Methods and compositions for quadrivalent influenza vaccines

Abstract

Provided herein are RNA molecules encoding viral replication proteins and antigenic proteins or fragments thereof. Also provided herein are compositions comprising RNA molecules encoding viral replication proteins and antigenic proteins or fragments thereof and lipids. The RNA molecules and compositions comprising the same are useful for inducing an immune response. The present disclosure provides RNA molecules useful for inducing an immune response. Both self-replicating RNA molecules and messenger RNA (mRNA) molecules, as well as polynucleotides encoding them, are provided.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 482,560, filed January 31, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporating a sequence listing The sequence listing entitled 207653-603601_SL.xml, created on January 8, 2024, and having a size of 367,117 bytes, is incorporated herein by reference in its entirety.

[0003] The present disclosure relates generally to inducing an immune response against infectious agents such as influenza viruses, and more specifically to RNA molecules and liponanoparticles as vaccines. [Background technology]

[0004] Influenza viruses cause epidemics almost every winter. Influenza infection results in a range of disease states, from asymptomatic infection to severe viral pneumonia. The severity of the disease is influenced by the age of the host, its immune status, and / or the site of infection. Vaccination plays an important role in controlling seasonal influenza epidemics.

[0005] Self-replicating ribonucleic acid (RNA), such as RNA derived from viral replicons, and messenger RNA (mRNA), are useful for expressing proteins, including heterologous proteins, for various purposes, such as the expression of therapeutic proteins and antigens for vaccines. A desirable property of replicons is their ability to sustainably express proteins. Few treatments are available for infections caused by viruses and eukaryotes, and resistance to antibiotics for the treatment of bacterial infections is increasing. Furthermore, rapid responses, including rapid vaccine development, are required to effectively control emerging infectious diseases and pandemics. Therefore, there is a need for prevention and / or treatment of infectious diseases, such as influenza. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides RNA molecules useful for inducing an immune response. Both self-replicating RNA molecules and messenger RNA (mRNA) molecules, as well as the polynucleotides that encode them, are provided.

[0007] In some aspects, provided herein are compositions comprising one or more RNA molecules, wherein the one or more RNA molecules collectively encode a hemagglutinin (HA) polypeptide and a neuraminidase (NA) polypeptide for each of four different influenza virus strains. In some embodiments, the one or more RNA molecules comprise eight RNA molecules (e.g., one for each NA and one for each HA). In some embodiments, two or more influenza polypeptides (e.g., two HAs, two NAs, or an HA and an NA) are encoded on a single RNA molecule such that fewer than eight RNA molecules encode the HA and NA polypeptides of the four different strains (e.g., seven, six, five, four, three, or two RNA molecules). In some embodiments, the HA and NA polypeptides are encoded by the same RNA molecule for each of the four influenza virus strains. In some embodiments, the HA and NA polypeptides of the four different strains are all encoded on the same RNA molecule.

[0008] In some embodiments, the HA and NA polypeptides of a first influenza virus strain are encoded by a first RNA molecule, the HA and NA polypeptides of a second influenza virus strain are encoded by a second RNA molecule, the HA and NA polypeptides of a third influenza virus strain are encoded by a third RNA molecule, and the HA and NA polypeptides of a fourth influenza virus strain are encoded by a fourth RNA molecule. In some embodiments, the first, second, third, and fourth RNA molecules are present in an equimolar ratio.

[0009] In some embodiments, each of the one or more RNA molecules further encodes one or more viral replication proteins. In still further embodiments, the one or more viral replication proteins are alphavirus proteins. In some embodiments, each of the one or more RNA molecules encodes, in 5' to 3' order, (i) one or more viral replication proteins, (ii) one of the NA polypeptides, and (iii) one of the HA polypeptides. In further embodiments, the alphavirus proteins are derived from Venezuelan Equine Encephalitis Virus (VEEV). In some embodiments, the one or more viral replication proteins comprise alphavirus nonstructural protein 1 (nsP1), alphavirus nonstructural protein 2 (nsP2), alphavirus nonstructural protein 3 (nsP3), alphavirus nonstructural protein 4 (nsP4), or any combination thereof. In some embodiments, the one or more viral replication proteins comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the RNA sequence encoded by SEQ ID NO:13.

[0010] In some embodiments, the sequences encoding at least one HA polypeptide and NA polypeptide of the one or more RNA molecules are preceded by a subgenomic promoter (sgP). In some embodiments, each HA polypeptide comprises an antigenic fragment of its respective HA protein. In yet further embodiments, each NA polypeptide comprises an antigenic fragment of its respective NA protein. In some embodiments, the four different influenza virus strains comprise one or more of H1N1, H3N2, Victoria-B, or Yamagata-B. In further embodiments, the four different influenza virus strains comprise Victoria B / Austria / 1359417 / 2021, H3N2 A / Darwin / 6 / 2021, H1N1 A / Wisconsin / 588 / 2019, and Yamagata B / PHUKET / 3073 / 2013.

[0011] In some embodiments, each of the one or more RNA molecules further comprises a 5' untranslated region (UTR). In some embodiments, at least one 5' UTR comprises a viral 5' UTR, a non-viral 5' UTR, or a combination of viral and non-viral 5' UTR sequences. In further embodiments, at least one 5' UTR comprises an alphavirus 5' UTR. In yet further embodiments, the alphavirus 5' UTR comprises a Venezuelan Equine Encephalitis Virus (VEEV) 5' UTR sequence. In some embodiments, at least one 5' UTR comprises an RNA sequence encoded by SEQ ID NO: 14. In some embodiments, each of the one or more RNA molecules further comprises a 3' untranslated region (UTR). In further embodiments, at least one 3' UTR comprises a viral 3' UTR, a non-viral 3' UTR, or a combination of viral and non-viral 3' UTR sequences. In yet further embodiments, at least one 3' UTR comprises an alphavirus 3' UTR sequence. In some embodiments, the alphavirus 3'UTR comprises a Venezuelan Equine Encephalitis Virus (VEEV) 3'UTR sequence. In some embodiments, at least one 3'UTR comprises an RNA sequence encoded by SEQ ID NO: 15. In some embodiments, the RNA molecule further comprises a polyA tail.

[0012] In some embodiments, the one or more RNA molecules are self-replicating RNA molecules. In further embodiments, the one or more RNA molecules comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4.

[0013] In some aspects, provided herein are compositions comprising one or more DNA molecules encoding one or more RNA molecules of any of the compositions disclosed herein. In some embodiments, each of the one or more DNA molecules comprises a promoter. In some embodiments, the promoter of each of the one or more DNA molecules is located 5' of the 5'UTR. In further embodiments, the promoter is a T7 promoter.

[0014] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 5. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 9.

[0015] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 6. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 10.

[0016] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 7. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 11.

[0017] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 8. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO: 12.

[0018] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:9.

[0019] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:10.

[0020] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:11.

[0021] In some aspects, the disclosure provides a composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:12.

[0022] In some aspects, the present disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4.

[0023] In some embodiments, the compositions disclosed herein further comprise an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid has the structure of Formula I:

[0024] [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 and R 6 are each independently a straight-chain or branched-chain C 1~ C 31 Alkyl, C 2~ C 31 Alkenyl or C 2~ C 31 L is selected from the group consisting of alkynyl and cholesteryl; 5 and L6 are each independently a linear C 1~ C 20 Alkyl and C 2~ C 20 alkenyl; and X 5 is -C(O)O-, thereby -C(O)OR 6 is formed, or -OC(O)-, whereby -OC(O)-R 6 is formed, and X 6 is -C(O)O-, thereby -C(O)OR 5 is formed, or -OC(O)-, whereby -OC(O)-R 5 is formed, and X 7 is S or O, and L 7 is absent or is lower alkyl, and R 4 is a linear or branched chain C 1~ C6 alkyl, and R 7 and R 8 are each independently hydrogen and a straight or branched chain C 1~ C6 alkyl.

[0025] In some embodiments, the ionizable cationic lipid is selected from Table 6 herein.

[0026] In some embodiments, the ionizable cationic lipid is ATX-126.

[0027] [ka]

[0028] In some embodiments, the ionizable cationic lipid is ATX-240.

[0029] [ka]

[0030] In some embodiments of compositions comprising an ionizable cationic lipid disclosed herein, the composition comprises a nitrogen to phosphate ratio (N:P) of about 5:1 to 7:1.

[0031] In one aspect, provided herein is a method of vaccinating a subject against influenza, the method comprising administering to the subject any of the compositions disclosed herein.

[0032] In one aspect, the present disclosure provides for the use of any of the compositions disclosed herein in the preparation of a medicament for vaccinating a subject against influenza.

[0033] In one aspect, the disclosure provides a composition comprising: (i) a polynucleotide having a length of about 5,000 to about 20,000 nucleotides; and (ii) an ionizable cationic lipid, wherein the composition comprises a nitrogen-to-phosphate (N:P) ratio of about 5:1 to about 7:1. In some embodiments, the nitrogen-to-phosphate (N:P) ratio is about 7:1.

[0034] In some embodiments, the RNA molecules or compositions described herein are used to induce an immune response against any of the antigens disclosed herein.

[0035] In some embodiments, the RNA molecules or compositions described herein are used in the manufacture of a medicament for inducing an immune response to any of the antigens disclosed herein. [Brief explanation of the drawings]

[0036] [Figure 1]1 shows a schematic diagram of various saRNA constructs for expression of neuraminidase (NA) and hemagglutinin (HA) antigens, according to some embodiments. Each of the depicted constructs contains one or more IRESs from coxsackievirus B3 (CVB3), encephalomyocarditis virus (EMCV), or enterovirus 71 (EV71), as indicated. [Figure 2] A general schematic diagram of an exemplary tetravalent seasonal influenza vaccine is shown, which contains four separate RNAs, each encoding a replicase backbone, a subgenomic promoter (sgP), and neuraminidase (NA) and hemagglutinin (HA) antigens from the same respective influenza strain. [Figure 3A] 1 shows exemplary results for hemagglutin (HA) inhibitory titers in the serum of vaccinated female BALB / c mice. [Figure 3B] 1 shows exemplary results for hemagglutin (HA) inhibitory titers in the serum of vaccinated female BALB / c mice. [Figure 4] 1 shows exemplary results of a Meso Scale Discovery (MSD) assay showing levels of HA-specific IgG in the serum of female BALB / c mice 28 days after vaccination with an N1-pseudouridine-modified mRNA vaccine encoding HA from the H3N2 A / Cambodia / e0826360 / 2020 influenza strain using two different constructs containing CODEX and hCAI codon optimization. [Figure 5A] Exemplary levels (AU / mL) of HA-specific binding IgG antibodies in vaccinated female BALB / c mice are shown. [Figure 5B] Exemplary levels (AU / mL) of HA-specific binding IgG antibodies in vaccinated female BALB / c mice are shown. [Figure 5C]Exemplary levels (AU / mL) of HA-specific binding IgG antibodies in vaccinated female BALB / c mice are shown. [Figure 5D] Exemplary levels (AU / mL) of HA-specific binding IgG antibodies in vaccinated female BALB / c mice are shown. [Figure 6A] 1 shows exemplary results for weight loss and immunogenicity in vaccinated female BALB / c mice. [Figure 6B] 1 shows exemplary results for weight loss and immunogenicity in vaccinated female BALB / c mice. [Figure 7A] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7B] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7C] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7D] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7E] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7F] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7G] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 7H] 1 shows exemplary results of tolerability and / or immunogenicity in animals vaccinated with various vaccine compositions. [Figure 8A] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8B]1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8C] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8D] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8E] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8F] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8G] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 8H] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 9A] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 9B] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 9C] 1 shows exemplary results for the immunogenicity of vaccines with different designs. [Figure 9D] 1 shows exemplary results for the immunogenicity of vaccines with different designs. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure relates to RNA, e.g., self-replicating RNA and messenger RNA (mRNA), and the nucleic acids encoding them, for expression of transgenes, e.g., antigenic proteins. Also provided herein are methods of administering RNA (e.g., to a host, e.g., a mammalian subject), whereby the RNA is translated in vivo and a heterologous protein-coding sequence is expressed, e.g., which may elicit an immune response in the recipient against the heterologous protein-coding sequence or provide a therapeutic effect including induction of an immune response, where the heterologous protein-coding sequence is a therapeutic protein or an antigenic protein. The RNA provided herein, e.g., self-replicating RNA and messenger RNA (mRNA), is useful as a vaccine that can be rapidly produced and may be effective at low and / or single doses. The present disclosure further relates to methods of inducing an immune response using the RNA provided herein.

[0038] In some embodiments, the immune response can be elicited against influenza, including, but not limited to, immunogens derived from influenza A strains (e.g., H1N1 or H3N2) or influenza B strains (e.g., Victoria or Yamagata).

[0039] Self-replicating RNAs are described, for example, in U.S. Patent Application Publication No. 2018 / 0036398, the contents of which are incorporated herein by reference in their entirety.

[0040] definition As used herein, the term "fragment," when referring to a protein or nucleic acid, means, for example, any sequence that is shorter than the full-length protein or nucleic acid. Thus, any sequence of a nucleic acid or protein other than the full-length nucleic acid or protein sequence can be a fragment. In some aspects, a protein fragment comprises an epitope. In other aspects, a protein fragment is an epitope.

[0041] As used herein, the term "nucleic acid" refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analog. DNA or RNA molecules can be double-stranded or single-stranded and can be of any size. Exemplary nucleic acids include, but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), mitochondrial DNA, chloroplast DNA, viral DNA, mRNA, tRNA, rRNA, long non-coding RNA, siRNA, microRNA (miRNA or miR), hnRNA, and viral RNA. Exemplary nucleic acid analogs include peptide nucleic acids, morpholino and locked nucleic acids, glycol nucleic acids, and threose nucleic acids. As used herein, the term "nucleic acid molecule" is meant to include, for example, fragments of nucleic acid molecules, as well as any full-length or non-fragmented nucleic acid molecule. As used herein, the terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably unless the context clearly indicates otherwise.

[0042] As used herein, the term "polynucleotide" refers to a nucleic acid sequence comprising at least two nucleotide monomers. The term "polynucleotide" can refer to a polymer of DNA, RNA, a nucleic acid analog, or a combination thereof. A "polynucleotide" can be double-stranded or single-stranded and can be of any size. A polynucleotide can be a separate nucleic acid molecule or a portion of a nucleic acid molecule. Thus, the term "polynucleotide" can refer to a nucleic acid molecule or a region of a nucleic acid molecule.

[0043] As used herein, the term "protein" refers to any polymeric chain of amino acids. The terms "peptide" and "polypeptide" can be used interchangeably with the term protein, unless the context clearly indicates otherwise, and can also refer to a polymeric chain of amino acids. The term "protein" encompasses natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. Proteins can be monomeric or polymeric. The term "protein" encompasses fragments and variants thereof (including fragments of variants) unless contradicted by context.

[0044] Generally, "sequence identity" or "sequence homology," which may be used interchangeably, refer to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence of the polypeptide encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. As used herein, the term "percent sequence identity" or "percent identity" (which also includes "percent homology") refers to the percentage of amino acid residues or nucleotides in a sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Thus, two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity" (also referred to as "percent homology"). Percent identity to a reference sequence (e.g., a nucleic acid or amino acid sequence), which may be a sequence within a longer molecule (e.g., a polynucleotide or polypeptide), can be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, advanced BLAST computer programs, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. This program can be used to determine percent identity over the entire length of the compared sequences. Default parameters are provided to optimize searches using a short query sequence, such as the blastp program. This program also allows the use of a SEG filter to mask off segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from approximately 80% to 100% and integer values ​​therebetween. The percent identity between the reference sequence and the claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. Generally, an exact match indicates 100% identity over the length of the reference sequence.Further programs and methods for comparing sequences and / or assessing sequence identity include the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at ebi.ac.uk / Tools / psa / emboss needle / , optionally with default settings), the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner available at ebi.ac.uk / Tools / psa / emboss water / , optionally with default settings), the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Drive, Madison, Wis.). In some embodiments, references to percent sequence identity refer to sequence identity measured using BLAST (Basic Local Alignment Search Tool). In other embodiments, ClustalW is used for multiple sequence alignment. Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0045] As used herein, "homologous sequences" refers to sequences that share sequence and / or structural similarity (Pearson, 2013, An Introduction to Sequence Similarity ("Homology") Searching, Current Protocols Bioinformatics, 42:3.1.1-3.1.8). Thus, homologous sequences share a common evolutionary ancestor or are derived from a common sequence. Homologous sequences may also share structural or sequence similarity with intermediate sequences. Homologous sequences may have similar functions, i.e., functional similarity. Homology can be inferred based on nucleic acid and / or amino acid sequences, with protein similarity searches generally having higher sensitivity than nucleic acid sequence searches. Homology can also be inferred for amino acid sequences that contain similar amino acids, i.e., amino acids with similar physiochemical properties, rather than identical amino acids, at least over a region of the sequence. The terms "homologous sequence," "homologue," and "homologous nucleic acid" and / or "homologous protein" can be used interchangeably unless the context clearly indicates otherwise.

[0046] As used herein, the term "drug" or "medication" means a pharmaceutical formulation or composition described herein.

[0047] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps, of the type described herein that will become apparent to those skilled in the art upon reading this disclosure, and so forth.

[0048] As used herein, "about," when referring to a measurable value such as an amount, time period, etc., is meant to encompass a variation of ±20%, or ±10%, or ±5%, or even ±1% from the specified value, where such variation is appropriate for the disclosed method or for practicing the disclosed method.

[0049] The term "expression" refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA or other RNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and the encoded polypeptides may be collectively referred to as "gene products."

[0050] As used herein, the terms "self-replicating RNA," "self-transcribed and self-replicating RNA," "self-amplifying RNA" (saRNA or samRNA), and "replicon" may be used interchangeably unless the context clearly indicates otherwise. Generally, the term "replicon" or "viral replicon" refers to a self-replicating subgenomic RNA derived from a viral genome that contains viral genes encoding nonstructural proteins important for viral replication and lacks viral genes encoding structural proteins. A self-replicating RNA can encode an additional subgenomic RNA that is incapable of self-replicating. A self-replicating RNA may also be referred to as "STARR™" RNA.

[0051] As used herein, "operably linked," "operably linked," "operably linked," or their grammatical equivalents refer to the juxtaposition of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, etc., where the elements are in a relationship permitting them to operate in an expected manner. For example, a regulatory element, which may include a promoter and / or enhancer sequence, is operably linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. Intervening residues can be present between the regulatory element and the coding region so long as this functional relationship is maintained.

[0052] RNA molecule In some aspects, provided herein are RNA molecules collectively encoding hemagglutinin (HA) and neuraminidase (NA) polypeptides of each of four different influenza virus strains, as well as polynucleotides encoding the RNA molecules. In some embodiments, the one or more RNA molecules comprise eight RNA molecules (e.g., one for each NA and one for each HA). In some embodiments, two or more influenza polypeptides (e.g., two HAs, two NAs, or an HA and an NA) are encoded on a single RNA molecule such that fewer than eight RNA molecules encode the HA and NA polypeptides of the four different strains (e.g., seven, six, five, four, three, or two RNA molecules). In some embodiments, the HA and NA polypeptides are encoded by the same RNA molecule for each of the four influenza virus strains. In some embodiments, the HA and NA polypeptides are encoded by the same RNA molecule for each of the four different influenza virus strains.

[0053] Also provided herein is an RNA molecule for expressing an antigen, comprising a 5'UTR comprising the sequence of SEQ ID NO: 14 and a 3'UTR comprising the sequence of SEQ ID NO: 15, or a 5'UTR comprising the sequence of SEQ ID NO: 17 and a 3'UTR comprising the sequence of SEQ ID NO: 19, wherein T is replaced with U.

[0054] In some embodiments, the RNA molecule comprises a poly-A tail. The poly-A tail may be directly transcribed from a template polynucleotide or may be added post-transcriptionally. The poly-A tail may have any of a variety of lengths, such as between 20 and 300 nucleotides. In some embodiments, the poly-A tail is approximately 130 nucleotides in length. In some embodiments, the poly-A tail is encoded by a polynucleotide having the sequence of SEQ ID NO: 16. In some embodiments, the poly-A tail is encoded by a polynucleotide having the sequence of nucleotides 31-130 of SEQ ID NO: 16. In some embodiments, the poly-A tail is less than 100 nucleotides in length (e.g., between 20 and 99 nucleotides in length). In some embodiments, the poly-A tail is less than 90, 80, 75, 70, 65, 60, 55, or 50 nucleotides in length.

[0055] The RNA molecule may encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If the immunogens are expressed as separate polypeptides from a replicon, one or more of them may be provided with an upstream IRES or additional viral promoter elements. Alternatively, multiple immunogens can be expressed from a polyprotein encoding each immunogen fused to a short autocatalytic protease (e.g., foot-and-mouth disease virus 2A protein) or as inteins.

[0056]

[0010] In some aspects, provided herein are compositions comprising one or more RNA molecules collectively encoding a hemagglutinin (HA) polypeptide and a neuraminidase (NA) polypeptide for each of four different influenza virus strains. In some embodiments, the four different influenza virus strains include one or more of H1N1, H3N2, Victoria-B, or Yamagata-B. In further embodiments, the four different influenza virus strains include Victoria B / Austria / 1359417 / 2021, H3N2 A / Darwin / 6 / 2021, H1N1 A / Wisconsin / 588 / 2019, and Yamagata B / PHUKET / 3073 / 2013.

[0057] In some embodiments, the HA and NA polypeptides are encoded by the same RNA molecule for each of four different influenza virus strains. In some aspects, the HA and NA polypeptides of a first influenza virus strain are encoded by a first RNA molecule, the HA and NA polypeptides of a second influenza virus strain are encoded by a second RNA molecule, the HA and NA polypeptides of a third influenza virus strain are encoded by a third RNA molecule, and the HA and NA polypeptides of a fourth influenza virus strain are encoded by a fourth RNA molecule. In some embodiments, the first, second, third, and fourth RNA molecules are present in an equimolar ratio. In some embodiments, each of the one or more RNA molecules further encodes one or more viral replication proteins. In some embodiments, the one or more viral replication proteins are alphavirus proteins. In still further embodiments, the alphavirus proteins are derived from Venezuelan equine encephalitis virus (VEEV).

[0058] In one aspect, each of the one or more RNA molecules of any of the compositions disclosed herein encodes, in 5' to 3' order, (i) one or more viral replication proteins, (ii) one of the NA polypeptides, and (iii) one of the HA polypeptides. In some embodiments, the one or more viral replication proteins comprise alphavirus nonstructural protein 1 (nsP1), alphavirus nonstructural protein 2 (nsP2), alphavirus nonstructural protein 3 (nsP3), alphavirus nonstructural protein 4 (nsP4), or any combination thereof. In further embodiments, the one or more viral replication proteins comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the RNA sequence encoded by SEQ ID NO:13.

[0059] In some embodiments, the sequence encoding at least one HA polypeptide and NA polypeptide of the one or more RNA molecules is preceded by a subgenomic promoter (sgP).

[0060] In one embodiment, each HA polypeptide of any of the compositions disclosed herein comprises an antigenic fragment of its respective HA protein. In another embodiment, each NA polypeptide of any of the compositions disclosed herein comprises an antigenic fragment of its respective NA protein.

