RNA solution as a retentate. In some aspects, both “ultrafiltration” and “diafiltration” refer to a membrane filtration process. Ultrafiltration typically uses membranes having pore sizes of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 µm. In some aspects, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO may be at least, at most, exactly, or between (inclusive or exclusive) any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, and 10000 kDa. A skilled artisan will understand that filtration membranes may comprise different suitable materials, including, e.g., polymers, cellulose, ceramic, etc., depending upon the application. In some aspects, membrane filtration may be more desirable for large volume purification process. In some aspects, ultrafiltration and diafiltration of the IVT mixture for purifying RNA may include (1) Direct Flow Filtration (DFF), also known as “dead-end” filtration, that applies a feed stream perpendicular to the membrane face and attempts to pass 100% of the fluid through the membrane, and / or (2) Tangential Flow Filtration (TFF), also known as crossflow filtration, where a feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is retained and / or recirculated back to the feed tank. In some aspects, the filtering of the IVT mixture is conducted via TFF comprising an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some aspects, the first diafiltration step is conducted in the presence of ammonium sulfate. The first diafiltration step may be configured to remove a majority of impurities from the IVT mixture. In some aspects, the second diafiltration step is conducted without ammonium sulfate. The second diafiltration step may be configured to transfer the RNA into a DS buffer formulation. A filtration membrane with an appropriate MWCO may be selected for ultrafiltration in the TFF process. The MWCO of a TFF membrane determines which solutes may pass through the membrane into the filtrate and which are retained in the retentate. The MWCO of a TFF membrane may be selected such that substantially all of the solutes of interest (e.g., desired synthesized RNA species) remain in the retentate, whereas undesired components (e.g., excess ribonucleotides, small nucleic acid fragments such as digested or hydrolyzed DNA template, peptide fragments such as digested proteins and / or other impurities) pass into the filtrate. In some aspects, the retentate comprising desired synthesized RNA species may be re-circulated to a feed reservoir to be re-filtered in additional cycles. In some aspects, a TFF membrane may have a MWCO of at least, at most, exactly, or between (inclusive or exclusive) any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or more. In some aspects, a TFF membrane may have a MWCO of at least, at most, exactly, or between (inclusive or exclusive) any two of 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or more. In some aspects, a TFF membrane may have a MWCO of or of about 250-350 kDa. In some aspects, a TFF membrane (e.g., a cellulose-based membrane) may have a MWCO of or of about 30-300 kDa; 50-300 kDa, 100-300 kDa, or 200-300 kDa. Diafiltration may be performed either discontinuously, or alternatively, continuously. For example, in continuous diafiltration, a diafiltration solution may be added to a sample feed reservoir at the same rate as filtrate is generated. In this way, the volume in the sample reservoir remains constant but small molecules (e.g., salts, solvents, etc.) that may freely permeate through a membrane are removed. Using solvent removal as an example, each additional diafiltration volume (DV) reduces the solvent concentration further. In discontinuous diafiltration, a solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the desired concentration of small molecules (e.g., salts, solvents, etc.) remaining in the reservoir is reached. Each additional diafiltration volume (DV) reduces the small molecule (e.g., solvent) concentration further. Continuous diafiltration typically requires a minimum volume for a given reduction of molecules to be filtered. Discontinuous diafiltration, on the other hand, permits fast changes of the retentate condition, such as pH, salt content, and the like. In some aspects, the first diafiltration step is conducted with at least, at most, exactly, or between (inclusive or exclusive) any two of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more diavolumes. In some aspects, the second diafiltration step is conducted with at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more diavolumes. In some aspects, the first diafiltration step is conducted with 5 diavolumes, and second diafiltration step is conducted with 10 diavolumes. In some aspects, for ultrafiltration and / or diafiltration, the IVT mixture is filtered at a rate of at least, at most, exactly, or between (inclusive or exclusive) any two of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L / m2of filter area per hour, or more. The concentrated RNA solution may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / mL single stranded RNA. The bioburden of the concentrated RNA solution via filtration to obtain an RNA product solution may also be reduced, in some aspects. The filtration for reducing bioburden may be conducted using one or more filters. The one or more filters may include a filter with a pore size that is or is not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.2 µm, 0.45 µm, 0.65 µm, 0.8 µm, or any other pore size configured to remove bioburdens. As one example, reducing the bioburden may include draining a retentate tank containing retentate obtained from the ultrafiltration and / or diafiltration to obtain the retentate. Reducing the bioburden may include flushing a filtration system for ultrafiltration and / or diafiltration using a wash buffer solution to obtain a wash pool solution comprising residue RNA remaining in the filtration system. The retentate may be filtered to obtain a filtered retentate. The wash pool solution may be filtered using a first 0.2 µm filter to obtain a filtered wash pool solution. The retentate may be filtered using the first 0.2 µm filter or another 0.2 µm filter. The filtered wash pool solution and the filtered retentate may be combined to form a combined pool solution. The combined pool solution may be filtered using a second 0.2 µm filter to obtain a filtered combined pool solution, which is further filtered using a third 0.2 µm filter to produce an RNA product solution. A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, and / or analytical HPLC. In some aspects, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. In some aspects, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size and / or to assess degradation. Degradation of the nucleic acid may be assessed by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). In some aspects, 1, 2, 3, 4, 5, or more of the foregoing assessment methods may be excluded. RNA ENCAPSULATION The RNA in an RNA product solution may be encapsulated, and the RNA solution may further comprise at least one encapsulating agent. In one aspect, the encapsulating agent comprises a lipid, a lipid nanoparticle (LNP), lipoplexes, polymeric particles, polyplexes, monolithic delivery systems, or a combination thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing elements may be excluded as an encapsulating agent. In one aspect, the encapsulating agent is a lipid, and produced is lipid nanoparticle (LNP)- encapsulated RNA. