Nucleic acids and their use

Abstract

This specification discloses a self-amplifying RNA (saRNA) molecule encoding an influenza virus antigen and a method of using the same molecule. [Figure 1] TIFF2026514290000060.tif76164

Description

[Technical Field]

[0001] Cross-referencing with the application This application claims the interests of U.S. Provisional Patent Application No. 63 / 484,186, filed on 9 February 2023, U.S. Provisional Patent Application No. 63 / 484,747, filed on 13 February 2023, and U.S. Provisional Patent Application No. 63 / 621,102, filed on 15 January 2024, each of which is incorporated herein by reference in its entirety.

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

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

[0004] The genome of influenza A virus contains eight linear, negatively polarized, single-stranded RNA molecules (seven for influenza C virus), which encode several polypeptides, including RNA-dependent RNA polymerase proteins (PB2, PB1, and PA), nucleoproteins (NP) that form the nucleocapsid, matrix proteins (M1, M2, which are also surface-exposed proteins 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).

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

[0006] Challenges with conventional vaccines for treating and preventing influenza and other infectious diseases include the limited scope of vaccines, which provide protection only against closely related subtypes. Furthermore, the length of time required to complete current standard influenza virus vaccine manufacturing processes hinders the rapid development and production of suitable vaccines in pandemic situations. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] An improved composition, preferably an immunogenic composition, is needed for combating influenza. [Means for solving the problem]

[0008] In particular, an unaddressed need for improved compositions, preferably immunogenic compositions, against influenza is presented herein. In one embodiment, the disclosure relates to a composition comprising a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from an alphavirus; a first subgenome promoter derived from an alphavirus; a first open reading frame encoding a first target gene derived from influenza virus hemagglutinin (HA); a second subgenome promoter derived from an alphavirus; a second open reading frame encoding a second target gene derived from an influenza virus; a 3' untranslated region (3'UTR); and a self-amplified RNA (saRNA) comprising a 3' poly-A sequence.

[0009] In another aspect, the disclosure relates to a composition comprising a self-amplified RNA (saRNA) including a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from an alphavirus; a subgenome promoter derived from an alphavirus; an open reading frame encoding a gene of interest derived from an influenza virus; a 3' untranslated region (3'UTR); and a 3' poly-A sequence, wherein at least 5% of the entire population of specific nucleotides within the molecule are replaced by one or more modified or non-native nucleotides.

[0010] In preferred embodiments, the saRNA polynucleotide has clinical-grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%. In some embodiments, the integrity of the purified RNA polynucleotide is determined by known methods such as capillary electrophoresis to be 60% or higher, 70% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. In some embodiments, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition, at least one of these, up to one of these, or between two of these, are full-length RNA transcripts. A “full-length” RNA molecule includes a 5' cap and a polyA tail.

[0011] In some embodiments, the disclosure provides an isolated polynucleotide (e.g., a replicon) or set of polynucleotides comprising a first polynucleotide encoding an innate immune inhibitor, such as an influenza non-structural (NS1) protein, and a second polynucleotide encoding a heterologous target mRNA, such as an influenza HA protein. Such polynucleotides can drive enhanced and sustained expression of the heterologous target mRNA in cells.

[0012] Some aspects of this disclosure relate to a polynucleotide or set of polynucleotides comprising a first nucleic acid molecule encoding an influenza non-structural (NS1) protein and a second nucleic acid molecule encoding a heterologous target mRNA.

[0013] In some embodiments, a first nucleic acid molecule encoding the influenza NS1 protein and a second nucleic acid molecule encoding the target mRNA are located within the first vector (which may be referred to herein as being in the "cis" state). In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein is located within the first vector, and the second nucleic acid molecule encoding the target mRNA is located within the second vector (which may be referred to herein as being in the "trans" state).

[0014] In some embodiments, a first nucleic acid molecule encoding the influenza NS1 protein is expressed under the control of a first promoter, such as a subgenome promoter derived from an alphavirus. In some embodiments, a second nucleic acid molecule encoding the target mRNA is expressed under the control of a second promoter, such as a subgenome promoter derived from an alphavirus. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different.

[0015] In some embodiments, a first nucleic acid molecule encoding the influenza NS1 protein and a second nucleic acid molecule encoding the target mRNA are expressed under the control of a first promoter, where the first promoter drives the expression of both the influenza NS1 protein and the target mRNA. In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are linked by an IRES sequence. In some embodiments, the first vector, the second vector, or both contain one or more regulatory elements.

[0016] In some embodiments, the expression 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 NS1 protein. In some embodiments, 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 NS1 protein. In some embodiments, the increased expression of the target mRNA persists for at least approximately 6 hours, at least approximately 12 hours, at least approximately 18 hours, at least approximately 24 hours, at least approximately 30 hours, at least approximately 36 hours, at least approximately 42 hours, or at least approximately 48 hours.

[0017] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. The invention can be better understood by referring to one or more of these figures in combination with a detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0018] [Figure 1A] This graph shows functional anti-HA antibodies elicited by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by HAI. Figure 1A shows the results for 3 weeks after priming. Female Balb / c mice were immunized on day 0 with 20 ng of LNP-formulated bicistronic saRNA vaccine preparation, 20 ng of LNP-formulated monocistronic saRNA vaccine preparation, 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 encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody response to A / Wisconsin / 588 / 2019 was measured on day 21 (3 weeks after immunization) by HAI or 1-Day MNT assay. HAI titers are reported (geometric mean and geometric SD). [Figure 1B] This graph shows functional anti-HA antibodies elicited by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by HAI. Figure 1B shows the results two weeks after boost. Female Balb / c mice were immunized on day 0 with 20 ng of LNP-formulated bicistronic saRNA vaccine preparation, 20 ng of LNP-formulated monocistronic saRNA vaccine preparation, 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 encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody response to A / Wisconsin / 588 / 2019 was measured on day 21 (three weeks after immunization) by HAI or 1-Day MNT assay. HAI titers are reported (geometric mean and geometric SD). [Figure 2A]This graph shows the neutralizing antibodies elicited by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by 1-Day MNT. Figure 2A shows the results for 3 weeks after priming. Female Balb / c mice were immunized on day 0 with 20 ng of LNP-formulated bicistronic saRNA vaccine preparation, 20 ng of LNP-formulated monocistronic saRNA vaccine preparation, 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 encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody response to A / Wisconsin / 588 / 2019 was measured on day 21 (3 weeks after immunization) by HAI or 1-Day MNT assay. 50% neutralizing titers are reported (geometric mean and geometric SD). [Figure 2B] This graph shows the neutralizing antibodies elicited by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by 1-Day MNT. Figure 2B shows the results two weeks after boost. Female Balb / c mice were immunized on day 0 with 20 ng of LNP-formulated bicistronic saRNA vaccine preparation, 20 ng of LNP-formulated monocistronic saRNA vaccine preparation, 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 encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody response to A / Wisconsin / 588 / 2019 was measured on day 21 (three weeks after immunization) by HAI or 1-Day MNT assay. 50% neutralizing titers are reported (geometric mean and geometric SD). [Figure 3] This graph shows the neutralizing antibodies extracted by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by 3-Day MNT. [Figure 4A]This graph shows functional anti-NA antibodies elicited by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by NAI. Figure 4A shows the results three weeks after priming. Female Balb / c mice were immunized on day 0 with 20 ng of LNP-formulated bicistronic saRNA vaccine preparation, 20 ng of LNP-formulated monocistronic saRNA vaccine preparation, 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 encoding A / Wisconsin / 588 / 2019(H1N1)NA. Antibody response to A / Wisconsin / 588 / 2019 was measured by NAI on day 21 (three weeks after immunization). Geometric mean titers and geometric standard deviations are reported. [Figure 4B] This graph shows functional anti-NA antibodies elicited by immunization of mice with LNP-formulated saRNAs encoding influenza HA and / or NA, as measured by NAI. Figure 4B shows the results two weeks after boost. Female Balb / c mice were immunized on day 0 with 20 ng of LNP-formulated bicistronic saRNA vaccine preparation, 20 ng of LNP-formulated monocistronic saRNA vaccine preparation, 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 encoding A / Wisconsin / 588 / 2019(H1N1)NA. Antibody response to A / Wisconsin / 588 / 2019 was measured by NAI on day 21 (three weeks after immunization). Geometric mean titers and geometric standard deviations are reported. [Figure 5] This graph shows serum cytokines and chemokines 24 hours after immunization of Balb / c mice with influenza saRNA-HA vaccine preparations containing various amounts of modified nucleosides. [Figure 6]This graph shows functional HAIs and neutralizing antibodies elicited by immunization of Balb / c mice with LNP-formulated saRNA-HA vaccine preparations containing various amounts of modified nucleosides. [Figure 7] This graph shows serum cytokines and chemokines 24 hours after immunization of C57BL6 / J mice with influenza saRNA-HA vaccine preparations containing various amounts of modified nucleosides. [Figure 8] This graph shows the functional HAI and neutralizing antibodies elicited by immunization of C57NL6 / J mice with LNP-formulated saRNA-HA vaccine preparations containing varying amounts of modified nucleosides. Female Balb / c mice were immunized on day 0 using either 20 ng of LNP-formulated tetravalent saRNA, which consists 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 adjuvant-added tetravalent inactivated vaccine (QIV; FluAd). On day 42 (two weeks after the second dose), antibody responses to each vaccine component were measured by HAI or 1-Day MNT assay. HAI and 50% neutralizing titers are reported (geometric mean and geometric SD). [Figure 9] This graph shows functional HAIs and neutralizing antibodies extracted by immunization of mice with LNP-formulated quadrivalent bicistronic saRNAs encoding HA and NA from four seasonal influenza strains. [Figure 10]This graph shows functional NAI antibodies elicited by immunization of mice using LNP-formulated quadrivalent bicistronic saRNA encoding HA and NA from four seasonal influenza strains. Female Balb / c mice were immunized on day 0 with either 20 ng of LNP-formulated quadrivalent saRNA composed 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 with 2.4 μg of an approved adjuvant-added quadrivalent inactivated vaccine (QIV; FluAd). On day 42 (two weeks after the second dose), antibody responses to each vaccine component were measured by NAI. NAI titers have been reported for three of the four strains (geometric mean and geometric SD). Due to technical issues with the NAI assay for H3N2, it was not possible to report either saRNA or QIV titers. [Figure 11] Geometric mean titer and 95% CI:HAI - Vaccine preparations 1, 2, and control group - Graph showing evaluable populations for immunogenicity. Abbreviations: GMT = Geometric mean titer; HAI = Hemagglutination inhibition; QIV = Quadrivalent influenza vaccine; Vax Prep = Vaccine preparation. Note: V1 = Before vaccination on day 1; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks. Note: Dots indicate individual antibody levels. Note: The number / GMT in each bar indicates the number of participants with valid and definitive assay results for the specified assay at a given sampling time, and the corresponding geometric mean titer. The mean of two samples taken before vaccination on day 1 was used to calculate GMT. Note: Approved QIV-15A includes participants who received approved QIV and whose VRD data was tested concurrently with the VRD data test for group C1. Note: Approved QIV-18 includes participants who received approved QIV and whose VRD data was tested concurrently with the VRD data test for groups C2-C5 and C7. Note: Placebo includes trial participants who were randomly assigned to receive a placebo. [Figure 12] Geometric mean titer and 95% CI:HAI - vaccine preparations 3, 4, 7, and control group - graph showing evaluable populations for immunogenicity. Abbreviations: GMT = Geometric mean titer; HAI = Hemagglutination inhibition; QIV = Quadrivalent influenza vaccine; Vax Prep = Vaccine preparation. Note: V1 = Before vaccination on day 1; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks. Note: Dots indicate individual antibody levels. Note: The number / GMT in each bar indicates the number of participants with valid and definitive assay results for the specified assay at a given sampling time, and the corresponding geometric mean titer. The mean of two samples taken before vaccination on day 1 was used to calculate GMT. Note: Approved Q1V-18 includes trial participants who received approved QIV, whose VRD data was tested concurrently with the VRD data trials for groups C2-C5 and C7. Note: Placebo includes trial participants who were randomly assigned to receive placebo. [Figure 13] Geometric mean titer and 95% CI: This graph shows the populations that can be evaluated for immunogenicity in relation to HAI-vaccine preparations 5, 6 and the control group. Abbreviations: GMT = Geometric mean titer; HAI = Hemagglutination inhibition; QIV = Quadrivalent influenza vaccine; Vax Prep = Vaccine preparation. Note: VI = Before vaccination on day 1; V3 = 1 week, V4 = 2 weeks; V5 = 4 weeks. Note: Dots indicate individual antibody levels. Note: The number / GMT in each bar indicates the number of participants with valid and definitive assay results for the given assay at a given sampling time, and the corresponding geometric mean titer. The mean of two samples taken before vaccination on day 1 was used to calculate GMT. Note: Approved QIV-18 includes participants who received approved QIV, whose VRD data were tested concurrently with the VRD data testing for groups C2-C5 and C7. Note: Approved QIV-15B includes participants who received approved QIV, whose VRD data were tested concurrently with the VRD data testing for group C6. Note: Placebo includes trial participants who were randomly assigned to receive a placebo. [Figure 14]This graph shows that trans-addition of modNS1 increases saRNA HA expression and cell viability in HeLa cells. HeLa cells were transfected with gradually increasing doses of modNS1 or modGFP (as a control) in combination with 25 ng of saRNA-HA-Wisconsin. The total number of cells for which % HA-positive cells, protein expression levels (MFI), and cell viability were assessed was measured at 24 hours. Throughout this specification, RMM59 refers to saRNA HA Wisconsin. [Figure 15] This graph shows the results of transfecting HeLa cells with a total of 25 ng of saRNA-HA-Wisconsin, either in combination with low-escalation modNS1 (1 ng to 25 ng) for the blue values ​​(i.e., "RMM59 + NS1"), or in combination with saRNA (a dose-balanced control) for the red values ​​(i.e., "RMM59 + (X)RMM59"). Cells were fixed at 24 hours and stained with FI6 HA human monoclonal antibody and goat anti-human A488 secondary antibody. (%) HA-positive cells and cell counts were measured. [Figure 16] This graph shows the results from a test of saRNA encoding HA Wisconsin but containing 50% m1ψ. The test was performed under the same conditions as described in Figure 15. [Figure 17] This graph shows that saRNA replication can be improved in human monocytes. Human CD14+ cells (stem cells) were transfected with GFP-expressing saRNA at different doses (5 ng to 100 ng) in combination with modNS1 at escalating doses (0 to 50 ng), and modRNA-GFP was used as a control. The total percentage of positive cells was measured by FACS at 24 hours, and cell viability was also measured. [Figure 18] This graph shows the total percentage of positive cells measured using Aqua live / dead staining. [Figure 19A]This graph shows the results of transfecting 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. HA-positive cells (%) and protein expression levels (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 19B] Figure 19A shows different saRNA configurations (monocistronic and bicistronic) either alone or in combination with modNS1. [Figure 20A] This graph shows the results of adding a fixed total amount of RNA (100 ng) to HeLa cells under different conditions. U RNA (i.e., unmodified RNA), a combination of normal modRNA and modNS1, or modGFP (control) were added in different ratios, and the number of %HA(+) cells (Figure 20A) and the number of cells (Figure 20B) were assessed at 24 hours. Confocal images in HeLa cells were observed comparing those with 100 ng of RMM71 (uRNA HA Wisconsin) and those with a combination of 50 ng of uRNA and 50 ng of modNS1 (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 20B] This graph shows the results of adding a fixed total amount of RNA (100 ng) to HeLa cells under different conditions. U RNA (i.e., unmodified RNA), a combination of normal modRNA and modNS1, or modGFP (control) were added in different ratios, and the number of %HA(+) cells (Figure 20A) and the number of cells (Figure 20B) were assessed at 24 hours. Confocal images in HeLa cells were observed comparing those with 100 ng of RMM71 (uRNA HA Wisconsin) and those with a combination of 50 ng of uRNA and 50 ng of modNS1 (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 20C]This is a comparison diagram of the different configurations tested. [Figure 21] This graph shows the percentage of HA-positive cells after administration of each active RNA molecule. It further shows the total number of viable cells after administration of each active RNA molecule. See Example 10 for the method. Trans-delivery of modNS1 significantly increases antigen expression by saRNA, promoting the preservation of cell viability. [Figure 22] This graph shows functional anti-HA antibodies extracted in mice after a single dose of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA, as measured by HAI. [Figure 23] This graph shows functional anti-NA antibodies extracted in mice after a single dose of saRNA-LNP or FluAd encoding HA / NA, as measured by NAI. [Figure 24] This graph shows the viral neutralizing antibodies drawn in mice after a single dose of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA, as measured by 1-Day MNT. [Figure 25] This graph shows functional anti-HA antibodies drawn in mice after two doses of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA, as measured by HAI. [Figure 26] This graph shows functional anti-NA antibodies extracted in mice after two doses of saRNA-LNP or FluAd encoding HA / NA, as measured by NAI. [Figure 27] This graph shows the viral neutralizing antibodies drawn in mice after two doses of saRNA-LNP, Quad modRNA, or FluAd encoding HA / NA, as measured by 1-Day MNT. [Modes for carrying out the invention]

[0019] Embodiments of this disclosure provide compositions comprising self-amplified RNA (saRNA) polynucleotides encoding influenza virus antigens. Influenza virus RNA vaccines presented herein can be used to induce a balanced immune response comprising both cellular and humoral immunity.