[0061] In some embodiments, each of the one or more RNA molecules of any of the compositions disclosed herein further comprises a 5' untranslated region (UTR). In further embodiments, at least one 5' UTR comprises a viral 5' UTR, a non-viral 5' UTR, or a combination of viral and non-viral 5' UTR sequences. In yet further embodiments, at least one 5' UTR comprises an alphavirus 5' UTR. In yet further embodiments, the alphavirus 5' UTR comprises a Venezuelan equine encephalitis virus (VEEV) 5' UTR sequence.

[0062] In some embodiments, at least one 5'UTR of any of the compositions disclosed herein comprises an RNA sequence encoded by SEQ ID NO:14.

[0063] In some embodiments, each of the one or more RNA molecules of any of the compositions disclosed herein further comprises a 3' untranslated region (UTR). In some embodiments, at least one 3'UTR comprises a viral 3'UTR, a non-viral 3'UTR, or a combination of viral and non-viral 3'UTR sequences. In further embodiments, at least one 3'UTR comprises an alphavirus 3'UTR sequence. In yet further embodiments, the alphavirus 3'UTR comprises a Venezuelan equine encephalitis virus (VEEV) 3'UTR sequence. In yet further embodiments, at least one 3'UTR comprises an RNA sequence encoded by SEQ ID NO: 15.

[0064] In some embodiments, the RNA molecule of any of the compositions disclosed herein further comprises a poly-A tail. The poly-A tail may be directly transcribed from a template polynucleotide or may be added post-transcriptionally. The poly-A tail may have any of a variety of lengths, such as between 20 and 300 nucleotides. In some embodiments, the poly-A tail is approximately 130 nucleotides in length. In some embodiments, the poly-A tail is encoded by a polynucleotide having the sequence of SEQ ID NO: 16. In some embodiments, the poly-A tail is encoded by a polynucleotide having the sequence of nucleotides 31-130 of SEQ ID NO: 16. In some embodiments, the poly-A tail is less than 100 nucleotides in length (e.g., between 20 and 99 nucleotides in length). In some embodiments, the poly-A tail is less than 90, 80, 75, 70, 65, 60, 55, or 50 nucleotides in length.

[0065] In some aspects, the RNA molecules disclosed herein are self-replicating RNA molecules. In some embodiments, the RNA molecules comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4. In some embodiments, the RNA molecule comprises a first RNA molecule having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an RNA sequence encoded by SEQ ID NO: 1. In some embodiments, the first RNA molecule has at least 90% sequence identity to the RNA sequence encoded by SEQ ID NO:1. In some embodiments, the first RNA molecule has at least 95% sequence identity to the RNA sequence encoded by SEQ ID NO:1. In some embodiments, the first RNA molecule comprises an RNA sequence encoded by SEQ ID NO:1. In some embodiments, the RNA molecule comprises a second RNA molecule that has at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO:2. In some embodiments, the second RNA molecule has at least 90% sequence identity to the RNA sequence encoded by SEQ ID NO:2. In some embodiments, the second RNA molecule has at least 95% sequence identity to the RNA sequence encoded by SEQ ID NO:2. In some embodiments, the second RNA molecule comprises an RNA sequence encoded by SEQ ID NO:2.In some embodiments, the RNA molecule comprises a third RNA molecule that has at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO:3. In some embodiments, the third RNA molecule has at least 90% sequence identity to the RNA sequence encoded by SEQ ID NO:3. In some embodiments, the third RNA molecule has at least 95% sequence identity to the RNA sequence encoded by SEQ ID NO:3. In some embodiments, the third RNA molecule comprises an RNA sequence encoded by SEQ ID NO:3. In some embodiments, the RNA molecule comprises a fourth RNA molecule that has at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO:4. In some embodiments, the fourth RNA molecule has at least 90% sequence identity to the RNA sequence encoded by SEQ ID NO:4. In some embodiments, the fourth RNA molecule has at least 95% sequence identity to the RNA sequence encoded by SEQ ID NO:4. In some embodiments, the fourth RNA molecule comprises an RNA sequence encoded by SEQ ID NO:4. In some embodiments, the compositions provided herein comprise a combination of first, second, third, and fourth RNA molecules. In some embodiments, the composition comprises a first RNA molecule encoded by SEQ ID NO:1, a second RNA molecule encoded by SEQ ID NO:2, a third RNA molecule encoded by SEQ ID NO:3, and a fourth RNA molecule encoded by SEQ ID NO:4, each of which may comprise a poly-A tail encoded by SEQ ID NO:16 at their 3' ends. In some embodiments, the first, second, third, and fourth RNA molecules are present in the combination in equimolar amounts. In some embodiments, the poly-A coding sequence in one or more (e.g., all) of SEQ ID NOs:1-4 is replaced with another sequence encoding a poly-A tail as described herein.

[0066] In some embodiments, the compositions disclosed herein comprise a DNA molecule encoding any of the RNA molecules of the compositions disclosed herein. In some embodiments, each of the DNA molecules comprises a promoter. In further embodiments, the promoter of each DNA molecule is located 5' of the 5'UTR. In yet further embodiments, the promoter is a T7 promoter.

[0067] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO: 5. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to the RNA sequence encoded by SEQ ID NO: 5. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to the RNA sequence encoded by SEQ ID NO: 5. In some embodiments, the RNA molecule comprises the RNA sequence encoded by SEQ ID NO: 5. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO:9, which may be present on the same or a different RNA molecule. In some embodiments, the additional sequence is at least 90% identical to the RNA sequence encoded by SEQ ID NO:9. In some embodiments, the additional sequence is at least 95% identical to the RNA sequence encoded by SEQ ID NO:9. In some embodiments, the additional sequence is an RNA sequence encoded by SEQ ID NO:9.

[0068] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by SEQ ID NO:6. In some embodiments, the RNA molecule further comprises a sequence having at least 90% sequence identity to an RNA sequence encoded by SEQ ID NO:6. In some embodiments, the RNA molecule further comprises a sequence having at least 95% sequence identity to an RNA sequence encoded by SEQ ID NO:6. In some embodiments, the RNA molecule comprises an RNA sequence encoded by SEQ ID NO:6. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO: 10, which may be present on the same or a different RNA molecule. In some embodiments, the additional sequence is at least 90% identical to the RNA sequence encoded by SEQ ID NO: 10. In some embodiments, the additional sequence is at least 95% identical to the RNA sequence encoded by SEQ ID NO: 10. In some embodiments, the additional sequence is the RNA sequence encoded by SEQ ID NO: 10.

[0069] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by SEQ ID NO:7. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to an RNA sequence encoded by SEQ ID NO:7. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to an RNA sequence encoded by SEQ ID NO:7. In some embodiments, the RNA molecule comprises an RNA sequence encoded by SEQ ID NO:7. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO:11, which may be present on the same or a different RNA molecule. In some embodiments, the additional sequence is at least 90% identical to the RNA sequence encoded by SEQ ID NO:11. In some embodiments, the additional sequence is at least 95% identical to the RNA sequence encoded by SEQ ID NO:11. In some embodiments, the additional sequence is an RNA sequence encoded by SEQ ID NO:11.

[0070] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by SEQ ID NO: 8. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to an RNA sequence encoded by SEQ ID NO: 8. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to an RNA sequence encoded by SEQ ID NO: 8. In some embodiments, the RNA molecule comprises an RNA sequence encoded by SEQ ID NO: 8. In some embodiments, the composition further comprises a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO: 12, which may be present on the same or a different RNA molecule. In some embodiments, the additional sequence is at least 90% identical to the RNA sequence encoded by SEQ ID NO: 12. In some embodiments, the additional sequence is at least 95% identical to the RNA sequence encoded by SEQ ID NO: 12. In some embodiments, the additional sequence is the RNA sequence encoded by SEQ ID NO: 12.

[0071] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by SEQ ID NO:9. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to an RNA sequence encoded by SEQ ID NO:9. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to an RNA sequence encoded by SEQ ID NO:9. In some embodiments, the RNA molecule comprises an RNA sequence encoded by SEQ ID NO:9.

[0072] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by SEQ ID NO: 10. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to an RNA sequence encoded by SEQ ID NO: 10. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to an RNA sequence encoded by SEQ ID NO: 10. In some embodiments, the RNA molecule comprises an RNA sequence encoded by SEQ ID NO: 10.

[0073] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the RNA sequence encoded by SEQ ID NO: 11. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to the RNA sequence encoded by SEQ ID NO: 11. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to the RNA sequence encoded by SEQ ID NO: 11. In some embodiments, the RNA molecule comprises the RNA sequence encoded by SEQ ID NO: 11.

[0074] In some aspects, the disclosure provides compositions comprising an RNA molecule comprising a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by SEQ ID NO: 12. In some embodiments, the RNA molecule comprises a sequence having at least 90% sequence identity to an RNA sequence encoded by SEQ ID NO: 12. In some embodiments, the RNA molecule comprises a sequence having at least 95% sequence identity to an RNA sequence encoded by SEQ ID NO: 12. In some embodiments, the RNA molecule comprises an RNA sequence encoded by SEQ ID NO: 12.

[0075] In some aspects, the RNA molecules disclosed herein are self-replicating RNA molecules. In some embodiments, the RNA molecules comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the RNA sequence encoded by any of SEQ ID NOs: 151-161. In some embodiments, the RNA molecule comprises a first RNA molecule having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to an RNA sequence encoded by any one of SEQ ID NOs: 151-161. In some embodiments, the first RNA molecule has at least 90% sequence identity to an RNA sequence encoded by any one of SEQ ID NOs: 151-161. In some embodiments, the first RNA molecule has at least 95% sequence identity to an RNA sequence encoded by any one of SEQ ID NOs: 151-161. In some embodiments, the first RNA molecule comprises an RNA sequence encoded by any one of SEQ ID NOs: 151-161.

[0076] Codon optimization In some embodiments, the compositions provided herein encode one or more viral replication proteins that contain codon-optimized sequences. As a result, the protein-coding sequences provided herein may be substantially altered and still encode the same protein. As used herein, the term "codon optimization" means that a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by selecting different codons without changing the amino acid sequence of the encoded protein. Thus, codon optimization generally refers to the replacement of codons with synonymous codons to optimize protein expression while maintaining the same amino acid sequence of the translated protein. Codon optimization of a sequence can, for example, increase the protein expression level of the encoded protein (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22:346-53) and provide other benefits. Variables such as codon usage preference, as measured by the codon adaptation index (CAI), such as the presence or frequency of U and other nucleotides, mRNA secondary structure, cis-regulatory sequences, GC content, and other variables, can correlate with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285). Polynucleotides can be codon-optimized before modifying the miRNA binding site. The miRNA binding site can be modified to replace one or more codons with synonymous codons.

[0077] Any codon optimization method can be used to codon-optimize the polynucleotides and nucleic acid molecules provided herein, and any variable can be altered by codon optimization. Therefore, any combination of codon optimization methods can be used. Exemplary methods include the high codon adaptation index (CAI) method, the low U method, and others. The CAI method selects the most frequently used synonymous codons for the entire protein-coding sequence. As an example, the most frequently used codons for each amino acid can be estimated from 74,218 protein-coding genes from the human genome. The low U method targets U-containing codons that can be replaced with synonymous codons with fewer U moieties, generally without changing other codons. If there is more than one option for replacement, the more frequently used codon can be selected. Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon-optimized.

[0078] In some embodiments, the nucleotide sequence of any region of an RNA or DNA template described herein may be codon-optimized, including, for example, a nucleotide sequence encoding a viral replication protein, a hemagglutinin protein, a neuraminidase protein, or any combination thereof. Preferably, the primary cDNA template may include a reduced occurrence or frequency of a particular nucleotide in the template strand. For example, the occurrence of a nucleotide in the template may be reduced to a level of less than 25% of that nucleotide in the template. In a further example, the occurrence of a nucleotide in the template may be reduced to a level of less than 20% of that nucleotide in the template. In some examples, the occurrence of a nucleotide in the template may be reduced to a level of less than 16% of that nucleotide in the template. Preferably, the occurrence of a nucleotide in the template may be reduced to a level of less than 15% of that nucleotide in the template, preferably less than 12%.

[0079] In some embodiments, the reduced nucleotide is uridine. For example, the present disclosure provides nucleic acids with altered uracil content, wherein at least one codon in the wild-type sequence is replaced with an alternative codon to generate a uracil-altered sequence. The altered uracil sequence may have at least one of the following properties: (i) an increase or decrease in the overall uracil content (i.e., the percentage of uracil of the total nucleotide content in a section of nucleic acid, e.g., an open reading frame nucleic acid); (ii) localized increases or decreases in uracil content (i.e., changes in uracil content are restricted to specific subsequences); (iii) a change in uracil distribution without a change in overall uracil content; (iv) changes in uracil clustering (e.g., number of clusters, location of clusters, or distance between clusters); or (v) combinations thereof.

[0080] In some embodiments, the percentage of uracil nucleobases in nucleic acid sequence is reduced relative to the percentage of uracil nucleobases in wild-type nucleic acid sequence.For example, 30% of nucleobases can be uracil in wild-type sequence, but the nucleobases that are uracil are preferably less than 15%, preferably less than 12%, preferably less than 10% of the nucleobases in the nucleic acid sequence of the present disclosure.The percentage of uracil content can be determined by dividing the number of uracils in sequence by the total number of nucleotides and multiplying by 100.

[0081] In some embodiments, the percentage of uracil nucleobases in a subsequence of a nucleic acid sequence is reduced relative to the percentage of uracil nucleobases in the corresponding subsequence of the wild-type sequence. For example, a wild-type sequence may have a 5'-terminal region (e.g., 30 codons) with a local uracil content of 30%, and the uracil content in that same region may be reduced to preferably 15% or less, preferably 12% or less, preferably 10% or less in a nucleic acid sequence of the present disclosure. These subsequences may also be part of the wild-type sequence of the heterologous 5' and 3' UTR sequences of the present disclosure.

[0082] In some embodiments, codons in the nucleic acid sequences of the present disclosure are reduced or altered in number, size, location, or distribution of uracil clusters, which may, for example, have deleterious effects on protein translation. While lower uracil content is desirable in certain aspects, the uracil content, particularly localized uracil content, of some subsequences of the wild-type sequence can be greater than the wild-type sequence and still maintain beneficial characteristics (e.g., increased expression).

[0083] In some embodiments, uracil-modified sequences induce a lower Toll-like receptor (TLR) response when compared to wild-type sequences. Some TLRs recognize and respond to nucleic acids. Double-stranded (ds) RNA, frequently a viral component, has been shown to activate TLR3. Single-stranded (ss) RNA activates TLR7. RNA oligonucleotides, e.g., RNA with phosphorothioate internucleotide linkages, are ligands for human TLR8. DNA containing unmethylated CpG motifs characteristic of bacterial and viral DNA activates TLR9.

[0084] As used herein, the term "TLR response" is defined as the recognition of single-stranded RNA by the TLR7 receptor, and preferably includes RNA degradation and / or physiological responses triggered by the recognition of single-stranded RNA by the receptor. Methods for determining and quantifying RNA binding to TLR7 are known in the art. Similarly, methods for determining whether an RNA elicits a TLR7-mediated physiological response (e.g., cytokine secretion) are well known in the art. In some embodiments, the TLR response may be mediated by TLR3, TLR8, or TLR9 instead of TLR7. Inhibition of TLR7-mediated responses can be achieved through nucleoside modifications. RNA naturally contains over 100 different nucleoside modifications. For example, human rRNA has 10-fold more pseudouracil ('P) and 25-fold more 2'-O-methylated nucleosides than bacterial rRNA. Bacterial RNA does not contain nucleoside modifications, but mammalian RNA has modified nucleosides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine, and many 2'-O-methylated nucleosides in addition to N7-methylguanosine (m7G).

[0085] In some embodiments, the uracil content of the polynucleotides disclosed herein is less than about 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total nucleobases in a reference sequence. In some embodiments, the uracil content of the polynucleotides disclosed herein is between about 5% and about 25%. In some embodiments, the uracil content of the polynucleotides disclosed herein is between about 15% and about 25%.

[0086] In some embodiments, the increased or decreased nucleotides are nucleotides other than or in addition to uracil. A sequence with altered nucleotide content can have (i) a local increase or decrease in C content (i.e., the change in cytosine content is limited to a specific subsequence), (ii) a local increase or decrease in G content (i.e., the change in guanosine content is limited to a specific subsequence), or (iii) a combination thereof.

[0087] In some embodiments, the polynucleotides of the nucleic acid molecules provided herein comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range of identity therebetween to the sequence of SEQ ID NO: 13. In some embodiments, the polynucleotides of the nucleic acid molecules provided herein comprise the sequence of SEQ ID NO: 13.

[0088] Intergenic region In some embodiments, the polynucleotides provided herein (e.g., a polynucleotide encoding an NA polypeptide and a polynucleotide terminating an HA polypeptide) are contained in the same (i.e., a single) or separate nucleic acid molecule. In some embodiments, the polynucleotides of the nucleic acid molecules provided herein are contained in a single nucleic acid molecule. In some embodiments, the polynucleotide encoding the NA polypeptide is located 5' of the polynucleotide encoding the HA polypeptide. In some embodiments, the polynucleotides encoding the NA polypeptide and the HA polypeptide are contained in separate nucleic acid molecules.

[0089] In some embodiments, the first polynucleotide (e.g., a polynucleotide encoding one or more viral replication proteins, or a polynucleotide encoding an NA polypeptide) and the second polynucleotide (e.g., a polynucleotide encoding an NA polypeptide or an HA polypeptide) are contained in the same (i.e., single) nucleic acid molecule. In some embodiments, the nucleic acid molecules herein comprise first, second, and third polynucleotides (e.g., a polynucleotide encoding one or more viral replication proteins, a polynucleotide encoding an NA polypeptide, and a polynucleotide encoding an HA polypeptide). Contiguously located polynucleotides of the nucleic acid molecules provided herein can be contiguous, i.e., adjacent to each other with no intervening nucleotides. In one aspect, an intergenic region is located between contiguous polynucleotides (e.g., between a first polynucleotide and a second polynucleotide, and / or a second polynucleotide and a third polynucleotide). As used herein, the terms "intergenic region" and "intergenic sequence" can be used interchangeably unless the context clearly indicates otherwise.

[0090] The intergenic region located between the polynucleotides can be of any length and have any nucleotide sequence. For example, an intergenic region between two polynucleotides may have about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, about 50 nucleotides, about 51 nucleotides, about 52 nucleotides, about 53 nucleotides, about 54 nucleotides, about 55 nucleotides, about 56 nucleotides, about 57 nucleotides, about 58 nucleotides, about 59 nucleotides, about 60 nucleotides, about 61 nucleotides, about 62 nucleotides, about 63 nucleotides, about 6 5 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 600 nucleotides, It can comprise about 700 nucleotides, about 800 nucleotides, about 900, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, about 5,000 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, and any number or range therebetween.In one embodiment, the intergenic region between two polynucleotides comprises about 10 to 100 nucleotides, about 10 to 200 nucleotides, about 10 to 300 nucleotides, about 10 to 400 nucleotides, or about 10 to 500 nucleotides. In another embodiment, the intergenic region between two polynucleotides comprises about 1 to 10 nucleotides, about 1 to 20 nucleotides, about 1 to 30 nucleotides, about 1 to 40 nucleotides, or about 1 to 50 nucleotides. In yet another embodiment, the region comprises about 44 nucleotides.

[0091] In one embodiment, the intergenic region between the two polynucleotides comprises a viral sequence. The intergenic region between the two polynucleotides can comprise sequences from any virus, such as, for example, alphavirus and rubivirus.In one embodiment, the intergenic region between the two polynucleotides is an alphavirus sequence, e.g., Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus (SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), Barmah Forest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (Sagiyama Virus), or any of the following: Virus (SAGV), Bebaru Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus (SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV), Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV), Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV), Salmonid Alphavirus (SAV), and Buggy Creek Virus (BV). In another embodiment, the intergenic region between the two polynucleotides comprises sequences from Venezuelan Equine Encephalitis Virus (VEEV).In yet another embodiment, the intergenic region between the two polynucleotides comprises a sequence having at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range of identity therebetween to the sequence of SEQ ID NO: 150. In a further embodiment, the intergenic region between the two polynucleotides comprises the sequence of SEQ ID NO: 150. In yet a further embodiment, the intergenic region between the two polynucleotides is a second intergenic region comprising a sequence having at least 85% identity to the sequence of SEQ ID NO: 150.

[0092] Natural and Modified Nucleotides The self-replicating RNA of the present disclosure can contain one or more chemically modified nucleotides. Examples of nucleic acid monomers include unnatural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the base or sugar moiety. In nature, most polynucleotides contain nucleotides that are "unmodified" or "natural" nucleotides, including the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). These bases are typically anchored at the 1' position to ribose or deoxyribose. The use of RNA polynucleotides containing chemically modified nucleotides has been shown to improve RNA expression, expression rate, half-life, and / or expressed protein concentration. RNA polynucleotides containing chemically modified nucleotides are also useful for optimizing protein localization, thereby avoiding adverse biological responses, such as immune responses and / or degradation pathways.

[0093] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N4-alkylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4,N4-dialkylcytidine.

[0094] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine; N4-methylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4,N4-dimethylcytidine.

[0095] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkylester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine, and 6-alkyluridine.

[0096] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "5MeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.

[0097] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'-O-methyluridine, and 3,2'-O-dimethyluridine.

[0098] Examples of modified or chemically modified nucleotides include N6-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N6-methyl-purine, 1-thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0099] Examples of modified or chemically modified nucleotides include Nl-alkylguanosine, N2-alkylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-alkylguanosine, xanthosine, inosine, and Nl-alkylinosine.

[0100] Examples of modified or chemically modified nucleotides include Nl-methylguanosine, N2-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-methylguanosine, xanthosine, inosine, and Nl-methylinosine.

[0101] An example of a modified or chemically modified nucleotide is pseudouridine. Examples of pseudouridines include Nl-alkylpseudouridine, Nl-cycloalkylpseudouridine, Nl-hydroxypseudouridine, Nl-hydroxyalkylpseudouridine, Nl-phenylpseudouridine, Nl-phenylalkylpseudouridine, Nl-aminoalkylpseudouridine, N3-alkylpseudouridine, N6-alkylpseudouridine, N6-alkoxypseudouridine, N6-hydroxypseudouridine, N6-hydroxyalkylpseudouridine, N6-morpholinopseudouridine, N6-phenylpseudouridine, and N6-halopseudouridine. Examples of pseudouridines include Nl-alkyl-N6-alkylpseudouridine, Nl-alkyl-N6-alkoxypseudouridine, Nl-alkyl-N6-hydroxypseudouridine, Nl-alkyl-N6-hydroxyalkylpseudouridine, Nl-alkyl-N6-morpholinopseudouridine, Nl-alkyl-N6-phenylpseudouridine, and Nl-alkyl-N6-halopseudouridine. In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents.

[0102] Examples of pseudouridines include Nl-methylpseudouridine (also referred to herein as "N1MPU"), Nl-ethylpseudouridine, Nl-propylpseudouridine, Nl-cyclopropylpseudouridine, Nl-phenylpseudouridine, Nl-aminomethylpseudouridine, N3-methylpseudouridine, N1-hydroxypseudouridine, and N1-hydroxymethylpseudouridine.

[0103] Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

[0104] Examples of modified and chemically modified nucleotide monomers include any such nucleotide known in the art, such as 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxyribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted deoxy abasic monomer residues.

