Immunogenic composition against influenza

Abstract

The present invention relates to compositions and methods for the preparation, manufacture, and therapeutic use of ribonucleic acid vaccines comprising polynucleotide molecules encoding one or more influenza antigens, such as hemagglutinin antigens.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 582513 filed on 13 September 2023, U.S. Provisional Patent Application No. 63 / 611106 filed on 15 December 2023, U.S. Provisional Patent Application No. 63 / 559863 filed on 29 February 2024, U.S. Provisional Patent Application No. 63 / 562257 filed on 6 March 2024, U.S. Provisional Patent Application No. 63 / 568754 filed on 22 March 2024, U.S. Provisional Patent Application No. 63 / 634399 filed on 15 April 2024, and U.S. Provisional Patent Application No. 63 / 656098 filed on 4 June 2024, each of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to compositions and methods for the preparation, manufacture, and therapeutic use of ribonucleic acid vaccines comprising polynucleotide molecules encoding one or more influenza antigens, such as hemagglutinin antigens. [Background technology]

[0003] Influenza viruses belong to the Orthomyxoviridae family and are classified into three types (A, B, and C) based on antigenic differences between their nucleoprotein (NP) and matrix (M) proteins.

[0004] The genome of the influenza A virus contains eight molecules of linear, negatively polarized single-stranded RNA (seven for the influenza C virus) that encode several polypeptides, including RNA-driven RNA polymerase proteins (PB2, PB1, and PA) and nucleoprotein (NP) that form the nucleocapsid; matrix proteins (M1, M2, which are also surface-exposed proteins embedded in the viral membrane); two surface glycoproteins that protrude from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA); and non-structural proteins (NS1 and NS2).

[0005] Hemagglutinin is the major envelope glycoprotein of influenza A and B viruses, and hemagglutinin-esterase (HE) of influenza C virus is a protein homologous to HA.

[0006] Challenges in the treatment and prevention of influenza and other infections using traditional vaccines include the limited scope of vaccines, which provide protection only against closely related subtypes. Furthermore, the length of time required to complete current standard influenza virus vaccine production processes hinders the rapid development and production of suitable vaccines in pandemic situations. [Overview of the project] [Problems that the invention aims to solve]

[0007] An improved immunogenic composition against influenza is needed. [Means for solving the problem]

[0008] In particular, an unmet need for improved immunogenic compositions against influenza is provided herein. In one embodiment, the disclosure relates to an improved polypeptide derived from influenza virus, wherein the polypeptide has mutations in the fusion peptide and the fusion peptide proximal region (FPPR) compared to the corresponding wild-type influenza polypeptide. In preferred embodiments, the polypeptide is derived from an influenza hemagglutinin polypeptide. In some embodiments, the polypeptide is derived from hemagglutinin of influenza B virus.

[0009] In some embodiments, the influenza hemagglutinin polypeptide may be derived from hemagglutinin of influenza viruses from the B / Yamagata lineage (represented by B / Yamagata / 16 / 88) or the B / Victoria lineage (represented by B / Victoria / 2 / 87). In some embodiments, the polypeptide is derived from B / Brisbane / 60 / 08, B / Iowa / 06 / 2017, or B / Lee / 40.

[0010] In some embodiments, the polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs. 10 to 68.

[0011] As used herein, the terms “unnatural,” “not naturally occurring,” and “mutant” are used interchangeably in the context of organisms, polypeptides, or nucleic acids. In this context, the terms “unnatural,” “not naturally occurring,” and “mutant” mean a polypeptide or nucleic acid having at least one change or mutation at an amino acid position or nucleic acid position compared to the respective wild-type polypeptide or nucleic acid. Non-limiting examples of at least one change include one or more amino acid or nucleotide insertions, one or more amino acid or nucleotide deletions, or one or more amino acid or nucleotide substitutions. In some embodiments, the polypeptides and / or nucleic acids of this disclosure, for example, polypeptides containing the amino acid sequence of the influenza B virus hemagglutinin protein or nucleic acids encoding such polypeptides, are not naturally occurring and contain deletions at specific positions of the respective wild-type sequences compared to the respective wild-type sequences. Furthermore, when “residues X-Y” refer to a particular sequence herein, those skilled in the art will understand that this means a contiguous sequence of the indicated amino acid residues in the respective particular sequence. In some embodiments, the similar polypeptides of this disclosure have about 40%, at least about 40%, about 45%, at least about 45%, about 50%, at least about 50%, about 55%, at least about 55%, about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% identical amino acids.In some embodiments, similar polypeptides of this disclosure have about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% functionally identical amino acids. "Percent identity" (% identity) between two sequences is determined when the sequences are aligned for maximum homology, without gaps or truncations as described in the alignment parameters. For example, exemplary parameters for determining the relationship between two or more amino acid sequences using the BLAST algorithm may be as provided in BLASTP. Nucleic acid sequence alignment can be performed using BLASTN. Modifications can be made to alignment parameters to increase or decrease the rigor of comparisons, for example, for determining the relevance of two or more sequences. Additional sequences appended to polypeptide sequences, including, but not limited to, immunodetection tags, purification tags, and localization sequences (present or absent), do not affect the identity percentage. Algorithms such as Align, BLAST, ClustalW, and others can be used to compare and determine the similarity or identity of raw sequences to another sequence, and to determine the presence or significance of gaps in sequences to which weights or scores can be assigned. Such algorithms are similarly applicable to determining the similarity or identity of nucleotide or amino acid sequences and may be useful for identifying orthologues of a gene of interest. Parameters for sufficient similarity to determine relevance are calculated based on statistical similarity, or the chance of finding similar matches in random polypeptides, and well-known methods for calculating the significance of determined matches. Those skilled in the art can also visually optimize computer comparisons of two or more sequences as needed. Related gene products or proteins can be expected to have high similarity, e.g., 45%–100% sequence identity.Unrelated proteins may have an identity (about 5%) that is essentially the same as what would be expected to occur by chance when a sufficiently sized database is scanned. For example, alignment can be performed using the Needleman-Wunsch algorithm implemented by the BALIGN tool. Default parameters can be used for alignment, and BLOSUM62 can be used as the scoring matrix. In some cases, it may be useful to use the BLAST algorithm to understand sequence identity between amino acid motifs in template and target sequences. Thus, in some embodiments, BLAST is used to identify or understand the identity of shorter amino acid extensions (e.g., sequence motifs) between template and target proteins. BLAST discovers similar sequences using a heuristic method that approximates the Smith-Waterman algorithm by locating short matches between two sequences. The BLAST algorithm can identify library sequences that are similar to query sequences above a certain threshold. As used herein, an amino acid position (or simply an amino acid) "corresponding" to an amino acid position in another polypeptide sequence is the amino acid position that is referenced and aligned when the polypeptide is aligned. Gaps in sequence homology within a protein sequence can be aligned with relevant sequences and domains, for example, by BLAST, allowing polypeptides to be aligned to maximum homology. Alternatively, in some cases, when polypeptide sequences are aligned for maximum homology, the corresponding amino acids may be the closest amino acids to the identified amino acid within the same biochemical group as the identified amino acid, i.e., the closest acidic amino acid, the closest basic amino acid, the closest aromatic amino acid, etc."Substantially identical" with respect to a nucleic acid sequence (e.g., a gene, RNA, or cDNA) or an amino acid sequence (e.g., a protein or polypeptide) means having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid identity with respect to the reference sequence, respectively.

[0012] In one embodiment, the disclosure relates to a nucleic acid encoding an influenza polypeptide, preferably a polypeptide derived from a hemagglutinin polypeptide, comprising a fusion peptide and proximal region (FPPR) comprising a deletion of at least 3 to 7 amino acid residues between amino acid positions 369 and 382, ​​more preferably between positions 352 and 382, ​​corresponding to the amino acid positions of SEQ ID NO: 9. In some preferred embodiments, the disclosure relates to an influenza polypeptide, preferably a nucleic acid encoding a polypeptide derived from a hemagglutinin polypeptide, comprising a fusion peptide and proximal region (FPPR) comprising a deletion of at least 3 to 7 amino acid residues between amino acid positions 352 and 382, ​​corresponding to the amino acid positions of SEQ ID NO: 9.

[0013] In one embodiment, the present disclosure relates to an immunogenic composition comprising (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in lipid nanoparticles (LNPs). In some embodiments, the first and second antigens comprise hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen comprises HA derived from a subtype of influenza virus different from the influenza virus antigenic polypeptide or immunogenic fragment of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, which is derived from an influenza virus but from a strain of influenza virus different from both the first and second antigens. In some embodiments, the first, second, and third RNA polynucleotides are formulated in lipid nanoparticles.

[0014] In some embodiments, the composition further comprises (iv) a fourth antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, which is derived from an influenza virus but from a strain of influenza virus different from the first, second, and third antigens, and which has an open reading frame encoding the fourth antigen. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in lipid nanoparticles.

[0015] In some embodiments, each RNA polynucleotide includes a modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0016] In some embodiments, each RNA polynucleotide includes a 5' terminal cap, a 5' UTR, a 3' UTR, and a 3' polyadenylated tail. In some embodiments, the 5' terminal cap is

[0017] [ka] This includes: In some embodiments, the 5'UTR includes SEQ ID NO: 1. In some embodiments, the 3'UTR includes SEQ ID NO: 2. In some embodiments, the 3' polyadenylated tail includes SEQ ID NO: 3.

[0018] In some embodiments, the RNA polynucleotides have an integrity of higher than 85%. In some embodiments, the RNA polynucleotides have a purity of higher than 85%.

[0019] In some embodiments, the lipid nanoparticles contain 20-60 mol% ionizable cationic lipids, 5-25 mol% neutral lipids, 25-55 mol% cholesterol, and 0.5-5 mol% PEG-modified lipids.

[0020] In some embodiments, cationic lipids are

[0021] [ka] Includes.

[0022] In some embodiments, PEG-modified lipids are

[0023] [ka] Includes.

[0024] In some embodiments, the first antigen is HA or an immunogenic fragment or variant derived from influenza A subtype H1, and the second antigen is HA or an immunogenic fragment or variant derived from a different H1 strain than the first antigen. In some embodiments, the first and second antigens are HA or an immunogenic fragment or variant derived from influenza A subtype H3, and both antigens are derived from different strains of H3 influenza virus.

[0025] In some embodiments, the first and second antigens are HA derived from influenza A subtype H1 or an immunogenic fragment or variant thereof, the third and fourth antigens are influenza A subtype H3 or an immunogenic fragment or variant thereof, the first and second antigens are derived from different strains of the H1 virus, and the third and fourth antigens are derived from different strains of the H3 influenza virus.

[0026] In some embodiments, at least the first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first, second, and third RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in a single LNP.

[0027] In some embodiments, each RNA polynucleotide is formulated in a single LNP, and each single LNP encapsulates an RNA polynucleotide encoding a single antigen. In some embodiments, the first RNA polynucleotide is formulated in the first LNP, and the second RNA polynucleotide is formulated in the second LNP. In some embodiments, the first RNA polynucleotide is formulated in the first LNP, the second RNA polynucleotide is formulated in the second LNP, and the third RNA polynucleotide is formulated in the third LNP. In some embodiments, the first RNA polynucleotide is formulated in the first LNP, the second RNA polynucleotide is formulated in the second LNP, the third RNA polynucleotide is formulated in the third LNP, and the fourth RNA polynucleotide is formulated in the fourth LNP.

[0028] In another aspect, the present disclosure relates to any immunogenic composition described herein for use in eliciting an immune response to influenza.

[0029] In another aspect, the present disclosure relates to a method for eliciting an immune response to influenza disease, comprising administering an effective amount of any immunogenic composition described herein.

[0030] In another embodiment, the disclosure relates to a method for purifying RNA polynucleotides synthesized by transcription in vitro. The method includes ultrafiltration and diafiltration. In some embodiments, the method does not include a chromatography step. In some embodiments, the purified RNA polynucleotides are substantially free of impurities including short avotive RNA species, long avotive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvent and / or residual salts. In some embodiments, the residual plasmid DNA is 500 ng or less of DNA / mg RNA. In some embodiments, the yield of purified mRNA is about 70% to about 99%. In some embodiments, the purity of purified mRNA is about 60% to about 100%. In some embodiments, the purity of purified mRNA is about 85% to 95%.

[0031] In some embodiments, the Disclosure provides nucleic acids encoding the polypeptides described herein. In some embodiments, the Disclosure provides expression constructs comprising the nucleic acids described herein. In some embodiments, the Disclosure provides a method for inducing an immune response to influenza B virus in a subject requiring such response, comprising administering to the subject an immunologically effective amount of a polypeptide or protein trimer described herein, an immunogenic composition described herein, or a combination thereof.

[0032] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings together with a detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0033] [Figure 1] Functional anti-HA antibodies extracted by immunization of mice with monovalent or tetravalent LNP-formulated modRNA encoding influenza HA, as measured by MNT. [Figure 2]An example of the mutant (#24) (also referred to herein as either Δ355-363 or pBV-024 (SEQ ID NO: 33)) shows increased potency compared to wild-type FluB. [Figure 3] Correlation of in vitro expression in human HeLa cells determined using the influenza virus monoclonal antibody CR8071 versus the polyclonal antibody B295-HA variant. Selected variants are labeled with their sequence numbers. [Figure 4] Correlation of in vitro expression in human HeLa cells measured using monoclonal antibodies CR8071 and CR9114 of influenza virus HA variants. Selected variants are labeled with their sequence numbers. CR8071 and CR9114 recognize different epitopes of HA. [Figure 5] Functional anti-HA antibodies extracted by immunization of mice with monovalent or tetravalent LNP-formulated modRNA encoding influenza HA, as measured by MNT. In vivo immunogenicity extracted by influenza B virus HA variants, as determined by neutralizing antibody titer 3 weeks after dose 1. [Figure 6] In vivo immunogenicity was elicited by the HA variant of influenza B virus, as measured by neutralizing antibody titers, two weeks after dose 2. [Figure 7] Correlation between in vivo immunogenicity drawn by the HA variant of influenza B virus, as measured by neutralizing antibody titer two weeks after dose 2, and its in vitro expression in human cells. DS: formulation raw material; DR: formulation. [Figure 8]Figures 8A-C: The same design principle can be applied to the HA of other influenza B virus strains to improve immunogenicity against these virus strains. In this example, an HA variant with a mutation equivalent to SEQ ID NO: 47 was manipulated in the Washington and Colorado sublineages of the Victory strain of influenza B virus, and the Phuket sublineage of the Yamagata strain. Improvement in immunogenicity, as measured by neutralizing antibody titers, was observed. [Figure 9] Figures 9A-C: CleanCap AG saRNA functioned better in THP-1 than enzymatically capped saRNA in terms of the number of cells expressing the antigen encoded by the saRNA and the geometric mean fluorescence intensity (GMFI) (antigen copy number) of the antigen in cells transfected with saRNA. Figure 9C depicts A / Wisc / 588 / 19 HA expression in THP-1. THP-1 cells were transfected with saRNA-TC83-A / Wisc / 588 / 19 HA-40A or 2-cistronic saRNA-TC83-A / Wisc / 588 / 19 HA-NA-80A without nucleoside modification, m5c, Hm5C, or 2'Ome-G integration (11 points, 2-fold dilution series starting from 1000 ng). The number of HA-expressing cells (%HA+ cells) (Figure 9A), total HA expression per HA-positive cell (geometric mean fluorescence intensity (GMFI)) (Figure 9B), and the number of viable cells (% viable cells) (Figure 9C) were determined by flow cytometry 22 hours after transfection. The results are presented as the mean ± standard deviation for each group, derived from representative experiments. [Figure 10] Figure 10A depicts the inclusion rate (%) and LNP size (d.nm) of LNPs before dialysis and after filtration. Figure 10B depicts the percentage of positive expression in HEK293T cells when comparing LNP formulations in the presence of egg sphingomyelin (ESM) and cholesterol to a benchmark LNP formulation in the absence of ESM and cholesterol. [Figure 11]Figure 11A depicts the testing of various LNP formulations and the measurement of LNP size (d.nm), with the tested samples listed in Table 54. Successful use of sitosterol and SM combinations can be achieved using PBS as a buffer. Figure 11B depicts the fractions of positive expression in HEK293T for each of the samples listed in Table 52. Figure 11C depicts the mean fluorescence intensity (MFI) for each of the samples listed in Table 52. [Figure 12] Figure 12A illustrates the change in LNP size for samples with various cationic lipid and ESM ratios. The tested samples are listed in Table 54. Figure 12B illustrates the change in encapsulation efficiency (EE) (%) for samples with various cationic lipid and ESM ratios. The tested samples are listed in Table 54. [Figure 13] Figure 13A depicts the percentage of positive expression in HEK293T cells for samples with various cationic lipid and ESM ratios. The tested samples are listed in Table 55. Figure 13B depicts the MFI of positive-expressing cells derived from the samples listed in Table 55. [Figure 14]HA-specific antibody titers were measured by hemagglutination inhibition assay (HAI) (Figure 14A) and microneutralization assay (MNT) (Figure 14B). Splenocytes were harvested two weeks after the second immunization and stimulated with peptides targeting the H1N1 HA protein derived from the vaccine strain (A / Wisconsin / 588 / 2019). CD4+ T cells expressing IFN-γ, IL-4, IL-2, TNF-α and / or CD154, as well as CD8+ T cells expressing IFN-γ, TNF-α and / or CD107a, were evaluated by intracellular cytokine staining. Immunization with two doses of mIRV induced a higher percentage of IFN-γ-producing CD4+ T cells than IL-4-producing CD4+ T cells, which exhibited a Th1-biased response (Figure 14C), while two doses of QIV induced a higher percentage of IL-4+CD4+ T cells than IFN-γ+CD4+ T cells, which exhibited a Th2-biased response (Figure 14D). Furthermore, a potent multifunctional (IFN-γ+, IL-2+, TNF-α+, CD154+)CD4+ T cell response was observed with the mIRV vaccine but not with QIV (Figure 14E). In addition, immunization with mIRV induced higher levels of IFN-γ+CD8+ T cells (Figure 14F) and multifunctional (IFN-γ+, TNF-α+, CD107a+)CD8+ T cells (Figure 14G) compared to QIV. [Figure 15] HA-specific antibodies were measured by HAI (Figure 15A-B) and MNT (Figure 15C-D). T cell immunity was quantified by measuring peripheral CD4+ and CD8+ T cells expressing cytokines after ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with an HA peptide pool derived from the H1N1 vaccine strain (Figure 15E-F). [Figure 16] Mice were immunized with two doses of qIRV or licensed adjuvanted qIV at 28-day intervals. Two weeks after the second immunization, functional antibodies against each of the four vaccine-encoded strains were measured by HAI (Figure 16A) and MNT (Figure 16B). [Figure 17]Two weeks after the second immunization, viral neutralization titers against vaccine-matched strains were measured by MNT (Figure 17). Neutralization titers drawn by mIRV, tIRV, or qIRV against the shared vaccine strains (H1N1, H3N2, and B / Vic) were not statistically different (Figures 17A-C), indicating the absence of interference. Neutralization titers drawn by mIRV and qIRV against B / Yamagata were also not statistically different (Figure 17D). [Figure 18] Hematological findings were consistent with the inflammatory leukocyte morphology, including higher neutrophil counts on days 3 and 17; more highly segmented neutrophils on day 17; and higher monocytes, eosinophils, and / or larger unstained cells on days 3 and 17 (Figure 18A). Findings consistent with the acute phase response were also observed for both vaccines, including higher fibrinogen on day 17; higher globulin and / or lower albumin on days 3 and 17; and higher alpha-2 macroglobulin (A2M) and alpha-1-glycoprotein (A1AGP) on days 3 and 17 (Figures 18B-D). [Figure 19] The mean influenza challenge virus viral load per protocol population, measured by qRT-PCR over days, was described. The tested treatment groups were monovalent modRNA HA (N=55), QIV control group (N=48), and placebo (N=52). [Figure 20] This diagram plots the area under the curve of qRT-PCR-mediated influenza challenge virus viral load per protocol population over time. The tested treatment groups were monovalent modRNA HA (N=55), QIV control group (N=48), and placebo (N=52). Whiskers represent minimum and maximum values, boxes represent the interquartile range along with a line representing the median, diamonds represent the mean, and circles represent values ​​for each individual. [Figure 21] Forest plots of vaccine efficacy for moderate influenza infection, as confirmed by qRT-PCR, per protocol population, i.e., QIV comparison criteria and monovalent modRNA HA. [Figure 22] Tables 57 and 58 respectively describe the 50% reversal value results at 3 weeks of dose 1 against the H1N1 A / California strain derived from the formulations listed in Tables 57 and 58. [Figure 23] Tables 57 and 58 describe the 50% reversal value results at 3 weeks of dose 1 for the RSV M37 strain derived from the formulations listed in Tables 57 and 58, respectively. [Figure 24] Tables 57 and 58 respectively describe the 50% reversal value results at 3 weeks of dose 1 for the RSV B18537 strain derived from the formulations listed in Tables 57 and 58. [Figure 25] Figure 25A depicts the 50% neutralization titer results after 3 weeks of dose 1 for influenza B / Colorado strains derived from the Bv / Colorado / 06 / 2017-HA-Δ370-374 sample listed in Table 61. Figure 25B depicts the 50% neutralization titer results after 3 weeks of dose 1 for influenza B / Washington strains derived from the Bv / Washington / 02 / 2019-HA-Δ369-373 sample listed in Table 61. Figure 25C depicts the 50% neutralization titer results after 3 weeks of dose 1 for influenza B / Phuket strains derived from the By / Phuket / 3073 / 2013-HA-Δ371-375 sample listed in Table 61. Figure 25D depicts the 50% neutralization titer results at 3 weeks for influenza B / Austria strains derived from mutant B / Austria samples listed in Table 62. [Figure 26]Figure 26A depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza B / Colorado strains derived from the Bv / Colorado / 06 / 2017-HA-Δ370-374 sample listed in Table 61. Figure 26B depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza B / Washington strains derived from the Bv / Washington / 02 / 2019-HA-Δ369-373 sample listed in Table 61. Figure 26C depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza B / Phuket strains derived from the By / Phuket / 3073 / 2013-HA-Δ371-375 sample listed in Table 61. Figure 26D depicts the 50% neutralization titer results at 2 weeks after dose 2 for influenza B / Austria strains derived from mutant B / Austria samples listed in Table 62. [Figure 27] This report describes the 50% neutralization titer results at 2 weeks after dose 2 against influenza B / Austria strains derived from mutant B / Austria samples. A Flu B modRNA construct carrying the deletion Δ(369-373) within the HA fusion peptide resulted in higher neutralization titers in vivo compared to the WT benchmark control. [Figure 28] Each of these describes the 50% neutralization titer results at 2 weeks after dose 2 for influenza B / Austria strains derived from the samples listed in Table 60. [Figure 29] Each of these describes the 50% neutralization value results at 2 weeks after dose 2 for the influenza H1N1 A / Wisconsin / 67 / 2022 strain derived from the samples listed in Table 63. [Figure 30] Figure 30A depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza H1N1 A / Wisconsin / 67 / 2022 strain derived from the samples listed in Table 63. Figure 30B depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza H3N2 A / Darwin / 06 / 2021 strain derived from the samples listed in Table 63. [Figure 31]Each figure depicts the 50% neutralization value at 3 weeks after dose 1 for influenza Bv / Austria strains derived from the samples listed in Table 63. In the figures, "?" represents "Δ". [Figure 32] Figure 32A depicts the 50% neutralization titer results after 3 weeks of dose 1 for influenza H1N1 A / Wisconsin / 67 / 2022 strain derived from the samples listed in Table 63, where "?" represents "Δ". Figure 32B depicts the 50% neutralization titer results after 3 weeks of dose 1 for influenza H3N2 A / Darwin / 06 / 2021 strain derived from the samples listed in Table 63, where "?" represents "Δ". [Figure 33] Each figure depicts the 50% neutralization value at 2 weeks after dose 2 for influenza Bv / Austria strains derived from the samples listed in Table 63. In the figures, "?" represents "Δ". [Figure 34] Figure 34A depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza H1N1 A / Wisconsin / 67 / 2022 strain derived from the samples listed in Table 63. In the figure, "?" represents "Δ". Figure 34B depicts the 50% neutralization titer results after 2 weeks of dose 2 for influenza H3N2 A / Darwin / 06 / 2021 strain derived from the samples listed in Table 63. In the figure, "?" represents "Δ". [Modes for carrying out the invention]

[0034] Embodiments of this disclosure provide RNA (e.g., mRNA) vaccines containing polynucleotides encoding influenza virus antigens. Using the influenza virus RNA vaccines provided herein, a balanced immune response, including both cellular and humoral immunity, can be induced without many of the risks associated with DNA vaccination. In some embodiments, the virus is a strain of influenza A or influenza B, or a combination thereof.