[0020] Any embodiment discussed herein may be implemented with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the methods of the Disclosure can be implemented using the compositions of the Disclosure.

[0021] Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate specific embodiments of this disclosure, it should be understood that they are provided merely as examples, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description.

[0022] Throughout this application, the term "approximately" is used to indicate a value that includes variations in errors inherent to the measurement or quantification method.

[0023] The use of the words "a" or "an" can mean "one" when used in conjunction with the term "comprising," but it can also mean "one or more," "at least one," and "one or more than one."

[0024] The phrase "and / or" means "and" or "or". For example, A, B, and / or C can include A only, B only, C only, 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".

[0025] The words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude additional, undescribed elements or method steps.

[0026] The phrase "basically all" is defined as "at least 95%", meaning that if basically all members of a group possess a certain trait, then at least 95% of the members of that group possess that trait. In some cases, "basically all" means that 95%, 96%, 97%, 98%, 99%, or 100% of the members of that group, at least one of these, or any two of these, possess that trait.

[0027] Compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this Spec. Compositions and methods "consisting essentially of" any of the disclosed components or steps are limited to designated materials or steps that do not substantially affect the basic and novel features of the claimed disclosure.

[0028] A. Self-amplifying RNA (saRNA) In some embodiments, the RNA molecule, for example, the first RNA molecule, is saRNA. “saRNA,” “self-replicating RNA,” and “replicon” refer to RNA that has the ability to replicate on its own. Self-replicating RNA molecules can be constructed by using replication elements derived from one or more viruses, such as alphaviruses, and substituting a structural viral polypeptide with a nucleotide sequence encoding the polypeptide of interest. Self-replicating RNA molecules are generally positive-chain molecules that can be directly translated after being delivered to a cell, resulting in RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts of the delivered RNA. The delivered RNA results in the production of numerous daughter RNAs. These daughter RNAs, as well as the subgenome transcripts on the same linear chain, can be translated to result in the in situ expression of the target gene they encode, e.g., a viral antigen, or they can be transcribed to result in further transcripts with the same sense as the delivered RNA, which are then translated to result in the in situ expression of the target protein, e.g., an antigen. As a result of this series of transcriptions, the number of introduced saRNAs is amplified, and therefore the gene of interest being encoded, such as a viral antigen, can become the cell's primary polypeptide product.

[0029] In some embodiments, the self-amplifying RNA comprises at least one gene selected from among viral replicases, viral proteases, viral helicases, and other non-structural viral proteins. In some embodiments, the self-amplifying RNA may also comprise 5' and 3' tractive replication sequences, as well as, optionally, heterologous sequences encoding a desired amino acid sequence (e.g., the antigen of interest). A subgenome promoter directing the expression of the heterologous sequence may be included in the self-amplifying RNA. The heterologous sequence (e.g., the antigen of interest) may also be fused in-frame to other coding regions within the self-amplifying RNA and / or placed under the control of an internal ribosome entry site (IRES).

[0030] In some embodiments, the self-amplified RNA molecule is not encapsulated in a virus-like particle. The self-amplified RNA molecules described herein can be designed so that they cannot induce the production of infectious viral particles. This can be achieved, for example, by removing one or more viral genes that encode structural proteins necessary for the production of viral particles in the self-amplified RNA. For example, if the self-amplified RNA molecule is based on an alphavirus, such as Sindbis virus (SIN), Semlyki ​​forest virus, and Venezuelan encephalitis virus (VEE), one or more genes encoding viral structural proteins, such as a capsid and / or envelope glycoprotein, can be removed.

[0031] In some embodiments, the self-amplifying RNA molecule described herein comprises (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., encoding a viral antigen. In some embodiments, the polymerase may be an alphaviral replicase comprising, for example, one of the alphaviral proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof. In some embodiments, the self-amplifying RNA molecule described herein may 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).

[0032] A saRNA construct may encode at least one non-structural protein (NSP) located at the 5' or 3' of the sequence encoding at least one peptide or polypeptide of interest. In some embodiments, the sequence encoding at least one NSP is located at the 5' of the sequence encoding the peptide or polypeptide of interest. Thus, the sequence encoding at least one NSP may be located at the 5' end of the RNA construct. In some embodiments, the at least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4. In some embodiments, the saRNA construct encodes nsP1, nsP2, nsP3, and nsP4. As is known in the art, nsP1 is the membrane anchor of the viral capping enzyme and replication complex (RC). nsP2 is an RNA helicase and protease responsible for ns polyprotein processing. nsP3 interacts with several host proteins and can modulate protein poly-ADP-ribosylation and mono-ADP-ribosylation. nsP4 is the central viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphaviral replicase containing, for example, one or more of the alphaviral proteins nsP1, nsP2, nsP3, and nsP4.

[0033] While the natural alphaviral genome encodes structural virion proteins in addition to non-structural replicase polypeptides, in some embodiments, the self-amplified RNA molecule does not encode alphaviral structural proteins. In some embodiments, the self-amplified RNA can lead to the production of its own genomic RNA copies in cells, but not to the production of RNA containing virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike wild-type alphavirus, the self-amplified RNA molecule cannot persist as an infectious form on its own. The alphaviral structural proteins necessary for persistence in wild-type virus can be omitted from the self-amplified RNA of this disclosure, and one or more genes encoding the immunogen of interest can be placed in their place, so that the subgenomic transcript encodes an immunogen rather than alphaviral structural virion proteins.

[0034] In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame may encode a replicase, and the second (3') open reading frame may encode a polypeptide containing the antigen of interest. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, for example, to encode further antigens or accessory polypeptides.

[0035] In some embodiments, the second RNA or saRNA molecule further comprises (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

[0036] The self-amplifying RNA molecules described herein may also be designed to induce the production of attenuated or toxic infectious viral particles, or to produce viral particles capable of subsequent infections in a single round.

[0037] In some embodiments, the saRNA molecule is based on an alphavirus. Alphaviruses include a range of genetically, structurally, and serologically related arthropod-borne viruses belonging to the family Togaviridae. Exemplary viruses and viral subtypes belonging to the genus Alphavirus include Sindbis virus, Semryqui forest virus, Ross River virus, and Venezuelan encephalitis virus. Therefore, the self-amplified RNA described herein can incorporate RNA replicases derived from any one of the Alphaviridae viruses: Semryqui forest virus (SFV), Sindbis virus (SIN), Venezuelan encephalitis virus (VEE), Ross River virus (RRV), or other viruses. In some embodiments, the self-amplified RNA described herein can incorporate sequences derived from mutant or wild-type viral sequences; for example, the attenuated TC83 variant of VEEV is used in the saRNA.

[0038] Alphavirus-based saRNAs are (+)-strand saRNAs that, after being delivered to a cell, can be translated, thereby leading to the translation of a replicase (or replicase-transcriptase). The replicase is translated into a polyprotein that self-cleaves, resulting in a replication complex that creates a genomic (-)-strand copy of the delivered (+)-strand RNA. These (-)-strand transcripts can themselves be transcribed, resulting in further copies of the (+)-strand parental RNA, as well as subgenomic transcripts encoding the desired gene product. Thus, translation of the subgenomic transcripts leads to in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs can include replicases derived from Sindbis virus, Semlik Forest virus, Eastern Equine Encephalitis virus, Venezuelan Equine Encephalitis virus, or their variant strains.

[0039] In some embodiments, the self-amplifying RNA molecule is derived from or based on viruses other than alphaviruses, such as positive-strand RNA viruses, particularly picornaviruses, flaviviruses, rubiviruses, pestiviruses, hepaciviruses, caliciviruses, or coronaviruses. Suitable wild-type alphavirus sequences are well known and available from sequencing service providers such as the American Type Culture Collection, Rockville, Md. Representative examples of appropriate alphaviruses include Aura (ATCC VR-368), Beval virus (ATCC VR-600, ATCC VR-1240), Kabasou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern Equine Encephalomyelitis Virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Geta virus (ATCC VR-369, ATCC VR-1243), Kyjilagash (ATCC VR-927), Mayaro (ATCC VR-66), Mayarovirus (ATCC VR-1277), Middleberg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), and Ndum (ATCC VR-371), Pixnavirus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semlik Forest virus (ATCC VR-67, ATCC VR-1247), Sindbisvirus (ATCC VR-68, ATCC VR-1248), Tonate virus (ATCC VR-925), Trinichi virus (ATCC VR-469), Una virus (ATCC VR-374), Venezuelan horse encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250, ATCC VR-1249, ATCC VR-532), Western horse encephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC Examples include VR-1252), Wataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some embodiments, one or more alphaviruses in this list may be excluded.

[0040] In some embodiments, the self-amplified RNA molecules described herein are larger than other types of RNA (e.g., saRNA). Generally, the self-amplified RNA molecules described herein contain at least about 4 kb. For example, the self-amplified RNA may be any one of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 16 kb, at least one of these, up to one of these, or between two of these. In some cases, the self-amplified RNA may contain at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In certain cases, self-amplified RNA is approximately 4kb-12kb, 5kb-12kb, 6kb-12kb, 7kb-12kb, 8kb-12kb, 9kb-12kb, 10kb-12kb, 11kb-12kb, 5kb-11kb, 5kb-10kb, 5kb-9kb, 5kb-8kb, 5kb-7kb, 5kb-6kb, and 6kb. The ranges are approximately b - 12kb, 6kb - 11kb, 6kb - 10kb, 6kb - 9kb, 6kb - 8kb, 6kb - 7kb, 7kb - 11kb, 7kb - 10kb, 7kb - 9kb, 7kb - 8kb, 8kb - 11kb, 8kb - 10kb, 8kb - 9kb, 9kb - 11kb, 9kb - 10kb, or 10kb - 11kb.

[0041] In some embodiments, the self-amplified RNA molecule may encode a single polypeptide antigen, or, optionally, two or more polypeptide antigens linked together such that each sequence retains its identity (e.g., is sequentially linked) when expressed as an amino acid sequence. Thus, polypeptides generated from the self-amplified RNA can be produced as fusion polypeptides, or they can be engineered to produce separate polypeptide or peptide sequences. In some embodiments, the saRNA molecule may encode one polypeptide of interest or more, for example, one antigen or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides. Alternatively, or in addition to this, a single saRNA molecule may encode one or more polypeptides of interest or more, for example, one antigen, and may be a bicistronic or tricistronic RNA molecule encoding different or identical antigens.

[0042] As used herein, the term "linked" refers to the covalent or noncovalent linkage between a first amino acid sequence or polynucleotide sequence and a second amino acid sequence or polynucleotide sequence. The first amino acid or polynucleotide sequence may be directly linked or parallel to the second amino acid or polynucleotide sequence, or the first and second sequences may be covalently linked by an intervening sequence. The term "linked" encompasses not only the fusion of the first RNA molecule at its 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 RNA molecule may be linked to the second RNA molecule by a phosphate diester bond or a linker, which may be, for example, a polynucleotide.

[0043] In some embodiments, the self-amplified RNA described herein may encode one or more polypeptide antigens comprising various epitopes. In some embodiments, the self-amplified RNA described herein may encode epitopes capable of eliciting either a helper T cell response or a cytotoxic T cell response, or both.

[0044] In some embodiments, saRNA molecules are purified by filtration, such as ultrafiltration, diafiltration, or tangential flow ultrafiltration / diafiltration.

[0045] Some embodiments of this disclosure relate to compositions comprising a self-amplifying RNA molecule comprising a 5' cap, a 5' untranslated region, a sequence encoding RNA-dependent RNA polymerase (also referred to as "replicase"), a subgenome promoter such as one derived from an alphavirus, a coding region including an open reading frame encoding a gene of interest (e.g., an antigen derived from an influenza virus), a 3' untranslated region, and a 3' poly-A sequence. In some embodiments, at least 5% of the entire population of specific nucleotides within the saRNA molecule are replaced by one or more modified or non-native nucleotides.

[0046] In some embodiments, the saRNA molecule is free of modified nucleotides, for example, without modified nucleic acid bases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, with the exception of an optional 5' cap which may contain, for example, 7-methylguanosine, the 5' cap is described further below. In some embodiments, the RNA may contain a 5' cap containing 7'-methylguanosine, and the first one, two, or three 5' ribonucleotides may be methylated at the 2' position of ribose.

[0047] The efficacy of the product depends on the expression of the delivered saRNA, requiring a sufficiently intact RNA molecule. RNA integrity is one measure of RNA quality that quantifies intact RNA. The method can also detect potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. Initial specifications are set to ensure sufficient RNA integrity in the drug product preparation. In some embodiments, the RNA polynucleotide has an integrity of at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA polynucleotide has an integrity of about 95% or more. In some embodiments, the RNA polynucleotide has an integrity of about 98% or more. In some embodiments, the RNA polynucleotide has an integrity of about 99% or more.

[0048] In preferred embodiments, the saRNA polynucleotide has clinical-grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%. In some embodiments, the purity of the RNA polynucleotide is between about 80% and about 99%. In some embodiments, the purity of the RNA polynucleotide is between about 90% and about 99%. In some embodiments, the purified mRNA has clinical-grade purity without further purification. In some embodiments, clinical-grade purity is achieved by methods including tangential flow filtration (TFF) purification. In some embodiments, clinical-grade purity is achieved without further purification selected from high-performance liquid chromatography (HPLC) purification, ligand or binding-based purification, and / or ion-exchange chromatography. In some embodiments, the method for producing RNA polynucleotide removes long incomplete RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual solvent, and / or residual salts. In some embodiments, short incomplete transcript contaminants contain fewer than 15 bases. In some embodiments, the short-chain incomplete transcript contaminants contain approximately 8 to 12 bases. In some embodiments, the method of the present invention also removes ribonuclease inhibitors.

[0049] In some embodiments, the purified saRNA polynucleotides, as determined by capillary electrophoresis, contain or are substantially free of protein contaminants of 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. In some embodiments, the purified RNA polynucleotides, as determined by high-performance liquid chromatography (HPLC), contain or are substantially free of salt contaminants of less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the purified RNA polynucleotides, as determined by known methods such as high-performance liquid chromatography (HPLC), contain or are substantially free of short-chain incomplete transcript contaminants of 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. In some embodiments, the purified RNA polynucleotides have an integrity of 60% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, as determined by known methods such as capillary electrophoresis.