[0105] Examples of modified and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides, and 3'-inverted thymidines.

[0106] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoronucleotides, and 2'-O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0107] Examples of modified and chemically modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNA.

[0108] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.

[0109] Examples of modified and chemically modified nucleotide monomers include N6-methyl adenosine nucleotides.

[0110] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers having the modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.

[0111] Examples of modified and chemically modified nucleotide monomers include 2'-O-aminopropyl substituted nucleotides.

[0112] Examples of modifications and chemically modified nucleotide monomers include replacing the 2'-OH group of the nucleotide with 2'-R, 2'-OR, 2'-halogen, 2'-SR, or 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0113] The above exemplary base modifications can be combined with further modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers can be found in nature.

[0114] Preferred nucleotide modifications include N1-methylpseudouridine and 5-methoxyuridine.

[0115] Viral replication proteins and polynucleotides encoding them In some embodiments, RNA molecules encoding one or more viral replication proteins are provided herein. As used herein, the term "replication protein" or "viral replication protein" refers to any protein or any protein subunit of a protein complex that functions in the replication of a viral genome. Generally, viral replication proteins are nonstructural proteins. The viral replication proteins encoded by the nucleic acid molecules provided herein can function in the replication of any viral genome. The viral genome can be a single-stranded positive-strand RNA genome, a single-stranded negative-strand RNA genome, a double-stranded RNA genome, a single-stranded positive-strand DNA genome, a single-stranded negative-strand DNA genome, or a double-stranded DNA genome. The viral genome can comprise a single nucleic acid molecule or more than one nucleic acid molecule. The nucleic acid molecules provided herein can encode one or more viral replication proteins from any virus or virus family, including, for example, animal viruses and plant viruses. The viral replication proteins encoded by the polynucleotides contained in the nucleic acid molecules provided herein can be expressed from self-replicating RNA.

[0116] In some embodiments, the RNA molecules provided herein can encode one or more togavirus replication proteins. In some aspects, one or more viral replication proteins encoded by the RNA molecules provided herein are alphavirus proteins. In some embodiments, one or more viral replication proteins encoded by the RNA molecules provided herein are rubivirus proteins. The RNA molecules provided herein can encode any alphavirus replication protein and any rubivirus replication protein. Exemplary replication proteins from alphaviruses include Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki Forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'nyong-nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BFV), Getah virus (GETV), Sagiyama virus (SAGV), Bebaru virus (BEV), and others. Exemplary rubivirus replication proteins include proteins from rubella virus, ...

[0117] Viral replication proteins encoded by the RNA molecules provided herein can be expressed as one or more polyproteins or as separate or single proteins. Generally, a polyprotein is a precursor protein that is cleaved to generate individual or separate proteins. Thus, proteins derived from a precursor polyprotein can be expressed from a single open reading frame (ORF). As used herein, the term "ORF" refers to a nucleotide sequence beginning with a start codon, generally ATG, and ending with a stop codon, such as TAA, TAG, or TGA. It will be understood that T occurs in DNA and U occurs in RNA. Thus, the start codon ATG in DNA corresponds to AUG in RNA, and the stop codons TAA, TAG, and TGA in DNA correspond to UAA, UAG, and UGA in RNA. It will be further understood that for any sequence provided in this disclosure, T occurs in DNA, while U occurs in RNA. Thus, for any sequence provided herein, T occurring in DNA is replaced with U in RNA molecules, and U occurring in RNA is replaced with T in DNA molecules.

[0118] The protease that cleaves the polyprotein can be a viral protease or a cellular protease. In some embodiments, the RNA molecules provided herein encode one or more viral replication proteins, including an alphavirus nsP1 protein, an alphavirus nsP2 protein, an alphavirus nsP3 protein, an alphavirus nsP4 protein, or any combination thereof. In other embodiments, the RNA molecules provided herein encode one or more viral replication proteins, including an alphavirus nsP1 protein, an alphavirus nsP2 protein, an alphavirus nsP3 protein, or any combination thereof, and an alphavirus nsP4 protein. In some embodiments, the polyprotein is a VEEV polyprotein. In other embodiments, the alphavirus nsP1, nsP2, nsP3, and nsP4 proteins are VEEV proteins.

[0119] In one embodiment, the RNA molecules provided herein lack a stop codon between the sequence encoding the nsP3 protein and the sequence encoding the nsP4 protein. Thus, in some embodiments, the polynucleotide of the RNA molecules provided herein encodes a P1234 polyprotein comprising nsP1, nsP2, nsP3, and nsP4. The RNA molecules provided herein may also comprise a stop codon between the sequence encoding the nsP3 protein and the sequence encoding the nsP4 protein. Thus, in some embodiments, the polynucleotide of the nucleic acid molecules provided herein encodes a P123 polyprotein comprising nsP1, nsP2, and nsP3, and a P1234 polyprotein comprising nsP1, nsP2, nsP3, and nsP4, e.g., as a result of stop codon readthrough. In one embodiment, the nsP2 and nsP3 proteins comprise mutations. Exemplary mutations include the G1309R and S1583G mutations in the VEEV protein. In another embodiment, the nsP1, nsP2, and nsP4 proteins are VEEV proteins and the nsP3 protein is a Chikungunya virus (CHIKV) nsP3 protein.

[0120] In some embodiments, the RNA molecules provided herein encode one or more viral replication proteins comprising a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the sequence of SEQ ID NO: 13. In some embodiments, the one or more viral replication proteins comprise the sequence of SEQ ID NO: 13.

[0121] 5' Untranslated Region (5' UTR) The nucleic acid molecules provided herein may further comprise untranslated regions (UTRs). For example, untranslated regions, including 5'UTRs and 3'UTRs, can affect RNA stability and / or the efficiency of RNA translation, e.g., the translation of cellular and viral mRNAs. 5'UTRs and 3'UTRs can also affect the stability and translation of viral genomic RNAs and self-replicating RNAs, including virally derived self-replicating RNAs or replicons. Exemplary viral genomic RNAs whose stability and / or translation efficiency can be affected by 5'UTRs and 3'UTRs include the genomic nucleic acids of positive-strand RNA viruses. Both the genomic nucleic acids of positive-strand RNA viruses and self-replicating RNAs (including virally derived self-replicating RNAs or replicons) can be translated upon infection or introduction into cells.

[0122] In some aspects, the nucleic acid molecules provided herein further comprise a 5' untranslated region (5'UTR). Any 5'UTR sequence can be included in the nucleic acid molecules provided herein. In some embodiments, the nucleic acid molecules provided herein comprise a viral 5'UTR. In one aspect, the nucleic acid molecules provided herein comprise a non-viral 5'UTR. Any non-viral 5'UTR, such as a 5'UTR of a transcript expressed in any cell or organ, including muscle, skin, subcutaneous tissue, liver, spleen, lymph nodes, antigen-presenting cells, and others, can be included in the nucleic acid molecules provided herein. In another aspect, the nucleic acid molecules provided herein comprise a 5'UTR comprising viral and non-viral sequences. Thus, the 5'UTR included in the nucleic acid molecules provided herein can comprise a combination of viral and non-viral 5'UTR sequences. In some aspects, the 5'UTR included in the nucleic acid molecules provided herein is located upstream or 5' of a polynucleotide encoding one or more viral replication proteins. In some embodiments, the 5' UTR is located 5' or upstream of a polynucleotide of a nucleic acid molecule provided herein that encodes one or more viral replication proteins, which in turn is located 5' or upstream of one or more additional polynucleotides of a nucleic acid molecule provided herein (e.g., one or more of the polynucleotides encoding an NA polypeptide or an HA polypeptide).

[0123] In one embodiment, the 5' UTR of a nucleic acid molecule provided herein comprises an alphavirus 5' UTR. The 5' UTR from any alphavirus can be any of the following: Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki Forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'nyong-nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BFV), Getah virus (GETV), Sagiyama virus (SAGV), Bebaru virus (BEBV), and the like. The nucleic acid molecules provided herein may include 5' UTR sequences from HIV-1, HIV-2, HIV-3, HIV-4, HIV-5, HIV-6, HIV-1, HIV-2, HIV-2-related viruses, including HIV-1, HIV-2-related viruses, HIV-1, HIV-2-related viruses, HIV-2-related viruses, HIV-3, HIV-4, HIV-5, HIV-1-related viruses, HIV-1, HIV-2 ..., HIV-1-related viruses, HIV-1, HIV-1, HIV-1-related viruses, HIV-1, HIV-1, HIV-1-related viruses, HIV-1, HIV-1, HIV-1-related viruses, HIV-1, HIV-1, HIV-1-related viruses, HIV-1, HIV-1, HIV-1 In another aspect, the 5' UTR comprises a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range therebetween, identity to the sequence of SEQ ID NO: 14, for example. In some embodiments, the 5' UTR comprises the sequence of SEQ ID NO: 14.

[0124] In some embodiments, the 5'-UTR comprises a sequence selected from human IL-6, alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human transthyretin, human haptoglobin, human alpha1-antichymotrypsin, human antithrombin, human alpha1-antitrypsin, human albumin, human beta globin, human complement C3, human complement C5, SynK (a thylakoid potassium channel protein from the cyanobacterium Synechocystis sp.), mouse beta globin, mouse albumin, and the 5'-UTR of tobacco etch virus, or a fragment of any of the foregoing. Preferably, the 5'-UTR is derived from tobacco etch virus (TEV). In one embodiment, the 5' UTR comprises a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range therebetween, identity to the sequence of SEQ ID NO: 17. In another embodiment, the 5' UTR comprises the sequence of SEQ ID NO: 17 or SEQ ID NO: 14.

[0125] The mRNA or any other RNA described herein can include any 5' UTR sequence provided herein. For example, the RNA described herein can include a 5' UTR sequence derived from a gene expressed by Arabidopsis thaliana. In some embodiments, the 5' UTR sequence of the gene expressed by Arabidopsis thaliana is AT1G58420. Examples of 5' UTRs and 3' UTRs are described in U.S. Patent Application Publication No. 20190002906 (A1), the contents of which are incorporated herein by reference. Exemplary 5' UTR sequences include SEQ ID NOs: 17 and 22-50, as shown in Table 1.

[0126] [Table 1]

[0127] Further exemplary 5'UTR sequences of SEQ ID NOs: 65-111 are shown in Table 2.

[0128] [Table 2-1]

[0129] [Table 2-2]

[0130] [Table 2-3]

[0131] [Table 2-4]

[0132] 3' Untranslated Region (3'UTR) In some aspects, the nucleic acid molecules provided herein further comprise a 3' untranslated region (3'UTR). Any 3'UTR sequence can be included in the nucleic acid molecules provided herein. In one aspect, the nucleic acid molecules provided herein comprise a viral 3'UTR. In another aspect, the nucleic acid molecules provided herein comprise a non-viral 3'UTR. Any non-viral 3'UTR, for example, the 3'UTR of a transcript expressed in any cell or organ, including muscle, skin, subcutaneous tissue, liver, spleen, lymph nodes, antigen-presenting cells, and others, can be included in the nucleic acid molecules provided herein. In some aspects, the nucleic acid molecules provided herein comprise a 3'UTR comprising viral and non-viral sequences. Thus, the 3'UTR included in the nucleic acid molecules provided herein can comprise a combination of viral and non-viral 3'UTR sequences. In one aspect, the 3'UTR is located 3' or downstream of a polynucleotide of a nucleic acid molecule provided herein that comprises a first transgene encoding a first antigenic protein or fragment thereof (e.g., an NA polypeptide or an HA polypeptide). In some aspects, the 3' UTR is located 3' or downstream of a polynucleotide of a nucleic acid molecule provided herein that includes a first transgene encoding a first antigenic protein or fragment thereof, which is in turn located 3' or downstream of one or more additional polynucleotides of a nucleic acid molecule provided herein (e.g., a polynucleotide encoding another antigenic protein or fragment thereof, and / or a polynucleotide encoding one or more viral replication proteins).

[0133] In one embodiment, the 3' UTR of a nucleic acid molecule provided herein comprises an alphavirus 3' UTR. The 3' UTR from any alphavirus can be any of the following: Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki Forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'nyong-nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BFV), Getah virus (GETV), Sagiyama virus (SAGV), Bebaru virus (BEBV), and the like. The nucleic acid molecules provided herein may include 3' UTR sequences from HIV-1, HIV-2, HIV-3, HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-3 virus (HIV-3), HIV-4 virus (HIV-4), HIV-5, HIV-6, HIV-7, HIV-8, HIV-1 virus (HIV-1), HIV-1 virus (HIV-2), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-3 virus (HIV-3), HIV-4 virus (HIV-4), HIV-5, HIV-6, HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-3 virus (HIV-3), HIV-4 virus (HIV-4), HIV-5, HIV-6 virus (HIV-1), HIV-1 virus (HIV-2), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-3 virus (HIV-3), HIV-4 virus (HIV-4), HIV-5, HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-3 virus (HIV-3), HIV-4 virus (HIV-4), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-1 virus (HIV-1), HIV-2 virus (HIV-2), HIV-3 virus (HIV-1), HIV-3 virus (HIV-1), HIV-4 virus (HIV-1), HIV-4 virus (HIV-1), HIV-4 virus (HIV-1), HIV-4 virus (HIV-1), HIV-4 virus (HIV-1), HIV-5, HIV-1 virus (HIV-1), HIV-1 virus (HIV-1), HIV-2 virus ( In some embodiments, the 3' UTR comprises a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range of identity therebetween, for example, to the sequence of SEQ ID NO: 15. In yet another aspect, the 3' UTR further comprises a polyA sequence. In some embodiments, the 3' UTR comprises the sequence of SEQ ID NO: 15. In some embodiments, the 3' UTR comprises, for example, the sequence of SEQ ID NO: 16. In some embodiments, the 3' UTR comprises a polyA tail of about 20-300 nucleotides. In some embodiments, the polyA tail comprises about 20-300 consecutive A nucleotides.In some embodiments, the poly-A tail comprises about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300 nucleotides, and any number or range therebetween. In some embodiments, the nucleotides of the poly-A tail are A nucleotides. In some embodiments, the 3'UTR comprises a poly-C tail.

[0134] In some embodiments, the 3'-UTR comprises a sequence selected from the 3'-UTRs of alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human haptoglobin, human antithrombin, human alpha globin, human beta globin, human complement C3, human growth factor, human hepcidin, MALAT-1, mouse beta globin, mouse albumin, and Xenopus beta globin, or a fragment of any of the foregoing. In some embodiments, the 3'-UTR is derived from Xenopus beta globin. Any 3'-UTR provided herein may comprise a poly-A tail, as described in further detail below. In some embodiments, the 3' UTR comprises a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range of identity therebetween to the sequence of SEQ ID NO: 18 or SEQ ID NO: 19. The 3' UTRs provided herein can be included in any RNA molecule provided herein, including self-replicating RNA and mRNA molecules. Exemplary 3' UTR sequences include SEQ ID NOs: 51-57, as shown in Table 3.

[0135] [Table 3]

[0136] Further exemplary 3'UTR sequences of SEQ ID NOs: 112-149 are shown in Table 4.

[0137] [Table 4-1]

[0138] [Table 4-2]

[0139] [Table 4-3]

[0140] [Table 4-4]

[0141] [Table 4-5]

[0142] [Table 4-6]

[0143] Triple stop codon In some embodiments, RNA molecules provided herein, including self-replicating RNA and mRNA, may contain a sequence immediately downstream of the coding region (i.e., ORF) that creates a triple stop codon. A triple stop codon is a sequence of three consecutive stop codons. Triple stop codons can ensure complete isolation of the expression cassette and may be incorporated to increase translation efficiency. In some embodiments, RNA molecules of the present disclosure may contain any triple combination of the sequences UAG, UGA, or UAA immediately downstream of the ORF described herein. The triple combination may be three of the same codons, three different codons, or any other permutation of three stop codons.

[0144] Translational enhancers and Kozak sequences For translation initiation, proper interactions between the ribosome and mRNA must be established to determine the precise location of the translation initiation region. However, the ribosome must also dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream of the mRNA initiation sequence increase the yield of protein biosynthesis. Several studies have investigated the effects of translation enhancers. In some embodiments, the RNA molecules described herein, such as self-replicating RNAs or mRNAs, contain translation enhancer sequences. These translation enhancer sequences increase the translation efficiency of the self-replicating RNAs or mRNAs of the present disclosure, thereby providing increased production of the protein encoded by the RNA. Translation enhancer regions can be located in the 5' or 3' UTR of the self-replicating RNA or mRNA sequence. Examples of translation enhancer regions include the naturally occurring enhancer regions from the TEV 5' UTR and the Xenopus beta-globin 3' UTR. Exemplary 5'UTR enhancer sequences include, but are not limited to, those derived from mRNAs encoding human heat shock proteins (HSPs), including HSP70-P2, HSP70-M1, HSP72-M2, HSP17.9, and HSP70-P1. Exemplary translation enhancer sequences for use in accordance with embodiments of the present disclosure are represented by SEQ ID NOs: 58-62, as shown in Table 5.

[0145] [Table 5]

[0146] In some embodiments, the self-replicating RNA or mRNA of the present disclosure comprises a Kozak sequence. As understood in the art, a Kozak sequence is a short consensus sequence centered around the translation start site of eukaryotic mRNA that allows for efficient initiation of translation of the self-replicating RNA or mRNA. See, e.g., Kozak, Marilyn (1988) Mol. and Cell Biol, 8:2737-2744; Kozak, Marilyn (1991) J. Biol. Chem, 266:19867-19870; Kozak, Marilyn (1990) Proc. Natl. Acad. Sci. USA, 87:8301-8305; and Kozak, Marilyn (1989) J. Cell Biol, 108:229-241. This ensures that proteins are accurately translated from the genetic message and mediates ribosome assembly and translation initiation. The ribosomal translation machinery recognizes the AUG start codon in the context of a Kozak sequence. The Kozak sequence may be inserted upstream of the coding sequence of the protein of interest and downstream of the 5'UTR, or may be inserted upstream of the coding sequence of the protein of interest and downstream of the 5'UTR. In some embodiments, the self-replicating RNA or mRNA described herein comprises a Kozak sequence having the sequence GCCACC (SEQ ID NO: 63). The self-replicating RNA or mRNA described herein may comprise a partial Kozak sequence "p" having the nucleotide sequence GCCA (SEQ ID NO: 64).

[0147] antigenic proteins In some embodiments, the nucleic acid molecules provided herein (e.g., RNA molecules, and polynucleotides encoding them) can encode antigenic proteins or fragments thereof. In some embodiments, the RNA molecules provided herein encode the entire HA polypeptide or an antigenic fragment thereof. In some embodiments, the RNA molecules provided herein encode the entire NA polypeptide or an antigenic fragment thereof. The RNA molecules provided herein can encode homologs of any antigenic protein provided herein. Any antigenic protein can be encoded by the nucleic acid molecules provided herein. In one aspect, the antigenic protein is a viral protein, a bacterial protein, a fungal protein, a protozoan protein, or a parasitic protein. The RNA molecules provided herein can be expressed from self-replicating RNA or subgenomic RNA derived from mRNA.

[0148] In some aspects, the antigenic protein, when administered to a mammalian subject, elicits an immune response against a pathogen, optionally the pathogen being a virus, bacterium, fungus, protozoan, or any other type of pathogen. In other aspects, the antigenic protein is expressed on the outer surface of the pathogen, while in further aspects, the antigen can be a non-surface antigen, useful, for example, as a T-cell epitope. The immune response can include an antibody response (usually involving IgG) and / or a cell-mediated immune response. Polypeptide immunogens typically elicit an immune response that recognizes the corresponding pathogen polypeptide, although in some embodiments, the polypeptide can act as a mimotope to elicit an immune response that recognizes a carbohydrate. The immunogen can be a surface polypeptide, such as an adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, etc.

[0149] Any viral, bacterial, fungal, protozoan, parasitic, or other protein can be encoded by the RNA molecules provided herein. Proteins from any infectious agent can be encoded by the RNA molecules provided herein. As used herein, the term "infectious agent" refers to any agent that can infect organisms, including humans and animals, and cause disease or deterioration in health. The terms "infectious agent" and "infectious pathogen" can be used interchangeably unless the context clearly indicates otherwise.

[0150] In one aspect, the antigenic protein encoded by the nucleic acid molecules provided herein is an influenza virus protein or fragment thereof. In another aspect, the nucleic acid molecule encodes one or more influenza virus proteins or fragments thereof from any strain or subtype of influenza virus. Exemplary influenza virus proteins that can be encoded by the nucleic acid molecules provided herein include proteins from any human or animal virus, including influenza A virus, influenza B virus, influenza C virus, influenza D virus, or any combination thereof. Exemplary influenza proteins include hemagglutinin (HA), neuraminidase (NA), M2, M1, NP, NS1, NS2, PA, PB1, PB2, and PB1-F2. Hemagglutinin proteins from any influenza virus subtype, such as H1-H18 and any neohemagglutinin, and neuraminidase proteins from any influenza virus subtype, such as N1-N11 and any neoneuraminidase, can be antigenic proteins encoded by the polynucleotides of the nucleic acid molecules provided herein. Any suitable fragment of an influenza virus protein can be encoded by a polynucleotide of a nucleic acid molecule provided herein, including, for example, one or more helper T lymphocyte (HTL) epitopes, one or more cytotoxic T lymphocyte (CTL) epitopes, or any combination thereof.

[0151] In some aspects, the polynucleotides of the nucleic acid molecules provided herein encode markers, including reporters or selectable markers. Reporters and markers can include, for example, fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), luciferase enzymes such as firefly and Renilla luciferase, and antibiotic selectable markers.

[0152] In some embodiments, the nucleic acid molecules provided herein comprise at least two transgenes (e.g., at least two transgenes in addition to one or more viral replication proteins). Any number of transgenes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transgenes, can be included in the nucleic acid molecules provided herein. In one embodiment, the polynucleotide of the nucleic acid molecule provided herein encodes an antigenic protein or fragment thereof or an immunomodulatory protein. In one embodiment, the nucleic acid molecule further comprises an internal ribosomal entry site (IRES), a sequence encoding a 2A peptide, or a combination thereof, located between the transgenes. As used herein, the term "2A peptide" refers to a small (generally 18-22 amino acids) sequence that enables efficient stoichiometric production of distinct protein products within a single reading frame via ribosomal skipping events within the 2A peptide sequence. As used herein, the term "internal ribosome entry site" or "IRES" refers to a nucleotide sequence that allows initiation of protein translation of a messenger RNA (mRNA) sequence in the absence or without the use of an AUG start codon. An IRES can be found anywhere in an mRNA sequence, for example, at or near the beginning, at or near the middle, or at or near the end of the mRNA sequence. In another aspect, the nucleic acid molecules provided herein contain one or more subgenomic promoters (e.g., upstream of each HA or NA encoded by the nucleic acid molecule). The subgenomic promoter located between transgenes can be an additional subgenomic promoter, such as a second, third, fourth, etc. subgenomic promoter located between the second and third transgenes, between the third and fourth transgenes, between the fourth and fifth transgenes, etc.