[0035] In one embodiment, the present disclosure relates to an immunogenic composition comprising (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in lipid nanoparticles (LNPs). In some embodiments, the first and second antigens comprise hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen comprises HA derived from a subtype of influenza virus different from the influenza virus antigenic polypeptide or immunogenic fragment of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, which is derived from an influenza virus but from a strain of influenza virus different from both the first and second antigens. In some embodiments, the first, second, and third RNA polynucleotides are formulated in lipid nanoparticles.

[0036] In some embodiments, the composition further comprises (iv) a fourth antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, which is derived from an influenza virus but from a strain of influenza virus different from the first, second, and third antigens, and which has an open reading frame encoding the fourth antigen. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in lipid nanoparticles.

[0037] In some embodiments, RNA polynucleotides are mixed in a desired ratio in a single container and subsequently formulated in lipid nanoparticles. Surprisingly, the inventors discovered that an initial input of different RNA polynucleotides in known ratios formulated in a single LNP process resulted in an LNP encapsulating different RNA polynucleotides in approximately the same ratio as the input ratio. This result was surprising considering the possibility of a manufacturing process that selects one RNA polynucleotide over another when encapsulating RNA polynucleotides in an LNP. Such embodiments may be referred to herein as “pre-mixing.” Thus, in some embodiments, the first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, and fifth RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, fifth, and sixth RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, fifth, sixth, and seventh RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh, and eighth RNA polynucleotides are formulated in a single LNP.

[0038] In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide is greater than 1:1.

[0039] In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide is greater than 1:1.

[0040] In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fifth RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fifth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide is greater than 1:1.In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide in the RNA polynucleotide mix prior to formulation in LNP is approximately 1:50, approximately 1:25, approximately 1:10, approximately 1:5, approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1, approximately 10:1, approximately 25:1, or approximately 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide is greater than 1:1.

[0041] Self-amplifying RNA (saRNA) In some embodiments, the RNA molecule, such as the first RNA molecule, is saRNA. “saRNA,” “self-replicating RNA,” and “replicon” refer to RNA that has the ability to replicate itself. Self-replicating RNA molecules can be produced by using replication elements derived from a virus or multiple viruses, e.g., alphaviruses, and by substituting a structural viral polypeptide with a nucleotide sequence encoding the polypeptide of interest. Self-replicating RNA molecules are typically positive-chain molecules that can be directly translated after delivery to a cell, and this translation provides RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts derived from the delivered RNA. The delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can be translated themselves to result in the in situ expression of the gene encoding the target, e.g., a viral antigen, or they can be transcribed to result in further transcripts with the same sense as the delivered RNA, and translated to result in the in situ expression of the protein of interest, e.g., an antigen. The overall result of this series of transcriptions is an amplification of the number of saRNAs introduced, and therefore the gene encoding the target, such as a viral antigen, can become the cell's primary polypeptide product.

[0042] In some embodiments, the self-amplifying RNA comprises at least one gene selected from among viral replicases, viral proteases, viral helicases, and other non-structural viral proteins. In some embodiments, the self-amplifying RNA may also comprise 5' and 3' end-pulling replication sequences and, optionally, heterologous sequences encoding a desired amino acid sequence (e.g., the antigen of interest). A subgenome promoter directing the expression of the heterologous sequence may be incorporated into the self-amplifying RNA. Optionally, the heterologous sequence (e.g., the antigen of interest) may be fused in-frame to other coding regions in the self-amplifying RNA and / or under the control of an internal ribosome entry site (IRES).

[0043] In some embodiments, the self-amplifying RNA molecule is not encapsulated within a virus-like particle. The self-amplifying RNA molecules described herein can be designed so as not to induce the production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins necessary for the production of viral particles within the self-amplifying RNA. For example, if the self-amplifying RNA molecule is based on an alphavirus such as Sindbis virus (SIN), Semlik Forest virus, and Venezuelan encephalitis virus (VEE), one or more genes encoding viral structural proteins such as the capsid and / or envelope glycoprotein may be omitted.

[0044] In some embodiments, the self-amplifying RNA molecule described herein comprises (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, such as a viral antigen. In some embodiments, the polymerase may be an alphaviral replicase comprising, for example, one of the alphaviral proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof. In some embodiments, the self-amplifying RNA molecule described herein may comprise one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine). In some embodiments, the self-amplifying RNA molecule does not comprise modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0045] The saRNA construct may encode at least one non-structural protein (NSP) located at the 5' or 3' end of the sequence encoding at least one peptide or polypeptide of interest. In some embodiments, the sequence encoding at least one NSP is located at the 5' end of the sequence encoding the peptide or polypeptide of interest. Thus, the sequence encoding at least one NSP may be located at the 5' end of the RNA construct. In some embodiments, the at least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4. In some embodiments, the saRNA construct encodes nsP1, nsP2, nsP3, and nsP4. As is known in the Art, nsP1 is a viral capping enzyme and a membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and a protease responsible for processing ns polyproteins. nsP3 interacts with several host proteins and can modulate protein poly and mono ADP-ribosylation. nsP4 is a core viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphaviral replicase containing, for example, one or more of the alphaviral proteins nsP1, nsP2, nsP3, and nsP4.

[0046] While the natural alphavirus genome encodes structural virion proteins in addition to non-structural replicase polypeptides, in some embodiments, the self-amplifying RNA molecule does not encode alphavirus structural proteins. In some embodiments, the self-amplifying RNA may produce copies of its own genomic RNA in the cell, but not RNA containing virions. Although not bound by theory or mechanism, the inability to produce these virions means that, unlike wild-type alphavirus, the self-amplifying RNA molecule cannot persist itself in the infectious form. The alphavirus structural proteins required for persistence in wild-type viruses may not be present in the self-amplifying RNA of this disclosure, and their position may be occupied by a gene encoding the immunogen of interest, such that the subgenome transcript encodes an immunogen rather than a structural alphavirus virion protein.

[0047] In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame may encode a replicase; the second (3') open reading frame may encode a polypeptide containing the antigen of interest. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames that encode, for example, further antigens or accessory polypeptides.

[0048] In some embodiments, the second RNA or saRNA molecule further comprises (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

[0049] If necessary, the self-amplifying RNA molecules described herein may also be designed to induce the production of attenuated or virulent infectious viral particles, or viral particles capable of causing a single subsequent infection.

[0050] In some embodiments, the saRNA molecule is based on an alphavirus. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and viral subtypes within the genus Alphavirus include Sindbis virus, Semryqui forest virus, Ross River virus, and Venezuelan encephalitis virus. Therefore, the self-amplifying RNA described herein may include an RNA replicase derived from any one of the following viruses: Semryqui forest virus (SFV), Sindbis virus (SIN), Venezuelan encephalitis virus (VEE), Ross River virus (RRV), or another virus belonging to the Alphaviridae family. In some embodiments, the self-amplifying RNA described herein may include sequences derived from mutant or wild-type viral sequences, for example, the attenuated TC83 mutant of VEEV is used in the saRNA.

[0051] Alphavirus-based saRNAs are (+)-strand saRNAs that can be translated after delivery to cells, resulting in the translation of a replicase (or replicase-transcriptase). The replicase is translated into a polyprotein that self-cleaves, resulting in a replication complex that produces a genomic (-)-strand copy of the (+)-strand delivery RNA. These (-)-strand transcripts may transcribe themselves to give further copies of the (+)-strand parental RNA, or to give subgenomic transcripts encoding the desired gene product. Thus, translation of the subgenomic transcripts results in in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs may be replicases derived from Sindbis virus, Semlik Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, or their mutant variants.

[0052] In some embodiments, the self-amplifying RNA molecule is derived from or based on viruses other than alphaviruses, such as positive-strand RNA viruses, particularly picornaviruses, flaviviruses, rubiviruses, pestiviruses, hepaciviruses, caliciviruses, or coronaviruses. Suitable wild-type alphavirus sequences are well known and available from sequence depositaries, such as the American Type Culture Collection, Rockville, Md.Representative examples of suitable alphaviruses include Aura virus (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou virus (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern Equine Encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan virus (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach virus (ATCC VR-927), Mayaro virus (ATCC VR-66), and Mayarovirus (ATCC VR-1277), Middleburg virus (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu virus (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate virus (ATCC VR-925), Triniti virus (ATCC Examples include VR-469), Una virus (ATCC VR-374), Venezuelan horse encephalomyelitis virus (ATCC VR-69, ATCC VR-923, ATCC VR-1250, ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis virus (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa virus (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some embodiments, one or more alphaviruses in the list may be excluded.

[0053] In some embodiments, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., saRNA). Typically, the self-amplifying RNA molecules described herein contain at least about 4 kb. For example, the self-amplifying RNA may be equal to any one, at least one, as many as one, or any two of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 16 kb. In some cases, the self-amplifying RNA may contain at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In certain cases, self-renewing RNA is approximately 4kb-12kb, 5kb-12kb, 6kb-12kb, 7kb-12kb, 8kb-12kb, 9kb-12kb, 10kb-12kb, 11kb-12kb, 5kb-11kb, 5kb-10kb, 5kb-9kb, 5kb-8kb, 5kb-7kb, 5kb-6kb, and 6kb. The ranges are approximately b - 12kb, 6kb - 11kb, 6kb - 10kb, 6kb - 9kb, 6kb - 8kb, 6kb - 7kb, 7kb - 11kb, 7kb - 10kb, 7kb - 9kb, 7kb - 8kb, 8kb - 11kb, 8kb - 10kb, 8kb - 9kb, 9kb - 11kb, 9kb - 10kb, or 10kb - 11kb.

[0054] In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more polypeptide antigens linked together (e.g., in series) in such a way that each sequence maintains its identity when expressed as an amino acid sequence. The polypeptide produced from the self-amplifying RNA can then be produced as a fusion polypeptide or manipulated in a manner that yields separate polypeptide or peptide sequences. In some embodiments, the saRNA molecule may encode one or more polypeptides of interest, such as one antigen or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides. Alternatively, a single saRNA molecule may also encode one or more polypeptides of interest, such as antigens, e.g., two-cistronic or three-cistronic RNA molecules encoding different or identical antigens. An exemplary dicistronic saRNA encoding HA and NA includes the sequence described in SEQ ID NO: 70 (published as SEQ ID NO: 9 in PCT / IB2023 / 057034, published as WO2024 / 013625). Another exemplary dicistronic saRNA includes the sequence described in SEQ ID NO: 71 (published as SEQ ID NO: 10 in PCT / IB2023 / 057034, published as WO2024 / 013625). See, for example, international patent application PCT / IB2023 / 057034, filed on July 7, 2023, titled "Self-amplifying rna encoding an influenza virus antigen" (Pfizer Inc.), published as WO2024 / 013625, which is incorporated in its entirety by reference and describes the saRNA molecule and the dicistronic saRNA.

[0055] As used herein, the term “linked” refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently linked to a second amino acid sequence or polynucleotide sequence. The first amino acid sequence or polynucleotide sequence may be directly linked to or juxtaposed with the second amino acid sequence or polynucleotide sequence, or the intervening sequence may covalently link the first sequence to the second sequence. The term “linked” includes not only the fusion of the first RNA molecule to the second RNA molecule at its 5' or 3' end, but also the insertion of the entire first RNA molecule into any two nucleotides in the second RNA molecule. The first second RNA molecule may be linked to the second RNA molecule by a phosphodiester bond or a linker. The linker may be, for example, a polynucleotide.

[0056] In some embodiments, the self-amplifying RNA described herein may encode one or more polypeptide antigens comprising a range of epitopes. In some embodiments, the self-amplifying RNA described herein may encode an epitope capable of eliciting a helper T cell response, a cytotoxic T cell response, or both.

[0057] In some embodiments, saRNA molecules are purified by filtration, such as ultrafiltration, diafiltration, or filtration, which may be carried out via tangential ultrafiltration / diafiltration, for example.

[0058] Some embodiments of this disclosure relate to compositions comprising a self-amplifying RNA molecule comprising a 5' cap, a 5' untranslated region, a coding region including a sequence encoding RNA-dependent RNA polymerase (also referred to herein as “replicase”), a subgenome promoter such as one derived from an alphavirus, an open reading frame encoding a gene of interest (e.g., an antigen derived from an influenza virus), a 3' untranslated region, and a 3' polyA sequence. In some embodiments, at least 5% of the entire population of a particular nucleotide in the saRNA molecule are replaced with one or more modified or non-native nucleotides.

[0059] In some embodiments, the saRNA molecule is free of modified nucleotides, for example, free of modified nucleic acid bases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, except for any 5' cap, which may include, for example, 7-methylguanosine, as further described below.

[0060] In some embodiments, the saRNA molecule is free of modified nucleotides, e.g., no modified nucleic acid bases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, except for an optional 5' cap which may contain, for example, 7-methylguanosine, as further described below. In some embodiments, the RNA may contain a 5' cap containing 7'-methylguanosine, and the first 1,2 or 3,5' ribonucleotide may be methylated at the 2' position of ribose. In alternative embodiments, each RNA polynucleotide encoding a particular antigen is formulated within individual LNPs such that each LNP encapsulates an RNA polynucleotide encoding the same antigen. Such embodiments may be referred to herein as “post-mixing.” Therefore, in some embodiments, the first RNA polynucleotide is formulated in the first LNP, the second RNA polynucleotide in the second LNP, the third RNA polynucleotide in the third LNP, the fourth RNA polynucleotide in the fourth LNP, the fifth RNA polynucleotide in the fifth LNP, the sixth RNA polynucleotide in the sixth LNP, the seventh RNA polynucleotide in the seventh LNP, and the eighth RNA polynucleotide in the eighth LNP.

[0061] In some embodiments, the molar ratio of the first LNP to the second LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the second LNP is greater than 1:1.

[0062] In some embodiments, the molar ratio of the first LNP to the third LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the third LNP is greater than 1:1.

[0063] In some embodiments, the molar ratio of the first LNP to the fourth LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the fourth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP in the LNP mix prior to formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP is greater than 1:1.

[0064] Surprisingly, the inventors discovered that the resulting ratios of RNA polynucleotides are equivalent regardless of the process, whether they attempt to mix multiple RNA polynucleotides before formulation in LNPs (pre-mixing) or to formulate RNA polynucleotides encoding specific antigens in individual LNPs and then mix multiple LNPs for different antigens (post-mixing). As a result of this finding, particularly when individual LNPs encapsulate RNA for a single antigen, healthcare professionals may have the option of mixing and administering antigens in different ratios depending on the influenza season.

[0065] In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein does not contain a head domain. In some embodiments, the hemagglutinin protein contains a portion of the head domain. In some embodiments, the hemagglutinin protein does not contain a cytoplasmic domain. In some embodiments, the hemagglutinin protein contains a portion of the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein contains a portion of the transmembrane domain.

[0066] Some embodiments provide an influenza vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein, formulated within cationic lipid nanoparticles, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the hemagglutinin protein is selected from H1, H7, and H10. In some embodiments, the RNA polynucleotide further encodes a neuraminidase (NA) protein. In some embodiments, the hemagglutinin protein is derived from an influenza A virus or an influenza B virus strain or a combination thereof. In some embodiments, the influenza virus is selected from H1N1, H3N2, H7N9, and H10N8.

[0067] In some embodiments, the virus is a strain of influenza A or influenza B, or a combination thereof. In some embodiments, the strain of influenza A or influenza B is associated with birds, pigs, horses, dogs, humans, or non-human primates. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or a fragment thereof. In some embodiments, the hemagglutinin protein is H7 or H10, or a fragment thereof. In some embodiments, the hemagglutinin protein includes a portion of the head domain (HA1). In some embodiments, the hemagglutinin protein includes a portion of the cytoplasmic domain. In some embodiments, the hemagglutinin protein is truncated. In some embodiments, the protein is a truncated hemagglutinin protein including a portion of the transmembrane domain. In some embodiments, the virus is selected from the group consisting of H7N9 and H10N8. Protein fragments, functional protein domains, and homologous proteins are also considered to be within the range of the polypeptide of interest. For example, any protein fragment of a reference protein having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids, or longer than 100 amino acids (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence, but otherwise identical) is provided herein.

[0068] In some embodiments, the influenza RNA composition includes RNA encoding an antigenic fusion protein. Thus, the encoded antigen or multiple antigens may include two or more proteins (e.g., a protein and / or protein fragments) linked together. Alternatively, the protein to which the protein antigen is fused does not promote a strong immune response to itself, but rather promotes a strong immune response to the influenza antigen. In some embodiments, the antigenic fusion protein retains functional properties derived from each of the original proteins.

[0069] Some embodiments provide a method for preventing or treating influenza virus infection, which includes administering any of the vaccines described herein to a subject. In some embodiments, the antigen-specific immune response includes a T-cell response. In some embodiments, the antigen-specific immune response includes a B-cell response. In some embodiments, the antigen-specific immune response includes both a T-cell response and a B-cell response. In some embodiments, the method for eliciting an antigen-specific immune response includes a single dose of the vaccine. In some embodiments, the vaccine is administered to a subject by intradermal, intramuscular, subcutaneous, intranasal, or oral administration.

[0070] In some embodiments, an RNA (e.g., mRNA) polynucleotide or a portion thereof may encode one or more polypeptides or fragments of influenza strains as an antigen.

[0071] mRNA vaccines disclosed herein This disclosure relates in general to mRNA vaccines. Several mRNA vaccine platforms are available in the prior art. The basic structure of in vitro transcribed (IVT) mRNA is very similar to that of “mature” eukaryotic mRNA, and includes (i) a protein-coding open reading frame (ORF), (ii) 5' and 3' untranslated regions (UTR) flanking it, and at the terminal end, (iii) a 7-methylguanosine 5' cap structure and (iv) a 3' poly(A) tail. Non-coding structural features play an important role in the pharmacological action of mRNA and can be individually optimized to modulate mRNA stability, translation efficiency, and immunogenicity. By incorporating modified nucleosides, mRNA transcripts with reduced immunostimulatory activity, referred to as “nucleoside-modified mRNA,” can be produced, thus providing an improved safety profile. Furthermore, modified nucleosides can circumvent the direct antiviral pathway, which is induced by IFN types and programmed to degrade and inhibit invading mRNA, making it possible to design mRNA vaccines with potently enhanced stability and translational capacity. For example, replacing uridine with pseudouridine in IVT mRNA reduces the activity of 2'-5' oligoadenylate synthetase, which modulates mRNA cleavage by RNase L. In addition, a decrease in the activity of the enzyme protein kinase R, which is associated with the inhibition of the mRNA translation process, can be measured.

[0072] In addition to the incorporation of modified nucleotides, other methods have been shown to increase the translational capacity and stability of mRNA. One example is the development of "sequence-engineered mRNA." In this case, mRNA expression can be potently increased by optimizing the sequence of the mRNA's ORF and UTR, for example, by enriching the GC content or by selecting the UTR of a naturally occurring long-lived mRNA molecule. Another method is the design of "self-amplifying mRNA" constructs. These are mostly derived from alphaviruses and contain ORFs replaced with the target antigen, along with additional ORFs encoding viral replicases. The latter drives intracellular amplification of mRNA and can therefore significantly increase antigen expression.

[0073] Furthermore, several modifications are made to the terminal structure of mRNA. Anti-reverse cap (ARCA) modification ensures the correct cap orientation at the 5' end, allowing almost the entire fraction of mRNA to efficiently bind to ribosomes. Other cap modifications, such as phosphorothioate cap analogs, can further improve affinity for eukaryotic translation initiation factor 4E and increase resistance to RNA cap removal complexes.

[0074] Conversely, modifying the structure of mRNA can further improve its efficacy in inducing an innate immune response, but this will impair its translational ability. Stabilizing mRNA, either by using a phosphorothioate backbone or by precipitation with the cationic protein protamine, may reduce antigen expression, but it can yield more potent immunostimulatory activity.

[0075] In one embodiment, the present invention relates to an immunogenic composition comprising mRNA molecules encoding one or more polypeptides or fragments of influenza strains as antigens.