[0050] B. Modified nucleic acid bases Modified nucleic acid bases that can be incorporated into modified nucleosides and nucleotides and may be present within 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); g6A(N6-glycinylcarbamoyladenosine); t6A(N6-threonylcarbamoyladenosine); ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamo Iladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p)(2'-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine); m1Im (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); 5-forC (5-phonylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl -2'-O-methylcytidine); k2C (lysidine); 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'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHaW (hydroxywybutosine);OHyWx (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queosine); oQ (epoxyqueosine); galQ (galtactosylqueosine); manQ (mannosylqueosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (alkaeosine); 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);m cm5Um(S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine);nm5s2U(5-aminomethyl-2-thiouridine);mnm5U(5-methylaminomethyluridine);mnm5s2U(5-methylaminomethyl-2-thiouridine);mnm5se2U(5-methylaminomethyl-2-selenouridine);ncm5U(5-carbamoylmethyluridine);ncm5Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm5U(5-carboxy Methylaminomethyluridine); cnmm5Um(5-carboxymethyl-1-aminomethyl-2-LOmethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm(2'-O-methylinosine); m4C(N4-methylcytidine); m4Cm(N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U(3-methyluridine); cm5U(5-carboxymethyluridine); m6Am(N6,TO-dimethyladenosine);rn62Am(N6,N6,0-2-trimethyladenosine);m2,7G(N2,7-dimethylguanosine);m2,2,7G(N2,N2,7-trimethylguanosine);m3Um(3,2-O-dimethyluridine);m5D(5-methyldihydrouridine);f5Cm(5-formyl-2'-O-methylcytidine);m1Gm(1,2'-O-dimethylguanosine);m1Am(1,2-O-dimethyladenosine)irinomethyluridine);tm5s2U(S-tau Linomethyl-2-thiouridine); imG-14(4-demethylguanosine); imG2(isoguanosine); ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkine Ruuracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7 Examples include -deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (debasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. In some embodiments, one or more of the modified nucleosides in the list may be excluded.

[0051] Additional exemplary modified nucleotides include N-1-methylpseudridine; any one of pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some embodiments, the modified nucleotide is N-1-methylpseudridine.

[0052] In some embodiments, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0053] In some embodiments, the RNA molecule contains a modified nucleotide selected from any one of the following: pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of 5-methyluridine, N1-methylpseudridine, 5-methoxyuridine, and 5-methylcytosine.

[0054] In some embodiments, at least 10% of the entire population of a particular nucleotide within the saRNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a particular nucleotide within the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a particular nucleotide within the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a particular nucleotide within the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, essentially all of the population of a particular nucleotide within the molecule is replaced by one or more modified or non-natural nucleotides.

[0055] In some embodiments, at least some or all of the entire population of specific nucleotides within the saRNA molecule are replaced by two modified or non-natural nucleotides. In some embodiments, the two modified or non-natural nucleotides are 1:99;2:98;3:97;4:96;5:95;6:94;7:93;8:92;9:91;10:90;11:89;12:88;13:87;14:86;15:85;16:84;17:83;18:82, 19:81;20:80;21:79;22:78;23:77;24:76;25:75;26:74;2 7:73;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;56:4 4;57:43;58:42;59:41;60:40;61:39;62:38;63:37;64:36;65:35;66:34;67:33;68:32;69:31;70:30;71:29;72:28;73:27;74:26;75:25;76:24;77:23;78:22;79:21;80:20;81:19;82:18;83:17;84:16;85:15;8 The ratios are provided as any one of the ranges from 1:99 to 99:1, including 6:14; 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 that can be derived within those ranges, at least one of these, at most one of these, or a ratio between two of these.

[0056] In some embodiments, at least 10% of the entire population of a first specific nucleotide in the saRNA molecule disclosed herein is replaced by one or more modified or non-natural nucleotides, and at least 10% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 75% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, essentially all of the entire population of a first specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the molecule is replaced by one or more modified or non-natural nucleotides.

[0057] In some embodiments, at least 25% of the entire population of uridine nucleotides in the saRNA molecule is replaced by N1-methylpseudridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule is replaced by N1-methylpseudridine. In some embodiments, at least 75% of the entire population of uridine nucleotides in the molecule is replaced by N1-methylpseudridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced by N1-methylpseudridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule is replaced by 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced by 5-methoxyuridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule is replaced by 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced by 5-methyluridine. In some embodiments, at least 50% of the entire population of cytosine nucleotides in the molecule is replaced by 5-methylcytosine. In some embodiments, virtually all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule are replaced by 2-thiouridine. In some embodiments, virtually all uridine nucleotides in the molecule are replaced by 2-thiouridine.

[0058] In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule is replaced by N1-methylpseudridine, and essentially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule is replaced by 5-methoxyuridine, and essentially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the molecule is replaced by 5-methyluridine, and essentially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine.

[0059] In some embodiments, virtually all uridine nucleotides in the molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudridine.

[0060] C.UTR The 5' untranslated region (UTR) is a regulatory region of DNA located at the 5' end of a protein-coding sequence that is transcribed into mRNA but not translated into protein. The 5'UTR may contain various regulatory elements, such as 5' cap structures, stem-loop structures, and internal ribosome entry sites (IRESs), which may play a role in regulating translation initiation. The 3'UTR is located downstream of the protein-coding sequence and may be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. In some embodiments, the UTR is derived from naturally abundant mRNA in specific tissues targeted for mRNA expression (e.g., lymphoid tissue). In some embodiments, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR can increase protein synthesis by increasing the time mRNA remains in the polysome during translation (message stability) and / or the rate at which ribosomes initiate translation of the message (message translation efficiency). Therefore, the UTR sequence can be used to extend protein synthesis in a tissue-specific manner. In some embodiments, the 5'UTR and 3'UTR sequences are derived by computer. In some embodiments, the 5'UTR and 3'UTR are derived from mRNA that is naturally abundant in the tissue. The tissue may be, for example, the liver, stem cells, or lymphoid tissue. Examples of lymphoid tissue include any one 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 alphaviruses. In some embodiments, the 5'UTR and 3'UTR are derived from wild-type alphaviruses. Examples of alphaviruses are listed below.

[0061] In some embodiments, the first RNA molecule includes 5'UTR and 3'UTR derived from naturally abundant mRNA in tissues. In some embodiments, the first RNA molecule includes 5'UTR and 3'UTR derived from alphavirus. In some embodiments, the second RNA or saRNA molecule includes 5'UTR and 3'UTR derived from alphavirus. In some embodiments, the second RNA or saRNA molecule includes 5'UTR and 3'UTR derived from wild-type alphavirus. In some embodiments, the RNA molecule includes a 5' cap.

[0062] D. Open Reading Frame (ORF) The 5' and 3' UTRs may be operably ligated to an ORF, which may be a sequence of codons that can be translated into the polypeptide of interest. As described above, an RNA molecule may contain one (monocistronic), two (bisistronic), or more (multicistronic) open reading frames (ORFs).

[0063] In some embodiments, the ORF encodes a viral non-structural gene. In some embodiments, the ORF further comprises one or more subgenome promoters. In some embodiments, the RNA molecule comprises a subgenome promoter operably linked to the ORF. In some embodiments, the subgenome promoter comprises a cis-acting regulatory element. In some embodiments, the cis-acting regulatory element is B 2 It is located immediately downstream of (5'-3'). In some embodiments, the cis-acting regulatory element is B 2(5'-3') is located immediately downstream of guanine, which is immediately downstream of . In some embodiments, the cis-acting regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauauau, auauauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug. In some embodiments, the second RNA or saRNA molecule may comprise (i) an ORF encoding a replicase capable of transcribing RNA from the second RNA or saRNA molecule, and (ii) an ORF encoding at least one antigen or polypeptide of interest. The polymerase may be an alphavirus replicase comprising, for example, any one of the alphavirus non-structural proteins nsP1, nsP2, nsP3, and nsP4, or any combination thereof. In some embodiments, the RNA molecule comprises the alphavirus non-structural protein nsP1. In some embodiments, the RNA molecule comprises the alphavirus non-structural protein nsP2. In some embodiments, the RNA molecule includes the alphavirus non-structural protein nsP3. In some embodiments, the RNA molecule includes the alphavirus non-structural protein nsP4. In some embodiments, the RNA molecule includes the alphavirus non-structural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule includes the alphavirus non-structural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4.

[0064] In some embodiments, the open reading frame of an RNA (e.g., saRNA) composition is codon-optimized. In some embodiments, the open reading frame encoding an influenza polypeptide or a fragment thereof is codon-optimized.

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

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

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

[0068] In some embodiments, the at least one antigenic polypeptide is one of the predefined antigenic subdomains of HA, referred to as HA1, HA2, or a combination of HA1 and HA2, as well as at least one antigenic polypeptide selected from neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2).

[0069] In some embodiments, the at least one antigenic polypeptide is HA or a derivative thereof containing an antigenic sequence derived from HA1 and / or HA2, as well as at least one antigenic polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0070] In some embodiments, the antigenic polypeptide is HA or a derivative thereof containing an antigenic sequence derived from HA1 and / or HA2, as well as at least two antigenic polypeptides selected from HA, NA, NP, M1, M2, NS1, and NS2.

[0071] 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 as described herein include, but are 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 influenza NS1 proteins including these Varians.

[0072] In some embodiments, influenza 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, variants thereof, or combinations thereof. In some embodiments, influenza NS1 is H1N2 NS1. In some embodiments, influenza NS1 is H2N2 NS1. In some embodiments, influenza NS1 is H3N2 NS1. In some embodiments, influenza NS1 is H7N9 NS1. In some embodiments, influenza NS1 is H7N7 NS1. In some embodiments, influenza NS1 is H9N2 NS1. In some embodiments, influenza NS1 is H7N2 NS1. In some embodiments, influenza NS1 is H7N3 NS1. In some embodiments, influenza NS1 is H5N2 NS1. In some embodiments, influenza NS1 is H10N7 NS1. In some embodiments, influenza NS1 is H1N1 NS1. In some embodiments, influenza is H1N1 TX91 variant NS1. In some embodiments, influenza NS1 encoded by the first nucleic acid molecule is GenBank(NCBI)AF389122: MDPNTVSSSFQ VDCFLWHVRK RVADQELGDA PFLDRLRRDQ KSLRGRGSTL GLDIETATRA 60 GKQIVERILK EESDEALKMT MASVPASRYL TDMTLEEMSR DWSMLIPKQK VAGPLCIRMD 120 QAIMDKNIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLPSLPGHT AEDVKNAVGV 180 LIGGLEWNDN TVRVSETLQR FAWRSSNENG RPPLTPKQKR EMAGTIRSEV 230 (Sequence ID 33) It includes 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 respect to the amino acid sequence encoded by the nucleotide sequence shown.

[0073] 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.

[0074] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding multiple influenza virus proteins or their immunogenic fragments.

[0075] 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).

[0076] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18 or any combination of any or all of these HA1, HA2, or a combination of both), and at least one other RNA (e.g., saRNA) polynucleotide having an open reading frame encoding a protein selected from HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from influenza virus.

[0077] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding any one 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 combination of any or all of these), and at least two other RNA (e.g., saRNA) polynucleotides having two open reading frames encoding two proteins selected from HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from influenza virus.

[0078] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding any one 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 combination of any or all of these), and at least three other RNA (e.g., saRNA) polynucleotides having three open reading frames encoding three proteins selected from HA protein, NP protein, NA protein, M protein, M2 protein, NS1 protein, and NS2 protein obtained from influenza virus.

[0079] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding any one 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 combination of any or all of these), and at least four other RNA (e.g., saRNA) polynucleotides having four open reading frames encoding four proteins selected from HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from influenza virus.

[0080] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding any one 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 combination of any or all of these), and at least five other RNA (e.g., saRNA) polynucleotides having five open reading frames encoding five proteins selected from HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from influenza virus.

[0081] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding the HA protein or an immunogenic fragment thereof obtained from the influenza virus (e.g., at least one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any combination thereof), the HA protein, the NP protein or an immunogenic fragment thereof, the NA protein or an immunogenic fragment thereof, the M1 protein or an immunogenic fragment thereof, the M2 protein or an immunogenic fragment thereof, the NS1 protein or an immunogenic fragment thereof, and the NS2 protein or an immunogenic fragment thereof.

[0082] In some embodiments, the influenza RNA composition includes saRNA encoding an antigenic fusion protein. Thus, the encoded antigen(s) may comprise two or more conjugated proteins (e.g., a protein and / or protein fragments). Alternatively, the protein fused with the protein antigen may not promote a potent immune response to itself, but it may promote a potent immune response to the influenza antigen. In some embodiments, the antigenic fusion protein retains the functional properties derived from each of the original proteins.

[0083] F.5' Cap In some embodiments, the saRNA molecules described herein include a 5' cap. In some embodiments, the 5' cap portion is a natural 5' cap.

[0084] A “natural 5' cap” is defined as a cap containing 7-methylguanosine attached to the 5' end of an mRNA molecule via a 5'-5' triphosphate linkage. In some embodiments, the 5' cap portion is a 5' cap analogue. In some embodiments, the 5' end of RNA is capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis (e.g., simultaneous capping during transcription) or engineered enzymatically after RNA transcription (e.g., post-transcriptional capping), where “N” is any ribonucleotide. In some embodiments, the 5' end molecule of RNA is capped with a modified ribonucleotide by an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification of the RNA molecule, e.g., tangential flow filtration. Exemplary enzymatic reactions for capping may involve the use of vaccinia virus capping enzymes (VCEs) comprising mRNA triphosphatase, guanylyl-transferase, and guanine-7-methyltransferase to catalyze the construction of an N7-monomethylated cap 0 structure. The cap 0 structure may help maintain the stability and translational efficacy of the RNA molecule. The 5' cap of the RNA molecule may also be further modified with a 2'-O-methyltransferase, resulting in the formation of a cap 1 structure (m7Gppp[m2'-O]N), thereby further increasing translational efficacy. In some embodiments, a cap 0 structure can be obtained by enzymatically capping the 5' end of the RNA molecule using vaccinia guanyyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine. An inverted 7-methylguanosine cap is added via a 5'-5' triphosphate crosslink. Alternatively, by using 2'-O-methyltransferase and vaccinia guanyyltransferase, a cap 1 structure can be obtained in addition to the cap 0 structure, in which the 2'OH group of the second-to-last nucleotide is methylated. S-adenosyl-L-methionine (SAM) is a cofactor used as a methyl transfer reagent.Non-limiting examples of 5' cap structures include, among others, those having enhanced binding of cap-binding polypeptides, extended half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' cap removal compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can create a standard 5'-5'-triphosphate linkage between the 5' terminal nucleotide of mRNA and the guanine cap nucleotide, where the cap guanine includes N7 methylation and the 5' terminal nucleotide of mRNA includes 2'-O-methylation. Such a structure is referred to as a cap 1 structure. This cap results in higher translational qualification and cellular stability and reduced activation of pro-inflammatory cytokines in cells, compared to, for example, other 5' cap analog structures known in the art. Cap structures are not limited to these, but include 7mG(5')ppp(5')N,pN2p (cap 0) and 7mG(5')ppp(5')N1mpNp (cap 1). Cap 0 is N7-methylguanosine linked to the 5' nucleotide by a 5'-5' triphosphate linkage, and is generally referred to as the m7G cap or m7Gppp. In cells, the cap 0 structure may help to result in efficient translation of cap-bearing mRNA. Cap 1 is generated by additional methylation at the 2'O position of the start nucleotide. Alternatively, cap 1 is referred to as m7GpppNm-, where Nm refers to any nucleotide having a 2'O methylation. In some embodiments, the 5' terminal cap may include a cap analog, for example, the 5' terminal cap may include a guanine analog. Examples of guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the capping region may comprise a single cap or a series of nucleotides forming a cap.In this embodiment, the cap addition region may be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or fewer nucleotides, at least any one of these, at most any one of these, or the length between any two of these. In some embodiments, there is no cap. In some embodiments, the first and second operable regions may be 3 to 40, for example, 5 to 30, 10 to 20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or fewer nucleotides, at least any one of these, at most any one of these, or the length between any two of these, and may include one or more signals and / or restriction sequences in addition to start and / or stop codons.