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and / or the cationic polymer combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles. A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. A lipid is a substance that is insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids and are encompassed by the compositions and methods of the present disclosure. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently. In some aspects, LNPs may be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular Rnases and / or may be engineered for systemic delivery of the RNA to target cells. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intravenously administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intramuscularly administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intradermally administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intranasally administered to a subject in need thereof. In one aspect, the RNA in the RNA product solution is at a concentration of < 1 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.05 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 1 mg / mL. In another aspect, the RNA concentration is from or from about 0.05 mg / mL to about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least 10 mg / mL. In another aspect, the RNA is at a concentration of at least 50 mg / mL. In some aspects, the RNA is or is not at a concentration of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.30 mg / mL, 0.40 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or more. The present disclosure provides for an RNA product solution and a lipid preparation mixture or compositions thereof comprising at least one RNA encoding, e.g., an antigen (e.g., an RSV prefusion F protein) complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes and / or nanoliposomes. In some aspects, the composition comprises a lipid nanoparticle. A lipid nanoparticle or LNP refers to particles of any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of RNA. In some aspects, lipid nanoparticles are included in a formulation that may be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some aspects, the lipid nanoparticles of the present disclosure comprise a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids, or combinations thereof. In some aspects, the LNPs comprise at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and / or at least one polymer conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, 1, 2, 3, or more of the foregoing excipients may be excluded from the LNPs. In some aspects, the LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipid(s). For example, the LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise or do not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 45 to 55 mole percent (mol%) cationic (e.g., ionizable) lipid(s). For example, LNPs may comprise or not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipid(s). For example, the LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non- cationic) lipid(s). In some aspects, the LNPs comprise 5 to 15 mol% neutral (e.g., non-cationic) lipid(s). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid). For example, the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 35 to 40 mol% structural lipid(s) (e.g., a steroid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, or 40 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 0.5-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). For example, the lipid nanoparticle may comprise 0.5- 10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5- 15 mol%, 5-10 mol%, or 10-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, the lipid LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, the LNPs comprise 1 to 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)- modified lipid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 1.5, or 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, the LNPs comprise 20-75 mol% cationic (e.g., ionizable) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%), 0.5-25 mol% neutral (e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11%, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25%, and 25%), 5-55 mol% structural lipid(s) (e.g., a sterol) e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5-20 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)- modified lipid) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11%, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some aspects, 1, 2, 3, or more of the lipids may be excluded from the LNPs. In some non-limiting aspects, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 60 / 7.5 / 31 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.5 / 7.5 / 31.5 / 3.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 47.5 / 10 / 40.7 / 1.8 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid). In some aspects, the active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle and / or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. The nucleic acid (e.g., mRNA) or a portion thereof may also be associated and complexed with the lipid nanoparticle. A lipid nanoparticle may comprise any lipid capable of forming a particle to which the nucleic acids are attached, and / or in which the one or more nucleic acids are encapsulated. In some aspects, provided RNA molecules (e.g., mRNA) may be formulated with LNPs. In some aspects, the lipid nanoparticles may or may not have a mean diameter of or of about 1 to 500 nm (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some aspects, the lipid nanoparticles have a mean diameter of or of from about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, exactly, or between (inclusive or exclusive) of 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. The term “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, “mean diameter,” “diameter,” or “size” for particles is used synonymously with the value of the Z-average. LNPs described herein may exhibit a polydispersity index less than or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, exactly, or between (inclusive or exclusive) of 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. The polydispersity index is, in some aspects, calculated based on dynamic light scattering measurements by the so-called cumulant analysis referred to in the definition of “average diameter.” Under certain prerequisites, it may be taken as a measure of the size distribution of an ensemble of nanoparticles. In some aspects, an LNP of the disclosure comprises or does not comprise an N:P ratio of or of from about 2:1 to about 30:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 6:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 3:1. As used herein “N:P” is the ratio of cationic lipid to RNA. In some aspects, an LNP of the disclosure comprises or does not comprise a wt / wt ratio of the cationic lipid component to the RNA of or of from about 5:1 to about 100:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, or 100:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 20:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 10:1. In certain aspects, nucleic acids (e.g., RNA molecules), when present in provided LNPs, are resistant in aqueous solution to degradation with a nuclease. In some aspects, LNPs are liver- targeting lipid nanoparticles. In some aspects, LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some aspects, cationic LNPs may comprise at least one cationic