[0076] In some embodiments, the mRNA molecule includes nucleoside-modified mRNA. mRNA useful in this disclosure typically includes a first region of linked nucleosides encoding the polypeptide of interest (e.g., coding region), a first flanking region (e.g., 5-UTR) located at the 5' end of the first region, a second flanking region (e.g., 3-UTR) located at the 3' end of the first region, at least one 5' cap region, and a 3' stabilization region. In some embodiments, the mRNA of this disclosure further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the mRNA of this disclosure may contain one or more intronic nucleotide sequences that can be cleaved from a polynucleotide. In some embodiments, the mRNA of this disclosure may include a 5' cap structure, a chain termination nucleotide, a stem-loop, a poly-A sequence, and / or a polyadenylation signal. Any one of the regions of the nucleic acid may contain one or more alternative components (e.g., alternative nucleosides). For example, the 3' stabilizing region may contain alternative nucleosides such as L-nucleosides, reversed thymidines, or 2'-O-methylnucleosides, and / or the coding region, 5'UTR, 3'UTR, or capping region may contain alternative nucleosides such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methylpseudridine), and / or 5-substituted cytidines (e.g., 5-methylcytidine).

[0077] The compositions described herein comprise at least one RNA polynucleotide, such as mRNA (e.g., modified mRNA). The mRNA is transcribed in vitro from template DNA, for example, referred to as an "in vitro transcription template." In some embodiments, the in vitro transcription template encodes the 5' untranslated (UTR) region, contains an open reading frame, and encodes the 3' UTR and poly-A tail. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0078] The "5' untranslated region" (UTR) refers to the region of mRNA that does not encode a polypeptide and is located immediately upstream (i.e., at the 5' end) of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome).

[0079] In a preferred embodiment, 5'UTR includes SEQ ID NO: 1.

[0080] The "3' untranslated region" (UTR) refers to the region of mRNA that does not encode a polypeptide and is located immediately downstream (i.e., at the 3' end) of a stop codon (i.e., a codon in an mRNA transcript that signals the termination of translation).

[0081] In a preferred embodiment, 3'UTR includes SEQ ID NO: 2.

[0082] An "open reading frame" is a continuous extension of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide.

[0083] The "poly-A tail" is a region of mRNA downstream of the 3'UTR containing multiple consecutive adenosine monophosphates, for example, the immediately downstream (i.e., 3' side) region. The poly-A tail may contain 10 to 300 adenosine monophosphates. For example, the poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the poly-A tail contains 50 to 250 adenosine monophosphates. In relevant biological settings (e.g., in cells, in vivo), the poly(A) tail functions to protect mRNA from enzymatic degradation in the cytoplasm, for example, and assists in transcription termination, mRNA export from the nucleus, and translation.

[0084] In a preferred embodiment, the 3'-polyadenylated tail includes SEQ ID NO: 3.

[0085] In some embodiments, the polynucleotide contains 200 to 3,000 nucleotides. For example, the polynucleotide may contain 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides.

[0086] In some embodiments, the LNP may contain one or more RNAs, and the one or more RNAs, lipids, and their amounts can be selected to yield a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of the number of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. Generally, a low N:P ratio is preferred. The one or more RNAs, lipids, and their amounts can be selected to yield an N:P ratio of about 2:1 to about 30:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In some particular embodiments, the N:P ratio may be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be approximately 5.0:1, 5.5:1, 5.67:1, 6.0:1, 6.5:1, or 7.0:1. For example, the N:P ratio could be approximately 5.67:1.

[0087] The mRNAs of this disclosure may contain one or more naturally occurring components, including any of the standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides, including (a) the 5'UTR, (b) the open reading frame (ORF), (c) the 3'UTR, (d) the poly-A tail, and any combination of (a, b, c, or d) above, contain the naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0088] The mRNAs of this disclosure may comprise one or more of the alternative components described herein that confer useful properties, including increased stability and / or the absence of substantial induction of the innate immune response in cells into which the polynucleotides are introduced. For example, modRNA may exhibit reduced degradation in cells into which it is introduced compared with the corresponding unmodified mRNA. These alternative species may enhance the efficiency of protein production, intracellular retention of polynucleotides, and / or the viability of contacted cells, as well as have reduced immunogenicity.

[0089] The mRNAs of this disclosure may contain one or more modified (e.g., altered or substituted) nucleic acid bases, nucleosides, nucleotides, or combinations thereof. mRNAs useful for LNPs may include any useful modifications or alterations to nucleic acid bases, sugars, or nucleoside linkages (e.g., to linking phosphates / to phosphate diester linkages / to phosphate diester backbone). In certain embodiments, the alterations (e.g., one or more alterations) are present in each of the nucleic acid bases, sugars, and nucleoside linkages. The alterations according to this disclosure may be alterations of ribonucleic acid (RNA), e.g., substitution of 2'-OH to 2'-H in a ribofuranosyl ring, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. Additional alterations are described herein.

[0090] The mRNA of this disclosure may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or one or more or all of A, G, U, and C) may or may not be uniformly modified in the mRNA or in a given given sequence region. In some cases, all nucleotides X in the mRNA (or in a given sequence region thereof) are modified, where X may be one of nucleotides A, G, U, or C, or one of any combination of A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0091] Various sugar modifications and / or nucleoside linkages (e.g., skeletal structures) can be located at various positions within the polynucleotide. Those skilled in the art will understand that nucleotide analogs or other modifications may be located at any position(s) within the polynucleotide where the function of the polynucleotide is not substantially impaired. The modifications may be at the 5' or 3' end. In some embodiments, the polynucleotide includes a modification at the 3' end. Polynucleotides make up approximately 1% to 100% (in relation to the overall nucleotide content, or in relation to one or more types of nucleotides, i.e., one or more of A, G, U, or C) or any percentage in between (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 10%). It may contain alternative nucleotides in the following percentages: up to 0%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%. It will be understood that the presence of standard nucleotides (e.g., A, G, U, or C) accounts for any remaining percentage.

[0092] A polynucleotide may contain at least zero, at most 100%, or any percentage in between, for example, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, or at least 90% of alternative nucleotides. For example, a polynucleotide may contain alternative uracil or alternative pyrimidines such as cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the polynucleotide is replaced with alternative uracil (e.g., 5-substituted uracil). The alternative uracil can be replaced with a single compound having a unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some cases, at least 5%, 10%, 25%, 50%, 80%, 90%, or 100% of the cytosines in a polynucleotide are replaced by substituted cytosines (e.g., 5-substituted cytosines). These substituted cytosines can be replaced by a single compound having a unique structure, or by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0093] In some cases, nucleic acids do not substantially induce the innate immune response of cells into which polynucleotides (e.g., mRNA) are introduced. The characteristics of the induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.), and / or 3) termination or reduction of protein translation.

[0094] In some embodiments, mRNA comprises one or more alternative nucleosides or nucleotides. Alternative nucleosides and nucleotides may comprise alternative nucleic acid bases. The nucleic acid bases of nucleic acids are organic bases such as purines or pyrimidines or their derivatives. The nucleic acid bases may be standard bases (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleic acid bases can be modified or completely replaced to result in polynucleotide molecules with enhanced properties, such as increased stability, including resistance to nucleases. Non-standard or modified bases may include, but are not limited to, one or more substitutions or modifications, one or more fused or ring-opening, oxidation, and / or reduction, including alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions.

[0095] In some embodiments, the nucleic acid base is a uracil substitute. Exemplary nucleic acid bases and nucleosides having a uracil substitute include pseudouridine (ψ), pyridine-4-onyribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil) Uracil, 3-methyl-uracil (mU), 5-methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudruridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-uracil Bonylmethyl-2-thiouracil (mcm5s2U), 5-aminomethyl-2-thiouracil (nmVu), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnmVu), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyluracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnmVu ), 5-propynyl-uracil, 1-propynyl-pseudracil, 5-taurinomethyl-uracil (xm5U), 1-taurinomethyl-pseudraidine, 5-taurinomethyl-2-thio-uracil (xm5s2U), 1-taurinomethyl-4-thio-pseudraidine, 5-methyl-uracil (m5U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseudraidine (mV), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudraidine (m xj / ), 4-thio-1-methyl-pseudraidine, 3-methyl-pseudraidine (m(| / ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-l-methyl-1-deaza-pseuduridine, dihydrouracil (D), dihydropseuduridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseuduridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseuduridine, 4-methoxy-2-thio-pseuduridine, N1-methyl-pseuduridine, 3-(3-amino-3-carboxypropyl)uracil (acp U), 1-methyl-3-(3-amino-3-carboxypropyl)pseuduridine (acp ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thiouracil (inm5s2U), 5,2'-0-dimethyluridine (m5Um), 2-thio-2'-0-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyluridine (memUm), 5-carbamoylmethyl-2'-0-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyluridine (cmnm5Um), 3,2'-0-dimethyluridine (m Examples include Um), and 5-(isopentenylaminomethyl)-2'-0-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propenylamino)]uracil.

[0096] In some embodiments, the nucleic acid base is a cytosine substitute. Exemplary nucleic acid bases and nucleosides having a cytosine substitute include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methylcytosine (m4C), 5-methylcytosine (m5C), 5-halocytosine (e.g., 5-iodocytosine), 5-Hydroxymethylcytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza- Isocytidine, Zebralin, 5-Aza-Zebralin, 5-Methyl-1-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxycytosine, 2-Methoxy-5-Methylcytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-dimethylcytidine (m5Cm), N4- Examples include acetyl-2'-0-methylcytidine (ac4Cm), N4,2'-0-dimethylcytidine (m4Cm), 5-formyl-2'-0-methylcytidine (f5Cm), N4,N4,2'-0-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxycytosine, 5-(3-azidopropyl)cytosine, and 5-(2-azidoethyl)cytosine.

[0097] In some embodiments, the nucleic acid base is an alternative adenine. Exemplary nucleic acid bases and nucleosides having an alternative adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-a Mino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-1-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl) Adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-H Examples include droxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (ml Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0098] In some embodiments, the nucleic acid base is a guanine substitute. Exemplary nucleic acid bases and nucleosides having a guanine substitute include inosine (I), 1-methyl-inosine (mil), waiosine (imG), methylwaiosine (mimG), 4-demethylwaiosine (imG-14), isowyosine (imG2), waibutosine (yW), peroxywaibutosine (o2yW), hydroxywaibutosine (OHyW), and undermodified hydroxywaibutosine (OHyW). * ), 7-deaza-guanine, quosin (Q), epoxyquosin (oQ), galactosylquosin (galQ), mannosylquosin (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl), alkaeosin (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (mlG), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-di Examples include methyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl-2'-0-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-0-methyl-guanosine (m22Gm), 1-methyl-2'-0-methyl-guanosine (mlGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'-0-methyl-inosine (Im), 1,2'-0-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl-guanine.

[0099] Nucleic acid bases that substitute for nucleotides may independently be purines, pyrimidines, or purine or pyrimidine analogs. For example, the nucleic acid base may be a substitute for adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleic acid bases include, for example, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines. Naturally occurring and synthetic derivatives of the bases may also be included, including guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinon, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazine-2-one, 1,2,4-triazine, pyridazine; or 1,3,5-triazine. When nucleotides are represented using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives, for example, A includes adenine or adenine analogs, such as 7-deazaadenine.

[0100] mRNA can contain a 5' cap structure. The 5' cap structure of polynucleotides is involved in nuclear export and increased polynucleotide stability, binding to cap-binding proteins (CBPs) of mRNA. This binding, in turn, facilitates the association of CBPs and poly(A)-binding proteins to form mature circular mRNA species, thereby contributing to polynucleotide stability and translational competency in cells. The cap also assists in the removal of 5' proximal introns during mRNA splicing.

[0101] An endogenous polynucleotide molecule can be capped at its 5' end, thereby creating a 5'ppp-5' triphosphate linkage between the terminal guanosine cap residue and the transcribed sense nucleotide at the 5' end of the polynucleotide. This 5' guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugar of the transcribed nucleotide at the 5' end and / or anteterminal end of the polynucleotide can also be 2'-O-methylated. By removing the 5' cap through hydrolysis and cleavage of the guanylate cap structure, polynucleotide molecules such as mRNA molecules can be targeted for degradation.

[0102] Modifications to the polynucleotide can generate a non-hydrolyzable cap structure that prevents cap removal and thus increases the polynucleotide half-life. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, alternative nucleotides can be used during the cap addition reaction. For example, Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage to the 5'-ppp-5' cap.

[0103] Additional alternative guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, can be used. Additional modifications, though not limited to those mentioned above, include 2'-O-methylation of the ribose sugar at the 2'-hydroxyl group of the ribose sugar at the 5' end and / or pre-5' end of the polynucleotide (as described above). Multiple distinct 5' cap structures can be used to generate the 5' cap of the mRNA molecule.

[0104] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5' caps, while retaining cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically, and / or linked to polynucleotides. For example, an anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, where one guanosine contains an N7-methyl group and a 3'-O-methyl group (i.e., N7, 3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7G-3'mppp-G, which may be equivalent to 3'0-Me-m7G(5')ppp(5')G). The other, unmodified 3'-0 atom of guanosine becomes ligated to the 5' terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3'-0 methylated guanosine provides the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on the guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'triphosphate-5'guanosine, m7Gm-ppp-G).

[0105] The cap may be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified with a boranophosphate group or a phosphoroselenoate group at different phosphate positions, such as the dinucleotide cap analog described in U.S. Patent No. 8,519,110, whose cap structure is incorporated herein by reference.

[0106] Alternatively, the cap analog may be an N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogs include the N7-(4-chlorophenoxyethyl)-G(5)ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5)ppp(5')G cap analogs (see, for example, Kore et al., Bioorganic & Medicinal Chemistry 2013 21:4570~4574, whose cap structures are incorporated herein by reference, for various cap analogs and methods for synthesizing cap analogs). In other cases, a useful cap analog for the polynucleotides of this disclosure is a 4-chloro / bromophenoxyethyl analog.

[0107] Capping analogs enable simultaneous capping of polynucleotides during in vitro transcription, but up to 20% of the transcript remains uncapped. This, along with structural differences in the endogenous 5' cap structure between polynucleotides produced by the endogenous cell transcription mechanism and those produced by the capping analog, leads to decreased translational competency and reduced cellular stability.

[0108] To generate a more authentic 5' cap structure, alternative polynucleotides may also be capped post-transcriptionally using an enzyme. As used herein, the phrase “more authentic” refers to a feature that closely reflects or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a “more authentic” feature better represents an endogenous, wild-type, natural, or physiological cellular function and / or structure compared to a synthetic feature or a prior art analog, or surpasses the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more authentic 5' cap structures useful for the polynucleotides of this disclosure include, among other things, those having enhanced binding affinity to cap-binding proteins, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' cap removal compared to synthetic 5' cap structures known in the art (or compared to wild-type, natural, or physiological 5' cap structures). For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can create a standard 5'-5'-triphosphate linkage between the 5' terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, where the cap guanosine contains N7-methylation and the 5' terminal nucleotide of the polynucleotide contains 2'-O-methylation. Such a structure is referred to as a cap 1 structure. This cap results in higher translational competency, cellular stability, and reduced activation of pro-inflammatory cytokines in cells, compared, for example, with other 5' cap analog structures known in the art. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0109] Alternative polynucleotides can be capped post-transcriptionally, and this process is more efficient, allowing capping of nearly 100% of mRNA. This is in contrast to the -80% rate achieved when capping analogs are ligated to polynucleotides during the in vitro transcription reaction. The 5' end cap can contain either an endogenous cap or a capping analog. The 5' end cap can contain a guanosine analog. Useful guanosine analogs include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, polynucleotides contain a modified 5' cap. Modification of the 5' cap can increase the stability of the polynucleotide, increase its half-life, and improve its translational efficiency. Modified 5' caps are not limited to these, but may include one or more of the following modifications: modifications at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), substitution of the methylene moiety (CH2) of the sugar ring oxygen (which forms a carbocyclic ring), modifications at the triphosphate bridge portion of the cap structure, or modifications at the nucleic acid base (G) portion.

[0110] A 5'UTR can be provided to the mRNA as an adjoint region. The 5'UTR may be homologous or heterologous to the coding region found in the polynucleotide. Multiple 5'UTRs may be included in the adjoint region, and their sequences may be the same or different. Any part of the adjoint region can be codon-optimized, including not optimizing any part, and each part may independently contain one or more different structural or chemical alterations before and / or after codon optimization.

[0111] In one embodiment, the ORF encoding the antigen of this disclosure is codon-optimized. Codon optimization methods are known in the art. For example, the ORF of any one or more sequences provided herein can be codon-optimized. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organisms to ensure proper folding; to bias the GC content to increase mRNA stability or reduce secondary structures; to minimize tandem repeat codons or base runs that may impair gene construction or expression; to customize transcriptional and translational regulatory regions; to insert or remove protein transport sequences; to remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; to add, remove or shuffle protein domains; to insert or delete restriction sites; to modify ribosome binding sites and mRNA degradation sites; to adjust the translation rate to properly fold various domains of the protein; or to reduce or remove problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art. Non-limiting examples include services and / or patented methods from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.). In some embodiments, open reading frame (ORF) sequences are optimized using optimization algorithms. To modify one or more properties of mRNA, heterologous 5'UTRs can be engineered for the coding region of mRNA. The mRNA can then be administered to cells, tissues, or organisms, and outcomes such as protein levels, localization, and / or half-life can be measured to evaluate the potentially beneficial effects on the mRNA brought about by the heterologous 5'UTR. Variants of the 5'UTR in which one or more nucleotides, including A, T, C, or G, are added to or removed from the terminal can be utilized. The 5'UTR can also be codon-optimized or modified in any manner described herein.

[0112] mRNA may include stem loops, such as histone stem loops, for example, but not limited to these. The stem loop may be a nucleotide sequence approximately 25 or 26 nucleotides long. The histone stem loop may be located 3' to the coding region (e.g., at the 3' end of the coding region). In a non-limiting example, the stem loop may be located at the 3' end of the polynucleotides described herein. In some cases, mRNA may contain more than one stem loop (e.g., two stem loops). The stem loop may be located at the second terminal region of the polynucleotide. In a non-limiting example, the stem loop may be located within the untranslated region of the second terminal region (e.g., the 3'UTR). In some cases, mRNA containing histone stem loops can be stabilized by adding a 3' stabilization region (e.g., a 3' stabilization region containing at least one chain-terminating nucleoside). Rather than being constrained by theory, the addition of at least one chain termination nucleoside can slow the degradation of a polynucleotide and thus increase its half-life. In other cases, mRNA containing a histone stem loop can be stabilized by making a modification to the 3' region of the polynucleotide that can prevent and / or inhibit the addition of oligo(U). In yet another case, mRNA containing a histone stem loop can be stabilized by adding oligonucleotides terminated with 3'-deoxynucleoside, 2',3'-dideoxynucleoside, 3'-O-methylnucleoside, 3-O-ethylnucleoside, 3'-arabinoside, and other alternative nucleosides known in the art and / or described herein. In some cases, the mRNAs of this disclosure may contain a histone stem loop, a polyA region, and / or a 5' cap structure. The histone stem loop may be before and / or after the polyA region. Polynucleotides comprising a histone stem-loop and a poly(A) region sequence may include the chain termination nucleosides described herein. In other cases, the polynucleotides of this disclosure may include a histone stem-loop and a 5' cap structure.Examples of 5' cap structures include, but are not limited to, those described herein and / or known in the art. In some cases, the conserved stem-loop region may contain the miR sequence described herein. In a non-limiting example, the stem-loop region may contain the seed sequence of the miR sequence described herein. In another non-limiting example, the stem-loop region may contain the miR-122 seed sequence.

[0113] mRNA may contain at least one histone stem-loop and a polyA region or polyadenylation signal. In certain cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode a pathogen antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode a therapeutic protein. In some cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode a tumor antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode an allergen antigen or an autoimmune autoantigen.

[0114] mRNA may contain a polyA sequence and / or a polyadenylation signal. The polyA sequence may consist of all or most of adenine nucleotides or their analogs or derivatives. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the nucleic acid. During RNA processing, a long chain of adenosine nucleotides (polyA region) is usually added to the messenger RNA (mRNA) molecule to increase molecular stability. Immediately after transcription, the 3' end of the transcript is cleaved, releasing a 3'-hydroxyl group. The chain of adenosine nucleotides is then added to the RNA by polyA polymerase. This process, called polyadenylation, involves the addition of a polyA region between 100 and 250 residues in length. The unique length of the polyA region can provide certain advantages to the alternative polynucleotides of this disclosure. Generally, the length of the polyA region of this disclosure is at least 30 nucleotides. In another embodiment, the polyA region is at least 35 nucleotides long. In yet another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 70 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides.In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1700 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 1900 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In some cases, the polyA region may be 80, 120, or 160 nucleotides long on the alternative polynucleotide molecules described herein. In other cases, the polyA region may be 20, 40, 80, 100, 120, 140, or 160 nucleotides long on the alternative polynucleotide molecules described herein. In some cases, the polyA region is designed relative to the total length of the alternative polynucleotide. This design may be based on the length of the coding region of the alternative polynucleotide, the length of a specific feature or region of the alternative polynucleotide (e.g., mRNA), or the length of the final product expressed from the alternative polynucleotide. When relative to any feature of the alternative polynucleotide (e.g., a portion of the mRNA containing the polyA region), the polyA region may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the additional feature.The polyA region can also be designed as a fraction of the alternative polynucleotide to which it belongs. In this regard, the polyA region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the polyA region.

[0115] In certain cases, expression can be enhanced using engineered binding sites and / or conjugation of mRNA to poly(A)-binding proteins. The engineered binding sites may be sensor sequences that can act as ligand binding sites in the local microenvironment of the mRNA. As a non-limiting example, the mRNA may contain at least one engineered binding site to alter the binding affinity of poly(A)-binding proteins (PABPs) and their analogues. By incorporating at least one engineered binding site, the binding affinity of PABPs and their analogues can be increased.