[0085] In some embodiments, the 5' cap is of formula I:

[0086] [Chemical formula] (where R 1 and R 2 are each independently H or Me, and B 1 and B 2 are each independently guanine, adenine, or uracil) represented by. In some embodiments, B 1 and B 2 are naturally occurring bases. In some embodiments, R 1 is methyl and R 2 is hydrogen. In some embodiments, B 1 is guanine. In some embodiments, B 1 is adenine. In some embodiments, B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, B2 It is uracil, and the B inside the molecule 2 At least 5% of the entire population of uracil nucleotides downstream of it are replaced by one or more modified or non-natural nucleotides.

[0087] In some embodiments, the nucleotide immediately downstream of the 5' cap (in the direction from 5' to 3') contains guanine. In some embodiments, B 1 It is adenine, and B 2 is uracil. In some embodiments, B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen. In some cases, saRNA does not contain a 5' cap. In some cases, the 5' cap is not represented by formula I. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and this embodiment corresponds to CleanCap AU, B 2 The uracil nucleotide downstream of B may be substituted, 2 In some embodiments, the inclusion of uracil has been shown to improve saRNA functionality. In some embodiments, the RNA molecule further comprises (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the RNA molecule encodes at least one antigen. In some embodiments, the RNA molecule contains at least 7,000 nucleotides. In some embodiments, the RNA molecule contains at least 8,000 nucleotides. In some embodiments, at least 80% of the entire RNA molecule is full length. In some embodiments, the alphavirus is Venezuelan encephalitis virus. In some embodiments, the alphavirus is Semliki forest virus.

[0088] In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and at least 50% of the entire population of uridine nucleotides in the molecule are replaced by N1-methylpseudridine, and essentially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and at least 50% of the entire population of uridine nucleotides in the molecule are replaced by 5-methoxyuridine, and essentially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and at least 50% of the entire population of uridine nucleotides in the molecule are replaced by 5-methyluridine, and essentially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 The is hydrogen, and essentially all uridine nucleotides in the molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudolidine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2is uracil, and R 1 is methyl, and R 2 The is hydrogen, and essentially all uridine nucleotides in the molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudolidine. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 It is hydrogen, and essentially all uridine nucleotides in the molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudolidine.

[0089] In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap is

[0090] [ka] Includes. In some embodiments, the 5' cap is CLEANCAP® Reagent AG(3'OMe), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG for capping concurrently with mRNA transcription.

[0091] [ka] This includes. In alternative embodiments, the 5' cap is CLEANCAP® AU for self-amplifying mRNA, CLEANCAP® Reagent AU for capping concurrently with mRNA transcription, m7G(5')ppp(5')(2'OMeA)pU,

[0092] [ka] Includes.

[0093] G. PolyA tail As used herein, “poly-A tail” refers to a sequence of consecutive adenine residues that can be attached to the 3' end of an RNA molecule. The poly-A tail can increase the half-life of an RNA molecule. The poly-A tail can play a crucial regulatory role in enhancing translation efficiency and regulating mRNA quality control and degradation efficiency. Short sequences or high polyadenylation can signal RNA degradation. Exemplary designs include a poly-A tail of approximately 40 to 80 adenine residues. In some embodiments, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly-A tail sequence. In some embodiments, for example, for a second RNA or saRNA molecule, the RNA molecule further includes a poly-A polymerase recognition sequence (e.g., AAUAAA) near the 3' end. A “full-length” RNA molecule includes a 5' cap and a poly-A tail.

[0094] In some embodiments, the polyA tail contains a length of 5 to 400 nucleotides. The nucleotide length of the polyA tail may be any one of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400, at least one of these, up to one of these, or any two of these. In some embodiments, the RNA molecule includes a poly-A tail containing a sequence of about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly-A tail containing a sequence of more than 30 adenosine nucleotides ("As"). In some embodiments, the RNA molecule includes a poly-A tail containing about 40As. In some embodiments, the RNA molecule includes a poly-A tail containing about 80As. As used herein, the term "about" refers to a deviation of ±10% of the value (one or more) it is associated with. In some embodiments, the 3' poly-A tail has a sequence of at least 10 consecutive adenosine residues and up to 300 consecutive adenosine residues. In some embodiments, the RNA molecule contains at least 20 consecutive adenosine residues and up to 40 consecutive adenosine residues. In some embodiments, the RNA molecule contains approximately 40 consecutive adenosine residues. In some embodiments, the RNA molecule contains approximately 80 consecutive adenosine residues.

[0095] H. Composition In some cases, the compositions described herein include at least one saRNA described herein. Some embodiments of this disclosure provide an influenza virus (influenza) vaccine (or composition or immunogenic composition) comprising at least one saRNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to influenza).

[0096] In some embodiments, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition (capped and uncapped) are capped, with at least one of these, up to one of these, or two of these capped.

[0097] In some embodiments, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition are full-length RNA transcripts, at least one of these, up to one of these, or between two of these. Purity can be determined, as described herein, for example, by reverse-phase HPLC or electrophoresis based on a bioanalyzer tip, and, for example, by the peak area of ​​the full-length RNA molecule relative to the total peak. In some embodiments, a fragment analyzer (FA) can be used to quantify and purify RNA. The fragment analyzer automates capillary electrophoresis and HPLC.

[0098] In some embodiments, the composition is substantially free of one or more impurities or contaminants, including linear DNA templates and / or reversed-phase complement transcripts, and includes, for example, RNA molecules that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, at least 98% pure, or at least 99% pure, at least one of these, up to one of these, or between two of these.

[0099] In some embodiments, the composition contains a first RNA molecule in an amount greater than the amount of the second RNA molecule. In some embodiments, the composition contains 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 contains a first RNA molecule in an amount at least about 1 to 100 times the amount of the second RNA molecule.

[0100] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable vehicle.

[0101] In some embodiments, the composition further comprises a lipid-based delivery system, thereby delivering RNA molecules into the cell, where the RNA molecules can replicate and / or express the polypeptide of interest that is encoded. The delivery system may have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some embodiments, the composition further comprises neutral lipids, cationic lipids, cholesterol, and polyethylene glycol (PEG) to form nanoparticles containing RNA molecules. In some embodiments, the composition further comprises one of cationic lipids, liposomes, lipid nanoparticles, polyplexes, cocreates, viromosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsomes, polycationic peptides, and cationic nanoemulsions. In some embodiments, RNA molecules are encapsulated in, bound to, or adsorbed to any one of the following: cationic lipids, liposomes, lipid nanoparticles, polyplexes, cocreates, viromosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsionosomes, polycationic peptides, and cationic nanoemulsions, or any combination thereof.

[0102] In some cases, the compositions described herein include at least two RNA molecules, a first saRNA molecule and a second RNA molecule, as described herein. To provide protection against more than one strain of influenza, a mixed vaccine composition can be administered that includes RNA (e.g., saRNA) encoding at least one antigenic polypeptide protein (or antigenic moiety thereof) of a first influenza virus or organism, and further includes a second RNA molecule encoding at least one antigenic polypeptide protein (or antigenic moiety thereof) of a second influenza virus or organism. The RNA (e.g., saRNA) may be co-formulated, for example, in a single lipid nanoparticle (LNP), or it may be formulated in separate LNPs that are administered simultaneously.

[0103] In some embodiments, the second RNA molecule includes one of the following: a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a polyA sequence, or any combination thereof. In some embodiments, the second RNA molecule includes a 5' cap portion. In some embodiments, the second RNA molecule includes a 5' UTR and a 3' UTR. In some embodiments, the second RNA molecule includes a 5' UTR, an open reading frame, and a 3' UTR, but does not further include a 5' cap. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' UTR, a coding region, a 3' UTR, and a 3' polyA sequence. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' UTR, a non-coding region, a 3' UTR, and a 3' polyA sequence. In some embodiments, the second RNA molecule includes a non-coding region and does not further include one of the following: a 5' cap portion, a 5' UTR, a 3' UTR, and a 3' polyA sequence. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' untranslated region (5'UTR), a modified nucleotide, an open reading frame, a 3' untranslated region (3'UTR), and a 3' polyA sequence.

[0104] Some aspects of this disclosure relate to compositions comprising (i) a first RNA molecule encoding a gene of interest derived from influenza, and (ii) a second RNA molecule containing modified or unnatural nucleotides. In some cases, the first RNA molecule is one of the saRNA molecules described herein. In some cases, the first RNA molecule includes a 5' cap, a 5' untranslated region, a coding region for unstructured proteins including RNA replicase, a subgenome promoter, an open reading frame encoding the gene of interest, a 3' untranslated region, and a 3' poly-A sequence. In some cases, at least 5% of the entire population of specific nucleotides in the first RNA molecule are replaced by one or more modified or unnatural nucleotides. In some cases, the saRNA molecule contains natural unmodified nucleotides and does not contain modified or unnatural nucleotides. In some cases, the 5' cap is represented by formula I, where R 1 and R 2 Each of them is independently H or Me, and B 1 and B 2 Each of these is independently a 5' untranslated region, a non-structural protein coding region derived from an alphavirus, a subgenome promoter such as one derived from an alphavirus, an open reading frame encoding the gene of interest, a 3' untranslated region, and a 3' polyA sequence, each being guanine, adenine, or uracil. In some embodiments, B 1 and B 2 R is a naturally occurring base. In some embodiments, 1 is methyl, and R 2 is hydrogen. In some embodiments, B 1 is guanine. In some embodiments, B 1 is adenine. In some embodiments, B 2 is adenine. In some embodiments, B 2 It is uracil. In some embodiments, the nucleotide immediately downstream of the 5' cap (in the direction from 5' to 3') contains guanine.

[0105] In some embodiments, B 1It is adenine, and B 2 is uracil. In some embodiments, B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen. In some embodiments, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 This is hydrogen, and this embodiment corresponds to CLEANCAP AU (Trilink), B 2 The uracil nucleotide downstream of B may be substituted, 2 In some embodiments, it has been shown that including uracil results in increased saRNA functionality.

[0106] In some embodiments, at least 10% of the entire population of a particular nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a particular nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a particular nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a particular nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, essentially all of the particular nucleotide population in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, one or more replaced modified or non-natural nucleotides include two modified or non-natural nucleotides provided in a ratio ranging from 1:99 to 99:1, or any derivable range within that range. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 10% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 25% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 50% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and at least 75% of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the entire population of a first specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides, and essentially all of the entire population of a second specific nucleotide in the first or second RNA molecule is replaced by one or more modified or non-natural nucleotides.

[0107] In some embodiments, at least 25% of the entire population of uridine nucleotides in the first RNA molecule is replaced by N1-methylpseudridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule is replaced by N1-methylpseudridine. In some embodiments, at least 75% of the entire population of uridine nucleotides in the first RNA molecule is replaced by N1-methylpseudridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced by N1-methylpseudridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule are replaced by 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule are replaced by 5-methoxyuridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule are replaced by 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule are replaced by 5-methyluridine. In some embodiments, at least 50% of the entire population of cytosine nucleotides in the first RNA molecule are replaced by 5-methylcytosine. In some embodiments, virtually all of the cytosine nucleotides in the first RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule are replaced by 2-thiouridine. In some embodiments, virtually all of the uridine nucleotides in the first RNA molecule are replaced by 2-thiouridine.

[0108] In some embodiments, at least 25% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudridine. In some embodiments, at least 75% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the second RNA molecule are replaced by N1-methylpseudridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 5-methoxyuridine. In some embodiments, virtually all uridine nucleotides in the second RNA molecule are replaced by 5-methoxyuridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 5-methyluridine. In some embodiments, virtually all uridine nucleotides in the second RNA molecule are replaced by 5-methyluridine. In some embodiments, at least 50% of the entire population of cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, virtually all of the cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced by 2-thiouridine. In some embodiments, virtually all of the uridine nucleotides in the second RNA molecule are replaced by 2-thiouridine.

[0109] In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudridine, and virtually all cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 5-methoxyuridine, and virtually all cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 5-methyluridine, and virtually all cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine.

[0110] In some embodiments, virtually all uridine nucleotides in the second RNA molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the second RNA molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the second RNA molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudridine.

[0111] In some embodiments, virtually all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine, and at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced with N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine, and virtually all uridine nucleotides in the second RNA molecule are replaced with N1-methylpseudridine. In some embodiments, virtually all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine, and at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced with 5-methoxyuridine. In some embodiments, virtually all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine, and at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced with 5-methyluridine, and virtually all cytosine nucleotides in the second RNA molecule are replaced with 5-methylcytosine. In some embodiments, virtually all uridine nucleotides in the first RNA molecule are replaced with N1-methylpseudridine, and virtually all uridine nucleotides in the second RNA molecule are replaced with approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudridine.

[0112] I. How to use Various genotypes, strains, and isolates of influenza virus can be treated and / or prevented using saRNA compositions. Some embodiments provide a method for preventing or treating influenza virus infection, comprising administering one 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 a T cell response and a B cell response. In some embodiments, the method for eliciting an antigen-specific immune response involves a single administration of the saRNA composition. In some embodiments, the saRNA composition is administered to the subject by intradermal, intramuscular, subcutaneous, intranasal inoculation, or oral administration.

[0113] In some embodiments, an RNA (e.g., saRNA) polynucleotide or a portion thereof may encode one or more polypeptides or fragments of an influenza strain as an antigen.

[0114] Some aspects of this disclosure relate to a method for inducing an immune response in a subject, comprising administering an effective amount of the composition disclosed herein to a subject in need of such response. Some aspects of this disclosure relate to a method for vaccinating a subject, comprising administering an effective amount of the composition disclosed herein to a subject in need of such response. Some aspects of this disclosure relate to a method for administering an effective amount of the composition disclosed herein to a subject in need of such response. In some embodiments, the composition disclosed herein elicits an immune response, including an antibody response. In some embodiments, the composition disclosed herein elicits an immune response, including a T cell response.

[0115] Some embodiments of this disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject one of the RNA (e.g., saRNA) compositions presented herein in an effective amount to produce 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 influenza vaccines.

[0116] In some embodiments, the antigen-specific immune response includes a T-cell response or a B-cell response. In some embodiments, a method for inducing an antigen-specific immune response includes administering a single dose (without a booster dose) of the influenza RNA (e.g., saRNA) composition of the Disclosure to a subject. In some embodiments, the method further includes administering a second (booster) dose of the influenza RNA (e.g., saRNA) composition to a subject. Additional doses of the influenza RNA (e.g., saRNA) composition may be administered.

[0117] In some embodiments, subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after the first or second (booster) dose of the vaccine. Seroconversion is the period during which specific antibodies are produced and detectable in the blood. After seroconversion has occurred, the virus may be detected in blood tests for antibodies. During infection or immunization, the antigen enters the bloodstream, and the immune system responds by beginning to produce antibodies. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are not thought to be present. During seroconversion, antibodies are present but not yet detectable. After seroconversion, antibodies can be detected in the blood at any point in time, thereby indicating a previous or current infection.

[0118] In some embodiments, the influenza RNA (e.g., saRNA) composition is administered by intradermal injection, intramuscular injection, or intranasal injection.

[0119] Some embodiments of this disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an effective amount of an influenza RNA (e.g., saRNA) composition that elicits an antigen-specific immune response in the subject. In some embodiments, the antigen-specific immune response in the subject can be determined by assaying the antibody titer (the titer of an antibody that binds to an influenza antigenic polypeptide) after administering one of the influenza RNA (e.g., saRNA) compositions of this disclosure to the subject. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log compared to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1 to 3 log compared to a control.