lipid, at least one polymer conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid). In certain aspects, the RNA solution and lipid preparation mixture or compositions thereof may have at least, at most, exactly, between (inclusive or exclusive) of, or about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a particular lipid, lipid type, or non- lipid component such as lipid-like materials and / or cationic polymers and / or an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or as would be known to one of skill in the art. LNPs described herein can be generated using components, compositions, and methods as are generally known in the art, see , e.g., PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491, all of which are incorporated by reference herein in their entirety. Other non-limiting examples of methods for preparing LNPs can be found in, e.g., WO 2022 / 032154, the disclosure of which is incorporated by reference herein in its entirety. For example, methods of preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term “colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” refers only to the particles in the mixture and not the entire suspension. For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer, methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media). In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included. Reverse phase evaporation is an alternative method to film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that subsequently turns into a liposomal suspension. The term “ethanol injection technique” refers to a process in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example, lipid vesicle formation such as liposome formation. Generally, the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some aspects, formed as follows: an ethanol solution comprising lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In some aspects, the RNA lipoplex particles described herein are obtainable without a step of extrusion. The term “extruding” or “extrusion” refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores. Other methods for preparing a colloid having organic solvent free characteristics may also be used according to the present disclosure. In some aspects, LNP-encapsulated RNA may be produced by rapid mixing of an RNA solution described herein (e.g., the RNA product solution) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some aspects, suitable buffering agents comprise tris, histidine, citrate, acetate, phosphate, and / or succinate. In some aspects, 1, 2, 3, or more of the foregoing buffering agents are excluded. The pH of a liquid formulation relates to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which, for example, in some aspects, may result in much higher encapsulation efficiency than is achieved in the absence of interactions between nucleic acids and at least one of the lipid components. In certain aspects, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species, such as RNA species. In an LNP formulation, it is possible that each nucleic acid species is separately formulated as an individual LNP formulation. In that case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations). In some aspects, a composition such as a pharmaceutical composition comprises more than one individual LNP formulation. Respective pharmaceutical compositions are referred to as mixed LNP formulations. Mixed LNP formulations according to the invention are obtainable by forming, separately, individual LNP formulations, as described above, followed by a step of mixing of the individual LNP formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing LNPs is obtainable. Individual LNP populations may be together in one container, comprising a mixed population of individual LNP formulations. Alternatively, it is possible that different nucleic acid species are formulated together as a combined LNP formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. As opposed to a mixed LNP formulation, a combined LNP formulation will typically comprise LNPs that comprise more than one RNA species. In a combined LNP composition, different RNA species are typically present together in a single particle. A. CATIONIC POLYMERIC MATERIALS Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L- lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some aspects herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. In some aspects, such synthetic materials may be suitable for use as cationic materials herein. A “polymeric material,” as used herein, is given its ordinary meaning, e.g., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some aspects, such repeat units may all be identical; alternatively, in some cases, there may be more than one type of repeat unit present within the polymeric material. In some cases, a polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties may also be present in the polymeric material, for example targeting moieties such as those described herein. Those skilled in the art are aware that, when more than one type of repeat unit is present within a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is said to be a “copolymer.” In some aspects, a polymer (or polymeric moiety) utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer may be arranged in any fashion. For example, in some aspects, repeat units may be arranged in a random order; alternatively or additionally, in some aspects, repeat units may be arranged in an alternating order, or as a “block” copolymer, e.g., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks. In certain aspects, a polymeric material for use in accordance with the present disclosure is biocompatible. Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations. In certain aspects, a biocompatible material is biodegradable, e.g., is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body. In certain aspects, a polymeric material may be or comprise protamine or polyalkyleneimine, in particular protamine. As those skilled in the art are aware term “protamine” is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term “protamine” is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin. In some aspects, the term “protamine” as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and / or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources. In some aspects, a polyalkyleneimine comprises polyethylenimine and / or polypropylenimine. In some aspects, the polyalkyleneimine is polyethyleneimine (PEI). In some aspects, the polyalkyleneimine is a linear polyalkyleneimine, e.g., linear polyethyleneimine (PEI). Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some aspects, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid may be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated. In some aspects, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and / or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials. B. LIPIDS & LIPID-LIKE MATERIALS The terms “lipid” and “lipid-like material” are used herein to refer to molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH. The term “lipid” refers to a group of organic compounds that are characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids as well as sterol-containing metabolites such as cholesterol, and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramides phosphosphingolipids (e.g., sphingomyelins, phosphocholine), and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of...