[0116] Furthermore, by using alternative nucleotides at the 3' end of the poly-A region, multiple distinct mRNAs can be ligated to PABP (poly-A binding protein) through the 3' end. Transfection experiments can be performed in relevant cell lines, and protein production can be assayed by ELISA at 12, 24, 48, 72, and 7 days post-transfection. As a non-limiting example, transfection experiments can be used to evaluate the effect on the binding affinity of PABP or its analogues as a result of the addition of at least one engineered binding site. In certain cases, the poly-A region can be used to modulate translation initiation. While we do not wish to be constrained by theory, the poly-A region may mobilize PABP, which in turn interacts with the translation initiation complex, and therefore the poly-A region may be essential for protein synthesis. In some cases, as disclosed herein, the poly-A region can also be used for protection from 3'-5' exonuclease digestion. In some cases, the mRNA may contain a poly-AG quadruplex. A G quadruplex is an array of four guanosine nucleotides linked by a cyclic hydrogen bond, and can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G quadruplex is incorporated at the end of the poly-A region. The resulting mRNA can be assayed at various time points for other parameters, including stability, protein production, and half-life. It has been found that the poly-AG quadruplex results in protein production equivalent to at least 75% of the protein production seen when using the 120-nucleotide poly-A region alone. In some cases, the mRNA can include a poly-A region and can be stabilized by adding a 3' stabilization region. mRNA with a poly-A region may further include a 5' cap structure. In other cases, the mRNA may include a poly-AG quadruplex. mRNA with a poly-AG quadruplex may further include a 5' cap structure. In some cases, the poly-A region or the poly-AG quadruplex can be used as a 3' stabilization region to stabilize the mRNA.In other cases, 3' stabilizing regions that can be used in this disclosure include chain termination nucleosides, such as 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleosides, or O-methylnucleosides. In other cases, mRNA containing a polyA region or polyAG quadruplex can be stabilized by modifications that can prevent and / or inhibit the addition of oligo(U) to the 3' region of the polynucleotide. In other cases, mRNA containing a poly-A region or poly-AG quadruple strand is converted into a 3'-deoxynucleoside, a 2',3'-dideoxynucleoside. It can be stabilized by adding oligonucleotides terminated with 3-O-methyl nucleoside, 3'-O-ethyl nucleoside, 3'-arabinoside, and other nucleosides known in the art and / or alternative nucleosides described herein.

[0117] In one embodiment, the mRNA vaccine of this disclosure contains lipids. Lipids and modRNA can combine to form nanoparticles. Encapsulating mRNA in lipids can create lipid nanoparticles (LNPs), which can aid in the entry and stability of RNA / lipid nanoparticles into cells.

[0118] Lipid nanoparticles may contain lipid components as well as one or more additional components, such as therapeutic and / or prophylactic agents. LNPs can be designed for one or more specific applications or targets. The elements of an LNP can be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, a specific formulation of an LNP can be selected for a specific application or target, for example, depending on the efficacy and toxicity of a particular combination of elements. The efficacy and tolerability of an LNP formulation may be affected by the stability of the formulation.

[0119] Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within the mammalian body.

[0120] To increase selectivity for specific body targets, the biochemical properties of lipid nanoparticles can be modified. For example, particle size can be adjusted based on the window sizes of different organs. The therapeutic and / or prophylactic agents contained in LNPs can also be selected based on the desired delivery target(s). For example, therapeutic and / or prophylactic agents can be selected for specific indications, conditions, diseases, or disorders, and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., localized or specific delivery). In certain embodiments, LNPs may contain mRNA encoding a target polypeptide, which can be translated in cells to produce the target polypeptide. Such compositions can be designed to be specifically delivered to specific organs. In some embodiments, a composition can be designed to be specifically delivered to the mammalian liver. In some embodiments, a composition can be designed to be specifically delivered to lymph nodes. In some embodiments, a composition can be designed to be specifically delivered to the mammalian spleen.

[0121] LNPs may comprise one or more components described herein. In some embodiments, the LNP formulations of this disclosure comprise at least one lipid nanoparticle component. The lipid nanoparticles may comprise the lipid components as well as one or more additional components, such as therapeutic and / or prophylactic agents, such as nucleic acids. LNPs can be designed for one or more specific applications or targets. The elements of an LNP can be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, a particular formulation of an LNP can be selected for a specific application or target, for example, depending on the efficacy and toxicity of a particular combination of elements. The efficacy and tolerability of an LNP formulation may be affected by the stability of the formulation.

[0122] In some embodiments, polymers may be included and / or used for encapsulation or partial encapsulation of LNPs. The polymers may be biodegradable and / or biocompatible. The polymers may be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, as polymers, poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L- Poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyacid anhydride, polyorthoester, poly(ester) Polyalkylenes such as amides, polyamides, poly(ester ethers), polycarbonates, polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), poly(ethylene terephthalate) and other polyalkylene terephthalates, polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyhalogenated vinyls such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, cellulose derivatives such as nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose, poly(methyl(meth)acrylate)) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate) Examples include polymers of acrylic acid such as poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoic acid, polypropylene fumarate, polyoxymethylene, poloxamer, poloxamine, poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerin.

[0123] Examples of surface modifiers, though not limited to these, include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, Clerodendrum, bromhexine, carbocysteine, eprazinon, mesna, ambroxol, sobrelol, domiodol, letosteine, stepronin, thiopronin, gelzolin, thymosin β4, dorunase alpha, neltenexin, and erdosteine), and DNA-degrading enzymes (e.g., rhDNase). Surface modifiers can be positioned within nanoparticles and / or on the surface of LNPs (e.g., by coating, adsorption, covalent linking, or other processes).

[0124] LNPs may contain one or more functionalized lipids. For example, lipids can be functionalized with alkyne groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner using one or more groups useful for facilitating membrane permeation, cell recognition, or imaging. One or more useful antibodies can also be conjugated to the surface of the LNP. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0125] In addition to these components, lipid nanoparticles may contain any substance useful in a pharmaceutical composition. For example, lipid nanoparticles may contain, but are not limited to, one or more pharmaceutically acceptable excipients or adjuncts such as solvents, dispersion media, diluents, dispersion aids, suspension aids, surfactants, buffers, preservatives, and others.

[0126] Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), and polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45]). Examples include polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and / or combinations thereof.

[0127] Examples of preservatives, but not limited to these, include antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcoholic preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants, but not limited to these, include alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcoholic preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and / or phenylethyl alcohol. Examples of acidic preservatives, though not limited to these, include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®. Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

[0128] Examples of buffers, though not limited to these, include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dsodium phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, aminosulfonic acid buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, Tris buffer, and / or combinations thereof.

[0129] In some embodiments, the formulation containing LNP may further contain salts such as chloride salts. In some embodiments, the formulation containing LNP may further contain sugars such as disaccharides. In some embodiments, the formulation further contains sugars but does not contain salts such as chloride salts. In some embodiments, the LNP may further contain one or more small hydrophobic molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols. The carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0130] The characteristics of LNPs can depend on their constituent components. For example, an LNP containing cholesterol as a structural lipid may have different characteristics than an LNP containing a different structural lipid. As used herein, the term “structural lipid” refers to sterols, and also to lipids containing a sterol moiety. As defined herein, “sterol” is a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.

[0131] Lipid nanoparticle compositions may contain one or more structural lipids. Incorporation of structural lipids into lipid nanoparticles may help reduce the aggregation of other lipids within the particles. Structural lipids may be selected from, but are not limited to, cholesterol, fecosterol, sitosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol. In some preferred embodiments, the sterol is

[0132] [ka] Includes. In some preferred embodiments, sterols are

[0133] [ka] It includes. In some preferred embodiments, the sterol includes stigmasterol. In some preferred embodiments, the sterol includes citostanol.

[0134] In some embodiments, the structural lipids are sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, beta-sitosterol, stigmastanol, beta-sitostanol, ergosterol, lupeol, cycloartenol, Δ5-avenacerol, Δ7-avenacerol, or Δ7-stigmasterol, and include their analogues, salts, or esters. In some embodiments, the sterol component of the LNP of the Disclosure is a single phytosterol. In some embodiments, the phytosterol component of the LNP of the Disclosure is a mixture of different phytosterols (e.g., 2, 3, 4, 5, or 6 different phytosterols). In some embodiments, the phytosterol component of the LNP of the Disclosure is a blend of one or more phytosterols and one or more zoosterols, e.g., a blend of a phytosterol (e.g., a sitosterol such as beta-sitosterol) and cholesterol. In some embodiments, the phytosterol is β-sitosterol, campesterol, sigmasterol, or any combination thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, one or more structural lipids comprise a mixture of β-sitosterol, campesterol, and stigmasterol. In some embodiments, one or more structural lipids comprise a mixture of β-sitosterol and cholesterol. In one embodiment, the structural lipid is selected from β-sitosterol and cholesterol. In some embodiments, the structural lipid is β-sitosterol. In some embodiments, the structural lipid is cholesterol.

[0135] In some embodiments, one or more structural lipids contain about 35% to about 85% β-sitosterol, about 5% to about 35% stigmasterol, and about 5% to about 35% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 80% β-sitosterol, about 10% to about 30% stigmasterol, and about 10% to about 30% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 70% β-sitosterol, about 10% to about 25% stigmasterol, and about 10% to about 25% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 70% β-sitosterol, about 15% to about 25% stigmasterol, and about 15% to about 25% campesterol. In some embodiments, one or more structural lipids contain about 35% to about 45% β-sitosterol, about 20% to about 30% stigmasterol, and about 20% to about 30% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 50% β-sitosterol, about 25% to about 35% stigmasterol, and about 25% to about 35% campesterol. In some embodiments, one or more structural lipids contain about 65% to about 75% β-sitosterol, about 5% to about 15% stigmasterol, and about 5% to about 15% campesterol. In some embodiments, one or more structural lipids contain about 35% to about 85% β-sitosterol, about 5% to about 35% stigmasterol, and 0% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 80% β-sitosterol, about 10% to about 30% stigmasterol, and 0% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 70% β-sitosterol, about 10% to about 25% stigmasterol, and 0% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 70% β-sitosterol, about 15% to about 25% stigmasterol, and 0% campesterol.In some embodiments, one or more structural lipids contain about 35% to about 45% β-sitosterol, about 20% to about 30% stigmasterol, and 0% campesterol. In some embodiments, one or more structural lipids contain about 40% to about 50% β-sitosterol, about 25% to about 35% stigmasterol, and 0% campesterol. In some embodiments, one or more structural lipids contain about 65% to about 75% β-sitosterol, about 5% to about 15% stigmasterol, and 0% campesterol. Therefore, in some preferred embodiments, the composition does not contain campesterol.

[0136] In some embodiments, the composition comprises one or more structural lipids, including about 10% to about 30% cholesterol, about 10% to about 30% β-sitosterol, about 10% to about 30% stigmasterol, and 0% campesterol. See, for example, Table 41. In some embodiments, the composition further comprises about 30% to 50% cationic lipids and about 5% to 25% phospholipids.

[0137] In some embodiments, the mol% of one or more structural lipids is between about 1% and 50% of the mol% of the compounds having any of the structures of the aforementioned compounds present in the lipid nanoparticles. In some embodiments, the mol% of one or more structural lipids is between about 10% and 40% of the mol% of the compounds having any of the structures of the aforementioned compounds present in the lipid nanoparticles. In some embodiments, the mol% of one or more structural lipids is between about 20% and 30% of the mol% of the compounds having any of the structures of the aforementioned compounds present in the lipid nanoparticles. In some embodiments, the mol% of one or more structural lipids is between about 30% of the mol% of the compounds having any of the structures of the aforementioned compounds present in the lipid nanoparticles.

[0138] In some embodiments, the lipid nanoparticle compositions described herein may contain about 20 mol% to about 60 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions contain about 30 mol% to about 50 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions contain about 35 mol% to about 45 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions contain about 37 mol% to about 42 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions contain about 35, about 36, about 37, about 38, about 39, or about 40 mol% of structural lipids. In some embodiments, the nanoparticles contain about 39 to about 40 mol% of structural lipids.

[0139] In some embodiments, the LNP of the present invention includes an N:P ratio of about 2:1 to about 30:1. In some embodiments, the LNP of the present invention includes an N:P ratio of about 6:1. In some embodiments, the LNP of the present invention includes an N:P ratio of about 3:1, 4:1, or 5:1.

[0140] In some embodiments, the characteristics of LNPs may depend on the absolute or relative amounts of their constituent components. For example, LNPs containing a high molar fraction of phospholipids may have different characteristics from LNPs containing a low molar fraction of phospholipids. The properties may also vary depending on the method and conditions for preparing the lipid nanoparticles. Generally, phospholipids consist of a phospholipid moiety and one or more fatty acid moieties.

[0141] The phospholipid moiety can be selected from a non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin (SM). Further examples of phospholipid moieties for lipid nanoparticles include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphospha Tidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethylPE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diemcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin,Lipids selected from the group consisting of phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. In some embodiments, the lipid nanoparticles include sphingomyelin. In some embodiments, the nanoparticle composition comprises multiple lipid nanoparticles, the lipid nanoparticles including (a) sphingomyelin at about 5-40 mol% of the total lipids present in the nanoparticle composition; (b) cationic lipids; (c) steroids; (d) lipids conjugated to polymers; and (e) nucleic acids. In some embodiments, sphingomyelin is at about 10-40 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin is at about 10-30 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 10-25 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 10-20 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 10-15 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 10 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 15 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 20 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 10-20 mol% of the total lipids present in the nanoparticle composition, and cationic lipids account for approximately 40-50 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin accounts for approximately 10-15 mol% of the total lipids present in the nanoparticle composition, and cationic lipids account for approximately 45 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin constitutes about 10-15 mol% of the total lipids present in the nanoparticle composition.Cationic lipids constitute approximately 40 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin constitutes approximately 10 mol% of the total lipids present in the nanoparticle composition, and cationic lipids constitute approximately 50 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin constitutes approximately 10 mol% of the total lipids present in the nanoparticle composition, and cationic lipids constitute approximately 45 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin constitutes approximately 15 mol% of the total lipids present in the nanoparticle composition, and cationic lipids constitute approximately 45 mol% of the total lipids present in the nanoparticle composition. In some embodiments, sphingomyelin is a sphingomyelin compound. In some embodiments, sphingomyelin is selected from SM-01, SM-02, SM-03, SM-04, SM-05, SM-06, and SM-07. In some embodiments, the molar percentage of sphingomyelin in the total lipids present in the nanoparticle composition is the same as the molar percentage of DSPC in the total lipids present in the reference nanoparticle composition. In some embodiments, sphingomyelin is present at about 5-40 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 10-40 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 10-30 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 10-25 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 10-20 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 10-15 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 10 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present at about 15 mol% of the total lipids present in the composition. In some embodiments, sphingomyelin is present in the composition at approximately 20 mol% of the total lipids. In some embodiments, sphingomyelin is present in the composition at approximately 5 mol%, approximately 6 mol%, approximately 7 mol%, approximately 8 mol%, approximately 9 mol%, approximately 10 mol%, of the total lipids.about 11 mol%, about 11.5 mol%, about 12 mol%, about 12.5 mol%, about 13 mol%, about 13.5 mol%, about 14 mol%, about 14.5 mol%, about 15 mol%, about 15.5 mol%, about 16 mol%, about 16.5 mol%, about 17 mol%, about 17.5 mol%, about 18 mol%, about 18.5 mol%, about 19 mol%, about 19.5 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 30 mol%, about 35 mol%, or about 40 mol%. In some embodiments, the sphingomyelin in the composition has the following structure:

[0142] [Chemical Formula] (wherein R is alkyl or alkenyl) is a sphingomyelin compound having In some embodiments, R is C 11 to C 23 alkyl. In some embodiments, R is C 11 to C 19 alkyl. In some embodiments, R is C 13 to C 19 alkyl. In some embodiments, R is C 15 to C 19 alkyl. In some embodiments, R is C 11 alkyl (e.g., -(CH2) 10 -CH3). In some embodiments, R is C 13 alkyl (e.g., -(CH2) 12 -CH3). In some embodiments, R is C 14 alkyl (e.g., -(CH2) 13 -CH3). In some embodiments, R is C 15 alkyl (e.g., -(CH2) 14 -CH3). In some embodiments, R is C 16 alkyl (e.g., -(CH2) 15 -CH3). In some embodiments, R is C 17 alkyl (e.g., -(CH2) 16-CH3) is. In some embodiments, R is C 18 Alkyl (e.g., -(CH2)) 17 -CH3) is. In some embodiments, R is C 19 Alkyl (e.g., -(CH2)) 18 -CH3) is. In some embodiments, R is C 20 Alkyl (e.g., -(CH2)) 19 -CH3) is. In some embodiments, R is C 21 Alkyl (e.g., -(CH2)) 20 -CH3) is. In some embodiments, R is C 22 Alkyl (e.g., -(CH2)) 21 -CH3) is. In some embodiments, R is C 23 Alkyl (e.g., -(CH2)) 22 -CH3). In one embodiment, the alkyl is a linear alkyl. In one embodiment, the alkyl is a branched alkyl. In some embodiments, the alkyl is unsubstituted. In some embodiments, the sphingomyelin provided herein is selected from the SM-01, SM-02, SM-03, SM-06 and SM-07 molecules shown in Table 53. In some embodiments, R is C 11 ~C 23 It is an alkenyl. In one embodiment, R is C 13 ~C 19 It is an alkenyl. In one embodiment, R is C 15 ~C 19 It is an alkenyl. In one embodiment, R is C 11 It is an alkenyl. In one embodiment, R is C 13 It is an alkenyl. In one embodiment, R is C 14 It is an alkenyl. In one embodiment, R is C 15 It is an alkenyl. In some embodiments, R is C 16 It is an alkenyl. In one embodiment, R is C 17 It is an alkenyl. In one embodiment, R is C 18 It is an alkenyl. In one embodiment, R is C 19 It is an alkenyl. In one embodiment, R is C 20It is an alkenyl. In one embodiment, R is C 21 It is an alkenyl. In one embodiment, R is C 22 It is an alkenyl. In one embodiment, R is C 23 It is an alkenyl. In one embodiment, the alkenyl has one double bond. In one embodiment, the double bond has a Z configuration. In one embodiment, the double bond is at position 9 of the alkenyl R group. In one embodiment, the alkenyl is a linear alkenyl. In one embodiment, the alkenyl is a branched alkenyl. In one embodiment, the alkenyl is unsubstituted.

[0143] The fatty acid portion can be selected from a non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Certain phospholipids can facilitate fusion with membranes. In some embodiments, cationic phospholipids can be made to interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with membranes makes it possible to pass one or more elements (e.g., therapeutic agents) of a lipid-containing composition (e.g., LNP) through membrane permeability, for example, to deliver one or more elements to a target tissue. Non-natural phospholipid species, including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also intended. In some embodiments, phospholipids can be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Exposure of the alkyne groups to an azide under suitable reaction conditions can lead to copper-catalyzed cycloaddition. Such reactions may be useful in functionalizing the lipid bilayer of a nanoparticle composition to facilitate membrane permeability or cell recognition, or in conjugating the nanoparticle composition with useful components such as a targeting or imaging moiety (e.g., a dye). Examples of phospholipids, but not limited to these, include glycerophospholipids such as phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Sphingophospholipids such as sphingomyelin are also examples of phospholipids. In some embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC.

[0144] Lipid nanoparticles can be characterized by various methods. For example, the morphology and size distribution of LNPs can be investigated using a microscope (e.g., a transmission electron microscope or a scanning electron microscope). Zeta potentials can be measured using dynamic light scattering or potentiometric measurements (e.g., potentiometric titration). Particle size can also be determined using dynamic light scattering. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to measure the characteristics of multiple LNPs, such as particle size, polydispersity index, and zeta potential.

[0145] The average size of LNPs can range from tens of nanometers to hundreds of nanometers, measured, for example, by dynamic light scattering (DLS). For example, the average size could range from approximately 40 nm to approximately 150 nm, such as approximately 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the LNP may be approximately 50 nm to approximately 100 nm, approximately 50 nm to approximately 90 nm, approximately 50 nm to approximately 80 nm, approximately 50 nm to approximately 70 nm, approximately 50 nm to approximately 60 nm, approximately 60 nm to approximately 100 nm, approximately 60 nm to approximately 90 nm, approximately 60 nm to approximately 80 nm, approximately 60 nm to approximately 70 nm, approximately 70 nm to approximately 100 nm, approximately 70 nm to approximately 90 nm, approximately 70 nm to approximately 80 nm, approximately 80 nm to approximately 100 nm, approximately 80 nm to approximately 90 nm, or approximately 90 nm to approximately 100 nm. In a particular embodiment, the average size of the LNP may be approximately 70 nm to approximately 100 nm. In a particular embodiment, the average size may be approximately 80 nm. In other embodiments, the average size may be approximately 100 nm.

[0146] LNPs can be relatively uniform. The polydispersity index can be used to indicate the uniformity of LNPs, for example, the particle size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The polydispersity index of LNPs can range from about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs can range from about 0.10 to about 0.20.

[0147] The zeta potential of LNPs can be used to indicate the interfacial dynamic potential of a composition. For example, the zeta potential can describe the surface charge of LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally preferred because highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of LNPs is approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, and approximately -5mV. It could be from V to approximately +10mV, from approximately -5mV to approximately +5mV, from approximately -5mV to approximately 0mV, from approximately 0mV to approximately +20mV, from approximately 0mV to approximately +15mV, from approximately 0mV to approximately +10mV, from approximately 0mV to approximately +5mV, from approximately +5mV to approximately +20mV, from approximately +5mV to approximately +15mV, or from approximately +5mV to approximately +10mV.

[0148] The efficiency of encapsulation of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agents encapsulated in the LNPs or otherwise associated with them after preparation. High encapsulation efficiency (e.g., close to 100%) is desirable. Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agents in a solution containing lipid nanoparticles with the amount of therapeutic and / or prophylactic agents before and after disintegration of the lipid nanoparticles using one or more organic solvents or surfactants. Free therapeutic and / or prophylactic agents (e.g., RNA) in solution can be quantified using fluorescence. With respect to the lipid nanoparticles described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents may be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0149] LNP may optionally include one or more coatings. For example, LNP can be formulated into capsules, films, or tablets with coatings. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0150] Formulations comprising amphiphilic polymers and lipid nanoparticles can be formulated as whole or in part as pharmaceutical compositions. A pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid nanoparticles. For example, a pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid nanoparticles comprising one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or adjuncts, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and drugs are available, for example, in Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjuncts may be used in any pharmaceutical composition unless any of them may be incompatible with one or more components of the LNPs or one or more amphiphilic polymers in the formulations of this disclosure. If a combination of an excipient or accessory with a component of the formulation's LNP or amphiphilic polymer could result in any undesirable biological or other adverse effects, then that excipient or accessory may be incompatible with the component of the formulation's LNP or amphiphilic polymer.

[0151] In some embodiments, one or more excipients or adjuncts may constitute more than 50% of the total mass or volume of the pharmaceutical composition containing LNP. For example, one or more excipients or adjuncts may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, pharmaceutically acceptable excipients are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for use in humans and animals. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and / or International Pharmacopoeia. The relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical compositions according to this disclosure will vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route of administration of the composition. For example, a pharmaceutical composition may contain one or more lipid nanoparticles between 0.1% and 100% (wt / wt). Another example is a pharmaceutical composition which may contain one or more amphiphilic polymers between 0.1% and 15% (wt / vol) (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / v).

