Target gene RNA preparation

Abstract

The present disclosure relates to RNA molecules with multivalent properties against multiple virus strains or antigens, and methods for making the same.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of and priority to PCT / CN2022 / 126258, filed October 19, 2022, the entire contents of which are incorporated herein by reference for all purposes. [Background technology]

[0002] RNA is a single-stranded polynucleotide molecule that can encode the expression of specific proteins, such as disease-specific antigens, once it reaches the cytoplasm. The successful synthesis of RNA by in vitro transcription (IVT) has initiated research into its use as a therapeutic agent. Currently, RNA vaccines against COVID-19 are widely used and have also been shown to be effective against SARS-CoV-2. However, current vaccines generally target only one variant.

[0003] As new variants of viruses (e.g., SARS-CoV-2) continue to emerge, there is a need for vaccines that can target not only these new variants but also combinations of variants. Accordingly, there is a need for multivalent vaccines to address variants of human infections. Provided herein, in certain embodiments, are improved RNA vaccines with broad protection and safety against multiple viral variants. Summary of the Invention [Problem to be solved by the invention]

[0004] Described herein are RNA vaccines that provide broad protection against multiple viral variants, such as SARS-CoV-2. These RNA vaccines are designed to effectively integrate different sequences of genes expressing proteins from different viral strains into a single sequence platform, enabling the creation of RNA vaccines with broad protection. Furthermore, the presence of an oligomerization domain, such as a T4-foldon domain, promotes multimerization to express multiple antigens, enhancing stability and further enhancing immune efficacy. Additionally, compared to conventional vaccines, the RNA vaccines described herein avoid complex blend preparation processes, overdosing, excipient burden, and toxic safety issues. Also provided herein are methods for preparing RNA vaccines, for example, by providing RNA with an LNP composition. [Means for solving the problem]

[0005] In one aspect, provided herein is a purified ribonucleic acid (RNA) molecule comprising: a) a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from a first viral variant; b) a second nucleotide sequence encoding a multimerization domain; and c) a third nucleotide sequence encoding a second antigen or antigenic fragment thereof from a second viral variant, wherein the first viral variant and the second viral variant are different, and the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5' to 3' direction.

[0006] In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA is self-replicating RNA. In some embodiments, the RNA is not self-replicating RNA.

[0007] In some embodiments, the virus is selected from the group consisting of influenza virus, rabies virus, respiratory syncytial virus (RSV), and coronavirus.

[0008] In some embodiments, the virus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0009] In some embodiments, the virus is SARS-CoV-2.

[0010] In some embodiments, the first viral variant or the second viral variant is selected from the group consisting of Wuhan-Hu-1, alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, and omicron strains.

[0011] In some embodiments, the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu-1 and Delta strains.

[0012] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a viral envelope protein, a viral spike protein, a viral membrane protein, or a viral capsid protein. The first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a viral spike protein.

[0013] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a receptor binding domain (RBD).

[0014] In some embodiments, the RBD domain is an RBD domain from a Wuhan-Hu-1 mutant or a delta mutant.

[0015] In some embodiments, the first antigen or antigenic fragment thereof comprises an RBD domain from a delta mutant and the second antigen or antigenic fragment thereof comprises an RBD domain of a Wuhan-Hu-1 mutant.

[0016] In some embodiments, the virus is an influenza A virus.

[0017] In some embodiments, the influenza A virus is selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3.

[0018] In some embodiments, the first viral variant or the second viral variant is selected from the group consisting of H1N1(PR8) and H3N2(Udorn).

[0019] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises an RNA polymerase subunit, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein NS1, or nonstructural protein NEP.

[0020] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof comprises a hemagglutinin (HA) protein.

[0021] In some embodiments, the HA protein is derived from H1N1(PR8) or H3N2(Udorn).

[0022] In some embodiments, the first antigen or antigenic fragment thereof comprises an HA protein from H1N1(PR8) and the second antigen or antigenic fragment thereof comprises an HA protein from H3N2(Udorn).

[0023] In some embodiments, the multimerization domain is selected from the group consisting of a dimerization domain, a trimerization domain, and a tetramerization domain.

[0024] In some embodiments, the multimerization domain is selected from the group consisting of enterobacteriaceae phage T4, GCN4pII, GCN4-pLI, and p53.

[0025] In some embodiments, the multimerization domain comprises a leucine zipper or a fibritin foldon domain.

[0026] In some embodiments, the multimerization domain comprises a trimerization domain.

[0027] In some embodiments, the fibritin foldon domain is a trimerization domain derived from enterobacteriaceae phage T4.

[0028] In some embodiments, the trimerization domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:1.

[0029] In some embodiments, the RNA molecules described herein further comprise a sequence encoding a first linker that connects the first antigen or antigenic fragment thereof to the multimerization domain.

[0030] In some embodiments, the RNA molecules described herein further comprise a sequence encoding a second linker that links a second antigen or antigenic fragment thereof to the multimerization domain.

[0031] In some embodiments, the first linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids.

[0032] In some embodiments, the second linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids.

[0033] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO:9), (G2S)n (SEQ ID NO:10), (G3S)n (SEQ ID NO:11), (G4S)n (SEQ ID NO:32), and (G)n (SEQ ID NO:33), where n is an integer from 2 to 20. In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO:9), (G2S)n (SEQ ID NO:10), (G3S)n (SEQ ID NO:11), (G4S)n (SEQ ID NO:32), and (G)n (SEQ ID NO:33), where n is an integer from 2 to 20.

[0034] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), where n is an integer from 2 to 6.

[0035] In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), where n is an integer from 2 to 6.

[0036] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO:36) and (GGGGGPGGGGP)n (SEQ ID NO:37), where n is an integer between 1 and 3. In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO:36) and (GGGGGPGGGGP)n (SEQ ID NO:37), where n is an integer between 1 and 3.

[0037] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8.

[0038] In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8.

[0039] In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0040] In some embodiments, the first linker encodes GGSG (SEQ ID NO: 38). In some embodiments, the second linker encodes GGSG (SEQ ID NO: 38).

[0041] In some embodiments, the first linker encodes GGSLGGGGGSGS (SEQ ID NO: 39). In some embodiments, the second linker encodes GGSLGGGGGSGS (SEQ ID NO: 39).

[0042] In some embodiments, the first linker is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0043] In some embodiments, the second linker is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0044] In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-4, 7, and 8.

[0045] In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-4, 7, and 8.

[0046] In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19, 20, 22, and 23.

[0047] In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19, 20, 22, and 23.

[0048] In some embodiments, the purified RNA further comprises m7GpppNm- (where Nm represents any nucleotide having a 2'O methylation (Cap1) at the 5' end of the first nucleotide sequence).

[0049] In some embodiments, the purified RNA further comprises a 5' UTR at the 3' end of Cap1 and the 5' end of the first nucleotide sequence.

[0050] In some embodiments, the purified RNA further comprises a sequence encoding a signal peptide at the 3' end of the 5' UTR and the 5' end of the first nucleotide sequence.

[0051] In some embodiments, the purified RNA further comprises a sequence encoding a 3' UTR at the 3' end of the second nucleotide sequence.

[0052] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 26-31.

[0053] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:26.

[0054] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:27.

[0055] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:28.

[0056] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:29.

[0057] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:30.

[0058] In one aspect, provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:31.

[0059] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' to the 3' end: a) m7GpppNm-, where Nm represents any nucleotide with 2'O-methylation (Cap1); b) a 5'UTR; c) a first nucleotide sequence encoding a signal peptide; and d) a second nucleotide sequence encoding a first antigen or antigenic fragment thereof from a wild-type SARS-CoV-2 virus or a first virus variant selected from the group consisting of a Wuhan-Hu-1 variant, an Omicron variant, and a Delta variant. e) a third nucleotide sequence encoding a first linker; f) a fourth nucleotide sequence encoding a T4-foldon domain; g) a fifth nucleotide sequence encoding a second linker; h) a sixth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from a wild-type SARS-CoV-2 virus or a second virus variant selected from the group consisting of the Wuhan-Hu-1 variant, the Omicron variant, and the Delta variant; i) a 3'UTR; and j) a polyA tail.

[0060] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding a first antigen or an antigenic fragment thereof from a wild-type SARS-CoV-2 virus or a first virus variant selected from an omicron mutant; e) a third nucleotide sequence encoding a first linker; f) a fourth nucleotide sequence encoding a T4-foldon domain; g) a fifth nucleotide sequence encoding a second linker; h) a sixth nucleotide sequence encoding a second antigen or an antigenic fragment thereof from a wild-type SARS-CoV-2 virus; i) a 3'UTR; and j) a poly-A tail.

[0061] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from a wild-type SARS-CoV-2 virus; e) a third nucleotide sequence encoding a first linker; f) a fourth nucleotide sequence encoding a T4-foldon domain; g) a fifth nucleotide sequence encoding a second linker; h) a sixth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from an omicron mutant; i) a 3'UTR; and j) a poly-A tail.

[0062] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' to the 3' end, a) m7GpppNm- (where Nm represents any nucleotide with 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from an influenza virus selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; and d) optionally, a second nucleotide sequence encoding a first linker. a nucleotide sequence encoding a T4-foldon domain; e) optionally a third nucleotide sequence encoding a T4-foldon domain; f) optionally a fourth nucleotide sequence encoding a second linker; g) optionally a fifth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from an influenza virus selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; h) a 3'UTR; and i) a poly-A tail.

[0063] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an H1N1 (PR8) influenza virus; d) a second nucleotide sequence encoding a first linker; e) a third nucleotide sequence encoding a T4-foldon domain; f) a fourth nucleotide sequence encoding a second linker; g) a fifth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from an influenza virus selected from an H3N2 (Udorn) influenza virus; h) a 3'UTR; and i) a poly-A tail.

[0064] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an H1N1(PR8) influenza virus; d) a 3'UTR; and e) a poly-A tail.

[0065] In one aspect, provided herein is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a nucleotide sequence encoding a first antigen or an antigenic fragment thereof from an H3N2 (Udorn) influenza virus; d) a 3'UTR; and e) a poly-A tail.

[0066] In one aspect, there is provided herein an isolated polypeptide encoded by a purified RNA molecule according to any of the preceding claims.

[0067] In one aspect, provided herein is a composition comprising any of the purified RNA molecules described herein and a delivery vehicle.

[0068] In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0069] In some embodiments, the LNP comprises an ionizable lipid.

[0070] In some embodiments, the LNPs comprise an ionizable lipid:cholesterol:DSPC:DMG-PEG2000 ratio of about 48:40:10:2, and an LNP:RNA (N:P) ratio of about 8:1.

[0071] In some embodiments, the composition has a particle size of about 300 nanometers (nm) or less.

[0072] In some embodiments, the composition comprises a particle size of about 50 to about 300 nm. In some embodiments, the composition comprises a particle size of about 150 nm or less. In some embodiments, the composition comprises a particle size of about 140 nm or less. In some embodiments, the composition comprises a particle size of about 50 nm to about 140 nm. In some embodiments, the composition comprises a particle size of about 125 nm.

[0073] In some embodiments, the RNA is lyophilized.

[0074] In some embodiments, the RNA is adsorbed to the surface of the LNP.

[0075] In some embodiments, the RNA is encapsulated by LNPs.

[0076] In some embodiments, the RNA encapsulated by the LNPs is lyophilized.

[0077] In one aspect, provided herein is a kit comprising any of the purified RNA molecules described herein, a delivery vehicle, and instructions.

[0078] In one aspect, provided herein is a method of treating or preventing a disease in a subject, comprising administering to the subject an effective amount of any of the compositions described herein.

[0079] In some embodiments, the disease is caused by a virus selected from the group consisting of influenza virus, rabies virus, respiratory syncytial virus (RSV), and coronavirus.

[0080] In some embodiments, the disease is caused by a coronavirus. In some embodiments, the coronavirus is selected from the group consisting of 229E (an alpha coronavirus), NL63 (an alpha coronavirus), OC43 (a beta coronavirus), HKU1 (a beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the coronavirus is SARS-CoV-2.

[0081] In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state is at a temperature of 2-8°C.

[0082] In some embodiments, the RNA and LNPs are injected intramuscularly into the subject.

[0083] In some embodiments, lyophilized RNA encapsulated in LNPs is mixed with sterile water.

[0084] In some embodiments, lyophilized RNA encapsulated in LNPs and sterile water are injected intramuscularly into a subject.

[0085] In some embodiments, RNA adsorbed to the surface of LNPs in liquid form or lyophilized RNA encapsulated in LNPs is administered to a subject at least twice.

[0086] In some embodiments, the liquid LNPs or lyophilized RNA encapsulated in LNPs are administered once every one, two, three, or four weeks.

[0087] In one aspect, provided herein is a method of stimulating an immune response in a subject, comprising administering to the subject an effective amount of any of the compositions described herein.

[0088] In one aspect, provided herein is a method for preparing an RNA-LNP composition, the method comprising mixing an ethanol phase comprising one or more lipids with an aqueous phase comprising any of the purified RNA molecules described herein, and purifying the RNA-LNPs produced from step a).

[0089] In some embodiments, one or more lipids comprise an ionizable lipid. In some embodiments, one or more lipids comprise cholesterol. In some embodiments, one or more lipids comprise a phospholipid. In some embodiments, one or more lipids are PEGylated.

[0090] In some embodiments, the N:P ratio is from about 6.5 to about 9.

[0091] In some embodiments, the aqueous phase consists of Tris, sodium chloride, and sucrose.

[0092] In one aspect, provided herein is a kit comprising any of the compositions described herein, a delivery vehicle, and instructions. [Brief explanation of the drawings]

[0093] [Figure 1A] Figure 1A shows an exemplary schema of proteins encoded by the RNA constructs described herein, which are designed to simultaneously express different receptor binding domain (RBD) proteins from various coronavirus strains via a linker and T4-foldon oligomerization domain. Figure 1A shows SEQ ID NOS: 38-39, in order of appearance, respectively. [Figure 1B] FIG. 1 shows a schematic diagram of an RNA structure with two antigen sequences with an oligomerization domain between them. [Figure 2] 1 shows a Western blot probed for the RBD protein of SARS-CoV-2 from a sample of BHK-21 cells transfected with 2 micrograms (μg) of a lipid nanoparticle (LNP)-encapsulated RNA vaccine described herein. [Figure 3] 1 shows the treatment protocol for BALB / c mice vaccinated with the RNA-LNP vaccine described herein, encoding the RBD protein of SARS-CoV-2. [Figure 4] A bar graph of geometric mean antibody titers (GMTs) is shown for BALB / c mice vaccinated with the RNA-LNP vaccine described herein encoding the RBD protein of SARS-CoV-2 on the indicated days. [Figure 5] A bar graph of SARS-CoV-2 neutralizing antibodies showing cross-protection against different SARS-CoV-2 strains is shown. [Figure 6] 1 shows an exemplary schematic diagram of an mRNA described herein. [Figure 7] Schematic diagram of mouse immunization. [Figure 8] Figure 8A shows a bar graph of antigen-specific binding antibodies against the SARS-CoV-2 RBD polypeptide of the SARS-CoV-2 original strain, measured by ELISA, at different time points. Figure 8B shows a bar graph of antigen-specific binding antibodies against the SARS-CoV-2 RBD polypeptide of the Omicron BA.1 strain, measured by ELISA, at different time points. * indicates below the lowest starting dilution. ** indicates above the highest dilution. [Figure 9] Graph showing mouse serum neutralizing antibody titers against SARS-CoV-2 pseudotyped viruses (original strain, beta strain, delta strain, Omicron BA.1 strain, BA.2.12.1 strain, and BA.4 / .5 strain). [Figure 10] Graph showing the percentage of IFN-γ-secreting splenocytes isolated from mice immunized with mRNA-LNP vaccines by ICS upon stimulation with antigen peptide pools. *** indicates p<0.001 compared to the control group. [Figure 11] FIG. 1 shows a group of schematic diagrams of an RNA structure with two antigen sequences with an oligomerization domain between them. [Figure 12]A bar graph shows the geometric mean antibody titers (GMT) of BALB / c mice vaccinated with RNA-LNP vaccines (having Structure 1, Structure 2, or Structure 3) encoding the SARS-CoV-2 RBD-WT and RBD-BA.1 antigen proteins. Serum samples were collected on day 28. Each bar shown is the geometric mean IgG antibody titer of individual mouse sera (N=4) in each group. [Figure 13] A bar graph of hemagglutination inhibition (HAI) titers in BALB / c mice vaccinated with RNA-LNP vaccines (having Structure 4, Structure 5, or Structure 6) encoding the indicated influenza antigens is shown. Serum samples were collected on day 28. Each bar shown is the geometric mean (N=5) of HAI antibody titers for individual mouse sera in each group. DETAILED DESCRIPTION OF THE INVENTION

[0094] Conventional vaccines usually use a single antigen sequence to provide immunity against a specific virus. Viral mutations may occur, and these mutations may render the immunity provided by conventional vaccines meaningless. Therefore, it is advantageous to create a vaccine that can provide immunity against multiple virus variants. The RNA molecules described herein contain multiple antigen sequences, thereby providing immunity against multiple virus variants.