[0120] In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject increases by at least twofold compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject increases by at least fivefold compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject increases by at least tenfold compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject increases by at least two to tenfold compared to the control.

[0121] In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in subjects that have not been administered the RNA (e.g., saRNA) composition of the Disclosure. In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in subjects that have been administered attenuated live or inactivated influenza, or the control is the anti-antigenic polypeptide antibody titer produced in subjects that have been administered recombinant or purified influenza protein vaccine.

[0122] In some embodiments, an RNA (e.g., saRNA) composition is formulated into an effective amount to produce an antigen-specific immune response in a target.

[0123] In some embodiments, the effective dose is a total dose of 1 μg to 1000 μg of saRNA, or 1 μg to 100 μg. In some embodiments, the effective dose is a total dose of 30 μg. In some embodiments, the effective dose is a dose of 10 μg administered to the subject in a total of two doses. In some embodiments, the effective dose is a dose of 10 μg administered to the subject in a total of two doses. In some embodiments, the effective dose is a dose of 15 μg administered to the subject in a total of two doses. In some embodiments, the effective dose is a dose of 30 μg administered to the subject in a total of two doses.

[0124] In some embodiments, the method involves administering the saRNA composition described herein to a subject in doses between 10 μg / kg and 400 μg / kg. In some embodiments, the dose of saRNA polynucleotide per single dose is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg. The dosages are g, 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the saRNA composition is administered by intradermal or intramuscular injection. In some embodiments, the saRNA composition is administered on day 0. In some embodiments, the second dose of the saRNA composition is administered on day 21.

[0125] In some embodiments, the target age is approximately 5 years or younger. For example, the target age may be between approximately 1 year and approximately 5 years (e.g., approximately 1, 2, 3, 5, or 5 years), or between approximately 6 months and approximately 1 year (e.g., approximately 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, the target age is approximately 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month). In some embodiments, the target age is approximately 6 months or younger.

[0126] In some embodiments, subjects were born at full term (e.g., approximately 37–42 weeks). In some embodiments, subjects were born prematurely 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, subjects were born at approximately 32 weeks of gestation or earlier. In some embodiments, subjects were born prematurely between approximately 32 and 36 weeks of gestation. For such subjects, RNA (e.g., mRNA) vaccines may be administered later in life, for example, between approximately 6 months and 5 years of age, or later.

[0127] In some embodiments, the subjects are young adults between approximately 20 and 50 years of age (for example, approximately 20, 25, 30, 35, 40, 45, or 50 years of age).

[0128] In some embodiments, the subjects are elderly individuals aged approximately 60, 70, or older (e.g., approximately 60, 65, 70, 75, 80, 85, or 90 years).

[0129] In some embodiments, the subject is already exposed to influenza (e.g., Chlamydia trachomatis), is infected with influenza (e.g., Chlamydia trachomatis), or is at risk of being infected with influenza (e.g., Chlamydia trachomatis).

[0130] In some embodiments, the subject is already exposed to a betacoronavirus (e.g., SARS-CoV-2), is infected with a betacoronavirus (e.g., SARS-CoV-2), or is at risk of being infected with a betacoronavirus (e.g., SARS-CoV-2).

[0131] In some embodiments, the subject has already received at least one dose of an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2), selected from, for example, COMIRNATY®, Pfizer-BioNTech's COVID-19 vaccine, Moderna's mRNA-1273 COVID-19 vaccine, and Janssen's COVID-19 vaccine; the subject has already received at least two doses of an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2); the subject has already received, for example, COMIRNATY®, Pfizer-BioNTech's COVID-19 vaccine, and Moderna's mRNA-1273 The subject has received at least one dose of an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2), selected from any one of the COVID-19 vaccines and Janssen's COVID-19 vaccines, or the subject is at risk of infection with a beta-coronavirus (e.g., SARS-CoV-2) and has received, concurrently with, or within 12 to 48 hours of, an immunogenic composition against a beta-coronavirus (e.g., SARS-CoV-2), selected from any one of the following: COMIRNATY®, Pfizer-BioNTech's COVID-19 vaccine, Moderna's mRNA-1273 COVID-19 vaccine, and Janssen's COVID-19 vaccine, in parallel with, simultaneously with, or within 12 to 48 hours of, any one of the immunogenic compositions against influenza disclosed herein.

[0132] In some embodiments, the subjects are in an immunodeficient state (having impaired immune system function, e.g., immunodeficiency or autoimmune disorder).

[0133] Aspects of the present disclosure provide a saRNA composition comprising one or more saRNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the saRNA polynucleotides exist as a formulation for in vivo administration to a host, conferring an antibody titer to an acceptable percentage of human subjects that exceeds a standard antibody prevalence for the first antigen (e.g., HA). In some embodiments, the antibody titer produced by the saRNA composition of the present disclosure is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is higher than that produced by a protein vaccine. In other embodiments, the neutralizing antibody titer produced by the saRNA composition is higher than that produced by an adjuvant-added protein vaccine. In yet another embodiment, the neutralizing antibody titer produced by the saRNA composition is 1,000-10,000, 1,200-10,000, 1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000, 2,000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500. The neutralizing titer is generally expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.

[0134] J. Nucleic acids In certain embodiments, nucleic acid sequences can exist in various contexts, such as isolated segments, recombinant vectors of sequences to be incorporated, or recombinant polynucleotides encoding polypeptides such as one or both antigen or antibody chains, or fragments thereof, derivatives, mutant proteins, or variants; hybridization probes for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; polynucleotides sufficient for use as PCR primers or sequencing primers; and antisense nucleic acids for inhibiting the expression of the aforementioned polynucleotides, mRNA, saRNA, and complementary sequences. The nucleic acid encodes an epitope to which an antibody can bind. Nucleic acids encoding fusion proteins containing these polypeptides are also provided. The nucleic acid may be single-stranded or double-stranded and may include RNA and / or DNA nucleotides and their artificial nucleotides (e.g., peptide nucleic acids).

[0135] The term "polynucleotide" refers to nucleic acid molecules that are recombinant or isolated from the entire genome. The term "polynucleotide" encompasses oligonucleotides (nucleic acids with a length of 100 residues or less), recombinant vectors, and, for example, plasmids, cosmids, phages, and viruses. In certain embodiments, polynucleotides include regulatory sequences substantially isolated from their naturally occurring gene- or protein-coding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic DNA, cDNA, or synthetic DNA), their analogues, or combinations thereof. Additional coding or non-coding sequences may be present within a polynucleotide, but are not required.

[0136] In this regard, the term “gene” is used to refer to nucleic acids (including any sequences necessary for accurate transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller, engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. Nucleic acids encoding all or part of a polypeptide may contain a continuous nucleic acid sequence encoding all or part of such polypeptide. It is also intended that a particular polypeptide may be encoded by nucleic acids containing variations that have slightly different nucleic acid sequences but nevertheless encode the same or substantially similar polypeptide.

[0137] In certain embodiments, there exist polynucleotide variants having substantial identity with respect to the sequences disclosed herein; variants having sequence identity between 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, at least one of these, up to one of these, or two of these, compared to the polynucleotide sequences presented herein, using the methods described herein (e.g., BLAST analysis using standard parameters). In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90% identity with respect to the amino acid sequences described herein over the entire length of the sequence, or a nucleotide sequence complementary to the aforementioned isolated polynucleotide. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 95% identity with respect to the amino acid sequences described herein over the entire length of the sequence, or a nucleotide sequence complementary to the aforementioned isolated polynucleotide.

[0138] In some embodiments, the polynucleotide includes a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 12. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 13. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 14. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 15. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 16. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 17. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 18. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 19. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 20. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to 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 more sequence identity with respect to SEQ ID NO: 22.

[0139] In some embodiments, the polynucleotides are: a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13; and at least 70%, 75% to SEQ ID NO: 14. Polynucleotide sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 16 A polynucleotide sequence having sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 17; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 19; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 21; and a polyA tail containing at least 20 consecutive adenines.

[0140] In some embodiments, the polynucleotides are: a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13; and a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90% to SEQ ID NO: 14. Polynucleotide sequences having 95%, 96%, 97%, 98%, or 99% or more sequence identity; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 16; to SEQ ID NO: 17 Polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 18; Polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 19; A polynucleotide sequence having sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 20; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 21; and a polyA tail containing at least 20 consecutive adenines.

[0141] In some embodiments, the polynucleotide includes 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 polyA tail containing at least 20 consecutive adenines.

[0142] In some embodiments, the polynucleotide includes 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 more 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 more sequence identity with SEQ ID NO: 20; a polynucleotide sequence having SEQ ID NO: 21; and a polyA tail containing at least 20 consecutive adenines.

[0143] In some embodiments, the polynucleotide includes a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 23. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 24. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 25. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 26. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 27. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 28. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 29. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 30. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 31. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to SEQ ID NO: 32.

[0144] In some embodiments, the polynucleotides are: a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more 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 more sequence identity with SEQ ID NO: 25 Sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 26; Polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27; A polynucleotide sequence having 96%, 97%, 98%, or 99% or more sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 29; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 31; and a polyA tail containing at least 20 consecutive adenines.

[0145] In some embodiments, the polynucleotides are: a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more 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 more sequence identity with SEQ ID NO: 24; and a less Sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 26; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or polynucleotide sequences having 99% or more sequence identity; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 28; polynucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 29; A polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to sequence number 30; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with respect to sequence number 31; and a polyA tail containing at least 20 consecutive adenines.

[0146] In some embodiments, the polynucleotide comprises a 5'UTR sequence having 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 polyA tail containing at least 20 consecutive adenines.

[0147] In some embodiments, the polynucleotide includes a 5'UTR sequence having 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 more sequence identity with respect to SEQ ID NO: 30; a polynucleotide sequence having SEQ ID NO: 31; and a polyA tail containing at least 20 consecutive adenines.

[0148] Nucleic acid segments, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, and other coding segments; therefore, their overall length can vary considerably. Nucleic acids can be of any length. Nucleic acids may be, for example, one of the following nucleotide lengths: 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, at least one of these, up to one of these, or between two of these, and / or may include one or more additional sequences, such as regulatory sequences, and / or may be part of a larger nucleic acid, such as a vector. Therefore, nucleic acid fragments of almost any length are intended to be usable, and the overall length is limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence having additional heterologous coding sequences to enable therapeutic benefits such as the purification, transport, secretion, post-translational modification, or targeting or efficacy of the polypeptide. As described above, tags or other heterologous polypeptides can be attached to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0149] As used herein, “modNS1” refers to a polynucleotide encoding the NS1 protein, where the polynucleotide comprises N1-methylpseudridine (m1ψ). In preferred embodiments, modNS1 comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail.

[0150] K. Lipid delivery In some embodiments, the saRNA composition includes lipids. Lipids and saRNA can be combined to form nanoparticles. By encapsulating mRNA in lipids to form lipid nanoparticles (LNPs), the entry and stability of RNA / lipid nanoparticles into cells can be aided.

[0151] Lipid nanoparticles may contain lipid components and one or more additional components, such as therapeutic and / or prophylactic agents. LNPs can be designed for one or more specific applications or targets. The 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, a specific formulation of an LNP can be selected for a specific application or target, for example, depending on the efficacy and toxicity of a particular combination of components. The stability of the formulation may also affect the efficacy and tolerability of the LNP formulation.

[0152] Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within the mammalian body.

[0153] The physiological and chemical properties of lipid nanoparticles can be modified to increase their selectivity for specific bodily targets. For example, particle size can be adjusted based on the window sizes of different organs. The therapeutic and / or prophylactic agents included in the LNP can also be selected based on the desired delivery target(s). For example, therapeutic and / or prophylactic agents can be selected for specific indications, conditions, diseases, or disorders, and / or for delivery (e.g., localization or specific delivery) to specific cells, tissues, organs, or systems or groups thereof. In certain embodiments, the LNP may contain mRNA encoding a target polypeptide, which can be translated intracellularly to produce the target polypeptide. Such compositions can be designed to be specifically delivered to specific organs. In some embodiments, the composition can be designed to be specifically delivered to the mammalian liver. In some embodiments, the composition can be designed to be specifically delivered to lymph nodes. In some embodiments, the composition can be designed to be specifically delivered to the mammalian spleen.

[0154] LNPs may comprise one or more components described herein. In some embodiments, the LNP formulations of this disclosure comprise at least one lipid nanoparticle component. The lipid nanoparticles may comprise the lipid component and one or more additional components, such as therapeutic and / or prophylactic agents, such as nucleic acids. LNPs can be designed for one or more specific applications or targets. 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, a specific formulation of an LNP can be selected for a specific application or target, for example, depending on the efficacy and toxicity of a particular combination of components. The stability of the formulation may also affect the efficacy and tolerability of the LNP formulation.

[0155] In some embodiments, for example, a polymer can be used to incorporate and / or encapsulate or partially encapsulate an LNP. The polymer may be biodegradable and / or biocompatible. The polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, as polymers, poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide, polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyacid anhydride, polyorthoester, poly(ester) Polyamides, polyamides, poly(ester ethers), polycarbonates, polyalkylenes, e.g., polyethylene and polypropylene, polyalkylene glycols, e.g., poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates, e.g., poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters, e.g., poly(vinyl acetate), polyhalogenated vinyls, e.g., poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses, e.g., alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acid, e.g., poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly (Butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and their copolymers and mixtures, polydioxanone and its copolymers Examples include polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), their derivatives, and polyglycerin.

[0156] Examples of surface modifiers, though not limited to these, include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, Clerodendrum trichotomum, bromhexine, carbocysteine, eprazinon, mesna, ambroxol, sobrelol, domiodol, letosteine, stepronin, thiopronin, gelzolin, thymosin β4, dorunase alpha, neltenexin, and erdosteine), and DNA-degrading enzymes (e.g., rhDNase). Surface modifiers can be positioned inside nanoparticles and / or on the surface of LNPs (e.g., by coating, adsorption, covalent linking, or other processes).

[0157] LNPs may also contain one or more functionalized lipids. For example, lipids can be functionalized with alkyne groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner using one or more groups useful for facilitating membrane permeation, cell recognition, or imaging. One or more useful antibodies can also be conjugated to the surface of LNPs. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0158] In addition to these components, lipid nanoparticles may contain any substance useful in a pharmaceutical composition. For example, lipid nanoparticles may contain, but are not limited to, one or more pharmaceutically acceptable excipients or adjuncts such as solvents, dispersion media, diluents, dispersion aids, suspension aids, surfactants, buffers, preservatives, and others.

[0159] Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), and polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45]). Examples include polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and / or combinations thereof.

[0160] Examples of preservatives, but not limited to these, include antioxidants, chelating agents, free radical scavengers, antibacterial preservatives, antifungal preservatives, alcoholic preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants, but not limited to these, include alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and / or phenylethyl alcohol. Examples of acidic preservatives, though not limited to these, include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®. Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

[0161] Examples of buffers, though not limited to these, include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, Tris buffer, and / or combinations thereof.

[0162] In some embodiments, the formulation containing LNP may further contain salts such as chloride salts. In some embodiments, the formulation containing LNP may further contain sugars such as disaccharides. In some embodiments, the formulation further contains sugars but does not contain salts such as chloride salts. In some embodiments, the LNP may further contain one or more hydrophobic low molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. The carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0163] The characteristics of LNPs can depend on their constituent components. For example, an LNP containing cholesterol as a structural lipid may have different characteristics than an LNP containing a different structural lipid. As used herein, the term “structural lipid” refers to sterols, and also to lipids containing a sterol moiety. As defined herein, “sterol” is a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is a cholesterol analog. In some embodiments, the structural lipid is alpha-tocopherol.

