Nucleic acid and use thereof

Abstract

Disclosed herein are self-amplifying RNA (saRNA) molecules encoding influenza virus antigens and methods of use thereof.

Description

[0001] cross-reference requests

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 484,186, filed February 9, 2023; U.S. Provisional Application No. 63 / 484,747, filed February 13, 2023; and U.S. Provisional Application No. 63 / 621,102, filed January 15, 2024, all of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to compositions of ribonucleic acid (RNA) vaccines, as well as methods for preparing, manufacturing, and therapeutic uses of RNA vaccines, wherein the RNA vaccines comprise polynucleotide molecules encoding one or more influenza antigens (such as hemagglutinin antigen). Background Technology

[0004] Influenza viruses belong to the family Orthomyxoviridae and are classified into three types (type A, type B, and type C) based on the antigenic differences between their nucleoprotein (NP) and matrix protein (M).

[0005] The genome of influenza A virus contains eight linear, negatively polar, single-stranded RNA molecules (seven for influenza C virus), which encode a variety of polypeptides, including: RNA-directed RNA polymerase proteins (PB2, PB1, and PA) and nucleoproteins (NP), which together constitute the nucleocapsid; matrix proteins (M1 and M2, where M2 is also a surface-exposed protein embedded in the viral membrane); two surface glycoproteins protruding from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA); and non-structural proteins (NS1 and NS2).

[0006] Hemagglutinin is the main envelope glycoprotein of influenza A and B viruses, while hemagglutinin esterase (HE) of influenza C virus is a protein homologous to hemagglutinin (HA).

[0007] A major challenge in using traditional vaccines to treat and prevent influenza and other infections is the limited spectrum of protection, offering protection only against closely related subtypes. Furthermore, the lengthy time required to complete existing standard influenza vaccine production processes hinders the ability to rapidly develop and produce adaptive vaccines in pandemic scenarios.

[0008] There is a need for improved anti-influenza compositions, preferably immunogenic compositions. Summary of the Invention

[0009] This disclosure provides improved anti-influenza compositions, preferably immunogenic compositions, to meet unmet needs. In one aspect, this disclosure relates to a composition comprising self-amplified RNA (saRNA), said saRNA comprising: a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from influenza A virus; a first subgenome promoter derived from influenza A virus; a first open reading frame encoding a first target gene derived from influenza virus hemagglutinin (HA); a second subgenome promoter derived from influenza A virus; a second open reading frame encoding a second target gene derived from influenza virus; a 3' untranslated region (3'UTR); and a 3' polyadenylate sequence.

[0010] In another aspect, this disclosure relates to a composition comprising self-amplified RNA (saRNA), said saRNA comprising: a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from alphavirus; a subgenomic promoter derived from alphavirus; an open reading frame encoding a target gene derived from influenza virus; a 3' untranslated region (3'UTR); and a 3' polyadenylate sequence; wherein at least 5% of the total population of a particular nucleotide in said molecule has been replaced by one or more modified or non-natural nucleotides.

[0011] In a preferred embodiment, the saRNA polynucleotide has clinical-grade purity. In some embodiments, the purity of the RNA polynucleotide is from about 60% to about 100%. In some embodiments, the integrity of the purified RNA polynucleotide is greater than 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99%, as determined by known methods (such as, for example, capillary electrophoresis). In some embodiments, RNA molecules comprising any one, at least one, at most one, or between any two of the following total RNA molecules in the composition are full-length RNA transcripts: 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%. A "full-length" RNA molecule is defined as a molecule containing a 5' cap and a polyadenylated tail.

[0012] In some aspects, this disclosure provides isolated polynucleotides (e.g., replicons) or a group of polynucleotides comprising: a first polynucleotide encoding an innate immunosuppressant (e.g., an influenza virus nonstructural (NS1) protein); and a second polynucleotide encoding a heterologous target mRNA (e.g., an influenza virus HA protein). Such polynucleotides are capable of driving enhanced and persistent expression of said heterologous target mRNA in cells.

[0013] Some aspects of this disclosure relate to a polynucleotide or a group of polynucleotides, including: a first nucleic acid molecule encoding an influenza virus non-structural (NS1) protein; and a second nucleic acid molecule encoding a heterologous target mRNA.

[0014] In some respects, a first nucleic acid molecule encoding the influenza virus NS1 protein and a second nucleic acid molecule encoding the target mRNA are present in a first vector (referred to herein as "cis"). In other respects, a first nucleic acid molecule encoding the influenza virus NS1 protein is present in a first vector, and a second nucleic acid molecule encoding the target mRNA is present in a second vector (referred to herein as "trans").

[0015] In some respects, the first nucleic acid molecule encoding the influenza virus NS1 protein is expressed under the control of a first promoter, such as a subgenomic promoter, like a subgenomic promoter derived from alpha virus. In some respects, the second nucleic acid molecule encoding the target mRNA is expressed under the control of a second promoter, such as a subgenomic promoter, like a subgenomic promoter derived from alpha virus. In some respects, the first and second promoters are the same. In some respects, the first and second promoters are different.

[0016] In some respects, a first nucleic acid molecule encoding the influenza virus NS1 protein and a second nucleic acid molecule encoding the target mRNA are expressed under the control of a first promoter, wherein the first promoter drives the expression of both the influenza virus NS1 protein and the target mRNA. In some respects, the first nucleic acid molecule encoding the influenza virus NS1 protein and the second nucleic acid molecule encoding the target mRNA are linked by an IRES sequence. In some respects, the first vector, the second vector, or both contain one or more regulatory elements.

[0017] In some respects, the expression level of the target mRNA is increased compared to the expression of the target mRNA in the absence of a first nucleic acid molecule encoding the influenza virus NS1 protein. In some respects, the expression of the target mRNA is increased by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% compared to the expression of the target mRNA in the absence of a first nucleic acid molecule encoding the influenza virus NS1 protein. In some respects, the increase in target mRNA expression persists for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, or at least about 48 hours. Attached Figure Description

[0018] The following figures form part of this specification and are used to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of the accompanying figures in conjunction with the detailed description of the specific embodiments presented herein.

[0019] Figure 1A-Figure 1B — Functional anti-HA antibodies induced after immunization with saRNA encoding influenza virus HA and / or NA, formulated with LNP, as measured by HAI; Figure 1A It describes the three weeks following the initial vaccination; Figure 1B Two weeks post-boost immunization, female Balb / c mice were immunized intramuscularly (IM) on day 0 with the following formulations: 20 ng of LNP-formulated bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA and saRNA-NA, and 200 ng of a modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody responses against A / Wisconsin / 588 / 2019 were measured by HAI or a one-day MNT assay on day 21 (3 weeks post-immunization). HAI titers (geometric mean plus geometric standard deviation (SD)) were reported.

[0020] Figures 2A-2B - Neutralizing antibodies induced in mice immunized with saRNA encoding influenza virus HA and / or NA formulated with LNP, measured by one-day MNT. Figure 2A It describes the three weeks following the initial vaccination; Figure 2BTwo weeks post-boost immunization, female Balb / c mice were immunized intramuscularly (IM) on day 0 with the following formulations: 20 ng of LNP-formulated bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA and saRNA-NA, and 200 ng of a modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody responses against A / Wisconsin / 588 / 2019 were measured by HAI or a one-day MNT assay on day 21 (3 weeks post-immunization). 50% neutralizing titers (geometric mean plus geometric standard deviation) were reported.

[0021] Figure 3 - Neutralizing antibodies induced in mice by immunization with saRNA encoding influenza virus HA and / or NA formulated with LNP, measured by 3-day MNT.

[0022] Figures 4A-4B - Functional anti-NA antibodies induced in mice by immunization with saRNA encoding influenza virus HA and / or NA formulated with LNP, as measured by NAI. Figure 4A It describes the three weeks following the initial vaccination; Figure 4B Two weeks after booster immunization, female Balb / c mice were immunized intramuscularly (IM) on day 0 with the following formulations: 20 ng of LNP-formulated bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA and saRNA-NA, and 200 ng of a modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody responses against A / Wisconsin / 588 / 2019 were measured by NAI on day 21 (3 weeks post-immunization). Geometric mean titers plus geometric standard deviations are reported.

[0023] Figure 5 – Serum cytokines and chemokines in Balb / c mice 24 hours after immunization with an influenza saRNA-HA vaccine formulation containing varying amounts of modified nucleosides.

[0024] Figure 6 - Functional HAI and neutralizing antibodies induced in Balb / c mice after immunization with saRNA-HA vaccine formulations containing different amounts of modified nucleosides.

[0025] Figure 7 - Serum cytokines and chemokines in C57BL6 / J mice 24 hours after immunization with an influenza saRNA-HA vaccine formulation containing different amounts of modified nucleosides.

[0026] Figure 8 - Functional HAI and neutralizing antibodies induced by immunization of C57NL6 / J mice with LNP-formulated saRNA-HA vaccine formulations containing varying amounts of modified nucleosides; female Balb / c mice were immunized on day 0 by intramuscular (IM) injection of the following formulations: 20 ng of LNP-formulated tetravalent saRNA containing four bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage); or 2.4 μg of an approved adjuvanted tetravalent inactivated vaccine (QIV; FluAd). Antibody responses against each vaccine component were measured on day 42 (2 weeks after the second dose) by HAI or one-day MNT assay. HAI and 50% neutralizing titers (geometric mean plus geometric standard deviation) were reported.

[0027] Figure 9 - Functional HAI and neutralizing antibodies induced in mice by immunization with tetravalent bicistronic saRNA (encoding HA and NA from four seasonal influenza virus strains) formulated with LNP.

[0028] Figure 10 - Functional NAI antibodies induced by immunization of mice with LNP-formulated tetravalent bicistronic saRNAs encoding HA and NA from four seasonal influenza virus strains; female Balb / c mice were immunized on day 0 by intramuscular (IM) injection of the following formulations: 20 ng LNP-formulated tetravalent saRNA containing four bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage); or 2.4 μg of an approved adjuvanted tetravalent inactivated vaccine (QIV; FluAd). On day 42 (2 weeks after the second dose), antibody responses against each vaccine component were measured by NAI. NAI titers (geometric mean plus geometric standard deviation) for 3 out of 4 viral strains were reported. Due to technical issues with NAI determination for the H3N2 viral strains, H3N2 NAI titers for saRNA and QIV could not be reported.

[0029] Figure 11 – Geometric mean titer and 95% confidence interval (CI): HAI – Vaccine formulations 1, 2 and control group – Evaluable immunogenic populations.

[0030] Abbreviations: GMT = Geometric Mean Titer; HAI = Hemagglutination Inhibition; QIV = Quadrivalent Influenza Vaccine; Vax Prep = Vaccine Preparation.

[0031] Note: V1 = Day 1 before vaccination; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks.

[0032] Note: Dots represent individual antibody levels.

[0033] Note: The number / GMT in each bar chart represents the number of participants who obtained valid and definitive results for a given assay at a given sampling time point, and the corresponding geometric mean titer. GMT is calculated using the average of two samples collected on day 1 prior to vaccination.

[0034] Note: Approved QIV-15A includes participants in the study who received the approved QIV, and their VRD data were tested simultaneously with the VRD data of the C1 group.

[0035] Note: Approved QIV-18 includes participants in the study who received approved QIVs, whose VRD data were tested concurrently with VRD data from the C2-C5 and C7 groups.

[0036] Note: Placebo includes participants who are randomly assigned to receive a placebo in the study.

[0037] Figure 12 – Geometric mean titer and 95% confidence interval: HAI – vaccine formulations 3, 4, 7 and control group – evaluable immunogenic population.

[0038] Abbreviations: GMT = Geometric Mean Titer; HAI = Hemagglutination Inhibition; QIV = Quadrivalent Influenza Vaccine; Vax Prep = Vaccine Preparation.

[0039] Note: V1 = Day 1 before vaccination; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks.

[0040] Note: Dots represent individual antibody levels.

[0041] Note: The number / GMT in each bar chart represents the number of participants who obtained valid and definitive results for a given assay at a given sampling time point, and the corresponding geometric mean titer. GMT is calculated using the average of two samples collected on day 1 prior to vaccination.

[0042] Note: Approved Q1V-18 includes participants in the study who received approved QIV, whose VRD data were tested concurrently with VRD data from groups C2-C5 and C7.

[0043] Note: The placebo group includes participants who were randomly assigned to receive a placebo in the study.

[0044] Figure 13 – Geometric mean titer and 95% confidence interval: HAI – vaccine formulations 5 and 6 and control group – evaluable immunogenic population.

[0045] Abbreviations: GMT = Geometric Mean Titer; HAI = Hemagglutination Inhibition; QIV = Quadrivalent Influenza Vaccine; Vax Prep = Vaccine Preparation.

[0046] Note: VI = Day 1 before vaccination; V3 = 1 week, V4 = 2 weeks; V5 = 4 weeks.

[0047] Note: Dots represent individual antibody levels.

[0048] Note: The number / GMT in each bar chart represents the number of participants who obtained valid and definitive test results for a given assay at a given sampling time point, and the corresponding geometric mean titer. GMT is calculated using the average of two samples collected on day 1 prior to vaccination.

[0049] Note: Approved QIV-18 includes participants in the study who received approved QIVs, whose VRD data were tested concurrently with VRD data from the C2-C5 and C7 groups.

[0050] Note: Approved QIV-15B includes participants in the study who received approved QIVs, and their VRD data were tested simultaneously with the VRD data of the C6 group.

[0051] Note: Placebo includes participants who are randomly assigned to receive a placebo in the study.

[0052] Figure 14 – Trans-addition of modNS1 increased saRNA-HA expression and cell viability in HeLa cells. 25 ng of saRNA-HA-Wisconsin was co-transfected into HeLa cells with incremental amounts of modNS1 or modGFP (as a control). Cell viability was assessed at 24 hours by measuring the percentage of HA-positive cells (%), protein expression level (MFI), and total cell count. RMM59 in this paper refers to saRNA-HA Wisconsin.

[0053] Figure 15– A total of 25 ng of saRNA-HA-Wisconsin was co-transfected into HeLa cells with low increments of modNS1 (1 ng to 25 ng) (blue, i.e., “RMM59+NS1”) or saRNA (as a matched dose level control) (red, i.e., “RMM59+(X)RMM59”). Cells were fixed at 24 h and stained with FI6 HA human monoclonal antibody and goat anti-human A488 secondary antibody. The percentage (%) of HA-positive cells and the cell number were measured.

[0054] Figure 16 - Test results for saRNA encoding HA Wisconsin but containing 50% m1ψ. Test conditions and Figure 15 The same as described above.

[0055] Figure 17 -saRNA replication in human monocytes was improved. Human CD14+ cells (stem cells) were transfected with different doses of GFP-expressing saRNA (5 ng to 100 ng) in combination with escalating doses of modNS1 (0 to 50 ng), with modRNA GFP used as a control. The total percentage of positive cells (%) and cell viability were measured by FACS at 24 hours.

[0056] Figure 18 - Shows the total percentage (%) of positive cells as measured using Aqua live / dead cell staining.

[0057] Figure 19 AB diagram – Figure 19 Figure A: HeLa cells were transfected individually with monocistronic saRNA expressing HA or bicistronic saRNA expressing both HA and NS1, or in combination with 10 ng or 25 ng modNS1. The percentage of HA-positive cells (%) and protein expression level (MFI) were measured. Cells were fixed after 24 hours and stained with rabbit anti-HA polyclonal antibody and goat anti-rabbit A647 secondary antibody. Figure 19 Figure B in the middle: as shown Figure 19 Figure A shows a schematic diagram of different saRNA conformations (monocistan and bicistronic) transfected alone or in combination with modNS1.

[0058] Figure 20 The AC diagram shows the addition of a constant total amount of 100 ng of RNA to HeLa cells under each condition. Different proportions of U RNA (i.e., unmodified RNA) or a combination of conventional modRNA with modNS1 or modGFP (control) were added, and the percentage of HA(+) cells was assessed at 24 hours. Figure 20 (Figure A) and cell number ( Figure 20(Figure B). Confocal images of HeLa cells were observed (data not shown) to compare 100 ng RMM71 (uRNA HA Wisconsin) and 50 ng nguRNA combined with 50 ng modNS1. Cells were fixed at 24 h and stained with FI6HA human monoclonal antibody and goat anti-human A647 secondary antibody. Figure 20 Figure C shows a comparison of different test configurations.

[0059] Figure 21 – The percentage of HA-positive cells after administration of various active RNA molecules was depicted, and the total number of viable cells after administration of various active RNA molecules was further depicted. See Example 10 for the method. Trans-delivery of modNS1 significantly increased the expression of saRNA antigen and helped maintain cell viability.

[0060] Figure 22 -Measure the functional anti-HA antibodies induced in mice that have received a dose of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA by HAI.

[0061] Figure 23 - Measure the functional anti-NA antibodies induced in mice that have received a dose of saRNA-LNP or FluAd encoding HA / NA by NAI.

[0062] Figure 24 -Measure virus-neutralizing antibodies induced in mice that received a dose of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA by 1-day MNT.

[0063] Figure 25 - Measure the functional anti-HA antibodies induced in mice that received two doses of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA by HAI.

[0064] Figure 26 – The functional anti-NA antibodies induced in mice that received two doses of saRNA-LNP or FluAd encoding HA / NA were measured by NAI.

[0065] Figure 27 –Measure virus-neutralizing antibodies induced in mice that received two doses of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA by 1-day MNT. Detailed Implementation

[0066] The embodiments disclosed herein provide compositions comprising self-amplifying RNA (saRNA) polynucleotides encoding influenza virus antigens. The influenza virus RNA vaccines provided herein can be used to induce balanced immune responses, including cellular and humoral immunity.

[0067] It is anticipated that any embodiments discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0068] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments indicate particular implementations of this disclosure, they are given by way of example only, as various changes and modifications within the spirit and scope of this disclosure will be readily apparent to those skilled in the art from this detailed description.

[0069] Throughout this application, the term “about” is used to indicate that a value includes the inherent error variation of the measurement or quantitative method.

[0070] When the word “a” or “a kind” is used with the term “including”, it may mean “a / a kind”, but it is also consistent with the meaning of “one or more / one or more kinds”, “at least one / at least one kind” and “one or more than one / a kind or more kinds”.

[0071] The phrase “and / or” means “and” or “or”. For example, A, B and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C. In other words, “and / or” functions as an inclusive “or”.

[0072] The words “comprise” (and any form of inclusion, such as “comprise” and “comprises”), “have” (and any form of having, such as “have” and “has”), “include” (and any form of inclusion, such as “includes” and “include”), or “contain” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or methodological steps.

[0073] The phrase “substantially all” is defined as “at least 95%”; if substantially all members of a group possess a certain characteristic, then at least 95% of the members in the group possess that characteristic. In some cases, substantially all means that any one, at least one, or between any two of the members in the group possesses that characteristic, equivalent to 95%, 96%, 97%, 98%, 99%, or 100%.

[0074] The composition and its method of use may “comprising” any ingredient or step disclosed in full in this specification, “consistently composed of any ingredient or step disclosed in full in this specification,” or “composed of any ingredient or step disclosed in full in this specification.” Compositions and methods “consisting substantially of any disclosed ingredient or step” limit the scope of the claims to specific materials or steps that do not materially affect the essential and novel features of the claimed disclosure.

[0075] A. Self-amplifying RNA (saRNA)

[0076] In some implementations, the RNA molecule (such as the first RNA molecule) is saRNA. "saRNA," "self-amplifying RNA," and "replicon" refer to RNA capable of self-replication. Self-amplifying RNA molecules can be generated by using replication elements derived from one or more viruses (e.g., alphaviruses) and replacing the viral structural polypeptide with a nucleotide sequence encoding a target polypeptide. Self-amplifying RNA molecules are typically positive-strand molecules that are directly translated upon delivery to the cell. This translation provides an RNA-dependent RNA polymerase, which subsequently produces antisense and sense transcripts from the delivered RNA. The delivered RNA results in the production of multiple progeny RNAs. These progeny RNAs, along with collinear subgenomic transcripts, can be self-translated to provide in situ expression of the encoded target gene (e.g., a viral antigen); or they can be transcribed to produce additional transcripts synonymous with the delivered RNA, which, upon translation, can express the target protein (e.g., the antigen) in situ. The overall result of this transcriptional sequence is an amplification of the number of introduced saRNAs, thus enabling the encoded target gene (e.g., a viral antigen) to become the major polypeptide product of the cell.

[0077] In some embodiments, the self-amplifying RNA contains at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases, and other non-structural viral proteins. In some embodiments, the self-amplifying RNA may also contain 5' and 3' pull-to-replication sequences, and a heterologous sequence optionally encoding a desired amino acid sequence (e.g., a target antigen). The self-amplifying RNA may contain a subgenomic promoter that directs the expression of the heterologous sequence. Optionally, the heterologous sequence (e.g., the target antigen) may be fused within the coding frame of other regions in the self-amplifying RNA, and / or may be controlled by an internal ribosome entry site (IRES).

[0078] In some implementations, the self-amplifying RNA molecule is not encapsulated by virus-like particles. The self-amplifying RNA molecule described herein can be programmed to prevent the induction of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins essential for the production of viral particles from the self-amplifying RNA. For instance, when the self-amplifying RNA molecule is based on an alphavirus (such as Sindbis virus (SIN), Semliki forest virus, and Venezuelan equine encephalitis virus (VEE)), one or more genes encoding viral structural proteins (such as capsid and / or envelope glycoproteins) can be omitted.

[0079] In some embodiments, the self-amplifying RNA molecule described herein encodes: (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule; and (ii) a target polypeptide, such as a viral antigen. In some embodiments, the polymerase may be an alphavirus replicase, for example, any one or any combination of alphavirus proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the self-amplifying RNA molecule described herein may contain one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine). In some embodiments, the self-amplifying RNA molecule does not contain modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0080] The saRNA construct may encode at least one non-structural protein (NSP) located at the 5' or 3' end of a sequence encoding at least one target peptide or polypeptide. In some embodiments, the sequence encoding at least one NSP is located at the 5' end of a sequence encoding the target peptide or polypeptide. Therefore, 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. It is known in the art that nsP1 is a membrane anchor of the viral capping enzyme and replication complex (RC). nsP2 is an RNA helicase and a protease responsible for processing ns polyproteins. nsP3 interacts with a variety of host proteins and may regulate poly(ADP) ribosylation and mono(ADP) ribosylation of proteins. nsP4 is a core viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphavirus replicase, for example, comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0081] Although the natural alphavirus genome encodes structural virosomal 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 can lead to the production of its own genomic RNA copies in the cell, but not to the production of RNA containing virions. The inability to produce these virions, without being bound by theory or mechanism, means that, unlike wild-type alphaviruses, the self-amplifying RNA molecule cannot persist in an infectious form. The alphavirus structural proteins necessary for the perpetuation of wild-type viruses may not be present in the self-amplifying RNA of this disclosure, but may be replaced by genes encoding the target immunogen, such that the subgenomic transcript encodes the immunogen instead of structural alphavirus virosomal proteins.

[0082] 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 target antigen. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, for example, to encode other antigens or to encode additional polypeptides.

[0083] 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-amplified RNA molecule is selected to ensure compatibility with the encoded replicase.

[0084] Optionally, the self-amplifying RNA molecules described herein can also be designed to induce the production of attenuated or highly virulent infectious viral particles, or to produce viral particles capable of a single round of subsequent infection.

[0085] In some embodiments, the saRNA molecule is based on alphaviruses. Alphaviruses comprise a group of genetically, structurally, and serologically related arthropod-borne viruses of the family Ophioviridae. Exemplary viruses and subtypes within the genus Alphavirus include Sindbisvirus, Sindeliki Forest Virus, Ross River Virus, and Venezuelan Equine Encephalitis Virus. Therefore, the self-amplifying RNA described herein can be incorporated with an RNA replicase derived from any of Sindeliki Forest Virus (SFV), Sindbisvirus (SIN), Venezuelan Equine Encephalitis Virus (VEE), Ross River Virus (RRV), or other viruses belonging to the family Alphaviridae. In some embodiments, the self-amplifying RNA described herein can be incorporated with a sequence derived from a mutated or wild-type viral sequence; for example, the attenuated TC83 mutant of VEEV has been used as saRNA.