Claims

CLAIMS 1. An immunogenic composition comprising a) a fatty acid, including a derivative or salt thereof; b) one or more lipid nanoparticles (LNP), wherein the LNP comprises: i. an ionizable cationic lipid; ii. cholesterol, a cholesterol analog or a mixture of cholesterol and a cholesterol analog; iii. a neutral lipid; and iv. a polymer-conjugated lipid: and c) one or more ribonucleic acids (RNA) comprising at least one open reading frame (ORF) encoding a polypeptide of a gene of interest or an immunogenic fragment thereof, wherein the RNA is encapsulated in the LNP of item (b).

2. The immunogenic composition of claim 1, wherein the fatty acid is an unsaturated fatty acid.

3. The immunogenic composition of claim 2, wherein the unsaturated fatty acid is a monounsaturated fatty acid, a di-unsaturated fatty acid or a polyunsaturated fatty acid.

4. The immunogenic composition of claim 3, wherein the fatty acid is selected from oleic acid, arachidonic acid, eruric acid, linolenic acid, ricinoleic acid, palmitoleic acid, linoleic acid, or a derivative or salt thereof.

5. The immunogenic composition of claim 4, wherein the salt is selected from sodium, potassium, magnesium or calcium.

6. The immunogenic composition of claim 4, wherein the fatty acid is oleic acid.

7. The immunogenic composition of claim 5, wherein the fatty acid salt is sodium oleate.

8. The immunogenic composition of claim 4, wherein the fatty acid derivative is a derivative of ricinoleic acid.

9. The immunogenic composition of claim 1, wherein the fatty acid derivative is Kolliphor EL® or Cremaphor EL®.

10. The immunogenic composition according to any one of claims 1-9, wherein the immunogenic composition comprises a fatty acid to RNA weight ratio (O:R) of from about 1.5:1 to about 24:1.

11. The immunogenic composition of claim 10, wherein the immunogenic composition has an O:R of at least 1.5:

1.

12. The immunogenic composition of claim 11, wherein the immunogenic composition has an O:R selected from about 1.5:1, about 2:1, about 3:1, about 4:1, about 6:1, about 6.5:1, about 8:1, about 10:1, about 12:1, about 13:1 or about 24:

1.

13. The immunogenic composition of claim 12, wherein the immunogenic composition has an O:R of 8:

1.

14. The immunogenic composition according to any one of claims 1-13, wherein the cholesterol analog is selected from sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, β-sitosterol, stigmastanol, β-sitostanol, ergosterol, fecosterol, lupeol, cycloartenol, Δ5-avenasterol, Δ7-avenasterol or a Δ7-stigmasterol, tomatidine, ursolic acid or alpha-tocopherol, including analogs, salts or esters thereof.

15. The immunogenic composition of claim 14, wherein the cholesterol analog is β-sitosterol, stigmasterol or campesterol.