[0153] Any number of transgenes contained in the nucleic acid molecules provided herein can be expressed via any combination of 2A peptide and IRES sequences. For example, a second transgene located 3' of a first transgene can be expressed via a 2A peptide sequence or an IRES sequence. As another example, a second transgene located 3' of a first transgene and a third transgene located 3' of a second transgene can be expressed via a 2A peptide sequence located between the first and second transgenes and the second and third transgenes, via an IRES sequence located between the first and second transgenes and the second and third transgenes, via a 2A peptide sequence located between the first and second transgenes and an IRES located between the second and third transgenes, or via an IRES sequence located between the first and second transgenes and a 2A peptide sequence located between the second and third transgenes. Similar configurations and combinations of 2A peptide and IRES sequences located between transgenes are contemplated for any number of transgenes contained in the polynucleotide of a nucleic acid molecule provided herein. In addition to expression via 2A peptides and IRES sequences, two or more transgenes contained in the nucleic acid molecules provided herein can also be expressed from separate subgenomic RNAs.

[0154] The polynucleotides of the nucleic acid molecules provided herein can encode an immunomodulatory protein, or a functional fragment or functional variant thereof. Any immunomodulatory protein, or a functional fragment or functional variant thereof, can be encoded by a polynucleotide.

[0155] As used herein, the term "functional variant" or "functional fragment" refers to a molecule, including, for example, a nucleic acid or protein, comprising a nucleotide and / or amino acid sequence in which one or more nucleotides and / or amino acids are altered compared to the nucleotide and / or amino acid sequence of a parent or reference molecule. For proteins, functional variants are still capable of functioning in a manner similar to the parent molecule. In other words, modifications in the amino acid and / or nucleotide sequence of a parent molecule do not significantly affect or alter the functional characteristics of the molecule encoded by the nucleotide sequence or containing the amino acid sequence. Functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis. Functional variants may also include, but are not limited to, derivatives that are substantially similar in primary structural sequence but contain, for example, chemical and / or biochemical in vitro or in vivo modifications not found in the parent molecule. Such modifications include, inter alia, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, ubiquitination, and the like.

[0156] In one aspect, the transgene included in the nucleic acid molecules provided herein encodes a cytokine, chemokine, or interleukin. A transgene encoding a cytokine, chemokine, or interleukin can be included in the nucleic acid molecules provided herein in addition to a transgene encoding HA, NA, both HA and NA, any other antigenic protein, or any combination thereof. Exemplary cytokines include interferons, TNF-α, TGF-β, G-CSF, and GM-CSF. Exemplary chemokines include CCL3, CCL26, and CXCL7. Exemplary interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, and IL-23. Any transgene or combination of transgenes encoding any cytokine, chemokine, interleukin, or combination thereof can be included in the nucleic acid molecules provided herein.

[0157] In one aspect, the first and second transgenes comprised in the nucleic acid molecules provided herein encode a viral protein, a bacterial protein, a fungal protein, a protozoan protein, a parasitic protein, an immunomodulatory protein, or any combination thereof. In yet another aspect, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more transgenes comprised in the polynucleotide of the nucleic acid molecules provided herein encode a viral protein, a bacterial protein, a fungal protein, a protozoan protein, a parasitic protein, an immunomodulatory protein, or any combination thereof.

[0158] In other embodiments, the second transgene encodes a second influenza virus protein. In yet other embodiments, the first and second transgenes both encode influenza virus proteins that may be derived from the same influenza virus strain.

[0159] RNA and DNA molecules RNA Molecules - Exemplary Features The nucleic acid molecules provided herein may be DNA molecules or RNA molecules. It will be understood that T occurring in DNA is replaced with U in RNA, and vice versa. In one aspect, the nucleic acid molecules provided herein are RNA molecules, and a first polynucleotide is located 5' of a second polynucleotide (which may optionally be located 5' of a third polynucleotide, etc.). A sequence presented herein as an RNA sequence can be encoded by a corresponding DNA sequence in which U is replaced with T. Similarly, a sequence presented herein as a DNA sequence can be converted to the corresponding RNA sequence encoded thereby by replacing T with U. Generally, an RNA sequence "encoded" by a DNA sequence herein refers to an RNA having the same 5' to 3' orientation and base order (except for U replacing T), and not to the reverse complement. Both DNA and RNA versions of a given sequence are contemplated herein unless the context clearly dictates otherwise. In some cases, a DNA sequence may include elements not found in the RNA encoded thereby (e.g., a promoter sequence). In some cases, the RNA sequence may contain elements not found in the DNA construct encoding the RNA (e.g., a polyA tail if added post-transcriptionally).

[0160] The RNA molecule provided herein can be a self-replicating RNA. In one embodiment, the RNA molecule provided herein comprises a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range therebetween, or 100% identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The RNA molecule provided herein can also be an mRNA. It will be understood that T in the sequences provided herein is replaced with U in the RNA molecule.

[0161] The RNA molecules provided herein can be generated by in vitro transcription (IVT) of the DNA molecules provided herein. In one embodiment, the RNA molecules provided herein are self-replicating RNA molecules. In another embodiment, the RNA molecules provided herein are mRNA molecules. In yet another embodiment, the RNA molecules provided herein further comprise a 5' cap. Any 5' cap can be included in the RNA molecules provided herein, including a 5' cap having a Cap1 structure, a Cap1(m6A) structure, a Cap2 structure, or a Cap0 structure. A population or a plurality of RNA molecules provided herein can have the same 5' cap or different 5' caps. For example, a population or a plurality of RNA molecules can have a 5' cap having a Cap1 structure, a Cap1(m6A) structure, a Cap2 structure, a Cap0 structure, or any combination thereof.

[0162] In one embodiment, the RNA molecules provided herein comprise a 5' cap having a Cap1 structure. In yet another embodiment, the RNA molecules provided herein are self-replicating RNA molecules comprising a 5' cap having a Cap1 structure. In a further embodiment, the RNA molecules provided herein comprise a cap having a Cap1 structure, in which m7G is linked to the 5' end of the 5' UTR via a 5'-5' triphosphate. In yet a further embodiment, the RNA molecules provided herein comprise a cap having a Cap1 structure, in which m7G is linked to the 5' end of the 5' UTR comprising the sequence of SEQ ID NO: 14 via a 5'-5' triphosphate. Any capping method can be used, including, but not limited to, using vaccinia capping enzyme (New England Biolabs, Ipswich, Mass.) and co-transcriptional capping, or capping at the beginning or shortly after in vitro transcription (IVT), for example, by including a capping agent as part of the IVT reaction. (Nuc. Acids Symp. (2009) 53:129).

[0163] Only RNA molecules, such as mRNAs with a cap structure and self-replicating RNAs that can function as mRNAs, are active in cap-dependent translation, and "decapitation" of mRNAs results in almost complete loss of their template activity for protein synthesis (Nature, 255:33-37, (1975), J. Biol. Chem., vol. 253:5228-5231 (1978), and Proc. Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

[0164] Another element of eukaryotic mRNA is the presence of 2'-O-methylnucleoside residues at transcription position 1 (Cap 1), and in some cases, at transcription positions 1 and 2 (Cap 2). 2'-O-methylation of mRNA provides greater efficiency of mRNA translation in vivo (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves the nuclease stability of 5'-capped mRNA. mRNAs with Cap 1 (and Cap 2) are distinctive marks that allow cells to recognize authentic mRNA 5' ends and, in some cases, distinguish transcripts emanating from infectious genetic elements (Nucleic Acid Research 43:482-492 (2015)).

[0165] Some examples of 5' cap structures and methods for preparing mRNAs containing the same are provided in International Publication Nos. WO 2015 / 051169(A2), WO 2015 / 061491, U.S. Patent Application Publication No. 2018 / 0273576, and U.S. Patent Nos. 8,093,367, 8,304,529, and 10,487,105. In some embodiments, the 5' cap is m7GpppAmpG, as known in the art. In some embodiments, the 5' cap is m7GpppG or m7GpppGm, as known in the art. Structural formulas for embodiments of 5' cap structures are provided below.

[0166] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises a 5' cap having the structure of formula (Cap I):

[0167] [ka] In the formula, B 1 is a natural or modified nucleobase, and R 1 and R 2are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; n is 0 or 1; and the mRNA represents an mRNA of the present disclosure linked at its 5' end. In some embodiments, B 1 , G, m 7 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B 1 is A or m 6 A and R 1 is OCH3, G is guanine, m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine.

[0168] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises a 5' cap having the structure of formula (Cap II):

[0169] [ka] In the formula, B 1 and B 2 are each independently a natural or modified nucleobase; R 1 , R 2 , and R 3 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, B 1 , G, m 7 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B 1 is A or m 6 A and R 1is OCH3, G is guanine, m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine.

[0170] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises a 5' cap having the structure of formula (Cap III):

[0171] [ka]

[0172] wherein B1, B2, and B3 are each independently a natural or modified nucleobase; R1, R2, R3, and R4 are each independently selected from halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; the mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, at least one of R1, R2, R3, and R4 is OH. In some embodiments, B1 is G, m7G, or A. In some embodiments, B1 is A or m6A, R1 is OCH3, G is guanine, m7G is 7-methylguanine, A is adenine, and m6A is N6-methyladenine. In some embodiments, n is 1.

[0173] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7GpppG 5' cap analog having the structure of formula (Cap IV):

[0174] [ka] In the formula, R 1 , R 2 , and R 3are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , and R 3 In some embodiments, the 5' cap is at least one of m 7 GpppG and R 1 , R 2 , and R 3 are each OH, n is 1, and each L is phosphate. In some embodiments, n is 1. In some embodiments, the 5' cap is m7GpppGm and R 1 and R 2 are each OH and R 3 is OCH3, each L is a phosphate, and n is 1.

[0175] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7Gpppm7G 5' cap analog having the structure of formula (Cap V).

[0176] [ka] In the formula, R 1 , R 2 , and R 3 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , and R 3 In some embodiments, n is 1.

[0177] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7Gpppm7GpN, 5' cap analog, where N is a natural or modified nucleotide, and the 5' cap analog has the structure of formula (Cap VI):

[0178] [ka] In the formula, B 3 is a natural or modified nucleobase, and R 1 , R 2 , R 3 , and R 4 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 3. In some embodiments, R 1 , R 2 , R 3 , and R 4 At least one of B is OH. 1 , G, m 7 G, or A. In some embodiments, B 1 is A or m 6 A and R 1 is OCH3, G is guanine, m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine. In some embodiments, n is 1.

[0179] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7Gpppm7GpG 5' cap analog having the structure of Formula (Cap VII):

[0180] [ka] In the formula, R 1, R 2 , R 3 , and R 4 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , R 3 , and R 4 In some embodiments, n is 1.

[0181] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7Gpppm7Gpm7G 5' cap analog having the structure of Formula (Cap VIII):

[0182] [ka] In the formula, R 1 , R 2 , R 3 , and R 4 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , R 3 , and R 4 In some embodiments, n is 1.

[0183] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7GpppA 5' cap analog having the structure of formula (Cap IX):

[0184] [ka] In the formula, R 1 , R 2 , and R 3 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , and R 3 In some embodiments, n is 1.

[0185] In some embodiments, a self-replicating RNA or mRNA of the disclosure comprises an m7GpppApN 5' cap analog, where N is a natural or modified nucleotide, and the 5' cap has the structure of formula (Cap X).

[0186] [ka] In the formula, B 3 is a natural or modified nucleobase, and R 1 , R 2 , R 3 , and R 4 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , R 3 , and R 4 At least one of B is OH. 3 , G, m 7 G, A, or m 6 A is guanine, G is guanine, and m 7G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine. In some embodiments, n is 1.

[0187] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7GpppAmpG 5' cap analog having the structure of formula (Cap XI).

[0188] [ka] In the formula, R 1 , R 2 , and R 4 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , and R 4 In some embodiments, the compound of formula Cap XI is 7 GpppAmpG and R 1 , R 2 , and R 4 are each OH, n is 1, and each L is a phosphate linkage. In some embodiments, n is 1.

[0189] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7GpppApm7G 5' cap analog having the structure of Formula (Cap XII).

[0190] [ka] In the formula, R 1 , R 2 , R 3 , and R 4are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , R 3 , and R 4 In some embodiments, n is 1.

[0191] In some embodiments, a self-replicating RNA or mRNA of the present disclosure comprises an m7GpppApm7G 5' cap analog having the structure of Formula (Cap XIII).

[0192] [ka] In the formula, R 1 , R 2 , and R 4 are each independently selected from halogen, OH, and OCH; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; each L is linked by a diester bond; mRNA represents an mRNA of the present disclosure linked at its 5' end; and n is 0 or 1. In some embodiments, R 1 , R 2 , and R 4 In some embodiments, n is 1.

[0193] Polyadenine (polyA) tail Polyadenylation is the addition of a poly(A) tail, a chain of adenine nucleotides typically about 100–120 monomers long, to mRNA or RNA capable of functioning as mRNA. In eukaryotes, polyadenylation is part of the process of generating mature mRNA for translation and begins when gene transcription terminates. The 3'-most segment of a newly produced pre-mRNA is first cleaved by a set of proteins. These proteins then synthesize a poly(A) tail at the 3' end. The poly(A) tail is important for nuclear export, translation, and mRNA stability. The tail shortens over time, and if short enough, the mRNA is enzymatically degraded. However, in some cell types, mRNAs with short poly(A) tails are stored in the cytosol for later activation by re-polyadenylation. In some embodiments, the poly(A) tail is either transcribed from a template polynucleotide (e.g., a template encoding the entire saRNA) or added post-transcriptionally. In some embodiments, an RNA molecule of the present disclosure comprises a poly-A tail encoded by the sequence of SEQ ID NO: 16. In some embodiments, the poly-A tail is encoded by a polynucleotide having the sequence of nucleotides 31-130 of SEQ ID NO: 16. In some embodiments, the poly-A tail is less than 100 nucleotides in length (e.g., 20-99 nucleotides in length). In some embodiments, the poly-A tail is less than 90, 80, 75, 70, 65, 60, 55, or 50 nucleotides in length.

[0194] In some embodiments, the RNA molecules of the present disclosure comprise a 3' tail region that can serve to protect the RNA from exonuclease degradation. The tail region can be a 3' poly(A) and / or a 3' poly(C) region. Preferably, the tail region is a 3' poly(A) tail. Any self-replicating RNA and any mRNA provided herein, as well as any 3' UTR of any self-replicating RNA or mRNA, can comprise a poly(A) tail. As used herein, a "3' poly(A) tail" is a polymer of consecutive adenine nucleotides that can range in size from, for example, 10 to 250 consecutive adenine nucleotides, 60 to 125 consecutive adenine nucleotides, 90 to 125 consecutive adenine nucleotides, 95 to 125 consecutive adenine nucleotides, 95 to 121 consecutive adenine nucleotides, 100 to 121 consecutive adenine nucleotides, 110 to 121 consecutive adenine nucleotides, 112 to 121 consecutive adenine nucleotides, 114 to 121 consecutive adenine nucleotides, or 115 to 121 consecutive adenine nucleotides. In some aspects, the 3' poly(a) tails described herein comprise about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, 240, 250, 260, 270, 280, 290, 300 consecutive adenine nucleotides, and any number or range therebetween.Preferably, the 3' poly(A) tails described herein are 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, In some embodiments, the 3' poly(A) tail described herein comprises 0, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 consecutive adenine nucleotides. In some embodiments, the 3' poly(A) tail described herein comprises 94 consecutive adenine nucleotides. In some embodiments, the 3' poly(A) tail described herein comprises 106 consecutive adenine nucleotides. In some embodiments, the 3' poly(A) tail described herein comprises 117 consecutive adenine nucleotides. In some embodiments, the 3' poly(A) tail described herein comprises 141 consecutive adenine nucleotides. The 3' poly(A) tail can be added using a variety of methods known in the art (e.g., using poly(A) polymerase to add a tail to synthetic RNA or in vitro transcribed RNA). Other methods include using a transcription vector encoding a poly(A) tail or using a ligase (e.g., by splint ligation using T4 RNA ligase and / or T4 DNA ligase), whereby poly(A) can be ligated to the 3' end of the sense RNA. In some embodiments, a combination of any of the above methods is utilized.

[0195] dna molecule In one aspect, provided herein is a DNA molecule encoding an RNA molecule disclosed herein. In another aspect, the DNA molecule provided herein further comprises a promoter. As used herein, the term "promoter" refers to a regulatory sequence that initiates transcription. A promoter can be operably linked to one or more polynucleotides of a DNA molecule provided herein, wherein the one or more polynucleotides of the DNA molecule correspond to one or more encoded polynucleotides of an RNA molecule provided herein. Generally, the promoter included in the DNA molecule provided herein comprises a promoter for in vitro transcription (IVT). Any promoter suitable for in vitro transcription, such as a T7 promoter, a T3 promoter, an SP6 promoter, and others, can be included in the DNA molecule provided herein. In one aspect, the DNA molecule provided herein comprises a T7 promoter. In another aspect, the promoter is located 5' of the 5'UTR included in the DNA molecule provided herein. In yet another aspect, the promoter is a T7 promoter located 5' of the 5'UTR included in the DNA molecule provided herein. In yet another aspect, the promoter overlaps with the 5'UTR.The promoter and 5'UTR may be about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, or The overlapping sequences may be about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, about 50 nucleotides, or more nucleotides.

[0196] In some embodiments, the DNA molecules provided herein comprise a promoter for in vivo transcription. Generally, the promoter for in vivo transcription is an RNA polymerase II (RNA pol II) promoter. Any RNA pol II promoter can be included in the DNA molecules provided herein, including constitutive promoters, inducible promoters, and tissue-specific promoters. Exemplary constitutive promoters include the cytomegalovirus (CMV) promoter, the EF1α promoter, the SV40 promoter, the PGK1 promoter, the Ubc promoter, the human beta-actin promoter, the CAG promoter, and others. Any tissue-specific promoter can be included in the DNA molecules provided herein. In one embodiment, the RNA pol II promoter is a muscle-specific promoter, a skin-specific promoter, a hypodermis-specific promoter, a liver-specific promoter, a spleen-specific promoter, a lymph node-specific promoter, or any other promoter with tissue specificity. The DNA molecules provided herein can also comprise an enhancer. Any enhancer that increases transcription can be included in the DNA molecules provided herein.

[0197] Design and synthesis of RNA and DNA molecules The RNA molecules provided herein can include any combination of RNA sequences provided herein, including, for example, any 5' UTR sequence, any sequence encoding a polyprotein including nsP1, nsP2, nsP3, and nsP4, any sequence encoding any transgene disclosed herein (e.g., an antigenic influenza polypeptide or fragment thereof), and any 3' UTR sequence provided herein. In some embodiments, the RNA molecules provided herein are self-replicating RNA molecules. Self-replicating RNA molecules can encode one or more viral replication proteins, including, for example, nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecules provided herein are mRNA molecules. Generally, mRNA molecules do not include sequences encoding a polyprotein for RNA replication.

[0198] In some aspects, the RNA molecules provided herein comprise modified nucleotides. For example, 0% to 100%, 1% to 100%, 25% to 100%, 50% to 100%, and 75% to 100% of the uracil nucleotides of the RNA molecule can be modified. In some aspects, 1% to 100% of the uracil nucleotides are N1-methylpseudouridine or 5-methoxyuridine. In some embodiments, 100% of the uracil nucleotides are N1-methylpseudouridine. In some embodiments, 100% of the uracil nucleotides are 5-methoxyuridine.

[0199] The RNA molecules of the present disclosure, such as self-replicating RNA or mRNA, may be obtained by any suitable means. Methods for producing RNA molecules are known in the art and will be readily apparent to those skilled in the art. The RNA molecules of the present disclosure may be prepared according to any available technique, including, but not limited to, chemical synthesis, in vitro transcription (IVT), or enzymatic or chemical cleavage of longer precursors.

[0200] In some embodiments, RNA molecules, such as self-replicating RNA or mRNA, of the present disclosure are generated from a primary complementary DNA (cDNA) construct. The cDNA construct can be generated on an RNA template by the action of a reverse transcriptase (e.g., an RNA-dependent DNA polymerase). The process of designing and synthesizing a primary cDNA construct described herein generally includes the steps of gene construction, RNA generation (with or without modification), and purification. In the IVT method, a target polynucleotide sequence encoding an RNA molecule of the present disclosure is first selected for incorporation into a vector that is amplified to generate a cDNA template. Optionally, the target polynucleotide sequence and / or any flanking sequences can be codon-optimized. The cDNA template is then used to generate the RNA molecule of the present disclosure via in vitro transcription (IVT). After generation, the RNA molecule of the present disclosure can undergo purification and cleanup processes, which are provided in more detail below.

[0201] Gene construction steps may include, but are not limited to, gene synthesis, vector amplification, plasmid purification, plasmid linearization and cleanup, and cDNA template synthesis and cleanup. Once a protein of interest is selected for production, a primary construct is designed. Within the primary construct, a first region of linked nucleosides encoding a polypeptide of interest may be constructed using the open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript. The ORF may include a wild-type ORF, an isoform, a variant, or a fragment thereof. As used herein, "open reading frame" or "ORF" refers to a nucleic acid sequence (DNA or RNA) capable of encoding a polypeptide of interest. ORFs often begin with an ATG start codon and terminate with a nonsense or stop codon or signal.

[0202] The cDNA template can be transcribed using an in vitro transcription (IVT) system to generate the RNA molecules of the present disclosure. This system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. The NTPs can be selected from, but are not limited to, those described herein, including natural and unnatural (modified) NTPs. The polymerase can be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, and mutant polymerases (e.g., polymerases capable of incorporating modified nucleic acids).

[0203] The primary cDNA template or the transcribed RNA sequence may also be subjected to a capping and / or tailing reaction. The capping reaction can be carried out by methods known in the art to add a 5' cap to the 5' end of the primary construct. Capping methods include, but are not limited to, using vaccinia capping enzyme (New England Biolabs, Ipswich, Mass.) or capping at the start of in vitro transcription, for example, by including a capping agent as part of the IVT reaction (Nuc. Acids Symp. (2009) 53:129). The poly(A) tailing reaction can be carried out by methods known in the art, such as, but not limited to, 2'O-methyltransferase, and by methods described herein. If the primary construct generated from cDNA does not contain polyT, it may be beneficial to carry out the poly(A) tailing reaction before washing the primary construct.

[0204] Codon-optimized cDNA constructs encoding transgenes for nonstructural proteins and self-replicating RNAs are particularly suitable for generating the self-replicating RNA sequences described herein. For example, such cDNA constructs can be used as a basis for in vitro transcription of polyribonucleotides encoding the protein of interest as part of the self-replicating RNA. Codon-optimized cDNA constructs can also be used to generate the mRNAs provided herein.

[0205] The present disclosure also provides expression vectors, preferably comprising a nucleotide sequence encoding a self-replicating RNA or mRNA operably linked to at least one regulatory sequence, which are art-recognized and selected to direct expression of the encoded polypeptide.

[0206] Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed.

[0207] The present disclosure also provides polynucleotides (e.g., DNA, RNA, cDNA, mRNA, etc.) directed to the self-replicating RNA or mRNA of the present disclosure, which can be operably linked to one or more regulatory nucleotide sequences in an expression construct, such as a vector or plasmid. In certain embodiments, such a construct is a DNA construct. The regulatory nucleotide sequence is generally appropriate for the host cell used for expression. Many types of appropriate expression vectors and suitable regulatory sequences are known in the art for various host cells.

[0208] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, transcriptional start and stop sequences, translational start and stop sequences, and an enhancer or activator sequence. Constitutive or inducible promoters known in the art are contemplated by embodiments of the present disclosure. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter.