[0086] Alphavirus-based saRNAs are (+) strand saRNAs that, upon delivery to the cell, are translated, triggering the translation of a replicase (or replicase-transcriptionase). The replicase is translated into a polyprotein, which cleaves itself to form a replication complex, producing a genomic (-) strand copy of the delivered (+) strand RNA. These (-) strand transcripts can themselves be transcribed, producing additional copies of the (+) strand parental RNA and subgenomic transcripts encoding the desired gene product. Therefore, the translation of these subgenomic transcripts leads to in situ expression of the desired gene product in infected cells. Suitable alphavirus saRNAs can utilize replicases from Sindbis virus, Semliki forest virus, eastern equine encephalitis virus, Venezuelan equine encephalitis virus, or their mutant variants.

[0087] In some implementations, the self-amplified RNA molecule is derived from or based on viruses other than alphaviruses, such as positive-sense RNA viruses, particularly picornaviruses, flaviviruses, rubella viruses, hepatitis C viruses, caliciviruses, or coronaviruses. Suitable wild-type alphavirus sequences are well-known and available from sequence collections, such as the American Type Culture Collection (Rockville, Md.). Representative examples of suitable alphaviruses include Aura virus (ATCC VR-368), Bibaru virus (ATCC VR-600, ATCC VR-1240), Kabasu virus (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern Equine Encephalomyelitis Virus (ATCC VR-65, ATCC VR-1242), Morganburg virus (ATCC VR-924), Getta virus (ATCC VR-369, ATCC VR-1243), Kyzylagach virus (ATCC VR-927), Mayaro virus (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg virus (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndum virus (ATCC VR-371), and Pixuna virus (ATCC VR-372, ATCC VR-378). The list includes: VR-1245, Ross River virus (ATCCVR-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), Trinity virus (ATCC VR-469), Una virus (ATCC VR-374), Venezuelan equine 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), Wataroa virus (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some respects, one or more alphaviruses on the list can be excluded.

[0088] In some implementations, 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 comprise at least about 4 kb. For example, the self-amplifying RNA can be equal to any, at least, at most, or between any two of the following: 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, and 16 kb. In some cases, the self-amplifying RNA may comprise 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 some embodiments, the self-amplifying RNA is approximately 4kb to approximately 12kb, approximately 5kb to approximately 12kb, approximately 6kb to approximately 12kb, approximately 7kb to approximately 12kb, approximately 8kb to approximately 12kb, approximately 9kb to approximately 12kb, approximately 10kb to approximately 12kb, approximately 11kb to approximately 12kb, approximately 5kb to approximately 11kb, approximately 5kb to approximately 10kb, approximately 5kb to approximately 9kb, approximately 5kb to approximately 8kb, approximately 5kb to approximately 7kb, approximately 5kb to approximately 6kb, approximately... 6kb to about 12kb, about 6kb to about 11kb, about 6kb to about 10kb, about 6kb to about 9kb, about 6kb to about 8kb, about 6kb to about 7kb, about 7kb to about 11kb, about 7kb to about 10kb, about 7kb to about 9kb, about 7kb to about 8kb, about 8kb to about 11kb, about 8kb to about 10kb, about 8kb to about 9kb, about 9kb to about 11kb, about 9kb to about 10kb, or about 10kb to about 11kb.

[0089] In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen, or optionally, two or more polypeptide antigens linked together in a manner that preserves the identity of each sequence when expressed as an amino acid sequence (e.g., tandem linking). The polypeptide generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered to produce independent polypeptide or peptide sequences. In some embodiments, the saRNA molecule may encode one or more target polypeptides, such as one antigen or more than one antigen, such as two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. Alternatively, or additionally, a saRNA molecule may also encode more than one target polypeptide or multiple target polypeptides, such as antigens, for example, bicistronic or tricistronic RNA molecules encoding different or the same antigens.

[0090] As used herein, the term "link" refers to the covalent or non-covalent connection of a first amino acid sequence or polynucleotide sequence to a second amino acid sequence or polynucleotide sequence. The first amino acid sequence or polynucleotide sequence may be directly linked or juxtaposed with the second amino acid sequence or polynucleotide sequence, or alternatively, an insertion sequence may covalently link the first and second sequences. The term "link" includes not only the fusion of the first RNA molecule with the second RNA molecule at the 5' or 3' end, but also the insertion of the entire first RNA molecule into any two nucleotides of the second RNA molecule. The first and second RNA molecules may be linked via a phosphodiester bond or a linker. A linker may be, for example, a polynucleotide.

[0091] In some embodiments, the self-amplifying RNA described herein may encode one or more polypeptide antigens comprising a series of epitopes. In some embodiments, the self-amplifying RNA described herein may encode epitopes capable of inducing helper T cell responses or cytotoxic T cell responses, or both.

[0092] In some implementations, the saRNA molecule is purified, for example by filtration, which may be performed via, for example, ultrafiltration, percolation, or, for example, tangential flow ultrafiltration / percolation.

[0093] Some embodiments of this disclosure relate to a composition comprising a self-amplifying RNA molecule including a 5' cap, a 5' untranslated region, a coding region containing a sequence encoding an RNA-dependent RNA polymerase (also known as a "replicaase"), a subgenomic promoter (such as a promoter derived from an alpha virus), an open reading frame encoding a target gene (e.g., an antigen derived from an influenza virus), a 3' untranslated region, and a 3' polyadenylated nucleotide sequence. In some embodiments, at least 5% of a specific total nucleotide population in the saRNA molecule has been substituted with one or more modified or non-natural nucleotides.

[0094] In some embodiments, the saRNA molecule does not contain modified nucleotides, for example, it does not contain modified nucleobases, 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 will be further described below. In some embodiments, the RNA may include a 5' cap containing 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.

[0095] The efficacy of the product depends on the expression of the delivered saRNA, which requires sufficiently intact RNA molecules. RNA integrity is an RNA quality indicator for quantifying intact RNA. This method can also detect potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. Initial specifications are set to ensure sufficient RNA integrity in the pharmaceutical formulation. In some embodiments, the integrity of the RNA polynucleotide is at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the integrity of the RNA polynucleotide is about 95% or higher. In some embodiments, the integrity of the RNA polynucleotide is about 98% or higher. In some embodiments, the integrity of the RNA polynucleotide is about 99% or higher.

[0096] In a preferred embodiment, the saRNA polynucleotide has clinical-grade purity. In some embodiments, the RNA polynucleotide has a purity of about 60% to about 100%. In some embodiments, the RNA polynucleotide has a purity of about 80% to about 99%. In some embodiments, the RNA polynucleotide has a purity of about 90% to about 99%. In some embodiments, the purified mRNA has clinical-grade purity and requires no 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, wherein the further purification is selected from high-performance liquid chromatography (HPLC) purification, ligand-based or binding-based purification, and / or ion exchange chromatography. In some embodiments, the method for producing the RNA polynucleotide removes long null RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual solvents, and / or residual salts. In some embodiments, the short null transcript contaminant contains less than 15 bases. In some embodiments, the short null transcript contaminant contains about 8-12 bases. In some embodiments, the method of the present invention also removes RNAse inhibitors.

[0097] In some embodiments, the purified saRNA polynucleotides, as determined by capillary electrophoresis, contain 5% or less, 4% or less, 3% or less, 2% or less, 1% or less protein contaminants, or are substantially free of protein contaminants. In some embodiments, the purified RNA polynucleotides, as determined by high-performance liquid chromatography (HPLC), contain less than 5%, less than 4%, less than 3%, less than 2%, less than 1% salt contaminants, or are substantially free of salt contaminants. In some embodiments, the purified RNA polynucleotides, as determined by known methods (such as, for example, high-performance liquid chromatography (HPLC)), contain 5% or less, 4% or less, 3% or less, 2% or less, 1% or less short ineffective transcript contaminants, or are substantially free of short ineffective transcript contaminants. In some embodiments, the purified RNA polynucleotides have 60% or greater, 70% or greater, 80% or greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater integrity, as determined by known methods (such as, for example, capillary electrophoresis).

[0098] B. Modified nucleobases

[0099] Modified nucleosides and nucleotides that can be incorporated into RNA molecules include, for example: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (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); (Alkenyl) adenosine); g6A (N6-glycylcarbamoyl adenosine); t6A (N6-threonylcarbamoyl adenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyl adenosine); m6t6A (N6-methyl-N6-threonylcarbamoyl adenosine); hn6A (N6-hydroxyn-valinecarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine); Ar(p)(2'-O-riboadenosine (phosphate)); I (inosine); miI (1-methylinosine); m'Im (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac 4C (N4-acetylcytidine); £5C (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2-O-methylcytidine); k2C (lysine); m1G (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'-trimethylguanosine); Gr(p)(2'-O-ribosylguanosine (phosphate)); yW (huaitin); o2yW (peroxyhuaitin); OHyW (hydroxyhuaitin); OHyW* (low Modified hydroxyguanosine; imG (huao glycoside); mimG (methylguanosine); Q (pigmentoside); oQ (epoxypigmentoside); galQ (galactosylpigmentoside); manQ (mannosylpigmentoside); preQo (7-cyano-7-deazoguanosine); preQi (7-aminomethyl-7-deazoguanosine); G* (archaeoside); 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); nmm5U (5-carbamoylmethyluridine); nmm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine); cmnm5s2U (5-carboxymethylamino... Methyl-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 (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyladenosine); tm5s2U (S-taurate methyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine);ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxoadenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 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 Pyrimidine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-denitroguanine, 8-azaguanine, 7-denitro-7-substituted guanine, 7-denitro-7-(C2-C6)ynylguanine, 7-denitro-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-denitropurine, 7-denitro-7-substituted purine, 7-denitro-8-substituted purine, hydrogen (debase residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. In some respects, one or more modified nucleosides in the list may be excluded.

[0100] Other exemplary modified nucleotides include any one of N-1-methylpseuuridine, pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some embodiments, the modified nucleotide is N-1-methylpseuuridine.

[0101] In some implementations, the RNA molecule may include aminophosphate, thiophosphate, and / or methylphosphonate bonds.

[0102] In some embodiments, the RNA molecule includes a modified nucleotide selected from any of the following: pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, and 2′-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of: pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, and 2′-O-methyluridine. In some implementations, the modified or non-natural nucleotide is selected from the group consisting of 5-methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine.

[0103] In some embodiments, at least 10% of the total population of a specific nucleotide in the saRNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of a specific nucleotide in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of a specific nucleotide in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of a specific nucleotide in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, substantially all of the specific nucleotide population in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0104] In some embodiments, at least a portion or all of a specific total population of nucleotides in the saRNA molecule has been replaced by two modified or unnatural nucleotides. In some embodiments, the two modified or unnatural nucleotides are provided at a ratio equal to any one, at least one, at most one, or between any two of 1:99 to 99:1, including: 1:99; 2:98; 3:97; 4:96; 5:95; 6:94; 7:93; 8:92; 9:91; 10:90; 11:89; 12:88; 13:87; 14:86; 15:85; 16:84; 17:83; 18 :82; 19:81; 20:80; 21:79; 22:78; 23:77; 24:76; 25:75; 26:74; 27:73; 28:72; 29:71; 30:70; 31:69; 32:68; 33:67; 34:66; 35:65; 36:64; 37:63; 38:62; 39:61; 40:60; 41:59; 42:58; 43:57; 44:56; 45:55 ; 46:54; 47:53; 48:52; 49:51; 50:50; 51:49; 52:48; 53:47; 54:46; 55:45; 56:44; 57:43; 58:42; 59:41; 60:40; 61:39; 62:38; 63:37; 64:36; 65:35; 66:34; 67:33; 68:32; 69:31; 70:30; 71:29; 72:28; 7 3:27; 74:26; 75:25; 76:24; 77:23; 78:22; 79:21; 80:20; 81:19; 82:18; 83:17; 84:16; 85:15; 86:14; 87:13; 88:12; 89:11; 90:10; 91:9; 92:8; 93:7; 94:6; 95:5; 96:4; 97:3; 98:2; and 99:1, or any range derived therefrom.

[0105] In some embodiments, at least 10% of the total first specific nucleotide population in the saRNA molecule disclosed herein has been substituted with one or more modified or unnatural nucleotides, and at least 10% of the total second specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the total first specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides, and at least 25% of the total second specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the total first specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides, and at least 50% of the total second specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the total first specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides, and at least 75% of the total second specific nucleotide population in the molecule has been substituted with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the total first specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides, and substantially all of the total second specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the total first specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides, and at least 25% of the total second specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the total first specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides, and at least 50% of the total second specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the total first specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides, and at least 75% of the total second specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the total first specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides, and substantially all of the total second specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the total first specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides, and at least 50% of the total second specific nucleotide population in the molecule has been replaced by one or more modified or unnatural nucleotides.In some embodiments, at least 50% of the total first specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides, and at least 75% of the total second specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the total first specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides, and substantially all of the total second specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the total first specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides, and at least 75% of the total second specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the total first specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides, and substantially all of the total second specific nucleotide group in the molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, substantially all of the first specific nucleotide total population in the molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the second specific nucleotide total population in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0106] In some embodiments, at least 25% of the total population of uridine nucleotides in the saRNA molecule is substituted with N1-methylpseudoruri. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule is substituted with N1-methylpseudoruri. In some embodiments, at least 75% of the total population of uridine nucleotides in the molecule is substituted with N1-methylpseudoruri. In some embodiments, substantially all uridine nucleotides in the molecule are substituted with N1-methylpseudoruri. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule is substituted with 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the molecule are substituted with 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule is substituted with 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the molecule are substituted with 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the molecule is substituted with 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the molecule are substituted with 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced by 2-thiouridine. In some embodiments, substantially all of the uridine nucleotides in the molecule have been replaced by 2-thiouridine.

[0107] In some embodiments, at least 50% of the total uridine nucleotide population in the molecule is replaced by N1-methylpseudouridine, and substantially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the total uridine nucleotide population in the molecule is replaced by 5-methoxyuridine, and substantially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the total uridine nucleotide population in the molecule is replaced by 5-methyluridine, and substantially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine.

[0108] In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseuuridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseuuridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseuuridine.

[0109] C.UTR

[0110] The 5' untranslated region (UTR) is a DNA regulatory region located at the 5' end of a protein-coding sequence. It is transcribed into mRNA but not translated into protein. The 5' UTR can contain various regulatory elements, such as 5' cap structures, stem-loop structures, and internal ribosome entry sites (IRES), which can play a role in controlling translation initiation. The 3' UTR, located downstream of the protein-coding sequence, can participate in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. In some embodiments, the UTR is derived from mRNAs naturally abundant in a specific tissue (e.g., lymphoid tissue), and mRNA expression targets that specific tissue. In some embodiments, the UTR increases protein synthesis. Without being bound by mechanistic or theoretical constraints, the UTR can increase protein synthesis by increasing the time mRNA remains in translational polysomes (information stability) and / or increasing the rate at which ribosome-initiated information translation occurs (information translation efficiency). Therefore, the UTR sequence can prolong protein synthesis in a tissue-specific manner. In some embodiments, the 5' UTR and 3' UTR sequences are derived computationally. In some embodiments, the 5′UTR and 3′UTR are derived from naturally abundant mRNA in a tissue. This tissue can be, for example, the liver, stem cells, or lymphoid tissue. Lymphoid tissue can include, for example, any of the following: 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 alphavirus. In some embodiments, the 5′UTR and 3′UTR are derived from wild-type alphavirus. Examples of alphaviruses are described below.

[0111] In some embodiments, the first RNA molecule comprises the 5′UTR and 3′UTR of mRNA naturally abundant in the tissue. In some embodiments, the first RNA molecule comprises the 5′UTR and 3′UTR of alphavirus. In some embodiments, the second RNA or saRNA molecule comprises the 5′UTR and 3′UTR of alphavirus. In some embodiments, the second RNA or saRNA molecule comprises the 5′UTR and 3′UTR of wild-type alphavirus. In some embodiments, the RNA molecule comprises a 5′ cap.

[0112] D. Open Reading Frame (ORF)

[0113] The 5′ and 3′ UTRs can be operatively linked to an ORF, which can be a codon sequence capable of being translated into a target polypeptide. As described above, an RNA molecule can contain one (monociston), two (bisiston), or more (polyciriston) open reading frames (ORFs).

[0114] In some embodiments, the ORF encodes a non-structural viral gene. In some embodiments, the ORF also contains one or more subgenomic promoters. In some embodiments, the RNA molecule contains a subgenomic promoter operatively linked to the ORF. In some embodiments, the subgenomic promoter contains a cis-regulatory element. In some embodiments, the cis-regulatory element is located at B... 2 Immediately downstream (5'-3'). In some embodiments, the cis-acting control element is located at B. 2 The immediate downstream of guanine (5'-3'). In some embodiments, the cis-regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug. In some embodiments, the second RNA or saRNA molecule may include: (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 target antigen or polypeptide. The polymerase may be an alphavirus replicase, for example, including any one or a combination of non-structural alphavirus proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule contains the alphavirus non-structural protein nsP1. In some embodiments, the RNA molecule contains the alphavirus non-structural protein nsP2. In some embodiments, the RNA molecule contains the alphavirus non-structural protein nsP3. In some embodiments, the RNA molecule comprises the alphavirus non-structural protein nsP4. In some embodiments, the RNA molecule comprises the alphavirus non-structural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule comprises the alphavirus non-structural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule comprises any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not contain nsP4.

[0115] In some embodiments, the open reading frame (OPF) of the RNA (e.g., saRNA) composition is codon-optimized. In some embodiments, the OPF encoding an influenza peptide or a fragment thereof is codon-optimized.

[0116] E. Genes encoding antigenic polypeptides

[0117] 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 include a head domain. In some embodiments, the hemagglutinin protein includes a portion of a head domain. In some embodiments, the hemagglutinin protein does not include a cytoplasmic domain. In some embodiments, the hemagglutinin protein includes a portion of a cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein includes a portion of a transmembrane domain.

[0118] Some embodiments provide an influenza vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein, and a pharmaceutically acceptable carrier or excipient, formulated in cationic lipid nanoparticles. In some embodiments, the hemagglutinin protein is selected from H1, H7, and H10. In some embodiments, the RNA polynucleotide also encodes a neuraminidase (NA) protein. In some embodiments, the hemagglutinin protein is derived from an influenza A virus strain or an influenza B virus strain, or a combination thereof. In some embodiments, the influenza virus is selected from H1N1, H3N2, H7N9, and H10N8.

[0119] In some embodiments, the virus is an influenza A virus strain or an influenza B virus strain, or a combination thereof. In some embodiments, the influenza A virus strain or influenza B virus strain 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 a head domain (HA1). In some embodiments, the hemagglutinin protein includes a portion of a cytoplasmic domain. In some embodiments, the hemagglutinin protein is a truncated hemagglutinin protein. In some embodiments, the protein is a truncated hemagglutinin protein including a portion of a 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 scope of the target polypeptide. For example, this document provides any protein fragment of a reference protein (a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical) with a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.

[0120] In some embodiments, at least one antigenic polypeptide is one of the defined antigenic subdomains of HA, referred to as HA1, HA2, or a combination of HA1 and HA2, and 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).

[0121] In some embodiments, at least one antigenic polypeptide is HA or a derivative thereof, comprising an antigen sequence from HA1 and / or HA2, and at least one antigenic polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0122] In some embodiments, at least one antigenic polypeptide is HA or a derivative thereof, comprising an antigen sequence from HA1 and / or HA2, and at least two antigenic polypeptides selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0123] As used herein, the term "non-structural protein" refers to a protein encoded by a virus but not part of the viral particle. More specifically, non-structural proteins described herein include, for example, influenza virus NS1 proteins, including but not limited to influenza A virus NS1, influenza B virus NS1, influenza C virus NS1, H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, combinations thereof, or variants thereof.

[0124] In some embodiments, influenza virus NS1 is H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, their variants, or combinations thereof. In some embodiments, influenza virus NS1 is H1N2 NS1. In some embodiments, influenza virus NS1 is H2N2 NS1. In some embodiments, influenza virus NS1 is H3N2 NS1. In some embodiments, influenza virus NS1 is H7N9 NS1. In some embodiments, influenza virus NS1 is H7N7 NS1. In some embodiments, influenza virus NS1 is H9N2 NS1. In some embodiments, influenza virus NS1 is H7N2 NS1. In some embodiments, influenza virus NS1 is H7N3 NS1. In some embodiments, the influenza virus NS1 is H5N2 NS1. In some embodiments, the influenza virus NS1 is H10N7 NS1. In some embodiments, the influenza virus NS1 is H1N1 NS1. In some embodiments, the influenza virus NS1 is the H1N1 TX91 variant NS1. In some embodiments, the influenza virus NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence encoded by the nucleotide sequence listed in GenBank (NCBI) AF389122.

[0125] MDPNTVSSFQ VDCFLWHVRK RVADQELGDA PFLDRLRRDQ KSLRGRGSTL GLDIETATRA 60

[0126] GKQIVERILK EESDEALKMT MASVPASRYL TDMTLEEMSR DWSMLIPKQK VAGPLCIRMD 120

[0127] QAIMDKNIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLPSLPGHT AEDVKNAVGV 180

[0128] LIGGLEWNDN TVRVSETLQR FAWRSSNENG RPPLTPKQKR EMAGTIRSEV 230 (SEQ ID NO: 33).

[0129] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an influenza virus protein or an immunogenic fragment thereof.

[0130] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding a variety of influenza virus proteins or immunogenic fragments thereof.

[0131] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) 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 thereof).

[0132] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) 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 of any one or any combination of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and / or H18), and at least one other RNA (e.g., saRNA) polynucleotide having an open reading frame encoding a protein selected from HA, NP, NA, M1, M2, NS1, and NS2 proteins obtained from influenza viruses.

[0133] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any combination or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and / or H18), and at least two other RNA (e.g., saRNA) polynucleotides having two open reading frames encoding two proteins selected from HA, NP, NA, M1, M2, NS1 and NS2 proteins obtained from influenza viruses.

[0134] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., any one or any combination of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and / or H18, or at least one of all of them), and at least three other RNA (e.g., saRNA) polynucleotides having three open reading frames encoding three proteins selected from HA, NP, NA, M, M2, NS1, and NS2 proteins obtained from influenza viruses.

[0135] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any combination or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and / or H18), and at least four other RNA (e.g., saRNA) polynucleotides having four open reading frames encoding four proteins selected from HA, NP, NA, M1, M2, NS1 and NS2 proteins obtained from influenza viruses.

[0136] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any combination or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and / or H18), and at least five other RNA (e.g., saRNA) polynucleotides having five open reading frames encoding five proteins selected from HA, NP, NA, M1, M2, NS1 and NS2 proteins obtained from influenza viruses.

[0137] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof obtained from an influenza virus (e.g., any one or any combination of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and / or H18, or at least one of all of them), an HA protein, 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.

[0138] In some embodiments, the influenza RNA composition comprises saRNA encoding an antigen fusion protein. Therefore, the encoded one or more antigens may comprise two or more proteins linked together (e.g., proteins and / or protein fragments). Alternatively, the protein fused to the protein antigen does not promote a strong immune response to itself, but rather promotes a strong immune response to the influenza virus antigen. In some embodiments, the antigen fusion protein retains the functional properties of each original protein.

[0139] F.5' cap

[0140] In some embodiments, the saRNA molecule described herein includes a 5' cap. In some embodiments, the 5'-cap portion is a natural 5'-cap.