16. The immunogenic composition of claim 15, wherein the cholesterol analog is β-sitosterol.

17. The immunogenic composition according to any one of claims 1-16, wherein the molar ratio of the cholesterol analog to cholesterol in the mixture of the cholesterol analog and cholesterol is 6:4, 1:1 or 4:

6.

18. The immunogenic composition of claim 17, wherein the mixture of the cholesterol analog and cholesterol has a molar ratio of 6:4 of cholesterol analog to cholesterol.

19. The immunogenic composition of claim 18, wherein the mixture comprises β-sitosterol and cholesterol.

20. The immunogenic composition of claim 19, wherein the mixture comprises a molar ratio of β-sitosterol:cholesterol of 6:

4.

21. The immunogenic composition of any one of claims 1- 20, wherein the ionizable cationic lipid is selected from N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 2-hexyldecyl 6-[(2-{[4-(heptylcarbonylamino)butyl]-N-methylamino}ethyl)[5-(2- hexyldecyloxycarbonyl)pentyl]amino]hexanoate (ALC-0515), 2-(7-(4-hydroxybutyl)-2,7- diazaspiro[3.5]nonan-2-yl)propane-1,3-diyl bis(2-heptylnonanoate) (PF-9032), heptadecane- 9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM-102), or a mixture thereof.

22. The immunogenic composition of claim 21, wherein the ionizable cationic lipid is ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) having the structure:.

23. The immunogenic composition of claim 21, wherein the ionizable cationic lipid is 2- hexyldecyl 6-[(2-{[4-(heptylcarbonylamino)butyl]-N-methylamino}ethyl)[5-(2- hexyldecyloxycarbonyl)pentyl]amino]hexanoate (ALC-0515) having the structure:.

24. The immunogenic composition of claim 21, wherein the ionizable cationic lipid is 2-(7-(4- hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diyl bis(2-heptylnonanoate) (PF- 9032) having the structure:.

25. The immunogenic composition of any one of claims 1-24, wherein the neutral lipid is a phospholipid.

26. The immunogenic composition of claim 25, wherein the phospholipid is a sphingolipid.

27. The immunogenic composition of claim 26, wherein the sphingolipid is a sphingomyelin.

28. The immunogenic composition of any one of claims 1-27, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16- O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2- oleoylphosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidyl serine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diemcoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), l,2-dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE); l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, or a mixture thereof.

29. The immunogenic composition of claim 28, wherein the neutral lipid is selected from 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl- oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE), or a mixture thereof.

30. The immunogenic composition of claim 29, wherein the neutral lipid is 1,2- Distearoyl-sn- glycero-3-phosphocholine (DSPC).

31. The immunogenic composition of claim 28, wherein the neutral lipid is egg sphingomyelin (ESM).

32. The immunogenic composition of any one of claims 1-31, wherein the polymer-conjugated lipid is a pegylated lipid.

33. The immunogenic composition of claim 32, wherein the pegylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) or a PEG dialkyoxypropylcarba PEGylated diacylglycerol (PEG-DAG), 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-((O- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG-ceramide, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) or α-(3’-[(1,2-di[myristyloxy]propanoxy) carbonylamino]propyl)-ω-methoxy, polyoxyethylene (PEG-C-DMG).

34. The immunogenic composition of claim 33, wherein the pegylated lipid is ALC-0159 (2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide) having the structure:.

35. The immunogenic composition of any one of claims 1-31, wherein the polymer-conjugated lipid is a terminally activated polyoxazoline (POZ)-conjugated lipid.

36. The immunogenic composition of claim 35, wherein the POZ-conjugated lipid is polyethyloxazoline-dimyristoylamide (PEOZ-dma) having the structure:,wherein n is an integer from 18-22.

37. The immunogenic composition of any one of claims 1-36, wherein the LNP comprises about 15-60 mol % ionizable cationic lipid, about 13-60 mol % cholesterol or mixture of cholesterol and a cholesterol analog, about 3-30 mol % neutral lipid, and about 0.5-10 mol % polymer- conjugated lipid.

38. The immunogenic composition according to any one of claims 1-37, wherein the lipid nanoparticle comprises about 20 mol % to about 60 mol % ionizable cationic lipids, about 18.5 mol % to about 48.5 mol % mixture of cholesterol and a cholesterol analog, about 0 mol % to about 30 mol % neutral lipids, and about 0 mol % to about 10 mol % polymer- conjugated lipid.