[0164] In some embodiments, the characteristics of LNPs may depend on the absolute or relative amounts of their constituent components. For example, LNPs containing phospholipids at a higher molar concentration fraction may have different characteristics than LNPs containing phospholipids at a lower molar concentration fraction. The characteristics may vary depending on the method and conditions for preparing the lipid nanoparticles. Generally, phospholipids consist of a phospholipid moiety and one or more fatty acid moieties.

[0165] The phospholipid moiety can be selected from an unspecified group, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety can be selected from an unspecified group, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Certain phospholipids may facilitate fusion with membranes. In some embodiments, cationic phospholipids may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or intracellular membrane). The fusion of phospholipids with a membrane may enable one or more components (e.g., therapeutic agents) of a lipid-containing composition (e.g., LNP) to pass through the membrane, thereby enabling, for example, delivery of one or more components to a target tissue. Non-natural phospholipid species, including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also intended. In some embodiments, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under suitable reaction conditions, the alkyne groups can undergo cycloaddition catalyzed by copper upon exposure to an azide. Such reactions may be useful in functionalizing the lipid bilayer of a nanoparticle composition to facilitate membrane permeability or cell recognition, or in conjugating the nanoparticle composition with useful components such as a targeting or imaging moiety (e.g., a dye). Phospholipids, though not limited to these, include glycerophospholipids such as phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Sphingophospholipids such as sphingomyelin are also examples of phospholipids.In some embodiments, the phospholipids useful or potentially useful in the present invention are analogs or variants of DSPCs.

[0166] Lipid nanoparticles can be characterized by various methods. For example, the morphology and size distribution of LNPs can be investigated using a microscope (e.g., a transmission electron microscope or a scanning electron microscope). Zeta potentials can be measured using dynamic light scattering or potentiometric measurements (e.g., potentiometric titration). Particle size can also be determined using dynamic light scattering. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to measure multiple LNP characteristics such as particle size, polydispersity index, and zeta potential.

[0167] The average size of LNPs can range from tens of nanometers to hundreds of nanometers, measured, for example, by dynamic light scattering (DLS). For example, the average size could range from approximately 40 nm to approximately 150 nm, such as approximately 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the LNP may range from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In a particular embodiment, the average size of the LNP may range from about 70 nm to about 100 nm. In a particular embodiment, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

[0168] LNPs can be relatively uniform. The polydispersity index can be used to indicate the uniformity of LNPs, for example, the particle size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. LNPs may have polydispersity indices ranging from about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs may range from about 0.10 to about 0.20.

[0169] The zeta potential of LNPs can be used to indicate the interfacial dynamic potential of a composition. For example, the zeta potential can describe the surface charge of LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally preferred because more highly charged species may interact undesirably with cells, tissues, and other components in the body. In some embodiments, the zeta potential of LNPs is approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, and approximately -5mV. It can range from V to approximately +10mV, from approximately -5mV to approximately +5mV, from approximately -5mV to approximately 0mV, from approximately 0mV to approximately +20mV, from approximately 0mV to approximately +15mV, from approximately 0mV to approximately +10mV, from approximately 0mV to approximately +5mV, from approximately +5mV to approximately +20mV, from approximately +5mV to approximately +15mV, or from approximately +5mV to approximately +10mV.

[0170] The encapsulation efficiency of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agent encapsulated in the LNP after preparation or otherwise associated with the LNP, compared to the initial amount prepared. A high encapsulation efficiency (e.g., close to 100%) is desirable. Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing lipid nanoparticles before and after the disintegration of the lipid nanoparticles with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For lipid nanoparticles described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents may be at least 50%, e.g., 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 certain embodiments, the encapsulation efficiency can be at least 90%.

[0171] LNP may contain one or more types of coatings. For example, LNP can be formulated as a capsule, film, or tablet having a coating. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0172] Formulations comprising amphiphilic polymers and lipid nanoparticles can be formulated as whole or in part as pharmaceutical compositions. A pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid nanoparticles. For example, a pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid nanoparticles comprising one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or adjuncts, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and drugs are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjuncts may be used in any pharmaceutical composition, except where any conventional excipient or adjunct may be incompatible with one or more components of the LNPs or one or more amphiphilic polymers in the formulations of this disclosure. If the combination of an excipient or accessory with a component or amphiphilic polymer may result in any undesirable biological effect or other adverse effect, the excipient or accessory may be incompatible with the component or amphiphilic polymer of the formulation's LNP.

[0173] In some embodiments, one or more excipients or adjuncts may constitute more than 50% of the total mass or volume of the pharmaceutical composition containing LNP. For example, one or more excipients or adjuncts may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, pharmaceutically acceptable excipients are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for use in humans and animals. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and / or International Pharmacopoeia. The relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical compositions according to this disclosure will vary depending on the identity, size, and / or condition of the object being treated, and further depend on the route through which the composition is administered. For example, a pharmaceutical composition may contain 0.1% to 100% (wt / wt) of one or more lipid nanoparticles. Another example is a pharmaceutical composition may contain 0.1% to 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / v).

[0174] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the Disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures below 4°C, e.g., between approximately -150°C and approximately 0°C or between approximately -80°C and approximately -20°C (e.g., approximately -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C)). For example, a pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., by freeze-drying) that is refrigerated for storage and / or transport at, for example, approximately -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C). In certain embodiments, the disclosure also relates to methods for increasing the stability of lipid nanoparticles by adding an effective amount of amphiphilic polymer and by storing the lipid nanoparticles and / or their pharmaceutical composition at a temperature of 4°C or less, for example, between about -150°C and about 0°C or between about -80°C and about -20°C (for example, about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C).

[0175] In some embodiments, the lipid components of LNPs include cationic lipids, phospholipids, PEG lipids, and structural lipids. In certain embodiments, the lipid components of lipid nanoparticles include about 30 mol% to about 60 mol% 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, provided that the total mol% does not exceed 100%. In some embodiments, the lipid components of lipid nanoparticles include about 35 mol% to about 55 mol% 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 certain embodiments, the lipid components include about 50 mol% of the aforementioned cationic lipids, about 10 mol% phospholipids, about 38.5 mol% structural lipids, and about 1.5 mol% PEG lipids. In another embodiment, the lipid composition includes about 40 mol% of the aforementioned cationic lipids, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipids may be PEG-DMG, and / or the structural lipids may be cholesterol.

[0176] In some embodiments, the ionized lipid is of formula (I):

[0177] [ka] A compound of or its N-oxide, or a salt or isomer thereof, where R1 is C5-30 alkyl, C5-20 alkenyl, -R * Selected from the group consisting of YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR”, -YR”, and -R *R2 and R3 are selected from the group consisting of OR, or together with the atom to which they are bonded, they form a heterocyclic or carbocyclic compound; R4 is hydrogen, a C3-6 carbocyclic compound, -(CH2) n Q, -(CH2) n Selected from the group consisting of CHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl groups, where Q is a carbocyclic compound, heterocyclic compound, -OR, -0(CH2) n N(R)2, -C(0)0R, -0C(0)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(0)N(R)2, -N(R)C(0) R, -N(R)S(0)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -N(R)Re, N(R)S(0)2R8, -0(CH2) nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -0C(0)N(R)2J-N(R)C(0)0R, -N(0R)C(0)R, -N(0R)S(0)2R, -N(0R)C( 0)0R, -N(0R)C(0)N(R)2, -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 is selected from -C(0)N(R)0R and -C(R)N(R)2C(0)0R, 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 R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(0)0-, -OC(O)-, and -0C(0)-M” -C(0)0-, -C(0)N(R')-, -N(R')C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R')0-, -S(0)2-, -SS-, aryl groups, and heteroaryl groups are selected, where 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; R 8 is selected from the group consisting of C3-6 carbocyclic compounds and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(0)2R, -S(0)2N(R)2, C2-6 alkenyl, C3-6 carbocyclic compounds and heterocycles; 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 C1-13 alkyl, C2-13 alkenyl, and -R * Selected from the group consisting of 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 carbocyclic compound; 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, where R4 is (CH2)nQ, -(CH2) n When CHQR, -CHQR, or -CQ(R)2, (i) Q is not -N(R)2 and n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7 membered heterocycloalkyl and n is 1 or 2. In some embodiments, the ionizable lipid is

[0178]

Chemical formula

[0179] In some embodiments, the compound has the following structure (I):

[0180]

Chemical formula

[0181]

Chemical formula

[0182] The lipid components of a lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may also be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids can be selected from a non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG. In some embodiments, the PEG-modified lipid is of formula (IV):

[0183] [ka] (In the formula, R 8 and R 9 Each of these is independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain may be interrupted by one or more ester bonds; and w has an average value ranging from 30 to 60. It is a PEG lipid that has [specific properties].

[0184] L. Preparations In one embodiment, the present disclosure relates to an immunogenic composition comprising (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen, wherein the aforementioned antigen comprises at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen, wherein the aforementioned second antigen comprises at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in lipid nanoparticles (LNPs). In some embodiments, the first and second antigens comprise hemagglutinin (HA) or an immunogenic fragment or variant thereof. In some embodiments, the first antigen comprises HA derived from a subtype of influenza virus different from the influenza virus antigenic polypeptide or immunogenic fragment of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is derived from an influenza virus but from a strain of influenza virus different from both the first and second antigens. In some embodiments, the first, second and third RNA polynucleotides are formulated in lipid nanoparticles.

[0185] In some embodiments, the composition further comprises (iv) a fourth RNA polynucleotide having an open reading frame encoding a fourth antigen, wherein the aforementioned antigen comprises at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and the fourth antigen is derived from influenza virus, but from a strain of influenza virus different from that of the first, second, and third antigens. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in lipid nanoparticles.

[0186] In some embodiments, RNA polynucleotides are mixed in a desired ratio in a single container and then formulated into lipid nanoparticles. In some embodiments, different RNA polynucleotides are initially input in known ratios to be formulated in a single LNP process, resulting in an LNP in which different RNA polynucleotides are encapsulated in approximately the same ratio as the input ratio. Such embodiments may be referred to herein as “premixing”. 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, the first, second, third, fourth, fifth, sixth, seventh, and eighth RNA polynucleotides are formulated into a single LNP.

[0187] In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide is greater than 1:1.

[0188] In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide is greater than 1:1.

[0189] In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide in the mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first 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 mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first 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 mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first 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 mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first 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 mixture of RNA polynucleotides before formulation in LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide is greater than 1:1.

[0190] In alternative embodiments, each RNA polynucleotide encoding a specific antigen is formulated within an individual LNP, so that each LNP contains an RNA polynucleotide encoding the same antigen. Such embodiments may be referred to herein as “post-mixing.” Thus, in some embodiments, the first RNA polynucleotide is formulated within the first LNP, the second RNA polynucleotide within the second LNP, the third RNA polynucleotide within the third LNP, the fourth RNA polynucleotide within the fourth LNP, the fifth RNA polynucleotide within the fifth LNP, the sixth RNA polynucleotide within the sixth LNP, the seventh RNA polynucleotide within the seventh LNP, and the eighth RNA polynucleotide within the eighth LNP.

[0191] In some embodiments, the molar ratio of the first LNP to the second LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the second LNP is greater than 1:1.

[0192] In some embodiments, the molar ratio of the first LNP to the third LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the third LNP is greater than 1:1.

[0193] In some embodiments, the molar ratio of the first LNP to the fourth LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the fourth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP in the mixture of LNPs before formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP is greater than 1:1.

[0194] In some embodiments, the relative amount of RNA encoding the influenza B virus antigen can be increased compared to RNA encoding the influenza A virus (e.g., an immune response including a higher neutralizing titer against influenza B virus (e.g., a higher neutralizing titer compared to a composition containing equal amounts of RNA encoding the influenza A antigen and RNA encoding the influenza B antigen (e.g., determined by a pseudovirus neutralizing assay described herein))). The disclosure also provides exemplary doses of RNA that can produce a potent immune response against either type of influenza virus (e.g., a clinically significant level of neutralizing titer and / or seroconversion rate (e.g., (i) a neutralizing titer equivalent to or exceeding those previously shown to prevent influenza symptoms, and / or (ii) a neutralizing titer and / or seroconversion rate equivalent to or exceeding those induced by a relevant comparator (e.g., a commercially approved influenza vaccine or influenza RNA vaccine))). In some embodiments, a composition containing more RNA encoding influenza B antigen than RNA encoding influenza A antigen produces an immune response against influenza B virus and influenza A virus, respectively, that is equivalent to or better than that induced by a non-RNA influenza vaccine (e.g., an approved vaccine) and / or an RNA vaccine containing equal amounts of RNA encoding influenza A antigen and RNA encoding influenza B antigen.

[0195] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is approximately 0.1 to 0.2 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is approximately 0.1 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is approximately 0.12 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is approximately 0.14 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is approximately 0.16 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is approximately 0.18 mg / ml. In some embodiments, approximately 30 μg of RNA is administered by administering approximately 200 μL of the RNA preparation. In some embodiments, the RNA in the pharmaceutical RNA preparation is diluted before administration (for example, to a concentration of approximately 0.05 mg / ml). In some embodiments, the administration volume is between approximately 200 μL and approximately 300 μL. In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in approximately 10 mM Tris buffer and approximately 10% sucrose.

[0196] In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.1 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.12 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.14 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.16 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.18 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. Such formulations can be diluted prior to administration as needed to administer different doses of RNA while keeping the total injection volume relatively constant. For example, a dose of about 10 μg of RNA can be administered by diluting such a pharmaceutical RNA preparation 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0197] In some embodiments, the vaccine is formulated in a vial (e.g., a glass vial). In some embodiments, the glass vial is sealed with a bromobutyl elastomer stopper and an aluminum seal with a flip-off plastic cap.

[0198] In some embodiments, the composition includes RNA encoding an influenza virus antigen (e.g., HA protein) that is recommended by the relevant health authorities to be included in a season-adjusted vaccine (e.g., cell-based, recombinant, or attenuated live virus). In some embodiments, the composition includes multiple RNAs encoding antigens (e.g., HA protein) of each influenza virus that is recommended by the relevant health authorities to be included in a season-adjusted vaccine (e.g., cell-based, recombinant, or attenuated live virus). In some embodiments, the influenza virus is influenza A, influenza B, or 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 an H1N1, H3N2, H5N1, or H5N8 virus. In some embodiments, the influenza A virus is an H1N1 virus (e.g., A / Wisconsin / 588 / 2019 or A / Sydney / 5 / 2021). In some embodiments, the influenza A virus is an H3N2 virus. In some embodiments, the H3N2 virus is A / Cambodia / e0826360 / 2020 or A / Darwin / 6 / 2021. In some embodiments, the influenza B virus is a 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 embodiments, the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013.

[0199] In some embodiments, the compositions described herein include a multivalent influenza vaccine. In some embodiments, the multivalent influenza vaccine includes 2 to 50 distinct RNA molecules (e.g., 2 to 40, 2 to 30, or 2 to 20 RNA molecules), each of which, in some embodiments, may encode a different antigenic polypeptide (or a different version of a particular antigenic polypeptide) related to influenza, as described, for example, 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 include a trivalent influenza vaccine. In some embodiments, the trivalent influenza vaccine includes RNA encoding antigenic polypeptides related to two type A viruses and one type B virus expected to be prevalent in the relevant jurisdiction. In some embodiments, the compositions described herein include a quadrivalent influenza vaccine. In some embodiments, the quadrivalent influenza vaccine includes RNA encoding antigenic polypeptides related to two type A viruses and two type B viruses expected to be prevalent in the relevant jurisdiction. In some embodiments, the compositions described herein include an octavalent influenza vaccine. In some embodiments, the octavalent influenza vaccine includes RNA encoding two antigenic polypeptides (e.g., HA protein and NA protein or immunogenic fragments thereof, associated with each of two type A viruses and two type B viruses that are expected to be prevalent in the relevant jurisdiction).In some embodiments, the compositions disclosed herein include a quadrivalent influenza vaccine comprising RNA containing a nucleotide sequence encoding an HA protein associated with an H1N1 virus (e.g., A / Wisconsin / 588 / 2019), RNA containing a nucleotide sequence encoding an HA protein associated with an H3N2 virus (e.g., A / Cambodia / e0826360 / 2020), RNA containing a nucleotide sequence encoding an HA protein associated with a B / Victoria influenza virus (e.g., B / Washington / 02 / 2019), and a HA protein associated with a B / Yamagata influenza virus (e.g., B / Phuket / 3073 / 2013).