[0152] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the Disclosure are cooled or frozen for storage and / or transport (e.g., at temperatures below 4°C, e.g., between approximately -150°C and approximately 0°C or between approximately -80°C and approximately -20°C (e.g., stored at approximately -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C)). For example, a pharmaceutical composition comprising one or more types of amphiphilic polymers and one or more types of lipid nanoparticles is stored and / or transported at, for example, approximately -20°C, -30°C, -40°C , a solution or solid (e.g., by freeze-drying) cooled to -50°C, -60°C, -70°C, or -80°C. In certain embodiments, the disclosure also relates to methods for increasing the stability of lipid nanoparticles by adding an effective amount of amphiphilic polymer, and by storing the lipid nanoparticles and / or their pharmaceutical composition at temperatures below 4°C, for example, between about -150°C and about 0°C, or between about -80°C and about -20°C, for example, about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C.

[0153] The chemical properties of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of this disclosure can be characterized by various methods. In some embodiments, mRNA integrity can be investigated using electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reversed-phase liquid chromatography).

[0154] The efficacy of the product depends on the expression of the delivered RNA, which requires a sufficiently intact RNA molecule. RNA integrity is a measure of RNA quality that quantifies intact RNA. The method can also detect potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. The initial specifications are set to ensure sufficient RNA integrity in the formulation preparation. In some embodiments, the RNA polynucleotide has an integrity of at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA polynucleotide has an integrity of about 95% or higher. In some embodiments, the RNA polynucleotide has an integrity of about 98% or higher. In some embodiments, the RNA polynucleotide has an integrity of about 99% or higher.

[0155] In preferred embodiments, the RNA polynucleotide has clinical-grade purity. In some embodiments, the purity of the RNA polynucleotide is about 60% to about 100%. In some embodiments, the purity of the RNA polynucleotide is about 80% to about 99%. In some embodiments, the purity of the RNA polynucleotide is about 90% to about 99%. In some embodiments, the purified mRNA has clinical-grade purity without further purification. In some embodiments, clinical-grade purity is achieved by a method including tangential flow filtration (TFF) purification. In some embodiments, clinical-grade purity is achieved without further purification selected from high-performance liquid chromatography (HPLC) purification, ligand or binding-based purification, and / or ion exchange chromatography. In some embodiments, the method for producing RNA polynucleotide removes long avotive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual solvent, and / or residual salts. In some embodiments, short avotive transcription contaminants contain less than 15 bases. In some embodiments, short avotive transcription contaminants contain about 8 to 12 bases. In some embodiments, the method of the present invention also removes RNAse inhibitors.

[0156] In some embodiments, the purified RNA polynucleotides, as determined by capillary electrophoresis, contain or are substantially free of protein contaminants at concentrations of 5%, 4%, 3%, 2%, and 1% or less. In some embodiments, the purified RNA polynucleotides, as determined by high-performance liquid chromatography (HPLC), contain or are substantially free of salt contaminants at concentrations of less than 5%, 4%, 3%, 2%, and 1% or less. In some embodiments, the purified RNA polynucleotides, as determined by known methods such as high-performance liquid chromatography (HPLC), contain or are substantially free of short avotive transcription contaminants at concentrations of 5%, 4%, 3%, 2%, and 1% or less. In some embodiments, the purified RNA polynucleotides have an integrity of 60% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, as determined by known methods such as capillary electrophoresis.

[0157] Modified nucleic acid bases Modified nucleic acid bases that can be incorporated into modified nucleosides and nucleotides and may be present in RNA molecules include, for example, m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A ( 2-Methylthio-N6-isopentenyladenosine); io6A(N6(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A(N6-threonylcarbamoyladenosine); ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamo Ilyadenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p)(2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); £5C (5-phonylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl2TO Methylcytidine); k2C (lysidine); mlG (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wibutosine); o2yW (peroxywibutosine); OHyW (hydroxywibutosine); OHyW *(Low-modified hydroxywibutosin); imG (wiosin); mimG (methylguanosine); Q (queosin); oQ (epoxyqueosin); galQ (galtactosylqueosin); manQ (mannosylqueosin); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G *(Archeosin); D(Dihydrouridine); m5Um(5,2'-O-dimethyluridine); s4U(4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um(2-thio-2'-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U(5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U(uridine 5-oxyacetic acid); mcmo5U(uridine 5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U(5-methoxycarbonylmethyluridine); mcm5Um(S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U(5-methylaminomethyluridine); mnm5s2U(5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine) ncm5U(5-carbamoylmethyluridine);ncm5Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm5U(5-carboxymethylaminomethyluridine);cnmm5Um(5-carboxymethylaminomethyl-2-LOmethyluridine);cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine);m62A(N6,N6-dimethyladenosine);Tm(2'-O-methylinosine);m4C(N4-methylcytidine);m4Cm(N4,2-O-dimethylcytidine);hm5C (5-hydroxymethylcytidine); m3U(3-methyluridine); cm5U(5-carboxymethyluridine); m6Am(N6,TO-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2'7G(N2,7-dimethylguanosine); m2'2'7G(N2,N2,7-trimethylguanosine); m3Um(3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm(5-formyl-2'-O-methylcytidine); mlGm(l,2'-O-dimethylguanosine);m'Am(1,2-O-dimethyladenosine)irinomethyluridine); tm5s2U(S-taurinomethyl-2-thiouridine)); imG-14(4-demethylguanosine); imG2(isoguanosine); ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1~C6)-alkyluracil, 5-methyluracil, 5-(C2~C; e )-Alkenyluracil, 5-(C2~C e )-Alkinyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1~C6)-alkylcytosine, 5-methylcytosine, 5-(C2~C6)-alkenylcytosine, 5-(C2~C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine Examples include 7-deaza-7-(C2~C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (debased residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. In some embodiments, one or more modified nucleosides in the list may be excluded.

[0158] Further exemplary modified nucleotides include any one of N-1-methylpseudridine, pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some embodiments, the modified nucleotide is N-1-methylpseudridine.

[0159] In some embodiments, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate bonds.

[0160] In some embodiments, the RNA molecule contains a modified nucleotide selected from one of the following: pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methyl-1-pseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of 5-methyluridine, N1-methylpseudridine, 5-methoxyuridine, and 5-methylcytosine.

[0161] In some embodiments, at least 10% of the entire population of a particular nucleotide in the saRNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a particular nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a particular nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a particular nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, essentially the entire population of a particular nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.

[0162] In some embodiments, at least a portion or all of the entire population of specific nucleotides in the saRNA molecule is replaced by two modified or non-natural nucleotides. In some embodiments, the two modified or non-natural nucleotides are 1:99;2:98;3:97;4:96;5:95;6:94;7:93;8:92;9:91;10:90;11:89;12:88;13:87;14:86;15:85;16:84;17:83;18:82;19:81;20:80;21:79;22:78;23:77;24:76;25:75;26 :74;27:73;28:72;29:71;30:70;31:69;32:68;33:67;34:66;35:65;36:64;37:63;38:62;39:61;40:60;41:59;42:58;43:57;44:56;45:55;46:54;47:53;48:52;49:51;50:50;51:49;52:48;53:47;54:46; 55:45;56:44;57:43;58:42;59:41;60:40;61:39;62:38;63:37;64:36;65:35;66:34;67:33;68:32;69:31;70:30;71:29;72:28;73:27;74:26;75:25;76:24;77:23;78:22;79:21;80:20;81:19;82:18;83:1 Provided in equal ratios between 1:99 to 99:1, or any range derived therefrom, including 7;84:16;85:15;86:14;87:13;88:12;89:11;90:10;91:9;92:8;93:7;94:6;95:5;96:4;97:3;98:2; and 99:1, or any range derived therefrom, at least one, at most one, or any two of them.

[0163] In some embodiments, at least 10% of the total population of a first specific nucleotide in the saRNA molecule disclosed herein are replaced with one or more modified or non-natural nucleotides, and at least 10% of the total population of a second specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of a first specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides, and at least 25% of the total population of a second specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of a first specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides, and at least 50% of the total population of a second specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of a first specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides, and at least 75% of the total population of a second specific nucleotide in the molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, essentially the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.

[0164] In some embodiments, at least 25% of the total population of uridine nucleotides in the saRNA molecule is replaced with N1-methylpseudridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule is replaced with N1-methylpseudridine. In some embodiments, at least 75% of the total population of uridine nucleotides in the molecule is replaced with N1-methylpseudridine. In some embodiments, essentially all of the uridine nucleotides in the molecule are replaced with N1-methylpseudridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule is replaced with 5-methoxyuridine. In some embodiments, essentially all of the uridine nucleotides in the molecule are replaced with 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule is replaced with 5-methyluridine. In some embodiments, essentially all of the uridine nucleotides in the molecule are replaced with 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced with 2-thiouridine.

[0165] In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with N1-methylpseudridine, and essentially all of the cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with 5-methoxyuridine, and essentially all of the cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with 5-methyluridine, and essentially all of the cytosine nucleotides in the molecule are replaced with 5-methylcytosine.

[0166] In some embodiments, essentially all uridine nucleotides in the molecule are replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced with about 75% 5-methoxyuridine and about 25% N1-methylpseudridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced with about 25% 5-methoxyuridine and about 75% N1-methylpseudridine.

[0167] UTR The 5' untranslated region (UTR) is a regulatory region of DNA located at the 5' end of a protein-coding region that is transcribed into mRNA but not translated into protein. The 5'UTR may contain various regulatory elements, such as a 5' cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which can play a role in regulating translation initiation. The 3'UTR, located downstream of the protein-coding sequence, may be involved in regulatory processes including transcript cleavage, stability, and polyadenylation, translation, and mRNA localization. In some embodiments, the UTR derives from mRNA that is naturally abundant in specific tissues where mRNA expression is targeted (e.g., lymphoid tissue). In some embodiments, the UTR increases protein synthesis. While not bound by mechanism or theory, the UTR may increase protein synthesis by increasing the time mRNA remains in the translation polysome (message stability) and / or the rate at which ribosomes initiate translation on the message (message translation efficiency). Therefore, UTR sequences may extend protein synthesis in a tissue-specific manner. In some embodiments, the 5'UTR and 3'UTR sequences are computer-derived. In some embodiments, the 5'UTR and 3'UTR are derived from naturally abundant mRNA in tissues. The tissue may be, for example, the liver, stem cells, or lymphoid tissue. Lymphoid tissue may include, for example, lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or natural killer cells), macrophages, monocytes, dendritic cells, neutrophils, eosinophils, and reticulocytes. In some embodiments, the 5'UTR and 3'UTR are derived from alphaviruses. In some embodiments, the 5'UTR and 3'UTR are derived from wild-type alphaviruses. Examples of alphaviruses are described below.

[0168] In some embodiments, the first RNA molecule includes 5'UTR and 3'UTR derived from naturally abundant mRNA in tissues. In some embodiments, the first RNA molecule includes 5'UTR and 3'UTR derived from alphavirus. In some embodiments, the second RNA molecule or saRNA molecule includes 5'UTR and 3'UTR derived from alphavirus. In some embodiments, the second RNA molecule or saRNA molecule includes 5'UTR and 3'UTR derived from wild-type alphavirus. In some embodiments, the RNA molecule includes a 5' cap.

[0169] Open Reading Frame (ORF) The 5' and 3' UTRs can be operably ligated to an ORF, which may be a sequence of codons that can be translated into the polypeptide of interest. As described above, an RNA molecule may contain one (monocistronic), two (dipocistronic), or more (polycistronic) open reading frames (ORFs).

[0170] In some embodiments, the ORF encodes an unstructured viral gene. In some embodiments, the ORF further comprises one or more subgenome promoters. In some embodiments, the RNA molecule comprises a subgenome promoter operably linked to the ORF. In some embodiments, the subgenome promoter comprises a cis-acting regulatory element. In some embodiments, the cis-acting regulatory element is B 2 It is located immediately downstream of (5'-3'). In some embodiments, the cis-acting modulating element is B 2(5'-3') is located immediately downstream of guanine, which is immediately downstream of . In some embodiments, the cis-active regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauauau, auauauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug. In some embodiments, the second RNA or saRNA molecule may comprise (i) an ORF encoding a replicase capable of transcribing RNA from the second RNA or saRNA molecule and (ii) an ORF encoding at least one antigen or polypeptide of interest. The polymerase may be an alphaviral replicase comprising, for example, one of the unstructured alphaviral proteins nsP1, nsP2, nsP3, and nsP4, or a combination thereof. In some embodiments, the RNA molecule comprises the alphaviral unstructured protein nsP1. In some embodiments, the RNA molecule comprises the alphaviral unstructured protein nsP2. In some embodiments, the RNA molecule includes the alphavirus non-structural protein nsP3. In some embodiments, the RNA molecule includes the alphavirus non-structural protein nsP4. In some embodiments, the RNA molecule includes the alphavirus non-structural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule includes the alphavirus non-structural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4.

[0171] In some embodiments, the open reading frame of an RNA (e.g., saRNA) composition is codon-optimized. In some embodiments, the open reading frame encoding an influenza polypeptide or a fragment thereof is codon-optimized.

[0172] 5' Cap In some embodiments, the saRNA molecules described herein include a 5' cap. In some embodiments, the 5' cap portion is a natural 5' cap.

[0173] A “natural 5' cap” is defined as a cap containing 7-methylguanosine attached to the 5' end of an mRNA molecule via a 5'-to-5' triphosphate bond. In some embodiments, the 5' cap portion is a 5' cap analog. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide or a derivative thereof having the structure m7G(5')ppp(5')N(cap 0 structure) (where “N” is any ribonucleotide), which may be incorporated during RNA synthesis (e.g., simultaneous transcription capping) or enzymatically manipulated after RNA transcription (e.g., post-transcription capping). In some embodiments, the 5' end of the RNA molecule is capped with a modified ribonucleotide by an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification of the RNA molecule, e.g., tangential flow filtration. Exemplary enzymatic reactions for capping may involve the use of vaccinia virus capping enzymes (VCEs) comprising mRNA triphosphatase, guanylyl-transferase, and guanine-7-methyltransferase to catalyze the construction of an N7-monomethylated cap 0 structure. The cap 0 structure may help maintain the stability and translation efficiency of the RNA molecule. The 5' cap of the RNA molecule can be further modified with a 2'-O-methyltransferase, resulting in the formation of a cap 1 structure (m7Gppp[m2'-O]N), which may further increase translation efficiency. In some embodiments, the RNA molecule can be enzymatically capped at the 5' end using vaccinia-guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to obtain a cap 0 structure. The inverted 7-methylguanosine cap is added via a 5'-to-5' triphosphate crosslink. Alternatively, using 2'O-methyltransferase together with vaccinia-guanylyltransferase yields a cap 1 structure in which the 2'OH group is methylated on the second-to-last nucleotide, in addition to the cap 0 structure. S-adenosyl-L-methionine (SAM) is a cofactor used as a methyl transfer agent.Non-limiting examples of 5' cap structures include, among others, those having enhanced binding of cap-binding polypeptides, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' cap removal compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can create a canonical 5'-5' triphosphate bond between the 5' terminal nucleotide of the mRNA and a guanine cap nucleotide in which the guanine of the cap contains N7 methylation and the 5' terminal nucleotide of the mRNA contains 2'-O-methyl. Such structures are called Cap1 structures. This cap results in higher translational competency and cellular stability and reduced activation of pro-inflammatory cytokines in cells, compared to, for example, other 5' cap analog structures known in the art. Cap structures, though not limited to these, include 7mG(5')ppp(5')N, pN2p (cap 0) and 7mG(5')ppp(5')N1mpNp (cap 1). Cap 0 is typically N7-methylguanosine linked to the 5' nucleotide via a 5'-to-5' triphosphate bond, referred to as m7G cap or m7Gppp. In cells, the cap 0 structure may help result in efficient translation of the cap-carrying mRNA. Further methylation at the 2'O position of the start nucleotide generates cap 1 or points to m7GpppNm- (where Nm represents any nucleotide having 2'O methylation). In some embodiments, the 5' terminal cap may contain a cap analog, for example, the 5' terminal cap may contain a guanine analog. Examples of guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the capping region may comprise a single cap or a series of nucleotides forming a cap.In this embodiment, the capping region may be equal in length to one, at least one, at most one, or any two of the following nucleotides: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or less. In some embodiments, the cap is absent. In some embodiments, the first and second usable regions are equal to one, at least one, at most one, or any two of the following lengths of nucleotides: 3 to 40, for example, 5 to 30, 10 to 20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or less, and may include one or more signal and / or restriction sequences in addition to start and / or stop codons.

[0174] In some embodiments, the 5' cap is formula I:

[0175] [ka] (In the formula, R 1 and R 2 Each of them is independently H or Me, and B 1 and B 2 (Each of these is independently guanine, adenine, or uracil.) It is represented by B. In some embodiments, B 1 and B 2 R is a naturally occurring base. In some embodiments, 1 is methyl, and R 2 is hydrogen. In some embodiments, B 1 is guanine. In some embodiments, B 1 is adenine. In some embodiments, B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, B 2 is uracil, B2 At least 5% of the total population of uracil nucleotides in the downstream molecule are replaced by one or more modified or non-natural nucleotides.

[0176] In some embodiments, the nucleotide immediately downstream of the 5' cap (in the direction from 5' to 3') contains guanine. In some embodiments, B 1 It is adenine, and B 2 is uracil. In some embodiments, B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen. In some cases, saRNA does not have a 5' cap. In some cases, the 5' cap is not represented by formula I. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' toward 3') contains guanine and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen; this embodiment corresponds to CleanCap AU and B 2 =Uracil content is B as needed. 2 In some embodiments, the function of the saRNA has been shown to be improved by substituting downstream uracil nucleotides. In some embodiments, the RNA molecule further comprises (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the RNA molecule encodes at least one antigen. In some embodiments, the RNA molecule contains at least 7,000 nucleotides. In some embodiments, the RNA molecule contains at least 8,000 nucleotides. In some embodiments, at least 80% of the total RNA molecule is full length. In some embodiments, the alphavirus is Venezuelan encephalitis virus. In some embodiments, the alphavirus is Semliki forest virus.

[0177] In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') comprises guanine, and B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with N1-methylpseudouridine, and essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') comprises guanine, and B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with 5-methoxyuridine, and essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') comprises guanine, and B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, at least 50% of the entire population of uridine nucleotides in the molecule are replaced with 5-methyluridine, and essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') comprises guanine, and B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, and essentially all uridine nucleotides in the molecule are replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') comprises guanine, and B 1 is adenine, B 2is uracil, and R 1 is methyl, and R 2 It is hydrogen, and essentially all uridine nucleotides in the molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudolidine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' toward 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 It is hydrogen, and essentially all uridine nucleotides in the molecule are replaced by 25% 5-methoxyuridine and about 75% N1-methylpseudolidine.

[0178] In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap is

[0179] [ka] Includes. In some embodiments, the 5' cap is CLEANCAP® reagent AG(3'OMe), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG for simultaneous transcription capping of mRNA.

[0180] [ka] This includes. In an alternative embodiment, the 5' cap is CLEANCAP® AU for self-amplifying mRNA, CLEANCAP® reagent AU for simultaneous transcription capping of mRNA, m7G(5')ppp(5')(2'OMeA)pU, [ka] Includes.

[0181] Poly A tail As used herein, “poly-A tail” refers to an extension of consecutive adenine residues that may be attached to the 3' end of an RNA molecule. Poly-A tails can increase the half-life of RNA molecules. Poly-A tails can play a significant regulatory role in enhancing translation efficiency and in mRNA quality control and degradation efficiency. Short sequences or high polyadenylation can signal for RNA degradation. Exemplary designs include a poly-A tail of approximately 40 to 80 adenine residues. In some embodiments, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly-A tail sequence. In some embodiments, for a second RNA or saRNA molecule, the RNA molecule further includes a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. A “full-length” RNA molecule is one that includes a 5' cap and a poly-A tail.

[0182] In some embodiments, the polyA tail contains a length of 5 to 400 nucleotides. The length of the polyA tail may be equal to one, at least one, as many as one, or any two of the following: 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400. In some embodiments, the RNA molecule includes a poly-A tail containing about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly-A tail containing a sequence of more than 30 adenosine nucleotides ("A"). In some embodiments, the RNA molecule includes a poly-A tail containing about 40 A. In some embodiments, the RNA molecule includes a poly-A tail containing about 80 A. As used herein, the term "about" refers to a ±10% deviation of the value it binds. In some embodiments, the 3' poly-A tail has an extension of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some embodiments, the RNA molecule contains at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some embodiments, the RNA molecule contains about 40 consecutive adenosine residues. In some embodiments, the RNA molecule contains about 80 consecutive adenosine residues.

[0183] composition In some cases, the compositions described herein include at least one saRNA described herein. Some embodiments of this disclosure provide an influenza virus (influenza) vaccine (or composition or immunogenic composition) comprising at least one saRNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment that can induce an immune response to influenza).

[0184] In some embodiments, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules (capped and uncapped) in the composition are capped, with at least one of these percentages, at most one of these percentages, or an equal percentage between any two of these percentages.

[0185] In some embodiments, full-length RNA transcripts are equal to 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition, with at least one, at most one, or any two being equal. Purity can be determined, for example, by reverse-phase HPLC or electrophoresis based on a bioanalyzer tip, as described herein, and can be measured, for example, by the peak area of ​​the full-length RNA molecule relative to the total peaks. In some embodiments, RNA can be quantified and purified using a fragment analyzer (FA). The fragment analyzer automates capillary electrophoresis and HPLC.

[0186] In some embodiments, the composition is substantially free of one or more impurities or contaminants, including linear DNA templates and / or reverse complement transcripts, and contains, for example, RNA molecules that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, at least 98% pure, or at least 99% pure, at least one of any of these, at most one of any of these, or equal to any two of these.

[0187] In some embodiments, the composition contains an amount of first RNA molecules greater than the amount of second RNA molecules. In some embodiments, the composition contains an amount of first RNA molecules at least about 1 to 2 times greater than the amount of second RNA molecules. In some embodiments, the composition contains an amount of first RNA molecules at least about 1 to 100 times greater than the amount of second RNA molecules.

[0188] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable vehicle.

[0189] In some embodiments, the composition further comprises a lipid-based delivery system capable of delivering RNA molecules into the interior of cells, and subsequently replicating and / or expressing the encoded polypeptide of interest. The delivery system may have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some embodiments, the composition further comprises neutral lipids, cationic lipids, cholesterol, and polyethylene glycol (PEG) to form nanoparticles containing RNA molecules. In some embodiments, the composition further comprises one of cationic lipids, liposomes, lipid nanoparticles, polyplexes, cochleates, viromosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsomes, polycationic peptides, and cationic nanoemulsions. In some embodiments, RNA molecules are encapsulated, bound to, or adsorbed within any one of the following: cationic lipids, liposomes, lipid nanoparticles, polyplexes, spiral condensates, viromosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsionosomes, polycationic peptides, and cationic nanoemulsions, or any combination thereof.