[0141] A “natural 5’-cap” is defined as a cap containing a 7-methylguanosine nucleotide linked 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 analogue. In some embodiments, the 5’ end of the RNA is capped with a modified ribonucleotide having the structure m7G(5’)ppp(5’)N (cap0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered post-transcriptionally (e.g., post-transcriptional capping), where “N” is any ribonucleotide. In some embodiments, the 5’ end of the RNA molecule is capped with a modified ribonucleotide via a post-transcriptional enzymatic reaction. In some embodiments, capping is performed after RNA molecule purification (e.g., tangential flow filtration). Exemplary enzymatic reactions for capping may include the use of vaccinia virus capping enzyme (VCE), which comprises an mRNA triphosphatase, guanylate transferase, and guanine-7-methyltransferase, catalyzing the construction of an N7-monomethylated cap 0 structure. The cap 0 structure can help maintain the stability and translation efficiency of the RNA molecule. The 5'-cap of the RNA molecule can be further modified by a 2'-O-methyltransferase to generate a cap 1 structure (m7Gppp[m2'-O]N), which can further improve translation efficiency. In some embodiments, the RNA molecule can be enzymatically capped at the 5' end using vaccinia virus guanylate transferase, guanosine triphosphate, and S-adenosyl-L-methionine to produce the cap 0 structure. A reverse 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, a cap 1 structure is obtained using a 2'-O-methyltransferase and vaccinia virus guanylate transferase, wherein, in addition to the cap 0 structure, the 2'OH group is methylated on the penultimate nucleotide. S-Adenosyl-L-methionine (SAM) is a cofactor used as a methyltransfer agent. Non-limiting examples of 5′ caps are those structures that, compared to synthetic 5′ caps (or wild-type, natural, or physiological 5′ caps) known in the art, exhibit enhanced binding to cap-binding peptides, increased half-life, decreased 5′ endonuclease sensitivity, and / or reduced 5′ uncapping. For example, recombinant vaccinia virus capping enzymes and recombinant 2′-O-methyltransferases can establish a typical 5′-5′-triphosphate bond between the 5′ terminal nucleotide of mRNA and the guanine cap nucleotide, wherein the guanine cap contains N7 methylation, and the 5′ terminal nucleotide of mRNA contains 2′-O-methylation. This structure is referred to as the cap 1 structure. Compared to other 5′ cap analog structures known in the art, such as those mentioned above, this cap structure improves translational efficiency and cell stability, and reduces the activation of pro-inflammatory cytokines. The cap structure includes, but is not limited to, 7mG(5′)ppp(5′)N, pN2p (cap 0) and 7mG(5′)ppp(5′)N1mpNp (cap 1).Cap 0 is an N7-methylguanosine linked to a 5′ nucleotide via a 5′ to 5′ triphosphate bond, commonly referred to as m7G cap or m7Gppp. In cells, the cap 0 structure can facilitate efficient translation of the mRNA carrying this cap. Additional methylation at the 2′O site of the starting nucleotide generates cap 1, or m7GpppNm-, where Nm represents any nucleotide with 2′O methylation. In some embodiments, the 5′ cap comprises a cap analog, for example, a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methylguanosine, 2′-fluoroguanosine, 7-deazoguanosine, 8-oxoguanosine, 2-aminoguanosine, LNAguanosine, and 2-azidoguanosine. In some embodiments, the capping region may contain a single cap or a series of nucleotides forming the cap. In this embodiment, the length of the capped region can be equal to any one, at least one, at most one, or between any two of the following nucleotides: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or at least two, ten, or fewer nucleotides. In some embodiments, there is no cap. In some implementations, the length of the first and second operating regions may be equal to any one, at least one, at most one, or between any two of 3 to 40 nucleotides, for example, 5-30, 10-20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides, or at least 4 or 30 or fewer nucleotides, and may also include one or more signal and / or restriction sequences in addition to start and / or stop codons.

[0142] In some implementations, the 5' cap is represented by formula I:

[0143]

[0144] Where R 1 and R 2 Each is independently H or Me, and B 1 and B 2 Each is independently guanine, adenine, or uracil. In some embodiments, B... 1 and B 2 R is a naturally occurring base. In some implementations, R... 1 It is methyl, and R 2 It is hydrogen. In some implementations, B 1 It is guanine. In some implementations, B... 1 It is adenine. In some implementations, B 2 It is adenine. In some implementations, B2 It is uracil. In some implementations, B 2 It is uracil, and the B in the molecule 2 At least 5% of the total downstream uracil nucleotide population has been replaced by one or more modified or non-natural nucleotides.

[0145] In some embodiments, the nucleotide immediately downstream of the 5' cap (in the 5' to 3' direction) contains guanine. In some embodiments, B... 1 It is adenine, B 2 It is uracil. In some implementations, B 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 It is hydrogen. In some cases, saRNA does not contain a 5' cap. In some cases, the 5' cap is not represented by Formula I. In some embodiments, the nucleotide immediately downstream (5' to 3' direction) of the 5' cap contains guanine, B. 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 It is hydrogen; this embodiment corresponds to CleanCap AU, and in some embodiments, it contains B. 2 =Uracil, while optionally replacing B 2 Downstream uracil nucleotides have been shown to improve the functionality of saRNA. In some embodiments, the RNA molecule further comprises: (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the RNA molecule encodes at least one antigen. In some embodiments, the RNA molecule comprises at least 7000 nucleotides. In some embodiments, the RNA molecule comprises at least 8000 nucleotides. In some embodiments, at least 80% of the total RNA molecule is full-length. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus. In some embodiments, the alphavirus is Semliki forest virus.

[0146] In some implementations, the nucleotide immediately downstream of the 5' cap (5' to 3') includes guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 The molecule is hydrogen-rich, and at least 50% of the total population of uridine nucleotides has been replaced by N1-methylpseudouridine, and substantially all cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, the nucleotides immediately downstream of the 5' cap (5' to 3') include guanine, B... 1 It is adenine, B 2For uracil, R 1 It is methyl, R 2 The molecule is hydrogen-rich, and at least 50% of the total population of uracil nucleotides in the molecule has been replaced by 5-methoxyuracil, and substantially all cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') includes guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 The molecule is hydrogen-rich, and at least 50% of the total population of uracil nucleotides in the molecule has been replaced by 5-methyluracil, and essentially all cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') includes guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 The uridine nucleotides in the molecule are essentially all replaced by hydrogen, with approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudouridine. In some embodiments, the nucleotides immediately downstream of the 5' cap (5' to 3') include guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 The uridine nucleotides in the molecule are essentially all replaced by hydrogen, with approximately 75% being 5-methoxyuridine and approximately 25% being N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') includes guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl, and R 2 The uridine nucleotides in the molecule are replaced by hydrogen, with approximately 25% of 5-methoxyuridine and approximately 75% of N1-methylpseuuridine.

[0147] In some embodiments, the 5' cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap comprises:

[0148]

[0149] In some implementations, the 5' cap contains capping material for mRNA co-transcription. Reagent AG(3'OMe), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG,

[0150]

[0151] In an alternative implementation, the 5' cap contains components for self-amplifying mRNA. Capping for mRNA co-transcription Reagent AU, m7G(5')ppp(5')(2'OMeA)pU,

[0152]

[0153] G. Polyadenylate tail

[0154] As used herein, a “polyadenylated tail” refers to a continuous adenine residue that can be attached to the 3' end of an RNA molecule. The polyadenylated tail can extend the half-life of the RNA molecule. It can play a key regulatory role in improving translation efficiency and modulating the efficiency of mRNA quality control and degradation. Short sequences or high polyadenylation can signal RNA degradation. Exemplary designs include a polyadenylated tail of approximately 40 to approximately 80 adenine residues. In some embodiments, the RNA molecule also includes a nuclease recognition site sequence immediately downstream of the polyadenylated tail sequence. In some embodiments, such as for second RNA or saRNA molecules, the RNA molecule also includes a polyadenylated polymerase recognition sequence (e.g., AAUAAA) near its 3' end. A “full-length” RNA molecule refers to an RNA molecule containing a 5' cap and a polyadenylated tail.

[0155] In some implementations, the length of the polyadenylated tail comprises 5-400 nucleotides. The length of the polyadenylated tail nucleotide can be equal to any one, at least one, at most 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 polyadenylated tail comprising a sequence of about 25 to about 400 adenosine nucleotides, about 50 to about 400 adenosine nucleotides, about 50 to about 300 adenosine nucleotides, about 50 to about 250 adenosine nucleotides, about 60 to about 250 adenosine nucleotides, or about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule includes a polyadenylated tail comprising a sequence of more than 30 adenosine nucleotides (“A”). In some embodiments, the RNA molecule includes a polyadenylated tail comprising about 40 A's. In some embodiments, the RNA molecule includes a polyadenylated tail comprising about 80 A's. The term “about” as used herein refers to ±10% deviation of the value to which it is attached. In some embodiments, the 3' polyadenylated tail has a fragment 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.

[0156] H. Composition

[0157] In some cases, the compositions described herein comprise 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 virus antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response against an influenza virus).

[0158] In some embodiments, any one, at least one, at most one, or between any two of the following percentages of the total RNA molecules (capped and uncapped) in the composition are capped: 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%.

[0159] In some embodiments, any one, at least one, at most one, or between any two of the following total RNA molecules in the composition are full-length RNA transcripts: 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%. Purity can be determined as described herein, for example by reversed-phase HPLC or Bioanalyzer-based electrophoresis, and measured by, for example, the ratio of the peak area of ​​full-length RNA molecules to the total peak area. In some implementations, a fragment analyzer (FA) can be used to quantify and purify RNA. The fragment analyzer automates capillary electrophoresis and HPLC.

[0160] In some embodiments, the composition is substantially free of one or more impurities or contaminants, including linear DNA templates and / or reverse transcription products, and for example includes RNA molecules with a purity of any one, at least one, at most one, or between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; RNA molecules with a purity of at least 98% or at least 99%.

[0161] In some embodiments, the composition comprises a first RNA molecule in a greater amount than a second RNA molecule. In some embodiments, the composition comprises a first RNA molecule in an amount at least about 1 to 2 times the amount of the second RNA molecule. In some embodiments, the composition comprises a first RNA molecule in an amount at least about 1 to 100 times the amount of the second RNA molecule.

[0162] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable mediator.

[0163] In some embodiments, the composition further comprises a lipid-based delivery system that delivers RNA molecules into the cell, where they can replicate and / or express a target polypeptide. The delivery system may have an adjuvant effect, enhancing the immunogenicity of the encoded antigen. In some embodiments, the composition further comprises neutral lipids, cationic lipids, cholesterol, and polyethylene glycol (PEG), forming nanoparticles encapsulating RNA molecules. In some embodiments, the composition further comprises any one of cationic lipids, liposomes, lipid nanoparticles, polymer complexes, helical structures, virions, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, chylosomes, polycationic peptides, and cationic nanoemulsions. In some embodiments, the RNA molecule is encapsulated, bound to, or adsorbed onto any one or a combination of cationic lipids, liposomes, lipid nanoparticles, polymer complexes, helical structures, virions, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, chylosomes, polycationic peptides, and cationic nanoemulsions.

[0164] In some cases, the compositions described herein comprise at least two RNA molecules: a first saRNA molecule and a second RNA molecule as described herein. For protection against more than one influenza virus strain, a combined vaccine composition may be administered comprising RNA (e.g., saRNA) encoding at least one antigenic polypeptide protein (or an antigenic moiety thereof) of a first influenza virus or organism, and a second RNA molecule encoding at least one antigenic polypeptide protein (or an antigenic moiety thereof) of a second influenza virus or organism. The RNA (e.g., saRNA) may be co-formulated, for example, co-formulated in a single lipid nanoparticle (LNP), or may be formulated in separate LNPs for co-administration.

[0165] In some embodiments, the second RNA molecule comprises any one or any combination thereof of a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a polyadenylated nucleotide sequence. In some embodiments, the second RNA molecule comprises a 5' cap portion. In some embodiments, the second RNA molecule comprises a 5' UTR and a 3' UTR. In some embodiments, the second RNA molecule comprises a 5' UTR, an open reading frame, and a 3' UTR, but does not comprise a 5' cap. In some embodiments, the second RNA molecule comprises a 5' cap portion, a 5' UTR, a coding region, a 3' UTR, and a 3' polyadenylated nucleotide sequence. In some embodiments, the second RNA molecule comprises a 5' cap portion, a 5' UTR, a non-coding region, a 3' UTR, and a 3' polyadenylated nucleotide sequence. In some embodiments, the second RNA molecule comprises a non-coding region and does not further comprise any one of a 5' cap portion, a 5' UTR, a 3' UTR, and a 3' polyadenylated nucleotide sequence. In some implementations, the second RNA molecule comprises a 5' cap portion, a 5' untranslated region (5'UTR), modified nucleotides, an open reading frame, a 3' untranslated region (3'UTR), and a 3' polyadenylate sequence.

[0166] Some aspects of this disclosure relate to a composition comprising: (i) a first RNA molecule encoding a target gene derived from an influenza virus; and (ii) a second RNA molecule comprising modified or non-natural nucleotides. In some cases, the first RNA molecule is any of the saRNA molecules described herein. In some cases, the first RNA molecule comprises a 5' cap, a 5' untranslated region, a region encoding a non-structural protein containing RNA replicase, a subgenomic promoter, an open reading frame encoding the target gene, a 3' untranslated region, and a 3' polyadenylated nucleotide sequence. In some cases, at least 5% of a specific total nucleotide population in the first RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some cases, the saRNA molecule comprises natural, unmodified nucleotides and does not contain modified or non-natural nucleotides. In some cases, the 5' cap is represented by Formula I, wherein R 1 and R 2 Each independently represents H or Me, B 1 and B 2 Each of these components independently represents guanine, adenine, or uracil, a 5' untranslated region, a coding region for a non-structural protein derived from an alphavirus, a subgenomic promoter (such as an alphavirus-derived promoter), an open reading frame encoding the target gene, a 3' untranslated region, and a 3' polyadenylate sequence. In some embodiments, B... 1 and B 2 It is a naturally occurring base. In some implementations, R... 1 It is methyl, R 2It is hydrogen. In some embodiments, B1 is guanine. In some embodiments, B... 1 It is adenine. In some implementations, B 2 It is adenine. In some implementations, B 2 It is uracil. In some embodiments, the nucleotide immediately downstream of the 5' cap (in the 5' to 3' direction) contains guanine.

[0167] In some implementation schemes, B 1 It is adenine, B 2 It is uracil. In some implementations, B 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 It is hydrogen. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B. 1 It is adenine, B 2 For uracil, R 1 It is methyl, R 2 It is hydrogen; this embodiment corresponds to CLEANCAP AU(Trilink) and contains B. 2 =Uracil, with optional substitution of B 2 Downstream uracil nucleotides have been shown in some implementations to provide enhanced saRNA functionality.

[0168] In some embodiments, at least 10% of the total population of a specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the total population of a specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the total population of a specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the total population of a specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, substantially all of the total population of a specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or unnatural nucleotides. In some embodiments, the one or more modified or unnatural substituted nucleotides comprise two modified or unnatural nucleotides provided in a ratio of 1:99 to 99:1 or any range thereof. In some embodiments, at least 10% of the total population of a first specific nucleotide in the first or second RNA molecule has been 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 has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 25% of the total population of the second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of the second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotide in the first or second RNA molecule has been 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 has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of a second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 25% of the total population of a first specific nucleotide in the first or second RNA molecule has been 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 has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of a first specific nucleotide in the first or second RNA molecule has been 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 has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of a first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of a second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of a first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of a second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of a first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of a second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of a first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of a second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of a first specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of a second specific nucleotide in the first or second RNA molecule has been replaced by one or more modified or non-natural nucleotides.

[0169] In some embodiments, at least 25% of the total uridine nucleotide population in the first RNA molecule has been replaced by N1-methylpseudoruridine. In some embodiments, at least 50% of the total uridine nucleotide population in the first RNA molecule has been replaced by N1-methylpseudoruridine. In some embodiments, at least 75% of the total uridine nucleotide population in the first RNA molecule has been replaced by N1-methylpseudoruridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseudoruridine. In some embodiments, at least 50% of the total uridine nucleotide population in the first RNA molecule has been replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by 5-methoxyuridine. In some embodiments, at least 50% of the total uridine nucleotide population in the first RNA molecule has been replaced by 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by 5-methyluridine. In some embodiments, at least 50% of the total cytosine nucleotide population in the first RNA molecule has been replaced by 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the first RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule has been replaced by 2-thiouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by 2-thiouridine.

[0170] In some embodiments, at least 25% of the total uridine nucleotide population in the second RNA molecule has been replaced by N1-methylpseudoruridine. In some embodiments, at least 50% of the total uridine nucleotide population in the second RNA molecule has been replaced by N1-methylpseudoruridine. In some embodiments, at least 75% of the total uridine nucleotide population in the second RNA molecule has been replaced by N1-methylpseudoruridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by N1-methylpseudoruridine. In some embodiments, at least 50% of the total uridine nucleotide population in the second RNA molecule has been replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by 5-methoxyuridine. In some embodiments, at least 50% of the total uridine nucleotide population in the second RNA molecule has been replaced by 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by 5-methyluridine. In some embodiments, at least 50% of the total cytosine nucleotide population in the second RNA molecule has been replaced by 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 2-thiouridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by 2-thiouridine.

[0171] In some embodiments, at least 50% of the total uridine nucleotide population in the second RNA molecule has been replaced by N1-methylpseudouridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total uridine nucleotide population in the second RNA molecule has been replaced by 5-methoxyuridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total uridine nucleotide population in the second RNA molecule has been replaced by 5-methyluridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine.

[0172] In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseuuridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseuuridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseuuridine.

[0173] In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseudoruris, and at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by N1-methylpseudoruris. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseudoruris, and substantially all uridine nucleotides in the second RNA molecule have been replaced by N1-methylpseudoruris. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseudoruris, and at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseudoruris, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 5-methyluridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some implementations, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseuuridine, and substantially all uridine nucleotides in the second RNA molecule have been replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseuuridine.

[0174] I. How to Use

[0175] The saRNA composition can be used to treat and / or prevent influenza viruses, strains, and isolates of various genotypes. Some embodiments provide methods for preventing or treating influenza virus infection, including administering any of the saRNA compositions 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 T-cell and B-cell responses. In some embodiments, the method of generating an antigen-specific immune response involves a single administration of the saRNA composition. In some embodiments, the saRNA composition is administered to the subject via intradermal injection, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.

[0176] In some implementations, RNA (e.g., saRNA) polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof of an influenza virus strain as an antigen.

[0177] Some aspects of this disclosure relate to a method of inducing an immune response in a subject, including administering an effective amount of the composition disclosed herein to a subject in need. Some aspects of this disclosure relate to a method of vaccinating a subject, including administering an effective amount of the composition disclosed herein to a subject in need. Some aspects of this disclosure relate to a method including administering an effective amount of the composition disclosed herein to a subject in need. In some embodiments, the compositions disclosed herein induce an immune response including an antibody response. In some embodiments, the compositions disclosed herein induce an immune response including a T-cell response.

[0178] Some embodiments of this disclosure provide methods for inducing an antigen-specific immune response in a subject, including administering an effective amount of any RNA (e.g., saRNA) composition provided herein to the subject to generate an antigen-specific immune response. In some embodiments, the RNA (e.g., saRNA) composition is an influenza vaccine. In some embodiments, the RNA (e.g., saRNA) composition is a combination vaccine (broad-spectrum influenza vaccine) comprising a combination of multiple influenza vaccines.

[0179] In some embodiments, the antigen-specific immune response includes a T-cell response or a B-cell response. In some embodiments, a method for generating an antigen-specific immune response includes administering to a subject a single dose (without a booster dose) of the influenza RNA (e.g., saRNA) composition disclosed herein. In some embodiments, the method further includes administering to the subject a second dose (booster dose) of the influenza RNA (e.g., saRNA) composition. Additional doses of the influenza RNA (e.g., saRNA) composition may be administered.

[0180] In some implementations, subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after receiving a first or second (boost) dose of the vaccine. Seroconversion refers to the period during which specific antibodies are produced and detectable in the blood. After seroconversion occurs, the virus can be detected by a blood antibody test. During infection or immunity, antigens enter the bloodstream, and the immune system begins to respond by producing antibodies. Before seroconversion, the antigens themselves may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. At any time after seroconversion, antibodies can be detected in the blood, indicating a previous or current infection.

[0181] In some embodiments, the influenza RNA (e.g., saRNA) composition is administered to the subject via intradermal injection, intramuscular injection, or intranasal administration. In some embodiments, the influenza RNA (e.g., saRNA) composition is administered to the subject via intramuscular injection.

[0182] Some embodiments of this disclosure provide methods for inducing an antigen-specific immune response in a subject, including administering an effective amount of an influenza RNA (e.g., saRNA) composition to the subject to generate an antigen-specific immune response. In some embodiments, the antigen-specific immune response in the subject can be determined by measuring the antibody titer (the titer of antibodies binding to influenza antigen peptides) after administering any of the influenza RNA (e.g., saRNA) compositions of this disclosure to the subject. In some embodiments, the anti-antigen peptide antibody titer generated in the subject increases by at least one log relative to the control. In some embodiments, the anti-antigen peptide antibody titer generated in the subject increases by 1-3 logs relative to the control.

[0183] In some embodiments, the titer of anti-antigen peptide antibodies produced by the subject is at least 2-fold higher than that of the control. In some embodiments, the titer of anti-antigen peptide antibodies produced by the subject is at least 5-fold higher than that of the control. In some embodiments, the titer of anti-antigen peptide antibodies produced by the subject is at least 10-fold higher than that of the control. In some embodiments, the titer of anti-antigen peptide antibodies produced by the subject is 2-10-fold higher than that of the control.

[0184] In some embodiments, the control is the anti-antigen polypeptide antibody titer produced by a subject who has not been administered the RNA (e.g., saRNA) composition of this disclosure. In some embodiments, the control is the anti-antigen polypeptide antibody titer produced by a subject who has been administered a live attenuated vaccine or an inactivated influenza virus, or wherein the control is the anti-antigen polypeptide antibody titer produced by a subject who has been administered a recombinant or purified influenza protein vaccine.

[0185] In some embodiments, the RNA (e.g., saRNA) composition is formulated in an effective amount to generate an antigen-specific immune response in a subject.

[0186] In some embodiments, the effective amount is a total dose of saRNA ranging from 1 μg to 1000 μg or from 1 μg to 100 μg. In some embodiments, the effective amount is a total dose of 30 μg. In some embodiments, the effective amount is a dose of 10 μg, administered to the subject twice. In some embodiments, the effective amount is a dose of 10 μg, administered to the subject twice. In some embodiments, the effective amount is a dose of 15 μg, administered to the subject twice. In some embodiments, the effective amount is a dose of 30 μg, administered to the subject twice.

[0187] In some embodiments, the method includes administering the saRNA composition described herein to a subject at a dose ranging from 10 μg / kg to 400 μg / kg. In some embodiments, the dosage of the 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, 1... The dosages are 0-200 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 via 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.

[0188] In some implementations, the subjects are approximately 5 years of age or younger. For example, the subjects may be approximately 1 year to approximately 5 years of age (e.g., approximately 1, 2, 3, 5, or 5 years old), or approximately 6 months to approximately 1 year of age (e.g., approximately 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months). In some implementations, the subjects are approximately 12 months of age or younger (e.g., 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month). In some implementations, the subjects are approximately 6 months of age or younger.

[0189] In some implementations, the subject is born at full term (e.g., approximately 37–42 weeks). In some implementations, the subject is born preterm, for example, at approximately 36 weeks of gestation or earlier (e.g., approximately 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 weeks). For example, the subject may be born at approximately 32 weeks of gestation or earlier. In some implementations, the subject is born preterm at approximately 32 to approximately 36 weeks of gestation. For such subjects, the RNA (e.g., mRNA) vaccine may be administered at a later stage of life, for example, at approximately 6 months to approximately 5 years of age or older.

[0190] In some implementations, the subjects are young people aged from about 20 to about 50 years old (e.g., about 20, 25, 30, 35, 40, 45 or 50 years old).

[0191] In some implementations, the subjects are elderly subjects who are about 60, about 70, or older (e.g., about 60, 65, 70, 75, 80, 85, or 90 years old).

[0192] In some implementations, the subject has been exposed to influenza (e.g., Chlamydia trachomatis); the subject has been infected with influenza (e.g., Chlamydia trachomatis); or the subject is at risk of being infected with influenza (e.g., Chlamydia trachomatis).

[0193] In some implementations, the subject has been exposed to beta coronavirus (e.g., SARS-CoV-2); the subject is infected with beta coronavirus (e.g., SARS-CoV-2); or the subject is at risk of infection with beta coronavirus (e.g., SARS-CoV-2).

[0194] In some implementations, the subject has received at least one dose of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2), such as those selected from... The subject must have received at least two doses of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2); or be receiving at least one dose of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2), selected from [the list of vaccines / treatments]. The vaccine may be one of the following: Pfizer-BioNTech COVID-19 vaccine, Moderna mRNA-1273 COVID-19 vaccine, or Janssen COVID-19 vaccine; or the subject is receiving an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2), for example, selected from [a specific vaccine / container / component]. Anyone who is at risk of infection with a beta coronavirus (e.g., SARS-CoV-2) and is concurrently or simultaneously or within 12-48 hours with any of the influenza-resistant immunogenic compositions disclosed herein.

[0195] In some implementations, the subjects have weakened immune function (impaired immune system, such as having an immune system disorder or autoimmune disease).