[0209] The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In some embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0210] The present disclosure also provides host cells transfected with the self-replicating RNA, mRNA, or DNA described herein. The self-replicating RNA, mRNA, or DNA can encode any protein of interest, such as an antigen or any other viral glycoprotein, such as influenza virus hemagglutinin and neuraminidase. The host cell can be any prokaryotic or eukaryotic cell. For example, polypeptides encoded by the self-replicating RNA or mRNA can be expressed in bacterial cells, such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0211] Host cells transfected with an expression vector containing the self-replicating RNA or mRNA of the present disclosure can be cultured under appropriate conditions to allow expression of the self-replicating RNA or mRNA and translation of the polypeptide to occur. Once expressed, the self-replicating RNA generally undergoes self-amplification and translation. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide can be retained in the cytoplasm or membrane fraction, and the cells can be harvested, lysed, and the protein isolated. Cell culture includes host cells, medium, and other by-products. Suitable media for cell culture are well known in the art.

[0212] The expressed proteins described herein can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for particular epitopes of the polypeptide.

[0213] Compositions and pharmaceutical compositions In some embodiments, the present invention provides a composition comprising any of the RNA or DNA molecules provided herein. The compositions provided herein may comprise a lipid. In one aspect, the lipid is an ionizable cationic lipid. Any ionizable cationic lipid can be included in the composition comprising the nucleic acid molecules provided herein.

[0214] The compositions, molecules, and polynucleotides of the present disclosure can be used to immunize or vaccinate a subject against a viral infection, such as influenza. In some embodiments, the compositions, molecules, and polynucleotides of the present disclosure can be used to vaccinate or immunize a subject against any or all of four distinct influenza strains (e.g., two from influenza A subtypes, H1N1 and H3N2, and two from influenza B subtypes, Victoria and Yamagata).

[0215] Also provided herein in some embodiments is a pharmaceutical composition comprising any of the RNA molecules and DNA molecules provided herein and a lipid formulation. Any lipid can be included in the lipid formulation of the pharmaceutical compositions provided herein. In one aspect, the lipid formulation of the pharmaceutical compositions provided herein comprises an ionizable cationic lipid. Exemplary ionizable cationic lipids for the compositions and pharmaceutical compositions provided herein are listed in Table 6 below.

[0216] [Table 6-1]

[0217] [Table 6-2]

[0218] [Table 6-3]

[0219] [Table 6-4]

[0220] [Table 6-5]

[0221] [Table 6-6]

[0222] [Table 6-7]

[0223] [Table 6-8]

[0224] [Table 6-9]

[0225] [Table 6-10]

[0226] [Table 6-11]

[0227] In one aspect, the ionizable cationic lipid of the compositions provided herein has the structure:

[0228] [ka] or a pharmaceutically acceptable salt thereof.

[0229] In another aspect, the ionizable cationic lipid of the compositions provided herein has the structure:

[0230] [ka] or a pharmaceutically acceptable salt thereof.

[0231] Lipid formulation / LNP Therapies based on the intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. In fact, naked nucleic acid materials cannot be easily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocytic uptake, and their ability to activate immune responses—all characteristics that hinder their clinical development. When exogenous nucleic acid materials (e.g., mRNA) enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and removed from the blood circulation before having a chance to encounter target cells inside or outside the vascular system. It has been reported that the half-life of naked nucleic acids in the bloodstream is approximately several minutes (Kawabata K, Takakura Y, Hashida MPharm Res. 1995 Jun;12(6):825-30). Chemical modifications and appropriate delivery methods can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, thereby increasing the stability and efficacy of nucleic acid-based therapies. Furthermore, RNA or DNA are anionic hydrophilic polymers that are unfavorable for cellular uptake and are also anionic at the surface. Therefore, the success of nucleic acid-based therapies depends heavily on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0232] Furthermore, upon internalization into target cells, nucleic acid delivery vectors are challenged by intracellular barriers, including endosomal entrapment, lysosomal degradation, nucleic acid unpacking from the vector, translocation across the nuclear membrane (in the case of DNA), and release in the cytoplasm (in the case of RNA). Therefore, the success of nucleic acid-based therapy depends on the ability of the vector to deliver the nucleic acid to a target site inside the cell to achieve a desired activity, such as sufficient levels of gene expression.

[0233] While some gene therapies have successfully utilized viral delivery vectors (e.g., AAV), lipid-based formulations are increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and ease of large-scale production. One of the most notable advances in lipid-based nucleic acid therapy occurred in August 2018, when patisiran (ALN-TTR02) became the first siRNA therapeutic approved by the Food and Drug Administration (FDA) and the European Commission (EC). ALN-TTR02 is an siRNA formulation based on so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of patisiran, delivery of nucleic acid therapeutics, including mRNA, via lipid formulations remains under development.

[0234] Some art-recognized lipid-formulated delivery vehicles for nucleic acid therapeutics, according to various embodiments, include polymer-based carriers such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, both natural and synthetic exosomes, natural, synthetic, and semi-synthetic lamellar bodies, nanoparticles, micelles, and emulsions. These lipid formulations can vary in structure and composition, and as can be expected in a rapidly developing field, several different terms are used in the art to describe a single type of delivery vehicle. At the same time, terms related to lipid formulations vary in their intended meaning throughout the scientific literature, and this inconsistent use has led to confusion regarding the exact meaning of some terms related to lipid formulations. Among several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically detailed and defined herein for purposes of this disclosure.

[0235] In some embodiments, the compositions disclosed herein comprise a nitrogen-to-phosphate (N:P) ratio of about 3:1 to about 8:1 (e.g., about 5:1 to about 7:1). In some embodiments, the N:P ratio is about 7:1 or less. In some embodiments, the N:P ratio is about 6:1 or less. In some embodiments, the N:P ratio is about 5:1 or less.

[0236] In one aspect, the disclosure provides a composition comprising: (i) a polynucleotide having a length of about 5,000 to about 20,000 nucleotides; and (ii) an ionizable cationic lipid, wherein the composition comprises a nitrogen-to-phosphate (N:P) ratio of about 5:1 to about 7:1. In some embodiments, the nitrogen-to-phosphate (N:P) ratio is about 7:1. In some embodiments, the polynucleotide is a polynucleotide disclosed herein, which may have a length of at least 5,000 nucleotides (e.g., about 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, or more). In some embodiments, the polynucleotide has a length of about 7,500 nucleotides or more. In some embodiments, the polynucleotide has a length of about 10,000 nucleotides or more. In some embodiments, the polynucleotide has a length of about 15,000 nucleotides or more. In some embodiments, the ionizable cationic lipid is an ionizable cationic lipid disclosed herein (e.g., ATX-126 or ATX-240). In some embodiments, the N:P ratio is about 3:1 to about 8:1 (e.g., about 5:1 to about 7:1). In some embodiments, the N:P ratio is about 7:1 or less. In some embodiments, the N:P ratio is about 6:1 or less. In some embodiments, the N:P ratio is about 5:1 or less.

[0237] Liposomes Conventional liposomes are vesicles consisting of at least one bilayer and an internal aqueous compartment. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymersomes, niosomes, etc.). They generally exist as spherical vesicles and can range in size from 20 nm to several microns. Liposome formulations can be prepared as colloidal dispersions, or they can be lyophilized to reduce stability risks and improve the shelf life of liposome-based drugs. Methods for preparing liposome compositions are known in the art and are within the skill of those in the art.

[0238] Liposomes with only one bilayer are referred to as unilamellar, and liposomes with more than one bilayer are referred to as multilamellar. The most common types of liposomes are small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and multilamellar vesicles (MLVs). In contrast to liposomes, lysosomes, micelles, and reverse micelles are composed of a single lipid layer. While liposomes are generally considered to have a single internal compartment, some preparations may be multivesicular liposomes (MVLs), which consist of multiple discontinuous internal aqueous compartments separated by several non-concentric lipid bilayers.

[0239] Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, given that liposomes are essentially analogs of biological membranes and can be prepared from both natural and synthetic phospholipids (Int J Nanomedicine. 2014;9:1833-1843). In their use as drug delivery vehicles, liposomes have an aqueous core surrounded by a hydrophobic membrane, so that hydrophilic solutes dissolved in the core cannot easily pass through the bilayer, and hydrophobic compounds associate with the bilayer. Thus, liposomes can be loaded with hydrophobic and / or hydrophilic molecules. When liposomes are used to deliver nucleic acids, such as RNA, the nucleic acid is contained within the liposomal compartment in the aqueous phase.

[0240] Cationic Liposomes Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes that are made entirely or in part from positively charged lipids, or more specifically, lipids that contain both cationic groups and lipophilic moieties. In addition to the general characteristics of liposomes profiled above, the positively charged portion of the cationic lipids used in cationic liposomes offers several advantages and some unique structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic, and therefore will separate itself from the aqueous interior of the liposome and associate with other non-polar and hydrophobic species. Conversely, the cationic portion will associate with the aqueous medium, and more importantly, will associate with polar molecules and species that can be complexed in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being investigated for use in gene therapy, as they favor negatively charged nucleic acids through electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed herein below.

[0241] lipid nanoparticles In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNPs) have a structure containing a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase within them. Rather, lipids from the bilayer or monolayer shell are directly complexed with the internal compound, thereby encapsulating it within the solid core. Lipid nanoparticles are typically spherical vesicles with relatively uniform distribution of shape and size. Sources vary as to the size that qualifies a lipid particle as a nanoparticle, but there is some overlap in agreement that lipid nanoparticles can have diameters ranging from 10 nm to 1000 nm. However, more commonly, they are considered to be less than 120 nm or even less than 100 nm.

[0242] In the case of lipid nanoparticle nucleic acid delivery systems, the lipid shell is formulated to contain ionizable cationic lipids that can complex and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with an apparent pKa value of less than about 7 provide cationic lipids for complexing with the negatively charged backbone of nucleic acids, and have the advantage of being loaded into lipid nanoparticles at a pH value below the pKa of the positively charged ionizable lipid. Then, at physiological pH values, the lipid nanoparticles can adopt a relatively neutral exterior, allowing for a significant increase in the circulation half-life of the particles after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved tissue penetration for delivering therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0243] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for the delivery of nucleic acid drugs. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed with cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by a wide size distribution ranging from submicron scale to several microns. Lipoplexes such as Lipofectamine® reagent have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (conferred by cationic lipids) result in rapid plasma clearance, hemolysis and other toxicities, and immune system activation. In some embodiments, the nucleic acid molecules provided herein and the lipids or lipid formulations provided herein form lipid nanoparticles (LNPs).

[0244] In other aspects, the nucleic acid molecules provided herein are incorporated into lipid formulations (ie, lipid-based delivery vehicles).

[0245] In the context of the present disclosure, lipid-based delivery vehicles typically serve to transport desired RNA to target cells or tissues. The lipid-based delivery vehicle may be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, cationic liposome, or lipid nanoparticle containing the self-replicating RNA or mRNA of the present disclosure. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and the self-replicating RNA or mRNA of the present disclosure. In some embodiments, the lipid bilayer further comprises a neutral lipid or polymer. In some embodiments, the lipid formulation comprises a liquid medium. In some embodiments, the formulation further encapsulates a nucleic acid. In some embodiments, the lipid formulation further comprises a nucleic acid and a neutral lipid or polymer. In some embodiments, the lipid formulation encapsulates a nucleic acid.

[0246] The present disclosure provides lipid formulations comprising one or more RNA molecules encapsulated in lipid formulations.In some embodiments, the lipid formulation comprises liposomes.In some embodiments, the lipid formulation comprises cationic liposomes.In some embodiments, the lipid formulation comprises lipid nanoparticles.

[0247] In some embodiments, the self-replicating RNA or mRNA is completely encapsulated within the lipid portion of the lipid formulation, so that the RNA in the lipid formulation is resistant to nuclease degradation in aqueous solution.In other embodiments, the lipid formulations described herein are substantially non-toxic to animals, such as humans and other mammals.

[0248] Lipid formulations of the present disclosure also typically have a total lipid:RNA ratio (mass / mass) of about 1:1 to about 100:1, about 1:1 to about 50:1, about 2:1 to about 45:1, about 3:1 to about 40:1, about 5:1 to about 45:1, or about 10:1 to about 40:1, or about 15:1 to about 40:1, or about 20:1 to about 40:1, or about 25:1 to about 45:1, or about 30:1 to about 45:1, or about 32:1 to about 42:1, or about 34:1 to about 42:1. In some embodiments, the total lipid:RNA ratio (mass / mass) is about 30:1 to about 45:1. The ratio can be any value or subvalue within the recited range, including the endpoints.

[0249] The lipid formulations of the present disclosure typically have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about The lipid nanoparticles have an average diameter of 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter can be any value or subvalue within the stated range, including the endpoints. In addition, when present in the lipid nanoparticles of the present disclosure, nucleic acids are generally resistant to degradation by nucleases in aqueous solution.

[0250] In some embodiments, the lipid nanoparticles have a size of less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm, hi certain embodiments, the lipid nanoparticles have a size of about 55 nm to about 90 nm.

[0251] In some embodiments, the lipid formulation comprises a self-replicating RNA or mRNA, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid (e.g., one or more PEG-lipid conjugates) that inhibit particle aggregation. The lipid formulation can also comprise cholesterol. In one embodiment, the cationic lipid is an ionizable cationic lipid.

[0252] In nucleic acid-lipid formulations, RNA can be completely encapsulated within the lipid portion of the formulation, thereby protecting nucleic acid from nuclease degradation.In some embodiments, the lipid formulation comprising RNA is completely encapsulated within the lipid portion of the lipid formulation, thereby protecting nucleic acid from nuclease degradation.In certain embodiments, the RNA in the lipid formulation is not substantially degraded after the particles are exposed to nuclease at 37 ℃ for at least 20, 30, 45 or 60 minutes.In certain other embodiments, the RNA in the lipid formulation is not substantially degraded after the formulation is incubated in serum at 37 ℃ for at least 30, 45 or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours.In some embodiments, RNA is complexed with the lipid portion of the formulation. One of the advantages of the formulations of the present disclosure is that the nucleic acid-lipid compositions are substantially non-toxic to animals, including humans and other mammals.

[0253] In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that exhibits enhanced fluorescence when associated with nucleic acids. Encapsulation is determined by adding the dye to the lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon the addition of a small amount of nonionic surfactant. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation can be calculated as E = (I - I) / I, where I and I refer to the fluorescence intensity before and after the addition of surfactant.

[0254] In some embodiments, the present disclosure provides nucleic acid-lipid compositions comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid compositions comprise a plurality of RNA-liposomes. In some embodiments, the nucleic acid-lipid compositions comprise a plurality of RNA-cationic liposomes. In some embodiments, the nucleic acid-lipid compositions comprise a plurality of RNA-lipid nanoparticles.

[0255] In some embodiments, the lipid formulation comprises about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% The RNA contained within the lipid portion of the formulation may be fully encapsulated such that up to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction or range therein) of the RNA is encapsulated therein. The amount may be any value or subvalue within the recited range, including the endpoints. The RNA contained in any of the RNA-lipid compositions or RNA-lipid formulations provided herein may be self-replicating RNA or mRNA.

[0256] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0257] In some aspects, the nucleic acid molecules provided herein are lipid-formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In one aspect, the lipid formulation is (a) RNA of the present disclosure; (b) a cationic lipid; and (c) an aggregation-reducing agent (e.g., a polyethylene glycol (PEG) lipid or a PEG-modified lipid); (d) optionally a non-cationic lipid (such as a neutral lipid); and (e) optionally, a sterol.

[0258] In another aspect, the cationic lipid is an ionizable cationic lipid. Any ionizable cationic lipid can be included in the lipid formulation, including the exemplary cationic lipids provided herein.

[0259] In some embodiments, compositions comprising lipids and / or lipid formulations provided herein comprise an RNA molecule comprising (A) the sequence of SEQ ID NO: 1, (B) the sequence of SEQ ID NO: 2, (C) the sequence of SEQ ID NO: 3, or (D) the sequence of SEQ ID NO: 4. In some embodiments, compositions provided herein comprise lipid nanoparticles (LNPs). In some embodiments, compositions provided herein comprise lyophilized LNPs.

[0260] Provided herein, in some embodiments, is a lipid composition comprising: i. about 45 mol % to about 55 mol % of an ionizable cationic lipid having the structure of ATX-126:

[0261] [ka] Provided herein are lipid nanoparticle compositions comprising: ii. about 8 mol% to about 12 mol% DSPC, iii. about 35 mol% to about 42 mol% cholesterol, and iv. about 1.25 mol% to about 1.75 mol% PEG2000-DMG; and b. a lipid formulation comprising an RNA molecule having at least 80% identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, wherein the lipid formulation encapsulates the RNA molecule and the lipid nanoparticles have a size of about 60 to about 90 nm. In some embodiments, the lipid nanoparticle compositions provided herein are lyophilized.

[0262] Also in some embodiments, i. about 45 mol % to about 55 mol % of an ionizable cationic lipid having the structure of ATX-240:

[0263] [ka] Also provided herein is a lipid nanoparticle composition comprising: ii. about 8 mol% to about 12 mol% DSPC, iii. about 35 mol% to about 42 mol% cholesterol, and iv. about 1.25 mol% to about 1.75 mol% PEG2000-DMG; and b. a lipid formulation comprising an RNA molecule having at least 80% identity to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, wherein the lipid formulation encapsulates the RNA molecule and the lipid nanoparticles have a size of about 60 to about 90 nm. In some embodiments, the lipid nanoparticle compositions provided herein are lyophilized.

[0264] cationic lipids In one embodiment, the lipid nanoparticle formulation comprises (i) at least one cationic lipid, (ii) a helper lipid, (iii) a sterol (e.g., cholesterol), and (iv) a PEG-lipid. In another embodiment, the cationic lipid is an ionizable cationic lipid. In yet another embodiment, the lipid nanoparticle formulation comprises (i) at least one cationic lipid, (ii) a helper lipid, (iii) a sterol (e.g., cholesterol), and (iv) a PEG-lipid in a molar ratio of about 40-70% ionizable cationic lipid: about 2-15% helper lipid: about 20-45% sterol, and about 0.5-5% PEG-lipid. In a further embodiment, the cationic lipid is an ionizable cationic lipid.

[0265] In one embodiment, the lipid nanoparticle formulation comprises (i) at least one cationic lipid, (ii) a helper lipid, (iii) a sterol (e.g., cholesterol), and (iv) a PEG-lipid. In another embodiment, the cationic lipid is an ionizable cationic lipid. In yet another embodiment, the lipid nanoparticle formulation comprises (i) at least one cationic lipid, (ii) a helper lipid, (iii) a sterol (e.g., cholesterol), and (iv) a PEG-lipid in a molar ratio of about 40-70% ionizable cationic lipid: about 2-15% helper lipid: about 20-45% sterol, and about 0.5-5% PEG-lipid. In a further embodiment, the cationic lipid is an ionizable cationic lipid.

[0266] In the lipid formulations of the present disclosure, the cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, chloride, DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-Dilinoleylcarbamoyl 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt, DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (3-(N,N-Dilinoleylamino)-1,2-propanediol, DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (3-(N,N-D ioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or their analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z )-Octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino) (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28 31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), or any combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-dimethylammonium propane. Examples of suitable cationic lipids include, but are not limited to, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). Additionally, commercially available preparations of cationic lipids (e.g., LIPOFECTIN (containing DOTMA and DOPE, available from GIBCO / BRL) and Lipofectamine (containing DOSPA and DOPE, available from GIBCO / BRL)) can be used.

[0267] Other suitable cationic lipids are disclosed in WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493; U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U.S. Patent No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0268] The RNA-lipid formulations of the present disclosure can include a helper lipid, which can be referred to as a neutral helper lipid, a non-cationic lipid, a non-cationic helper lipid, an anionic lipid, an anionic helper lipid, or a neutral lipid. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been found to increase cellular uptake when a helper lipid is present in the formulation. (Curr. Drug Metab. 2014; 15(9): 882-92). For example, several studies have shown that neutral and zwitterionic lipids such as 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) are more fusogenic (i.e., facilitate fusion) than cationic lipids and can affect the polymorphic properties of lipid-nucleic acid complexes, promoting the lamellar-to-hexagonal phase transition and thus inducing cell membrane fusion and disruption ( Nanomedicine (Lond). 2014 Jan;9(1):105-20 ). Additionally, the use of helper lipids can help reduce any potential adverse effects from using many of the predominant cationic lipids, such as toxicity and immunogenicity.

[0269] Non-limiting examples of non-cationic lipids suitable for the lipid formulations of the present disclosure include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin, sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylcholine (POPC)), palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (DOPE)), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DOPE) dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,Examples of suitable phospholipids include dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably derived from fatty acids having C10 to C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0270] Further examples of non-cationic lipids include sterols such as cholesterol and their derivatives. As a helper lipid, cholesterol increases the charge spacing of the lipid layer in contact with the nucleic acid, more closely matching the charge distribution to that of the nucleic acid. (JRSoc.Interface.2012 Mar 7;9(68):548-561). Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, and cholesteryl decanoate, and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0271] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives.In other embodiments, the neutral lipid present in the lipid formulation comprises or consists of one or more phospholipids, for example, cholesterol-free lipid formulations.In still other embodiments, the neutral lipid present in the lipid formulation comprises or consists of cholesterol-free lipid formulations, for example, phospholipid-free lipid formulations.

[0272] Other examples of helper lipids include non-phosphorus-containing lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, and sphingomyelin.

[0273] Other suitable cationic lipids include those with alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids called amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. Generally, amino lipids with fewer saturated acyl chains are easier to size, especially when the complexes need to be sized to less than about 0.3 microns for sterilization filtration purposes. Amino lipids containing unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 may also be used. Other scaffolds may also be used to separate the amino group and fatty acid or fatty alkyl portion of the amino lipid.

[0274] In some embodiments, the lipid formulation comprises a cationic lipid having Formula I according to U.S. Patent Application Publication No. 20200163878(A1), the disclosure of which is also incorporated herein by reference in this regard.

[0275] In some embodiments, the amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood, of course, that the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all of the lipids be present in a charged or neutral form. Lipids with more than one protonatable or deprotonatable group, or lipids that are zwitterionic, are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of the protonatable group ranging from about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of the ionizable cationic lipid is about 6 to about 7.

[0276] In some embodiments, the lipid formulation comprises an ionizable cationic lipid of Formula I:

[0277] [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein R5 and R6 are each independently selected from the group consisting of straight or branched chain C1 to C31 alkyl, C2 to C31 alkenyl or C2 to C31 alkynyl and cholesteryl; L5 and L6 are each independently selected from the group consisting of straight chain C1 to C20 alkyl and C2 to C20 alkenyl; X5 is -C(O)O-, thereby forming -C(O)O-R6, or -OC(O) -, thereby forming -OC(O)-R6; X6 is -C(O)O-, thereby forming -C(O)O-R5, or -OC(O)-, thereby forming -OC(O)-R5; X7 is S or O; L7 is absent or lower alkyl; R4 is straight or branched C1-C6 alkyl; and R7 and R8 are each independently selected from the group consisting of hydrogen and straight or branched C1-C6 alkyl.

[0278] In some embodiments, X7 is S.