[0190] In some cases, the compositions described herein comprise at least two RNA molecules: a first RNA molecule and a second RNA molecule as described herein. To provide protection against more than one influenza strain, a mixed vaccine composition can be administered comprising RNA encoding at least one antigenic polypeptide protein (or its antigenic moiety) of a first influenza virus or organism, and further comprising a second RNA molecule encoding at least one antigenic polypeptide protein (or its antigenic moiety) of a second influenza virus or organism. The RNAs may be formulated, for example, in a single lipid nanoparticle (LNP) or in separate LNPs for co-administration.

[0191] In some embodiments, the second RNA molecule includes one of the following: a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a polyA sequence, or any combination thereof. In some embodiments, the second RNA molecule includes a 5' cap portion. In some embodiments, the second RNA molecule includes a 5' UTR and a 3' UTR. In some embodiments, the second RNA molecule includes a 5' UTR, an open reading frame, and a 3' UTR, but does not further include a 5' cap. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' UTR, a coding region, a 3' UTR, and a 3' polyA sequence. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' UTR, a non-coding region, a 3' UTR, and a 3' polyA sequence. In some embodiments, the second RNA molecule includes a non-coding region and does not further include one of the following: a 5' cap portion, a 5' UTR, a 3' UTR, and a 3' polyA sequence. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' untranslated region (5'UTR), modified nucleotides, an open reading frame, a 3' non-coding region (3'UTR), and a 3' polyA sequence.

[0192] Some aspects of this disclosure relate to compositions comprising (i) a first RNA molecule encoding a gene of interest derived from influenza; and (ii) a second RNA molecule containing modified or unnatural nucleotides. In some cases, the first RNA molecule is one of the saRNA molecules described herein. In some cases, the first RNA molecule includes a 5' cap, a 5' untranslated region, a coding region for unstructured proteins including RNA replicases, a subgenome promoter, an open reading frame encoding the gene of interest, a 3' untranslated region, and a 3' poly-A sequence. In some cases, at least 5% of the total population of specific nucleotides in the first RNA molecule are replaced by one or more modified or unnatural nucleotides. In some cases, the RNA molecule contains natural unmodified nucleotides and does not contain modified or unnatural nucleotides. In some cases, the 5' cap is represented by formula I (wherein R1 and R2 are each independently H or Me, B1 and B2 are each independently guanine, adenine, or uracil, the 5' untranslated region, the coding region for a non-structural protein derived from an alphavirus, a subgenome promoter such as one derived from an alphavirus, an open reading frame coding for the gene of interest, the 3' untranslated region, and the 3' poly-A sequence). In some embodiments, B1 and B2 are naturally occurring bases. In some embodiments, R1 is methyl and R2 is hydrogen. In some embodiments, B1 is guanine. In some embodiments, B1 is adenine. In some embodiments, B2 is adenine. In some embodiments, B2 is uracil. In some embodiments, the nucleotide immediately downstream of the 5' cap (in the direction from 5' to 3') contains guanine.

[0193] In some embodiments, B 1 It is adenine, and B 2 is uracil. In some embodiments, B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2is hydrogen. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' toward 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen; this embodiment corresponds to CLEANCAP AU (Trilink), B 2 =Uracil content is B as needed. 2 In some embodiments, it has been shown that substituting downstream uracil nucleotides improves the function of saRNA.

[0194] In some embodiments, at least 10% of the total population of specific nucleotides in the first or second RNA molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of specific nucleotides in the first or second RNA molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of specific nucleotides in the first or second RNA molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of specific nucleotides in the first or second RNA molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, essentially all of the specific nucleotides in the first or second RNA molecule are replaced with one or more modified or non-natural nucleotides. In some embodiments, the one or more modified or non-natural replacement nucleotides comprise two modified or non-natural nucleotides provided in a ratio ranging from 1:99 to 99:1, or any range derived therefrom. In some embodiments, at least 10% of the total population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 10% of the total population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the total population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 50% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides.

[0195] In some embodiments, at least 25% of the total population of uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine. In some embodiments, at least 75% of the total population of uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine. In some embodiments, essentially all of the uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule are replaced with 5-methoxyuridine. In some embodiments, essentially all of the uridine nucleotides in the molecule are replaced with 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule are replaced with 5-methyluridine. In some embodiments, essentially all of the uridine nucleotides in the first RNA molecule are replaced with 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the first RNA molecule are replaced with 5-methylcytosine. In some embodiments, essentially all of the cytosine nucleotides in the first RNA molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule are replaced with 2-thiouridine. In some embodiments, essentially all of the uridine nucleotides in the first RNA molecule are replaced with 2-thiouridine.

[0196] In some embodiments, at least 25% of the total population of uridine nucleotides in the second RNA molecule is replaced with N1-methylpseudridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule is replaced with N1-methylpseudridine. In some embodiments, at least 75% of the total population of uridine nucleotides in the second RNA molecule is replaced with N1-methylpseudridine. In some embodiments, essentially all of the uridine nucleotides in the second RNA molecule are replaced with N1-methylpseudridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule are replaced with 5-methoxyuridine. In some embodiments, essentially all of the uridine nucleotides in the second RNA molecule are replaced with 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule are replaced with 5-methyluridine. In some embodiments, essentially all of the uridine nucleotides in the second RNA molecule are replaced with 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine. In some embodiments, essentially all of the cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule are replaced with 2-thiouridine. In some embodiments, essentially all of the uridine nucleotides in the second RNA molecule are replaced with 2-thiouridine.

[0197] In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule are replaced with N1-methylpseudridine, and essentially all of the cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second molecule are replaced with 5-methoxyuridine, and essentially all of the cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second molecule are replaced with 5-methyluridine, and essentially all of the cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine.

[0198] In some embodiments, essentially all uridine nucleotides in the second RNA molecule are replaced with approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule are replaced with approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule are replaced with approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudridine.

[0199] In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudouridine, and at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudouridine, and essentially all uridine nucleotides in the second RNA molecule are replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudouridine, and at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudouridine, at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced with 5-methyluridine, and essentially all cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudouridine, and essentially all uridine nucleotides in the second RNA molecule are replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine.

[0200] Methods of Use RNA compositions can be used to treat and / or prevent influenza viruses of various genotypes, strains, and isolates. Some embodiments provide a method for preventing or treating influenza virus infection, comprising administering any of the RNA compositions described herein to a subject. In some embodiments, the antigen-specific immune response comprises a T cell response. In some embodiments, the antigen-specific immune response comprises a B cell response. In some embodiments, the antigen-specific immune response comprises both a T cell response and a B cell response. In some embodiments, the method for generating an antigen-specific immune response comprises a single administration of the RNA composition. In some embodiments, the RNA composition is administered to the subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration. In some embodiments, the nanoparticles have a net neutral charge at a neutral pH value. In some embodiments, the RNA (e.g., mRNA) vaccine is multivalent.

[0201] In some embodiments, the RNA polynucleotide or a portion thereof may encode one or more polypeptides of an influenza strain or a fragment thereof as an antigen.

[0202] Some aspects of the present disclosure are directed to a method of inducing an immune response in a subject, comprising administering an effective amount of the composition disclosed herein to a subject in need thereof. Some aspects of the present disclosure are directed to a method of vaccinating a subject, comprising administering an effective amount of the composition disclosed herein to a subject in need thereof. Some aspects of the present disclosure are directed to a method comprising administering an effective amount of the composition disclosed herein to a subject in need thereof. In some embodiments, the compositions disclosed herein elicit an immune response comprising an antibody response. In some embodiments, the compositions disclosed herein elicit an immune response comprising a T cell response.

[0203] Nucleic acid In certain embodiments, nucleic acid sequences may be present in various examples, such as integrated sequences or isolated segments of recombinant polynucleotides and recombinant vectors encoding one or both strands of an antigen or antibody, or fragments thereof, derivatives, mutant proteins, or variants thereof; hybridization probes for identifying, analyzing, mutagenerating, or amplifying polynucleotides encoding polypeptides; polynucleotides sufficient for use as PCR primers or sequencing primers; antisense nucleic acids for inhibiting polynucleotide expression; mRNA, saRNA, and complementary sequences of the foregoing as described herein. The nucleic acid encodes an epitope to which the antibody can bind. Nucleic acids encoding fusion proteins containing these peptides are also provided. The nucleic acid may be single-stranded or double-stranded and may include RNA and / or DNA nucleotides and their artificial variants (e.g., peptide nucleic acids).

[0204] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from whole-genome nucleic acids. The term "polynucleotide" includes oligonucleotides (nucleic acids with a length of 100 residues or less), recombinant vectors such as plasmids, cosmids, phages, and viruses. In certain embodiments, polynucleotides contain regulatory or protein-coding sequences substantially isolated from naturally occurring genes. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), their analogues, or combinations thereof. Further coding or non-coding sequences may, but are not required, be present within a polynucleotide.

[0205] In this regard, the term “gene” is used to refer to nucleic acids (including any sequences required for proper transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. As those skilled in the art will understand, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller manipulated nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or part of such polypeptide. It is also intended that a particular polypeptide may be encoded by a nucleic acid encoding the same or substantially similar polypeptide, but nevertheless containing variations having slightly different nucleic acid sequences.

[0206] In certain embodiments, there exist polynucleotide variants that have substantial identity to the sequences disclosed herein; polynucleotide sequences provided herein, when compared using the methods described herein (e.g., BLAST analysis using standard parameters), including those with sequence identity equal to 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, at least one, at most one, or any two of them, or higher. In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90% identity to the amino acid sequences described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 95% identity to the amino acid sequences described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0207] Nucleic acid segments, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences such as promoters, polyadenylation signals, further restriction enzyme sites, multicloning sites, and other coding segments, and as a result, their overall length may vary considerably. Nucleic acids can be of any length. These may be, for example, any one, at least one, or equal to any two of the following lengths of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000 nucleotides or longer, and may include one or more further sequences, such as regulatory sequences, and / or may be part of a larger nucleic acid, such as a vector. Therefore, nucleic acid fragments of almost any length may be used, and their full length is intended to be limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence containing further heterologous coding sequences, for example, to enable the purification, transport, secretion, or post-translational modification of the polypeptide, or for therapeutic benefits such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides may be appended to the modified polypeptide coding sequence, where "heterologous" means a polypeptide that is not the same as the modified polypeptide.

[0208] lipid delivery In some embodiments, the saRNA composition contains lipids. The lipids and saRNA can together form nanoparticles. In some embodiments, the LNP integrity of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the Disclosure is about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more.

[0209] In some embodiments, the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the Disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, and about 80% or more than the LNP integrity of LNP, LNP suspension, lyophilized LNP composition, or LNP formulation prepared by equivalent methods. More than 90%, more than 1x, more than 2x, more than 3x, more than 4x, more than 5x, more than 10x, more than 20x, more than 30x, more than 40x, more than 50x, more than 100x, more than 200x, more than 300x, more than 400x, more than 500x, more than 1000x, more than 2000x, more than 3000x, more than 4000x, more than 500x, more than 1000x, more than 2000x, more than 3000x, more than 4000x, more than 5000x, or more than 10000x higher.

[0210] In some embodiments, the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the Disclosure is approximately 12 months or more, approximately 15 months or more, approximately 18 months or more, approximately 21 months or more, approximately 24 months or more, approximately 27 months or more, approximately 30 months or more, approximately 33 months or more, approximately 36 months or more, approximately 48 months or more, approximately 60 months or more, approximately 72 months or more, approximately 84 months or more, approximately 96 months or more, approximately 108 months or more, and approximately 120 months or more.

[0211] In some embodiments, the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the Disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 time or more, about 2 times or more, about 3 times or more, about 4 times or more, and about 5 times or more than the Txo% of LNP, LNP suspension, lyophilized LNP composition, or LNP formulation prepared by equivalent methods.

[0212] In some embodiments, the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the Disclosure is approximately 12 months or more, approximately 15 months or more, approximately 18 months or more, approximately 21 months or more, approximately 24 months or more, approximately 27 months or more, approximately 30 months or more, approximately 33 months or more, approximately 36 months or more, approximately 48 months or more, approximately 60 months or more, approximately 72 months or more, approximately 84 months or more, approximately 96 months or more, approximately 108 months or more, and approximately 120 months or more.

[0213] In some embodiments, the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the Disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 time or more, about 2 times or more, about 3 times or more, about 4 times or more, and about 5 times or more longer than the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation prepared by an equivalent method.

[0214] As used herein, “Tx” refers to the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to decrease to about X of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For example, “T80%” refers to the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to decrease to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. As another example, "T1 / 2" refers to the amount of time that it takes for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to decrease to approximately half of the original integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.

[0215] Lipid nanoparticles may contain lipid components as well as one or more additional components, such as therapeutic and / or prophylactic agents, including nucleic acids. LNPs can be designed for one or more specific applications or targets. The elements of an LNP can be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, a specific formulation of an LNP can be selected for a specific application or target, for example, depending on the efficacy and toxicity of a particular combination of elements. The efficacy and tolerability of an LNP formulation may be affected by the stability of the formulation.

[0216] The lipid components of LNP may include, for example, cationic lipids, phospholipids (e.g., unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid components can be provided in specific fractions.

[0217] In some embodiments, the LNP further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof. Phospholipids, PEG lipids, and structural lipids suitable for the methods of this disclosure are further disclosed herein.

[0218] In some embodiments, the lipid components of LNPs include cationic lipids, phospholipids, PEG lipids, and structural lipids. In certain embodiments, the lipid components of lipid nanoparticles include about 30 mol% to about 60 mol% of cationic lipids, about 0 mol% to about 30 mol% of phospholipids, about 18.5 mol% to about 48.5 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid components of lipid nanoparticles include about 35 mol% to about 55 mol% of cationic lipid compounds, about 5 mol% to about 25 mol% of phospholipids, about 30 mol% to about 40 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids. In certain embodiments, the lipid components include about 50 mol% of the cationic lipids, about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another embodiment, the lipid composition comprises about 40 mol% of the cationic lipid, about 20 mol% of the phospholipid, about 38.5 mol% of the structural lipid, and about 1.5 mol% of the PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0219] The amount of the therapeutic agent and / or prophylactic agent in the LNP may depend on the size of the lipid nanoparticle, the composition, the desired target and / or application, or other properties, as well as the properties of the therapeutic agent and / or prophylactic agent. For example, the amount of RNA useful for the LNP may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of the therapeutic agent and / or prophylactic agent (i.e., pharmaceutical substance) and other elements (e.g., lipids) in the LNP may also vary. In some embodiments, the wt / wt ratio of the lipid component to the therapeutic agent and / or prophylactic agent in the LNP can be from about 5:1 to about 60:1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic agent and / or prophylactic agent can be from about 10:1 to about 40:1. In certain embodiments, the wt / wt ratio is about 20:1. The amount of the therapeutic agent and / or prophylactic agent in the LNP can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0220] In some embodiments, the ionizable lipid is a compound of formula (I):

[0221]

Chemical Formula

[0222] [ka] That is the case.

[0223] In some embodiments, the compound has the following structure (I):

[0224] [ka] or a pharmaceutically acceptable salt thereof, tautomer, prodrug or stereoisomer (wherein L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC (=O)NRa- or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRa The C(=O)O- or direct bond is present, and G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene, G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, Ra is H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl, R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4 or -NR5C(=O)R4, R4 is C1-C12 alkyl, R5 is H or C1-C6 alkyl, and x is 0, 1 or 2). In a preferred embodiment, the ionizable lipid is

[0225] [ka] That is the case.

[0226] The lipid components of a lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may also be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids can be selected from a non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. In some embodiments, PEG-modified lipids are modified forms of PEG-DMG. In some embodiments, PEG-modified lipids may have formula (IV):

[0227] [ka] (In the formula, R8 and R9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain may be interrupted by one or more ester bonds, and w has an average value ranging from 30 to 60.) It is a PEG lipid that has [specific properties].

[0228] formulation In one embodiment, the present disclosure relates to an immunogenic composition comprising (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in lipid nanoparticles (LNPs). In some embodiments, the first and second antigens comprise hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen comprises HA derived from a subtype of influenza virus different from the influenza virus antigenic polypeptide or immunogenic fragment of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, which is derived from an influenza virus but from a strain of influenza virus different from both the first and second antigens. In some embodiments, the first, second, and third RNA polynucleotides are formulated in lipid nanoparticles.

[0229] In some embodiments, the composition comprises an octavalent influenza vaccine comprising RNA encoding antigenic polypeptides associated with two type A viruses and two type B viruses that are expected to be prevalent in the relevant jurisdiction. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from HA from influenza A virus, RNA encoding an antigenic polypeptide derived from HA from influenza A virus, RNA encoding an antigenic polypeptide derived from HA from influenza B virus, RNA encoding an antigenic polypeptide derived from HA from influenza B virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza B virus, and RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza B virus.In some embodiments, the octavalent influenza vaccine includes RNA encoding an antigenic polypeptide derived from HA of influenza A virus, RNA encoding an antigenic polypeptide derived from HA of influenza A virus, RNA encoding an antigenic polypeptide derived from HA of influenza B virus, RNA encoding an antigenic polypeptide derived from HA of influenza B virus, RNA encoding an antigenic polypeptide derived from NA of influenza A virus, RNA encoding an antigenic polypeptide derived from NA of influenza A virus, RNA encoding an antigenic polypeptide derived from NA of influenza B virus, and RNA encoding an antigenic polypeptide derived from NA of influenza B virus. In some embodiments, the octavalent influenza vaccine includes RNA encoding an antigenic polypeptide associated with H1N1 influenza virus, RNA encoding an antigenic polypeptide associated with H3N2 influenza virus, RNA encoding an antigenic polypeptide associated with Victoria lineage influenza virus, and RNA encoding an antigenic polypeptide associated with Yamagata lineage influenza virus. In some embodiments, the octavalent influenza vaccine contains RNA associated with the influenza type predicted to be prevalent in the relevant jurisdiction (e.g., HA polypeptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses predicted to be prevalent in the relevant geography). RNA (e.g., mRNA) vaccines can be used in a variety of settings depending on the prevalence of infection or the degree or level of unmet medical needs. RNA vaccines can be used to treat and / or prevent influenza viruses of various genotypes, strains, and isolates. RNA vaccines typically have superior properties in that they produce much higher antibody titers and faster responses than commercially available antiviral therapeutic treatments.While we do not wish to be constrained by theory, RNA vaccines as mRNA polynucleotides are thought to be better designed to yield appropriate protein conformation during translation because they incorporate natural cellular mechanisms. Unlike traditional vaccines, which are manufactured ex vivo and can induce undesirable cellular responses, RNA (e.g., mRNA) vaccines are presented to cell lines in a more natural manner.

[0230] In one embodiment, a method is provided for purifying RNA polynucleotides synthesized by transcription in vitro. The method comprises ultrafiltration and diafiltration. In some embodiments, the method does not include a chromatography step. In some embodiments, the purified RNA polynucleotides are substantially free of impurities including short avotive RNA species, long avotive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvent and / or residual salts. In some embodiments, the residual plasmid DNA is 500 ng or less of DNA / mg RNA. In some embodiments, the purity of the purified mRNA is about 60% to about 100%. In another embodiment, a method is provided for producing LNPs encapsulated in RNA polynucleotides. The method comprises a buffer for exchanging the LNPs. The method further comprises concentrating the LNPs via a flat sheet cassette membrane. In preferred embodiments, the UFDF process does not use a hollow fiber membrane.

[0231] Situations may arise where people are at risk of infection by more than one strain of influenza virus. RNA (e.g., mRNA) therapeutic vaccines are particularly well-suited to combination vaccination methods due to several factors, including, but not limited to, the speed of production and the ability to rapidly adapt vaccines to recognized geographical threats. Furthermore, because vaccines utilize the human body to produce antigenic proteins, they are well-suited to the production of larger, more complex antigenic proteins, enabling proper folding, surface expression, antigen presentation, etc., in human subjects. To protect against more than one strain of influenza, a combination vaccine can be administered that contains RNA (e.g., mRNA) encoding at least one antigenic polypeptide protein (or its antigenic moiety) of a first influenza virus or organism, and further contains RNA encoding at least one antigenic polypeptide protein (or its antigenic moiety) of a second influenza virus or organism. The RNA (e.g., mRNA) can be co-formulated, for example, in a single lipid nanoparticle (LNP), or in separate LNPs for co-administration.

[0232] Some embodiments of this disclosure provide an influenza virus (influenza) vaccine (or composition or immunogenic composition) comprising at least one RNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to influenza).

[0233] In some embodiments, the antigenic polypeptide is one of the specified antigenic subdomains of HA, called HA1, HA2, or a combination of HA1 and HA2, as well as at least one antigenic polypeptide selected from neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2).

[0234] In some embodiments, the at least one antigenic polypeptide is HA or a derivative thereof containing an antigenic sequence derived from HA1 and / or HA2, as well as at least one antigenic polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0235] In some embodiments, the antigenic polypeptide is at least one antigenic polypeptide, which is HA or a derivative thereof containing an antigenic sequence derived from HA1 and / or HA2, as well as at least two antigenic polypeptides selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0236] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza virus protein or an immunogenic fragment thereof.

[0237] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding multiple influenza virus proteins or their immunogenic fragments.

[0238] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one HA1, HA2, or a combination of both).

[0239] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding the HA protein or its immunogenic fragment (e.g., at least one HA1, HA2, or a combination of both of any or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any combination thereof), and at least one other RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a protein selected from the HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from the influenza virus.

[0240] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding the HA protein or any one or all of the immunogenic fragments thereof (e.g., at least one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or a combination thereof), and at least two other RNA (e.g., mRNA) polynucleotides having open reading frames encoding two proteins selected from the HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from the influenza virus.

[0241] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding the HA protein or any one or all of the immunogenic fragments thereof (e.g., at least one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or a combination thereof), and at least three other RNA (e.g., mRNA) polynucleotides having open reading frames encoding three proteins selected from the HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from the influenza virus.

[0242] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding the HA protein or any one or all of the immunogenic fragments thereof (e.g., at least one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or a combination thereof), and at least four other RNA (e.g., mRNA) polynucleotides having open reading frames encoding four proteins selected from the HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from the influenza virus.