[0095] Nucleic acids of the present disclosure Provided herein in some embodiments is a purified ribonucleic acid (RNA) molecule comprising: a) a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from a first viral variant; b) a second nucleotide sequence encoding a multimerization domain; and c) a third nucleotide sequence encoding a second antigen or antigenic fragment thereof from a second viral variant, wherein the first viral variant and the second viral variant are different, and the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5' to 3' direction.

[0096] In some embodiments, the RNA molecules described herein are messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to a type of RNA molecule that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. Those skilled in the art will understand that, unless otherwise specified, the RNA molecule sequence will be written with the "T" of a typical DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" is replaced with a "U."

[0097] Thus, any RNA molecule encoded by a DNA identified by a particular sequence identification number can also include the corresponding RNA (e.g., mRNA or other coding RNA) sequence encoded by that DNA, in which each "T" in the DNA sequence is replaced with a "U."

[0098] It should be understood that the RNA molecules provided herein are synthetic molecules, i.e., not naturally occurring molecules. In other words, the RNA molecules of the present disclosure are isolated (i.e., purified) RNA molecules. As known in the art, an "isolated RNA molecule or polynucleotide" refers to a polynucleotide that is substantially physically separated from other cellular material (e.g., from the cell and / or system that produces the polynucleotide) or from other materials that would interfere with its use in the vaccines and therapeutics of the present disclosure. Isolated RNA molecules are substantially pure because they have been substantially separated from materials with which they may be associated in a living organism or viral system. Thus, the RNA molecule is not associated with a living organism or viral system, such as a cell or virus. The RNA molecule does not include viral components (e.g., viral capsids, viral enzymes, or other viral proteins necessary for viral-based replication), and the RNA molecule is not packaged, enclosed, bound, or otherwise associated within a virus or viral particle.

[0099] Any sequence can be codon-optimized. Methods of codon optimization are well known in the art. In some embodiments, codon optimization is used to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase RNA stability or reduce secondary structure, minimize tandem repeat codons or base runs that may impair gene constructs or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein, add, remove, or shuffle protein domains, insert or delete restriction sites, modify ribosome binding and RNA degradation sites, adjust translation rates to allow various domains of a protein to fold properly, or reduce or remove problematic secondary structure within a polynucleotide. In some embodiments, an optimization algorithm is used to optimize open reading frame (ORF) sequences. In some embodiments, a sequence encoding an antigen, e.g., the spike protein of SARS-CoV-2, is codon-optimized.

[0100] In some embodiments, the RNA comprises at least one RNA molecule encoding at least one antigenic polypeptide having at least one of the following modifications: at least one 5'-end cap; and formulation with lipid nanoparticles. 5'-capping of polynucleotides can be completed simultaneously with the in vitro transcription reaction using the following chemical RNA cap analogs: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, and m7G(5')ppp(5')G, according to the manufacturer's protocol, to generate a 5'-guanosine cap structure. 5'-capping of modified RNAs can also be completed post-transcriptionally using vaccinia virus capping enzyme to generate the "Cap0" structure: m7G(5')ppp(5')G. The Cap1 structure can be generated using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. The Cap2 structure can be generated from the Cap1 structure, followed by 2'-O-methylation of the third nucleotide from the 5' end using 2'-O-methyltransferase. The Cap3 structure can be generated from Cap2, followed by 2'-O-methylation of the fourth nucleotide from the 5' end using 2'-O-methyltransferase. The enzymes can be derived from recombinant sources.

[0101] In some embodiments, the RNA molecules described herein comprise a cap (e.g., m7G(Cap0), m7GpppNm- (where Nm represents any nucleotide with a 2'0-methylation (Cap1)), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or an anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide with a 2'0-methylation)). In some embodiments, the RNA molecules described herein comprise a m7G(Cap0) cap. In some embodiments, the RNA molecules described herein comprise a m7GpppNm- (where Nm represents any nucleotide with a 2'0-methylation (Cap1)) cap. In some embodiments, the RNA molecules described herein comprise a N6,2'-O-dimethyladenosine (m6AM) cap. In some embodiments, the RNA molecules described herein comprise an m7G(5')ppp(5')G (mCAP) cap. In some embodiments, the RNA molecules described herein comprise an anti-reverse cap analog (ARCA).

[0102] In some embodiments, the RNA molecules described herein comprise a 5' UTR. In some embodiments, the 5' UTR is 5' of a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from a first viral variant. In some embodiments, the RNA molecules described herein comprise a 3' UTR. In some embodiments, the 3' UTR is 3' of a second nucleotide sequence encoding a second antigen or antigenic fragment thereof from a second viral variant.

[0103] In some embodiments, the 5' UTR comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 80% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 85% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 90% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 95% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 96% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 97% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 98% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having at least about 99% identity to SEQ ID NO:24. In some embodiments, the 5' UTR comprises a sequence having 100% identity to SEQ ID NO:24.

[0104] In some embodiments, the 5' UTR is encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 80% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 85% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 90% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 95% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 96% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 97% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 98% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having at least about 99% identity to SEQ ID NO: 14. In some embodiments, the 5' UTR is encoded by a sequence having 100% identity to SEQ ID NO: 14.

[0105] In some embodiments, the 3' UTR comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 120% sequence identity to SEQ ID NO:25. In some embodiments, the 3' UTR comprises a sequence having at least about 80% identity to SEQ ID NO:25. In some embodiments, the 3' UTR comprises a sequence having at least about 85% identity to SEQ ID NO:25. In some embodiments, the 3' UTR comprises a sequence having at least about 90% identity to SEQ ID NO:25. In some embodiments, the 3' UTR comprises a sequence having at least about 95% identity to SEQ ID NO:25. In some embodiments, the 3' UTR comprises a sequence having at least about 96% identity to SEQ ID NO: 25. In some embodiments, the 3' UTR comprises a sequence having at least about 97% identity to SEQ ID NO: 25. In some embodiments, the 3' UTR comprises a sequence having at least about 98% identity to SEQ ID NO: 25. In some embodiments, the 3' UTR comprises a sequence having at least about 99% identity to SEQ ID NO: 25. In some embodiments, the 3' UTR comprises a sequence having 100% identity to SEQ ID NO: 25.

[0106] In some embodiments, the 3' UTR is encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 120% sequence identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 80% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 85% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 90% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 95% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 96% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 97% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 98% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having at least about 99% identity to SEQ ID NO: 15. In some embodiments, the 3' UTR is encoded by a sequence having 100% identity to SEQ ID NO: 15.

[0107] In some embodiments, the RNA molecules described herein comprise a nucleotide sequence encoding a signal peptide. In some embodiments, the signal peptide is encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 80% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 85% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 90% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 95% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 96% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 97% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 98% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having at least about 99% identity to SEQ ID NO:2. In some embodiments, the signal peptide is encoded by a sequence having 100% identity to SEQ ID NO:2.

[0108] In some embodiments, the RNA molecules described herein comprise a polyA tail (ie, SEQ ID NO: 16).

[0109] Provided herein, in certain embodiments, is a purified RNA molecule comprising, from the 5' end to the 3' end, a) m7GpppNm- (where Nm represents any nucleotide with 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a signal peptide; and d) a second nucleotide sequence encoding a first antigen or antigenic fragment thereof from a wild-type SARS-CoV-2 virus or a first virus variant selected from the group consisting of a Wuhan-Hu-1 variant, an Omicron variant, and a Delta variant. a sequence of nucleotides encoding a first linker; e) a third nucleotide sequence encoding a first linker; f) a fourth nucleotide sequence encoding a T4-foldon domain; g) a fifth nucleotide sequence encoding a second linker; h) a sixth nucleotide sequence encoding a second antigen or antigenic fragment thereof from a wild-type SARS-CoV-2 virus or a second virus variant selected from the group consisting of the Wuhan-Hu-1 mutant, the Omicron mutant, and the Delta mutant; i) a 3'UTR; and j) a polyA tail.

[0110] Provided herein in certain embodiments is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an omicron mutant; e) a third nucleotide sequence encoding a first linker; f) a fourth nucleotide sequence encoding a T4-foldon domain; g) a fifth nucleotide sequence encoding a second linker; h) a sixth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from a wild-type SARS-CoV-2 virus; i) a 3'UTR; and j) a polyA tail.

[0111] Provided herein in certain embodiments is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from a wild-type SARS-CoV-2 virus; e) a third nucleotide sequence encoding a first linker; f) a fourth nucleotide sequence encoding a T4-foldon domain; g) a fifth nucleotide sequence encoding a second linker; h) a sixth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from an omicron mutant; i) a 3'UTR; and j) a poly-A tail.

[0112] Provided herein in certain embodiments is a purified RNA molecule comprising, from the 5' end to the 3' end, a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an influenza virus selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; and d) optionally, a second nucleotide sequence encoding a first linker. e) optionally a third nucleotide sequence encoding a T4-foldon domain; f) optionally a fourth nucleotide sequence encoding a second linker; g) optionally a fifth nucleotide sequence encoding a second antigen or an antigenic fragment thereof derived from an influenza virus selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; h) a 3'UTR; and i) a poly-A tail.

[0113] Provided herein in certain embodiments is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having 2'O-methylation (Cap1)); b) a 5'UTR; c) a first nucleotide sequence encoding a first antigen derived from an H1N1 (PR8) influenza virus or an antigenic fragment thereof; d) a second nucleotide sequence encoding a first linker; e) a third nucleotide sequence encoding a T4-foldon domain; f) a fourth nucleotide sequence encoding a second linker; g) a fifth nucleotide sequence encoding a second antigen derived from an H3N2 (Udorn) influenza virus or an antigenic fragment thereof; h) a 3'UTR; and i) a poly-A tail.

[0114] Provided herein in certain embodiments is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1)); b) a 5'UTR; c) a nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an H1N1(PR8) influenza virus; d) a 3'UTR; and e) a poly-A tail.

[0115] Provided herein in certain embodiments is a purified RNA molecule comprising, from the 5' end to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide having 2'O-methylation (Cap1)); b) a 5'UTR; c) a nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an H3N2 (Udorn) influenza virus; d) a 3'UTR; and e) a poly-A tail.

[0116] In some embodiments, the purified RNA molecule comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to SEQ ID NO:26. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to SEQ ID NO:26.

[0117] In some embodiments, the purified RNA molecule comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to SEQ ID NO:27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to SEQ ID NO:27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to SEQ ID NO:27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to SEQ ID NO:27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to SEQ ID NO:27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to SEQ ID NO: 27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to SEQ ID NO: 27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to SEQ ID NO: 27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to SEQ ID NO: 27. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to SEQ ID NO: 27. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to SEQ ID NO: 27.

[0118] In some embodiments, the purified RNA molecule comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to SEQ ID NO:28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to SEQ ID NO:28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to SEQ ID NO:28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to SEQ ID NO:28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to SEQ ID NO:28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to SEQ ID NO: 28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to SEQ ID NO: 28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to SEQ ID NO: 28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to SEQ ID NO: 28. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to SEQ ID NO: 28. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to SEQ ID NO: 28.

[0119] In some embodiments, the purified RNA molecule comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to SEQ ID NO:29. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to SEQ ID NO:29.

[0120] In some embodiments, the purified RNA molecule comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to SEQ ID NO: 30. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to SEQ ID NO: 30.

[0121] In some embodiments, the purified RNA molecule comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to SEQ ID NO:31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to SEQ ID NO:31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to SEQ ID NO:31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to SEQ ID NO:31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to SEQ ID NO:31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to SEQ ID NO: 31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to SEQ ID NO: 31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to SEQ ID NO: 31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to SEQ ID NO: 31. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to SEQ ID NO: 31. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to SEQ ID NO: 31.

[0122] In some embodiments, the purified RNA molecule is encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 70% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 75% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 80% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 85% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 90% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 95% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 96% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 97% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 98% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 99% identity to SEQ ID NO: 12. In some embodiments, the purified RNA molecule is encoded by a sequence having 100% identity to SEQ ID NO: 12.

[0123] In some embodiments, the purified RNA molecule is encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 70% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 75% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 80% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 85% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 90% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 95% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 96% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 97% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 98% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having at least about 99% identity to SEQ ID NO: 13. In some embodiments, the purified RNA molecule is encoded by a sequence having 100% identity to SEQ ID NO: 13.

[0124] When transfected into mammalian cells, the modified RNA typically has a stability of 12 to 18 hours or more than 18 hours, for example, 24, 36, 48, 60, 72 hours, or more than 72 hours.

[0125] In addition to mRNA, the RNA molecules described herein can be one of several non-coding RNAs, such as ribosomal RNA (rRNA) or transfer RNA (tRNA). The term "RNA" or "RNA molecule" also includes other coding RNA molecules, such as viral RNA, retroviral RNA, self-replicating RNA (replicon RNA), small interfering RNA (siRNA), microRNA, small nuclear RNA (snRNA), small hairpin (sh)RNA, riboswitches, ribozymes, or aptamers.

[0126] In certain embodiments, the RNA molecule is a long RNA or a long RNA molecule. As used herein, the term "long RNA" typically refers to an RNA molecule, preferably as described herein, that contains at least 30 nucleotides. Alternatively, a long RNA may contain at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or at least 500 nucleotides. A long RNA may contain at least 1000 nucleotides or at least 2000 nucleotides. In the context of the present disclosure, a long RNA may comprise about 30 to about 50,000 nucleotides, about 30 to about 20,000 nucleotides, about 100 to about 20,000 nucleotides, about 200 to about 20,000 nucleotides, about 200 to about 15,000 nucleotides, about 500 to about 20,000 nucleotides, about 1,000 to about 15,000 nucleotides, about 1,500 to about 10,000 nucleotides, or about 2,000 to about 5,000 nucleotides. As used herein, the term "long RNA" is not limited to a particular type of RNA, but simply refers to the number of nucleotides contained in the RNA. In certain embodiments, the RNA used herein is a long RNA.

[0127] In the present disclosure, the RNA molecule may be a coding RNA molecule, encoding one or more proteins or peptides, for example but not limited to, from a therapeutically active protein or peptide selected from adjuvant proteins, from an antigen, for example a pathogenic antigen (e.g. an animal antigen, a viral antigen, a protozoan antigen, a bacterial antigen), from an allergen antigen, an autoimmune antigen or preferably a further antigen as defined herein, from an allergen, from an antibody, from an immunostimulatory protein or peptide, from an antigen-specific T cell receptor, or from any other protein or peptide suitable for a particular (therapeutic) application, wherein the coding RNA molecule can be delivered to a cell, tissue or organism and the protein can then be expressed in said cell, tissue or organism.

[0128] In certain embodiments, the RNA molecule of the present disclosure is an immunostimulatory RNA molecule capable of inducing an immune response, preferably an innate immune response. Such immunostimulatory RNA can be any RNA (double-stranded or single-stranded) as defined herein, e.g., coding RNA. In certain embodiments, the immunostimulatory RNA can be non-coding RNA. The immunostimulatory RNA can be single-stranded, double-stranded, or partially double-stranded RNA, optionally single-stranded RNA, or circular or linear RNA, preferably linear RNA. In various embodiments, the immunostimulatory RNA can be linear single-stranded RNA. More preferably, the immunostimulatory RNA is a long linear single-stranded RNA.

[0129] Immunostimulatory RNAs may also exist as short RNA oligonucleotides. As used herein, immunostimulatory RNAs may be selected from any class of RNA molecules found in nature or prepared synthetically and capable of inducing an innate immune response and supporting an antigen-induced adaptive immune response.

[0130] In some embodiments, the RNA molecule is self-replicating RNA.

[0131] When delivered to a vertebrate cell, a self-replicating RNA molecule (replicon) can direct the production of multiple daughter RNAs by transcription from itself (via antisense copies generated from itself) without any proteins. Thus, a self-replicating RNA molecule can generally be a positive-strand molecule that can be directly translated after delivery to a cell; this translation provides an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. In this way, the delivered RNA can direct the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can themselves be translated, resulting in the in situ expression of the encoded immunogen, or they can be transcribed to provide additional transcripts of the same sense as the delivered RNA, which can be translated, resulting in the in situ expression of the immunogen. The overall result of this transcription sequence is a significant amplification of the number of introduced replicon RNAs, thereby making the encoded immunogen the major polypeptide product of the cell.