[0279] In some embodiments, X5 is -C(O)O-, thereby forming -C(O)O-R6, and X6 is -C(O)O-, thereby forming -C(O)O-R5.

[0280] In some embodiments, R7 and R8 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.

[0281] In some embodiments, L5 and L6 are each independently a C1-C10 alkyl. In some embodiments, L5 is a C1-C3 alkyl and L6 is a C1-C5 alkyl. In some embodiments, L6 is a C1-C2 alkyl. In some embodiments, L5 and L6 are each a straight-chain C7 alkyl. In some embodiments, L5 and L6 are each a straight-chain C9 alkyl.

[0282] In some embodiments, R5 and R6 are each independently alkenyl. In some embodiments, R6 is alkenyl. In some embodiments, R6 is C2-C9 alkenyl. In some embodiments, the alkenyl contains a single double bond. In some embodiments, R5 and R6 are each alkyl. In some embodiments, R5 is branched alkyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of C9 alkyl, C9 alkenyl, and C9 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of C11 alkyl, C11 alkenyl, and C11 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of C7 alkyl, C7 alkenyl, and C7 alkynyl. In some embodiments, R5 is -CH((CH2)pCH3)2 or -CH((CH2)pCH3)((CH2)p-1CH3), where p is 4 to 8. In some embodiments, p is 5 and L5 is C1-C3 alkyl. In some embodiments, p is 6 and L5 is C3 alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5 is C1-C3 alkyl. In some embodiments, R5 consists of -CH((CH2)pCH3)((CH2)p-1CH3) and p is 7 or 8.

[0283] In some embodiments, R4 is ethylene or propylene. In some embodiments, R4 is n-propylene or isobutylene.

[0284] In some embodiments, L7 is absent, R4 is ethylene, X7 is S, and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is n-propylene, X7 is S, and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is ethylene, X7 is S, and R7 and R8 are each ethyl.

[0285] In some embodiments, X7 is S, X5 is -C(O)O-, thereby forming -C(O)O-R6, X6 is -C(O)O-, thereby forming -C(O)O-R5, L5 and L6 are each independently a straight chain C3-C7 alkyl, L7 is absent, R5 is -CH((CH2)pCH3)2, and R6 is a C7-C12 alkenyl. In some further embodiments, p is 6 and R6 is a C9 alkenyl.

[0286] In embodiments, any one or more lipids described herein may be explicitly excluded.

[0287] In some embodiments, the helper lipid comprises about 2 mol% to about 20 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, about 6 mol% to about 12 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction or range therein) of the total lipid present in the lipid formulation.

[0288] The lipid moiety, or cholesterol or cholesterol derivative in the lipid formulation, may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 35 mol%, or about 28 mol% to about 35 mol%, or about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, or about 37 mol% of the total lipid present in the lipid formulation.

[0289] In certain embodiments, the lipid portion of the lipid formulation is about 35 mol % to about 42 mol % cholesterol.

[0290] In some embodiments, the phospholipid component in the mixture may comprise about 2 mol% to about 20 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, about 6 mol% to about 12 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction or range therein) of the total lipid present in the lipid formulation.

[0291] In certain embodiments, the lipid portion of the lipid formulation includes, but is not limited to, 40 mol % to about 60 mol % ionizable cationic lipid, about 4 mol % to about 16 mol % DSPC, about 30 mol % to about 47 mol % cholesterol, and about 0.5 mol % to about 3 mol % PEG2000-DMG.

[0292] In certain embodiments, the lipid portion of the lipid formulation may include, but is not necessarily limited to, about 42 mol% to about 58 mol% ionizable cationic lipid, about 6 mol% to about 14 mol% DSPC, about 32 mol% to about 44 mol% cholesterol, and about 1 mol% to about 2 mol% PEG2000-DMG.

[0293] In certain embodiments, the lipid portion of the lipid formulation may include, but is not necessarily limited to, about 45 mol% to about 55 mol% ionizable cationic lipid, about 8 mol% to about 12 mol% DSPC, about 35 mol% to about 42 mol% cholesterol, and about 1.25 mol% to about 1.75 mol% PEG2000-DMG.

[0294] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ±5 mole %.

[0295] A lipid formulation comprising a cationic lipid compound or an ionizable cationic lipid compound may be, on a molar basis, about 30-70% cationic lipid compound, about 25-40% cholesterol, about 2-15% helper lipid, and about 0.5-5% polyethylene glycol (PEG) lipid, where the percentages are based on the total lipid present in the formulation. In some embodiments, the composition is about 40-65% cationic lipid compound, about 25-35% cholesterol, about 3-9% helper lipid, and about 0.5-3% PEG-lipid, where the percentages are based on the total lipid present in the formulation.

[0296] The formulation may be, for example, a lipid particle formulation containing 8-30% nucleic acid compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine; 2-30% cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine; or up to 90% cationic lipid and 2-10% helper lipid, or even 100% cationic lipid.

[0297] lipid conjugates The lipid formulations described herein can further comprise lipid conjugates.Conjugated lipids are useful in preventing particle aggregation.Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.In addition, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the end of the attached PEG chain (Front Pharmacol.2015 Dec 1;6:286).

[0298] In some embodiments, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) as a coating or surface ligand in lipid formulations (a technique called PEGylation) protects nanoparticles from the immune system and helps them escape RES uptake (Nanomedicine (London). 2011 Jun;6(4):715-28). PEGylation has been used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Surfactant-like PEG lipids (e.g., PEG-DSPE) can enter lipid formulations and form a hydration layer and a steric barrier on the surface. Based on the degree of PEGylation, the surface layer can generally be divided into two types: a brush-like layer and a mushroom-like layer. In PEG-DSPE-stabilized formulations, PEG adopts a mushroom-like conformation at low PEGylation levels (usually less than 5 mol%) and will shift to a brush-like conformation when the PEG-DSPE content increases beyond a certain level (Journal of Nanomaterials. 2011;2011:12). PEGylation results in a significant increase in the circulating half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15;13():507-30, J. Control Release. 2010 Aug 3;145(3):178-81).

[0299] Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyl (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), methoxypolyethylene glycol (PEG-DMG or PEG2000-DMG), PEG coupled to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.

[0300] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), as well as compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0301] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain embodiments, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons). In some embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight can be any value or subvalue within the recited range, including the endpoints.

[0302] In certain embodiments, PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.PEG can be directly conjugated to lipid, or can be linked to lipid via a linker moiety.For example, any linker moiety suitable for coupling PEG to lipid can be used, including non-ester-containing linker moieties and ester-containing linker moieties.In one embodiment, the linker moiety is a non-ester-containing linker moiety. Exemplary non-ester containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCHCHC(O)-), succinamidyl (-NHC(O)CHCHC(O)NH-), ether, and combinations thereof (e.g., linkers comprising both carbamate and amide linker moieties). In one aspect, a carbamate linker is used to couple PEG to a lipid.

[0303] In some embodiments, an ester-containing linker moiety is used to couple PEG to a lipid. Exemplary ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0304] Phosphatidylethanolamines with various acyl chain groups of varying chain length and saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of 0 to 100 are preferred. Phosphatidylethanolamines containing mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0305] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In some embodiments, the PEG has an average molecular weight of about 750 or about 2,000 daltons. In some embodiments, the terminal hydroxyl group of the PEG is replaced with a methyl group.

[0306] In addition to the above, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose (e.g., hydroxymethylcellulose or hydroxyethylcellulose).

[0307] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol%, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5% (or any fraction or range therein) of the total lipid present in the lipid formulation. The amount can be any value or subvalue within the recited range, including the endpoints.

[0308] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations of the present disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ±0.5 mole %. One skilled in the art will understand that the concentration of lipid conjugate may vary depending on the lipid conjugate used and the rate at which the lipid formulation becomes fusogenic.

[0309] In some embodiments, the lipid formulation for any of the compositions described herein comprises a lipoplex, liposome, lipid nanoparticle, polymer-based particle, exosome, lamellar body, micelle, or emulsion.

[0310] Mechanisms of action for cellular uptake of lipid formulations In some embodiments, lipid formulations for intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating target cells using the target cell's endocytic mechanism, delivering the contents of the lipid delivery vehicle into the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3):146-157, 2018) Prior to endocytosis, functionalized ligands, such as PEG-lipids, on the surface of the lipid delivery vehicle are shed from the surface, triggering internalization into the target cell. During endocytosis, a portion of the cell's plasma membrane surrounds the vector, enveloping it in a vesicle, which then pinches off from the plasma membrane, enters the cytosol, and ultimately travels through the endolysosomal pathway. For ionizable cationic lipid-containing delivery vehicles, increasing acidity as endosomes age results in vehicles with a strong positive charge on the surface. Interaction of the delivery vehicle with the endosomal membrane then leads to a membrane fusion event that leads to cytoplasmic delivery of the payload. For RNA payloads, the cell's own internal translation processes will then translate the RNA into an encoded protein, which can undergo further post-translational processing, including transport to a targeted organelle or location within the cell or excretion from the cell.

[0311] By controlling the composition and concentration of the lipid conjugate, the rate at which the lipid conjugate is exchanged from the lipid formulation and subsequently the rate at which the lipid formulation becomes fusogenic can be controlled. Furthermore, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which the lipid formulation becomes fusogenic. Other methods that can be used to control the rate at which the lipid formulation becomes fusogenic will be apparent to those skilled in the art upon reading this disclosure. Furthermore, by controlling the composition and concentration of the lipid conjugate, the particle size of the liposome or lipid can be controlled.

[0312] Manufacture of lipid formulations There are many different methods for preparing lipid formulations containing nucleic acids. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, double asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.

[0313] Thin Film Hydration In the thin film hydration (TFH) or Bangham method, lipids are dissolved in an organic solvent and then evaporated using a rotary evaporator to form a thin lipid layer. After hydration of the layer with an aqueous buffer containing the compound to be loaded, multilamellar vesicles (MLVs) are formed, which can be reduced in size by extrusion through a membrane or by sonication of the starting MLVs to produce small unilamellar vesicles or large unilamellar vesicles (LUVs and SUVs).

[0314] Double emulsion Lipid formulations can also be prepared by a double emulsion technique, which involves dissolving lipids in a water / organic solvent mixture. The organic solution containing aqueous droplets is mixed with an excess of aqueous medium, resulting in the formation of a water-in-oil-in-water (W / O / W) double emulsion. After vigorous mechanical shaking, some of the aqueous droplets collapse, resulting in large unilamellar vesicles (LUVs).

[0315] Reverse Phase Evaporation The reverse phase evaporation (REV) method also allows for the creation of nucleic acid-loaded LUVs. In this technique, a two-phase system is formed by dissolving phospholipids in an organic solvent and an aqueous buffer. The resulting suspension is then sonicated for a short time until the mixture becomes a clear, one-phase dispersion. The lipid formulation is obtained after evaporating the organic solvent under reduced pressure. This technique has been used to encapsulate a variety of large and small hydrophilic molecules, including nucleic acids.

[0316] Microfluidic preparation Unlike other bulk techniques, microfluidic methods offer the possibility of controlling the lipid hydration process. These methods can be classified as continuous-flow microfluidics and droplet-based microfluidics according to the way the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method, which operates in continuous flow mode, lipids are dissolved in isopropyl alcohol, which is then hydrodynamically focused at a microchannel intersection between two aqueous buffer streams. Vesicle size can be controlled by adjusting the flow rate, thus controlling the lipid solution / buffer dilution process. This method can be used to produce oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three inlet ports and one outlet port.

[0317] Double asymmetric centrifugation Dual asymmetric centrifugation (DAC) differs from more conventional centrifugation by using an additional rotation about its own vertical axis. The two overlapping motions created achieve efficient homogenization. The sample is pushed outward as in a regular centrifuge, and then pushed toward the center of the vial by the additional rotation. Mixing the lipid and NaCl solutions results in a viscous vesicular phospholipid gel (VPC), which is then diluted to obtain a lipid formulation dispersion. Lipid formulation size can be adjusted by optimizing the DAC speed, lipid concentration, and homogenization time.

[0318] Ethanol injection The ethanol injection (EI) method can be used for nucleic acid encapsulation. This method involves rapidly injecting an ethanol solution containing dissolved lipids into an aqueous medium containing the nucleic acid to be encapsulated using a needle. Vesicles spontaneously form when the phospholipids are dispersed throughout the medium.

[0319] Detergent dialysis Detergent dialysis can be used to encapsulate nucleic acids. Briefly, lipids and plasmids are solubilized in a detergent solution of appropriate ionic strength, and after removal of the detergent by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acids are then removed by ion exchange chromatography, and empty vesicles are removed by sucrose density gradient centrifugation. This technique is highly sensitive to the cationic lipid content and salt concentration of the dialysis buffer, and the method is difficult to scale up.

[0320] Spontaneous vesicle formation upon ethanol dilution Stable lipid formulations can also be generated by spontaneous vesicle formation by the ethanol dilution method, in which stepwise or dropwise ethanol dilution provides for the immediate formation of nucleic acid-loaded vesicles by controlled addition of lipids dissolved in ethanol to a rapidly mixing aqueous buffer containing nucleic acid.

[0321] Encapsulation in preformed liposomes Entrapment of nucleic acids can also be achieved by two different methods, starting from preformed liposomes: (1) simple mixing of cationic liposomes with nucleic acids, yielding electrostatic complexes called "lipoplexes," which can be successfully used to transfect cell cultures but are characterized by their low encapsulation efficiency and poor performance in vivo; and (2) destabilization of liposomes, in which absolute ethanol is slowly added to a suspension of cationic vesicles to a concentration of 40% v / v, followed by dropwise addition of nucleic acid to achieve loaded vesicles. However, the two main steps that characterize the encapsulation process are very sensitive, and the particles must be miniaturized.

[0322] excipients The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow for sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct, or RNA), (4) alter biodistribution (e.g., targeting the polynucleotide, primary construct, or RNA to a particular tissue or cell type), (5) increase translation of the encoded protein in vivo, and / or (6) alter the release profile of the encoded protein in vivo.

[0323] The pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods include bringing into association the active ingredient (i.e., nucleic acid) with an excipient and / or one or more other accessory ingredients. Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0324] Pharmaceutical compositions may further comprise pharmaceutically acceptable excipients, which as used herein includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, and the like, appropriate for the particular dosage form desired.

[0325] In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, excipients of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with primary DNA constructs or RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0326] Thus, the pharmaceutical compositions described herein can include one or more excipients, each in an amount that together increase the stability of the nucleic acid in the lipid formulation, increase cell transfection with the nucleic acid, increase expression of the encoded protein, and / or modify the release profile of the encoded protein. Additionally, the RNA of the present disclosure can be formulated using self-assembling nucleic acid nanoparticles.

[0327] Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006, incorporated herein by reference in its entirety). The use of any conventional excipient vehicle may be contemplated within the scope of embodiments of the present disclosure, except insofar as the conventional excipient vehicle may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other components of the pharmaceutical composition.

[0328] The pharmaceutical compositions of the present disclosure may further contain pharmaceutically acceptable carrier substances, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof, as needed to approximate physiological conditions. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0329] In certain embodiments of the present disclosure, RNA-lipid formulations can be administered in sustained release formulations, for example, in compositions that contain sustained release polymers.Active agent can be prepared with carriers that prevent rapid release, for example, controlled release vehicles such as polymers, microencapsulated delivery systems or bioadhesive gels.The long-term delivery of RNA in various compositions of the present disclosure can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate hydrogel and gelatin.

[0330] Methods for inducing an immune response In some embodiments, provided herein are methods for inducing an immune response in a subject. Any type of immune response, including adaptive and innate immune responses, can be induced using the methods provided herein. In one aspect, the immune response induced using the methods provided herein includes an antibody response, a cellular immune response, or both an antibody response and a cellular immune response.

[0331] Methods of inducing an immune response provided herein include administering to a subject an effective amount of any composition, RNA, or DNA molecule, i.e., nucleic acid molecule, provided herein. In one aspect, a method of inducing an immune response includes administering to a subject an effective amount of any composition comprising an RNA molecule and a lipid provided herein. In another aspect, a method of inducing an immune response includes administering to a subject an effective amount of any pharmaceutical composition comprising an RNA molecule and a lipid formulation provided herein. In some aspects, the RNA molecules, compositions, and pharmaceutical compositions provided herein are vaccines that can elicit, for example, a protective or therapeutic immune response.

[0332] As used herein, the term "subject" refers to any individual or patient on whom the methods disclosed herein are performed. The term "subject" can be used interchangeably with the terms "individual" or "patient." While a subject can be a human, a subject can also be an animal, as will be understood by those skilled in the art. Thus, other animals, including livestock including rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, etc., and mammals such as primates (including monkeys, chimpanzees, orangutans, and gorillas), are included within the definition of a subject. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an RNA molecule, composition, or pharmaceutical composition described herein sufficient to effect the intended application, including, but not limited to, induction of an immune response and / or disease treatment as defined herein. A therapeutically effective amount may vary depending on the intended application (e.g., induction of an immune response, treatment, in vivo application), or the subject or patient and disease state to be treated, such as the weight and age of the subject, species, severity of the disease state, mode of administration, etc., and these can be readily determined by one skilled in the art. The term also applies to a dose that induces a specific response in target cells. A specific dose will vary depending on the particular RNA molecule, composition, or pharmaceutical composition selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which it is delivered.

[0333] Exemplary doses of nucleic acid molecule that can be administered are about 0.01 μg, about 0.02 μg, about 0.03 μg, about 0.04 μg, about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg, about 0.09 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0μg, about 1.5μg, about 2.0μg, about 2.5μg, about 3.0μg, about 3.5μg, about 4.0μg, about 4.5μg, about 5.0μg, about 5.5μg, about 6.0μg, about 6.5μg, about 7.0μg, about 7.5μg, about 8.0μg, about 8.5μg, about 9.0μg, about 9.5μg, about 10μg, about 11μg, about 12μg, about 13μ about 14μg, about 1 5μg, about 16μg, about 17μg, about 18μg, about 19μg, about 20μg, about 21μg, about 22μg, about 23μg, about 24μg, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 35μg, about 40μg, about 45μg, about 50μg, about 55μg, about 60μg, about 65μg, about 70μg, about 75μg, about 80μg, about

[0013] Examples of nucleic acid molecules include 85 μg, about 90 μg, about 95 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1,000 μg, or more, and any number or range therebetween. In one embodiment, the nucleic acid molecule is an RNA molecule. In another embodiment, the nucleic acid molecule is a DNA molecule. In yet another embodiment, the nucleic acid molecule comprises one RNA molecule or one DNA molecule. In a further embodiment, the nucleic acid molecule comprises two, three, four, five, six, seven, eight, or more different RNA or DNA molecules. The nucleic acid molecule may have a unit dose containing from about 0.01 μg to about 1,000 μg or more of nucleic acid in a single administration.

[0334] In some embodiments, the compositions provided herein that may be administered include about 0.01 μg, about 0.02 μg, about 0.03 μg, about 0.04 μg, about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg, about 0.09 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, Approximately 0.9μg, approximately 1.0μg, approximately 1.5μg, approximately 2.0μg, approximately 2.5μg, approximately 3.0μg, approximately 3.5μg, approximately 4.0μg, approximately 4.5μg, approximately 5.0μg, approximately 5.5μg, approximately 6. 0μg, about 6.5μg, about 7.0μg, about 7.5μg, about 8.0μg, about 8.5μg, about 9.0μg, about 9.5μg, about 10μg, about 11μg, about 12μg, about 13μg, about 14μ g, about 15μg, about 16μg, about 17μg, about 18μg, about 19μg, about 20μg, about 21μg, about 22μg, about 23μg, about 24μg, about 25μg, about 26μg, about 27μg, Approximately 28μg, approximately 29μg, approximately 30μg, approximately 35μg, approximately 40μg, approximately 45μg, approximately 50μg, approximately 55μg, approximately 60μg, approximately 65μg, approximately 70μg, approximately 75μg, approximately 80μg, approximately 85 μg, about 90 μg, about 95 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1,000 μg or more, and any number or range of nucleic acids and lipids therebetween.In other aspects, the pharmaceutical compositions provided herein that may be administered include about 0.01 μg, about 0.02 μg, about 0.03 μg, about 0.04 μg, about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg, about 0.09 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about .9μg, about 1.0μg, about 1.5μg, about 2.0μg, about 2.5μg, about 3.0μg, about 3.5μg, about 4.0μg, about 4.5μg, about 5.0μg, about 5.5μg, about 6.0μ g, about 6.5μg, about 7.0μg, about 7.5μg, about 8.0μg, about 8.5μg, about 9.0μg, about 9.5μg, about 10μg, about 11μg, about 12μg, about 13μg, about 14μg, Approximately 15μg, approximately 16μg, approximately 17μg, approximately 18μg, approximately 19μg, approximately 20μg, approximately 21μg, approximately 22μg, approximately 23μg, approximately 24μg, approximately 25μg, approximately 26μg, approximately 27μg, approximately 2 8μg, about 29μg, about 30μg, about 35μg, about 40μg, about 45μg, about 50μg, about 55μg, about 60μg, about 65μg, about 70μg, about 75μg, about 80μg, about 85μg , about 90 μg, about 95 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1,000 μg, or more, and any number or range therebetween. In some embodiments, the composition or pharmaceutical composition comprises one RNA molecule or one DNA molecule. In further embodiments, the composition or pharmaceutical composition comprises two, three, four, five, six, seven, eight, or more different RNA molecules or DNA molecules.

[0335] In one aspect, the compositions provided herein may have a unit dose amount containing about 0.01 μg to about 1,000 μg or more of nucleic acid and lipid in a single administration. In another aspect, the pharmaceutical compositions provided herein may have a unit dose amount containing about 0.01 μg to about 1,000 μg or more of nucleic acid and lipid formulation in a single administration. A vaccine unit dose may correspond to a unit dose of a nucleic acid molecule, composition, or pharmaceutical composition provided herein that can be administered to a subject. In one aspect, the vaccine composition of the present disclosure has a unit dose amount containing about 0.01 μg to about 1,000 μg or more of nucleic acid and lipid formulation in a single administration. In another aspect, the vaccine composition of the present disclosure has a unit dose amount containing about 0.01 μg to about 50 μg of nucleic acid and lipid formulation in a single administration. In yet another aspect, the vaccine composition of the present disclosure has a unit dose amount containing about 0.2 μg to about 20 μg of nucleic acid and lipid formulation in a single administration.

[0336] The compositions of the present disclosure may be in solid dosage form, which can be reconstituted in liquid before administration. Solids can be administered as powders. Solids can be in the form of capsules, tablets, or gels. In some embodiments, the pharmaceutical composition comprises a lyophilized nucleic acid-lipid formulation. In some embodiments, the lyophilized composition may comprise one or more lyoprotectants, such as, but not limited to, glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol, hydroxypropyl-β-cyclodextrin, and / or polyethylene glycol. In some embodiments, the lyophilized composition comprises a poloxamer, potassium sorbate, sucrose, or any combination thereof. In certain embodiments, the poloxamer is poloxamer 188. In some embodiments, the lyophilized compositions described herein may comprise about 0.01 to about 1.0% w / w poloxamer. In some embodiments, the lyophilized compositions described herein may comprise about 1.0 to about 5.0% w / w potassium sorbate. The percentage can be any value or subvalue within the stated range, including the endpoints.