[0243] In some embodiments, the vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding the HA protein or any one or all of the immunogenic fragments thereof (e.g., at least one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or a combination thereof), and at least five other RNA (e.g., mRNA) polynucleotides having open reading frames encoding five proteins selected from the HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from the influenza virus.

[0244] In some embodiments, the vaccine comprises an HA protein or an immunogenic fragment thereof (e.g., at least one of any or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or a combination thereof), an HA protein obtained from an influenza virus, an NP protein or an immunogenic fragment thereof, an NA protein or an immunogenic fragment thereof, an M1 protein or an immunogenic fragment thereof, an M2 protein or an immunogenic fragment thereof, an NS1 protein or an immunogenic fragment thereof, and an NS2 protein or an immunogenic fragment thereof, having an open reading frame, and at least one RNA (e.g., mRNA) polynucleotide. In some embodiments, the influenza RNA composition comprises an saRNA encoding an antigenic fusion protein. Thus, the encoded antigen or multiple antigens may comprise two or more proteins (e.g., a protein and / or protein fragments) linked together. Alternatively, a protein fused with a protein antigen may not promote a strong immune response to itself, but rather a strong immune response to the influenza antigen. In some embodiments, antigenic fusion proteins retain the functional properties derived from their respective original proteins.

[0245] Some embodiments of this disclosure relate to the following novel influenza virus polypeptide sequences: H1HA10-Foldon_ΔNgly1; H1HA10TM-PR8(H1 A / Puerto Rico / 8 / 34 HA); H1HA10-PR8-DS(H1 A / Puerto Rico / 8 / 34 HA); pH1HA10-Cal04-DS(H1 A / California / 04 / 2009 HA); Pandemic H1HA10 derived from California 04; pH1HA10-ferritin; HA10; Pandemic H1HA10 derived from California 04; Pandemic H1HA10 derived from California 04 strain lacking Foldon and possessing the K68C / R76C mutation for trimerization; A / Puerto strain lacking Foldon and possessing the Y94D / N95L mutation for trimerization We provide H1HA10 derived from the Rico / 8 / 34 strain; H1HA10 derived from the A / Puerto Rico / 8 / 34 strain, which lacks Foldon and has the K68C / R76C mutation for trimerization; H1N1 A / Viet Nam / 850 / 2009; H3N2 A / Wisconsin / 67 / 2005; H7N9 (A / Anhui / 1 / 2013); H9N2 A / Hong Kong / 1073 / 99; and H10N8 A / JX346 / 2013.

[0246] Some embodiments of this disclosure provide an influenza virus (influenza) vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment of the above novel influenza virus polypeptide sequence (e.g., an immunogenic fragment capable of inducing an immune response to influenza). In some embodiments, the influenza vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide comprising a modified sequence that is at least 75% (e.g., any number between 75% and 100%) identical to the amino acid sequence of the above novel influenza virus sequence. The modified sequence may be at least 75% (e.g., any number between 75% and 100%) identical to the amino acid sequence of the above novel influenza virus sequence.

[0247] Some embodiments of this disclosure provide isolated nucleic acids containing sequences encoding the novel influenza virus polypeptide sequences described above; expression vectors containing the nucleic acids; and host cells containing the nucleic acids. This disclosure also provides methods for producing polypeptides of any of the novel influenza virus sequences described above. The methods may include culturing host cells in a culture medium under conditions that enable nucleic acid expression of the novel influenza virus sequences described above, and purifying the novel influenza virus polypeptides from the cultured cells or cell culture medium. This disclosure also provides antibody molecules, including full-length antibodies and antibody derivatives, against the novel influenza virus sequences.

[0248] In some embodiments, the open reading frame of an RNA (e.g., mRNA) vaccine is codon-optimized. In some embodiments, the open reading frame encoding an influenza polypeptide or a fragment thereof is codon-optimized. Some embodiments provide the use of an influenza vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment thereof, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of uracil in the open reading frame has chemical modification, and optionally the vaccine is formulated in lipid nanoparticles. In some embodiments, 100% of uracil in the open reading frame has chemical modification. In some embodiments, the chemical modification is at position 5 of uracil. In some preferred embodiments, the chemical modification is N1-methylpseudridine.

[0249] In some embodiments, the RNA (e.g., mRNA) vaccine further comprises an adjuvant.

[0250] In some embodiments, at least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that binds to a cell receptor.

[0251] In some embodiments, at least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that causes fusion of the viral membrane with the cell membrane.

[0252] In some embodiments, at least one RNA polynucleotide encodes at least one influenza antigenic polypeptide responsible for the binding of the virus to the infected cell.

[0253] Some embodiments of the present disclosure provide a vaccine comprising at least one ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide, at least one 5' terminal cap, and at least one chemical modification, formulated within lipid nanoparticles.

[0254] In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap is

[0255] [ka] This includes. In some embodiments, at least one chemical modification is selected from pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the chemical modification is located at the 5-position of uracil. In some embodiments, the chemical modification is N1-methylpseudridine. In some embodiments, the chemical modification is N1-ethyl pseudouridine.

[0256] In some embodiments, lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. In some embodiments, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyric acid (DLin-MC3-DMA), di((Z)-nona-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine (L530).

[0257] Some embodiments of the present disclosure provide a vaccine comprising at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) of the uracil in the open reading frame is chemically modified, and optionally the vaccine is formulated in lipid nanoparticles (e.g., the lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids).

[0258] In some embodiments, 100% of the uracil in the open reading frame has a chemical modification. In some embodiments, the chemical modification is at position 5 of uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, 100% of the uracil in the open reading frame has N1-methylpseudouridine at position 5 of uracil.

[0259] In some embodiments, the open reading frame of the RNA (e.g., mRNA) polynucleotide encodes at least one influenza antigenic polypeptide. In some embodiments, the open reading frame encodes at least two, at least five, or at least ten antigenic polypeptides. In some embodiments, the open reading frame encodes at least 100 antigenic polypeptides. In some embodiments, the open reading frame encodes 1 to 100 antigenic polypeptides.

[0260] In some embodiments, the vaccine comprises at least two RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one influenza virus antigenic polypeptide. In some embodiments, the vaccine comprises at least five or at least ten RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide or its antigenic fragment. In some embodiments, the vaccine comprises at least 100 RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide. In some embodiments, the vaccine comprises 2 to 100 RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide.

[0261] Also provided herein are influenza RNA (e.g., mRNA) vaccines formulated in nanoparticles (e.g., lipid nanoparticles) according to any one of the preceding paragraphs.

[0262] In some embodiments, the nanoparticles have an average diameter of 50 to 200 nm. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. In some embodiments, the lipid nanoparticles have a molar ratio of approximately 20 to 60% cationic lipids, 0.5 to 15% PEG-modified lipids, 25 to 55% sterols, and 25% non-cationic lipids. In some embodiments, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol.

[0263] In some embodiments, the nanoparticles have a polydispersity value of less than 0.4 (e.g., less than 0.3, 0.2, or 0.1).

[0264] In some embodiments, the nanoparticles have a net neutral charge at a neutral pH value.

[0265] In some embodiments, RNA (e.g., mRNA) vaccines are polyvalent.

[0266] Some embodiments of this disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an amount effective to produce an antigen-specific immune response in which one of the RNA (e.g., mRNA) vaccines provided herein. In some embodiments, the RNA (e.g., mRNA) vaccine is an influenza vaccine. In some embodiments, the RNA (e.g., mRNA) vaccine is a combination vaccine (broad-spectrum influenza vaccine) comprising a combination of influenza vaccines.

[0267] In some embodiments, the antigen-specific immune response includes a T-cell response or a B-cell response.

[0268] In some embodiments, a method for eliciting an antigen-specific immune response includes administering a single dose (without a booster dose) of the influenza RNA (e.g., mRNA) vaccine of the present disclosure to a subject.

[0269] In some embodiments, the method further includes administering a second (booster) dose of the influenza RNA (e.g., mRNA) vaccine to the subject. Further doses of the influenza RNA (e.g., mRNA) vaccine may be administered.

[0270] In some embodiments, subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after a first or second (booster) dose of vaccine. Seroconversion is the period during which specific antibodies are produced and detectable in the blood. After seroconversion has occurred, the virus can be detected in blood tests for antibodies. During infection or immunization, the antigen enters the bloodstream, and the immune system responds by beginning to produce antibodies. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are not considered to be present. During seroconversion, antibodies are present but not yet detectable. At any time after seroconversion, antibodies are detectable in the blood and indicate a previous or current infection.

[0271] In some embodiments, the influenza RNA (e.g., mRNA) vaccine is administered to the subject by intradermal injection, intramuscular injection, or intranasal administration.

[0272] Some embodiments of this disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an amount of influenza RNA (e.g., mRNA) vaccine effective in producing an antigen-specific immune response in the subject. In some embodiments, the antigen-specific immune response in the subject can be determined by assaying the antibody titer (the titer of an antibody that binds to an influenza antigenic polypeptide) after administration of any of the influenza RNA (e.g., mRNA) vaccines of this disclosure to the subject. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log compared to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1 to 3 log compared to a control.

[0273] In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by at least twofold compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by at least fivefold compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by at least tenfold compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by two to tenfold compared to the control.

[0274] In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in subjects who have never been administered the RNA (e.g., mRNA) vaccine of the Disclosure. In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in subjects administered with attenuated live or inactivated influenza, or the control is the anti-antigenic polypeptide antibody titer produced in subjects administered with recombinant or purified influenza protein vaccine. In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in subjects administered with influenza virus-like particle (VLP) vaccine.

[0275] The RNA (e.g., mRNA) vaccines of this disclosure are administered to a subject in an effective dose (an amount effective in inducing an immune response). In some embodiments, the effective dose is equivalent to a dose reduced to less than half, less than a quarter, less than one-tenth, less than one-hundredth, or less than one-thousandth of the standard therapeutic dose of recombinant influenza protein vaccine, in which case the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-antigenic polypeptide antibody titer produced in a control subject administered with a standard therapeutic dose of recombinant influenza protein vaccine, purified influenza protein vaccine, attenuated live influenza vaccine, inactivated influenza vaccine, or influenza VLP vaccine. In some embodiments, the effective dose is equivalent to a dose reduced to 1 / 2 to 1 / 1000 of the standard therapeutic dose of recombinant influenza protein vaccine, in which case the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-antigenic polypeptide antibody titer produced in a control subject administered a standard therapeutic dose of recombinant influenza protein vaccine, purified influenza protein vaccine, attenuated live influenza vaccine, inactivated influenza vaccine, or influenza VLP vaccine.

[0276] In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in subjects administered with a virus-like particle (VLP) vaccine containing the structural protein of influenza.

[0277] In some embodiments, RNA (e.g., mRNA) vaccines are formulated in an effective amount to elicit an antigen-specific immune response in a target.

[0278] In some embodiments, the effective dose is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective dose is a total dose of 100 μg. In some embodiments, the effective dose is a 25 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is a 100 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is a 400 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is a 500 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is 1 μg to 1000 μg, or a total dose of 1 μg to 100 μg of saRNA. In some embodiments, the effective dose is a total dose of 30 μg. In some embodiments, the effective dose is a 10 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is a 10 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is a 15 μg dose administered to the subject in a total of two doses. In some embodiments, the effective dose is a 30 μg dose administered to the subject in a total of two doses. In some embodiments, the method comprises administering the subject a dose of the saRNA composition described herein in doses ranging from 10 μg / kg to 400 μg / kg.

[0279] In some embodiments, the dose of saRNA polynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg per dose. The dosages are 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the saRNA composition is administered to the subject by intradermal or intramuscular injection. In some embodiments, the saRNA composition is administered to the subject on day 0. In some embodiments, a second dose of the saRNA composition is administered to the subject on day 21.

[0280] In some embodiments, the efficacy (or effectiveness) of the RNA (e.g., mRNA) vaccine is higher than 60%. In some embodiments, the vaccine RNA (e.g., mRNA) polynucleotide contains at least one influenza antigenic polypeptide.

[0281] Vaccine efficacy can be evaluated using standard analyses. For example, vaccine efficacy can be measured by double-blind, randomized, controlled clinical trials. Vaccine efficacy can be expressed as a proportional reduction in disease incidence (AR) between an unvaccinated (ARU) trial cohort and an vaccinated (ARV) trial cohort, and can be calculated from the relative risk (RR) of disease within the vaccinated group using the following formula: Efficacy = (ARU - ARV) / ARU x 100; and Efficacy = (1 - RR) x 100.

[0282] Similarly, vaccine efficacy can be assessed using standard analyses. Vaccine efficacy is an assessment of how much a vaccine (which may already be proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and harms of a vaccination program, as well as the vaccine itself, under natural conditions rather than controlled clinical trials. Vaccine efficacy is proportional to vaccine efficacy (potency) but is also influenced by how well the target group in the population is immunized, as well as other vaccine-unrelated factors that affect "real-world" outcomes such as hospitalization, outpatient visits, or costs. For example, a retrospective case-control analysis can be used to compare vaccination rates between a set of infection cases and appropriate controls. Vaccine efficacy can be expressed as a rate difference using the odds ratio (OR) for developing an infection despite vaccination: efficacy = (1-OR) x 100. In some embodiments, the efficacy (or effectiveness) of RNA (e.g., mRNA) vaccines is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0283] In some embodiments, the vaccine immunizes the target against influenza for up to two years. In some embodiments, the vaccine immunizes the target against influenza for more than two years, more than three years, more than four years, or for five to ten years.

[0284] In some embodiments, the subjects are approximately 5 years of age or younger. For example, the subjects may be approximately 1 to 5 years of age (e.g., approximately 1, 2, 3, 5, or 5 years), or approximately 6 months to 1 year of age (e.g., approximately 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, the subjects are approximately 12 months of age or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month). In some embodiments, the subjects are approximately 6 months of age or younger. In some embodiments, the RNA composition is administered to the subjects by intradermal or intramuscular injection. In some embodiments, the RNA composition is administered to the subjects on day 0. In some embodiments, a second dose of the RNA composition is administered to the subjects on day 21.

[0285] In some embodiments, subjects were born at full term (e.g., approximately 37–42 weeks). In some embodiments, subjects were born prematurely, for example, before approximately 36 weeks of gestation (e.g., approximately 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 weeks). For example, subjects may be born before approximately 32 weeks of gestation. In some embodiments, subjects were born prematurely between approximately 32 and 36 weeks of gestation. In such subjects, the RNA (e.g., mRNA) vaccine may be administered later in life, for example, between approximately 6 months and approximately 5 years of age or older.

[0286] In some embodiments, the subjects are young adults aged approximately 20 to 50 years (for example, approximately 20, 25, 30, 35, 40, 45, or 50 years).

[0287] In some embodiments, the target population is elderly individuals aged approximately 60, 70 or older (for example, approximately 60, 65, 70, 75, 80, 85, or 90 years).

[0288] In some embodiments, the subject has been exposed to influenza (e.g., Chlamydia trachomatis); the subject is infected with influenza (e.g., Chlamydia trachomatis); or the subject is at risk of infection by influenza (e.g., Chlamydia trachomatis).

[0289] In some embodiments, the subject has been exposed to a beta-coronavirus (e.g., SARS-CoV-2); the subject is infected with a beta-coronavirus (e.g., SARS-CoV-2); or the subject is at risk of infection by a beta-coronavirus (e.g., SARS-CoV-2).

[0290] In some embodiments, the subject has received at least one dose of an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2) selected from, for example, COMIRNATY®, Pfizer-BioNTech's COVID-19 vaccine, Moderna's mRNA-1273 COVID-19 vaccine, and Janssen's COVID-19 vaccine; the subject has received at least two doses of an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2); the subject has received, for example, COMIRNATY®, Pfizer-BioNTech's COVID-19 vaccine, and Moderna's mRNA-1273 A subject has received at least one dose of an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2) selected from either the COVID-19 vaccine or Janssen's COVID-19 vaccine; or a subject at risk of infection with a beta-coronavirus (e.g., SARS-CoV-2) has been administered, concurrently with or within 12 to 48 hours thereof, any immunogenic composition against an influenza virus as disclosed herein, including, for example, COMIRNATY®, Pfizer-BioNTech's COVID-19 vaccine, Moderna's mRNA-1273 COVID-19 vaccine, and Janssen's COVID-19 vaccine.

[0291] In some embodiments, the subjects are in an immunodeficient state (having a damaged immune system, e.g., having an immune disorder or autoimmune disorder).

[0292] In some embodiments, the nucleic acid vaccines described herein are chemically modified. In other embodiments, the nucleic acid vaccines are unmodified.

[0293] Further embodiments provide compositions and methods for vaccinating a subject, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first viral antigenic polypeptide, wherein the RNA polynucleotides do not contain stabilizing elements and the adjuvant is not co-formulated or co-administered with the vaccine.

[0294] In other embodiments, the present invention is a composition or method for vaccinating a subject, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the nucleic acid vaccine is administered to the subject in doses of 10 μg / kg to 400 μg / kg. In some embodiments, the dose of RNA polynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, The dosages are 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day 0. In some embodiments, the second dose of the nucleic acid vaccine is administered to the subject on day 21.

[0295] In some embodiments, a dose of 25 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dose of 100 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dose of 50 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dose of 75 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dose of 150 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dose of 400 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dose of 200 micrograms of RNA polynucleotides is included in the nucleic acid vaccine administered to the subject. In some embodiments, RNA polynucleotides accumulate at a 100-fold higher level in local lymph nodes compared to distal lymph nodes. In other embodiments, the nucleic acid vaccine is chemically modified, and in other embodiments, the nucleic acid vaccine is not chemically modified.

[0296] Aspects of the present disclosure provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotides do not contain a stabilizing element, and a pharmaceutically acceptable carrier or excipient, wherein the adjuvant is not included in the vaccine. In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence with increased GC content compared to a wild-type sequence.

[0297] Aspects of the present disclosure provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide that confers an antibody titer superior to a standard of seroprotection against a first antigen to an acceptable percentage of human subjects, wherein the RNA polynucleotides are present in a formulation for in vivo administration to a host. In some embodiments, the antibody titer induced by the mRNA vaccine of the present disclosure is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is higher than that of a protein vaccine. In other embodiments, the neutralizing antibody titer induced by the mRNA vaccine of the present disclosure is higher than that of an adjuvanted protein vaccine. In further embodiments, the neutralizing antibody titers induced by the mRNA vaccine of this disclosure are 1,000–10,000, 1,200–10,000, 1,400–10,000, 1,500–10,000, 1,000–5,000, 1,000–4,000, 1,800–10,000, 2,000–10,000, 2,000–5,000, 2,000–3,000, 2,000–4,000, 3,000–5,000, 3,000–4,000, or 2,000–2,500. The neutralizing titer is generally expressed as the highest serum dilution required to achieve a 50% reduction in plaque number.

[0298] Also provided are nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotides are present in the formulation for in vivo administration to a host in order to elicit a higher antibody titer that lasts longer than that elicited by the mRNA vaccine encoding the first antigenic polypeptide, and are formulated with a stabilizing element or with an adjuvant. In some embodiments, the RNA polynucleotides are formulated to produce neutralizing antibodies within one week of a single dose. In some embodiments, the adjuvant is selected from cationic peptides and immunostimulatory nucleic acids. In some embodiments, the cationic peptide is a protamine.

[0299] The embodiment provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, which may include at least one chemical modification or may not include a modified nucleotide, wherein the RNA polynucleotides are present in the formulation for in vivo administration to the host such that the level of antigen expression in the host significantly exceeds the level of antigen expression brought about by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.

[0300] Other embodiments provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, which may include at least one chemical modification or may not include a modified nucleotide, wherein the vaccine has less than one-tenth of the RNA polynucleotides required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotides are present in a dose of 25 to 100 micrograms.

[0301] Aspects of the present disclosure also provide units of vaccines for use, comprising 10 μg to 400 μg of one or more RNA polynucleotides, formulated for delivery to human subjects, having at least one chemical modification, or not having a modified nucleotide, and having an open reading frame encoding a first antigenic polypeptide, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

[0302] Aspects of the present disclosure provide a method for creating, maintaining, or restoring antigenic memory to a viral strain in an individual or population of individuals, comprising administering to the individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide comprising two or more codon-optimized open reading frames encoding a set of reference antigenic polypeptides, which may include at least one chemical modification or may not include a modified nucleotide, and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual by a route selected from the group consisting of intramuscular, intradermal, and subcutaneous administration. In some embodiments, the administration step comprises contacting the muscle tissue of interest with a device suitable for injecting the composition. In some embodiments, the administration step comprises contacting the muscle tissue of interest with a device suitable for injecting the composition, in conjunction with electroporation.

[0303] In some embodiments, a method is provided for inducing an antigen-specific immune response in a subject. The method comprises administering to the subject an amount of an influenza RNA composition effective in producing an antigen-specific immune response. In some embodiments, the antigen-specific immune response comprises a T-cell response or a B-cell response. In some embodiments, the antigen-specific immune response comprises both a T-cell response and a B-cell response. In some embodiments, the method for producing an antigen-specific immune response comprises a single dose of vaccine. In some embodiments, the method further comprises administering to the subject a booster dose of vaccine. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0304] Aspects of this disclosure provide a method for vaccinating a subject, comprising administering a single dose of a nucleic acid vaccine, ranging from 25 μg / kg to 400 μg / kg, to the subject, the nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide in an amount effective for vaccinating the subject.

[0305] Other embodiments provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide and comprising at least one chemical modification, wherein the vaccine has less than one-tenth of the RNA polynucleotides required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotides are present in a dose of 25 to 100 micrograms.

[0306] Another embodiment provides a nucleic acid vaccine comprising an LNP-formulated RNA polynucleotide that is unmodified and has an open reading frame encoding a first antigenic polypeptide, wherein the vaccine contains less than one-tenth of the RNA polynucleotide required for an unmodified mRNA vaccine not formulated in the LNP to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dose of 25 to 100 micrograms.

[0307] The data presented in the examples demonstrate a significant enhancement of the immune response using the formulations of this disclosure. Both chemically modified RNA vaccines and unmodified RNA vaccines are useful according to the present invention. Surprisingly, contrary to prior art reports that it is preferable to use chemically unmodified mRNA formulated in a carrier to produce the vaccine, it is described herein that chemically modified mRNA-LNP vaccines required a much lower effective mRNA dose than unmodified mRNA, i.e., one-tenth of that of unmodified mRNA when formulated in a carrier other than LNP. Both the chemically modified RNA vaccines and unmodified RNA vaccines of this disclosure yield a better immune response than mRNA vaccines formulated in different lipid carriers.