[0132] One suitable system for achieving such self-replication is the use of alphavirus replicons. These replicons can be positive-strand RNA that, after delivery to a cell, triggers the translation of a replicase (or replicase-transcriptase). The replicase can be translated into a polyprotein that self-cleaves to provide a replication complex that generates genomic negative-strand copies of the positive-stranded RNA. These negative-stranded transcripts can themselves be transcribed to produce additional copies of the positive-stranded parent RNA and also to produce subgenomic transcripts encoding immunogens. Translation of the subgenomic transcripts can thus result in in situ expression of the immunogen by infected cells. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and the like. Mutant or wild-type virus sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used as a replicon.

[0133] The self-replicating RNA molecule can encode (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an immunogen. The polymerase can be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsp1, nsp2, nsp3, and nsp4.

[0134] While naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, the self-replicating RNA molecules described herein may lack one, more, or all of the alphavirus structural proteins. Thus, while a self-replicating RNA can direct the production of its own genomic RNA copies within a cell, it cannot direct the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecules do not perpetuate themselves in an infectious form. Alphavirus structural proteins used for persistence in wild-type viruses are typically absent from the self-replicating RNA described herein; their place is occupied by a gene encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.

[0135] Thus, a self-replicating RNA molecule can have two open reading frames: a first (5') open reading frame that encodes a replicase and a second (3') open reading frame that encodes an immunogen. In some embodiments, the RNA has an additional (e.g., downstream) open reading frame, e.g., to encode a further immunogen (see below) or to encode an accessory polypeptide.

[0136] The self-replicating RNA molecule can have a variety of lengths, but is typically 5,000 to 25,000 nucleotides in length, for example, 8,000 to 15,000 nucleotides, or 9,000 to 12,000 nucleotides.

[0137] The self-replicating RNA molecules described herein can have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of RNA molecules useful in the present disclosure can have a 5' triphosphate group. In capped RNA, this can be linked to the 7-methylguanosine via a 5'-5' bridge. In some embodiments, the RNA cap includes m7G (Cap0), m7GpppNm- (where Nm represents any nucleotide with 2'O-methylation (Cap1)), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or an anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide with 2'O-methylation).

[0138] The self-replicating RNA molecule may have a 3' poly-A tail. It may also contain a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.

[0139] Self-replicating RNA molecules are typically single-stranded. Single-stranded RNA can generally induce adjuvant effects by binding to TLR7, TLR8, RNA helicase, and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA formed during replication of single-stranded RNA or within the secondary structure of single-stranded RNA.

[0140] The self-replicating RNA molecules described herein can be prepared by in vitro transcription (IVT). IVT can use cDNA templates that are generated and propagated in bacteria in the form of plasmids or that are synthetically generated (e.g., by gene synthesis and / or polymerase chain reaction (PCR) manipulation). For example, a DNA-dependent RNA polymerase (e.g., bacteriophage T7, T3, or SP6 RNA polymerase) can be used to transcribe the RNA from the DNA template. Appropriate capping and poly(A) addition can be used as needed (although the replicon's poly(A) is typically encoded within the DNA template). These RNA polymerases can have stringent requirements for the 5' nucleotide to be transcribed, and in some embodiments, these requirements must match those of the encoded replicase to ensure that the IVT-transcribed RNA can efficiently function as a substrate for the self-encoded replicase.

[0141] In some embodiments, the RNA molecule is not self-replicating RNA.

[0142] In some embodiments, RNA of the present disclosure includes purified RNA molecules, e.g., messenger RNA (mRNA). RNA is transcribed in vitro, e.g., from a DNA template, referred to as an "in vitro transcription template."

[0143] In vitro transcription of RNA is known in the art and is described, for example, in International Publication WO 2014 / 152027, the entirety of which is incorporated herein by reference. For example, in some embodiments, RNA transcripts are generated using a non-amplified linearized DNA template in an in vitro transcription reaction to produce the RNA transcript. In some embodiments, the RNA transcript is capped by enzymatic capping. In some embodiments, the RNA transcript is purified by chromatographic methods, for example, using an oligo-dT substrate. In some embodiments, the use of DNase is omitted. In some embodiments, the RNA transcript is synthesized from a non-amplified linear DNA template encoding a gene of interest by an enzymatic in vitro transcription reaction utilizing T7 phage RNA polymerase and nucleotide triphosphates of desired chemistry. Any number of RNA polymerases or variants may be used in the methods of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, such as, but not limited to, a mutant polymerase, such as, but not limited to, a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase, and / or a polymerase capable of incorporating modified nucleic acids and / or nucleotides, including chemically modified nucleic acids and / or nucleotides.

[0144] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a poly-A tail. The composition and length of the particular nucleic acid sequence of the in vitro transcription template depends on the RNA encoded by the template.

[0145] antigen The RNA molecules described herein can be designed to contain or encode a substance that elicits an immune response in a subject. After administration of the RNA molecule, the RNA is translated in vivo. The RNA can induce an immune response against an antigen, a virus, and / or a viral antigen. The immune response can include an antibody response. The RNA typically induces an immune response that recognizes the corresponding antigen, such as a viral polypeptide. The RNA typically includes one or more sequences of a surface polypeptide, such as an adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, or receptor binding domain (RBD). In some embodiments, the RNA induces an immune response against the receptor binding domain (RBD) of SARS-CoV-2.

[0146] In some embodiments, the RNA molecules provided herein comprise a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from a first viral variant, a second nucleotide sequence encoding a multimerization domain, and a third nucleotide sequence encoding a second antigen or antigenic fragment thereof from a second viral variant, wherein the first viral variant and the second viral variant are different, and the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5' to 3' direction.

[0147] In some embodiments, the virus is selected from an orthomyxovirus, such as influenza virus, a rhabdovirus, such as rabies virus, a picornavirus, such as poliovirus, a poxvirus, such as vaccinia virus, a rotavirus, a respiratory syncytial virus (RSV), and a coronavirus, such as COVID-19.

[0148] In some embodiments, the virus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2.

[0149] In some embodiments, the first viral variant or the second viral variant is selected from the group consisting of Wuhan-Hu-1, alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, and omicron. In some embodiments, the first viral variant or the second viral variant is selected from the group consisting of Wuhan-Hu-1 and delta.

[0150] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a viral envelope protein, a viral spike protein, a viral membrane protein, or a viral capsid protein. In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a viral spike protein.

[0151] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a receptor binding domain (RBD). In some embodiments, the RBD domain is an RBD domain from a Wuhan-Hu-1 mutant or a delta mutant. In some embodiments, the first antigen or antigenic fragment thereof comprises an RBD domain from a delta mutant, and the second antigen or antigenic fragment thereof comprises an RBD domain from a Wuhan-Hu-1 mutant.

[0152] In some embodiments, the virus is an influenza A virus. In some embodiments, the influenza A virus is selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3.

[0153] In some embodiments, the first viral variant or the second viral variant is selected from the group consisting of H1N1(PR8) and H3N2(Udorn).

[0154] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises an RNA polymerase subunit, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein NS1, or nonstructural protein NEP.

[0155] In some embodiments, the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof comprises a hemagglutinin (HA) protein.

[0156] In some embodiments, the HA protein is derived from H1N1(PR8) or H3N2(Udorn).

[0157] In some embodiments, the first antigen or antigenic fragment thereof comprises an HA protein from H1N1(PR8) and the second antigen or antigenic fragment thereof comprises an HA protein from H3N2(Udorn).

[0158] In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen or antigenic fragment thereof is encoded by a nucleotide sequence having the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8.

[0159] In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen or antigenic fragment thereof comprises a sequence having the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23.

[0160] In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen or antigenic fragment thereof is encoded by a nucleotide sequence having the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8.

[0161] In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen or antigenic fragment thereof comprises a sequence having the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23.

[0162] In some embodiments, the first antigen sequence is encoded by a portion of the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen sequence is encoded by at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen sequence is encoded by at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the first antigen sequence comprises from about 50 nucleotides to about 72 nucleotides. In some embodiments, the first antigen sequence is encoded by at least about 50 nucleotides. In some embodiments, the first antigen sequence comprises up to about 72 nucleotides. In some embodiments, the first antigen sequence comprises about 50 to about 70 nucleotides, about 50 to about 67 nucleotides, about 50 to about 65 nucleotides, about 50 to about 62 nucleotides, about 50 to about 60 nucleotides, about 50 to about 57 nucleotides, about 50 to about 55 nucleotides, or about 50 to about 52 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 3 to 4, 7, and 8.

[0163] In some embodiments, the first antigen sequence comprises a portion of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen sequence comprises at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen sequence comprises at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the first antigen sequence comprises from about 50 nucleotides to about 72 nucleotides. In some embodiments, the first antigen sequence comprises at least about 50 nucleotides. In some embodiments, the first antigen sequence comprises up to about 72 nucleotides. In some embodiments, the first antigen sequence comprises about 50 to about 70 nucleotides, about 50 to about 67 nucleotides, about 50 to about 65 nucleotides, about 50 to about 62 nucleotides, about 50 to about 60 nucleotides, about 50 to about 57 nucleotides, about 50 to about 55 nucleotides, or about 50 to about 52 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23.

[0164] In some embodiments, the second antigen sequence is encoded by a portion of the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen sequence is encoded by at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen sequence is encoded by at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 3-4, 7, and 8. In some embodiments, the second antigen sequence comprises from about 50 nucleotides to about 72 nucleotides. In some embodiments, the second antigen sequence is encoded by at least about 50 nucleotides. In some embodiments, the second antigen sequence comprises up to about 72 nucleotides. In some embodiments, the second antigen sequence comprises about 50 to about 70 nucleotides, about 50 to about 67 nucleotides, about 50 to about 65 nucleotides, about 50 to about 62 nucleotides, about 50 to about 60 nucleotides, about 50 to about 57 nucleotides, about 50 to about 55 nucleotides, or about 50 to about 52 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 3 to 4, 7, and 8.

[0165] In some embodiments, the second antigen sequence comprises a portion of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen sequence comprises at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen sequence comprises at least 50, 52, 55, 57, 60, 62, 65, 67, or more than 70 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23. In some embodiments, the second antigen sequence comprises from about 50 nucleotides to about 72 nucleotides. In some embodiments, the second antigen sequence comprises at least about 50 nucleotides. In some embodiments, the second antigen sequence comprises up to about 72 nucleotides. In some embodiments, the second antigen sequence comprises about 50 to about 70 nucleotides, about 50 to about 67 nucleotides, about 50 to about 65 nucleotides, about 50 to about 62 nucleotides, about 50 to about 60 nucleotides, about 50 to about 57 nucleotides, about 50 to about 55 nucleotides, or about 50 to about 52 nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 19, 20, 22, and 23.

[0166] Linker sequence Linker sequences can be used within an RNA molecule to separate different components of the RNA molecules described herein. The linker sequence can link two antigens, or antigen fragments thereof, or an antigen and an oligomerization domain. It is understood that the linker sequence is not a sequence that naturally separates a first RNA molecule and a second RNA molecule when the first and second RNA molecules are naturally linked together.

[0167] In some embodiments, the RNA molecules described herein comprise a sequence encoding a first linker that links a first antigen or antigenic fragment thereof to a multimerization domain, hi some embodiments, the RNA molecules described herein comprise a sequence encoding a second linker that links a second antigen or antigenic fragment thereof to a multimerization domain.

[0168] In some embodiments, the first linker encodes an amino acid sequence containing at least 5 to about 50 amino acids. In some embodiments, one or both of the first linker and the second linker encodes an amino acid sequence containing about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 10 to about 50 amino acids, about 15 to about 50 amino acids, about 20 to about 50 amino acids, about 25 to about 50 amino acids, about 30 to about 50 amino acids, about 35 to about 50 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids.

[0169] In some embodiments, the second linker encodes an amino acid sequence containing at least 5 to about 50 amino acids. In some embodiments, one or both of the first linker and the second linker encodes an amino acid sequence containing about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 10 to about 50 amino acids, about 15 to about 50 amino acids, about 20 to about 50 amino acids, about 25 to about 50 amino acids, about 30 to about 50 amino acids, about 35 to about 50 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids.

[0170] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO:9), (GS)n (SEQ ID NO:10), (G3S)n (SEQ ID NO:11), (GS)n (SEQ ID NO:32), and (G)n (SEQ ID NO:33), where n is an integer between 2 and 20. In some embodiments, n is an integer between 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 6 and 20, 8 and 20, 10 and 20, 12 and 20, 14 and 20, 16 and 20, or 18 and 20.

[0171] In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO:9), (GS)n (SEQ ID NO:10), (GS)n (SEQ ID NO:11), (GS)n (SEQ ID NO:32), and (G)n (SEQ ID NO:33), where n is an integer between 2 and 20. In some embodiments, n is an integer between 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 6 and 20, 8 and 20, 10 and 20, 12 and 20, 14 and 20, 16 and 20, or 18 and 20.

[0172] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), where n is an integer from 2 to 6.

[0173] In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), where n is an integer from 2 to 6.

[0174] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), where n is an integer from 1 to 3.

[0175] In some embodiments, one or both of the first linker and the second linker encodes an amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), where n is an integer from 1 to 3.

[0176] In some embodiments, the first linker encodes an amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8. In some embodiments, z is 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0177] In some embodiments, the second linker encodes an amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8. In some embodiments, z is 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0178] In some embodiments, the first linker encodes GGSG (SEQ ID NO: 38). In some embodiments, the second linker encodes GGSG (SEQ ID NO: 38).

[0179] In some embodiments, the first linker encodes GGSLGGGGGSGS (SEQ ID NO: 39). In some embodiments, the second linker encodes GGSLGGGGGSGS (SEQ ID NO: 39).

[0180] In some embodiments, the first linker is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0181] In some embodiments, the second linker is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0182] In some embodiments, the first linker is encoded by the nucleotide sequence of SEQ ID NO:5 and the second linker is encoded by the nucleotide sequence of SEQ ID NO:6. In some embodiments, the first linker is encoded by the nucleotide sequence of SEQ ID NO:6 and the second linker is encoded by the nucleotide sequence of SEQ ID NO:5. In some embodiments, the first linker is encoded by the nucleotide sequence of SEQ ID NO:5 and the second linker is encoded by the nucleotide sequence of SEQ ID NO:5. In some embodiments, the first linker is encoded by the nucleotide sequence of SEQ ID NO:6 and the second linker is encoded by the nucleotide sequence of SEQ ID NO:6.

[0183] fusion proteins The present disclosure generally relates to strategies for multimerizing protein antigens in vaccine-related or other immunotherapeutic constructs, which in some embodiments involve creating a nucleic acid construct that includes an oligomerization motif and a linker sequence separating two or more antigens, thereby enabling the encoded fusion protein to form dimeric, trimeric, tetrameric, hexameric, heptameric, or octameric complexes from a single nucleic acid construct.

[0184] This protein multimerization strategy can be utilized with proteins including viral, bacterial, parasitic, autoimmune, and tumor antigens. This platform can be used to generate multimeric fusion proteins containing multiple copies of a single antigen of interest. For example, two, three, or four copies of the same antigen with a dimerization, trimerization, or tetramerization domain can be used to generate homodimers, homotrimers, or tetramers. When the oligomerization domains associate with each other, the constructs form tetramers containing four copies of the same antigen (when a dimerization domain is used), hexamers containing six copies of the same antigen (when a trimerization domain is used), or octamers containing eight copies of the same antigen (when a tetramerization domain is used).

[0185] Alternatively, this platform can be used to generate multimeric fusion proteins containing two or more different antigens of interest. For example, heterodimers (or heterotrimers containing two or three different antigens) can be generated, linking a first antigen with a second, different antigen. When the oligomerization domains associate with each other, the construct forms a tetramer (if a dimerization domain is used) that is a dimer of both the first and second antigens, a hexamer (if a trimerization domain is used in the construct) that is a dimer of at least the first and second antigens, or a trimer of the first, second, and third antigens, or an octamer (if a tetramerization domain is used). Alternatively, trimeric proteins can be formed when the original protein is presented in a monomeric form associated with the trimerization domain.

[0186] One aspect described herein relates to an RNA comprising a first antigen, an oligomerization or multimerization domain, and a second antigen. One aspect described herein relates to an RNA comprising a first antigen, a first linker sequence, an oligomerization domain, a second linker sequence, and a second antigen, wherein the first linker sequence connects the first antigen to the oligomerization domain and the second linker connects the oligomerization domain to the second antigen. In one embodiment, the first antigen and the second antigen are the same. In another embodiment, the first antigen and the second antigen are different. The first antigen and the second antigen may be viral, bacterial, parasitic, autoimmune, or tumor antigens. In one embodiment, the first antigen and the second antigen comprise a polypeptide and / or a polysaccharide. In one embodiment, the RNA forms a multimeric protein when expressed in a host cell. In another embodiment, the first antigen and the second antigen do not naturally exist as a multimeric protein.