39. The immunogenic composition of any of claims 37-38, wherein the immunogenic composition comprises a mol% ratio of ionizable cationic lipid:cholesterol:neutral lipid:polymer-conjugated lipid:fatty acid or derivative or salt thereof selected from the group: a) 42.22:36.18:8.89:1.6:11.11; b) 40.71:34.88:8.57:1.54:14.29; c) 38:32.56:8:1.44:20; d) 35.62:30.52:7.5:1.35:25; e) 31.67:27.13:6.67:1.2:33.34; f) 30.81:26.4:6.49:1.17:35.14; g) 28.5:24.42:6:1.08:40; h) 23.75;20.35:5:0.90:50; or i) 15.83:13.57:3.33:0.6:66.

67.

40. The immunogenic composition of any of claims 37-38, wherein the immunogenic composition comprises a mol% ratio of ionizable cationic lipid:cholesterol analog:cholesterol:neutral lipid:polymer conjugated lipid:fatty acid or fatty acid salt selected from the group: a) 42.22:21.71:14.47:8.89:1.6:11.11; b) 40.71:20.93:13.95:8.57:1.54:14.29; c) 38:13.02:19.54:8:1.44:20;d) 35.62:18.31:12.21:7.5:1.35:25; e) 31.67:10.85:16.28:6.67:1.2:33.34; f) 30.81:10.56:15.84:6.49:1.17:35.14; g) 28.5:14.65:9.77:6:1.08:40; h) 25.91:13.32:8.88:5.45:0.98:45.46; or i) 22.8:11.72:7.81:4.8:0.86:

52.

41. The immunogenic composition of any one of claims 37, 38 and 40, wherein the cholesterol analog is β-sitosterol.

42. The immunogenic composition of claim 41, wherein the LNP comprises a combination of lipids selected from: a) ALC-0315, beta-sitosterol, cholesterol, DSPC, and ALC-0159; b) ALC-0315, beta-sitosterol, cholesterol, ESM, and ALC-0159; c) ALC-0315, beta-sitosterol, cholesterol, DSPC, and PEOZ; d) ALC-0315, beta-sitosterol, cholesterol, ESM, and PEOZ; e) ALC-0515, beta-sitosterol, cholesterol, DSPC, and ALC-0159; f) ALC-0515, beta-sitosterol, cholesterol, ESM, and ALC-0159; g) ALC-0515, beta-sitosterol, cholesterol, DSPC, and PEOZ; h) ALC-0515, beta-sitosterol, cholesterol, ESM, and PEOZ; i) PF-9032, beta-sitosterol, cholesterol, DSPC, and ALC-0159; j) PF-9032, beta-sitosterol, cholesterol, ESM, and ALC-0159; k) PF-9032, beta-sitosterol, cholesterol, DSPC, and PEOZ; or l) PF-9032, beta-sitosterol, cholesterol, ESM, and PEOZ.

43. The immunogenic composition of claim 39, wherein the LNP comprises a combination of lipids selected from: a) ALC-0315, cholesterol, DSPC, and ALC-0159;b) ALC-0315, cholesterol, ESM, and ALC-0159; c) ALC-0315, cholesterol, DSPC, and PEOZ; d) ALC-0315, cholesterol, ESM, and PEOZ; e) ALC-0515, cholesterol, DSPC, and ALC-0159; f) ALC-0515, cholesterol, ESM, and ALC-0159; g) ALC-0515, cholesterol, DSPC, and PEOZ; h) ALC-0515, cholesterol, ESM, and PEOZ; i) PF-9032, cholesterol, DSPC, and ALC-0159; j) PF-9032, cholesterol, ESM, and ALC-0159; k) PF-9032, cholesterol, DSPC, and PEOZ; or l) PF-9032, cholesterol, ESM, and PEOZ.

44. The immunogenic composition according to any of claims 1-43, wherein the LNP size is at least 40 nm.

45. The immunogenic composition according to any of claims 1-44, wherein the LNP size is at most 180 nm.

46. The immunogenic composition according to any one of claims 1-45, wherein the RNA is modRNA or saRNA.

47. The immunogenic composition of claim 46, wherein the RNA comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.

48. The immunogenic composition of claim 47, wherein the 5’ cap is a 5’ cap analog.

49. The immunogenic composition according to any of claims 46 to 48, wherein the RNA further comprises one or more modified nucleotides.

50. The immunogenic composition of claim 49, wherein the modified nucleotide is selected from pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio- 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2- thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2′-O-methyl uridine or N1-Methylpseudourodine-5’-triphosphate (m1ΨTP). 51.The immunogenic composition of claim 50, wherein the modified nucleotide is N1- Methylpseudourodine-5’-triphosphate (m1ΨTP).