[0308] In other embodiments, the present invention includes a method for treating an elderly subject aged 60 years or older, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a viral antigenic polypeptide in an amount effective for vaccinating the subject.

[0309] In other embodiments, the present invention includes a method for treating a young subject under 17 years of age, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a viral antigenic polypeptide in an amount effective for vaccinating the subject.

[0310] In other embodiments, the present invention includes a method for treating an adult subject, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a viral antigenic polypeptide in an amount effective for vaccinating the subject.

[0311] In some embodiments, the present invention relates to a method of vaccinating a subject with a mixed vaccine comprising at least two nucleic acid sequences encoding an antigen, wherein the dose of the vaccine is a mixed therapeutic dose, where the dose of each individual nucleic acid encoding the antigen is less than the therapeutic dose. In some embodiments, the mixed dose is 25 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the mixed dose is 100 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the mixed dose is 50 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the mixed dose is 75 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the mixed dose is 150 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the mixed dose is 400 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject.

[0312] In a preferred embodiment, the vaccine of this disclosure (e.g., an mRNA vaccine encapsulated in an LNP) produces antigen-specific antibodies in the blood or serum of a vaccinated subject at levels, concentrations, and / or titers of prophylactic and / or therapeutic efficacy. Where defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in a subject, e.g., a human subject. In exemplary embodiments, the antibody titer is expressed as the reciprocal of the maximum dilution (in serial dilutions) that still yields a positive result. In exemplary embodiments, the antibody titer is determined or measured by an enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, the antibody titer is determined or measured by a neutralization assay, e.g., a microneutralization assay. In certain embodiments, the antibody titer measurement is expressed as a ratio, e.g., 1:40, 1:100, etc.

[0313] In exemplary embodiments of this disclosure, an effective vaccine produces antibody titers greater than 1:40, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, or greater than 1:10000. In exemplary embodiments, such antibody titers are produced or reached within 10 days after vaccination, within 20 days after vaccination, within 30 days after vaccination, within 40 days after vaccination, or within 50 days or more after vaccination. In exemplary embodiments, such titers are produced or reached after a single dose of the vaccine is administered to the subject. In other embodiments, such titers are produced or reached after multiple doses, for example, after the first and second doses (e.g., booster doses). In exemplary embodiments of this disclosure, antigen-specific antibodies are measured in units of μg / ml, or in units of IU / L (International Units per Liter) or mIU / ml (Milli-International Units per ml). In exemplary embodiments of this disclosure, effective vaccines are produced in concentrations exceeding 0.5 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.35 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, or 10 μg / ml. In exemplary embodiments of this disclosure, effective vaccines are produced in concentrations exceeding 10 mIU / ml, 20 mIU / ml, 50 mIU / ml, 100 mIU / ml, 200 mIU / ml, 500 mIU / ml, or 1000 mIU / ml. In exemplary embodiments, antibody levels or concentrations are produced or reached within 10 days, 20 days, 30 days, 40 days, or 50 days or more after vaccination. In exemplary embodiments, such levels or concentrations are produced or reached after a single dose of the vaccine is administered to the subject. In other embodiments, such levels or concentrations are produced or reached after multiple doses, for example, after the first and second doses (e.g., booster doses). In exemplary embodiments, antibody levels or concentrations are determined or measured by enzyme-linked immunosorbent assay (ELISA).In exemplary embodiments, antibody levels or concentrations are determined or measured by a neutralization assay, for example, a microneutralization assay.

[0314] In some embodiments, the Disclosure provides a composition comprising (a) a second mRNA encoding the HA antigen of a second influenza A virus, wherein the first mRNA encoding the hemagglutinin (HA) antigen of a first influenza A virus is of a different subtype; and (b) a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the third mRNA encoding the HA antigen of a first influenza B virus is of a different lineage; the composition further comprising lipid nanoparticles comprising 40-55 mol% ionizable amino lipids; 5-15 mol% neutral lipids; 35-45 mol% sterols; and 1-5 mol% PEG-modified lipids, and comprising a composition comprising administering to a human subject a composition comprising 50 or 25 μg-200 μg of mRNA in total. In some embodiments, the composition comprises 25 μg of mRNA in total. In some embodiments, the composition comprises 50 μg of mRNA in total. In some embodiments, the composition contains a total of 100 μg of mRNA. In some embodiments, the composition contains a total of 200 μg of mRNA. In preferred embodiments, the ratio of the first:second:third:fourth mRNAs is not 1:1:1:1. In preferred embodiments, the ratio of the first:second:third:fourth mRNAs is greater than 1:1:1:greater than 1, with influenza A:A:B:B, respectively. In preferred embodiments, the ratio of the first:second:third:fourth mRNAs is 1:1:2:2, with influenza A:A:B:B, respectively. In preferred embodiments, the ratio of the first:second:third:fourth mRNAs is 1:1:4:4, with influenza A:A:B:B, respectively. In some embodiments, each mRNA contains a 5' cap analog. In some embodiments, the 5' cap analog is a 5'7mG(5')ppp(5')NlmpNp cap. In some embodiments, each mRNA contains chemical modifications. In some embodiments, the chemical modification is 1-methylpseudridine. In some embodiments, the lipid nanoparticles contain 40-50 mol% ionizable aminolipids, 35-45 mol% sterols, 10-15 mol% neutral lipids, and 2-4 mol% PEG-modified lipids.In some embodiments, the lipid nanoparticles comprise 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol% of ionizable aminolipids. In some embodiments, the sterol is cholesterol. In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the PEG-modified lipid is 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol (PEG2000DMG). In some embodiments, the composition further comprises Tris buffer, sucrose, and sodium acetate. In some embodiments, the composition comprises 10 mM to 30 mM Tris buffer, 75 mg / mL to 95 mg / mL sucrose, and 5 mM to 15 mM sodium acetate, and optionally, the composition has a pH of 6 to 8. In some embodiments, the composition comprises 20 mM Tris buffer, 87 mg / mL sucrose, and 10.7 mM sodium acetate, and optionally, the composition has a pH of 7.5. In some embodiments, the composition contains 0.5 mg / mL of mRNA.

[0315] In some embodiments, the composition is administered intramuscularly to the deltoid muscle of a human subject, if necessary. In some embodiments, the human subject is 18–49 years of age. In some embodiments, the human subject is at least 50 years of age. In some embodiments, the human subject is 50–64 years of age. In some embodiments, the human subject is at least 65 years of age. In some embodiments, the human subject is serologically positive for at least one HA antigen. In some embodiments, the human subject is serologically positive for all HA antigens. In some embodiments, the subject is serologically negative for all HA antigens. In some embodiments, the HA antigens are selected according to or in accordance with standardized criteria used by the World Health Organization's Global Influenza Surveillance and Response System (GISRS). In some embodiments, the HA antigens are selected using a hemagglutinin inhibition (HAI) assay to identify circulating influenza viruses antigenically similar to influenza viruses derived from the previous season's vaccine, and, if necessary, influenza viruses are considered antigenically similar if their HAI titers differ by two or fewer dilutions. In some embodiments, the first mRNA encodes the HA antigen of influenza A of the H1 subtype, the second mRNA encodes the HA antigen of influenza A of the H3 subtype, the third mRNA encodes the HA antigen of influenza B of the B / Yamagata lineage, and the fourth mRNA encodes the HA antigen of influenza B of the B / Victoria lineage.

[0316] In some embodiments, the mRNA vaccine is administered in a dose effective to induce a neutralizing antibody response to influenza A H1N1, influenza A H3N2, influenza B / Yamagata, and influenza B / Victoria. In another aspect, the present disclosure provides a composition comprising (a) a second mRNA encoding the HA antigen of a second influenza A virus, wherein the first mRNA includes an open reading frame (ORF) encoding the hemagglutinin (HA) antigen of a first influenza A virus and the HA antigen of influenza A is of a different subtype; and (b) a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the third mRNA encoding the HA antigen of a first influenza B virus and the HA antigen of influenza B is of a different lineage, the composition further comprising lipid nanoparticles comprising 40-55 mol% ionizable amino lipids; 5-15 mol% neutral lipids; 35-45 mol% sterols; and 1-5 mol% PEG-modified lipids, wherein the ratio of the first:second:third:fourth mRNAs is greater than 1:1:1:greater than 1, and each is an influenza A:A:B:B strain.

[0317] In another aspect, the present disclosure provides a composition comprising (a) a second mRNA encoding the HA antigen of a second influenza A virus, wherein the first mRNA includes an open reading frame (ORF) encoding the hemagglutinin (HA) antigen of a first influenza A virus and the HA antigen of influenza A is of a different subtype; and (b) a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the third mRNA encoding the HA antigen of a first influenza B virus and the HA antigen of influenza B is of a different lineage, the composition further comprising lipid nanoparticles comprising 40-55 mol% ionizable amino lipids; 5-15 mol% neutral lipids; 35-45 mol% sterols; and 1-5 mol% PEG-modified lipids, wherein the ratio of the first:second:third:fourth mRNAs is greater than 1:1:1:greater than 1, and each is an influenza A:A:B:B strain.

[0318] Another aspect of the present disclosure provides a composition comprising (a) a second mRNA encoding the HA antigen of a second influenza A virus, wherein the first mRNA includes an open reading frame (ORF) encoding the hemagglutinin (HA) antigen of a first influenza A virus and the HA antigen of influenza A is of a different subtype; and (b) a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the third mRNA encoding the HA antigen of a first influenza B virus and the HA antigen of influenza B is of a different lineage, further comprising lipid nanoparticles comprising 40-55 mol% ionizable amino lipids; 5-15 mol% neutral lipids; 35-45 mol% sterols; and 1-5 mol% PEG-modified lipids, wherein the ratio of the first:second:third:fourth mRNAs is greater than 1:1:1:greater than 1, each being an influenza A:A:B:B strain, and the composition comprises a total of 100 μg of mRNA.

[0319] In another aspect, the present disclosure provides a composition comprising (a) a second mRNA encoding the HA antigen of a second influenza A virus, wherein the first mRNA includes an open reading frame (ORF) encoding the hemagglutinin (HA) antigen of a first influenza A virus and the HA antigen of influenza A is of a different subtype; and (b) a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the third mRNA encoding the HA antigen of a first influenza B virus and the HA antigen of influenza B is of a different lineage, further comprising lipid nanoparticles comprising 40-55 mol% ionizable amino lipids; 5-15 mol% neutral lipids; 35-45 mol% sterols; and 1-5 mol% PEG-modified lipids, wherein the ratio of the first:second:third:fourth mRNAs is greater than 1:1:1:greater than 1, and each is an influenza A:A:B:B strain, and the composition comprises a total of 200 μg of mRNA.

[0320] Another aspect of the present invention includes a method for vaccinating a human subject, comprising administering an mRNA vaccine comprising mRNA encoding at least four influenza antigens, each comprising at least two hemagglutinin (HA)A antigens of each different subtype and at least two HA B antigens of each different lineage in an amount effective to evoke an antigen-specific immune response in the subject.

[0321] In some embodiments, the influenza antigen is encoded by 1 to 4 mRNAs. In some embodiments, the mRNA comprises a single mRNA encoding at least 4 influenza antigens. In some embodiments, the mRNA comprises 4 mRNAs, each containing a single open reading frame (ORF) encoding one of the 4 influenza antigens. In some embodiments, the 4 influenza antigens include the HA antigen of influenza A of the H1 subtype, the HA antigen of influenza A of the H3 subtype, the HA antigen of influenza B of the B / Yamagata lineage, and the HA antigen of influenza B of the B / Victoria lineage.

[0322] Another aspect of the present disclosure provides a composition comprising an mRNA vaccine comprising mRNA encoding at least four influenza antigens, wherein the influenza antigens comprise at least two hemagglutinin (HA)A antigens of different subtypes and at least two HA B antigens of different lineages, and further comprising lipid nanoparticles comprising 40-55 mol% ionizable amino lipids; 5-15 mol% neutral lipids; 35-45 mol% sterols; and 1-5 mol% PEG-modified lipids.

[0323] In some embodiments, the influenza antigen is encoded by 1 to 4 mRNAs. In some embodiments, the mRNA comprises a single mRNA encoding at least 4 influenza antigens. In some embodiments, the mRNA comprises 4 mRNAs, each containing a single open reading frame (ORF) encoding one of the 4 influenza antigens. In some embodiments, the 4 influenza antigens include the HA antigen of influenza A of the H1 subtype, the HA antigen of influenza A of the H3 subtype, the HA antigen of influenza B of the B / Yamagata lineage, and the HA antigen of influenza B of the B / Victoria lineage.

[0324] In some embodiments, the four mRNAs are present in the composition in a ratio greater than 1:1:1:greater than 1, representing influenza A:A:B:B strains, respectively. In some embodiments, the composition contains a total of 25 μg to 200 μg of mRNA. In some embodiments, the percentage of subjects showing seroconversion for one of the four influenza antigens after a single dose on day 29 is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In some embodiments, the percentage of subjects showing seroconversion for one of the four influenza antigens after a single dose on day 29 is 100%.

[0325] In some embodiments, the percentage of subjects showing seroconversion for two of the four influenza antigens after a single dose on day 29 is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In some embodiments, the percentage of subjects showing seroconversion for two of the four influenza antigens after a single dose on day 29 is 100%.

[0326] In some embodiments, the percentage of subjects showing seroconversion for three of the four influenza antigens after a single dose on day 29 is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In some embodiments, the percentage of subjects showing seroconversion for three of the four influenza antigens after a single dose on day 29 is 100%.

[0327] In some embodiments, the percentage of subjects showing seroconversion for all four influenza antigens after a single dose on day 29 is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In some embodiments, the percentage of subjects showing seroconversion for all four influenza antigens after a single dose on day 29 is 100%.

[0328] In some embodiments, the percentage of subjects showing at least a twofold increase in geometric mean factor-free glucose (GMFR) after a single dose on day 29 is at least 80%, at least 90%, at least 95%, or 100%. In some embodiments, the percentage of subjects showing a twofold increase in GMFR after a single dose on day 29 is 100%. In some embodiments, the percentage of subjects showing a fourfold increase in GMFR after a single dose on day 29 is at least 80%, at least 90%, at least 95%, or 100%. In some embodiments, the percentage of subjects showing a fourfold increase in GMFR after a single dose on day 29 is 100%.

[0329] In some embodiments, GMFR includes the GMFR of H1N1 HAI titer. In some embodiments, GMFR includes the GMFR of H3N2 HAI titer. In some embodiments, GMFR includes the GMFR of B / Yamagata HAI titer. In some embodiments, GMFR includes the GMFR of B / Victoria HAI titer.

[0330] In some embodiments, serum antibody titers increased fourfold on days 29 and 57 after a single dose compared to baseline (day 0). In some embodiments, serum antibody titers decreased on day 181 compared to day 29 after a single dose.

[0331] In some embodiments, the microneutralizing antibody titer increased fourfold on day 29 and / or day 57 after a single dose compared to baseline (day 0). In some embodiments, the microneutralizing antibody titer decreased on day 181 compared to day 29 after a single dose.

[0332] In some embodiments, the composition contains approximately 25 μg to 250 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata). In some embodiments, the composition contains equal amounts of each mRNA encoding each of the four HA proteins (e.g., the mRNAs are present in the composition in a ratio greater than 1:1:1 for each influenza A:A:B:B strain). In some embodiments, the composition contains approximately 25 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata). In some embodiments, the composition contains approximately 50 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata). In some embodiments, the composition contains approximately 100 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata). In some embodiments, the composition contains approximately 150 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata). In some embodiments, the composition contains approximately 200 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata). In some embodiments, the composition contains approximately 250 μg of mRNA encoding four influenza HA proteins (e.g., HA proteins associated with strains A / H1N1, A / H3N2, B / Victoria, and B / Yamagata).

[0333] The composition may further contain a buffering agent, such as a Tris buffer. For example, the composition may contain 10 mM to 30 mM, 10 mM to 20 mM, or 20 mM to 30 mM of a Tris buffer. In some embodiments, the composition contains 10, 15, 20, 25, or 30 mM of a Tris buffer. In some embodiments, the composition contains 20 mM of a Tris buffer.

[0334] In some embodiments, the mRNA in the composition is formulated at a concentration of 0.1 to 1 mg / mL. In some embodiments, the mRNA in the composition is formulated at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / mL. In some embodiments, the mRNA in the composition is formulated at a concentration of 0.5 mg / mL.

[0335] In some embodiments, the composition contains sucrose. For example, the composition may contain sucrose in concentrations of 75 mg / mL to 95 mg / mL, 75 mg / mL to 85 mg / mL, or 85 mg / mL to 95 mg / mL. In some embodiments, the composition contains 75, 80, 85, 86, 87, 88, 89, 90, or 95 mg / mL of sucrose. In some embodiments, the composition contains 87 mg / mL of sucrose. In preferred embodiments, the composition does not contain sodium acetate.

[0336] The composition may have a pH value of 6 to 8. In some embodiments, the composition has a pH value of 6, 6.5, 7, 7.5, or 8. In some embodiments, the composition has a pH value of 7.5.

[0337] In some embodiments, the composition further comprises a mixture of lipids. The mixture of lipids typically forms lipid nanoparticles. The mRNA described herein is formulated in some embodiments together with lipid nanoparticles (for example, for administration to a subject). In some embodiments, the lipid mixture, and thus the lipid nanoparticles, comprises ionizable aminolipids; neutral lipids; sterols; and PEG-modified lipids.

[0338] For example, the lipid mixture / lipid nanoparticles may contain 20-60 mol% ionizable aminolipids, 5-25 mol% neutral lipids, 25-55 mol% sterols, and 0.5-15 mol% PEG-modified lipids. In some embodiments, the lipid nanoparticles contain 20-60 mol% ionizable aminolipids, 5-25 mol% neutral lipids, 25-55 mol% sterols, and 0.5-15 mol% PEG-modified lipids. In some embodiments, the lipid nanoparticles contain 40-55 mol% ionizable aminolipids, 5-15 mol% neutral lipids, 35-45 mol% sterols, and 1-5 mol% PEG-modified lipids. For example, lipid nanoparticles may contain (a) 47 mol% ionizable amino lipids, 11.5 mol% neutral lipids, 38.5 mol% sterols, and 3.0 mol% PEG-modified lipids; (b) 48 mol% ionizable amino lipids, 11 mol% neutral lipids, 38.5 mol% sterols, and 2.5 mol% PEG-modified lipids; (c) 49 mol% ionizable amino lipids, 10.5 mol% neutral lipids, 38.5 mol% sterols, and 2.0 mol% PEG-modified lipids; (d) 50 mol% ionizable amino lipids, 10 mol% neutral lipids, 38.5 mol% sterols, and 1.5 mol% PEG-modified lipids; or (e) 51 mol% ionizable amino lipids, 9.5 mol% neutral lipids, 38.5 mol% sterols, and 1.0 mol% PEG-modified lipids. In some embodiments, the lipid mixture, thus the lipid nanoparticles, contains ionizable aminolipids in amounts of 20-55 mol%, 20-50 mol%, 20-45 mol%, 20-40 mol%, 25-60 mol%, 25-55 mol%, 25-50 mol%, 25-45 mol%, 25-40 mol%, 30-60 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 35-60 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-60 mol%, 40-55 mol%, 40-45 mol%, 50-60 mol%, 55-55 mol%, or 55-60 mol%.In some embodiments, the lipid mixture, and thus the lipid nanoparticles, contain 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-15 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% of neutral lipids. In some embodiments, the lipid mixture, thus the lipid nanoparticles, contains 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol...

Claims

1. A polypeptide having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs. 10 to 68.

2. The polypeptide according to claim 1, comprising at least 80% identity with SEQ ID NO:

30.

3. The polypeptide according to claim 1, comprising at least 80% identity with SEQ ID NO:

32.

4. The polypeptide according to claim 1, comprising at least 80% identity with SEQ ID NO:

33.

5. The polypeptide according to claim 1, comprising at least 80% identity with SEQ ID NO:

41.

6. The polypeptide according to claim 1, comprising at least 80% identity with SEQ ID NO:

47.

7. A polynucleotide encoding a polypeptide that has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs. 10 to 68.

8. The polynucleotide according to claim 7, which is RNA.

9. The polynucleotide according to claim 8, further comprising at least one poly(A) sequence containing 30 to 200 adenosine nucleotides.

10. The polynucleotide according to claim 8, comprising at least one untranslated region selected from at least one heterologous 5'-UTR and at least one heterologous 3'-UTR.

11. The polynucleotide according to claim 8, comprising a nucleotide analog.

12. The polynucleotide according to claim 8, wherein the RNA comprises a 1-methylpseudridine substitution.

13. The polynucleotide according to claim 8, comprising a 5'-cap structure including a structure selected from the group consisting of m7G, cap 0, cap 1, cap 2, modified cap 0, and modified cap 1 structures.

14. A composition comprising an RNA polynucleotide containing at least one coding sequence that encodes a polypeptide having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 10 to 68.

15. The composition according to claim 14, further comprising (c) at least one heterologous untranslated region (UTR); and (c) at least one pharmaceutically acceptable carrier wherein the RNA is complexed with or associated with a lipid, and the lipid comprises a cationic lipid, a neutral lipid, a steroid, and a PEG lipid.

16. The composition according to any one of claims 14 to 15, further comprising a second RNA polynucleotide containing an open reading frame encoding a second antigen, wherein the first and second RNA polynucleotides are complexed or associated with a lipid.

17. The composition according to any one of claims 14 to 16, wherein the first and second antigens comprise hemagglutinin (HA), or an immunogenic fragment or variant thereof.

18. The composition according to claim 17, wherein the first and second antigens each comprise HA derived from different subtypes of influenza virus, or immunogenic fragments thereof.

19. A polypeptide having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs. 69 to 115.

20. A polynucleotide encoding a polypeptide that has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs. 69 to 115.

21. The polynucleotide according to claim 20, which is RNA.

22. The polynucleotide according to claim 21, further comprising: at least one poly(A) sequence containing 30 to 200 adenosine nucleotides; at least one untranslated region selected from at least one heterologous 5'-UTR and at least one heterologous 3'-UTR; a 5' cap; and a nucleotide analog.

23. The polynucleotide according to claim 22, wherein the RNA comprises a 1-methylpseudridine substitution.

24. A composition comprising RNA containing at least one coding sequence that encodes a polypeptide having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs. 69 to 115.

25. The composition according to claim 24, further comprising RNA with at least one heterologous untranslated region (UTR); and at least one pharmaceutically acceptable carrier, wherein the RNA is complexed with or associated with a lipid, and the lipid comprises a cationic lipid, a neutral lipid, a steroid, and a PEG lipid.

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