[0196] Several aspects of this disclosure provide saRNA compositions comprising one or more saRNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein said saRNA polynucleotide is present in a formulation for in vivo administration to a host, conferring antibody titers superior to acceptable percentages of serological protection against the first antigen (e.g., HA) in human subjects. In some embodiments, the antibody titers produced by the saRNA compositions of this disclosure are neutralizing antibody titers. In some embodiments, the neutralizing antibody titers are higher than those of protein vaccines. In other embodiments, the saRNA compositions produce neutralizing antibody titers higher than those of adjuvanted protein vaccines. In some further embodiments, the saRNA composition produces neutralizing antibody titers of 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. Neutralizing titers are typically expressed as the highest serum dilution required to reduce plaque number by 50%.

[0197] J. Nucleic Acids

[0198] In some embodiments, nucleic acid sequences can exist in various forms, such as: incorporation sequences or isolated fragments and recombinant vectors encoding polypeptides (such as one or both strands of an antigen or antibody, or fragments, derivatives, mutant proteins, or variants thereof); polynucleotides sufficient to serve as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting polynucleotide expression; and the aforementioned mRNA, saRNA, and complementary sequences. Nucleic acids encoding epitopes that antibodies can bind are also provided. Nucleic acids encoding fusion proteins containing these polypeptides are also provided. Nucleic acids can be single-stranded or double-stranded and can contain RNA and / or DNA nucleotides and their artificial variants (e.g., peptide nucleic acids).

[0199] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or has been isolated from the total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids with a length not exceeding 100 residues) and recombinant vectors, including, for example, plasmids, granules, bacteriophages, and viruses. In some respects, polynucleotides include regulatory sequences substantially separated from their naturally occurring gene or protein-coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic DNA, cDNA, or synthetic DNA), analogues thereof, or combinations thereof. Other coding or non-coding sequences may be present within a polynucleotide, but are not required to be.

[0200] In this regard, the term "gene" is used to refer to nucleic acids (including any sequence required for proper transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. Those skilled in the art will understand that the term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid fragments that express or can be modified to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or part of a polypeptide may comprise a continuous nucleic acid sequence encoding all or part of that polypeptide. Furthermore, it is also considered that a particular polypeptide may be encoded by a nucleic acid variant comprising a slightly different nucleic acid sequence but encoding the same or substantially similar polypeptide.

[0201] In some embodiments, there are polynucleotide variants that are substantially identical to the sequences disclosed herein; using the methods described herein (e.g., BLAST analysis using standard parameters), these variants, compared to the polynucleotide sequences provided herein, contain sequence identity equal to, at least, at most, or between any two of, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher. In some aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that is at least 90% identical to the amino acid sequence described herein over its entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that is at least 95% identical to the amino acid sequence described herein over its entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide.

[0202] In some embodiments, the polynucleotide comprises a 5' UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:12. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:13. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:14. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:15. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:16. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:17. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:18. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:19. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:20. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:21. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:22.

[0203] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:13; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:14; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:15; and a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:12; and ...2; and a polynucleotide sequence having at least NO:16 A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:17; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a ... NO: 21 is a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity; and a polyadenylate tail containing at least 20 consecutive adenines.

[0204] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:13; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:14; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:15; and a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:12; and ...2; and a polynucleotide sequence having at least NO:16 A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:17; A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:18; A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:19; A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:19; NO:20 is a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:21; and a polynucleotide sequence containing at least 20 consecutive adenine nucleotides.

[0205] In some embodiments, the polynucleotide comprises: a 5'UTR sequence having SEQ ID NO:12; a polynucleotide sequence having SEQ ID NO:13; a polynucleotide sequence having SEQ ID NO:14; a sequence having SEQ ID NO:15; a polynucleotide sequence having SEQ ID NO:16; a polynucleotide sequence having SEQ ID NO:17; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having SEQ ID NO:19; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having SEQ ID NO:21; and a polyadenylated tail comprising at least 20 consecutive adenine nucleotides.

[0206] In some embodiments, the polynucleotide comprises the following: a 5' UTR sequence having SEQ ID NO:12; a polynucleotide sequence having SEQ ID NO:13; a polynucleotide sequence having SEQ ID NO:14; a sequence having SEQ ID NO:15; a polynucleotide sequence having SEQ ID NO:16; a polynucleotide sequence having SEQ ID NO:17; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:18; a polynucleotide sequence having SEQ ID NO:19; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:20; a polynucleotide sequence having SEQ ID NO:21; and a polyadenylate tail comprising at least 20 consecutive adenine nucleotides.

[0207] In some embodiments, the polynucleotide comprises a 5' UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:23. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:24. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:25. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:26. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:27. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:28. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:29. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:30. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:31. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:32.

[0208] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:23; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:24; a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:25; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:26; and a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:23; and ...3; and a polynucleotide sequence having at least 7 NO:27 A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:29; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO:28 ... NO: 31 is a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity; and a polyadenylate tail containing at least 20 consecutive adenines.

[0209] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:23; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:24; a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:25; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:26; and a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:23; and ...3; and a polynucleotide sequence having at least 7 NO:27 is a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:29; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:30; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:27; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO:28 ... NO:31 is a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity; and a polyadenylate tail containing at least 20 consecutive adenines.

[0210] In some embodiments, the polynucleotide comprises a 5' UTR sequence SEQ ID NO: 23; a polynucleotide sequence having SEQ ID NO: 24; a polynucleotide sequence having SEQ ID NO: 25; a polynucleotide sequence having SEQ ID NO: 26; a polynucleotide sequence having SEQ ID NO: 27; a polynucleotide sequence having SEQ ID NO: 28; a polynucleotide sequence having SEQ ID NO: 29; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having SEQ ID NO: 31; and a polyadenylate tail comprising at least 20 consecutive adenine nucleotides.

[0211] In some embodiments, the polynucleotide comprises a 5' UTR sequence SEQ ID NO: 23; a polynucleotide sequence having SEQ ID NO: 24; a polynucleotide sequence having SEQ ID NO: 25; a polynucleotide sequence having SEQ ID NO: 26; a polynucleotide sequence having SEQ ID NO: 27; a polynucleotide sequence having SEQ ID NO: 28; a polynucleotide sequence having SEQ ID NO: 29; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO: 30; a polynucleotide sequence having SEQ ID NO: 31; and a polyadenylate tail comprising at least 20 consecutive adenine nucleotides.

[0212] Regardless of the length of the coding sequence itself, nucleic acid fragments can combine with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction endonuclease sites, multiple cloning sites, and other coding fragments, resulting in a wide variation in their total length. Nucleic acids can be of arbitrary length. Their length can be, for example, equal to any one, at least one, at most one, or between any two of the following: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides, and / or may contain one or more additional sequences, such as regulatory sequences, and / or may be part of a larger nucleic acid (e.g., a vector). Therefore, it is possible to use nucleic acid fragments of virtually any length, with the total length limited by the ease of preparation and its use in the intended recombinant nucleic acid protocol. In some cases, nucleic acid sequences can encode peptide sequences with additional heterologous coding sequences, for example, to allow for peptide purification, transport, secretion, post-translational modification, or for therapeutic benefits (such as targeting or efficacy). As described above, tags or other heterologous peptides can be added to the modified peptide coding sequence, where "heterologous" refers to a peptide different from the modified peptide.

[0213] As used herein, “modNS1” refers to a polynucleotide encoding the NS1 protein, wherein the polynucleotide contains N1-methylpseuuridine (m1ψ). In a preferred embodiment, the modNS1 comprises a 5' cap, a 5' UTR, a 3' UTR, and a polyadenylate tail.

[0214] K. Lipid delivery

[0215] In some embodiments, the saRNA composition comprises lipids. Lipids and saRNA can co-form nanoparticles. The lipids can encapsulate the mRNA in the form of lipid nanoparticles (LNPs) to facilitate the entry of the RNA / lipid nanoparticles into cells and maintain their stability.

[0216] Lipid nanoparticles may contain lipid components and one or more other components, such as therapeutic and / or preventative ingredients. LNPs can be designed for one or more specific applications or targets. The components 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 components. Similarly, specific formulations of LNPs can be selected for a specific application or target based, for example, the efficacy and toxicity of a specific combination of components. The efficacy and tolerability of LNP formulations can be affected by formulation stability.

[0217] Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and / or preventative substances (such as RNA) to specific cells, tissues, organs, systems, or groups thereof in mammals.

[0218] The physicochemical properties of lipid nanoparticles can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted according to the pore size of different organs. The therapeutic and / or preventative substances contained in the LNP can also be selected based on one or more desired delivery targets. For example, therapeutic and / or preventative substances can be selected for specific indications, conditions, diseases, or ailments and / or delivery to specific cells, tissues, organs, systems, or groups thereof (e.g., local or specific delivery). In some embodiments, the LNP may contain mRNA encoding a target polypeptide that can be translated intracellularly to produce the target polypeptide. Such compositions can be programmed for specific delivery to specific organs. In some embodiments, the composition can be programmed for specific delivery to the mammalian liver. In some embodiments, the composition can be programmed for specific delivery to lymph nodes. In some embodiments, the composition can be programmed for specific delivery to the mammalian spleen.

[0219] LNPs may comprise one or more of the components described herein. In some embodiments, the LNP formulations of this disclosure comprise at least one lipid nanoparticle component. The lipid nanoparticles may comprise a lipid component and one or more additional components, such as therapeutic and / or preventative substances, such as nucleic acids. LNPs may be designed for one or more specific applications or targets. The components of an LNP may 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 components. Similarly, specific formulations of LNPs may be selected for a specific application or target based on, for example, the efficacy and toxicity of a specific combination of components. The efficacy and tolerability of LNP formulations may be affected by formulation stability.

[0220] In some embodiments, for example, the polymer may be included in and / or used to encapsulate or partially encapsulate the LNP. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from (but is not limited to) polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrene, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. For example, polymers may include polycaprolactone (PCL), ethylene vinyl acetate polymer (EVA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), poly-L-lactic acid-glycolic acid copolymer (PLLGA), poly-D,L-lactide (PDLA), poly-L-lactide (PLLA), poly-D,L-lactide-caprolactone copolymer, poly-D,L-lactide-caprolactone-glycolic acid copolymer, poly-D,L-lactide-PEO-D,L-lactide copolymer, and poly-D,L- L-lactide-PPO-D, L-lactide copolymer, alkyl polycyanate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, polyhydroxy acid, polyanhydride, polyorthoester, polyesteramide, polyamide, polyester ether, polycarbonate, polyolefins such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol (PEG), polyalkylene oxide (PEO), polyalkylene terephthalates such as polyethylene terephthalate, polyvinyl alcohol (PVA), polyethylene ether, polyethylene esters such as polyvinyl acetate Esters, polyhalogenated ethylenes such as polyvinyl chloride (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derived celluloses such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic polymers such as poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(laurate methacrylate), poly(benzene methacrylate) Esters), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and their copolymers and mixtures, polydioxane and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, poloxamine, poly(orthoester), polybutyric acid, polyvaleric acid, poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), their derivatives, and polyglycerol.

[0221] Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, styrax, bromhexine, carboxymethylcysteine, ipramone, mesna, ambroxol, sobrorilol, domiocol, letostan, stipronin, thiopronin, gelling agents, thymosin β4, alfa streptocysteine, netticine, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be disposed within nanoparticles and / or on the surface of LNPs (e.g., through coating, adsorption, covalent bonding, or other processes).

[0222] LNPs may also contain one or more functionalized lipids. For example, lipids can be functionalized with alkyne groups, which can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. Specifically, lipid bilayers can be functionalized in this way with one or more groups that facilitate membrane permeation, cell recognition, or imaging. The surface of the LNP can also be conjugated to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0223] In addition to these components, lipid nanoparticles may also contain any substances that can be used in pharmaceutical compositions. For example, lipid nanoparticles may contain one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, surfactants, buffers, preservatives, and other substances.

[0224] Surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginate, sodium alginate, cholesterol, and lecithin) and sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate). Polyoxyethylene dehydrated sorbitol Polyoxyethylene dehydrated sorbitan monooleate Sorbitol monopalmitate Sorbitol monostearate Sorbitol Tristearate Glyceryl monooleate, sorbitan monooleate ), polyoxyethylene esters (e.g., polyoxyethylene monostearate) Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ether (e.g., polyoxyethylene lauryl ether) Polyvinylpyrrolidone, diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Hexadecyltrimethylammonium bromide, hexadecylpyridine chloride, benzalkonium chloride, sodium docusate, and / or combinations thereof.

[0225] Examples of preservatives may include, but are not limited to: antioxidants, chelating agents, free radical scavengers, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to: α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, thioglycerol, 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 ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, fumaric acid, malic acid, phosphoric acid, sodium ethylenediaminetetraacetate, tartaric acid, and / or trisodium ethylenediaminetetraacetate. Examples of antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzyl alcohol, bromonitrile glycol, cetrimonium bromide, hexadecylpyridine chloride, chlorhexidine, chlorobutanol, chlorocresol, xylene, cresol, ethanol, glycerin, hexamethylenediaminetetraacetic acid, imidazourea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric 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 alcohol preservatives include, but are not limited to: ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and / or phenethyl alcohol. Examples of acidic preservatives include, but are not limited to: vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to: tocopherol, tocopherol acetate, deoxyoxime mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, etc. Methylparaben, NEOLONE TM KATHON TM and / or Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

[0226] Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, aminosulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, Tris buffer and / or combinations thereof.

[0227] In some embodiments, formulations containing LNPs may also contain salts, such as chloride salts. In some embodiments, formulations containing LNPs may also contain sugars, such as disaccharides. In some embodiments, formulations also contain sugars but not salts, such as chloride salts. In some embodiments, LNPs may also contain one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogues).

[0228] The properties of a LNP may depend on its composition. For example, an LNP containing cholesterol as a structural lipid may have different properties than an LNP containing different structural lipids. As used herein, the term "structural lipid" refers to sterols, and also to lipids containing sterol moieties. "Sterol" as defined herein is the steroid subgroup 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 analogue of cholesterol. In some embodiments, the structural lipid is α-tocopherol.

[0229] In some embodiments, the properties of LNPs can depend on the absolute or relative amounts of their components. For example, an LNP containing a higher molar fraction of phospholipids can have different properties than an LNP containing a lower molar fraction of phospholipids. Properties can also vary depending on the preparation method and conditions of the lipid nanoparticles. Typically, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0230] The phospholipid portion may be selected from, for example, the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion may be selected from, for example, the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Specific phospholipids can promote membrane fusion. In some embodiments, cationic phospholipids can interact with one or more negatively charged phospholipids of the membrane (e.g., cell membrane or intracellular membrane). Phospholipid fusion with the membrane allows one or more components (e.g., therapeutic agents) of a lipid-containing composition (e.g., LNP) to cross the membrane, thereby allowing, for example, delivery of one or more components to a target tissue. Non-natural phospholipids, including modified and substituted natural species (including branched, oxidized, cyclized, and alkyne-based ones), are also considered. In some embodiments, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are substituted with triple bonds). Under suitable reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition reactions upon exposure to azides. Such reactions can be used to functionalize lipid bilayers of nanoparticle compositions to facilitate membrane permeation or cell recognition, or to conjugate nanoparticle compositions with useful components such as targeting or imaging components (e.g., dyes). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include sphingomyelins, such as neurosphingomyelin. In some embodiments, phospholipids useful or potentially useful in this invention are analogues or variants of DSPC.

[0231] Lipid nanoparticles (LNPs) can be characterized using a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and particle size distribution of LNPs. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure various properties of LNPs, such as particle size, polydispersity index, and zeta potential.

[0232] The average size of an LNP can range from tens of nanometers to hundreds of nanometers, for example, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the LNP can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average size of the LNP can be about 70 nm to about 100 nm. In one specific embodiment, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0233] LNPs can be relatively homogeneous. A polydispersity index can be used to indicate the homogeneity of an LNP, such as the particle size distribution of lipid nanoparticles. A smaller polydispersity index (e.g., less than 0.3) typically indicates a narrower particle size distribution. The polydispersity index of an LNP can be from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNP can be from about 0.10 to about 0.20.

[0234] The zeta potential of LNPs can be used to indicate the potential of the composition. For example, the zeta potential can describe the surface charge of the LNP. Lipid nanoparticles with relatively low charge (positive or negative) are often desirable because substances with higher charges may engage in undesirable interactions with cells, tissues, and other components of the body. In some implementations, the zeta potential of the LNP can be 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, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or from approximately +5mV to approximately +10mV.

[0235] The encapsulation efficiency of therapeutic and / or preventative substances describes the amount of therapeutic and / or preventative substances encapsulated or otherwise associated with LNPs after preparation, relative to the initially provided amount. A high encapsulation efficiency is desired (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or preventative substances in a solution containing lipid nanoparticles before and after degradation with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or preventative substances (e.g., RNA) in solution. For the lipid nanoparticles described herein, the encapsulation efficiency of therapeutic and / or preventative substances can be at least 50%, such as 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 can be at least 80%. In some implementations, the encapsulation efficiency can be at least 90%.

[0236] LNPs may optionally contain one or more layers of coating. For example, LNPs may be formulated into coated capsules, films, or tablets. Capsules, films, or tablets containing the compositions described herein may have any available size, tensile strength, hardness, or density.

[0237] Formulations comprising amphiphilic polymers and lipid nanoparticles can be formulated wholly or partially into pharmaceutical compositions. Pharmaceutical compositions 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, said lipid nanoparticles comprising one or more different therapeutic and / or preventative substances. Pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein. General guidance on the formulation and manufacture of pharmaceutical compositions and formulations can be found, for example, Remington's *The Science and Practice of Pharmacy*, 21st Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjuvants may be used in any pharmaceutical composition unless any conventional excipient or adjuvant may be incompatible with one or more components of LNP or one or more amphiphilic polymers in the formulations disclosed herein. If the combination of an excipient or auxiliary component with the amphiphilic polymer of a component or formulation of LNP may result in any adverse biological or other harmful effects, then the excipient or auxiliary component may be incompatible with the amphiphilic polymer of the component or formulation of LNP.

[0238] In some embodiments, one or more excipients or adjuvants may constitute more than 50% by mass or volume of the total pharmaceutical composition comprising LNP. For example, one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipients have a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipients are approved for human and veterinary use. 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), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the 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 other components in the pharmaceutical compositions disclosed herein will vary depending on the identity, size, and / or condition of the treated subject, and also depend on the route of administration of the composition. For example, a pharmaceutical composition may comprise 0.1% to 100% (by weight) one or more lipid nanoparticles. As another example, a pharmaceutical composition may comprise 0.1% to 15% (by weight) one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / v).

[0239] In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions of this disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures of 4°C or lower, such as temperatures between about -150°C and about 0°C, or temperatures between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C). For example, pharmaceutical compositions comprising one or more amphiphilic polymers and one or more lipid nanoparticles are solutions or solids (e.g., via lyophilization) that are refrigerated for use. Storage and / or transportation at, for example, about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In some embodiments, this disclosure also relates to a method for increasing the stability of lipid nanoparticles by adding an effective amount of an amphiphilic polymer and storing the lipid nanoparticles and / or their pharmaceutical compositions at temperatures of 4°C or lower (such as temperatures from about -150°C to about 0°C or from about -80°C to 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).

[0240] In some embodiments, the lipid component of the LNP includes cationic lipids, phospholipids, PEG lipids, and structural lipids. In some embodiments, the lipid component of the lipid nanoparticles includes about 30 mol% to about 60 mol% cationic lipids, about 0 mol% to about 30 mol% phospholipids, about 18.5 mol% to about 48.5 mol% structural lipids, and about 0 mol% to about 10 mol% PEG lipids, but the total mol% does not exceed 100%. In some embodiments, the lipid component of the lipid nanoparticles comprises about 35 mol% to about 55 mol% cationic lipid compounds, about 5 mol% to about 25 mol% phospholipids, about 30 mol% to about 40 mol% structural lipids, and about 0 mol% to about 10 mol% PEG lipids. In one specific embodiment, the lipid component comprises about 50 mol% of the cationic lipids, about 10 mol% phospholipids, about 38.5 mol% structural lipids, and about 1.5 mol% PEG lipids. In another embodiment, the lipid component 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.

[0241] In some implementations, the ionizable lipid is a compound of formula (I):

[0242]

[0243] Or its N-oxide, or its salt or isomer, wherein: Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" and -R"M'R"; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2 and unsubstituted C1-6 alkyl, wherein Q is selected from carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, - OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R )S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)Re, N(R)S(O)2R8, -O( CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2J-N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2 The following are given: -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(O)OR, where each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R')-, and -N (R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl and heteroaryl, wherein M” is a bond, C1-13 alkyl or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; Re is selected from the group consisting of C3-6 carbon ring and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbon ring and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H;Each R' is independently selected from the group consisting of C1-1s alkyl, C2-1s alkenyl, -R*YR", -YR", and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbon ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) when n is 1 or 2, Q is not a 5, 6, or 7-membered heterocyclic alkyl group. In some embodiments, the ionizable lipid is: ;

[0244]

[0245] In some embodiments, the compound has the following structure (I):

[0246]

[0247] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: one of L1 or L2 is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x—, —S—S—, —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—, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═ ...x—, SC(═O)x—, —NRaC(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O)x—, —C(═O R1 is an unsubstituted C1-C12 alkylene group or a C1-C12 alkenyl group; R2 is a C1-C24 alkylene group, a C1-C24 alkenyl group, a C3-C8 cycloalkylene group, or a C3-C8 cycloalkylene group; R3 is H or a C1-C12 alkyl group; R4 is a C1-C12 alkyl group; R5 is H or a C1-C6 alkyl group; and x is 0, 1, or 2. In a preferred embodiment, the ionizable lipid is:

[0248]

[0249] An asterisk (*) indicates a chiral center.

[0250] The lipid component of the lipid nanoparticle composition may contain one or more polyethylene glycol-containing molecules, such as PEG or PEG-modified lipids. Such substances are also referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from the 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 a polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropyl-3-amine. Such lipids are also referred to as PEGylated lipids. In some embodiments, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. In some embodiments, the PEG-modified lipid is a modified form of PEG DMG. In some embodiments, the PEG-modified lipid is a PEG lipid having formula (IV):

[0251]

[0252] R8 and R9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and the average value of w ranges from 30 to 60.

[0253] L. formulation

[0254] In one aspect, this disclosure relates to an immunogenic composition comprising: (i) a first RNA polynucleotide having an open reading frame encoding a first antigen, the first antigen comprising at least one influenza virus antigenic peptide or an immunogenic fragment thereof; and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen, the second antigen comprising at least one influenza virus antigenic peptide or an immunogenic fragment thereof, wherein the first RNA molecule and the second RNA polynucleotide 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 from an influenza virus subtype different from the influenza virus antigenic peptide or immunogenic fragment thereof of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen comprising at least one influenza virus antigenic peptide or immunogenic fragment thereof, wherein the third antigen is derived from an influenza virus, but from an influenza virus strain different from the first and second antigens. In some embodiments, the first, second, and third RNA polynucleotides are formulated in lipid nanoparticles.

[0255] In some embodiments, the composition further comprises (iv) a fourth RNA polynucleotide having an open reading frame encoding a fourth antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the fourth antigen is derived from an influenza virus strain different from the first, second, and third antigens. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in lipid nanoparticles.

[0256] In some embodiments, RNA polynucleotides are mixed in a single container at a desired ratio and then formulated into lipid nanoparticles. In some embodiments, different RNA polynucleotides are initially input and formulated in a single LNP process at a known ratio to produce an LNP encapsulating the different RNA polynucleotides at approximately the same ratio as the input ratio. Such embodiments may be referred to herein as “premixed”. Thus, in some embodiments, first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, first, second, third, and fourth RNA polynucleotides are formulated in a single LNP. In some embodiments, first, second, third, fourth, and fifth RNA polynucleotides are formulated in a single LNP. In some embodiments, first, second, third, fourth, fifth, and sixth RNA polynucleotides are formulated in a single LNP. In some embodiments, first, second, third, fourth, fifth, sixth, and seventh RNA polynucleotides are formulated in a single LNP. In some embodiments, first, second, third, fourth, fifth, sixth, seventh, and eighth RNA polynucleotides are formulated in a single LNP.

[0257] In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the RNA polynucleotide mixture 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, about 5:1, about 10:1, about 25:1, or about 50:1 before being formulated into the LNP. In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide is greater than 1:1.