[0187] In one embodiment, the RNA forms a multimeric protein when expressed in a host cell, hi another embodiment, the first and second antigens do not naturally occur as multimeric proteins.

[0188] In some embodiments, the oligomerization or multimerization domain is selected from the group consisting of a dimerization domain, a trimerization domain, and a tetramerization domain. In some embodiments, the oligomerization domain is a dimerization domain. In other embodiments, the oligomerization domain is a trimerization domain. In some embodiments, the oligomerization domain is a tetramerization domain.

[0189] In some embodiments, the multimerization domain is selected from the group consisting of enterobacteriaceae phage T4, GCN4pII, GCN4-pLI, and p53. In some embodiments, the multimerization domain comprises a leucine zipper or fibritin foldon domain. In some embodiments, the dimerization domain is a leucine zipper domain, including but not limited to, the yeast GCN4 leucine zipper domain or a derivative thereof. In some embodiments, the trimerization domain is a T4 bacteriophage fibritin motif. In some embodiments, the trimerization domain is a eukaryotic GNC4 transcription factor motif or a derivative thereof. In some embodiments, the fibritin foldon domain is a trimerization domain from enterobacteriaceae phage T4 or a derivative thereof. In some embodiments, the trimerization domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:1.

[0190] In some embodiments, the oligomerization domain is located between any antigens.

[0191] In one aspect, provided herein is a polypeptide encoded by any embodiment of an RNA described herein.

[0192] Given the disclosure of this application, one of skill in the art would be able to substitute any antigen of interest into the RNA constructs described herein.

[0193] Lipid Compositions and Methods of Preparation In some embodiments, the present invention provides a composition of the RNA molecule and a delivery vehicle described herein. Also provided are methods related to the RNA molecule and delivery vehicle described herein. In some embodiments, the RNA delivery vehicle is a nanoparticle (e.g., LNP).

[0194] The delivery vehicle can be a non-virion, i.e., a particle that is not a virion. Thus, in some embodiments, the delivery vehicle does not contain a protein capsid. By eliminating the need to produce a capsid, the delivery vehicle does not utilize a packaging cell line, which makes it easier to scale up for commercial manufacturing and minimizes the risk of unintentionally producing dangerous infectious viruses. Various materials are suitable delivery particles capable of delivering RNA to vertebrate cells in vivo. Examples of delivery materials include (i) amphiphilic lipids capable of forming liposomes, and (ii) non-toxic and biodegradable polymers capable of forming microparticles. Other delivery methods include, but are not limited to, exosomes and cationic nanoemulsions.

[0195] In the case of delivery by liposomes, RNA can be encapsulated or adsorbed, and in the case of delivery by polymeric microparticles, RNA can be encapsulated or adsorbed. The third delivery material is a particulate reaction product of a polymer, a crosslinker, RNA, and a charged monomer. In certain embodiments, the delivery particles described herein comprise liposomes that adsorb the RNA molecules described herein.

[0196] In some embodiments, RNA is encapsulated in LNP. This means that the RNA in the particle is separated from any external medium by delivery material, and it has been found that the encapsulation protects the RNA from RNase digestion. Encapsulation can take various forms. For example, in some embodiments, the delivery material forms an outer layer around the aqueous RNA-containing core. In some embodiments, the composition of RNA encapsulated by LNP is lyophilized.

[0197] In some embodiments, the RNA is adsorbed to the surface of the LNP, which in some embodiments means that, unlike the RNA genome of a natural virus, the RNA is not separated from any external medium by the delivery material.

[0198] In some embodiments, the LNP has a pH of about 5 to about 7. In some embodiments, the LNP has a pH of about 5.2 to about 6.8. In some embodiments, the LNP has a pH of about 5.4 to about 6.6. In some embodiments, the LNP has a pH of about 5.5 to about 6.5. In some embodiments, the LNP has a pH of about 5.2 to about 6.4. In some embodiments, the LNP has a pH of about 5 to about 6.2.

[0199] In some embodiments, the RNA of the present disclosure is formulated into lipid nanoparticles (LNPs) having a diameter of about 40 nanometers (nm) to about 600 nm. In some embodiments, the diameter of the LNP is about 50 to about 600 nm, about 100 to about 600 nm, about 150 to about 600 nm, about 200 to about 600 nm, about 250 to about 600 nm, or about 300 to about 600 nm. In some embodiments, the diameter of the LNP is about 50 to about 550 nm, about 50 to about 500 nm, about 50 to about 450 nm, about 50 to about 400 nm, about 50 to about 350 nm, or about 50 to about 300 nm.

[0200] In some embodiments, the diameter of the LNP is about 300 nm or less. In some embodiments, the diameter of the LNP is about 250 nm or less. In some embodiments, the diameter of the LNP is about 200 nm or less. In some embodiments, the diameter of the LNP is about 190 nm or less. In some embodiments, the diameter of the LNP is about 180 nm or less. In some embodiments, the diameter of the LNP is about 170 nm or less. In some embodiments, the diameter of the LNP is about 160 nm or less. In some embodiments, the diameter of the LNP is about 150 nm or less. In some embodiments, the diameter of the LNP is about 140 nm or less. In some embodiments, the diameter of the LNP is about 130 nm or less. In some embodiments, the diameter of the LNP is about 120 nm or less.

[0201] Further provided herein is a method of preparing an RNA-LNP composition, the method comprising: a) mixing an ethanol phase comprising one or more lipids with an aqueous phase comprising an RNA molecule described herein; and b) purifying the RNA-LNPs produced from step a).

[0202] In some embodiments, one or more lipids comprise an ionizable lipid. In some embodiments, one or more lipids comprise cholesterol. In some embodiments, one or more lipids comprise a phospholipid. In some embodiments, one or more lipids are PEGylated.

[0203] In some embodiments, the LNP:RNA (N:P) ratio is about 6 to about 10. In some embodiments, the N:P ratio is about 6.5 to about 9, about 7 to about 9, about 7.5 to about 9, about 8 to about 9, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, or about 6 to about 8.

[0204] In some embodiments, the aqueous phase comprises Tris, sodium chloride, sucrose, or a combination thereof. In some embodiments, the aqueous phase comprises Tris. In some embodiments, the aqueous phase comprises sodium chloride. In some embodiments, the aqueous phase comprises sucrose. In some embodiments, the aqueous phase comprises or consists of Tris, sodium chloride, and sucrose.

[0205] In some embodiments, the lipid nanoparticle (LNP) formulations described herein comprise, consist essentially of, or consist of (i) neutral phospholipids, (ii) a sterol, e.g., cholesterol, (iii) optionally, a PEGylated lipid, and (iv) an ionizable lipid, in molar ratios ranging from 5% to 20% neutral phospholipid, 18.5% to 58.5% sterol, 1% to 4% PEGylated lipid, and 30% to 70% ionizable lipid, with the sum of the lipid molar ratios being 100%, optionally 10:40.5:1.5:48.

[0206] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG2000:and ionized lipid in molar ratios ranging from 5% to 20% DSPC, 18.5% to 58.5% cholesterol, 1% to 4% DMG-PEG2000, and 30% to 70% ionized lipid, with the sum of the lipid molar ratios being 100%.

[0207] In some embodiments, the lipid is a cationic lipid, also referred to as an ionizable lipid. In some embodiments, useful cationic lipids generally contain a nitrogen atom that is positively charged under physiological conditions, for example, as a tertiary amine or quaternary amine. This nitrogen can be present in the hydrophilic head group of an amphiphilic surfactant. The lipid can be selected from, but is not limited to, 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA, e.g., bromide), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl (C16:0) trimethylammoniumpropane (DPTAP), and distearoyltrimethylammoniumpropane (DSTAP). Other useful cationic lipids include benzalkonium chloride (BAK), benzethonium chloride, cetramide (containing tetradecyltrimethylammonium bromide and, optionally, small amounts of dedecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide), cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, mixed alkyltrimethylammonium bromide, benzyldimethyldodecylammonium bromide, and methyltrimethylammonium bromide. ammonium chloride, benzyldimethylhexadecylammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkylammonium chloride, methyltrioctylammonium chloride, N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy)ethyl]benzenemethanaminium chloride (DEBDA), dialkyldimethylammonium salt, [1-(2,3-dioleyloxy)-propyl]-N,N,N,Trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (acyl groups = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3-(dimethylammonio)propane (acyl groups = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol, 1,2-dioleoyl 3-succinyl-sn-glycerol choline ester, cholesteryl (4'-trimethylammonio) butanoate), N-alkylpyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic boraform electrolytes (Cl2Me6; Cl2BU6), dialkylglycetylphosphorylcholine, lysolecithin, L-α dioleoylphosphatidylethanolamine, cholesterol hemisuccinate choline ester, lipopolyamines (dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanolamidospermine (DPPES), lipopoly-L (or D)-lysine (LPLL, LPDL) ), poly(L(or D)-lysine) conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate esters with pendant amino groups (C12GluPhCnN+), ditetradecyl glutamate esters with pendant amino groups (C12GluPhCnN|), cationic derivatives of cholesterol (including, but not limited to, cholesteryl-3β-oxysuxamidoethylenetrimethylammonium salt, cholesteryl-3β-oxysuxamidoethylenedimethylamine, cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt, and cholesteryl-3β-carboxyamidoethylenedimethylamine).

[0208] In some embodiments, the lipid has the following structure: [ka]

[0209] In some embodiments, the lipid has the following structure: [ka]

[0210] In some embodiments, the RNA delivery vehicle is a nanoparticle comprising at least one lipid. In some embodiments, the lipid can be a neutral lipid. In some embodiments, the lipid is a phospholipid. The phospholipids were DDPC, 1,2-didecanoyl-sn-glycero-3-phosphatidylcholine, DEPA, 1,2-dierucoyl-sn-glycero-3-phosphate, DEPC, 1,2-erucoyl-sn-glycero-3-phosphatidylcholine, DEPE, 1,2-dierucoyl-sn-glycero-3-phosphatidylethanolamine, DEPG, 1,2-dipalmitoylphosphatidylglycerol, DLOPC, 1,2-linoleoyl-sn-glycero-3-phosphatidylcholine, DLPA, 1,2-dilauroyl-sn-glycero-3-phosphate, DLPC, 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine, DLPE, 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine, and DLPG. 1,2-Dilauroyl-sn-glycero-3 (phosphorylglycerol), DLPS, 1,2-Dilauroyl-sn-glycero-3-phosphatidylserine, DMG, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPA, 1,2-Dimyristoyl-sn-glycero-3-phosphate, DMPC, 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine, DMPE, 1,2-Dimyristoyl-sn-glycero-3-phosphatidylethanolamine, DMPG, 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol, DMPS, 1,2-Dimyristoyl-sn-glycero-3-phosphatidylserine , DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate, DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, DOPG, 1,2-dipalmitoylphosphatidylglycerol, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine, DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, DPPE, 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, DPPG, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, DPPS, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylserine, DPyPE, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, DSPA, 1,2-distearoyl-sn-glycero-3-phosphate, DSPC, 1,2-distearoyl-sn-glycero-3-phosphatidyl Dylcholine, DSPE, 1,2-diostearoyl-sn-glycero-3-phosphatidylethanolamine, DSPG, 1,2-distearoyl-sn-glycero-3-phosphorylglycerol, DSPS, 1,2-diundecanoyl-sn-glycero-phosphocholine, DUPC, 1,2-distearoyl-sn-glycero-3-phosphatidylserine, EPC, Egg-PC, HEPC, Hydrogenated Egg PC, HSPC, High Purity Hydrogenated Soy PC, HSPC, Hydrogenated Soy PC, Lysopc Myristic, 1-Myristoyl-sn-glycero-3-phosphatidylcholine, LYSOPC PALMITIC, 1-Palmitoyl-sn-glycero-3-phosphatidylcholine, LYSOPC STEARIC, 1-stearoyl-sn-glycero-3-phosphatidylcholine, bovine milk-derived sphingomyelin, MPPC, 1-myristoyl,2-palmitoyl-sn-glycero-3-phosphatidylcholine, MSPC, 1-myristoyl,2-stearoyl-sn-glycero-3-phosphatidylcholine, PMPC, 1-palmitoyl,2-myristoyl-sn-glycero-3-phosphatidylcholine, POPC, 1-palmitoyl,2-oleoyl-sn-glycero-3-phosphatidylcholine, POPE, 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine, POPG, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)], PSPC, 1-palmitoyl,2-stearoyl-sn-glycero-3-phosphatidylcholine, SMPC, 1-stearoyl,2-myristoyl-sn-glycero-3-phosphatidylcholine, SOPC, 1-stearoyl,2-oleoyl-sn-glycero-3-phosphatidylcholine, SPPC, 1-stearoyl,It may be selected from, but is not limited to, 2-palmitoyl-sn-glycero-3-phosphatidylcholine.

[0211] In some embodiments, the RNA delivery vehicle is a nanoparticle comprising at least one lipid. In some embodiments, the lipid may be a PEGylated lipid (PEG). In some embodiments, the PEGylated lipid comprises a polyethylene glycol moiety. In some embodiments, the PEGylated lipid includes, but is not limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, or a combination thereof. In some embodiments, the PEGylated lipid is a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. In some embodiments, the PEGylated lipid is DMG-PEG, i.e., PEG-linked 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]. In some embodiments, the lipid is DMG-PEG2000, i.e., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), DMG-PEG3500, DMG-PEG5000, DTDAM-PEG2000 (ALC-0159), DTDAM-PEG5000, DMG-C-PEG2000, DMG-C-PEG5000, DSG-PEG2000, DSG-PEG5000, DPG-PEG2000, DPG-PEG5000, or any combination thereof.

[0212] In some embodiments, the lipid is a structured lipid. In some embodiments, the structured lipid includes, but is not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, or a combination thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid is cholesterol, a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0213] In certain embodiments, a pharmaceutically acceptable composition is provided comprising an RNA-LNP described herein and a pharmaceutically acceptable carrier.

[0214] In some embodiments, pharmaceutically acceptable salts of the lipids described herein include salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmitate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, trifluoroacetate, and undecanoate salts.

[0215] Treatment method Provided herein are compositions (eg, pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of diseases or conditions in humans and other mammals.

[0216] The disclosure herein provides a method for preparing a pharmaceutical composition by mixing lyophilized RNA with a liquid LNP solution. In certain embodiments, the liquid LNP solution is added to the lyophilized RNA. In certain embodiments, the lyophilized RNA and the liquid LNP solution are mixed at room temperature. In certain embodiments, the lyophilized RNA and the liquid LNP solution are mixed prior to clinical use. In some embodiments, the RNA adsorbed to the surface of the liquid LNP is administered to a subject at least twice. In some embodiments, the RNA adsorbed to the surface of the liquid LNP is administered to a subject two, three, or four times. In some embodiments, the RNA adsorbed to the surface of the liquid LNP is administered to a subject once every two, three, or four weeks.

[0217] In some embodiments, the lyophilized RNA encapsulated in LNPs is mixed with sterile water. In some embodiments, the lyophilized RNA encapsulated in LNPs and sterile water are injected intramuscularly into a subject. In some embodiments, the lyophilized RNA encapsulated in LNPs is administered to a subject at least twice. In some embodiments, the lyophilized RNA encapsulated in LNPs is administered to a subject two, three, or four times. In some embodiments, the lyophilized RNA encapsulated in LNPs is administered to a subject once every two, three, or four weeks. Prophylactic protection from an antigen can be achieved after administration of an RNA vaccine or therapeutic of the present disclosure. In certain embodiments, two administrations of the vaccine or therapeutic are sufficient. Although less desirable, it is possible to administer a vaccine or therapeutic to an infected individual to achieve a therapeutic response.

[0218] In some embodiments, the RNA is encapsulated in LNPs in a sterile buffer. In some embodiments, the sterile buffer is water. In some embodiments, the sterile buffer contains a salt. In some embodiments, the LNP-encapsulated RNA is administered to a subject at least twice. In some embodiments, the LNP-encapsulated RNA is equilibrated at room temperature for about 5-20 minutes or 10-15 minutes prior to administration. In some embodiments, the LNP-encapsulated RNA is administered to a subject two, three, or four times. In some embodiments, the LNP-encapsulated RNA is administered to a subject once every two, three, or four weeks.