[0337] In some embodiments, the lyophilized composition may comprise about 0.01 to about 1.0% w / w of nucleic acid molecules. In some embodiments, the composition may comprise about 1.0 to about 5.0% w / w of lipids. In some embodiments, the composition may comprise about 0.5 to about 2.5% w / w of TRIS buffer. In some embodiments, the composition may comprise about 0.75 to about 2.75% w / w of NaCl. In some embodiments, the composition may comprise about 5 to about 95% w / w sugar, about 10 to about 95% w / w sugar, about 15 to about 95% w / w sugar, about 20 to about 95% w / w sugar, about 25 to about 95% w / w sugar, about 30 to about 95% w / w sugar, about 35 to about 95% w / w sugar, about 40 to about 95% w / w sugar, about 45 to about 95% w / w sugar, about 50 to about 95% w / w sugar, about 55 to about 95% w / w sugar, about 60 to about 95% w / w sugar, about 65 to about 95% w / w sugar, about 70 to about 95% w / w sugar, about 75 to about 95% w / w sugar, about 80 to about 95% w / w sugar, or about 85 to about 95% w / w sugar. In some embodiments, the composition may comprise about 1 to about 50% w / w sugar, about 5 to about 50% w / w sugar, about 10 to about 50% w / w sugar, about 15 to about 50% w / w sugar, about 20 to about 50% w / w sugar, about 25 to about 50% w / w sugar, about 30 to about 50% w / w sugar, about 35 to about 50% w / w sugar, about 40 to about 50% w / w sugar, or about 45 to about 50% w / w sugar. In some embodiments, the composition comprises about 1 to about 20% w / w sugar, about 2 to about 20% w / w sugar, about 3 to about 20% w / w sugar, about 4 to about 20% w / w sugar, about 5 to about 20% w / w sugar, about 6 to about 20% w / w sugar, about 7 to about 20% w / w sugar, about 8 to about 20% w / w sugar, about 9 to about 20% w / w sugar, about 10 to about 20% w / w sugar, about 11 to about 20% w / w sugar, about 12 to about 20% w / w sugar, about 13 to about 20% w / w sugar, about 14 to about 20% w / w sugar, about 15 to about 20% w / w sugar, about 16 to about 20% w / w sugar, about 17 to about 20% w / w sugar, about 18 to about 20% w / w sugar, about 19 to about 20% w / w sugar, about 20 to about 20% w / w sugar, about 21 to about 20% w / w sugar, about 22 to about 22% w / w sugar, about 23 to about 23% w / w sugar, about 24 to about 24% w / w sugar, about 25 to about 25% w / w sugar, about 26 to about 26% w / w sugar, about 27 to about 27% w / w sugar, about 28 to about 28% w / w sugar, about 29 to about 29% w / w sugar, about 30 to about 30% w / w sugar, about 31 to about 31% w / w sugar, about 32 to about 32% w / w sugar, about 33 to about 33% w / w sugar, about 34 to about 34% w / w sugar, about 3 It may contain 0% w / w sugar, about 11 to about 20% w / w sugar, about 12 to about 20% w / w sugar, about 13 to about 20% w / w sugar, about 14 to about 20% w / w sugar, about 15 to about 20% w / w sugar, about 16 to about 20% w / w sugar, about 17 to about 20% w / w sugar, about 18 to about 20% w / w sugar, or about 19 to about 20% w / w sugar.In some embodiments, the composition may comprise about 1 to about 18% w / w sugar, about 2 to about 18% w / w sugar, about 3 to about 18% w / w sugar, about 4 to about 18% w / w sugar, about 5 to about 18% w / w sugar, about 6 to about 18% w / w sugar, about 7 to about 18% w / w sugar, about 8 to about 18% w / w sugar, about 9 to about 18% w / w sugar, about 10 to about 18% w / w sugar, about 11 to about 18% w / w sugar, about 12 to about 18% w / w sugar, about 13 to about 18% w / w sugar, about 14 to about 18% w / w sugar, about 15 to about 18% w / w sugar, about 16 to about 18% w / w sugar, or about 17 to about 18% w / w sugar. In some embodiments, the composition may comprise about 1 to about 16% w / w sugar, about 2 to about 16% w / w sugar, about 3 to about 16% w / w sugar, about 4 to about 16% w / w sugar, about 5 to about 16% w / w sugar, about 6 to about 16% w / w sugar, about 7 to about 16% w / w sugar, about 8 to about 16% w / w sugar, about 9 to about 16% w / w sugar, about 10 to about 16% w / w sugar, about 11 to about 16% w / w sugar, about 12 to about 16% w / w sugar, about 13 to about 16% w / w sugar, about 14 to about 16% w / w sugar, or about 15 to about 16% w / w sugar. In some embodiments, the compositions may comprise about 1 to about 12% w / w sugar, about 2 to about 12% w / w sugar, about 3 to about 12% w / w sugar, about 4 to about 12% w / w sugar, about 5 to about 12% w / w sugar, about 6 to about 12% w / w sugar, about 7 to about 12% w / w sugar, about 8 to about 12% w / w sugar, about 9 to about 12% w / w sugar, about 10 to about 12% w / w sugar, or about 11 to about 12% w / w sugar. The percentages may be any value or subvalue within the stated ranges, including the endpoints. The compositions provided herein may be lyophilized, liquid, frozen liquid, or liquid suspension.

[0338] In preferred embodiments, the dosage form of the pharmaceutical compositions described herein may be a liquid suspension of the RNA-lipid nanoparticles described herein. In some embodiments, the RNA in the RNA-lipid nanoparticles is self-replicating RNA. In some embodiments, the RNA in the RNA-lipid nanoparticles is mRNA. In some embodiments, the liquid suspension is in a buffer solution. In some embodiments, the buffer solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffer solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a sugar and glycerol or a combination of a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In certain embodiments, the composition comprises a HEPES, MOPS, TES, or TRIS buffer at a pH of about 7.0 to about 8.5. In some embodiments, the HEPES, MOPS, TES, or TRIS buffer can be at a concentration ranging from 7 mg / ml to about 15 mg / ml, and the pH or concentration can be any value or subvalue within the stated range, including the endpoints.

[0339] In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature below about 70°C. In some embodiments, the suspension is diluted with sterile water during intravenous administration. In some embodiments, intravenous administration comprises diluting the suspension with about 2 volumes to about 6 volumes of sterile water. In some embodiments, the suspension contains about 0.1 mg to about 3.0 mg of RNA / mL, about 15 mg / mL to about 25 mg / mL of ionizable cationic lipid, about 0.5 mg / mL to about 2.5 mg / mL of PEG-lipid, about 1.8 mg / mL to about 3.5 mg / mL of helper lipid, about 4.5 mg / mL to about 7.5 mg / mL of cholesterol, about 7 mg / mL to about 15 mg / mL of buffer, about 2.0 mg / mL to about 4.0 mg / mL of NaCl, about 70 mg / mL to about 110 mg / mL of sucrose, and about 50 mg / mL to about 70 mg / mL of glycerol. In some embodiments, the lyophilized RNA-lipid nanoparticle formulation can be resuspended in a buffer solution as described herein.

[0340] In some embodiments, compositions of the present disclosure are administered to a subject such that an RNA concentration of at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1.0 mg / kg, at least about 2.0 mg / kg, at least about 3.0 mg / kg, at least about 4.0 mg / kg, or at least about 5.0 mg / kg body weight is administered in a single dose or as part of a single treatment cycle. In some embodiments, compositions of the present disclosure are administered to a subject such that an RNA concentration of at least about 0.1 mg, at least about 0.5 mg, at least about 1.0 mg, at least about 2.0 mg, at least about 3.0 mg, at least about 4.0 mg, at least about 5.0 mg, at least about 6.0 mg, at least about 7.0 mg, at least about 8.0 mg, at least about 9.0 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, or at least about A total amount of at least about 55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, at least about 95 mg, at least about 100 mg, at least about 105 mg, at least about 110 mg, at least about 115 mg, at least about 120 mg, or at least about 125 mg of RNA is administered to the subject, in one or more doses up to a maximum dose of about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg of RNA.

[0341] Any route of administration can be included in the methods provided herein. In some aspects, the nucleic acid molecules, i.e., RNA or DNA molecules, compositions, and pharmaceutical compositions provided herein are administered, for example, intramuscularly, subcutaneously, intradermally, transdermally, intranasally, orally, sublingually, intravenously, intraperitoneally, topically, by aerosol, or via a pulmonary route, for example, by inhalation or spray. In some embodiments, the described pharmaceutical compositions are administered systemically. Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, intratracheal, or pulmonary, including inhalation, or intestinal administration; intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In certain embodiments, intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, the pharmaceutical composition is administered intravenously.

[0342] Pharmaceutical compositions can be administered to any desired tissue.In some embodiments, the RNA delivered is expressed in tissues different from the tissues to which lipid formulations or pharmaceutical compositions are administered.In preferred embodiments, RNA is delivered and expressed in the liver.

[0343] In other aspects, the nucleic acid molecules, ie, RNA or DNA molecules, compositions, and pharmaceutical compositions provided herein are administered intramuscularly.

[0344] In some aspects, the subject in which an immune response is induced is a healthy subject. As used herein, the term "healthy subject" refers to a subject that does not have a condition or disease, including, for example, an infectious disease or cancer, or does not have a condition or disease in which an immune response is induced. Thus, in some aspects, the nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered prophylactically, for example, to prevent an infectious disease. The nucleic acid molecules, compositions, or pharmaceutical compositions provided herein can also be administered therapeutically, i.e., to treat a condition or disease, such as an infection, after the onset of the condition or disease.

[0345] As used herein, the terms "treat," "treatment," "therapy," "therapeutic," and the like refer to achieving a desired pharmacological and / or physiological effect, including, but not limited to, palliating, delaying, or slowing the progression, reducing the effects or symptoms, preventing, inhibiting, or ameliorating the onset of a disease or disorder, and achieving beneficial or desired results, such as therapeutic and / or prophylactic benefit, with respect to a disease, disorder, or condition. As used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject, including a subject susceptible to or at risk for the disease but not yet diagnosed with it; (b) inhibiting the disease, i.e., halting its development; and (c) relieving the disease, i.e., causing regression of the disease. Therapeutic benefit includes eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in a subject, even though the subject may still be afflicted with the underlying disorder. In some embodiments, for prophylactic benefit, therapeutic compositions, including therapeutic or pharmaceutical compositions, are administered to subjects at risk of developing a particular disease or to subjects reporting one or more physiological symptoms of the disease, even if the disease has not been diagnosed. The methods of the present disclosure can be used on any mammal or other animal. In some embodiments, treatment results in a reduction or cessation of symptoms. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0346] The nucleic acid molecules, i.e., RNA or DNA molecules, compositions, and pharmaceutical compositions provided herein can be administered one or more times. Thus, the nucleic acid molecules, compositions, and pharmaceutical compositions provided herein can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The timing between two or more administrations can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks or more, and any number or range therebetween. In some embodiments, the timing between two or more administrations is 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or more, and any number or range therebetween. In other embodiments, the timing between two or more administrations can be 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more, and any number or range therebetween. The timing between the first administration and any subsequent administrations can be the same or different. In one embodiment, the nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered once.

[0347] More than one nucleic acid molecule, composition, or pharmaceutical composition can be administered in the methods provided herein. In one embodiment, two or more nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered simultaneously. In another embodiment, two or more nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered sequentially. Simultaneous administration and sequential administration can include any number and combination of nucleic acid molecules, compositions, or pharmaceutical compositions provided herein. Multiple nucleic acid molecules, compositions, or pharmaceutical compositions administered together or sequentially can contain transgenes encoding different antigenic proteins or fragments thereof. In this way, immune responses against different antigenic targets can be induced. Two, three, four, five, six, seven, eight, nine, ten, or more nucleic acid molecules, compositions, or pharmaceutical compositions containing transgenes encoding different antigenic proteins or fragments thereof can be administered simultaneously or sequentially. Any combination of nucleic acid molecules, compositions, and pharmaceutical compositions containing any combination of transgenes can be administered simultaneously or sequentially. In some embodiments, administration is simultaneous. In other embodiments, administration is sequential. The timing between two or more administrations can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks or more, and any number or range therebetween. In some embodiments, the timing between two or more administrations is 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more, and any number or range therebetween.In other aspects, the timing between two or more administrations can be 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more years, and any number or range therebetween. The timing between the first administration and any subsequent administrations can be the same or different. The nucleic acid molecules, compositions, and pharmaceutical compositions provided herein can be administered with any other vaccine or treatment.

[0348] After administering the composition to a subject, the protein product (e.g., antigen) encoded by the RNA of the present disclosure is detectable in the target tissue for at least about 1-7 days or longer. The amount of protein product necessary to achieve a therapeutic effect will vary depending on the antibody titer required to generate immunity to a pathogen or disease (e.g., influenza) in the patient. For example, the protein product may be present at a concentration of at least about 0.025-1.5 μg / ml (e.g., at least about 0.050 μg / ml, at least about 0.075 μg / ml, at least about 0.1 μg / ml, at least about 0.2 μg / ml, or at least about 0.4 μg / ml) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longer after administering the composition to the subject. In some embodiments, the agonist or antagonist may be detectable in the target tissue at a concentration (e.g., a therapeutic concentration) of at least about 0.3 μg / ml, at least about 0.4 μg / ml, at least about 0.5 μg / ml, at least about 0.6 μg / ml, at least about 0.7 μg / ml, at least about 0.8 μg / ml, at least about 0.9 μg / ml, at least about 1.0 μg / ml, at least about 1.1 μg / ml, at least about 1.2 μg / ml, at least about 1.3 μg / ml, at least about 1.4 μg / ml, or at least about 1.5 μg / ml.

[0349] In some embodiments, the compositions described herein may be administered once. In some embodiments, the compositions described herein may be administered twice.

[0350] In some embodiments, the composition may be administered in the form of a booster dose to a subject previously vaccinated against influenza.

[0351] In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject once a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject twice a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject three times a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject four times a month.

[0352] Alternatively, the compositions of the present disclosure may be administered in a local rather than systemic manner, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in a depot or sustained-release formulation. Local delivery can be affected in various ways depending on the tissue being targeted. For example, an aerosol containing a composition of the present disclosure can be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present disclosure can be injected, for example, at the site of injury, disease symptoms, or pain; the composition can be provided in a lozenge for oral, tracheal, or esophageal application; it can be supplied in the form of a liquid, tablet, or capsule for gastric or intestinal administration; it can be supplied in the form of a suppository for rectal or vaginal application; or it can be delivered to the eye using a cream, drops, or even an injection. Formulations containing compositions of the present disclosure complexed with therapeutic molecules or ligands can even be administered surgically, for example, in association with a polymer or other structure or substance that allows the composition to diffuse from the implantation site to surrounding cells. Alternatively, it can be applied surgically without the use of a polymer or support.

[0353] combination The RNA, such as the self-replicating RNA or mRNA provided herein, formulations thereof, or the encoded proteins described herein can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" is not intended to imply that the agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of the present disclosure. The compositions can be administered simultaneously with, prior to, or after one or more other desired therapeutic or medical procedures. Generally, each agent is administered at a dose and / or time schedule determined for that agent. Preferably, the therapeutic methods of the present disclosure involve delivering pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and / or alter their metabolism, inhibit their excretion, and / or alter their distribution in the body. As a non-limiting example, the RNA molecules of the present disclosure can be used in combination with pharmaceutical agents for immunizing or vaccinating a subject. Generally, it is expected that the drugs used in combination with the RNA molecules and formulations of the present disclosure will be used at levels that do not exceed the levels at which they are used individually.In some embodiments, the levels used in combination are lower than the levels at which they are used individually.In one embodiment, the combination can be administered individually or together according to a split-dose regimen known in the art.

[0354] range Throughout this disclosure, various aspects may be presented in a range format. It should be understood that any description in range format is merely for convenience and brevity and is not intended to be limiting. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.1, 2.2, 2.5, 3, 4, 4.75, 4.8, 4.85, 4.95, 5, 5.5, 5.75, 5.9, 5.00, and 6. This applies to any width range.

[0355] Illustrative Embodiments The present disclosure provides the following exemplary embodiments. Embodiment 1. A composition comprising one or more RNA molecules, wherein the one or more RNA molecules collectively encode a hemagglutinin (HA) polypeptide and a neuraminidase (NA) polypeptide of each of four different influenza virus strains. Embodiment 2. The composition of embodiment 1, wherein for each of four different influenza virus strains, the HA and NA polypeptides are encoded by the same RNA molecule. Embodiment 3. The composition of embodiment 1, wherein the HA and NA polypeptides of a first influenza virus strain are encoded by a first RNA molecule, the HA and NA polypeptides of a second influenza virus strain are encoded by a second RNA molecule, the HA and NA polypeptides of a third influenza virus strain are encoded by a third RNA molecule, and the HA and NA polypeptides of a fourth influenza virus strain are encoded by a fourth RNA molecule. Embodiment 4. The composition of embodiment 3, wherein the first, second, third, and fourth RNA molecules are present in an equimolar ratio. Embodiment 5. The composition of any one of embodiments 1-4, wherein each of the one or more RNA molecules further encodes one or more viral replication proteins. Embodiment 6 The composition of embodiment 5, wherein the one or more viral replication proteins is an alphavirus protein. Embodiment 7. The composition of embodiment 6, wherein each of the one or more RNA molecules encodes, in 5' to 3' order: (i) one or more viral replication proteins; (ii) one of the NA polypeptides; and (iii) one of the HA polypeptides. Embodiment 8 The composition of embodiment 6 or 7, wherein the alphavirus protein is from Venezuelan equine encephalitis virus (VEEV). Embodiment 9. The composition of any one of embodiments 6-8, wherein the one or more viral replication proteins comprises alphavirus nonstructural protein 1 (nsP1), alphavirus nonstructural protein 2 (nsP2), alphavirus nonstructural protein 3 (nsP3), alphavirus nonstructural protein 4 (nsP4), or any combination thereof. Embodiment 10. The composition of embodiment 9, wherein the one or more viral replication proteins comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the RNA sequence encoded by SEQ ID NO:13. Embodiment 11. The composition of any one of embodiments 5 to 10, wherein the sequence encoding at least one HA polypeptide and NA polypeptide of the one or more RNA molecules is preceded by a subgenomic promoter (sgP). Embodiment 12. The composition of any one of embodiments 1-11, wherein each HA polypeptide comprises an antigenic fragment of a respective HA protein. Embodiment 13 The composition of any one of embodiments 1-12, wherein each NA polypeptide comprises an antigenic fragment of a respective NA protein. Embodiment 14. The composition of any one of embodiments 1-13, wherein the four different influenza virus strains include one or more of H1N1, H3N2, Victoria-B, or Yamagata-B. Embodiment 15. The composition of embodiment 14, wherein the four different influenza virus strains include Victoria B / Austria / 1359417 / 2021, H3N2 A / Darwin / 6 / 2021, H1N1 A / Wisconsin / 588 / 2019, and Yamagata B / PHUKET / 3073 / 2013. Embodiment 16 The composition of any one of embodiments 1 to 15, wherein each of the one or more RNA molecules further comprises a 5' untranslated region (UTR). Embodiment 17. The composition of embodiment 16, wherein at least one 5'UTR comprises a viral 5'UTR, a non-viral 5'UTR, or a combination of viral and non-viral 5'UTR sequences. Embodiment 18. The composition of embodiment 17, wherein at least one 5'UTR comprises an alphavirus 5'UTR. Embodiment 19. The composition of embodiment 18, wherein the alphavirus 5'UTR comprises a Venezuelan equine encephalitis virus (VEEV) 5'UTR sequence. Embodiment 20 The composition of embodiment 16, wherein at least one 5'UTR comprises an RNA sequence encoded by SEQ ID NO:14. Embodiment 21 The composition of any one of embodiments 1 to 20, wherein each of the one or more RNA molecules further comprises a 3' untranslated region (UTR). Embodiment 22. The composition of embodiment 21, wherein at least one 3'UTR comprises a viral 3'UTR, a non-viral 3'UTR, or a combination of viral and non-viral 3'UTR sequences. Embodiment 23 The composition of embodiment 22, wherein at least one 3'UTR comprises an alphavirus 3'UTR sequence. Embodiment 24 The composition of embodiment 23, wherein the alphavirus 3'UTR comprises a Venezuelan equine encephalitis virus (VEEV) 3'UTR sequence. Embodiment 25. The composition of embodiment 21, wherein at least one 3'UTR comprises an RNA sequence encoded by SEQ ID NO: 15. Embodiment 26 The composition of any one of embodiments 1 to 25, wherein the RNA molecule further comprises a polyA tail. Embodiment 27 The composition of any one of embodiments 1 to 26, wherein one or more RNA molecules are self-replicating RNA molecules. Embodiment 28. The composition of embodiment 27, wherein the one or more RNA molecules comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4. Embodiment 29. A composition comprising one or more DNA molecules encoding one or more RNA molecules of the composition of any one of embodiments 1 to 28. Embodiment 30. The composition of embodiment 29, wherein each of the one or more DNA molecules comprises a promoter. Embodiment 31 The composition of embodiment 30, wherein the promoter of each of the one or more DNA molecules is located 5' of the 5'UTR. Embodiment 32 The composition of embodiment 31, wherein the promoter is a T7 promoter. Embodiment 33. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:5. Embodiment 34 The composition of embodiment 33, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:9. Embodiment 35. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:6. Embodiment 36 The composition of embodiment 35, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:10. Embodiment 37. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:7. Embodiment 38 The composition of embodiment 37, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:11. Embodiment 39. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:8. Embodiment 40 The composition of embodiment 39, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:12. Embodiment 41. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:9. Embodiment 42. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:10. Embodiment 43. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:11. Embodiment 44. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:12. Embodiment 45. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4. Embodiment 46 The composition of any one of embodiments 1 to 45, further comprising an ionizable cationic lipid. Embodiment 47. The ionizable cationic lipid has the structure of Formula I:

[0356] [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 and R 6 are each independently a straight-chain or branched-chain C 1~ C 31 Alkyl, C 2~ C 31 Alkenyl or C 2~ C 31 L is selected from the group consisting of alkynyl and cholesteryl; 5 and L 6 are each independently a linear C 1~ C 20 Alkyl and C 2~ C 20 alkenyl; and X 5 is -C(O)O-, thereby -C(O)OR 6 is formed, or -OC(O)-, whereby -OC(O)-R 6 is formed, and X 6 is -C(O)O-, thereby -C(O)OR 5 is formed, or -OC(O)-, whereby -OC(O)-R 5 is formed, and X 7 is S or O, and L 7 is absent or is lower alkyl, and R 4 is a linear or branched chain C 1~ C6 alkyl, and R 7 and R 8 are each independently hydrogen and a straight or branched chain C 1~ The composition of embodiment 46, wherein the C6 alkyl is selected from the group consisting of: Embodiment 48 The composition of embodiment 46, wherein the ionizable cationic lipid is selected from Table 6. Embodiment 49 The composition of embodiment 46, wherein the ionizable cationic lipid is ATX-126.

[0357] [ka] Embodiment 50 The composition of embodiment 46, wherein the ionizable cationic lipid is ATX-240.