[0258] In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the RNA polynucleotide mixture before formulation into 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 RNA polynucleotide to the third RNA polynucleotide is greater than 1:1.

[0259] In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide in the RNA polynucleotide mixture before formulation into 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, about 5:1, about 10:1, about 25:1, or about 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 mixture before formulation into 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 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 mixture before formulation into 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, about 5:1, about 10:1, about 25:1, or about 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 mixture before formulation into 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, about 5:1, about 10:1, about 25:1, or about 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 mixture before formulation into 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, about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide is greater than 1:1.

[0260] In an alternative implementation, each RNA polynucleotide encoding a specific antigen is formulated in a separate LNP such that each LNP encapsulates an RNA polynucleotide encoding the same antigen. Such implementations may be referred to herein as “post-mixing.” Thus, in some implementations, a first RNA polynucleotide is formulated in a first LNP; a second RNA polynucleotide is formulated in a second LNP; a third RNA polynucleotide is formulated in a third LNP; a fourth RNA polynucleotide is formulated in a fourth LNP; a fifth RNA polynucleotide is formulated in a fifth LNP; a sixth RNA polynucleotide is formulated in a sixth LNP; a seventh RNA polynucleotide is formulated in a seventh LNP; and an eighth RNA polynucleotide is formulated in an eighth LNP.

[0261] In some embodiments, the molar ratio of the first LNP to the second LNP in the LNP mixture before formulation into 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.

[0262] In some embodiments, the molar ratio of the first LNP to the third LNP in the LNP mixture before formulation into 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.

[0263] In some embodiments, before being incorporated into the LNP, the molar ratio of the first LNP to the fourth LNP in the LNP mixture 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, before being incorporated into the LNP, the molar ratio of the first LNP to the fifth LNP in the LNP mixture 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, before being formulated into the LNPs, the molar ratio of the first LNP to the sixth LNP in the LNP mixture 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, before being incorporated into the LNP, the molar ratio of the first LNP to the seventh LNP in the LNP mixture 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, before being incorporated into the LNP, the molar ratio of the first LNP to the eighth LNP in the LNP mixture 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 implementations, the molar ratio of the first LNP to the eighth LNP is greater than 1:1.

[0264] In some implementations, the relative amount of RNA encoding influenza B virus antigens may be increased compared to RNA encoding influenza A virus (e.g., including a higher neutralizing titer immune response against influenza B virus (e.g., a higher neutralizing titer compared to a composition containing equal amounts of RNA encoding influenza A antigens and RNA encoding influenza B antigens (e.g., determined by the pseudovirus neutralization assay described herein))). This disclosure also provides exemplary doses of RNA capable of generating a strong immune response against both types of influenza viruses (e.g., clinically relevant neutralizing titers and / or seroconversion rates, e.g., (i) comparable to or higher than neutralizing titers previously shown to prevent influenza symptoms, and / or (ii) comparable to or higher than neutralizing titers and / or seroconversion rates induced by relevant comparatives (e.g., commercially approved influenza vaccines or influenza RNA vaccines)). In some embodiments, compositions containing a larger amount of RNA encoding influenza B antigens generate immune responses against both influenza B and influenza A viruses that are comparable to or better than those induced by non-RNA influenza vaccines (e.g., approved vaccines) and / or RNA vaccines containing equal amounts of RNA encoding influenza A antigens and RNA encoding influenza B antigens.

[0265] In some embodiments, the concentration of RNA in the drug RNA preparation is about 0.1-0.2 mg / ml. In some embodiments, the concentration of RNA in the drug RNA preparation is about 0.1 mg / ml. In some embodiments, the concentration of RNA in the drug RNA preparation is about 0.12 mg / ml. In some embodiments, the concentration of RNA in the drug RNA preparation is about 0.14 mg / ml. In some embodiments, the concentration of RNA in the drug RNA preparation is about 0.16 mg / ml. In some embodiments, the concentration of RNA in the drug RNA preparation is about 0.18 mg / ml. In some embodiments, about 30 μg of RNA is administered by administering about 200 μL of the RNA preparation. In some embodiments, the RNA in the drug RNA preparation is diluted prior to administration (e.g., diluted to a concentration of about 0.05 mg / ml). In some embodiments, the administration volume is between about 200 μl and about 300 μl. In some embodiments, the RNA in the drug RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

[0266] In some embodiments, the drug RNA formulation comprises RNA at a concentration of about 0.1 mg / ml, formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the drug RNA formulation comprises RNA at a concentration of about 0.12 mg / ml, formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the drug RNA formulation comprises RNA at a concentration of about 0.14 mg / ml, formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the drug RNA formulation comprises RNA at a concentration of about 0.16 mg / ml, formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the drug RNA formulation comprises RNA at a concentration of about 0.18 mg / ml, formulated in about 10 mM Tris buffer and about 10% sucrose. Such formulations can be diluted as needed before administration to administer different doses of RNA while maintaining a relatively constant total injection volume. For example, a dose of about 10 μg of RNA can be administered by diluting such drug RNA preparations at a ratio of about 1:1 and applying about 200 μl of the diluted drug RNA preparation.

[0267] In some implementations, the vaccine is prepared in vials (e.g., glass vials). In some implementations, the glass vials are sealed with bromobutyl elastic stoppers and aluminum seals with flip-top plastic caps.

[0268] In some embodiments, the composition comprises RNA encoding an influenza virus antigen (e.g., HA protein) recommended by the relevant health authority for inclusion in seasonally adapted vaccines (e.g., cell-based, recombinant, or attenuated live virus). In some embodiments, the composition comprises multiple RNAs encoding antigens (e.g., HA proteins) of each influenza virus recommended by the relevant health authority for inclusion in seasonally adapted vaccines (e.g., cell-based, recombinant, or attenuated live virus). In some embodiments, the influenza virus is an influenza A virus, an influenza B virus, or an influenza C virus. In some embodiments, the influenza A virus is H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, or H10N8 virus. In some embodiments, the influenza A virus is H1N1, H3N2, H5N1, or H5N8. In some embodiments, the influenza A virus is H1N1 (e.g., A / Wisconsin / 588 / 2019 or A / Sydney / 5 / 2021). In some embodiments, the influenza A virus is H3N2. In some embodiments, the H3N2 virus is A / Cambodia / e0826360 / 2020 or A / Darwin / 6 / 2021. In some embodiments, the influenza B virus belongs to the B / Yamagata or B / Victoria lineage. In some embodiments, the B / Victoria lineage influenza virus is B / Washington / 02 / 2019. In some embodiments, the B / Victoria lineage virus is B / Austria / 1359417 / 2021. In some implementations, the B / Yamagata lineage influenza virus is designated as B / Phuket / 3073 / 2013.

[0269] In some embodiments, the compositions described herein comprise a multivalent influenza vaccine. In some embodiments, the multivalent influenza vaccine comprises 2 to 50 different RNA molecules (e.g., 2 to 40, 2 to 30, or 2 to 20 RNA molecules), and in some embodiments, each RNA molecule may encode a different influenza-associated antigenic polypeptide (or a different form of a specific antigenic polypeptide), for example, as described in Arevalo, Claudia P. et al., "A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes." Science 378.6622(2022):899-904. In some embodiments, the compositions described herein comprise a trivalent influenza vaccine. In some embodiments, the trivalent influenza vaccine comprises RNA encoding antigenic polypeptides associated with two influenza A viruses and one influenza B virus expected to circulate in the relevant jurisdiction. In some embodiments, the compositions described herein comprise a quadrivalent influenza vaccine. In some embodiments, the quadrivalent influenza vaccine comprises RNA encoding antigenic polypeptides associated with two influenza A viruses and two influenza B viruses expected to circulate in the relevant jurisdiction. In some embodiments, the compositions described herein comprise an octavalent influenza vaccine. In some embodiments, the octavalent influenza vaccine comprises RNA (e.g., HA and NA proteins, or immunogenic fragments thereof, associated with each of the two influenza A and two influenza B viruses expected to circulate in the relevant jurisdiction) encoding two antigenic polypeptides. In some embodiments, the compositions disclosed herein comprise a quadrivalent influenza vaccine comprising RNA containing nucleotide sequences encoding HA proteins associated with H1N1 virus (e.g., A / Wisconsin / 588 / 2019), RNA containing nucleotide sequences encoding HA proteins associated with H3N2 virus (e.g., A / Cambodia / e0826360 / 2020), RNA containing nucleotide sequences encoding HA proteins associated with B / Victoria lineage influenza virus (e.g., B / Washington / 02 / 2019), and RNA containing nucleotide sequences encoding HA proteins associated with B / Yamagata lineage influenza virus (e.g., B / Phuket / 3073 / 2013).

[0270] In some embodiments, the composition comprises a quadrivalent influenza vaccine containing RNA encoding antigenic peptides associated with two influenza A viruses and two influenza B viruses expected to circulate in the relevant jurisdiction. In some embodiments, the quadrivalent influenza vaccine contains RNA encoding an antigenic peptide associated with H1N1 influenza virus, an antigenic peptide associated with H3N2 influenza virus, an antigenic peptide associated with Victoria lineage influenza virus, and an antigenic peptide associated with Yamagata lineage influenza virus. In some embodiments, the quadrivalent influenza vaccine contains RNA associated with the influenza type expected to circulate in the relevant jurisdiction (e.g., HA peptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses expected to circulate in the relevant jurisdiction).

[0271] In some embodiments, the composition comprises an octavalent influenza vaccine containing RNA encoding antigenic peptides associated with two influenza A viruses and two influenza B viruses expected to circulate in the relevant jurisdiction. In some embodiments, the octavalent influenza vaccine contains RNA encoding an antigenic peptide derived from influenza A virus HA, RNA encoding an antigenic peptide derived from influenza A virus HA, RNA encoding an antigenic peptide derived from influenza B virus HA, RNA encoding an antigenic peptide derived from influenza B virus HA, RNA encoding an antigenic peptide derived from one antigenic peptide selected from influenza A virus NA, NP, M1, M2, NS1, and NS2, RNA encoding an antigenic peptide derived from one antigenic peptide selected from influenza A virus NA, NP, M1, M2, NS1, and NS2, RNA encoding an antigenic peptide derived from one antigenic peptide selected from influenza B virus NA, NP, M1, M2, NS1, and NS2, and RNA encoding an antigenic peptide derived from one antigenic peptide selected from influenza B virus NA, NP, M1, M2, NS1, and NS2. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic peptide derived from influenza A virus HA, RNA encoding an antigenic peptide derived from influenza A virus HA, RNA encoding an antigenic peptide derived from influenza B virus HA, RNA encoding an antigenic peptide derived from influenza B virus HA, RNA encoding an antigenic peptide derived from influenza A virus NA, RNA encoding an antigenic peptide derived from influenza A virus NA, RNA encoding an antigenic peptide derived from influenza B virus NA, and RNA encoding an antigenic peptide derived from influenza B virus NA. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic peptide associated with H1N1 influenza virus, RNA encoding an antigenic peptide associated with H3N2 influenza virus, RNA encoding an antigenic peptide associated with Victoria lineage influenza virus, and RNA encoding an antigenic peptide associated with Yamagata lineage influenza virus. In some embodiments, the octavalent influenza vaccine comprises RNA associated with the influenza type expected to circulate in the relevant jurisdiction (e.g., HA peptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses expected to circulate in the relevant jurisdiction).

[0272] In some embodiments, each RNA in the compositions disclosed herein encodes an antigenic polypeptide associated with an infectious agent expected to be prevalent in the relevant jurisdiction. Such compositions can reduce the number of vaccinations required.

[0273] In some embodiments, the nucleic acid-containing particle comprises two or more RNA molecules, each RNA molecule containing a nucleotide sequence encoding an antigen (e.g., HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particle comprises three or more RNA molecules, each RNA molecule containing a nucleotide sequence encoding an antigen (e.g., HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particle comprises four or more RNA molecules, each RNA molecule containing a nucleotide sequence encoding an antigen (e.g., HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particle includes an RNA molecule containing a nucleotide sequence encoding an antigenic polypeptide associated with H1N1 influenza virus, an RNA molecule containing a nucleotide sequence encoding an antigenic polypeptide associated with H3N2 influenza virus, an RNA molecule containing a nucleotide sequence encoding an antigenic polypeptide associated with B / Victoria lineage influenza virus, and an RNA molecule containing a nucleotide sequence encoding an antigenic polypeptide associated with B / Yamagata influenza virus. In some embodiments, each RNA in the composition containing a nucleotide sequence encoding an antigenic polypeptide associated with an influenza virus is formulated in the same nucleic acid-containing particle. In some embodiments, each RNA in the composition comprising a nucleotide sequence encoding an antigenic polypeptide associated with the influenza virus is formulated in a separate nucleic acid-containing particle.

[0274] In some embodiments, nucleic acid-containing particles containing two or more RNA molecules (e.g., LNPs as described herein in some embodiments) contain equal amounts (i.e., in a 1:1 ratio) of each RNA molecule.

[0275] In some embodiments, nucleic acid-containing particles containing two or more RNA molecules (e.g., in some embodiments, LNPs as described herein) contain different amounts of each RNA molecule. For example, in some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the amount of the first RNA molecule is 0.01 to 100 times that of the second RNA molecule (e.g., wherein the amount of the first RNA molecule is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times that of the second RNA molecule). In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 10 times that of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 5 times that of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 3 times that of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 2 times that of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 3 times that of the second RNA molecule.

[0276] In some embodiments, nucleic acid-containing particles containing three RNA molecules (e.g., LNPs as described herein) contain equal amounts of each RNA molecule (i.e., in a 1:1:1 ratio).

[0277] In some embodiments, nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) containing three RNA molecules contain different amounts of each RNA molecule. For example, in some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50; 1:0.01-40:0.01-40; 1:0.01-30:0.01-25; 1:0.01-25:0.01-25; 1:0.01-20:0.01-20; 1:0.01-15:0.01-1). 5; 1:0.01-10:0.01-9; 1:0.01-9:0.01-9; 1:0.01-8:0.01-8; 1:0.01-7:0.01-7; 1:0.01-6:0.01-6; 1:0.01-5:0.01-5; 1:0.01-4:0.01-4; 1:0.01-3:0.01-3; 1:0.01-2:0.01-2; or 1:0.01-1.5:0.01-1.5). In some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:1:3. In some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:3:3.

[0278] The term “dose” as used in this article generally refers to “dosage”, which refers to the amount of RNA administered per administration, i.e., per dose.

[0279] In some embodiments, the immunogenic composition or vaccine of this disclosure may be administered by a single dose or by booster doses.

[0280] In some embodiments, the treatment regimen described herein comprises at least one dose. In some embodiments, the treatment regimen comprises a first dose and at least one subsequent dose. In some embodiments, the dose of the first dose is the same as that of at least one subsequent dose. In some embodiments, the dose of the first dose is the same as that of all subsequent doses. In some embodiments, the dose of the first dose is different from that of at least one subsequent dose. In some embodiments, the dose of the first dose is different from that of all subsequent doses. In some embodiments, the treatment regimen comprises two doses. In some embodiments, the provided treatment regimen consists of two doses. In some embodiments, the treatment regimen comprises three doses.

[0281] In one embodiment, this disclosure contemplates administering a single dose. In one embodiment, this disclosure contemplates administering an initial immunization dose, followed by one or more booster doses. The booster dose, or the first booster dose, may be administered 7 to 28 days or 14 to 24 days after administering the initial immunization dose. In some embodiments, the first booster dose may be administered 1 week to 3 months after administering the initial immunization dose (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks). In some embodiments, subsequent booster doses may be administered at least 1 week or longer after the previous booster dose (including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer). In some embodiments, subsequent booster doses may be administered at intervals of approximately 5-9 weeks or 6-8 weeks. In some implementations, at least one subsequent booster dose (e.g., after the first booster dose) may be administered at least 3 months or longer after the previous dose (including, for example, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months or longer).

[0282] In some embodiments, a dose contains a total amount of 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg of RNA, such as about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg of RNA. In some embodiments, a dose contains a total amount of up to about 100 μg of RNA (e.g., modRNA). In some embodiments, a dose comprises 0.1 μg to 100 μg of one or more first RNAs and 0.1 μg to 100 μg of one or more second RNAs, wherein each of the one or more first RNAs comprises a nucleotide sequence encoding an antigenic polypeptide associated with a first infectious agent (e.g., coronavirus), and each of the one or more second RNAs comprises a nucleotide sequence encoding an antigenic polypeptide associated with a second infectious agent (e.g., influenza virus). In some embodiments, a dose comprises 3 to 60 μg of one or more first RNAs and 3 to 90 μg of one or more second RNAs. In some embodiments, a dose comprises 3 to 60 μg of one or more first RNAs and 3 to 90 μg of one or more second RNAs, wherein the dose comprises a total of up to 100 μg of RNA. In some embodiments, a dose comprises 3 to 30 μg of one or more first RNAs and 3 to 60 μg of one or more second RNAs, wherein the dose comprises a total of up to 100 μg of RNA. In some embodiments, a dose comprises 3 μg of one or more first RNAs and 3 μg of one or more second RNAs. In some embodiments, a dose comprises 3 μg of one or more first RNAs and 6 μg of one or more second RNAs. In some embodiments, a dose comprises 10 μg of one or more first RNAs and 10 μg of one or more second RNAs. In some embodiments, a dose comprises 10 μg of one or more first RNAs and 20 μg of one or more second RNAs. In some embodiments, a dose comprises 30 μg of one or more first RNAs and 30 μg of one or more second RNAs. In some embodiments, a dose comprises 30 μg of one or more first RNAs and 60 μg of one or more second RNAs. In some embodiments, a dose comprises 60 μg of one or more first RNAs and 30 μg of one or more second RNAs.

[0283] In some embodiments, the subsequent dose administered to an individual (e.g., as part of an initial or booster regimen) may have the same amount of RNA as the previously administered dose. In some embodiments, the amount of RNA in the subsequent dose administered to an individual (e.g., as part of an initial or booster regimen) may differ from the amount previously administered. For example, in some embodiments, the subsequent dose may be higher or lower than the previous dose, for example, based on considerations of various factors, including, for example, the immunogenicity and / or reactivity induced by the previous dose, the prevalence of the disease, etc. In some embodiments, the subsequent dose may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or higher than the previous dose. In some embodiments, the subsequent dose may be at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, or more higher than the previous dose. In some embodiments, the subsequent dose may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more higher than the previous dose. In some embodiments, subsequent doses may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or lower than previous doses. In some embodiments, each dose (e.g., in a given dose) may be administered in amounts of the RNA described herein, such as about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, or about 100 μg.

[0284] In some embodiments, each dose (e.g., in a given dose) may administer 60 μg or less, 55 μg or less, 50 μg or less, 45 μg or less, 40 μg or less, 35 μg or less, 30 μg or less, 25 μg or less, 20 μg or less, 15 μg or less, 10 μg or less, 5 μg or less, 3 μg or less, 2.5 μg or less, or 1 μg or less of the RNA described herein.

[0285] In some embodiments, each dose (e.g., in a given dose) may administer at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 40 μg, at least 50 μg, or at least 60 μg of the RNA described herein. In some embodiments, at least 3 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 10 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 15 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 20 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 25 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 30 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 50 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, at least 60 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, a combination of the above doses may be administered in a regimen comprising two or more doses (e.g., the preceding and subsequent doses may be in different amounts, as described herein). In some embodiments, a combination of the above doses may be administered in an initial regimen and a booster regimen (e.g., different doses may be given in the initial and booster regimens).

[0286] In some embodiments, each dose may administer 0.25 μg to 60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg of the RNA described herein. In some embodiments, 3 μg to 30 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, 3 μg to 20 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, 3 μg to 15 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, 3 μg to 10 μg of the RNA described herein may be administered in at least one given dose. In some embodiments, 10 μg to 30 μg of the RNA described herein may be administered in at least one given dose.

[0287] In some embodiments, the administration regimen to the subject may comprise multiple doses (e.g., at least two, at least three, or more). In some embodiments, the administration regimen to the subject may comprise a first dose and a second dose, administered at intervals of at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer. In some embodiments, such dosing intervals may be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer. In some embodiments, multiple doses may be administered at intervals of several days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days or more. In some embodiments, multiple doses may be administered at intervals of about 1 to about 3 weeks, or about 1 to about 4 weeks, or about 1 to about 5 weeks, or about 1 to about 6 weeks, or about 1 to more than 6 weeks. In some embodiments, the interval between two administrations can be from about 7 to about 60 days, such as from about 14 to about 48 days. In some embodiments, the minimum number of days between two administrations can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the maximum number of days between two administrations can be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 days or less. In some embodiments, the interval between two administrations can be about 21 to about 28 days. In some embodiments, the interval between two administrations can be about 19 to about 42 days. In some embodiments, the interval between two administrations can be about 7 to about 28 days. In some embodiments, the interval between two administrations can be about 14 to about 24 days. In some embodiments, the interval between two administrations can be about 21 to about 42 days.

[0288] In some implementations, the vaccination regimen includes a first dose and a second dose. In some implementations, the interval between the first and second doses is at least 21 days. In some implementations, the interval between the first and second doses is at least 28 days.

[0289] In some embodiments, the vaccination regimen includes a first dose and a second dose, wherein the amount of RNA administered in the first dose is the same as the amount of RNA administered in the second dose. In other embodiments, the vaccination regimen includes a first dose and a second dose, wherein the amount of RNA administered in the first dose is different from the amount of RNA administered in the second dose.

[0290] In some embodiments, the vaccination regimen includes a first dose and a second dose, wherein the amount of RNA administered in the first dose is less than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%–90% of the amount in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%–50% of the amount in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%–20% of the amount in the second dose. In some embodiments, the interval between the administration of the first and second doses is at least 2 weeks, including at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer. In some embodiments, the interval between the administration of the first and second doses is at least 3 weeks.

[0291] In some embodiments, the first dose contains less than about 30 μg of RNA, while the second dose contains at least about 30 μg of RNA. In some embodiments, the first dose contains about 1 μg of RNA (at least about 30 μg, e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or less than about 30 μg), while the second dose contains about 30 to about 100 μg of RNA (e.g., about 30, about 40, about 50, or about 60 μg). In some embodiments, the first dose contains about 1 to about 20 μg of RNA, about 1 to about 10 μg of RNA, or about 1 to about 5 μg of RNA, while the second dose contains about 30 to about 60 μg of RNA.

[0292] In some embodiments, the first dose contains about 1 to about 10 μg of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 μg of RNA) and the second dose contains about 30 to about 60 μg of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 μg of RNA).

[0293] In some embodiments, the first dose contains about 1 μg of RNA, and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA, and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 5 μg of RNA, and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA, and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 15 μg of RNA, and the second dose contains about 30 μg of RNA.

[0294] In some embodiments, the first dose contains about 1 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 5 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 6 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 15 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 20 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 25 μg of RNA, and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 30 μg of RNA, and the second dose contains about 60 μg of RNA.

[0295] In some embodiments, the first dose contains less than about 10 μg of RNA, while the second dose contains at least about 10 μg of RNA. In some embodiments, the first dose contains about 0.1 μg of RNA, at least less than about 10 μg (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10 μg of RNA), while the second dose contains about 10 to about 30 μg of RNA (e.g., about 10, about 15, about 20, about 25, or about 30 μg of RNA). In some embodiments, the first dose contains about 0.1 to about 10 μg of RNA, about 1 to about 5 μg of RNA, or about 0.1 to about 3 μg of RNA, and the second dose contains about 10 to about 30 μg of RNA.

[0296] In some embodiments, the first dose contains about 0.1 to about 5 μg of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5 μg of RNA) and the second dose contains about 10 to about 20 μg of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 μg of RNA).

[0297] In some embodiments, the first dose contains about 0.1 μg of RNA, and the second dose contains about 10 μg of RNA. In some embodiments, the first dose contains about 0.3 μg of RNA, and the second dose contains about 10 μg of RNA. In some embodiments, the first dose contains about 1 μg of RNA, and the second dose contains about 10 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA, and the second dose contains about 10 μg of RNA.

[0298] In some embodiments, the first dose contains less than about 3 μg of RNA, and the second dose contains at least about 3 μg of RNA. In some embodiments, the first dose contains about 0.1 μg of RNA (at least about 3 μg, e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5 μg), and the second dose contains about 3 to about 10 μg of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10 μg). In some embodiments, the first dose contains about 0.1 to about 3 μg of RNA, about 0.1 to about 1 μg of RNA, or about 0.1 to about 0.5 μg of RNA, and the second dose contains about 3 to about 10 μg of RNA.