[0219] In an embodiment of the present disclosure, a method for eliciting an immune response in a subject against an antigen is provided. The method comprises administering to the subject an RNA vaccine or therapeutic comprising an RNA molecule having an open reading frame encoding at least one antigen polypeptide and a delivery vehicle such as an LNP, thereby eliciting an immune response in the subject. An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to the antigen polypeptide.

[0220] As used herein, a "prophylactically effective dose" is a therapeutically effective dose that prevents viral infection at a clinically acceptable level. In some embodiments, a therapeutically effective dose is the dose listed on the package insert for a vaccine or therapeutic. As used herein, a conventional vaccine refers to a vaccine other than the RNA vaccine or therapeutic of the present disclosure. For example, conventional vaccines include, but are not limited to, live microbial vaccines, inactivated microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, VLP vaccines, etc. In an exemplary embodiment, a conventional vaccine is one that has received regulatory approval and / or is registered by a national drug regulatory agency, e.g., the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) in the United States.

[0221] Provided herein are methods of treating or preventing a disease in a subject, comprising administering to the subject an effective amount of an RNA-lipid composition described herein.

[0222] In some embodiments, the disease is caused by a virus selected from the group consisting of influenza virus, rabies virus, respiratory syncytial virus (RSV), and coronavirus.

[0223] In some embodiments, the disease is caused by a coronavirus. In some embodiments, the coronavirus is selected from the group consisting of 229E (an alpha coronavirus), NL63 (an alpha coronavirus), OC43 (a beta coronavirus), HKU1 (a beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the coronavirus is SARS-CoV-2.

[0224] In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state is at a temperature of 2-8°C.

[0225] In some embodiments, the RNA and LNPs are injected intramuscularly into the subject.

[0226] In some embodiments, lyophilized RNA encapsulated in LNPs is mixed with sterile water.

[0227] In some embodiments, lyophilized RNA encapsulated in LNPs and sterile water are injected intramuscularly into a subject.

[0228] In some embodiments, RNA adsorbed to the surface of LNPs in liquid form or lyophilized RNA encapsulated in LNPs is administered to a subject at least twice.

[0229] In some embodiments, RNA adsorbed to the surface of LNPs in liquid form or lyophilized RNA encapsulated in LNPs is administered once every one, two, three, or four weeks.

[0230] In some embodiments, the RNA vaccine or therapeutic is administered to a subject (e.g., parenterally and transmucosally (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal)). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. In some embodiments, the RNA vaccine or therapeutic is injected intramuscularly. In some embodiments, the RNA vaccine or therapeutic is injected intravenously. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0231] In some embodiments, the vaccine or therapeutic agent is administered to a subject at a dose of 1 μg to 100 μg, optionally 5, 30, or 50 μg. The exact amount will depend on the purpose of the treatment and can be ascertained by one of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0232] In some embodiments, the vaccine or therapeutic is administered to the subject multiple times. In some embodiments, the vaccine or therapeutic is administered to the subject at least twice. In some embodiments, the second dose is administered to the subject about 8 weeks after the initial or first dose. In some embodiments, the second dose is administered to the subject about 7 weeks after the first dose. In some embodiments, the second dose is administered to the subject about 6 weeks after the first dose. In some embodiments, the second dose is administered to the subject about 5 weeks after the first dose. In some embodiments, the second dose is administered to the subject about 4 weeks after the first dose. In some embodiments, the second dose is administered to the subject about 3 weeks after the first dose. In some embodiments, the second dose is administered to the subject about 2 weeks after the first dose. In some embodiments, the second dose is administered to the subject about 1 week after the first dose.

[0233] kit In some embodiments, the present disclosure also provides kits that include i) RNA, ii) a delivery vehicle, such as a liquid LNP solution, iii) instructions for mixing the RNA with the delivery carrier to prepare an immunogenic composition, and iv) instructions for administering the immunogenic composition to a mammalian subject, such as a human subject in need thereof, to stimulate an immune response to the antigen.

[0234] In some embodiments, the RNA is stored at about -50°C or below. In some embodiments, the RNA is stored at about -60°C or below. In some embodiments, the RNA is stored at about -70°C or below. In some embodiments, the RNA is stored at about -80°C or below. In some embodiments, the RNA-LNP vaccine is stored at about 10°C or below. In some embodiments, the RNA-LNP vaccine is stored at about 0°C or below. In some embodiments, the RNA-LNP vaccine is stored at about -10°C or below. In some embodiments, the RNA-LNP vaccine is stored at about -15°C or below. In some embodiments, the RNA-LNP vaccine is stored at about -20°C or below.

[0235] definition To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0236] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meaning in the chemical arts.

[0237] Where systems are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is also contemplated that there are systems of the invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the invention that consist essentially of, or consist of, the recited processing steps.

[0238] Whenever an element or component is referred to in this application as being included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or can be selected from a group consisting of two or more of the recited elements or components, or elements or components.

[0239] Furthermore, it should be understood that elements and / or features of the devices or methods described herein, whether expressly or implicitly stated herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, when a particular component of a system is referred to, that component can be used in various embodiments of the system and / or method of the invention, unless otherwise understood from the context. In other words, although embodiments are described and illustrated herein to facilitate clear and concise description and illustration of the application, it is intended and understood that the embodiments can be combined or separated in various ways without departing from the present teachings and invention. For example, it should be understood that all features described and illustrated herein are applicable to all aspects of the invention as described and illustrated herein.

[0240] The articles "a" and "an" are used in this disclosure to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, "an element" means one element or more than one element.

[0241] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0242] The phrase "at least one of" should be understood to include each of the listed objects individually and various combinations of two or more of the listed objects after this expression, unless otherwise understood from context and usage. The phrase "and / or" with respect to more than two listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0243] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents, should generally be understood to be open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless specifically stated or understood otherwise from the context.

[0244] When the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself, unless otherwise specified. The term "about," as used herein, means a deviation of ±10%, ±5%, ±3%, or ±2% from the nominal value, unless otherwise specified or inferred from the context.

[0245] At various places in this specification, variables or parameters are disclosed as groups or ranges. The description is expressly intended to include all individual combinations of each member of those groups and ranges. For example, integers in the range of 0 to 40 are specifically intended to individually disclose 0, 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, and 40, and integers in the range of 1 to 20 are specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0246] The use of any and all examples or exemplary language, such as "such as" or "including" herein, is intended merely to better describe the invention and does not limit the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0247] As a general matter, formulations specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not attached to a definition, the previous definition for the variable controls.

[0248] As used herein, the term "dried RNA" should be understood as RNA that has been lyophilized, spray-dried, or spray-freeze-dried, as defined herein, to obtain a temperature-stable dry RNA (powder).

[0249] "Cryoprotectants" are well known in the art and include, but are not limited to, sucrose, trehalose, and glycerol. Cryoprotectants that exhibit low toxicity in biological systems are commonly used.

[0250] The term "lyophilization," including the related terms "lyophilization," "lyophilizing," or "freeze-drying," relates to a process that allows the solvent (e.g., water) content of a frozen sample (preferably a solution containing RNA molecules and a cryoprotectant as described herein) to be reduced in one or more steps, typically by sublimation. In the context of the present disclosure, lyophilization is typically performed by freezing the sample and then drying the sample by sublimation, optionally by reducing the ambient pressure and / or by heating the sample so that the solvent sublimes directly from the solid phase into the gas phase.

[0251] The terms "polynucleotide," "nucleic acid," and "nucleotide" are used interchangeably herein to refer to a chain of nucleotides of any length, including DNA and RNA. In some embodiments, a nucleotide is a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or its analogues, or any substrate that can be incorporated into a chain by a DNA or RNA polymerase. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogues. If present, modifications to the nucleotide structure are imparted before or after assembly of the chain. In some embodiments, the nucleotide sequence can be interrupted by non-nucleotide components. In some embodiments, a polynucleotide is further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more natural nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates) and charged linkages (e.g., phosphorothioates, phosphorodithioates), those containing pendant moieties, e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine), those with intercalators (e.g., acridine, psoralen), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides), those containing alkylators, those with modified linkages (e.g., α-anomeric nucleic acids), as well as unmodified polynucleotides. In some embodiments, any of the hydroxy groups normally present on the sugars are replaced, e.g., with phosphonate or phosphate groups, protected with standard protecting groups, activated to form bonds with additional nucleotides, or attached to a solid support. In some embodiments, the 5' and 3' terminal OH are phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.In some embodiments, polynucleotides also contain ribose or deoxyribose sugar analogs, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. In some embodiments, one or more phosphodiester linkages are replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NRi ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages within a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA. In polynucleotides, when T is referred to, it means U (uracil) for RNA and T (thymine) for DNA.

[0252] The term "messenger RNA" (mRNA) refers to a polynucleotide that encodes (at least one) polypeptide (natural, non-natural, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0253] As used herein, the terms "terminus" or "terminus" when referring to a polypeptide, RNA molecule, or polynucleotide refer to the end of the polypeptide, RNA molecule, or polynucleotide, respectively. Such ends are not limited to the first or last position of a polypeptide, RNA molecule, or polynucleotide, but can include additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules can be characterized as having both an N-terminus (terminated by an amino acid having a free amino group (NH)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). Proteins are sometimes made up of multiple polypeptide chains (multimers, oligomers) held together by disulfide bonds or non-covalent forces. These proteins have multiple N- and C-termini. Alternatively, the termini of polypeptides can sometimes be modified to begin or end with non-polypeptide-based moieties, such as organic conjugates.

[0254] "5' untranslated region" (5'UTR) refers to the region of an RNA (e.g., an mRNA or other coding RNA) located immediately upstream (i.e., 5') from the start codon that does not encode a polypeptide (i.e., the first codon of an RNA, e.g., an mRNA or other coding RNA, a transcript translated by a ribosome).

[0255] "3' untranslated region" (3'UTR) refers to the region of an RNA (e.g., an mRNA or other coding RNA) located immediately downstream (i.e., 3') from a stop codon that does not encode a polypeptide (i.e., the codons of an RNA, e.g., an mRNA or other coding RNA, transcript that signal the end of translation).

[0256] An "open reading frame" is a contiguous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA), and typically encodes a polypeptide (e.g., a protein). The sequence may further include additional elements, e.g., 5' and 3' UTRs, although it will be understood that these elements, unlike ORFs, need not necessarily be present in the disclosed vaccines or therapeutics.

[0257] A "poly-A tail" is a region of an RNA (e.g., an mRNA or other coding RNA) downstream, e.g., directly downstream (i.e., 3'), of a 3' UTR that contains multiple consecutive adenosine monophosphates. A poly-A tail can contain 10 to 300 adenosine monophosphates (SEQ ID NO: 40). For example, a poly-A tail can 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 poly-A tail contains 50 to 250 adenosine monophosphates. In relevant biological settings (e.g., intracellular, in vivo), the poly(A) tail functions to protect the RNA from enzymatic degradation, for example, in the cytoplasm, and assists in transcription termination and / or export of the RNA from the nucleus and translation.

[0258] The term "dose" as used herein with reference to an immunogenic composition means the measured amount of immunogenic composition taken by (administered to) or received by) a subject at any given time.

[0259] The term "immunization" refers to the process of increasing a mammalian subject's response to an antigen, thus improving the subject's ability to resist or overcome infection and / or resist disease.

[0260] As used herein, the term "vaccination" means the introduction of a vaccine into a subject, preferably a mammalian subject such as a human.

[0261] The term "prophylactically effective dose," which includes the related terms "effective dose" and "therapeutically effective dose," as used herein means a dose that prevents infection by a virus at a clinically acceptable level.

[0262] The term "alkyl" refers to the radical of a straight-chain or branched-chain saturated hydrocarbon group having from 1 to 32 carbon atoms ("C1-C32 alkyl"). In some embodiments, the alkyl group has from 1 to 12 carbon atoms ("C1-C12 alkyl"). In some embodiments, the alkyl group has from 1 to 10 carbon atoms ("C1-C10 alkyl"). In some embodiments, the alkyl group has from 1 to 9 carbon atoms ("C1-C9 alkyl"). In some embodiments, the alkyl group has from 1 to 7 carbon atoms ("C1-C7 alkyl"). In some embodiments, the alkyl group has from 1 to 5 carbon atoms ("C1-C5 alkyl"). In some embodiments, the alkyl group has from 1 to 4 carbon atoms ("C1-C4 alkyl"). In some embodiments, the alkyl group has from 1 to 3 carbon atoms ("C1-C3 alkyl"). In some embodiments, the alkyl group has from 1 to 2 carbon atoms ("C1-C2 alkyl"). In some embodiments, the alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C1-C30 alkyl"). In some embodiments, an alkyl group has 1 to 22 carbon atoms ("C1-C22 alkyl"). In some embodiments, an alkyl group has 5 to 10 carbon atoms ("C5-C10 alkyl"). In some embodiments, an alkyl group has 7 to 17 carbon atoms ("C7-C17 alkyl"). In some embodiments, an alkyl group has 10 to 32 carbon atoms ("C10-C32 alkyl").

[0263] The term "alkenyl" refers to the radical of a straight- or branched-chain hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C2-C20 alkenyl"). In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C2-C10 alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-C5 alkenyl").

[0264] The term "suitable protective agent" includes the related terms "cryoprotectant" and "protectant" and does not cause or enhance RNA degradation.

[0265] Example Example 1. Synthesis of RNA molecules against various antigens This example describes the synthesis of purified RNA molecules (eg, mRNA) described herein.

[0266] The synthesis of the mRNA constructs described herein was carried out in three steps. First, the antigen-encoding plasmid DNA was amplified, extracted, and purified. Next, the plasmid was linearized and purified by chromatography and ethanol precipitation. Finally, mRNA was enzymatically synthesized in vitro using the linearized plasmid DNA as a template, and after purification, it was stored at -80°C. Schematics of exemplary mRNA structures are shown in Figure 1B and Figure 11.

[0267] Example 2. Preparation of mRNA-LNP vaccine This example describes a method for making a composition of an RNA molecule and a delivery vehicle (eg, an LNP).

[0268] Purified RNA molecules were formulated into lipid nanoparticles (LNPs) by rapidly mixing an ethanol phase and an aqueous phase using a microfluidic device. The aqueous phase contained 50 mM citrate buffer (pH 6.0) and a fixed amount of mRNA (approximately 50–150 μg / mL). The ethanol phase contained ionizable lipids (Immorna), cholesterol, 1,2-diastearoyl-sn-glycerol-3-phosphocholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). These four lipid components were mixed in a molar ratio of 40:48:10:2.0 (ionizable lipid:cholesterol:DSPC:DMG-PEG2000). The resulting LNPs were purified in citrate buffer (pH 6.0) and characterized for particle size, polydispersity index (PDI), and mRNA concentration. Finally, the pH was adjusted to 7.2. The mRNA-LNP was then diluted to the target concentration to obtain the mRNA-LNP vaccine product, which was stored at ≦-20°C or ≦-60°C.

[0269] In this disclosure, mRNA-LNPs containing 30 μg / mL of mRNA were prepared using the same method described in this section. The particle size and polydispersity index (PDI) of the mRNA-LNPs were measured by dynamic light scattering (DLS), and the purity of the mRNA was measured by agarose gel electrophoresis. The vaccine was equilibrated at room temperature for 15 minutes before use.

[0270] Example 3. In vitro transfection of mRNA vaccines This example describes the process followed to measure the transfection efficiency of an RNA molecule or composition and the ability of an mRNA vaccine to express a target protein.

[0271] To evaluate the in vitro transfection efficiency of the mRNA vaccine, 3 × 105 BHK-21 cells were seeded per well in a 6-well plate. 2 μg of LNP-encapsulated mRNA vaccine was transfected into BHK-21 cells. Intracellular SARS-CoV-2 RBD protein expression was detected by Western blotting. Briefly, 24 h posttransfection, mRNA vaccine-transfected cells were lysed in NP-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, leupeptin). The mixture was centrifuged at 13,000 rpm for 5 minutes at 4°C. The supernatant was collected, boiled with SDS at 95°C for 12 minutes, separated on a 6% SDS-PAGE gel, and transferred to a nitrocellulose filter membrane. After blocking with 5% BSA, the membrane was first blotted with a primary antibody (1:1000) (SARS-CoV-2 (2019-nCoV) spike protein rabbit PAb), then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000) (IgG(H+L) (HRP-labeled goat anti-rabbit IgG(H+L))), and visualized with a chemiluminescent reagent (chemiluminescent HRP substrate).

[0272] A few hours after transfection, the target RBD protein was detected in cell lysates using Western blotting. Based on the target RBD protein expression results shown in Figure 2, we can conclude that the mRNA-LNP vaccine can express the target protein.

[0273] Example 4. In Vivo Evaluation of mRNA-LNP Vaccines This example describes a timeline for testing mRNA-LNP compositions in mice.