[0200] In some embodiments, the composition comprising the quadrivalent influenza vaccine includes RNA encoding antigenic polypeptides associated with two type A viruses and two type B viruses expected to be prevalent in the relevant jurisdiction. In some embodiments, the quadrivalent influenza vaccine includes RNA encoding antigenic polypeptides associated with the H1N1 influenza virus, RNA encoding antigenic polypeptides associated with the H3N2 influenza virus, RNA encoding antigenic polypeptides associated with the Victoria lineage influenza virus, and RNA encoding antigenic polypeptides associated with the Yamagata lineage influenza virus. In some embodiments, the quadrivalent influenza vaccine includes RNA associated with influenza types expected to be prevalent in the relevant jurisdiction (e.g., HA polypeptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses expected to be prevalent in the relevant jurisdiction).

[0201] In some embodiments, the composition comprising the octavalent influenza vaccine comprises RNA encoding antigenic polypeptides related to two type A viruses and two type B viruses that are expected to be prevalent within the relevant jurisdiction. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from HA of influenza A virus, RNA encoding an antigenic polypeptide derived from HA of influenza A virus, RNA encoding an antigenic polypeptide derived from HA of influenza B virus, RNA encoding an antigenic polypeptide derived from HA of influenza B virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 of influenza A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 of influenza B virus, and RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 of influenza B virus. In some embodiments, the octavalent influenza vaccine includes RNA encoding an antigenic polypeptide derived from HA of influenza A virus, RNA encoding an antigenic polypeptide derived from HA of influenza A virus, RNA encoding an antigenic polypeptide derived from HA of influenza B virus, RNA encoding an antigenic polypeptide derived from HA of influenza B virus, RNA encoding an antigenic polypeptide derived from NA of influenza A virus, RNA encoding an antigenic polypeptide derived from NA of influenza A virus, RNA encoding an antigenic polypeptide derived from NA of influenza B virus, and RNA encoding an antigenic polypeptide derived from NA of influenza B virus.In some embodiments, the octavalent influenza vaccine includes RNA encoding an antigenic polypeptide associated with the H1N1 influenza virus, RNA encoding an antigenic polypeptide associated with the H3N2 influenza virus, RNA encoding an antigenic polypeptide associated with the Victoria lineage influenza virus, and RNA encoding an antigenic polypeptide associated with the Yamagata lineage influenza virus. In some embodiments, the octavalent influenza vaccine includes RNA associated with influenza types expected to be prevalent in the relevant jurisdiction (e.g., HA polypeptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses expected to be prevalent in the relevant jurisdiction).

[0202] In some embodiments, the RNA in the compositions disclosed herein each encodes an antigenic polypeptide associated with an infectious agent expected to be prevalent within the relevant jurisdiction. Such compositions can reduce the number of vaccinations required.

[0203] In some embodiments, the nucleic acid-containing particles include two or more RNA molecules, each containing a nucleotide sequence encoding an antigen (e.g., HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particles include three or more RNA molecules, each containing a nucleotide sequence encoding an antigen (e.g., HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particles include four or more RNA molecules, each containing a nucleotide sequence encoding an antigen (e.g., HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particles include 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 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, containing a nucleotide sequence encoding an antigenic polypeptide associated with an influenza virus, is formulated in separate nucleic acid-containing particles.

[0204] In some embodiments, nucleic acid-containing particles (e.g., LNPs as described herein in some embodiments) contain two or more RNA molecules in equal amounts (i.e., in a 1:1 ratio).

[0205] In some embodiments, nucleic acid-containing particles (e.g., LNPs as described herein in some embodiments) containing two or more RNA molecules contain each RNA molecule in different amounts. For example, in some embodiments, the nucleic acid-containing particles contain a first RNA molecule and a second RNA molecule, where the first RNA molecule is present in an amount of 0.01 to 100 times the amount of the second RNA molecule (e.g., 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 (15, 0.01~10, 0.01~9, 0.01~8, 0.01~7, 0.01~6, 0.01~5, 0.01~4, 0.01~3, 0.01~2, 0.01~1.5, 1~50, 1~4, 1~30, 1~25, 1~20, 1~15, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, or 1~1.5 times). In some embodiments, the nucleic acid-containing particles include a first RNA molecule and a second RNA molecule, where 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 particles include a first RNA molecule and a second RNA molecule, where 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 particles include a first RNA molecule and a second RNA molecule, where 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 particles include a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 2 times that of the second RNA molecule. In some embodiments, the nucleic acid-containing particles include a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 3 times that of the second RNA molecule.

[0206] In some embodiments, nucleic acid-containing particles (e.g., LNPs as described herein in some embodiments) contain each RNA molecule in equal amounts (i.e., in a 1:1:1 ratio).

[0207] In some embodiments, nucleic acid-containing particles (e.g., LNPs as described herein in some embodiments) containing three RNA molecules contain each RNA molecule in different amounts. For example, in some embodiments, the ratio of the first RNA molecule:second RNA molecule: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:second RNA molecule:third RNA molecule is 1:1:3. In some embodiments, the ratio of the first RNA molecule:second RNA molecule:third RNA molecule is 1:3:3.

[0208] As used herein, the term “dose” generally refers to the “amount of dose” in relation to the amount of RNA administered per single dose.

[0209] In some embodiments, the immunogenic composition or vaccine of this disclosure may be administered as a single dose or boosted by multiple doses.

[0210] In some embodiments, the regimen described herein includes at least one dose. In some embodiments, the regimen includes a first dose and at least one subsequent dose. In some embodiments, the amount of the first dose is the same as at least one subsequent dose. In some embodiments, the amount of the first dose is the same as all subsequent doses. In some embodiments, the amount of the first dose is different from at least one subsequent dose. In some embodiments, the amount of the first dose is different from all subsequent doses. In some embodiments, the regimen includes two doses. In some embodiments, the regimen provided consists of two doses. In some embodiments, the regimen includes three doses.

[0211] In one embodiment, the Disclosure envisions a single-dose administration. In one embodiment, the Disclosure envisions a priming dose followed by one or more booster doses. The booster dose or first booster dose may be administered 7 to 28 days or 14 to 24 days after the administration of the priming dose. In some embodiments, the first booster dose may be administered 1 week to 3 months after the administration of the priming 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, a subsequent booster dose may be administered at least one week after the preceding booster dose, including, for example, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, or later. In some embodiments, a subsequent booster dose may be administered at intervals of approximately five to nine weeks or six to eight weeks. In some embodiments, at least one subsequent booster dose (e.g., after the first booster dose) may be administered at least three months after the preceding dose, including, for example, 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, or later.

[0212] In some embodiments, the dose includes a total amount of RNA ranging from 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, for example, 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. In some embodiments, the dose includes a total amount of RNA (e.g., modRNA) up to about 100 μg. In some embodiments, the 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, where 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). In some embodiments, the dose comprises 3 to 60 μg of one or more first RNAs and 3 to 90 μg of one or more second RNAs, with a maximum total RNA of 100 μg. In some embodiments, the dose comprises 3 to 30 μg of one or more first RNAs and 3 to 60 μg of one or more second RNAs, with a maximum total RNA of 100 μg. In some embodiments, the dose comprises one or more first RNAs of 3 μg and one or more second RNAs of 3 μg. In some embodiments, the dose comprises one or more first RNAs of 3 μg and one or more second RNAs of 6 μg. In some embodiments, the dose comprises one or more first RNAs of 10 μg and one or more second RNAs of 10 μg. In some embodiments, the dose comprises one or more first RNAs of 10 μg and one or more second RNAs of 20 μg. In some embodiments, the dose comprises one or more first RNAs of 30 μg and one or more second RNAs of 30 μg.In some embodiments, the dose comprises 30 μg of one or more first RNAs and 60 μg of one or more second RNAs. In some embodiments, the dose comprises 60 μg of one or more first RNAs and 30 μg of one or more second RNAs.

[0213] In some embodiments, the amount of RNA in a subsequent dose given to an individual (e.g., as part of a primary or booster regimen) may be the same as the amount previously given to that individual. In some embodiments, the amount of RNA in a subsequent dose given to an individual (e.g., as part of a primary or booster regimen) may differ from the amount previously given to that individual. For example, in some embodiments, the subsequent dose may be greater or less than the previous dose based on consideration of various factors, including, for example, immunogenicity and / or reactability induced by the previous dose, disease prevalence, 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 more 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 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 than the previous dose. In some embodiments, the subsequent dose may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or less than the previous dose. In some embodiments, the RNA described herein can be administered in amounts of 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg per single dose (for example, in a given single dose), such as approximately 1 μg, approximately 2 μg, approximately 3 μg, approximately 10 μg, approximately 15 μg, approximately 20 μg, approximately 25 μg, approximately 30 μg, approximately 35 μg, approximately 40 μg, approximately 45 μg, approximately 50 μg, approximately 55 μg, approximately 60 μg, approximately 70 μg, approximately 80 μg, approximately 90 μg, or approximately 100 μg per single dose.

[0214] In some embodiments, the RNA described herein can be administered in amounts of 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 per single dose (for example, in a given single dose).

[0215] In some embodiments, the RNA described herein can be administered in amounts of 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 per single dose (for example, in a given single dose). In some embodiments, the RNA described herein can be administered in amounts of at least 3 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of at least 10 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of at least 15 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of at least 20 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of at least 25 μg in at least one dose of a given dose. In some embodiments, at least 30 μg of the RNA described herein can be administered in at least one dose of a given dose. In some embodiments, at least 50 μg of the RNA described herein can be administered in at least one dose of a given dose. In some embodiments, at least 60 μg of the RNA described herein can be administered in at least one dose of a given dose. In some embodiments, combinations of the above amounts can be administered in a regimen comprising two or more doses (for example, the amounts of the preceding and succeeding doses may differ as described herein). In some embodiments, combinations of the above amounts can be administered in a primary regimen and a booster regimen (for example, different doses may be given in the primary regimen and the booster regimen).

[0216] In some embodiments, the RNA described herein can be administered in amounts of 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 per single dose. In some embodiments, the RNA described herein can be administered in amounts of 3 μg to 30 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of 3 μg to 20 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of 3 μg to 15 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of 3 μg to 10 μg in at least one dose of a given dose. In some embodiments, the RNA described herein can be administered in amounts of 10 μg to 30 μg in at least one dose of a given dose.

[0217] In some embodiments, the regimen administered to the subject may consist of multiple doses (e.g., at least two doses, at least three doses, or more). In some embodiments, the regimen administered to the subject may consist of a first dose and a second dose, which are given at intervals of at least two weeks, at least three weeks, at least four weeks, or longer. In some embodiments, such multiple doses may be spaced at least one month, 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. In some embodiments, multiple doses may be administered at intervals of several days, for example, at intervals of 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 longer. In some embodiments, multiple doses may be administered at intervals of approximately 1 to 3 weeks, or approximately 1 to 4 weeks, or approximately 1 to 5 weeks, or approximately 1 to 6 weeks, or approximately 1 to more than 6 weeks. In some embodiments, the interval between multiple doses may be approximately 7 to 60 days, for example, approximately 14 to 48 days. In some embodiments, the minimum number of days between multiple doses may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In some embodiments, the maximum number of days between multiple doses may be approximately 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, or less.In some embodiments, multiple doses can be spaced approximately 21 to 28 days apart. In some embodiments, multiple doses can be spaced approximately 19 to 42 days apart. In some embodiments, multiple doses can be spaced approximately 7 to 28 days apart. In some embodiments, multiple doses can be spaced approximately 14 to 24 days apart. In some embodiments, multiple doses can be spaced approximately 21 to 42 days apart.

[0218] In some embodiments, the vaccination regimen includes a first dose and a second dose. In some embodiments, the first and second doses are administered at least 21 days apart. In some embodiments, the first and second doses are administered at least 28 days apart.

[0219] In some embodiments, the vaccination regimen comprises a first dose and a second dose, where the amount of RNA administered in the first dose is the same as the amount of RNA administered in the second dose. In some embodiments, the vaccination regimen comprises a first dose and a second dose, where the amount of RNA administered in the first dose is different from the amount of RNA administered in the second dose.

[0220] In some embodiments, the vaccination regimen includes a first dose and a second dose, where 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% to 90% of the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% to 50% of the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% to 20% of the amount of RNA administered in the second dose. In some embodiments, the first and second doses are administered at least two weeks apart, including at least three weeks apart, at least four weeks apart, at least five weeks apart, at least six weeks apart, or longer. In some embodiments, the first and second doses are administered at least three weeks apart.

[0221] In some embodiments, the first dose contains less than approximately 30 μg of RNA, and the second dose contains at least approximately 30 μg of RNA. In some embodiments, the first dose contains approximately 1 to less than approximately 30 μg of RNA (e.g., approximately 0.1, approximately 1, approximately 3, approximately 5, approximately 10, approximately 15, approximately 20, approximately 25, or less than approximately 30 μg of RNA), and the second dose contains approximately 30 to approximately 100 μg of RNA (e.g., approximately 30, approximately 40, approximately 50, or approximately 60 μg of RNA). In some embodiments, the first dose contains approximately 1 to approximately 20 μg of RNA, approximately 1 to approximately 10 μg of RNA, or approximately 1 to approximately 5 μg of RNA, and the second dose contains approximately 30 to approximately 60 μg of RNA.

[0222] In some embodiments, the first dose contains approximately 1 to 10 μg of RNA (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg of RNA), and the second dose contains approximately 30 to 60 μg of RNA (e.g., approximately 30, 35, 40, 45, 50, 55, or 60 μg of RNA).

[0223] In some embodiments, the first dose contains approximately 1 μg of RNA, and the second dose contains approximately 30 μg of RNA. In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 30 μg of RNA. In some embodiments, the first dose contains approximately 5 μg of RNA, and the second dose contains approximately 30 μg of RNA. In some embodiments, the first dose contains approximately 10 μg of RNA, and the second dose contains approximately 30 μg of RNA. In some embodiments, the first dose contains approximately 15 μg of RNA, and the second dose contains approximately 30 μg of RNA.

[0224] In some embodiments, the first dose contains approximately 1 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 5 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 6 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 10 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 15 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 20 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 25 μg of RNA, and the second dose contains approximately 60 μg of RNA. In some embodiments, the first dose contains approximately 30 μg of RNA, and the second dose contains approximately 60 μg of RNA.

[0225] In some embodiments, the first dose contains less than approximately 10 μg of RNA, and the second dose contains at least approximately 10 μg of RNA. In some embodiments, the first dose contains approximately 0.1 to less than approximately 10 μg of RNA (e.g., approximately 0.1, approximately 0.5, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, or less than approximately 10 μg of RNA), and the second dose contains approximately 10 to approximately 30 μg of RNA (e.g., approximately 10, approximately 15, approximately 20, approximately 25, or approximately 30 μg of RNA). In some embodiments, the first dose contains approximately 0.1 to approximately 10 μg of RNA, approximately 1 to approximately 5 μg of RNA, or approximately 0.1 to approximately 3 μg of RNA, and the second dose contains approximately 10 to approximately 30 μg of RNA.

[0226] In some embodiments, the first dose contains approximately 0.1 to 5 μg of RNA (e.g., approximately 0.1, 0.5, 1, 2, 3, 4, and 5 μg of RNA), and the second dose contains approximately 10 to 20 μg of RNA (e.g., approximately 10, 12, 14, 16, 18, and 20 μg of RNA).