[0299] In some embodiments, the first dose contains about 0.1 to about 1.0 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 μg of RNA), and the second dose contains about 1 to about 3 μg of RNA (e.g., about 1.0, about 1.5, about 2.0, about 2.5, or about 3.0 μg of RNA).

[0300] In some embodiments, the first dose contains about 0.1 μg of RNA, and the second dose contains about 3 μg of RNA. In some embodiments, the first dose contains about 0.3 μg of RNA, and the second dose contains about 3 μg of RNA. In some embodiments, the first dose contains about 0.5 μg of RNA, and the second dose contains about 3 μg of RNA. In some embodiments, the first dose contains about 1 μg of RNA, and the second dose contains about 3 μg of RNA.

[0301] In some embodiments, the vaccination regimen includes a first dose and a second dose, wherein the amount of RNA administered in the first dose is greater than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the second dose is 10%-90% of the amount administered in the first dose. In some embodiments, the amount of RNA administered in the second dose is 10%-50% of the amount administered in the first dose. In some embodiments, the amount of RNA administered in the second dose is 10%-20% of the amount administered in the first dose. In some embodiments, the interval between the administration of the first and second doses is at least 2 weeks, including at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer. In some embodiments, the interval between the administration of the first and second doses is at least 3 weeks.

[0302] In some embodiments, the first dose contains at least about 30 μg of RNA, and the second dose contains less than about 30 μg of RNA. In some embodiments, the first dose contains about 30 μg to about 100 μg of RNA (e.g., about 30 μg, about 40 μg, about 50 μg, or about 60 μg of RNA), and the second dose contains about 1 μg to about 30 μg of RNA (e.g., about 0.1 μg, about 1 μg, about 3 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, or about 30 μg of RNA). In some embodiments, the second dose contains about 1 μg to about 20 μg of RNA, about 1 μg to about 10 μg of RNA, or about 1 μg to about 5 μg of RNA. In some embodiments, the first dose contains about 30 to about 60 μg of RNA, and the second dose contains about 1 to about 20 μg of RNA, about 1 to about 10 μg of RNA, or about 0.1 to about 3 μg of RNA.

[0303] In some implementations, the first dose contains about 30 to about 60 μg of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 μg of RNA), and the second dose contains about 1 to about 10 μg of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 μg of RNA).

[0304] In some embodiments, the first dose contains about 30 μg of RNA and the second dose contains about 1 μg of RNA. In some embodiments, the first dose contains about 30 μg of RNA and the second dose contains about 3 μg of RNA. In some embodiments, the first dose contains about 30 μg of RNA and the second dose contains about 5 μg of RNA. In some embodiments, the first dose contains about 30 μg of RNA and the second dose contains about 10 μg of RNA. In some embodiments, the first dose contains about 30 μg of RNA and the second dose contains about 15 μg of RNA.

[0305] In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 1 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 3 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 5 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 6 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 10 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 15 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 20 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 25 μg of RNA. In some embodiments, the first dose contains about 60 μg of RNA and the second dose contains about 30 μg of RNA.

[0306] In some embodiments, the first dose contains at least about 10 μg of RNA, and the second dose contains less than about 10 μg of RNA. In some embodiments, the first dose contains about 10 to about 30 μg of RNA (e.g., about 10, about 15, about 20, about 25, or about 30 μg of RNA), and the second dose contains about 0.1 μg of RNA, at least less than about 10 μg (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10 μg of RNA). In some embodiments, the first dose contains about 10 to about 30 μg of RNA, or about 0.1 to about 3 μg of RNA, and the second dose contains about 1 to about 10 μg of RNA, or about 1 to about 5 μg of RNA.

[0307] In some implementations, the first dose contains about 10 to about 20 μg of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 μg of RNA), and the second dose contains about 0.1 to about 5 μg of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5 μg of RNA).

[0308] In some embodiments, the first dose contains about 10 μg of RNA and the second dose contains about 0.1 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA and the second dose contains about 0.3 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA and the second dose contains about 1 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA and the second dose contains about 3 μg of RNA.

[0309] In some embodiments, the first dose contains at least about 3 μg of RNA, and the second dose contains less than about 3 μg of RNA. In some embodiments, the first dose contains about 3 to about 10 μg of RNA (e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 μg of RNA), and the second dose contains 0.1 μg of at least about 3 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5 μg of RNA). In some embodiments, the first dose contains about 3 to about 10 μg of RNA, and the second dose contains about 0.1 to about 3 μg of RNA, about 0.1 to about 1 μg of RNA, or about 0.1 to about 0.5 μg of RNA.

[0310] In some embodiments, the first dose contains about 1 to about 3 μg of RNA (e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0 μg of RNA), and the second dose contains about 0.1 to 0.3 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 μg of RNA).

[0311] In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 0.1 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 0.3 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 0.6 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 1 μg of RNA.

[0312] In some implementations, the vaccination regimen comprises at least two doses, including, for example, at least three, at least four, or more doses. In some implementations, the vaccination regimen comprises three doses. In some implementations, the time interval between the first and second doses may be the same as the time interval between the second and third doses. In some implementations, the time interval between the first and second doses may be longer than the time interval between the second and third doses, for example, by several days or weeks (including, for example, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer). In some implementations, the time interval between the first and second doses may be shorter than the time interval between the second and third doses, for example, by several days or weeks (including, for example, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer). In some implementations, the time interval between the first and second doses may be shorter than the time interval between the second and third doses, for example, at least one month shorter (including, for example, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, or longer).

[0313] In some embodiments, the interval between the last dose of the initial immunization regimen and the first dose of the booster regimen is at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer. In some embodiments, the initial immunization regimen may consist of two doses. In some embodiments, the initial immunization regimen may consist of three doses. In some embodiments, the first and second doses (and / or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first and second doses (and / or other subsequent doses) may be administered in the deltoid muscle. In some embodiments, the first and second doses (and / or other subsequent doses) may be administered in the same arm.

[0314] In some embodiments, the mRNA composition described herein is administered in a two-dose series (e.g., 0.3 mL each) 21 days apart (e.g., by intramuscular injection). In some embodiments, the mRNA composition described herein is administered in a two-dose series (e.g., 0.2 mL each) 21 days apart (e.g., by intramuscular injection). In some embodiments, the mRNA composition described herein is administered in a three-dose series (e.g., 0.3 mL or less, including, for example, 0.2 mL) (e.g., by intramuscular injection), wherein the two doses are administered at least 3 weeks apart. In some embodiments, the first and second doses may be administered 3 weeks apart, while the second and third doses may be administered at a longer interval than the interval between the first and second doses, e.g., at least 4 weeks or longer (including at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks or longer). In some embodiments, each dose is about 60 μg. In some embodiments, each dose is about 50 μg. In some embodiments, each dose is about 30 μg. In some embodiments, each dose is about 25 μg. In some embodiments, each dose is about 20 μg. In some embodiments, each dose is about 15 μg. In some embodiments, each dose is about 10 μg. In some embodiments, each dose is about 3 μg.

[0315] In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 60 μg. In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 50 μg. In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 30 μg. In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 25 μg. In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 20 μg. In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 15 μg. In some embodiments, at least one dose of the vaccine regimen (e.g., primary vaccination and / or booster vaccination regimens) is about 10 μg. In some implementations, at least one dose administered in a vaccination regimen (e.g., primary vaccination regimen and / or booster vaccination regimen) is approximately 3 μg.

[0316] In one embodiment, each dose administers approximately 60 μg of the RNA described herein. In one embodiment, each dose administers approximately 50 μg of the RNA described herein. In one embodiment, each dose administers approximately 30 μg of the RNA described herein. In one embodiment, each dose administers approximately 25 μg of the RNA described herein. In one embodiment, each dose administers approximately 20 μg of the RNA described herein. In one embodiment, each dose administers approximately 15 μg of the RNA described herein. In one embodiment, each dose administers approximately 10 μg of the RNA described herein. In one embodiment, each dose administers approximately 5 μg of the RNA described herein. In one embodiment, each dose administers approximately 3 μg of the RNA described herein. In one embodiment, at least two such doses are administered. For example, a second dose may be administered approximately 21 days after the first dose.

[0317] In some embodiments, the efficacy of the RNA vaccine described herein (e.g., administered in two doses, wherein the second dose may be administered approximately 21 days after the first dose, and the dosage is, for example, approximately 30 μg per dose) begins to be at least 70%, at least 80%, at least 90%, or at least 95% starting 7 days after the administration of the second dose (e.g., starting 28 days after the administration of the first dose if the second dose is administered 21 days after the first dose). In some embodiments, such efficacy has been observed in individuals aged at least 50 years, at least 55 years, at least 60 years, at least 65 years, at least 70 years, or older. In some implementations, the efficacy of the RNA vaccine described herein (e.g., administered in two doses, wherein the second dose may be administered approximately 21 days after the first dose, and the dosage is, for example, approximately 30 μg per dose) begins 7 days after the administration of the second dose (e.g., from 28 days after the administration of the first dose if the second dose is administered 21 days after the first dose) in individuals aged at least 65 years (e.g., 65 to 80 years, 65 to 75 years, or 65 to 70 years) of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. Such efficacy may be observed over a period of up to 1 month, 2 months, 3 months, 6 months, or even longer.

[0318] In one embodiment, vaccine efficacy is defined as the percentage reduction in the number of subjects with evidence of infection (vaccinated subjects compared to unvaccinated subjects). In one embodiment, the methods and agents described herein are administered to a pediatric population. In several embodiments, the pediatric population includes or comprises subjects under 18 years of age, for example, 5 to under 18 years of age, 12 to under 18 years of age, 16 to under 18 years of age, 12 to under 16 years of age, 5 to under 12 years of age, or 6 months to under 12 years of age. In several embodiments, the pediatric population includes or comprises subjects under 5 years of age, for example, 2 to under 5 years of age, 12 months to under 24 months of age, 7 months to under 12 months of age, or 6 months of age. In some such embodiments, the mRNA composition described herein is administered to subjects under 2 years of age, for example, 6 months to under 2 years of age. In some such embodiments, the mRNA composition described herein is administered to subjects under 6 months of age (e.g., 1 month to under 4 months of age). In some embodiments, the dosing regimen (e.g., dosage and / or dosing schedule) for the pediatric population may vary according to different age groups. For example, in some embodiments, subjects aged 6 months to 4 years may be administered a primary immunization regimen comprising at least three doses, wherein the initial two doses are administered at an interval of at least 3 weeks (including, for example, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer), followed by a third dose at least 8 weeks after the second dose (including, for example, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer). In some such embodiments, at least one dose administered is 3 μg of the RNA described herein. In some embodiments, subjects aged 5 years and older may be administered a primary immunization regimen comprising at least two doses, wherein the two doses are administered at an interval of at least 3 weeks (including, for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer). In some such embodiments, at least one dose administered is 10 μg of the RNA described herein. In some implementations, immunocompromised subjects aged 5 years and older (e.g., in some implementations, subjects who have undergone solid organ transplantation or have been diagnosed with a condition considered equivalent to immunocompromise) may be administered a primary immunization regimen comprising at least three doses, wherein the initial two doses are administered at an interval of at least 3 weeks (including, for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or longer), followed by a third dose at least 4 weeks after the second dose (including, for example, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks or longer).

[0319] In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older at a dose of about 30 μg. In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older (including, for example, 18 years or older) at a dose greater than 30 μg, including, for example, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, or higher. In some such embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older at a dose of about 60 μg. In some such embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older at a dose of about 50 μg. In one embodiment, the pediatric population includes or comprises subjects aged 12 to under 18 years, including subjects aged 16 to under 18 years and / or subjects aged 12 to under 16 years. In this embodiment, treatment may include two vaccinations 21 days apart, wherein, in one embodiment, the vaccine is administered at a dose of 30 μg RNA per dose, for example, via intramuscular administration. In some embodiments, a higher dose is administered to older pediatric patients and adults (e.g., patients 12 years of age or older) compared to younger children or infants (e.g., children aged 2 to under 5 years, children aged 6 months to under 2 years, or children under 6 months). In some embodiments, a higher dose is administered to children aged 2 to under 5 years compared to toddlers and / or infants (e.g., children aged 6 months to under 2 years, or children under 6 months).

[0320] In one embodiment, the pediatric population includes or comprises subjects aged 5 to 18 years, including subjects aged 12 to 18 years and / or subjects aged 5 to 12 years. In this embodiment, treatment may include two vaccinations 21 days apart, wherein, in various embodiments, the vaccine is administered in an amount of 10 μg, 20 μg, or 30 μg of RNA per dose, for example, via intramuscular administration. In some such embodiments, the mRNA composition described herein is administered to subjects aged 5 to 11 years, at a dose of approximately 10 μg.

[0321] In one embodiment, the pediatric population includes or comprises subjects under 5 years of age, including subjects aged 2 to under 5 years, subjects aged 12 to under 24 months, subjects aged 7 to under 12 months, subjects aged 6 to under 12 months, and / or subjects under 6 months. In this embodiment, treatment may include two vaccinations, for example, 21 to 42 days apart, wherein, in various embodiments, the vaccine is administered in doses of 3 μg, 10 μg, 20 μg, or 30 μg of RNA, for example, via intramuscular administration. In some such embodiments, the mRNA composition described herein is administered to subjects aged 2 to under 5 years of age, in doses of about 3 μg. In some such embodiments, the mRNA composition described herein is administered to subjects aged about 6 months to about 5 years of age, in doses of about 3 μg.

[0322] In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, and at least one dose of about 60 μg is given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen). In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, and at least one dose of about 30 μg is given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen). In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, and at least one dose of about 15 μg is given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen). In some embodiments, the mRNA composition described herein is administered to subjects aged 5 to 12 years or younger, and at least one dose of about 10 μg is given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen). In some embodiments, the mRNA composition described herein is administered to subjects aged 2 to 5 years, and at least one dose in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg. In some embodiments, the mRNA composition described herein is administered to subjects aged 6 months to 2 years, and at least one dose in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg or less, including, for example, 2 μg, 1 μg or less. In some embodiments, the mRNA composition described herein is administered to infants under 6 months of age, and at least one dose in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg or less, including, for example, 2 μg, 1 μg, 0.5 μg or less.

[0323] In some implementations, the dose administered to a subject in need may include administration of the single mRNA composition described herein.

[0324] In some embodiments, the dose administered to the subject in need may include the administration of at least two or more (e.g., at least three or more) different drugs / formulations. For example, in some embodiments, at least two or more different drugs / formulations may comprise at least two different mRNA compositions described herein (e.g., in some embodiments, each composition comprises a different RNA construct).

[0325] In some embodiments, two or more RNAs are administered to the subject (e.g., as part of a primary or booster regimen), wherein the two or more RNAs are administered on the same day or at the same visit. In some embodiments, the two or more RNAs are administered in a separate composition, for example, by administering each RNA to different sites on the subject (e.g., by intramuscular administration to different arms of the subject or different sites on the same arm of the subject). In some embodiments, the two or more RNAs are mixed prior to administration (e.g., immediately prior to administration, such as by the administering physician). In some embodiments, the two or more RNAs are formulated together (e.g., by (a) mixing different LNP populations, each containing a different RNA; or (b) mixing the two or more RNAs prior to LNP formulation such that each LNP contains two or more RNAs).

[0326] In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, in equal amounts of each RNA (i.e., in a 1:1 ratio).

[0327] In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the amount of each RNA varies. For example, in some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the amount of said one or more first RNAs is 0.01 to 100 times that of said one or more second RNAs (e.g., wherein said one or more first RNAs are 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.0... times that of said one or more second RNAs). 1 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times). In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the concentration of said one or more first RNAs is 1 to 10 times the concentration of said one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the amount of the one or more first RNAs is 1 to 5 times the amount of the one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the concentration of the one or more first RNAs is 1 to 3 times the concentration of the one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the amount of the one or more first RNAs is 2 times the concentration of the one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or a composition comprising one or more first RNAs and one or more second RNAs, wherein the concentration of the one or more first RNAs is 3 times the concentration of the one or more second RNAs.

[0328] In some implementations, the subject is administered two first RNAs or a composition containing two first RNAs, each encoding an antigen or variant derived from an influenza virus strain, wherein the amount of each RNA is different. For example, in some implementations, the ratio of the two first RNAs is 1:0.01-100 (e.g., 1:0.01-50; 1:0.01-40; 1:0.01-30; 1:0.01-25; 1:0.01-20; 1:0.01-15; 1:0.01-10; 1:0.01-9; 1:0.01-8; 1:0.01-7; 1:0.01-6; 1:0.01-5; 1:0.01-4; 1:0.01-3; 1:0.01-2; 1:0.01-1.5; 1:0.1-10; 1:0.1-5; 1:0.1-3; 1:2-10; 1:2-5 or 1:2-3). In some embodiments, the subject is administered two first RNAs in a 1:3 ratio or a composition containing two first RNAs in a 1:3 ratio. In some embodiments, the subject is administered two first RNAs in a 1:2 ratio or a composition containing two first RNAs in a 1:2 ratio.

[0329] For example, in some implementations, the ratio of the three first RNAs is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50; 1:0.01-40:0.01-40; 1:0.01-30:0.01-30; 1:0.01-25:0.01-25; 1:0.01-20:0.01-20; 1:0.01-15:0.01-15; 1:0.01-10:0.01-10; 1:0.01-9:0.01-9; 1:0.01-8:0). 0.01-8; 1:0.01-7:0.01-7; 1:0.01-6:0.01-6; 1:0.01-5:0.01-5; 1:0.01-4:0.01-4; 1:0.01-3:0.01-3; 1:0.01-2:0.01-2; 1:0.01-1.5:0.01-1.5; 1:0.1-10:0.1-10; 1:0.1-5:0.1-5; 1:0.1-3:0.1-3; 1:2-10:2-10; 1:2-5:2-5 or 1:2-3:2-3). In some embodiments, the subject is administered three first RNAs in a ratio of 1:1:3 or a composition containing three first RNAs in a ratio of 1:1:3. In some embodiments, the subject is given three first RNAs in a ratio of 1:3:3 or a composition containing three first RNAs in a ratio of 1:3:3.

[0330] In some embodiments, a subject is administered two or more second RNAs or a composition comprising two or more second RNAs, wherein one or more second RNAs encode the HA protein of influenza A virus and one or more second RNAs encode the HA protein of influenza B virus. In some embodiments, one or more second RNAs encoding the HA protein of influenza A virus and one or more second RNAs encoding the HA protein of influenza B virus are present or administered in the same amount (i.e., at a 1:1 ratio). In some embodiments, one or more second RNAs encoding the HA protein of influenza A virus and one or more second RNAs encoding the HA protein of influenza B virus are administered in different amounts (e.g., at a ratio of 1:10 to 10:1, or at a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (total RNA encoding influenza A antigen: total RNA encoding influenza B antigen)).

[0331] In some embodiments, a subject is administered two second RNAs or a composition comprising two second RNAs, each encoding a different influenza virus type's HA protein (e.g., a second RNA encoding the HA protein of influenza A virus, and a second RNA encoding the HA protein of influenza B virus). In some embodiments, the two second RNAs are administered or present in the same amount (i.e., at a 1:1 ratio). In some embodiments, the two second RNAs are administered or present in different amounts (e.g., at a ratio of 1:10 to 10:1, or at a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (A:B).

[0332] In some embodiments, a subject is administered three second RNAs or a composition containing three second RNAs, each encoding the HA protein of a different influenza virus subtype (e.g., the HA protein of A / Wisconsin (H1N1) virus, A / Darwin (H3N2) virus, and B / Austria (Victoria) virus). In some embodiments, a subject is administered three second RNAs or a composition containing three second RNAs, wherein the amount of each of the three second RNAs is the same (i.e., in a 1:1:1 ratio). In some embodiments, a subject is administered three second RNAs or a composition containing three second RNAs, wherein one or more of the three second RNAs are in different amounts (e.g., in ratios of 1:1:2 to 1:1:10 (e.g., 1:1:2, 1:1:3, 1:1:4, or 1:1:5), or in ratios of 2:2:1 to 2:2:10 (e.g., 2:2:1, 3:3:1, 4:4:1, or 5:5:1)). In some embodiments, a subject is administered three second RNAs or a composition containing three second RNAs, wherein two of the second RNAs encode different HA proteins of influenza A virus, and one second RNA encodes an HA protein of influenza B virus. In some such embodiments, the second RNA encoding an HA protein of influenza B virus is present or administered in a higher amount than any of the second RNAs encoding an HA protein of influenza A virus (e.g., in some embodiments, the ratio of the two second RNAs encoding an HA protein of influenza A virus to the second RNA encoding an HA protein of influenza B virus is 1:1:1-10, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 (A:A:B)). In some embodiments, a subject is administered three second RNAs or a composition containing three second RNAs, wherein two encode an HA protein of influenza A virus, one encodes an HA protein of influenza B virus, and the ratio of the three second RNAs is 1:1:4 (A:A:B). In some embodiments, the two second RNAs encoding the HA protein of influenza A virus are each present in a higher amount or are each administered in a higher amount than the second RNA encoding the HA protein from influenza B virus (e.g., in some embodiments, the ratio of the two second RNAs encoding the HA protein from influenza A virus to the second RNA encoding the HA protein from influenza B virus is 1-10:1-10:1, 2:2:1, 3:3:1, 4:4:1 or 5:5:1 (A:A:B)).

[0333] In some embodiments, a subject is administered four second RNAs or a composition comprising four second RNAs, each encoding a HA protein of a different influenza virus subtype. In some such embodiments, the four second RNAs include two second RNAs encoding HA proteins of different influenza A viruses and two second RNAs encoding HA proteins of different influenza B viruses (e.g., HA proteins of H1N1 virus, H3N2 virus, B / Victoria lineage virus, and B / Yamagata lineage virus). In some embodiments, each of the two second RNAs encoding the influenza A virus HA protein and each of the two second RNAs encoding the influenza B virus HA protein are present in equal amounts (i.e., the ratio of the four second RNAs is 1:1:1:1). In some embodiments, the two second RNAs encoding the HA protein of influenza B virus are each applied in a higher amount or are present in a higher amount than either of the second RNAs encoding the HA protein from influenza A virus (e.g., in some embodiments, the ratio of the two second RNAs encoding the HA protein from influenza A virus to the two second RNAs encoding the HA protein from influenza B virus is 1:1:2-10:2-10, 1:1:2-5:2-5, 1:1:2:2, 1:1:3:3, 1:1:4:4, 1:1:5:5, 1:1:6:6, 1:1:7:7, 1:1:8:8, 1:1:9:9, 1:1:10:10 (A:A:B:B)). In some implementations, the subject is administered four secondary RNAs or a composition containing four secondary RNAs, two HA proteins encoding influenza A virus, and two HA proteins encoding influenza B virus, wherein the ratio of the four secondary RNAs is 1:1:5:5 (A:A:B:B). In some embodiments, the two second RNAs encoding the HA protein of influenza A virus are each administered or present in a higher amount than either of the second RNAs encoding the HA protein from influenza B virus (e.g., in some embodiments, the ratio of the two second RNAs encoding the HA protein from influenza A virus to the two second RNAs encoding the HA protein from influenza B virus is 2-10:2-10:1:1, 2-5:2-5:1:1, 2:2:1:1, 3:3:1:1, 4:4:1:1, 5:5:1:1, 6:6:1:1, 7:7:1:1, 8:8:1:1, 9:9:1:1, 10:10:1:1 (A:A:B:B)).

[0334] In some embodiments, a subject is administered four second RNAs or a composition containing four second RNAs, wherein three of the second RNAs encode the HA protein of different influenza A viruses, and one second RNA encodes the HA protein of an influenza B virus (e.g., A / Wisconsin (H1N1), A / Darwin (H3N2), A / Cambodia (H3N2), and B / Austria (Victoria)). In some such embodiments, each of the four second RNAs is administered or present in the same amount (i.e., in a 1:1:1:1 ratio). In some embodiments, the amount of the second RNA encoding the influenza B virus HA protein is higher than that of any one of the second RNAs encoding the influenza A virus HA protein (e.g., in some embodiments, the ratio of these second RNAs is 1:1:1:1-10, 1:1:1:1-5, 1:1:1:2, 1:1:1:3, 1:1:1:4, or 1:1:1:5 (A:A:A:B)). In some embodiments, the applied second RNA or the ratio of second RNA in the composition is 1:1:1:5 (A:A:A:B). In some embodiments, the amount of each second RNA encoding the influenza A virus HA protein is higher than the amount of the second RNA encoding the influenza B virus HA protein (e.g., in some embodiments, the ratio of second RNA is 1-10:1-10:1-10:1, 1-5:1-5:1-5:1, 2:2:2:1, 3:3:3:1, 4:4:4:1, or 5:5:5:1 (A:A:A:B)).