[0274] Animal studies were conducted at the Yangtze Delta Region Research Institute of Tsinghua University (Zhejiang). BALB / c mice (6-8 weeks old) were divided into groups of 4 or 5. On day 0 (primary injection) and days 14 or 21 (booster immunization), three groups of mice were immunized intramuscularly with mRNA-LNP vaccine (2 μg) and buffer vehicle, respectively. Serum was collected before the first vaccination and on days 14, 21, and 28. All collected samples were cryopreserved according to standard protocols.

[0275] Example 5. Quantification of antibody binding titers to SARS-CoV-2 spike protein by ELISA assay This example describes a process for measuring antibody titers from a sample.

[0276] Antibody binding titers against the SARS-CoV-2 spike protein (RBD, His tag) were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, 0.5 μg / mL of SARS-CoV-2 spike protein (RBD, His tag) diluted in carbonate-bicarbonate buffer was precoated onto a 96-well clear polystyrene microplate overnight at 4°C. After washing three times with PBS-T (0.05% Tween-20 in PBS), the coated plate was blocked with 300 μL of blocking buffer (15% normal goat serum and 2% bovine serum albumin in PBS-T) for 1 h at 37°C. Serum samples were serially diluted two-fold in blocking buffer, transferred to the plate, and incubated for 1 h at 37°C. After washing, the plate was incubated with HRP-conjugated rabbit anti-mouse IgG H+L for 1 h at 37°C. Plates were washed and incubated with TMB single-component substrate solution for 7 min at 37°C, and the reaction was stopped with ELISA stop solution. Absorbance was read at 450 nm in a microplate reader, and ELISA titers were determined using nonlinear four-parameter variable slope analysis in GraphPad Prism 8 software.

[0277] Example 6. Quantification of SARS-CoV-2 Neutralizing Antibodies by Pseudovirus-Based Assay This example describes a process for measuring neutralizing antibody titers from a sample.

[0278] Briefly, serum samples were inactivated in a 56°C water bath for 30 min to inactivate the complement system present in the innate immune system. The serum samples were then diluted 20-fold with DMEM medium and filtered through a 0.22 μm filter. The filtered serum samples were added to a 96-well plate and diluted 3-fold until a 4860-fold dilution was achieved. Pseudotyped viruses were rapidly thawed in a room-temperature water bath and then diluted to 1.3 × 10 TCID 50 / mL in DMEM complete medium. 50 μL of diluted virus was added to experimental wells. The 96-well plate was then placed in a 37°C, 5% CO 2 incubator for 1 h. Vero cells were lysed, 5 × 10 4 cells were added per well, and the samples were incubated at 37°C in a 5% CO 2 incubator for 24 h. After 24 h, the 96-well plate was removed and equilibrated at room temperature for 30 min. 150 μL of supernatant was removed from each well, and 100 μL / well of Bio-Lite Luciferase Detection Reagent was added. The plate was then shaken for 2 min. Luminescence (RLU) was immediately measured using a microplate reader. A cell control (CC) was prepared using the same procedure without adding pseudotyped virus, and a virus control (VC) was prepared using the same procedure without adding diluted serum samples. Neutralizing antibody titers were expressed as the reciprocal of the serum dilution ratio at 50% inhibition or the antibody concentration at 50% inhibition.

[0279] The inhibition rate is calculated by the following formula:

[0280] Inhibition rate %=(1-(sample average RLU-CC average RLU) / (VC average RLU-CC average RLU))×100% Example 7. Preparation and characterization of mRNA-LNP vaccines This example describes specific properties of mRNA-LNP compositions.

[0281] After thawing, the mRNA-LNP vaccine appears as a slightly milky dispersion, which is the expected appearance of a typical mRNA-LNP product.

[0282] In addition to appearance, important pharmaceutical properties of the LNP-mRNA vaccine are listed in Table 1.

[0283] [Table 1]

[0284] Example 8. Efficacy of mRNA-LNP vaccine in mice This example describes a study to measure antibody titers in mice.

[0285] We performed in vivo studies of antibody responses in BALB / c mice vaccinated with an mRNA-LNP vaccine encoding the SARS-CoV-2 RBD protein. Mice received a primary injection and a booster 2 weeks later (Figure 3).

[0286] Antibody titers are highly correlated with the protective effect and durability of vaccines. Therefore, in this study, they were used as a readout of efficacy and are expressed in the form of geometric mean titers (GMT). As shown in Figure 4, antibody titers increased significantly after two immunizations. SARS-CoV-2 neutralizing antibodies showing cross-protection against different strains are shown in Figure 5.

[0287] Example 9. Production of mRNA-LNP vaccine In this example, the manufacturing process of an mRNA-LNP vaccine is described.

[0288] The mRNA-LNP vaccine described in this example has a molecular weight of 586,246 Da and is composed of a gene of interest (GOI) encoding a polypeptide containing Cap1, a 5' untranslated region (5' UTR), a signal peptide (SP), the SARS-CoV-2 original strain RBD, a T4 foldon domain, the RBD of omicron BA.1 (but containing the L452R and F486V mutations), a 3' untranslated region (3' UTR), and a poly(A) tail. A schematic diagram of the mRNA is shown in Figure 6.

[0289] The final mRNA recombinant plasmid, pT7 6.1 Omicron Construct C (containing the L452R and F486V mutations), consisted of a T7 RNA polymerase promoter, a 5' untranslated region (5' UTR), a Kozak sequence, multiple restriction enzyme cleavage sites, a gene of interest (GOI), a 3' untranslated region (3' UTR), a poly(A) sequence, an ori (origin of replication), a kanamycin resistance gene (KanR), and an ampicillin resistance gene (AmpR) promoter. The GOI included sequences encoding the spike protein RBD polypeptide of the original SARS-CoV-2 strain and the spike protein RBD polypeptide of the Omicron BA.1 strain with two amino acid mutations (L452R and F486V). The integrity of the plasmid sequence was verified by sequencing.

[0290] The recombinant plasmid pT7 6.1 Omicron Construct C (containing the L452R and F486V mutations) was transformed into stable competent bacteria and plated on Luria Bertani (LB) plates containing kanamycin. One clone was selected, plated on LB liquid medium, and incubated overnight. After incubation, the plasmid was extracted and sequenced to confirm that it contained the correct gene sequence. The bacterial solution was aliquoted and stored in 10–20% glycerol and used as the seed strain.

[0291] A master cell bank (MCB) was created by inoculating one tube of the seed strain into LB liquid medium containing 50 μg / mL kanamycin. The incubation volume was 200 μL. The medium was incubated at 30°C for 8–14 hours and then harvested as the first passage. 10%–20% glycerol was added. The liquid was dispensed in 1 mL aliquots per tube and frozen at -60°C or below for use as the master cell bank (MCB).

[0292] A working cell bank (WCB) was generated from the MCB following the same procedure as for MCB preparation and stored at -60°C or below.

[0293] The manufacturing process for mRNA drug substances (DS) is divided into three major operational units: 1) plasmid DNA production, 2) in vitro transcription of the DNA template, and 3) mRNA purification.

[0294] First, one tube of WCB was selected and inoculated into LB medium to amplify the plasmid. The cells were incubated overnight (12–20 hours at 37±1°C) and then recovered. Supercoiled plasmid DNA was isolated using solutions P1, P2, and P3. The cell lysate was filtered and loaded onto a column packed with 6FF resin, and the first eluted peak was collected. Next, the supercoiled plasmid DNA was extracted and purified using affinity chromatography and anion exchange chromatography. Finally, absolute ethanol was added to recover the purified plasmid DNA from the elution peak of the anion exchange chromatography. The precipitated plasmid DNA was collected by centrifugation and resuspended in water for injection (WFI).

[0295] The DNA plasmid was linearized by incubation with the restriction endonuclease (BspQI). The linearized DNA was then purified by anion exchange chromatography and ethanol precipitation. mRNA DS was produced by in vitro enzymatic transcription using the linearized DNA plasmid as a template, followed by enzymatic capping and downstream purification. In this process, T7 RNA polymerase was induced using the fully linearized plasmid DNA as a template to transcribe and synthesize mRNA via the T7 promoter. After transcription, the template DNA was enzymatically digested with Turbo DNase, and then the mRNA buffer was exchanged into WFI using ultrafiltration and diafiltration (UF / DF). The enzymatic capping reaction (CAP) used vaccinia virus capping enzyme, which adds a 7-methylguanylate cap structure (Cap0) to the 5' end of the transcribed mRNA, and Cap2'-O methyltransferase, which converts Cap0 to Cap1. The Cap1 structure stabilizes the mRNA and enables efficient translation in vivo.

[0296] The transcribed capped mRNA was subsequently subjected to the following downstream processing steps: 1) affinity chromatography purification, 2) UF / DF, 3) cellulose chromatography purification, 4) UF / DF, and 5) 0.22 μm sterile filtration to obtain the mRNA bulk drug substance.

[0297] The final mRNA drug substance was contained in 18 mM citrate buffer (pH 5.8) containing 7% (w / v) sucrose. The mRNA drug substance was stored in a sealed PETG container at or below approximately -60°C. The shelf life is approximately 3 months.

[0298] The mRNA DS control strategy included general appearance, pH, polyA length distribution, identity (i.e., mRNA identity, sequence accuracy of target gene), mRNA content, in vitro potency, purity (i.e., mRNA purity, capping efficiency), impurities (i.e., residual kanamycin, residual protein, residual DNA template, residual double-stranded RNA), and safety (i.e., sterility and endotoxin content) testing.

[0299] The mRNA drug formulation (DP) consisted of lyophilized mRNA DP (vial 1) and LNP dispersion (vial 2). The LNP dispersion was synthesized using a microfluidic process, and the mRNA DP was lyophilized to a dry state to form the lyophilized mRNA DP. After reconstitution of the lyophilized mRNA with the LNP dispersion, the mRNA was sterilized without preservatives and prepared as a white to off-white suspension at a concentration of 50 μg / mL for intramuscular injection. The reconstituted vaccine contained 19 mmol / L citric acid and 6.5% (w / v) sucrose (pH 5.3–6.3).

[0300] The mRNA GMP batch DP was manufactured. In-process control tests were performed throughout the manufacturing process to monitor and record process parameters. Typical GMP batch sizes for the mRNA DP ranged from 500 to 1,200 mL (before lyophilization). Manufacturing batch sizes for the mRNA-LNP dispersion ranged from 1,250 to 3,000 mL. Prior to use, the lyophilized mRNA and LNP dispersions were equilibrated at room temperature (RT) for approximately 15 minutes. The LNP dispersion was then gently shaken for 5 to 10 seconds. 1.0 mL of the LNP dispersion was extracted with a needle-equipped syringe and added to the mRNA lyophilized powder. After addition, the vial was mixed by repeatedly inverting for approximately 30 seconds. After mixing, the reconstituted vaccine was observed as a white to off-white suspension. The vaccine should be administered within 1 hour of reconstitution.

[0301] LNPs were composed of four lipid components in a predetermined molar ratio: 1.5% 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 40.5% cholesterol, 10% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and a cationic ionizable lipid.

[0302] The container closure system for vial 1 consisted of a 2 mL borosilicate glass vial with a nominal fill volume of 0.5 mL / vial, a chlorinated butyl rubber stopper for lyophilization, and an aluminum plastic cap for antibiotic vials. The container closure system for vial 2 consisted of a 2 mL borosilicate glass vial with a nominal fill volume of 1.0 mL / vial, a halogenated butyl rubber stopper (brominated) for injection, and an aluminum plastic cap for antibiotic vials. Storage conditions were set at 2-8°C, away from direct sunlight.

[0303] The production of mRNA DP consisted of two segments: 1) production of mRNA DP and 2) production of LNP dispersion.

[0304] The production batch size of mRNA DP was 500–1,200 mL based on the yield of the mRNA DS manufacturing process. The mRNA bulk DS was first diluted with formulation buffer (18 mmol / L citric acid, 7.0% (w / v) sucrose, pH 5.8) to a concentration of approximately 100 μg / mL. After sterile filtration, the mRNA solution was filled into 2 mL borosilicate glass vials with a nominal volume of 0.5 mL per vial. Subsequently, the mRNA DP was produced by lyophilization.

[0305] The production batch size of mRNA-LNP dispersions ranged from 1,250 to 3,000 mL. Lipid self-assembly into nanoparticles was induced by rapidly mixing aqueous buffer and lipids dissolved in ethanol in a microfluidic mixer. The LNPs were then concentrated and buffer-exchanged into citrate buffer (10 mmol / L citric acid, pH 5.5). Sucrose stock solution was added to achieve a final concentration of 3% (w / v) sucrose. After lipid concentration adjustment and subsequent sterile filtration, the LNPs were filled into 2 mL borosilicate glass vials with a nominal volume of 1 mL per vial.

[0306] Aseptic process control of the freeze-drying and fill / finish processes was verified by mock filling of media. Process parameters were monitored and recorded throughout production. Process control was performed by in-process testing.

[0307] The control strategy for mRNA DP includes testing for general appearance, visible impurities, pH, osmolality, fill volume variation, moisture content, particle size, polydispersity index, identity, mRNA content, adsorption efficiency (%), in vitro potency, purity (i.e., mRNA purity and lipid purity), impurities (i.e., residual ethanol), and safety (e.g., endotoxin content and sterility). Among these, fill volume variation and moisture content were tested for the mRNA lyophilized powder, appearance was tested for both the mRNA lyophilized powder and after reconstitution with the mRNA-LNP dispersion, and other quality attributes were tested after reconstitution. The control strategy for mRNA-LNP dispersion included testing for general appearance, visible impurities, pH, fill volume variation, particle size, polydispersity index, zeta potential, lipid purity, lipid content and molar ratio, and safety (e.g., endotoxin content and sterility).

[0308] The mRNA DP and LNP dispersions were stored in glass vials at 2-8°C, avoiding direct sunlight, with an expected shelf life of 18 months.

[0309] Example 10. Evaluation of in vivo immunogenicity of mRNA-LNP vaccines in mice This example evaluates the in vivo efficacy of the mRNA vaccine described in Example 10, which encodes the receptor-binding domain (RBD) sequences of the spike protein of the SARS-CoV-2 original strain and mutant strain (Omicron BA.1). The encoded RBD sequence of the Omicron BA.1 mutant contains the amino acid substitutions L452R and F486V, which are specific to the two Omicron BA.4 / .5 strains. This example aims to evaluate the immunogenicity of the mRNA-LNP vaccine in mice. Vaccine immunogenicity was evaluated by measuring antigen-specific binding antibody titers by ELISA, pseudotype virus neutralizing antibody titers, and cellular immune responses after vaccination.

[0310] Experimental Method Animal experiments The immunization schedule and sampling / testing schedule are described in Tables 2 and 3.

[0311] [Table 2]

[0312] [Table 3]

[0313] Preparation of test article for administration: Prior to administration, the mRNA components and LNP dispersion were equilibrated to room temperature for approximately 15 minutes. The LNP dispersion was gently shaken for 5-10 seconds. 0.6 mL of the LNP dispersion was drawn up with a needle-equipped syringe and added to the mRNA lyophilized powder. After addition, the vial was inverted for approximately 30 seconds to mix thoroughly. The reconstituted vaccine was observed as a white to off-white suspension.

[0314] Mouse Immunization: A schematic diagram of the immunization process is shown in Figure 7. Briefly, 16 SPF-grade female mice were randomly divided into three groups: a control group (4 mice), a low-dose group (6 mice), and a high-dose group (6 mice). Mice were administered two doses at 21-day intervals, with the first dose being defined as the study start date (D0). Mice were administered intramuscularly (i.m.) into the right hind crural region. The dose volume for the negative control and low-dose groups was 0.08 mL / animal, and the dose volume for the high-dose group was 0.16 mL / animal. Multiple intramuscular injections were administered into the hind crural region, with each dose not exceeding 0.1 mL. Blood samples (50–100 μL per mouse) were collected, and the time points for spleen removal are shown in Figure 7.

[0315] Animals were examined daily for signs and symptoms, behavioral activity, posture, feeding status, coat, irritation, glandular secretions, excretions, respiratory status, and mortality. Mice were weighed periodically.

[0316] Serum preparation: Blood samples were collected in Eppendorf tubes and kept on ice. After centrifugation at 1,500 g for 10 min at 4°C, the supernatant was quickly transferred to a new tube and stored at a temperature below -70°C.