[0358] [ka] Embodiment 51. The composition of any one of embodiments 46 to 50, wherein the composition comprises a nitrogen to phosphate ratio (N:P) of about 5:1 to about 7:1. Embodiment 52. A method of vaccinating a subject against influenza, the method comprising administering to the subject the composition of any one of embodiments 1-51. Embodiment 53. Use of the composition of any one of embodiments 1 to 51 in the preparation of a medicament for vaccinating a subject against influenza. Embodiment 54. A composition comprising: (i) a polynucleotide having a length of about 5,000 to about 20,000 nucleotides; and (ii) an ionizable cationic lipid, wherein the composition comprises a nitrogen to phosphate ratio (N:P) of about 5:1 to about 7:1. Embodiment 55. The composition of embodiment 54, wherein the nitrogen to phosphate ratio (N:P) is about 7:1. [Example]

[0359] Example 1 Vaccine design and construction Four separate RNAs were designed for use in a tetravalent vaccine, each encoding hemagglutinin (HA) and neuraminidase (NA) from the same influenza strain on a single self-amplifying RNA (saRNA). Each antigen was preceded by a subgenomic promoter (sgP) sequence from Venezuelan equine encephalitis virus (VEEV), which facilitated replication of the entire saRNA and drove transcription of the HA and NA upon delivery into host cells. The saRNA also contained a poly(A) tail approximately 130 nucleotides long.

[0360] Figure 2 shows a schematic diagram of a vaccine combining four separate saRNA molecules, each encoding, in 5' to 3' order, (i) a viral replication protein (replicase portion), (ii) an NA polypeptide, and (iii) an HA polypeptide. Each polypeptide is preceded by a subgenomic promoter, and the NA and HA polypeptides are derived from the same influenza strain (H1N1, H3N2, B Victoria, or B Yamagata, as shown). The saRNAs shown in Figure 2 can be encoded by a DNA construct and prepared, for example, by in vitro transcription from a T7 promoter. The saRNA generally contains a poly(A) tail at the 3' end (not shown), which can be encoded by a DNA construct. Constructs and saRNAs encoding both the NA and HA polypeptides are referred to herein as "bicistronic."

[0361] Example 2 immunogenicity The immunogenicity of the bicistronic design was tested using two hemagglutinin antigens, each in either the 5' or "first" position or the 3' or "second" position. The saRNA constructs encoded hemagglutinins from H1 and H3 strains on a single saRNA, but in different orders. Female BALB / c mice were vaccinated with a single dose of one of the saRNA constructs, and post-vaccination HA inhibitory titers were measured for each virus (n = 5 / group). Serum was collected at the indicated time points (days 0-100 post-vaccination) and assessed for hemagglutinin inhibitory terminal titers against either H1N1 or H3N2 influenza HA. ​​When H1 was encoded in the "first" position, the H1N1 HA inhibitory titer was lower than that of constructs in which H1 was encoded in the second position (Figure 3A). Similarly, lower H3N2 HA inhibitory titers are observed when H3 is encoded in the "first" position compared to when H3 is encoded in the "second" position (Fig. 3B).

[0362] In summary, the data from this study demonstrated that when the HA antigen is located in the "second" position in a bicistronic vector, it is more immunogenic than when it is located in the "first" position.

[0363] Example 3 Codon-optimized sequences This example describes immunogenicity results for constructs with antigen sequences optimized using two different codon optimizations: one called "high codon adaptation index" (hCAI) and the other called "CODEX." To assess differences in immunogenicity, a conventional mRNA vaccine encoding hemagglutinin from the H3N2 A / Cambodia / e0826360 / 2020 strain of influenza was prepared. The only difference between the two vaccine formulations was the sequence encoding the HA antigen (the HA polypeptide sequence encoded by each codon-optimized sequence was the same).

[0364] Female BALB / c mice were vaccinated with either the hCAI-based or CODEX-based RNA vaccine at either a 2 μg or 10 μg dose, respectively. HA-specific IgG was assessed by a Meso Scale Discovery (MSD) ELISA-based assay using serum collected 28 days after vaccination. PBS was used as a control. The results are shown in Figure 4. Constructs using hCAI-based codon optimization achieved higher HA-specific responses than those using CODEX-based optimization.

[0365] Based on these results, hCAI codon optimization was used to generate the HA- and NA-encoding sequences of SEQ ID NOs: 1-4.

[0366] Example 4 Monovalent and tetravalent vaccine design Female BALB / c mice were vaccinated with either 0.5 μg of the indicated monovalent saRNA (encoding the NA and HA for the strains indicated in each of Figures 5A-5D) or a tetravalent vaccine in which each of the four saRNAs used in the monovalent vaccine was mixed in a 1:1:1:1 ratio and then formulated. Binding IgG antibody titers against the indicated strains were measured in serum obtained at each of the indicated time points. Figure 5A shows results for H1N1 A / Wisconsin / 588 / 2019 influenza. Figure 5B shows results for H3N2 A / Darwin / 6 / 2021 influenza. Figure 5C shows results for Victoria B / Austria / 1359417 / 2021 influenza. Figure 5D shows results for Yamagata B / Phuket / 3073 / 2013 influenza.

[0367] The data show that for most strains, a decrease in strain-specific HA antibody titers was observed with the tetravalent vaccine compared to the corresponding monovalent vaccine, however, the decrease was relatively small and the levels of antibody to each antigen were comparable.

[0368] Example 5 Immunogenicity and reactogenicity in formulations with different nitrogen:phosphate (N:P) ratios To evaluate the potential effect of the nitrogen-to-phosphate ratio in vaccine formulations on reactogenicity and tolerability, three N:P ratios were tested: 9:1, 7:1, and 5:1. Mice were vaccinated with modified conventional mRNA encoding H1N1 A / California / 07 / 2009 hemagglutinin formulated with the ionizable cationic lipid ATX-126 to achieve the indicated N:P ratios. Mice were tested for HA-specific antibodies (Figure 6B) and weight loss as a measure of reactogenicity (Figure 6A). Lower N:P ratios resulted in better-tolerated formulations while maintaining immunogenicity. A 7:1 ratio maintained an immunogenicity profile similar to that of the 9:1 formulation. Therefore, further screening of lipids was performed at the 7:1 ratio.

[0369] Example 6 Evaluation of vaccine formulations with different lipids We evaluated the tolerability and immunogenicity of vaccine formulations that varied by the amount of saRNA administered and the ionizable cationic lipid compound selected for the formulation. Tolerability was defined in mouse experiments as clinical observations (fur ruffling, activity, hunched posture) and weight loss. Because modified mRNA was better tolerated than unmodified RNA, and therefore the contribution of lipids to tolerability could be more easily assessed, formulations were first tested with modified mRNA. The top-ranking formulations were then tested with saRNA, and similar results were observed with modified conventional mRNA, except that the difference in the context of saRNA was smaller than with modified conventional mRNA.

[0370] Female BALB / c mice were immunized with modified conventional mRNA encoding hemagglutinin from A / California / 07 / 2009 formulated with various doses of different lipids. Serum collected on day 28 was used to determine HA-specific antibodies, while peak weight loss on day 3 was used as a measure of tolerability, and PBS was used as a control. Results for the indicated lipids and doses are shown in Figures 7A-7B. Figures 7A-7B show serum HA-specific antibody levels (reported in ECL units) versus percentage change in mouse body weight. Lipid compounds tested included ATX-126, ATX-2, ATX-194, ATX-95, ATX-221, ATX-88, ATX-239, and ATX-240. Vaccinations were administered at doses of 2 μg, 20 μg, 40 μg, or 80 μg. ATX-194 and ATX-95 showed comparable immunogenicity and tolerability (Figure 7A, no circle, center), while ATX-2 showed the least weight variation and the lowest immunogenicity (Figure 7A, no circle, bottom right). ATX-126 remained the most immunogenic ionizable lipid (Figure 7A), while formulations containing ATX-240 and ATX-239 had acceptable immunogenicity and were better tolerated at lower levels (Figure 7B).

[0371] Similar experiments were performed using saRNA instead of conventional mRNA in a top-tier formulation. Figure 7C shows the serum levels of bound IgG antibodies (AU / mL) versus the percentage change in body weight of immunized female BALB / c mice. Lipids tested in the saRNA experiments included ATX-126, ATX-221, ATX-239, and ATX-240. Vaccinations were performed at doses of 0.5 μg, 10 μg, 20 μg, and 40 μg. Serum collected on day 28 was used to determine HA-specific antibodies, while weight loss (peaking on day 3) was used as a measure of tolerability. PBS was used as a control. All vaccines demonstrated similar immunogenicity and tolerability at the lowest dose of 0.5 mg (Figure 7C, circle, bottom right of graph). At the 40 mg dose, ATX-126, ATX-239, and ATX-240 were most immunogenic, with ATX-221 being best tolerated (Figure 7C, diamonds). All vaccines were similarly immunogenic at the 20 mg dose, with ATX-221 being best tolerated (Figure 7C, triangles). At the 10 mg dose, similar immunogenicity was observed, with ATX-221 being best tolerated (Figure 7C, squares).

[0372] Female New Zealand White rabbits were vaccinated with 40 μg of saRNA formulated with either ATX-126 or ATX-240, and tolerability in rabbits was assessed using a surrogate for c-reactive protein (CRP) as a marker of systemic reactogenicity. Measurements were taken from pre-dose to 120 hours post-dose. As shown in Figure 7D, elevated CRP levels were observed at 48 hours with the ATX-126 formulation, while only a slight increase was observed with the ATX-240 formulation. The difference in CRP levels for the different formulations was confirmed in repeated studies using non-human primates (NHPs). Figure 7E shows serum c-reactive protein (CRP) levels (ng / mL) in non-human primates (NHPs) vaccinated with tetravalent modified conventional mRNA (encoding both HA and NA from the 2021 / 22 recommended strain) formulated with ATX-126, ATX-239, and ATX-240 at a dose of 30 μg each. Elevated CRP levels were observed only for formulations containing ATX-126. Formulations containing ATX-239 and -240, which contain the same drug substance, showed lower CRP levels 24 hours after administration.

[0373] Hemagglutinin-specific antibodies at day 28 from the same NHP experiment showed similar immunogenicity profiles among the three formulations tested. Figures 7F–7H show results for three of the four strains tested. Results report strain-specific HA IgG antibody titers for H1N1 A / Wisconsin / 588 / 2019 (Figure 7F), H3N2 A / Cambodia / e0826360 / 2020 (Figure 7G), and B Victoria B / Washington / 02 / 2019 (Figure 7H). Results for the Yamagata strain were similar. Results showed that the same trends observed in NHPs were also observed in mice, with conventional mRNA formulated with ATX-240 being slightly less immunogenic than mRNA formulated with ATX-126.

[0374] Example 7 Multicistronic design To test the feasibility of higher-order multicistronic expression cassettes, two saRNAs encoding HA and NA, each from two different strains (two tetracistronic constructs), were prepared and compared with bicistronic tetravalent formulations and conventional mRNAs.

[0375] To prepare the tetravalent influenza vaccine, HA and NA from FDA-recommended strains for cell-based vaccines for the 2021 / 2022 Northern Hemisphere influenza season were encoded in self-amplifying RNA (prepared at 2 μg doses) or conventional N1-pseudouridine-modified mRNA (prepared at 2 μg and 20 μg doses). The antigens in the conventional modified mRNA were encoded on separate RNAs and combined in equal amounts (by mass) during the lipid encapsulation process. The NA and HA from each strain were encoded on the same saRNA (four RNA groups, as shown in the RNA schematic in Figure 2), or two NAs and two HAs were encoded on the same saRNA (two RNA groups: NA2-NA1-HA3-HA1 and NA(Victoria)-NA(Yamagata)-HA(Victoria)-HA-Yamagata). Each antigen encoded by the saRNA is preceded by a subgenomic promoter. Figures 8A-8D show HA-specific IgG antibody responses for each influenza subtype (Figure 8A: H1N1 A / Wisconsin / 588 / 2019, Figure 8B: H3N2 A / Cambodia / e0826360 / 2020, Figure 8C: B Victoria B / Washington / 02 / 2019, Figure 8D: B Yamagata B / Phuket / 3073 / 2013), while Figures 8E-8H show NA-specific IgG responses for each influenza subtype (Figure 8E: H1N1 A / Wisconsin / 588 / 2019, Figure 8F: H3N2 A / Cambodia / e0826360 / 2020, Figure 8G: B Victoria B / Washington / 02 / 2019, Figure 8H: B Yamagata B / Phuket / 3073 / 2013).

[0376] It was found that up to four influenza antigens can be encoded on an mRNA molecule, and that when four antigens are encoded on one saRNA (the "2 samRNA" group), the response was lower than when only two antigens from the same strain, one HA and one NA, were encoded on one RNA (the "4 samRNA" group).

[0377] Example 8 Internal ribosome entry site (IRES) Internal ribosome entry sites (IRES) from several organisms were tested in bicistronic saRNAs. IRES sequences were inserted 3' of the subgenomic promoter (sgP) in various combinations. A schematic diagram of the tested constructs is shown in Figure 1, and the sequences of the various constructs are represented by SEQ ID NOS: 151-161. Female BALB / c mice (8-10 weeks old) were vaccinated with a single saRNA encoding both the NA and HA of H3N2 A / Cambodia / e0826360 / 2020, with the NA in the "first" position and the HA in the "second" position. The subgenomic promoter was located 5' of each antigen of interest, except as indicated in Figures 9A-9D. The IRES sequence was located between the sgP and the antigen of interest. Figures 9A-9B show hemagglutinin-specific IgG antibody responses from serum collected on the indicated days after vaccination (x-axis), while Figures 9C-9D show neuraminidase-specific IgG antibody responses from serum collected on the indicated days after vaccination (x-axis).

[0378] Constructs in which EMCV was located between the sgP and the antigen of interest generally showed slightly better responses, although these responses were not found to be significant with respect to the number of mice used. Furthermore, the results show that antigens preceded by the EMC IRES were typically slightly more immunogenic than those without the IRES sequence, but these differences were not significant.

[0379] array For SEQ ID NOs: 1-4, italicized letters indicate replicase sequences of nsP1-4, bold text indicates NA sequences, and bold and underlined text indicates HA sequences.

[0380] [Table 7-1]

[0381] [Table 7-2]

[0382] [Table 7-3]

[0383] [Table 7-4]

[0384] [Table 7-5]

[0385] [Table 7-6]

[0386] [Table 7-7]

[0387] [Table 7-8]

[0388] [Table 7-9]

[0389] Table 7-10

[0390] Table 7-11

[0391] Table 7-12

[0392] Table 7-13

[0393] Table 7-14

[0394] Table 7-15

[0395] Table 7-16

[0396] Table 7-17

[0397] Table 7-18

[0398] Table 7-19

[0399] Table 7-20

[0400] Table 7-21

[0401] Table 7-22

[0402] Table 7-23

[0403] Table 7-24

[0404] Table 7-25

[0405] Table 7-26

[0406] Table 7-27

[0407] Table 7-28

[0408] Table 7-29

[0409] Table 7-30

[0410] Table 7-31

[0411] Table 7-32

[0412] Table 7-33

[0413] Table 7-34

[0414] Table 7-35

[0415] Table 7-36

[0416] Table 7-37

[0417] Table 7-38

[0418] Table 7-39

[0419] Table 7-40

[0420] Table 7-41

[0421] Table 7-42

[0422] Table 7-43

[0423] Table 7-44

[0424] Table 7-45

[0425] Table 7-46

[0426] Table 7-47

[0427] Table 7-48

[0428] Table 7-49

[0429] Table 7-50

[0430] Table 7-51

[0431] Table 7-52

[0432] Table 7-53

[0433] Table 7-54

[0434] Table 7-55

[0435] Table 7-56

[0436] Table 7-57

[0437] Table 7-58

[0438] Table 7-59

[0439] Table 7-60

[0440] [Table 7-61]

[0441] [Table 7-62]

[0442] [Table 7-63]

[0443] 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.

[0444] Any and all references and citations made throughout this disclosure to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, and the like, are incorporated herein in their entirety for all purposes. Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A composition comprising one or more RNA molecules, wherein the one or more RNA molecules collectively encode hemagglutinin (HA) polypeptides and neuraminidase (NA) polypeptides of each of four different influenza virus strains.

2. 2. The composition of claim 1, wherein for each of the four different influenza virus strains, the HA polypeptide and the NA polypeptide are encoded by the same RNA molecule.

3. 2. The composition of claim 1, wherein the HA and NA polypeptides of a first influenza virus strain are encoded by a first RNA molecule, the HA and NA polypeptides of a second influenza virus strain are encoded by a second RNA molecule, the HA and NA polypeptides of a third influenza virus strain are encoded by a third RNA molecule, and the HA and NA polypeptides of a fourth influenza virus strain are encoded by a fourth RNA molecule.

4. The composition of claim 3 , wherein the first, second, third, and fourth RNA molecules are present in an equimolar ratio.

5. The composition of claim 1 , wherein each of the one or more RNA molecules further encodes one or more viral replication proteins.

6. 6. The composition of claim 5, wherein the one or more viral replication proteins are alphavirus proteins.

7. The composition of claim 6, wherein each of the one or more RNA molecules encodes, in 5' to 3' order, (i) the one or more viral replication proteins, (ii) one of the NA polypeptides, and (iii) one of the HA polypeptides.

8. 7. The composition of claim 6, wherein the alphavirus protein is derived from Venezuelan equine encephalitis virus (VEEV).

9. The composition of claim 6, wherein the one or more viral replication proteins comprise alphavirus nonstructural protein 1 (nsP1), alphavirus nonstructural protein 2 (nsP2), alphavirus nonstructural protein 3 (nsP3), alphavirus nonstructural protein 4 (nsP4), or any combination thereof.

10. 10. The composition of claim 9, wherein the one or more viral replication proteins comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to an RNA sequence encoded by SEQ ID NO:

13.

11. 6. The composition of claim 5, wherein the sequence encoding the HA polypeptide and the NA polypeptide of at least one of the one or more RNA molecules is preceded by a subgenomic promoter (sgP).

12. The composition of claim 1 , wherein each HA polypeptide comprises an antigenic fragment of a respective HA protein.

13. The composition of claim 1 , wherein each NA polypeptide comprises an antigenic fragment of a respective NA protein.

14. 2. The composition of claim 1, wherein the four different influenza virus strains include one or more of H1N1, H3N2, Victoria-B, or Yamagata-B.

15. 15. The composition of claim 14, wherein the four different influenza virus strains include Victoria B / Australia / 1359417 / 2021, H3N2 A / Darwin / 6 / 2021, H1N1 A / Wisconsin / 588 / 2019, and Yamagata B / PHUKET / 3073 / 2013.

16. 2. The composition of claim 1, wherein each of the one or more RNA molecules further comprises a 5' untranslated region (UTR).

17. 17. The composition of claim 16, wherein at least one 5'UTR comprises a viral 5'UTR, a non-viral 5'UTR, or a combination of viral and non-viral 5'UTR sequences.

18. 18. The composition of claim 17, wherein the at least one 5'UTR comprises an alphavirus 5'UTR.

19. 19. The composition of claim 18, wherein the alphavirus 5'UTR comprises a Venezuelan equine encephalitis virus (VEEV) 5'UTR sequence.

20. 17. The composition of claim 16, wherein at least one 5'UTR comprises an RNA sequence encoded by SEQ ID NO:

14.

21. 2. The composition of claim 1, wherein each of the one or more RNA molecules further comprises a 3' untranslated region (UTR).

22. 22. The composition of claim 21, wherein at least one 3'UTR comprises a viral 3'UTR, a non-viral 3'UTR, or a combination of viral and non-viral 3'UTR sequences.

23. 23. The composition of claim 22, wherein the at least one 3'UTR comprises an alphavirus 3'UTR sequence.

24. 24. The composition of claim 23, wherein the alphavirus 3'UTR comprises a Venezuelan equine encephalitis virus (VEEV) 3'UTR sequence.

25. 22. The composition of claim 21, wherein at least one 3'UTR comprises an RNA sequence encoded by SEQ ID NO:

15.

26. The composition of claim 1 , wherein the RNA molecule further comprises a polyA tail.

27. The composition of claim 1 , wherein the one or more RNA molecules are self-replicating RNA molecules.

28. 28. The composition of claim 27, wherein the one or more RNA molecules comprise a sequence having at least 80%, at least 85%, 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 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4.

29. A composition comprising one or more DNA molecules encoding the one or more RNA molecules of the composition of any one of claims 1 to 28.

30. 30. The composition of claim 29, wherein each of the one or more DNA molecules comprises a promoter.

31. 31. The composition of claim 30, wherein the promoter of each of the one or more DNA molecules is located 5' to a 5' UTR.

32. 32. The composition of claim 31, wherein the promoter is a T7 promoter.

33. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

5.

34. 34. The composition of claim 33, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

9.

35. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

6.

36. 36. The composition of claim 35, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

10.

37. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

7.

38. 38. The composition of claim 37, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

11.

39. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO:

8.

40. 40. The composition of claim 39, further comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

12.

41. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by SEQ ID NO:

9.

42. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

10.

43. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

11.

44. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to the RNA sequence encoded by SEQ ID NO:

12.

45. A composition comprising an RNA molecule comprising a sequence having at least 80% sequence identity to an RNA sequence encoded by any of SEQ ID NOs: 1-4.

46. 46. ​​The composition of any one of claims 1 to 28 or 33 to 45, further comprising an ionizable cationic lipid.

47. The ionizable cationic lipid has the structure of Formula I: 【Chemistry 1】 or a pharmaceutically acceptable salt or solvate thereof, wherein R ５ and R ６ each independently represents a linear or branched chain C １～ C ３１ Alkyl, C ２～ C ３１ Alkenyl or C ２～ C ３１ selected from the group consisting of alkynyl and cholesteryl; ５ and L ６ each independently represents a linear C １～ C ２０ Alkyl and C ２～ C ２０ alkenyl; X ５ is —C(O)O—, whereby —C(O)O—R ６ is formed, or —OC(O)—, whereby —OC(O)—R ６ is formed, and X ６ is —C(O)O—, whereby —C(O)O—R ５ is formed, or —OC(O)—, whereby —OC(O)—R ５ is formed, and X ７ is S or O, and L ７ is absent or is lower alkyl, and R ４ is a linear or branched chain C １～ C ６ alkyl, and R ７ and R ８ each independently represents hydrogen and a straight or branched chain C １～ C ６ 47. The composition of claim 46, wherein the alkyl is selected from the group consisting of alkyl.

48. 47. The composition of claim 46, wherein the ionizable cationic lipid is selected from Table 6.

49. 47. The composition of claim 46, wherein the ionizable cationic lipid is ATX-126. 【Chemistry 2】

50. 47. The composition of claim 46, wherein the ionizable cationic lipid is ATX-240. 【Transformation 3】

51. 47. The composition of claim 46, wherein the composition comprises a nitrogen to phosphate ratio (N:P) of from about 5:1 to about 7:

1.

52. 100. A method of vaccinating a subject against influenza, said method comprising administering to said subject a composition according to any one of claims 1 to 28 or 33 to 45.

53. 10. Use of a composition according to any one of claims 1 to 28 or 33 to 45 in the preparation of a medicament for vaccinating a subject against influenza.

54. 1. A composition comprising: (i) a polynucleotide having a length of about 5,000 to about 20,000 nucleotides; and (ii) an ionizable cationic lipid, wherein the composition comprises a nitrogen to phosphate ratio (N:P) of about 5:1 to about 7:

1.

55. 55. The composition of claim 54, wherein the nitrogen to phosphate ratio (N:P) is about 7:1.

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