[0335] In some embodiments, the subject is administered one or more second RNAs encoding the HA protein of influenza virus, or a composition containing one or more second RNAs encoding the HA protein of influenza virus (e.g., two, three, or four second RNAs, each encoding a different HA protein of influenza virus), in a total amount of 0.1 to 100 μg (e.g., 1 to 90 μg, 3 to 90 μg, 1 to 60 μg, 3 to 60 μg, 5 to 60 μg, 10 to 60 μg, 30 to 60 μg, 3 to 30 μg). In some embodiments, the subject is administered one or more second RNAs encoding the HA protein of influenza virus, or a composition containing one or more second RNAs encoding the HA protein of influenza virus, in a total amount of 3 μg, 5 μg, 6 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg.

[0336] In some implementations, a subject is given one amount of three or four second RNAs or a composition containing three or four second RNAs, each second RNA encoding the HA antigen of a different influenza virus strain (each "influenza component" corresponds to a second RNA encoding the HA antigen (e.g., the second RNA as described herein).

[0337] In some embodiments, the compositions described herein are characterized in that they produce influenza neutralizing antibody titers that are at least twice the neutralizing antibody titers produced by a reference vaccine for each influenza virus encoding its antigen (e.g., where the reference vaccine is a single-dose quadrivalent influenza RNA vaccine or an approved (non-RNA) influenza vaccine).

[0338] In some embodiments, the influenza vaccine is an alpha, beta, gamma, or delta influenza virus vaccine. In some embodiments, the vaccine is an influenza A, influenza B, influenza C, or influenza D virus vaccine. In some embodiments, the influenza A virus vaccine contains hemagglutinin selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18, or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any of them. In some embodiments, the influenza A vaccine contains or encodes a neuraminidase (NA) selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11, or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any of them. In some implementations, the influenza vaccine comprises at least one influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2 and / or polymerase basic protein 2 (PB2), or immunogenic fragments or variants thereof, or nucleic acids (e.g., RNA) encoding any of them.

[0339] Example

[0340] Example 1: Description of the manufacturing process

[0341] This section describes the manufacturing process and process controls for the influenza saRNA vaccine pharmaceutical material. The manufacturing process includes RNA synthesis via in vitro transcription (IVT) and purification via ultrafiltration / dialysis (UFDF-1). Subsequently, the RNA is enzymatically capped, purified by chromatography, and finally subjected to UFDF-2, followed by final filtration and dispensing.

[0342] The process has been scaled up to a starting IVT volume of 1.5 liters for the production of clinical materials. Aside from the changes required to scale up to 1.5 liters, there are no significant differences compared to the non-clinical toxicology / development process.

[0343] RT-ddPCR (identification of RNA sequences)

[0344] When RNA in a sample is subjected to a one-step reverse transcription (RT)-ddPCR assay, the influenza saRNA identity can be confirmed if the tested sample is positive for both the replicase sequence (confirming self-amplification of the RNA construct) and the target sequence (confirming the encoding of the influenza virus sequence). Digital droplet polymerase chain reaction (ddPCR) is a digital polymerase chain reaction (PCR) technique that uses a water-oil emulsion system to quantify target nucleic acids. The RNA sample is diluted to the final theoretical input concentration, which is within the linear range of the ddPCR assay. The reaction mixture containing reverse transcriptase, DNA polymerase, and sequence-specific primers and probes is dispensed into droplets, and a PCR reaction is performed separately in each compartment. The results are calculated by counting the amplified target sequence (positive droplets, measured by fluorescence amplitude above background) and the number of unamplified compartments (negative droplets). After checking the positive and negative controls and confirming their validity and acceptability, the identity is confirmed if the positive droplet count exceeds a set threshold.

[0345] Reversed-phase HPLC (Presence of pseudouridine)

[0346] After complete digestion of mRNA, the presence of pseudouridine was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The resulting single nucleosides exhibited characteristic elution patterns, including the separation of uridine and pseudouridine. The presence of pseudouridine was confirmed by comparison with uridine and pseudouridine reference standards and limit standards.

[0347] Capillary gel electrophoresis (RNA integrity)

[0348] RNA integrity is determined by capillary gel electrophoresis (CGE) based on the differences in migration of RNA molecules of different molecular weights under an applied electric field. RNA is treated with a denaturing agent, causing it to unfold and dissociate from non-covalent complexes. Under the influence of an electric field, denatured RNA species migrate towards the anode through the gel matrix according to their length and size. During migration, intercalating dyes bind to the RNA and its associated fragments, enabling fluorescence detection. Intact RNA separates from any fragmented species, thus quantifying RNA integrity by measuring the relative percentage time-corrected area of ​​the intact (main) peak.

[0349] qPCR (residual DNA template)

[0350] Residual DNA template levels were determined using quantitative polymerase chain reaction (qPCR) with fluorescence technology. A qPCR premix containing target-specific primers and fluorescent qPCR quantitative reagents was added to all sample wells. Samples were prepared at a series of dilutions and analyzed in real-time by qPCR. The measured fluorescence signal was proportional to the amount of PCR product. DNA quantification was performed during the exponential phase of the reaction at the cycle threshold (Ct), at which the amplification of the target sequence was first detected above a set signal threshold. This Ct point depends on the initial amount of DNA present in the sample. The DNA concentration in the test sample was determined by linear regression interpolation of a standard curve, taking dilution into account. Results were reported in ng DNA / mg RNA.

[0351] Table 1 provides batch details and batch analysis summary data for one batch of regulatory toxicology materials and one batch of GMP-grade pharmaceutical substances intended for clinical trials.

[0352] Table 1

[0353] S.4.4.6-2. Batch results of the drug substance TC83-delkozak-HA-SGP-NA-80A for influenza saRNA vaccine.

[0354]

[0355]

[0356] a. Identification was determined by reverse transcription quantitative polymerase chain reaction.

[0357] This standard applies only to clinical supplies.

[0358] Abbreviations: NTU = Turbidimetric Turbidity Unit; NT = Not Detected; TBP = To be provided in IND Supplemental Materials; ddPCR = Digital Droplet Polymerase Chain Reaction; qPCR = Quantitative Polymerase Chain Reaction; LAL = Limulus Amebocyte Lysate; NMT = Not Exceeding; EU = Endotoxin Unit; CFU = Colony Forming Unit.

[0359] Example 2: S.4.1 Drug Description and Composition

[0360] PF-07867246 (Constructor 6 (TC83-delkozak-HA-SGP-NA-80A) (SEQ ID NO:1)) is a preservative-free, sterile dispersion of liquid nanoparticles (LNPs) in an aqueous cryoprotectant buffer for intramuscular injection. The drug is formulated with 0.06 mg / mL RNA in 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamate (pH 7.4). The drug is packaged in 2 mL glass vials sealed with a chloroprene elastic stopper and an aluminum seal with a flip-top plastic cap (nominal capacity 0.5 mL). Table 2 lists the composition of the drug, including unit formulation, content per vial, function, and applicable quality standards for each component.

[0361] Table 2

[0362]

[0363]

[0364] Table 3

[0365] 3.6.TC83-delkozak-HA-SGP-NA-80A(A / Wisconsin / 588 / 2019)--VV00050(Constructor 6)

[0366] Nucleotide sequence 5'-->3'

[0367] Sequence length: 10936 nucleotides; 3113 A nucleotides; 2761 C nucleotides; 2796 G nucleotides; 2266 U nucleotides. UA = adenine; C = cytosine; G = guanine; U = uridine.

[0368]

[0369]

[0370]

[0371]

[0372] Example 3: S.4.1. Drug Description and Composition

[0373] PF-07871987 (Constructor 7, TC83 HA 40A 50U-50pU (SEQ ID NO:2)) is a preservative-free, sterile dispersion of liquid nanoparticles (LNPs) in an aqueous cryoprotectant buffer for intramuscular injection. The drug is formulated with 0.06 mg / mL RNA in 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamate (pH 7.4).

[0374] The medicine is contained in a 2mL glass vial, which is sealed with a chloroprene rubber stopper and an aluminum seal with a flip-top plastic cap (nominal capacity 0.5mL).

[0375] Table 4 lists the composition of the drug, including unit formulation, content per vial, function, and applicable quality standards for each ingredient.

[0376] Table 4:

[0377]

[0378]

[0379] Table 5

[0380] S.4.4.6-4. Batch results of influenza saRNA vaccine TC83-HA-40A 50U-50pU drug substance

[0381]

[0382] a. Identification was determined by reverse transcription quantitative polymerase chain reaction.

[0383] b. Non-clinical toxicology reports provided an approximate percentage of pseudouridine. Based on the reported presence, it can be inferred that its presence has been "confirmed".

[0384] This standard applies only to clinical supplies.

[0385] Abbreviations: NTU = Turbidimetric Turbidity Unit; NT = Not Detected; TBP = To be provided in IND Supplemental Materials; ddPCR = Digital Droplet Polymerase Chain Reaction; qPCR = Quantitative Polymerase Chain Reaction; LAL = Limulus Amebocyte Lysate (LAL) Reagent; NMT = Not More Than; EU = Endotoxin Unit; CFU = Colony Forming Unit.

[0386] Table 6 Sequence 3.1. TC83-HA-40-50U-50pU (A / Wisconsin / 588 / 2019) pKT177, containing 50% U + 50% pseudonucleotides.

[0387] Construct 7; Nucleotide sequence 5'-->3'; Sequence length: 9433 nucleotides; 2703 A; 2327 C; 2396 G; 2007 U; A = adenine; C = cytosine; G = guanine; (*) U = uridine or N1-methylpseudouridine; (*) The RNA obtained from this construct consists of 50% uridine and 50% N1-methylpseudouridine.

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394] Example 4: S.4.4.1 Component 7: TC83-HA-40A 50U-50PU(PF-07871987)

[0395] Table 7 provides batch details and batch analysis summary data for one batch of regulatory toxicology materials and one batch of GMP-grade pharmaceutical substances intended for clinical trials.

[0396] Table 7

[0397] S.4.4.6-7. Batch results of influenza saRNA vaccine TC83-HA-40A 50U-50pU drug substance.

[0398]

[0399] a. Identification was determined by reverse transcription quantitative polymerase chain reaction.

[0400] b. Non-clinical toxicology reports provided an approximate percentage of pseudouridine. Based on the reported presence, it can be inferred that its presence has been "confirmed".

[0401] This standard applies only to clinical supplies.

[0402] Abbreviations: NTU = Turbidimetric Turbidity Unit; NT = Not Detected; TBP = To be provided in IND Supplemental Materials; ddPCR = Digital Droplet Polymerase Chain Reaction; qPCR = Quantitative Polymerase Chain Reaction; LAL = Limulus Amebocyte Lysate (LAL) Reagent; NMT = Not Exceeding; EU = Endotoxin Unit; CFU = Colony Forming Unit

[0403] Example 5: Measurement Method

[0404] Blood coagulation inhibition assay

[0405] The primary serological assay used to measure vaccine-induced influenza immune responses is the hemagglutinin inhibition assay (HAI). HAI quantifies the presence of functional antibodies in serum that inhibit hemagglutinin (HA)-mediated erythrocyte aggregation in a reaction involving serum samples pretreated with receptor-destroying enzymes, influenza virus, and erythrocytes derived from turkey or guinea pigs. The HAI titer is the reciprocal of the highest serum dilution leading to loss of HA activity and appears as a teardrop shape when the microtiter plate is tilted. Titers measured multiple times for each sample are reported as the geometric mean titer (GMT). A HAI titer ≥1:40 is generally considered protective in humans.

[0406] Influenza virus micro-neutralization assay

[0407] The Micro-Neutralization Assay (MNT) quantitatively measures functional antibodies in serum that neutralize influenza virus activity, thereby preventing effective infection of host cell monolayers. A neutralization reaction occurs when influenza virus is incubated with a serum sample; this reaction mixture is then applied to a monolayer of Madin-Darby canine kidney cells (MDCK) to measure the degree of neutralization. Compared to a serum-free control, the MNT titer is reported as the reciprocal of the dilution resulting in a 50% or 90% reduction in infection. Day 1 MNT measures anti-HA neutralizing antibodies, and Day 3 MNT measures both anti-HA and anti-NA neutralizing antibodies.

[0408] Neuraminidase inhibition assay

[0409] The neuraminidase inhibition assay (NAI) quantifies the presence of functional antibodies in serum that prevent NA-mediated sialic acid cleavage in an enzyme-linked lectin assay. Briefly, antibody-containing serum is incubated with influenza virus, and the mixture is then transferred to a fetoglobulin-coated culture plate. Horseradish peroxidase-conjugated peanut lectin is bound to the exposed galactose moiety, and after substrate addition, the cleavage of sialic acid from the fetoglobulin is monitored by colorimetric reaction. The NAI titer, compared to a serum-free control, is the reciprocal of the highest serum dilution resulting in a 50% reduction in NA activity. Titers measured multiple times for each sample are reported as the geometric mean titer (GMT).

[0410] Example 6: Evaluation of Influenza Bicistronic HA-NA saRNA Vaccine Design in Mice

[0411] This study aimed to compare the immunogenicity of bicistronic saRNA vaccine candidates encoding influenza hemagglutinin (HA) and neuraminidase (NA) to determine the optimal bicistronic HA-NA saRNA vaccine design. All saRNA vectors used in this study were based on the TC-83 backbone, but the study was designed to evaluate immunogenicity with or without an exogenous kozak sequence upstream of the first target gene, as well as the impact of polyadenylate tail length (40 Å or 80 Å).

[0412] The key bicistronic design elements evaluated in this study include regulatory elements for driving the expression of a second target gene (subgenomic promoter (SGP) and internal ribosome entry site (IRES)) and the placement order of antigens on the vector (HA-NA or NA-HA).

[0413] Balb / c mice were immunized by intramuscular injection with a saRNA vaccine formulated with LNP that encodes the A / Wisconsin / 588 / 2019(H1N1)HA and / or NA antigens, which induced functional and neutralizing antibody responses.

[0414] Overall, all tested bicistronic saRNA vaccines induced similar titers, and these titers were also similar to those of saRNA vaccines composed of separately formulated saRNA-HA+saRNA-NA. These results confirm that the bicistronic saRNA approach induces titers and is feasible.

[0415] All saRNA vectors used in this study were based on the TC-83 backbone, but the study was designed to evaluate immunogenicity with or without an exogenous kozak sequence upstream of the first target gene, and the impact of polyadenylate tail length (40 Å or 80 Å). Key bicistronic design elements evaluated in this study included regulatory elements required to drive the expression of the second target gene, and the placement order of the antigen on the vector (HA-NA or NA-HA). The regulatory elements selected for comparison were the native VEEV subgenomic promoter (SGP; 61 nucleotides) and the internal ribosome entry site derived from encephalomyocarditis virus (IRES; 587 nucleotides). ModRNA vaccines encoding influenza virus HA or NA were also included as comparisons. Mice were immunized with saRNA or modRNA LNP formulations on days 0 and 28, and serum was collected on days 21 post-primary immunization and 14 post-boost immunization. Neutralizing antibodies and functional antibodies were measured on days 21 and 42 to determine immunogenicity.

[0416] This study designed 15 groups, as shown in Table 8, each containing a total of 10 female mice (mice strain: BALB / c). The mRNA drug was evaluated at a dose volume of 0.05 mL.

[0417] Table 8. Research Design

[0418]

[0419]

[0420] Table 9. Research Plan

[0421] Days program 0 Vax#1 21 Blood collection 28 Vax#2 42 Last blood draw

[0422] Table 10. Test samples and diluents for the study (provided for primary and booster immunizations)

[0423]

[0424]

[0425] M. Analytical and testing results of the test sample

[0426] Table 11. Drug Materials and Analytical Results

[0427]

[0428]

[0429] 2. Results and Discussion

[0430] Balb / c mice were intramuscularly injected with a saRNA vaccine formulated with LNP and encoding the A / Wisconsin / 588 / 2019(H1N1)HA and / or NA antigens, which induced functional and neutralizing antibody responses, such as HAI, 1-day MNT, 3-day MNT, and NAI (see Figures 1 and 2, respectively). Figure 3 As measured in Figure 4), a significant booster immunization effect was observed two weeks after the second immunization. The 3-day MNT result at day 42 indicated that NA contributed little to neutralization compared to HA in this assay. Overall, all evaluated bicistronic saRNA vaccine designs achieved similar titers, and these titers were also similar to those of saRNA vaccines composed of separately formulated HA and NA monocistronic saRNAs (HA / NA post-mixture) and modRNAs. The titers of saRNA vaccines expressing both antigens were similar to or slightly lower than those of controls containing only saRNA-HA or -NA. These data confirm the feasibility of the bicistronic saRNA approach.

[0431] The deletion of the Kozak sequence, the length of the polyadenylate tail, the regulatory element used to drive the second target gene (IRES compared to SGP), and the antigen placement order (HA-NA or NA-HA) did not have a significant effect on the induced titer.

[0432] Regarding Figure 1, female Balb / c mice were immunized intramuscularly with influenza saRNA vaccine constructs formulated with different LNPs and influenza modRNA comparators encoding A / Wisconsin / 588 / 2019(H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mixture consisted of a 1:1 mixture of separately formulated saRNA-HA and saRNA-NA. Functional antibody responses against A / Wisconsin / 588 / 2019 were measured by HAI on day 21 (3 weeks after primitivism) and day 42 (2 weeks after booster immunization).

[0433] Regarding Figure 2, female Balb / c mice were immunized intramuscularly with influenza saRNA vaccine constructs formulated with different LNPs and comparative influenza modRNAs encoding A / Wisconsin / 588 / 2019(H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mixture consisted of a 1:1 mixture of separately formulated saRNA-HA and saRNA-NA. Functional antibody responses against A / Wisconsin / 588 / 2019 were measured by a 1-day MNT assay on days 21 (3 weeks after primitivism) and 42 (2 weeks after booster immunization). Neutralizing titers of 50% were reported.

[0434] about Figure 3 Female Balb / c mice were immunized intramuscularly on days 0 and 28 with influenza saRNA vaccine constructs formulated with different LNPs and comparative influenza modRNAs encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA. The HA / NA post-mixture consisted of a 1:1 mixture of separately formulated saRNA-HA and saRNA-NA. Functional antibody responses against A / Wisconsin / 588 / 2019 were measured by a 3-day MNT assay on days 21 (3 weeks after primitivism) and 42 (2 weeks after booster immunization). Neutralizing titers of 50% were reported.

[0435] Regarding Figure 4, female Balb / c mice were immunized intramuscularly with influenza saRNA vaccine constructs formulated with different LNPs and influenza modRNA comparators encoding A / Wisconsin / 588 / 2019(H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mixture consisted of a 1:1 mixture of separately formulated saRNA-HA and saRNA-NA. Functional antibody responses against A / Wisconsin / 588 / 2019 were measured by NAI on days 21 (3 weeks after primitivism) and 42 (2 weeks after booster immunization).

[0436] 3. Conclusion

[0437] Seasonal influenza saRNA vaccines aim to express four different HA proteins and four different NA proteins to match the dominant circulating influenza virus strains for each season. This can be achieved by using eight separate saRNA components or four bicistronic saRNA components. To this end, the feasibility of the bicistronic saRNA approach was evaluated in mouse immunogenicity studies. A bicistronic saRNA vaccine candidate encoding both HA and NA antigens (TC83-delkozak-HA-SGP-NA-80A) was compared to monocistronic saRNA-HA or saRNA-NA controls encoding a single antigen (TC83-HA-40A or TC83-NA-40A), and also to a 1:1 mixture of separately formulated saRNA-HA and saRNA-NA components. ModRNA vaccine candidates encoding the same A / Wisconsin / 588 / 2019(H1N1) HA or NA antigen were also included as additional comparisons. Balb / c mice were immunized intramuscularly on day 0 with the following formulations: 20 ng of bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA and saRNA-NA, and 200 ng of modRNA comparator. All saRNA LNPs were placed in a 10 mM Tris / 10% sucrose + 20 mM glutamate (pH 7.4) matrix selected for clinical use. On day 21 (3 weeks post-primer), anti-HA antibody responses were induced by HAI and MNT measurements (Figures 1 and 2), and anti-NA antibody responses were also induced by NAI measurements (Figure 4). The titers of the bicistronic saRNA vaccine candidates were similar to those of the saRNA vaccine composed of a 1:1 mixture of monocistronic saRNA HA and saRNA-NA formulated separately. The titers of the saRNA vaccine formulations expressing both antigens were similar to or slightly lower than those produced by the saRNA control expressing only one antigen. These results confirm the feasibility of the bicistronic saRNA approach and demonstrate that, based on antibody titers after a single immunization, saRNA vaccines are more dose-efficient than modRNA vaccines in Balb / c mice.

[0438] Overall, the functional and neutralizing antibody titers generated by the evaluated bicistronic saRNA constructs were similar to those induced by saRNA vaccines composed of separately formulated HA and NA monocistronic saRNAs. The titers of saRNA vaccines expressing both antigens were similar to or only slightly lower than controls containing only saRNA-HA or -NA. These preliminary results confirm the feasibility of the bicistronic saRNA approach, independent of the regulatory elements used to drive the second target gene (IRES vs. SGP), antigen placement order (HA-NA or NA-HA), kozak sequence deletion, and polyadenylated tail length (40A vs. 80A).

[0439] Example 7: Immunogenicity of saRNA influenza vaccine containing modified nucleosides

[0440] To determine whether introducing modified bases could produce more tolerable and potent saRNA vaccines, saRNA formulations expressing influenza virus HA were prepared by replacing uridine with varying amounts of N1-methylpseuuridine, ranging from 0% to 100%. The effect of increasing the percentage of modified bases on in vitro antigen expression depended on cell type. Specifically, in immunocompetent human cell lines (such as HeLa), saRNA containing 25-75% N1-methylpseuuridine showed higher in vitro antigen expression than the unmodified (0%) saRNA control. However, regardless of cell type, 100% base-modified saRNA consistently produced lower levels of antigen, possibly due to impaired replicase function. Increasing the percentage of modified nucleosides incorporated into saRNA was also associated with decreased activation levels of different PRRs or RNA sensors (such as TLR3, TLR7, and RIG-1) in reporter cell lines (data not shown).

[0441] To assess immunogenicity, Balb / c mice were immunized on day 0 with 200 ng of saRNA vaccine formulation containing varying amounts of N1-methylpseuuridine via intramuscular injection. Serum cytokine and chemokine secretion was measured on day 1 post-vaccination; higher incorporation of modified nucleosides was associated with decreased innate immune activation. Figure 5 This may have improved vaccine tolerability. However, higher levels of modified nucleosides in saRNA were also associated with decreased neutralizing antibody titers 3 weeks post-vaccination. Figure 6 This may potentially reflect the effect on replicase activity. These results were confirmed in different C57BL6 / J mouse species. Figure 7 and Figure 8Based on mouse data, compared with the unmodified control, the saRNA construct incorporating 50% modified nucleosides significantly reduced the secretion of cytokines and chemokines, with a smaller decrease in antibody titers, reaching levels similar to or higher than the modRNA-HA baseline. Overall, the data indicate that saRNA can tolerate the incorporation of partially modified bases, thereby reducing early innate immune stimulation, while still evoking a strong adaptive humoral response.

[0442] Using 50% modified bases may partially affect the function of the replicase, but the enhanced tolerability could potentially improve human saRNA vaccines.

[0443] about Figure 5 Female Balb / c mice were immunized intramuscularly on day 0 with either an influenza saRNA vaccine formulation containing varying amounts (0% to 100%) of N1-methylpseuuridine, prepared with 200 ng of LNP, or a comparative influenza modRNA encoding A / Wisconsin / 588 / 2019(H1N1)HA, or HA. Serum cytokines and chemokines were measured 24 hours after the initial immunization using the mouse antiviral response group LEGENDplex assay. Data are presented as median and interquartile range.

[0444] about Figure 6 Female Balb / c mice were immunized on day 0 with either an influenza saRNA vaccine formulation containing varying amounts (0% to 100%) of N1-methylpseuuridine, prepared with 200 ng of LNP, or a modRNA comparator encoding the HA of A / Wisconsin / 588 / 2019 (H1N1). Antibody responses to A / Wisconsin / 588 / 2019 were measured on day 21 (3 weeks post-immunization) by HAI or a 1-day MNT assay. HAI and 50% neutralizing titers (geometric mean plus geometric standard deviation) were reported.