52. The immunogenic composition according to any of claims 46-51, wherein the RNA open reading frame is codon-optimized.

53. The immunogenic composition of according to any one of claims 1-52, wherein the RNA has an integrity greater than 85%.

54. The immunogenic composition of according to any one of claims 1-53, wherein the RNA has a purity of greater than 85%.

55. The immunogenic composition according to any one of claims 1-54, wherein at least 80% of the total RNA in the immunogenic composition is encapsulated.

56. The immunogenic composition according to any one of claims 1-55, wherein the percentage of intact mRNA encapsulated in the LNP is at least 80%.

57. The immunogenic composition according to any one of claims 1-56, wherein the LNP comprises an N:P ratio of from about 2:1 to about 30:

1.

58. The immunogenic composition of claim 57, wherein the LNP has an N:P ratio of at least 5.

59. The immunogenic composition of claim 57, wherein the LNP has an N:P ratio of at least 6.

60. The immunogenic composition of claim 57, wherein the LNP has an N:P ratio of 10.

61. The immunogenic composition of claim 57, wherein the LNP has an N:P ratio of 6.

62. The immunogenic composition according to any one of claims 1-61, wherein the immunogenic composition further comprises a buffer.

63. The immunogenic composition of claim 62, wherein the buffer is Tris.

64. The immunogenic composition of claim 63, wherein the buffer is 10 mM Tris.

65. The immunogenic composition according to any one of claim 62, wherein the buffer is phosphate buffered saline (PBS).

66. The immunogenic composition according to any one of claims 1-65, wherein the immunogenic composition further comprises sucrose.

67. The immunogenic composition of claim 66, wherein the immunogenic composition comprises 300 mM sucrose.

68. The immunogenic composition according to any one of claims 1-67, wherein the immunogenic composition does not comprise sodium chloride.

69. The immunogenic composition according to any one of claims 1-68, wherein the immunogenic composition has a pH from about 4 to about 11, from about 5 to about 10, from about 6 to about 9, from about 7 to about 8, or from about 7.3 to about 7.

5.

70. The immunogenic composition according to claim 69, wherein the immunogenic composition has a pH of 7.

4.

71. The immunogenic composition according to any one of claims 1-70, wherein the immunogenic composition has less than or equal to 12.5 EU / mL of bacterial endotoxins.

72. A combination formulation comprising the immunogenic composition of any one of claims 1- 71 and one or more additional compositions.

73. The combination formulation of claim 72, wherein the one or more additional compositions comprise one or more LNPs.

74. The combination formulation of claim 73, wherein the one or more additional compositions do not comprise a LNP.

75. The combination formulation of claim 74, wherein the one or more additional compositions comprise proteins or protein subunits, conjugated antigens, inactivated viral or bacterial strains or fragments thereof, adjuvants, or any combination thereof.

76. The combination formulation of claims 72-75, wherein the additional composition comprises TritonX.

77. The combination formulation of claims 72-76, wherein the immunogenic composition of any one of claims 1-71 and the additional composition are pre-mixed, post-mixed or co- lyophilized to form the combination formulation.

78. The immunogenic composition according to any one of claims 1-71 and the combination formulation of claims 72-77, wherein the immunogenic composition or combination formulation is liquid, frozen, or lyophilized.

79. The immunogenic composition according to any one of claims 1-71 and the combination formulation of claims 72-77, wherein the immunogenic composition or combination formulation has never been frozen.

80. The immunogenic composition or the combination formulation of any one of claims 1-79, wherein the immunogenic composition or combination formulation has a particle size which is less than that of an immunogenic composition which does not comprise a fatty acid, derivative or salt thereof.

81. The immunogenic composition or the combination formulation of any one of claims 1-79, wherein the immunogenic composition or combination formulation has a PDI which is less than that of an immunogenic composition which does not comprise a fatty acid, derivative or salt thereof.

82. The immunogenic composition or combination formulation of claims 80 or 81, wherein the particle size or PDI of the immunogenic composition is about 95% or less, about 90% or less, about 85% or less, about % 80 less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, as compared to that of an immunogenic composition which does not comprise a fatty acid, derivative or salt thereof.