[0317] Splenocyte isolation: Spleens were removed from mice, placed in PBS, and gently homogenized. The suspension was then filtered through a 70 μm cell strainer. Erythrocytes were lysed using erythrocyte lysis buffer according to the manufacturer's instructions. For immediate use, cells were washed twice with PBS and then suspended in RPMI-1640 complete medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. To preserve cells for later use, cell freezing medium containing 90% FBS (9 mL) and 10% DMSO (1 mL) was first prepared and stored at 4°C. The cells were then removed from the centrifuge tube and resuspended in freezing medium by pipetting up and down 20 times to break up cell-cell clumps. After adjusting the cell density to 2 × 106 / mL, the cell suspension was dispensed into cryotubes and stored overnight at -70°C in a cryobox. Finally, the tubes were transferred to a liquid nitrogen tank for storage.

[0318] Measurement of antigen-specific antibody titers: Binding antibody titers against SARS-CoV-2 spike protein RBD-Omicron strain (BA.1) His-tagged (RBD-O) or SARS-CoV-2 spike protein RBD-original strain His-tagged (RBD-WT) were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, 1 μg / mL RBD-O (100 μL) or 0.5 μg / mL RBD-WT (100 μL) was diluted in coating buffer (0.05 M carbonate buffer) and pre-coated onto a 96-well clear polystyrene microplate overnight at 4°C. After washing three times with PBS-T (0.05% Tween-20 in PBS), the coated plate was blocked with 300 μL of blocking buffer (15% normal goat serum and 2% bovine serum albumin in PBS-T) for 1 h at 37°C. Serum samples were serially diluted 2-fold in blocking buffer, transferred to the plate, and incubated for 1 h at 37°C. After washing, the plate was incubated with HRP-conjugated rabbit anti-mouse IgG H+L for 1 h at 37°C. The plate was washed and incubated with TMB single-component substrate solution for 7 min at 37°C, and the reaction was stopped with ELISA stop solution. Absorbance was read at 450 nm on a microplate reader, and ELISA titers were determined using GraphPad Prism 8 software based on a 2.1-fold blank value.

[0319] Measurement of neutralizing antibody titers: Briefly, serum samples to be tested were inactivated in a 56°C water bath for 30 min to inactivate the complement system. The serum samples were then diluted 20-fold with DMEM medium and filtered through a 0.22 μm filter. The filtered serum samples were added to a 96-well plate and serially diluted in 3-fold increments until the desired concentration was achieved. Pseudotyped viruses of various SARS-CoV-2 mutants of interest were rapidly lysed in a room-temperature water bath and then diluted to 1.3 x 104 TCID50 / mL in DMEM complete medium. 50 μL of diluted virus was then added to experimental wells. The 96-well plate was then placed in a 37°C, 5% CO2 incubator for 1 h. Subsequently, digested Vero cells were added to the wells at 5 x 104 cells / well and cultured at 37°C in a 5% CO2 incubator for 24 h. After 24 h, the 96-well plate was equilibrated at room temperature for 30 min. 150 μL of supernatant was removed from each well, 100 μL / well of Bio-Lite Luciferase Detection Reagent was added, and the plate was shaken for 2 min. Luminescence (RLU) was immediately measured using a microplate reader. For cell control (CC), the same procedure was repeated without adding pseudotyped virus. For virus control (VC), the same procedure was repeated without adding diluted serum samples. Neutralizing antibody titers were expressed as the reciprocal of the serum dilution ratio at 50% inhibition or the antibody concentration at 50% inhibition. The formula for calculating the inhibition rate was: Inhibition rate (%) = (1-(sample average RLU-CC average RLU) / (VC average RLU-CC average RLU))×100% Intracellular cytokine staining and flow cytometry Cell preparation and peptide stimulation: Cells were resuspended in RPMI 1640 medium containing 10% FBS and 1% PS, and 2 x 10 cells / 200 μL were added to a 96-well plate. Then, 25 μL of peptide solution (80 μg / mL dissolved in DMSO) was added to each well. Finally, 0.4 μL of protein transport inhibitor cocktail (500x stock) was added, and the plate was incubated overnight at 37°C in 5% CO2.

[0320] Surface marker staining: The cell suspension was centrifuged at 500g for 5 minutes. The pellet was resuspended in 200µL of FACS buffer (PBS containing 0.5% BSA), and the suspension was centrifuged at 500g for another 5 minutes at 4°C. This step was repeated. Antibodies such as anti-CD3, anti-CD44, and anti-CD8 were diluted 200-fold in 200µL of FACS buffer and used to resuspend the cells, followed by incubation on ice in the dark for 30 minutes. Next, the cells were centrifuged at 500g for 5 minutes at 4°C. The pellet was then resuspended in 200µL of FACS buffer and centrifuged again under the same conditions. This step was repeated. Finally, the cells were resuspended in 500µL of Foxp3 fixation / permeabilization (1 / 3 dilution) working solution and incubated in the dark at 4°C for 20 minutes.

[0321] Intracellular cytokine staining: The cell suspension was centrifuged at 600g for 5 min. The pellet was resuspended in 1 mL of 1x permeabilization buffer and centrifuged again. This step was repeated two more times. Antibodies containing anti-IFN-γ were diluted 200-fold in 200 μL of 1x permeabilization buffer, and the cells were resuspended and incubated on ice in the dark for 1 hr. After incubation, the cells were centrifuged at 600g for 5 min at 4°C. The pellet was then resuspended in 1 mL of FACS buffer and centrifuged again. This step was repeated once more. Finally, the stained cells were resuspended in 220 μL of FACS buffer and were ready for analysis on a flow cytometer.

[0322] Statistical analysis: All data are presented as the mean (SD) or mean ± SEM. Differences in the mean values ​​of two groups were assessed using a two-tailed Student's t-test. Differences in the mean values ​​between more than two groups were determined using ANOVA. P<0.05 (*), P<0.01 (**), and P<0.001 (***) indicated statistically significant differences. When the sample size was ≤2, individual data were listed and no statistical analysis was performed.

[0323] result General observation conditions: In general, no obvious abnormalities were observed in the appearance, signs, behavioral activity, posture, feeding status, coat, irritation response, gland secretions, or respiratory status of any animals during the test period.

[0324] Antibody binding titers to SARS-CoV-2 antigens: To evaluate the humoral immunogenicity of the mRNA-LNP vaccine, antigen-specific binding antibody titers to the SARS-CoV-2 RBD polypeptide were measured by ELISA using serum samples collected from mice immunized with the mRNA-LNP vaccine. As shown in Figures 8A and 8B, high titers of antigen-specific binding antibodies to the RBD polypeptides of the SARS-CoV-2 original and Omicron BA.1 strains were induced after a single injection of the mRNA-LNP vaccine. After 21 days, the mean titers of binding antibodies to the RBD of the original and Omicron BA.1 strains in the low-dose group reached 51,200 and 14,368, respectively. The high-dose group had higher antibody titers against both strains (114,940 and 36,204, respectively) than the low-dose group, demonstrating a clear dose-dependent immune effect. After a second booster injection, serum antibody titers further increased. Specifically, on day 28, the mean titers of binding antibodies against the RBD of the original strain and the RBD of the Omicron BA.1 strain in the low-dose group reached 2,919,297 and 579,262, respectively. Finally, on day 28, the antibody titers against both strains in the high-dose group were higher than those in the low-dose group (>3,276,800 and 1,638,400, respectively), confirming that a favorable dose-dependent immune response was maintained.

[0325] The above results indicate that the mRNA-LNP vaccine stimulates high titers of antigen-specific binding antibodies and exhibits strong humoral immunogenicity. The antibody titers induced by the mRNA-LNP vaccine were dose-dependent, with the titers of binding antibodies further increasing after the second injection.

[0326] SARS-CoV-2 pseudotype virus neutralizing antibodies: At the end of the study (D28), 7 days after the booster immunization, serum was collected from mice for neutralizing antibody testing. Neutralizing antibody titers against six SARS-CoV-2 pseudotype viruses (original, beta, delta, Omicron BA.1, Omicron BA.2.12.1, and Omicron BA.4 / .5) were measured. The results of neutralizing antibody titers (IC50, the titer at which 50% of pseudotype viruses are neutralized) are summarized in Table 4 and Figure 9. Under the study conditions, strong neutralizing antibody titers against all six pseudotype viruses were detected in all treated mice on day 28.

[0327] [Table 4]

[0328] The results show that the mRNA-LNP vaccine induced high-titer neutralizing antibodies against six SARS-CoV-2 pseudotyped viruses, including the original strain, beta strain, delta strain, Omicron BA.1 strain, Omicron BA.2.12.1 strain, and Omicron BA.4 / 5 strain, and showed a clear dose-dependent trend.

[0329] Combined with antigen-specific antibody titers and neutralizing activity, mRNA-LNP vaccines can stimulate broad protective humoral immunity against a range of targeted SARS-CoV-2 variants.

[0330] T cell responses: To evaluate the ability of mRNA-LNP vaccines to stimulate cellular immunity, the percentage of IFN-γ-producing T cells was measured by ICS in splenocytes isolated from mice immunized with the mRNA-LNP vaccine after overstimulation with an antigen peptide pool (15-mer peptides with 11 amino acid overlaps containing the full-length antigen sequence). As shown in Figure 10, in the low-dose group, on day 28, the percentage of IFN-γ-producing T cells in total splenocytes was 1.152% after stimulation with the antigen peptide pool and 0.278% after stimulation with a designated peptide pool containing only peptides with the L452R and F486V mutations (15-mer peptides with 11 amino acid overlaps containing the L452R and F486V mutations, a total of seven peptides). The percentage of IFN-γ-producing T cells in the high-dose group was 4.877% and 0.267%, respectively, showing a dose-dependent trend, comparable to or higher than that in the low-dose group.

[0331] The results showed that the mRNA-LNP vaccine can induce a significant cellular immune response in mice, as IFN-γ-producing T lymphocytes significantly increased upon stimulation with the antigen peptide pool, and the stimulated cellular immune response showed a dose-dependent tendency, indicating that the mRNA-LNP can induce a cellular immune response specific to the Omicron BA.4 / 5 strain.

[0332] conclusion These results demonstrate that immunization with the mRNA-LNP vaccine elicited enhanced antigen-specific binding antibody titers against the SARS-CoV-2 original strain and Omicron BA.1 strain, and that immunization with the mRNA-LNP vaccine enhanced potent pseudotype virus-neutralizing antibody titers against the original strain, beta strain, delta strain, Omicron BA.1 strain, Omicron BA.2.12.1 strain, and Omicron BA.4 / .5 strain, demonstrating significant cross-strain neutralizing activity against the targeted SARS-CoV-2 coronavirus variants. Immunization with the mRNA-LNP vaccine elicited potent T cell responses, as indicated by a significant increase in the frequency of IFN-γ-secreting T cells, and enhanced the cellular immune function of splenic T lymphocytes in immunized mice. Overall, mRNA-LNPs not only elicited strong cross-strain humoral immune responses against various targeted SARS-CoV-2 variants, but also generated strong cellular immunity, demonstrating that mRNA-LNP vaccines are broadly protective against primary SARS-CoV-2 infection and / or disease.

[0333] Example 11. Evaluation of in vivo immunogenicity of various mRNA-LNP vaccines in mice This example evaluates the in vivo efficacy of mRNA vaccines encoding viral antigens.

[0334] antigen-specific antibody reaction Blood was collected from the retroorbital sinus of immunized mice, and serum was prepared. Antibody binding titers against the SARS-CoV-2 spike protein RBD-His-tagged (RBD-BA.1) or SARS-CoV-2 spike protein RBD-His & Avi-tagged (RBD-WT) were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, 100 μL of RBD-BA.1 or RBD-WT antigen diluted in bicarbonate buffer was precoated onto a 96-well clear polystyrene microplate overnight at 4°C. After washing three times with PBS-T (0.05% Tween-20 in PBS), the coated plate was blocked with 300 μL of blocking buffer (15% normal goat serum and 2% bovine serum albumin in PBS-T) for 1 h at 37°C. Serum samples were serially diluted 2-fold in blocking buffer, transferred to the plate, and incubated for 1 h at 37°C. After washing, the plate was incubated with HRP-conjugated rabbit anti-mouse IgG H+L for 1 h at 37°C. The plate was washed and incubated with TMB single-component substrate solution for 7 min at 37°C, and the reaction was stopped with ELISA stop solution. Absorbance was read at 450 nm on a microplate reader, and ELISA titers were determined using nonlinear 4-parameter variable slope analysis in GraphPad Prism 8 software.

[0335] Determination of HAI titers The HAI procedure was derived from the WHO Manual for Laboratory Diagnosis and Virological Surveillance of Influenza. One volume of aliquot serum was incubated overnight at 37°C with three volumes of receptor-destroying enzyme II (RDE II) reconstituted in 0.85% NaCl, followed by heat inactivation at 56°C for 30 minutes the following day. RDE-treated serum was stored at 4°C. Red blood cells (RBCs) were washed to a final working concentration of 0.8%. Prior to the HAI assay, HAU tests (titer titration and back titration) were performed for each influenza virus by adding serially (2-fold) diluted virus and 50 μL of RBCs to a 96-well plate format. After mixing, the mixture was incubated at room temperature for 30 minutes, and the plate was tilted approximately 90 degrees. Agglutination and titer results were recorded and confirmed by back titration assay. For the HAI test, RDE-treated serum was serially (2-fold) diluted in a 96-well plate and mixed with 8 HAU of virus and 50 μL of RBCs. After 30 minutes of incubation, the plate was tilted and the results recorded.

[0336] result Preparation and characterization of mRNA-LNP vaccines After thawing, the mRNA-LNP vaccine appeared as a slightly milky dispersion, which is the appearance of a typical mRNA-LNP product.

[0337] In addition to appearance, the main pharmaceutical properties of the LNP-mRNA vaccines (different structures) were measured and are shown in Table 5. The particle size range of the four different structures was 106 nm to 124 nm, and the purity was approximately 95% ± 2%.

[0338] [Table 5]

[0339] Efficacy of mRNA-LNP vaccines (different structures) in mice The efficacy of three different mRNA-LNP vaccine structures (Structures 1-3) was compared in vivo. Compared with the original rigid short linker (SEQ ID NO: 5) of Structure-1, the flexible long linker (SEQ ID NO: 6) of Structure-2 connects the RBD (first antigen) of BA.1 (SEQ ID NO: 3) and the WT RBD (second antigen) (SEQ ID NO: 4). Furthermore, in Structure-3, the positions of the WT RBD (first antigen) and the BA.1 RBD (second antigen) sequences are swapped (Figure 12).

[0340] For COVID-19 vaccines, in vivo studies were performed by measuring antibody responses in BALB / c mice vaccinated with mRNA-LNP vaccines (Structure-1, Structure-2, or Structure-3) encoding antigens from the WT RBD and BA.1 RBD. Mice received a primary injection (day 0) and a booster 3 weeks later (day 21). Serum was collected 1 week after the booster (day 28).

[0341] Antibody titers correlate highly with the protective effect and durability induced by the vaccine. Therefore, in this study, they were used as a readout of efficacy and are expressed in the form of geometric mean titers (GMTs). Figure 12 shows the GMT results for long and short linkers and the efficacy of different GOI antigen configurations. Compared to Structure-1, Structure-2, which has a longer linker between the first antigen and the T4 foldon, was able to generate high antibody titers against both antigens, indicating that linker length does not affect the efficacy of the construct. The adoption of a longer, more flexible sequence in Structure-2 does not result in a decrease in antigen expression levels or induced antibody titers. Furthermore, the GMTs of Structure-3 against both WT-RBD and BA.1-RBD showed antibody levels similar to those of Structure-1. This indicates that changing the configuration of the GOI antigen within the design does not affect the final protein expression and the induced antibody immune protection. Therefore, the designs of Structure-2 and Structure-3 demonstrate broad adaptability to internal elements such as linker types and GOI placement for different virus variants.

[0342] HAI titers of strain-specific mouse sera To verify the suitability of this new structural platform, we designed construct-4, which encodes PR8 and H3N2 virus antigens for influenza vaccines instead of coronaviruses. Three constructs for influenza vaccines were designed to evaluate HAI titer levels, including the traditional PR8 vaccine construct (Structure-5), the H3N2 vaccine construct (Structure-6), and the combined PR8 antigen (SEQ ID NO:7) and H3N2 antigen (SEQ ID NO:8) construct (Figure 13). Unlike the schematic diagrams of coronavirus sequences, the signal peptides (sp) are not shown in the construct schematics because they are located within the PR8 or H3N2 antigen sequences.