[0445] about Figure 7 Female C57BL6 / J mice were immunized intramuscularly on day 0 with either an influenza saRNA vaccine formulation containing different amounts (0% or 50%) of N1-methylpseuuridine, prepared with 200 ng of LNP, or a comparative influenza modRNA encoding A / Wisconsin / 588 / 2019(H1N1)HA, or interquartile range. Serum cytokines and chemokines were measured 24 hours after the initial immunization using the mouse antiviral response group LEGENDplex assay. Data are expressed as median and interquartile range.

[0446] about Figure 8Female C57BL6 / J mice were immunized on day 0 with either an influenza saRNA vaccine formulation containing different amounts (0% or 50%) of N1-methylpseuuridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA, prepared with 200 ng LNP. Antibody responses against A / Wisconsin / 588 / 2019 were measured on day 21 (3 weeks post-immunization) by HAI or a 1-day MNT assay. HAI and 50% neutralizing titers (geometric mean plus geometric standard deviation) were reported.

[0447] Example 8: Immunogenicity of a tetravalent bicistronic saRNA influenza vaccine encoding HA and NA from four seasonal influenza virus strains

[0448] The major pharmacological effects of the influenza saRNA vaccine were evaluated in in vitro and in vivo nonclinical studies. In vitro and in vivo studies showed that the influenza virus HA and / or NA proteins encoded by the influenza saRNA vaccine induced robust functional and neutralizing antibody responses, as well as strong CD4+ and CD8+ T cell responses, with a significantly lower dose required in mice compared to modRNA. saRNA replication also led to innate immune activation, which may enhance adaptive immune responses to the expressed antigens. The influenza saRNA vaccine demonstrated efficient in vitro expression of HA and NA glycoproteins in cultured cells. Immunogenicity studies in mice, rats, and ferrets showed that different influenza saRNA vaccine formulations elicited strong functional, neutralizing antibody responses, as well as T cell responses. Innate immune activation was also confirmed in mice and rats, as demonstrated by measurements of serum cytokine and chemokine release 24 hours post-immunization. Immunogenicity studies in mice, benchmarked against the influenza modRNA vaccine, also support the use of a bicistronic influenza saRNA construct that expresses two independent influenza antigens (HA and NA) from the same saRNA vector. Immunogenicity studies in mice also showed that saRNAs can tolerate the incorporation of partially modified nucleosides, thereby reducing early innate immune stimulation, while still evoking a strong adaptive humoral response. Finally, immunogenicity studies in mice also support the use of four bicistronic influenza saRNA constructs, each encoding different HA and NA, to target four seasonal influenza virus strains.

[0449] The influenza saRNA vaccine candidates selected for preliminary POC testing contain the full-length, codon-optimized coding sequence of the HA or NA glycoprotein from a viral strain based on A / Wisconsin / 588 / 2019(H1N1) cells, and are recommended for use in the 2021, 2022, and 2022-2023 Northern Hemisphere and 2022 Southern Hemisphere influenza seasons.

[0450] Table 12. saRNA preparations

[0451]

[0452]

[0453] Using four bicistronic saRNA components, a seasonal influenza saRNA vaccine expressing four different HA and four different NA proteins can be prepared to match the dominant circulating influenza virus strains for each season. The feasibility of the tetravalent bicistronic saRNA approach was evaluated in a mouse immunogenicity study, compared to an adjuvanted seasonal tetravalent influenza vaccine (QIV; FluAd) approved in the Northern Hemisphere in 2021–22. BALB / c mice were immunized intramuscularly on days 0 and 28 with either a total dose of 0.8 μg of the tetravalent saRNA vaccine (0.2 μg of each component) or 2.4 μg of the approved QIV comparator. On day 42 (two weeks after the second immunization), HAI and MNT measurements showed that anti-HA antibody responses against each of the four components were induced. Figure 9 Furthermore, measurements of NAI revealed that it also induced an anti-NA antibody response. Figure 10 The tetravalent bicistronic saRNA vaccine candidate achieved HA and NA titers similar to or higher than those of the QIV comparative.

[0454] about Figure 9 Female Balb / c mice were immunized on day 0 by intramuscular injection with either 20 ng of a tetravalent saRNA formulated with LNP, consisting of four bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage), or 2.4 μg of an approved adjuvanted tetravalent inactivated vaccine (QIV; FluAd). Antibody responses against each vaccine component were measured on day 42 (2 weeks after the second dose) by HAI or a 1-day MNT assay. HAI and 50% neutralizing titers (geometric mean plus geometric standard deviation) were reported.

[0455] about Figure 10Female Balb / c mice were immunized intramuscularly on day 0 with either 20 ng of a tetravalent saRNA formulated with LNP, consisting of four bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage), or 2.4 μg of an approved adjuvanted tetravalent inactivated vaccine (QIV; FluAd). Antibody responses against each vaccine component were measured by NAI on day 42 (2 weeks after the second dose). NAI titers (geometric mean plus geometric standard deviation) for 3 of the 4 viral strains were reported. Due to technical issues with the NAI assay for H3N2 virus strains, the H3N2 NAI titers for both saRNA and QIV cannot be reported.

[0456] Example 9: Effects in the human body

[0457] C4861001 is an ongoing Phase 1 FIH study designed to evaluate the safety, tolerability, and immunogenicity of PF-07845104. As of the data cutoff date of November 15, 2022, 253 participants were randomized, of whom 248 received the vaccine. Five participants were unvaccinated (one from the 2.5 μg vaccine formulation 4, two from the 2.5 μg vaccine formulation 5, one from the 10 μg vaccine formulation 5, and one from the 2.5 μg vaccine formulation 6). The approved vaccine QIV was used as a comparison.

[0458] Safety and efficacy - Study C4861001

[0459] C4861001 is an ongoing Phase I FIH study, a randomized, placebo-controlled, observer-blinded, sponsor-unblinded, dose-exploration, and vaccine composition / formulation screening study in healthy adults. This study evaluates the safety, tolerability, and immunogenicity of single-dose monocistronic and ultimately bicistronic saRNA vaccine formulations against influenza. Participants aged 18 to 49 years were randomized in a 4:1 ratio to receive either the saRNA vaccine formulation or a placebo. An independent cohort of participants received an approved QIV vaccine as a control.

[0460] Table 13 provides details of the saRNA vaccine formulation.

[0461] Table 13. Vaccine Formulation Details

[0462]

[0463] Abbreviations: 50pU = 50% N1-methylpseudouridine; 50U = 50% uridine; HA = hemagglutinin; NA = neuraminidase; poly A = polyadenylate; SGP = subgenomic promoter; TRD = Trinidad donkey strain.

[0464] Note: This list is not exhaustive, and the formulations may be added or removed.

[0465] Vaccine formulations 1 and 2 and the control group - randomized participants

[0466] A total of 36 participants were randomly assigned to vaccine preparation group 1 and vaccine preparation group 2, 63 participants were assigned to placebo group, and 33 participants were assigned to control group.

[0467] Vaccine preparation group 1

[0468] In this group, 11 participants received the 1 μg vaccine, 13 participants received the 2.5 μg vaccine, and 12 participants received the 10 μg vaccine. One participant was randomly assigned to receive the 2.5 μg vaccine but was mistakenly given the 1 μg dose; therefore, for safety analysis purposes, this participant was included in the 1 μg group.

[0469] Five participants (one from the 1 μg group, two from the 2.5 μg group, and two from the 10 μg group) withdrew from the study after vaccination.

[0470] Two sets of vaccine formulations

[0471] In this group, 12 participants received 1 μg, 2.5 μg, and 10 μg of the vaccine, respectively.

[0472] One participant from the 1μg group lost follow-up after vaccination.

[0473] Vaccine formulations 3, 4, 7 and the control group - randomized participants

[0474] A total of 36 participants were randomly assigned to vaccine formulation 3, vaccine formulation 4, and vaccine formulation 7, while 63 participants were assigned to the placebo group. One participant from the vaccine formulation 4 2.5 μg group did not receive the vaccine.

[0475] 3 groups of vaccine preparations

[0476] In this group, 12 participants received 1 μg, 2.5 μg, and 10 μg of the vaccine, respectively. Two participants from the 1 μg group withdrew from the study after vaccination.

[0477] 4 groups of vaccine preparations

[0478] In this group, 12 participants received 1 μg and 10 μg of vaccine, and 11 participants received 2.5 μg of vaccine.

[0479] Two participants (one from the 1 μg group and one from the 10 μg group) withdrew from the study after receiving the vaccine.

[0480] 7 groups of vaccine preparations

[0481] In this group, 12 participants received 1 μg, 2.5 μg, and 10 μg of the vaccine, respectively.

[0482] Vaccine formulations 5 and 6 and the control group - randomized participants

[0483] A total of 38 participants were randomly assigned to vaccine formulation group 5, 35 participants to vaccine formulation group 6, 63 participants to the placebo group, and 33 participants to the control group. Three participants from vaccine formulation group 5 (two from the 2.5 μg group and one from the 10 μg group) and one participant from vaccine formulation group 6 (the 2.5 μg group) did not receive the vaccine.

[0484] 5 groups of vaccine preparations

[0485] In this group, 12 participants received either 1 μg or 2.5 μg of the vaccine, and 11 participants received 10 μg of the vaccine. Two participants from the 2.5 μg group and one participant from the 10 μg group did not receive the vaccine.

[0486] Two participants from the 1μg group withdrew from the study after receiving the vaccine.

[0487] 6 groups of vaccine preparations

[0488] In this group, 11 participants received either 1 μg or 2.5 μg of the vaccine, and 12 participants received 10 μg of the vaccine. One participant from the 2.5 μg group did not receive the vaccine.

[0489] Five participants (one from the 1 μg group and four from the 2.5 μg group) withdrew from the study after vaccination.

[0490] Immunogenicity

[0491] A total of 253 participants were randomly assigned to receive the vaccine, of whom 196 were evaluable for immunogenicity.

[0492] Four weeks after vaccine administration, a dose-dependent increase in HAI GMT was observed. Figure 11 , Figure 12 and Figure 13The HAI GMT is shown on day 1 (before immunization) and at 1, 2 and 4 weeks after vaccination.

[0493] Vaccine formulations 1 and 2

[0494] The proportion of participants who achieved seroconversion 4 weeks after receiving 10 μg of vaccine was higher than that of participants who achieved seroconversion after receiving 1 μg and 2.5 μg of vaccine (Table 14).

[0495] Vaccine formulations 3, 4 and 7

[0496] In the three vaccine formulation groups, the proportion of participants who achieved seroconversion 4 weeks after receiving the 10 μg vaccine was higher than the proportion of participants who achieved seroconversion after receiving the 1 μg and 2.5 μg vaccines (Table 15).

[0497]

[0498]

[0499]

[0500] In the four vaccine formulation groups, the proportion of participants who achieved seroconversion 4 weeks after receiving 1 μg of vaccine was higher than the proportion of participants who achieved seroconversion after receiving 2.5 μg and 10 μg of vaccine (Table 12).

[0501] In the 7 vaccine formulation groups, the proportion of participants who achieved seroconversion 4 weeks after receiving 1 μg of vaccine was the same as the proportion of participants who achieved seroconversion after receiving 2.5 μg of vaccine (Table 12).

[0502] Vaccine formulations 5 and 6

[0503] In vaccine formulation group 5, no participants achieved seroconversion after 4 weeks. In vaccine formulation group 6, the proportion of participants achieving seroconversion 4 weeks after receiving the 10 μg vaccine was higher than the proportion of participants achieving seroconversion after receiving the 1 μg and 2.5 μg vaccines (Table 13).

[0504] Example 10: Enhancing the efficacy of saRNA influenza virus through trans-co-delivery of MODRNA-NS1

[0505] We used a saRNA platform based on the Venezuelan equine encephalitis A virus genome, in which structural genes under the control of a subgenomic promoter (SGP) were replaced by vaccine antigens. We used influenza hemagglutinin (HA) derived from A / Wisconsin / 588 / 2019 (H1N1) as the vaccine antigen of interest. Upon delivery to cells, the saRNA expresses RNA-dependent RNA polymerase (RDRP), which replicates RNA and produces multiple copies of the gene expressed under the SGP. A potential strategy to evade innate immune response that may be induced upon saRNA delivery is based on the use of proteins capable of effectively inhibiting interferon (IFN)-induced and / or signaling cascades, similar to the mechanisms by which various RNA viruses evade innate immune recognition. In this study, unless explicitly stated otherwise, we used the influenza virus NS1 antigen (a well-known and effective IFN antagonist) from A / Puerto Rico / 08 / 1934 (H1N1) (AF389122 from Genbank (NCBI)) to enhance in vitro saRNA expression in HeLa cells (a cell line sensitive to saRNA). The NS1 sequence was codon-optimized by a commercial supplier. Our data show that trans-co-delivery of small amounts of NS1 in modRNA form significantly increased saRNA-HA expression levels and helped maintain overall cell viability. These results provide evidence that IFN antagonist proteins can effectively block naturally induced innate immune responses from saRNA and may potentially be used to enhance the immunogenicity and efficacy of saRNA vaccines. Figure 14 The study showed that trans-addition of modNS1 increased saRNA HA expression and cell viability in HeLa cells. As used herein, “modNS1” refers to a polynucleotide encoding the NS1 protein, wherein the polynucleotide contains N1-methylpseuuridine (m1ψ). Figure 14 The increased expression of saRNA antigen and cell viability following trans-co-delivery of modNS1 were depicted. HeLa cells were transfected with incremental doses of modNS1 or modGFP (as a control) in combination with 25 ng saRNA-HA-Wisconsin. Cell viability was assessed at 24 h by measuring the percentage of HA-positive cells (%), protein expression level (MFI), and total cell count. Confocal images (10x magnification) of HeLa cells transfected alone with 25 ng saRNA-HA-Wisconsin (left panel) or in combination with 25 ng modNS1 (right panel). Cells were fixed at 24 h and stained with FI6 HA human monoclonal antibody and goat anti-human A647 secondary antibody. We also showed that low doses of modNS1 were sufficient to increase HA expression of saRNAs containing unmodified or modified nucleosides. See also Figure 15 , Figure 16 and Figure 17 These figures illustrate how low doses of modNS1 increase antigen expression in both conventional saRNA and saRNA containing 50% m1ψA. A total of 25 ng of saRNA-HA-Wisconsin was transfected into HeLa cells in combination with low increments of modNS1 (1 ng to 25 ng) (blue, i.e., “RMM59+NS1”) or saRNA (as a matched dose level control) (red, i.e., “RMM59+(X)RMM59”). Cells were fixed at 24 h and stained with FI6 HA human monoclonal antibody and goat anti-human A488 secondary antibody. The percentage of HA-positive cells (%) and cell number were measured. Confocal images (10x magnification) of HeLa cells transfected alone or in combination with 25 ng of modNS1 were observed (data not shown). Table 17 summarizes the transfection amount, percentage of HA(+) cells (%), and absolute cell number obtained for each test condition.

[0506] Table 17

[0507]

[0508] *RMM59 → refers to saRNA HA Wisconsin.

[0509] Figure 16 Use saRNA that encodes HA Wisconsin but contains 50% m1ψ, such as Figure 15 The same conditions were tested as described in (and related Table 17), such as Figure 16 As shown in Table 18.

[0510] Table 18

[0511]

[0512]

[0513] *RMM59 → refers to saRNA HA Wisconsin.

[0514] Example 11: The presence of modNS1 and the expression of saRNA antigen in human monocytes

[0515] Figure 17Data showed that saRNA replication in human monocytes could be improved. Human CD14+ cells (stem cells) were transfected with different doses of GFP-expressing saRNA (5 ng to 100 ng) in combination with escalating doses of modNS1 (0 to 50 ng), with modRNA GFP used as a control. The total percentage of positive cells (%) and cell viability were measured by FACS at 24 hours. Figure 18 The total percentage (%) of positive cells is shown as measured using Aqua live / dead cell staining.

[0516] Example 12: Trans delivery modNS1 is superior to cis (bicistronic) delivery.

[0517] Figure 19 Figure A shows that the combination of monocistronic saRNA and modNS1, compared to the bicistronic approach, helps increase HA levels. Figure A illustrates transfection of HeLa cells with monocistronic saRNA expressing HA or bicistronic saRNA expressing both HA and NS1, either alone or in combination with 10 ng or 25 ng of modNS1. The percentage of HA-positive cells (%) and protein expression level (MFI) were measured. Cells were fixed at 24 hours and stained with rabbit polyclonal antibody against HA and goat anti-rabbit A647 secondary antibody. Figure 19 Figure B in the middle: as shown Figure 19 Figure A shows a schematic diagram of different saRNA conformations (monocistan and bicistronic) alone or in combination with modNS1.

[0518] Example 13: modNS1 increases antigen expression levels of uRNA but not MODRNA

[0519] Figure 20 The AB plot shows that NS1 can increase HA antigen expression in RNA containing conventional uridine. Under each condition, a total constant amount of RNA of 100 ng was added to HeLa cells. Different ratios of U RNA (i.e., unmodified RNA) or a combination of conventional modRNA and modNS1 or modGFP (control) were added, and the percentage of HA(+) cells was assessed at 24 hours. Figure 20 (Figure A) and cell number ( Figure 20 (Figure B in the middle). Confocal images of HeLa cells were observed, and the combination of 100 ng RMM71 (uRNA HAWisconsin) and 50 ng uRNA with 50 ng modNS1 was compared (data not shown). Cells were fixed at 24 hours and stained with FI6 HA human monoclonal antibody and goat anti-human A647 secondary antibody. Figure 20 Figure C: A schematic diagram comparing different configurations tested.

[0520] Example 14: Re-delivery of modNS1

[0521] Trans-delivery of modNS1 significantly increased saRNA antigen expression and helped maintain cell viability. See also Figure 21 .

[0522] Further findings based on this disclosure include: the combination of modNS1 with saRNA containing 50% modified nucleosides promotes more efficient antigen expression and helps maintain cell viability. modNS1 increases antigen expression of uRNA but not modRNA, suggesting that NS1 plays a role in preventing uridine-induced activation of cellular innate immune responses. Furthermore, modNS1 does not increase saRNA antigen expression in human monocytes.

[0523] Example 15: Immunogenicity of the octavalent influenza HA / NA saRNA vaccine in mice

[0524] This study aimed to test the immunogenicity of an octavalent saRNA-LNP vaccine encoding HA and NA antigens from four influenza virus strains in a mouse model. The saRNA constructs used in this study encode hemagglutinin (HA) and / or neuraminidase (NA) proteins from influenza virus strains A / Wisconsin / 588 / 2019, A / Cambodia / e0926360 / 2020, B / Phuket / 3073 / 2013, or B / Washington / 02 / 2019. Compared to the approved comparative vaccine FluAd, the octavalent saRNA-LNP vaccine induced comparable or higher levels of functional anti-HA, anti-NA, and virus-neutralizing antibodies.

[0525] The octavalent vaccine formulation, composed of monocistronic or bicistronic saRNA constructs, exhibited comparable immunogenicity in mice after two doses. The multivalent saRNA vaccine, mixed before (“premix”) or after (“postmix”) formulation with LNP, also demonstrated comparable immunogenicity in mice. Compared to mice treated with a monocistronic single antigen control, the octavalent saRNA-LNP vaccine did indeed produce moderate interference against certain viral strains, particularly influenza B virus strains. Overall, these data support continued evaluation of multivalent saRNA vaccines encoding influenza virus antigens.

[0526] The primary objective of this study was to evaluate the immunogenicity of an octavalent saRNA vaccine encoding hemagglutinin (HA) or neuraminidase (NA) proteins from influenza viruses in mice, compared with monocistronic and bicistronic saRNA vaccine controls. The octavalent vaccine consisted of eight monocistronic HA or NA saRNA constructs or four bicistronic HA-NA saRNA constructs. The octavalent saRNA vaccine was also compared with an approved quadrivalent inactivated influenza virus vaccine (containing eight HA / NA antigens from four viral strains) and a quadrivalent modified RNA (modRNA)-LNP vaccine encoding four HA proteins. A secondary objective of this study was to compare the immunogenicity of the octavalent vaccine constructs combined with lipid nanoparticles (LNPs) before or after formulation in mouse models.

[0527] This study tested saRNA (TC83-delkozak-80A) constructs encoding either a single HA or NA (monocistronic) protein, or both HA and NA (bicistronic) proteins, derived from H1N1 A / Wisconsin / 588 / 2019, H3N2A / Cambodia / e0926360 / 2020, B / Yam B / Phuket / 3073 / 2013, or B / Vic B / Washington / 02 / 2019. Octavalent formulations containing eight monocistronic (TC83-delkozak-HA-80A or TC83-delkozak-NA-80A) or four bicistronic (TC83-delkozak-HA-SGP-NA-80A) saRNAs formulated in LNPs were tested in mice. The octavalent saRNA vaccine is premixed before formulation in LNP (premix) or postmixed after formulation of each saRNA construct in LNP (postmix). This study included two alternative quadrivalent vaccine controls: a nucleoside-modified RNA (modRNA)-LNP vaccine encoding four HA proteins, and the approved comparative vaccine FluAd, composed of four inactivated influenza viruses. Monocistronic and bicistronic saRNA-LNP vaccines for each viral strain were also included as controls to assess any potential interference with antibody response observed in the octavalent vaccine formulation.

[0528] This study was designed with 20 groups, as shown in Table 19, each containing a total of 10 female mice (Balb / c strain). The study protocol assays used in this study are recorded below.

[0529] Table 19 Research Design [...

Claims

1. A composition comprising a first RNA molecule encoding a target or purpose gene derived from an influenza virus and a second RNA molecule encoding an influenza virus nonstructural (NS1) protein, wherein the first polynucleotide is self-amplifying RNA.

2. The composition according to claim 1, wherein the second RNA molecule comprises modified nucleotides.

3. The composition according to any one of claims 1-2, wherein the second RNA molecule does not contain a subgenomic promoter derived from an alphavirus.

4. The composition of claim 3, wherein the self-amplifying RNA comprises unmodified nucleotides.

5. The composition of claim 3, wherein the self-amplifying RNA comprises modified nucleotides.

6. The composition according to claim 5, wherein less than about 50% of the nucleic acid in the self-amplified RNA is modified nucleotides.

7. The composition of claim 7, wherein the first RNA molecule comprises: a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from alpha virus; a first subgenome promoter derived from alpha virus; a first open reading frame encoding a first target gene derived from influenza virus hemagglutinin (HA); a second subgenome promoter derived from alpha virus; a second open reading frame encoding a second target gene derived from influenza virus; a 3' untranslated region (3'UTR); and a 3' polyadenylate sequence, wherein the second target gene derived from influenza virus is an influenza virus non-structural (NS1) protein.

8. The composition according to claim 7, further comprising an IRES sequence.

9. The composition according to any one of claims 1-8, wherein the first RNA molecule and the second RNA molecule are not connected.

10. The composition according to any one of claims 1-9, wherein the influenza virus NS1 is selected from the group consisting of: influenza A virus NS1, influenza B virus NS1, influenza C virus NS1, and variants thereof.

11. The composition according to any one of claims 1-10, wherein the influenza virus NS1 is selected from the group consisting of: H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, and any combination thereof.

12. The composition according to any one of claims 1-11, wherein the influenza virus NS1 is encoded by an amino acid sequence having at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

33.

13. The composition according to any one of claims 1-12, wherein the expression of the target mRNA is increased relative to the expression of the target mRNA in the absence of a nucleic acid molecule encoding the influenza virus NS1 protein.

14. The composition according to any one of claims 1-13, wherein the expression of the target mRNA is increased by at least about 10% relative to the expression of the target mRNA in the absence of a nucleic acid molecule encoding the influenza virus NS1 protein.

15. The composition according to any one of claims 1-14, wherein the increase in expression of the target mRNA lasts for at least about 6 hours.

16. The composition according to any one of claims 1-15, wherein the RNA molecule is encapsulated in lipid nanoparticles.

17. The immunogenic composition according to any one of claims 1-16.

18. A method for expressing a target mRNA in a cell, comprising delivering the composition according to any one of claims 1-17 into the cell.

19. A method for expressing a target or objective gene derived from an influenza virus in a subject in need, comprising administering to the subject the composition according to any one of claims 1-18.

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