83. The immunogenic composition or the combination formulation of any one of claims 1-79, wherein the immunogenic composition or combination formulation has less adducts than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

84. The immunogenic composition or the combination formulation of any one of claims 1-79, wherein the immunogenic composition or combination formulation is more stable at 5ºC over time than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

85. The immunogenic composition or combination formulation of claim 84 , wherein the immunogenic composition or combination formulation is stable up to about 5 ºC for at least 3 months.

86. The immunogenic composition or combination formulation of claim 84 , wherein the immunogenic composition or combination formulation is stable up to about 5 ºC for at least 3 months, at least 6 months, at least 9 months or at least 12 months or longer.

87. The immunogenic composition or the combination formulation of any one of claims 1-79, wherein the immunogenic composition or combination formulation is more stable after one or more freeze / thaw cycles than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

88. The immunogenic composition or combination formulation of claim 79, wherein the immunogenic composition or combination formulation has more RNA integrity than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

89. The immunogenic composition or combination formulation of claim 79, wherein the immunogenic composition or combination formulation has higher RNA encapsulation than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

90. The immunogenic composition or combination formulation of claim 79, wherein the immunogenic composition or combination formulation has higher in vitro expression than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

91. The immunogenic composition or combination formulation of claim 79, wherein the immunogenic composition or combination formulation has higher immunogenicity than an immunogenic composition or combination formulation which does not comprise a fatty acid, derivative or salt thereof.

92. The immunogenic composition or combination formulation of claim 79, wherein the immunogenic composition or combination formulation is a vaccine.

93. A method of making the immunogenic composition of any one of claims 1-71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with the lipids of an LNP (organic phase); ii) filtering the mixture of step (i) via Tangential Flow Filtration / BBR; iii) dissolving a fatty acid salt in a buffer in an amount that will achieve the desired O:R ratio of the composition; iv) compounding the buffer solution of step (iii) with the filtered mixture of step (ii); and v) the compounded mixture of step (iv) undergoes terminal filtration,thereby making the immunogenic composition of any one of claims 1-71.

94. A method of producing a stable immunogenic composition of any one of claims 1-71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with the lipids of an LNP (organic phase); ii) filtering the mixture of step (i) via Tangential Flow Filtration / BBR; iii) mixing a fatty acid with a compound in a buffer to make a fatty acid salt in an amount that will achieve the desired O:R ratio of the composition; iv) adding the buffer solution of step (iii) to the filtered mixture of step (ii) and compounding; and v) terminal filtration of the compounded mixture of step (iv), thereby making the immunogenic composition of any one of claims 1-71.

95. A method of making the immunogenic composition of any one of claims 1-71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with an organic phase comprising the lipids of an LNP and a fatty acid in an amount that will achieve the desired O:R ratio of the composition; ii) filtering the mixture of step (i) via Tangential Flow Filtration / BBR; iii) compounding the filtered mixture of step (ii); and iv) the compounded mixture of step (iii) undergoes terminal filtration, thereby making the immunogenic composition of any one of claims 1-71.

96. A method of making the immunogenic composition of any one of claims 1-71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with the lipids of an LNP (organic phase); ii) mixing a fatty acid salt or a fatty acid with a compound to make a fatty acid salt with a buffer in an amount that will achieve the desired O:R ratio of the composition (quench buffer);iii) mixing the mixture of step (i) with the quench buffer of step (ii) and filtering via Tangential Flow Filtration / BBR; iv) compounding the filtered mixture of step (iii); and v) the compounded mixture of step (iv) undergoes terminal filtration, thereby making the immunogenic composition of any one of claims 1-71.

97. A method of making the immunogenic composition of any one of claims 1-71 comprising the steps of: i) mixing one or more nucleic acids (aqueous phase) with an organic phase comprising the lipids of an LNP and a fatty acid salt in an amount that will achieve the desired O:R ratio of the composition; ii) mixing the mixture of step (i) with a quench buffer having pH 7.4; iii) filtering the mixture of step (ii) via Tangential Flow Filtration / BBR; iv) compounding the filtered mixture of step (iii); and v) the compounded mixture of step (iv) undergoes terminal filtration, thereby making the immunogenic composition of any one of claims 1-71.

98. A method for delivering a ribonucleic acid to a subject in need thereof comprising administering to the subject the immunogenic composition or combination formulation of any one of claims 1-79.

99. The immunogenic composition or combination formulation of any one of claims 1-79 for use as a component of a medicament.

100. Use of the immunogenic composition or combination formulation of any one of claims 1-79 for the manufacture of a medicament.