[0343] After vaccination on days 0 and 21, mouse sera were collected on day 28, treated with RDE, and then incubated with each viral antigen and RBCs. Hemagglutination inhibition (HAI) levels were measured. The results are shown in Figure 13. The PR8 vaccine (Structure-5) and H3N2 vaccine (Structure-6), which have conventional mRNA constructs (Figure 13) and serve as positive controls, yield relatively low HAI titers of 1600 and 2425 against PR8 and H3N2 antigens, respectively. The third design, Structure-4, encodes both PR8 and H3N2 antigens in the same vector and yielded HAI titers of 2560 and 3200 against PR8 and H3N2 antigens, respectively (Figure 13). This result demonstrates that Structure-4 is highly compatible with different antigen GOIs without affecting the expression of individual antigens. Indeed, the HAI titers against both PR8 and H3N2 are higher than those of conventional single-GOI mRNA vectors. Thus, the multivariate RNA vector, Construct-4, can be used to express different viral variant antigens without compromising the expression of individual antigens within the new construct.

[0344] [Table 6-1]

[0345] [Table 6-2]

[0346] Table 6-3

[0347] Table 6-4

[0348] Table 6-5

[0349] Table 6-6

[0350] Table 6-7

[0351] Table 6-8

[0352] Table 6-9

[0353] Table 6-10

[0354] Table 6-11

[0355] Table 6-12

[0356] Table 6-13

[0357] Table 6-14

[0358] Table 6-15

[0359] Table 6-16

[0360] Table 6-17

[0361] Table 6-18

[0362] Table 6-19

[0363] Table 6-20

[0364] Table 6-21

[0365] Table 6-22

[0366] [Table 6-23]

[0367] [Table 6-24]

[0368] [Table 6-25]

[0369] [Table 6-26]

[0370] [Table 6-27]

[0371] [Table 6-28]

[0372] [Table 6-29]

[0373] [Table 6-30]

[0374] Incorporation by Reference The entire disclosure of each patent document and scientific article cited herein is incorporated by reference for all purposes.

[0375] equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure described herein. The scope of the present disclosure is, therefore, indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced within the scope of the present invention.

Claims

1. 1. A purified ribonucleic acid (RNA) molecule comprising: a) a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from a first virus variant; b) a second nucleotide sequence encoding a multimerization domain; and c) a third nucleotide sequence encoding a second antigen or antigenic fragment thereof from a second virus variant; wherein the first virus mutant and the second virus mutant are different; the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5' to 3' direction; Purified RNA molecules.

2. The RNA is messenger RNA (mRNA). The purified RNA of claim 1.

3. the RNA is self-replicating RNA; The purified RNA of claim 1.

4. the RNA is not self-replicating RNA; The purified RNA of claim 1.

5. The virus is selected from the group consisting of influenza virus, rabies virus, respiratory syncytial virus (RSV), and coronavirus. The purified RNA according to any one of claims 1 to 4.

6. The virus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); The purified RNA according to any one of claims 1 to 5.

7. The virus is SARS-CoV-2. The purified RNA of claim 6.

8. the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu-1, alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, and Omicron strains; The purified RNA of claim 6.

9. the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu-1 and Delta strains; The purified RNA of claim 8.

10. The first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a viral envelope protein, a viral spike protein, a viral membrane protein, or a viral capsid protein. The purified RNA of claim 9.

11. the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a viral spike protein; The purified RNA of claim 9.

12. the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a receptor binding domain (RBD); The purified RNA of claim 9.

13. the RBD domain is an RBD domain from a Wuhan-Hu-1 mutant or a delta mutant; The purified RNA of claim 12.

14. the first antigen or antigenic fragment thereof comprises an RBD domain derived from the delta mutant, and the second antigen or antigenic fragment thereof comprises an RBD domain of the Wuhan-Hu-1 mutant; The purified RNA of claim 13.

15. The virus is an influenza A virus. The purified RNA according to any one of claims 1 to 5.

16. the influenza A virus is selected from the group consisting of H1N1 (PR8), H2N2, H1N2, H3N2 (Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; The purified RNA of claim 15.

17. the first virus variant or the second virus variant is selected from the group consisting of H1N1(PR8) and H3N2(Udorn); 17. The purified RNA of claim 16.

18. the first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises an RNA polymerase subunit, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein NS1, or nonstructural protein NEP; 18. The purified RNA of claim 17.

19. The first antigen or antigenic fragment thereof, or the second antigen or antigenic fragment thereof, comprises a hemagglutinin (HA) protein.

19. The purified RNA of claim 18.

20. The HA protein is an HA protein derived from H1N1 (PR8) or H3N2 (Udorn).

20. The purified RNA of claim 19.

21. The first antigen or antigenic fragment thereof comprises an HA protein derived from H1N1 (PR8), and the second antigen or antigenic fragment thereof comprises an HA protein derived from H3N2 (Udorn).

21. The purified RNA of claim 20.

22. The multimerization domain is selected from the group consisting of a dimerization domain, a trimerization domain, and a tetramerization domain. The purified RNA according to any one of claims 1 to 21.

23. the multimerization domain is selected from the group consisting of enterobacteriaceae phage T4, GCN4pII, GCN4-pLI, and p53; The purified RNA according to any one of claims 1 to 22.

24. The multimerization domain comprises a leucine zipper or fibritin foldon domain. The purified RNA according to any one of claims 1 to 22.

25. The multimerization domain comprises a trimerization domain. The purified RNA according to any one of claims 1 to 22.

26. The fibritin foldon domain is the trimerization domain from Enterobacteriaceae phage T4.

26. The purified RNA of claim 25.

27. The trimerization domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1; 26. The purified RNA of claim 25.

28. Further comprising a sequence encoding a first linker that links the first antigen or antigen fragment thereof to the multimerization domain. A purified RNA molecule according to any one of claims 1 to 27.

29. further comprising a sequence encoding a second linker that links the second antigen or antigen fragment thereof to the multimerization domain; A purified RNA molecule according to any one of claims 1 to 28.

30. The first linker encodes an amino acid sequence containing at least 5 to about 50 amino acids.

29. The purified RNA molecule of claim 28.

31. The second linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids.

30. The purified RNA molecule of claim 29.

32. the first linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO:9), (GS)n (SEQ ID NO:10), (GS)n (SEQ ID NO:11), (GS)n (SEQ ID NO:32), and (G)n (SEQ ID NO:33), wherein n is an integer from 2 to 20; 29. The purified RNA molecule of claim 28.

33. the second linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO:9), (GS)n (SEQ ID NO:10), (GS)n (SEQ ID NO:11), (GS)n (SEQ ID NO:32), and (G)n (SEQ ID NO:33), wherein n is an integer from 2 to 20; 30. The purified RNA molecule of claim 29.

34. the first linker encodes an amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34), or (GGSGGE)n (SEQ ID NO: 35), where n is an integer from 2 to 6; 29. The purified RNA molecule of claim 28.

35. the second linker encodes an amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO:34), or (GGSGGE)n (SEQ ID NO:35), where n is an integer from 2 to 6; 30. The purified RNA molecule of claim 29.

36. the first linker encodes an amino acid sequence selected from the group consisting of (GGGSGSGGGGGS)n (SEQ ID NO:36) and (GGGGGPGGGGP)n (SEQ ID NO:37), where n is an integer from 1 to 3; 29. The purified RNA molecule of claim 28.

37. the second linker encodes an amino acid sequence selected from the group consisting of (GGGSGSGGGGGS)n (SEQ ID NO:36), and (GGGGGPGGGGP)n (SEQ ID NO:37), where n is an integer from 1 to 3; 30. The purified RNA molecule of claim 29.

38. the first linker encodes an amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8; 29. The purified RNA molecule of claim 28.

39. the second linker encodes an amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is 1 to 20 and n is at least 8; 30. The purified RNA molecule of claim 29.

40. X is serine, aspartic acid, glutamic acid, threonine, or proline; 40. A purified RNA molecule according to claim 38 or 39.

41. The first linker encodes GGSG (SEQ ID NO: 38), 29. The purified RNA of claim 28.

42. The second linker encodes GGSG (SEQ ID NO: 38).

30. The purified RNA of claim 29.

43. The first linker encodes GGSLGGGGGSGS (SEQ ID NO: 39), 29. The purified RNA of claim 28.

44. The second linker encodes GGSLGGGGGSGS (SEQ ID NO: 39), 30. The purified RNA of claim 29.

45. The first linker is encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6; 29. The purified RNA of claim 28.

46. The second linker is encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

30. The purified RNA of claim 29.

47. The first antigen or antigen fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NOs: 3 to 4, 7, and 8. The purified RNA according to any one of claims 1 to 46.

48. The second antigen or antigen fragment thereof is encoded by a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NOs: 3 to 4, 7, and 8. The purified RNA according to any one of claims 1 to 47.

49. The first antigen or antigen fragment thereof comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19, 20, 22, and 23; The purified RNA according to any one of claims 1 to 48.

50. The second antigen or antigen fragment thereof comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19, 20, 22, and 23; The purified RNA according to any one of claims 1 to 49.

51. The purified RNA further comprises m7GpppNm- (where Nm represents any nucleotide having a 2'O-methylation (Cap1) at the 5' end of the first nucleotide sequence); The purified RNA of any one of claims 1 to 50.

52. the purified RNA further comprises a 5' UTR at the 3' end of Cap1 and the 5' end of the first nucleotide sequence; 52. The purified RNA of claim 51.

53. the purified RNA further comprises a sequence encoding a signal peptide at the 3' end of the 5' UTR and the 5' end of the first nucleotide sequence; 53. The purified RNA of claim 52.

54. the purified RNA further comprises a sequence encoding a 3' UTR at the 3' end of the second nucleotide sequence; 54. The purified RNA of claim 53.

55. A sequence having at least 90% sequence identity with any one of SEQ ID NOs: 26 to 31; Purified RNA molecules.

56. comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 26; Purified RNA molecules.

57. comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 27; Purified RNA molecules.

58. comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 28; Purified RNA molecules.

59. comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 29; Purified RNA molecules.

60. comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 30; Purified RNA molecules.

61. comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 31; Purified RNA molecules.

62. A purified RNA molecule comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding a first antigen or antigenic fragment thereof from a wild-type SARS-CoV-2 virus or a first virus variant selected from the group consisting of a Wuhan-Hu-1 variant, an Omicron variant, and a Delta variant; e) a third nucleotide sequence encoding a first linker; and f) a fourth nucleotide sequence encoding a T4-foldon domain; and g) a fifth nucleotide sequence encoding a second linker; and h) a sixth nucleotide sequence encoding a second antigen or antigenic fragment thereof from a wild-type SARS-CoV-2 virus or a second virus variant selected from the group consisting of a Wuhan-Hu-1 variant, an Omicron variant, and a Delta variant; i) a 3′UTR; and j) a poly A tail; Purified RNA molecules.

63. Purified RNA comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding the first antigen or antigenic fragment thereof from an Omicron variant; e) a third nucleotide sequence encoding a first linker; and f) a fourth nucleotide sequence encoding a T4-foldon domain; and g) a fifth nucleotide sequence encoding a second linker; and h) a sixth nucleotide sequence encoding a second antigen or antigenic fragment thereof from a wild-type SARS-CoV-2 virus; and i) a 3′UTR; and j) a poly A tail; Purified RNA.

64. Purified RNA comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a first nucleotide sequence encoding a signal peptide; d) a second nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from a wild-type SARS-CoV-2 virus; and e) a third nucleotide sequence encoding a first linker; and f) a fourth nucleotide sequence encoding a T4-foldon domain; and g) a fifth nucleotide sequence encoding a second linker; and h) a sixth nucleotide sequence encoding a second antigen or antigenic fragment thereof from an Omicron variant; i) a 3′UTR; and j) a poly A tail; Purified RNA.

65. Purified RNA comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a first nucleotide sequence encoding a first antigen or antigenic fragment thereof derived from an influenza virus selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; d) optionally, a second nucleotide sequence encoding a first linker; and e) optionally, a third nucleotide sequence encoding a T4-foldon domain; and f) optionally, a fourth nucleotide sequence encoding a second linker; and g) optionally a fifth nucleotide sequence encoding a second antigen or antigenic fragment thereof derived from an influenza virus selected from the group consisting of H1N1(PR8), H2N2, H1N2, H3N2(Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; and h) a 3'UTR; and i) a polyA tail, Purified RNA.

66. Purified RNA comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a first nucleotide sequence encoding a first antigen or antigenic fragment thereof from an H1N1(PR8) influenza virus; d) a second nucleotide sequence encoding a first linker; and e) a third nucleotide sequence encoding a T4-foldon domain; and f) a fourth nucleotide sequence encoding a second linker; and g) a fifth nucleotide sequence encoding a second antigen or antigenic fragment thereof from an H3N2 (Udorn) influenza virus; and h) a 3'UTR; and i) a polyA tail, Purified RNA.

67. Purified RNA comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a nucleotide sequence encoding a first antigen or an antigenic fragment thereof derived from an H1N1(PR8) influenza virus; d) a 3′UTR; and e) a polyA tail; Purified RNA.

68. Purified RNA comprising, from the 5' to the 3' end: a) m7GpppNm- (where Nm represents any nucleotide with 2'O methylation (Cap1)); b) a 5'UTR; and c) a nucleotide sequence encoding a first antigen or an antigenic fragment thereof from an H3N2 (Udorn) influenza virus; d) a 3′UTR; and e) a polyA tail; Purified RNA.

69. 69. An isolated polypeptide encoded by the purified RNA molecule of any one of claims 1 to 68.

70. 69. A composition comprising the purified RNA molecule of any one of claims 1 to 68 and a delivery vehicle.

71. The delivery vehicle comprises a lipid nanoparticle (LNP).

71. The composition of claim 70.

72. The LNP comprises an ionized lipid.

72. The composition of claim 71.

73. The LNPs comprise an ionizable lipid:cholesterol:DSPC:DMG-PEG2000 ratio of about 48:40:10:2, and an LNP:RNA (N:P) ratio of about 8:

1.

73. The composition of claim 72.

74. the composition has a particle size of about 300 nanometers (nm) or less; The composition according to any one of claims 70 to 73.

75. the composition has a particle size of about 50 to about 300 nm; The composition according to any one of claims 70 to 73.

76. The composition has a particle size of about 150 nm or less. The composition according to any one of claims 70 to 73.

77. the composition has a particle size of about 50 nm to about 140 nm; The composition according to any one of claims 70 to 73.

78. The RNA is lyophilized. The composition according to any one of claims 70 to 77.

79. The RNA is adsorbed to the surface of the LNP.

79. The composition of claim 78.

80. The RNA is encapsulated by LNPs. The composition of any one of claims 70 to 78.

81. The RNA encapsulated by the LNPs is lyophilized.

81. The composition of claim 80.

82. 69. A kit comprising the purified RNA of any one of claims 1 to 68, a delivery vehicle, and instructions.

83. A method for treating or preventing a disease in a subject, comprising administering to the subject an effective amount of a composition according to any one of claims 70 to 81. method.

84. The disease is caused by a virus selected from the group consisting of influenza virus, rabies virus, respiratory syncytial virus (RSV), and coronavirus; 84. The method of claim 83.

85. The disease is caused by a coronavirus or an influenza A virus.

84. The method of claim 83.

86. The coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); 86. The method of claim 85.

87. The coronavirus is SARS-CoV-2.

87. The method of claim 86.

88. the influenza A virus is selected from the group consisting of H1N1 (PR8), H2N2, H1N2, H3N2 (Udorn), N2-NA, Wyo03, PC73, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N7, and H10N3; 86. The method of claim 85.

89. the influenza A virus is H1N1 (PR8) or H3N2 (Udorn); 86. The method of claim 85.

90. The composition is injected intramuscularly into the subject.

90. The method of any one of claims 83 to 89.

91. The composition is administered to the subject at least twice.

91. The method of any one of claims 83 to 90.

92. The composition is administered once every one, two, three, or four weeks.

92. The method of claim 91.

93. 82. A method of stimulating an immune response in a subject, comprising administering to the subject an effective amount of a composition according to any one of claims 70 to 81. method.

94. A method for preparing an RNA-LNP composition, comprising: a) mixing an ethanol phase comprising one or more lipids with an aqueous phase comprising the purified RNA molecule of any one of claims 1 to 62; b) purifying the RNA-LNPs produced from step a); method.

95. the one or more lipids comprise an ionizable lipid; 95. The method of claim 94.

96. the one or more lipids include cholesterol; 96. The method according to any one of claims 94 to 95.

97. the one or more lipids comprise a phospholipid; 97. The method of any one of claims 94 to 96.

98. the one or more lipids are PEGylated; 98. The method of any one of claims 94 to 97.

99. the N:P ratio is from about 6.5 to about 9; 99. The method of any one of claims 94 to 98.

100. The aqueous phase consists of Tris, sodium chloride, and sucrose.

100. The method of any one of claims 94 to 99.

101. 82. A kit comprising the composition of any one of claims 70 to 81, a delivery vehicle, and instructions.

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