[0227] In some embodiments, the first dose contains approximately 0.1 μg of RNA, and the second dose contains approximately 10 μg of RNA. In some embodiments, the first dose contains approximately 0.3 μg of RNA, and the second dose contains approximately 10 μg of RNA. In some embodiments, the first dose contains approximately 1 μg of RNA, and the second dose contains approximately 10 μg of RNA. In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 10 μg of RNA.

[0228] In some embodiments, the first dose contains less than approximately 3 μg of RNA, and the second dose contains at least approximately 3 μg of RNA. In some embodiments, the first dose contains approximately 0.1 to less than approximately 3 μg of RNA (e.g., approximately 0.1, 0.2, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, or 2.5 μg of RNA), and the second dose contains approximately 3 to approximately 10 μg of RNA (e.g., approximately 3, 4, 5, 6, or 7, 8, 9, or 10 μg of RNA). In some embodiments, the first dose contains approximately 0.1 to approximately 3 μg of RNA, approximately 0.1 to approximately 1 μg of RNA, or approximately 0.1 to approximately 0.5 μg of RNA, and the second dose contains approximately 3 to approximately 10 μg of RNA.

[0229] In some embodiments, the first dose contains approximately 0.1 to 1.0 μg of RNA (e.g., approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μg of RNA), and the second dose contains approximately 1 to 3 μg of RNA (e.g., approximately 1.0, 1.5, 2.0, 2.5, or 3.0 μg of RNA).

[0230] In some embodiments, the first dose contains approximately 0.1 μg of RNA, and the second dose contains approximately 3 μg of RNA. In some embodiments, the first dose contains approximately 0.3 μg of RNA, and the second dose contains approximately 3 μg of RNA. In some embodiments, the first dose contains approximately 0.5 μg of RNA, and the second dose contains approximately 3 μg of RNA. In some embodiments, the first dose contains approximately 1 μg of RNA, and the second dose contains approximately 3 μg of RNA.

[0231] In some embodiments, the vaccination regimen includes a first dose and a second dose, where 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% to 90% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10% to 50% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10% to 20% of the first dose. In some embodiments, the first and second doses are administered at least two weeks apart, including at least three weeks apart, at least four weeks apart, at least five weeks apart, at least six weeks apart, or longer. In some embodiments, the first and second doses are administered at least three weeks apart.

[0232] 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 to about 100 μg of RNA (e.g., about 30, about 40, about 50, or about 60 μg of RNA), and the second dose contains about 1 to about 30 μg of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30 μg of RNA). In some embodiments, the second dose contains about 1 to about 20 μg of RNA, about 1 to about 10 μg of RNA, or about 1 to 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.

[0233] In some embodiments, the first dose contains approximately 30 to 60 μg of RNA (e.g., approximately 30, 35, 40, 45, 50, 55, or 60 μg of RNA), and the second dose contains approximately 1 to 10 μg of RNA (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg of RNA).

[0234] In some embodiments, the first dose contains approximately 30 μg of RNA, and the second dose contains approximately 1 μg of RNA. In some embodiments, the first dose contains approximately 30 μg of RNA, and the second dose contains approximately 3 μg of RNA. In some embodiments, the first dose contains approximately 30 μg of RNA, and the second dose contains approximately 5 μg of RNA. In some embodiments, the first dose contains approximately 30 μg of RNA, and the second dose contains approximately 10 μg of RNA. In some embodiments, the first dose contains approximately 30 μg of RNA, and the second dose contains approximately 15 μg of RNA.

[0235] In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 1 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 3 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 5 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 6 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 10 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 15 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 20 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 25 μg of RNA. In some embodiments, the first dose contains approximately 60 μg of RNA, and the second dose contains approximately 30 μg of RNA.

[0236] 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 to about 10 μg of RNA (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.

[0237] In some embodiments, the first dose contains approximately 10 to 20 μg of RNA (e.g., approximately 10, 12, 14, 16, 18, or 20 μg of RNA), and the second dose contains approximately 0.1 to 5 μg of RNA (e.g., approximately 0.1, 0.5, 1, 2, 3, 4, or 5 μg of RNA).

[0238] In some embodiments, the first dose contains approximately 10 μg of RNA, and the second dose contains approximately 0.1 μg of RNA. In some embodiments, the first dose contains approximately 10 μg of RNA, and the second dose contains approximately 0.3 μg of RNA. In some embodiments, the first dose contains approximately 10 μg of RNA, and the second dose contains approximately 1 μg of RNA. In some embodiments, the first dose contains approximately 10 μg of RNA, and the second dose contains approximately 3 μg of RNA.

[0239] In some embodiments, the first dose contains at least about 3 μg of RNA, and the second dose contains less than 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, or about 7, about 8, about 9, or about 10 μg of RNA), and the second dose contains 0.1 to less than 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). It contains approximately 2.0 or 2.5 μg of RNA. In some embodiments, the first dose contains approximately 3 to 10 μg of RNA, and the second dose contains approximately 0.1 to 3 μg of RNA, approximately 0.1 to 1 μg of RNA, or approximately 0.1 to 0.5 μg of RNA.

[0240] In some embodiments, the first dose contains approximately 1 to approximately 3 μg of RNA (e.g., approximately 1, approximately 1.5, approximately 2.0, approximately 2.5, or approximately 3.0 μg of RNA), and the second dose contains approximately 0.1 to 0.3 μg of RNA (e.g., approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, or approximately 1.0 μg of RNA).

[0241] In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 0.1 μg of RNA. In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 0.3 μg of RNA. In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 0.6 μg of RNA. In some embodiments, the first dose contains approximately 3 μg of RNA, and the second dose contains approximately 1 μg of RNA.

[0242] In some embodiments, the vaccination regimen includes at least two doses, for example, including at least three doses, at least four doses, or more doses. In some embodiments, the vaccination regimen includes three doses. In some embodiments, 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 embodiments, 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 several weeks (for example, including at least three days, at least four days, at least five days, at least six days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more). In some embodiments, 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 several weeks (for example, including at least three days, at least four days, at least five days, at least six days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more). In some embodiments, 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 at least one month (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 more).

[0243] In some embodiments, the final dose of the primary regimen and the first dose of the booster regimen are administered at intervals of 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 primary regimen may consist of two doses. In some embodiments, the primary regimen may consist of three doses.

[0244] 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 into the deltoid muscle. In some embodiments, the first and second doses (and / or other subsequent doses) may be administered into the same arm.

[0245] In some embodiments, the mRNA composition described herein is administered in a series of two doses (e.g., 0.3 mL each) at intervals of 21 days (e.g., by intramuscular injection). In some embodiments, the mRNA composition described herein is administered in a series of two doses (e.g., 0.2 mL each) at intervals of 21 days (e.g., by intramuscular injection). In some embodiments, the mRNA composition described herein is administered in a series of three doses (e.g., 0.3 mL or less, including 0.2 mL) (e.g., by intramuscular injection), where the doses are given at intervals of at least 3 weeks. In some embodiments, the first and second doses may be administered at intervals of 3 weeks, while the second and third doses may be administered at longer intervals than the interval between the first and second doses, for example, at least 4 weeks or more (including at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or more). In some embodiments, each dose is approximately 60 μg. In some embodiments, each dose is approximately 50 μg. In some embodiments, each dose is approximately 30 μg. In some embodiments, each dose is approximately 25 μg. In some embodiments, each dose is approximately 20 μg. In some embodiments, each dose is approximately 15 μg. In some embodiments, each dose is approximately 10 μg. In some embodiments, each dose is approximately 3 μg.

[0246] In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 60 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 50 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 30 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 25 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 20 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 15 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 10 μg. In some embodiments, the at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is approximately 3 μg.

[0247] In one embodiment, the RNA described herein is administered in an amount of about 60 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 50 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 30 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 25 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 20 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 15 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 10 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 5 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 3 μg per dose. In one embodiment, at least two such doses are administered. For example, the second dose may be administered about 21 days after the first dose.

[0248] In some embodiments, the efficacy of the RNA vaccine described herein (for example, administered in two doses, where the second dose may be administered about 21 days after the first dose, and each dose may be about 30 μg) is at least 70%, at least 80%, at least 90%, or at least 95% starting 7 days after the second dose (for example, starting 28 days after the first dose if the second dose is administered 21 days after the first dose). In some embodiments, such efficacy is observed in a population of at least 50 years, at least 55 years, at least 60 years, at least 65 years, at least 70 years, or older. In some embodiments, the efficacy of the RNA vaccine described herein (for example, administered in two doses, where the second dose may be administered about 21 days after the first dose, and each dose may be about 30 μg) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% in a population aged at least 65 years, e.g., 65–80 years, 65–75 years, or 65–70 years, starting 7 days after the second dose (for example, starting 28 days after the first dose if the second dose is administered 21 days after the first dose). Such efficacy can be observed over a period of up to 1 month, 2 months, 3 months, 6 months, or longer.

[0249] In one embodiment, vaccine effectiveness is defined as a percentage reduction in the number of subjects showing signs of infection (vaccinated subjects versus unvaccinated subjects).

[0250] In one embodiment, the method and agents described herein are administered to a pediatric population. In various embodiments, the pediatric population includes or comprises subjects under 18 years of age, for example, 5 to under 18 years, 12 to under 18 years, 16 to under 18 years, 12 to under 16 years, 5 to under 12 years, or 6 months to under 12 years. In various embodiments, the pediatric population includes or comprises subjects under 5 years of age, for example, 2 to under 5 years, 12 months to under 24 months, 7 months to under 12 months, or under 6 months. 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. In some such embodiments, the mRNA composition described herein is administered to subjects under 6 months of age, for example, 1 month to under 4 months. In some embodiments, the dose regimen (e.g., dose and / or dose schedule) for the pediatric population can be varied for different age groups. For example, in some embodiments, administration to subjects aged 6 months to 4 years may be carried out according to a primary regimen comprising at least three doses, with the first two doses administered at least three weeks apart (e.g., including at least four weeks, at least five weeks, at least six weeks, or more), followed by a third dose administered at least eight weeks after the second dose (e.g., including at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, or more). In some such embodiments, at least one dose administered is 3 μg of RNA as described herein. In some embodiments, administration to subjects aged 5 years or older may be carried out according to a primary regimen comprising at least two doses, with the two doses administered at least three weeks apart (e.g., including at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more). In some such embodiments, at least one dose administered is 10 μg of RNA as described herein.In some embodiments, administration to immunocompromised subjects aged 5 years or older (e.g., subjects who have received a parenchymal organ transplant or who have been diagnosed with a condition considered equivalent to immunocompromised) may be carried out according to a primary regimen comprising at least three doses, with the first two doses administered at intervals of at least three weeks (e.g., including at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more), followed by the third dose administered at least four weeks after the second dose (e.g., at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, or more).

[0251] In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, with each dose being approximately 30 μg. In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older (e.g., including those aged 18 years or older), with each dose being greater than 30 μg, including, for example, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, or more. In some such embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, with each dose being approximately 60 μg. In some such embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, with each dose being approximately 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, the treatment may include two vaccinations administered 21 days apart, and in one embodiment, the vaccine is administered, for example, intramuscularly, at a dose of 30 μg of RNA per dose. In some embodiments, higher doses are administered to older pediatric patients and adults, e.g., patients 12 years of age or older, compared to younger children or infants, e.g., patients 2 to under 5 years of age, 6 months to under 2 years of age, or under 6 months of age. In some embodiments, higher doses are administered to children 2 to under 5 years of age, compared to toddlers and / or infants, e.g., 6 months to under 2 years of age, or under 6 months of age.

[0252] In one embodiment, the pediatric population includes or comprises subjects aged 5 to under 18 years, including subjects aged 12 to under 18 years and / or subjects aged 5 to under 12 years. In this embodiment, the treatment may include two vaccinations spaced 21 days apart, and in various embodiments, the vaccine is administered, for example, by intramuscular injection, in amounts of 10 μg, 20 μg, or 30 μg of RNA per dose. In some such embodiments, the mRNA composition described herein is administered to subjects aged 5 to 11 years, with each dose being approximately 10 μg.

[0253] In one embodiment, the pediatric population includes or comprises subjects under 5 years of age, including subjects 2 years to under 5 years of age, subjects 12 months to under 24 months of age, subjects 7 months to under 12 months of age, subjects 6 months to under 12 months of age, and / or subjects under 6 months of age. In this embodiment, the treatment may include two vaccinations spaced, for example, 21 days apart, with an interval of 21 to 42 days between them, and in various embodiments, the vaccine is administered, for example, by intramuscular administration, in amounts of 3 μg, 10 μg, 20 μg, or 30 μg of RNA per dose. In some such embodiments, the mRNA composition described herein is administered to subjects 2 years to under 5 years of age, with each dose being approximately 3 μg. In some such embodiments, the mRNA composition described herein is administered to subjects about 6 months to about 5 years of age, with each dose being approximately 3 μg.

[0254] In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being about 60 μg. In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being about 30 μg. In some embodiments, the mRNA composition described herein is administered to subjects aged 12 years or older, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being about 15 μg. In some embodiments, the mRNA composition described herein is administered to subjects aged 5 to under 12 years, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being about 10 μg. In some embodiments, the mRNA composition described herein is administered to subjects between 2 and 5 years of age, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being about 3 μg. In some embodiments, the mRNA composition described herein is administered to subjects between 6 months and 2 years of age, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being 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, with at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) being about 3 μg or less, including, for example, 2 μg, 1 μg, 0.5 μg, or less.

[0255] In some embodiments, the dose administered to a subject requiring it may include administration of a single mRNA composition as described herein.

[0256] In some embodiments, the dose administered to a subject requiring it may include the administration of at least two (e.g., at least three) different drug products / formulations. For example, in some embodiments, the at least two different drug products / formulations may include at least two different mRNA compositions described herein (e.g., in some embodiments, each containing a different RNA construct).

[0257] In some embodiments, two or more RNAs are administered to a subject (e.g., as part of either a primary or booster regimen), where the two or more RNAs are administered on the same day or during the same visit. In some embodiments, two or more RNAs are administered as separate compositions, for example, by administering each RNA to a different part of the subject (e.g., by intramuscular administration to a different arm of the subject or a different site on the same arm of the subject). In some embodiments, two or more RNAs are mixed before administration (e.g., immediately before administration, e.g., by the administering practitioner). In some embodiments, two or more RNAs are formulated together (e.g., (a) by mixing separate populations of LNPs, each containing a different RNA, or (b) by mixing two or more RNAs before LNP formulation such that each LNP contains two or more RNAs).

[0258] In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs in equal amounts (i.e., in a 1:1 ratio), or the composition comprises one or more first RNAs and one or more second RNAs in equal amounts (i.e., in a 1:1 ratio).

[0259] In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs in different amounts, or the composition contains one or more first RNAs and one or more second RNAs in different amounts. For example, in some embodiments, a subject is administered one or more first RNAs in an amount of 0.01 to 100 times the amount of one or more second RNAs, or the composition contains one or more first RNAs in an amount of 0.01 to 100 times the amount of one or more second RNAs (for example, the amount of one or more first RNAs is 0.01 to 50, 0.01 to 4, 0.01 to 100 times the amount of one or more second RNAs). (30, 0.01-25, 0.01-20, 0.01-15, 0.01-10, 0.01-9, 0.01-8, 0.01-7, 0.01-6, 0.01-5, 0.01-4, 0.01-3, 0.01-2, 0.01-1.5, 1-50, 1-4, 1-30, 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1-1.5 times). In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the concentration of one or more first RNAs is 1 to 10 times the concentration of one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the amount of one or more first RNAs is 1 to 5 times the amount of one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the concentration of one or more first RNAs is 1 to 3 times the concentration of one or more second RNAs.In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the amount of one or more first RNAs is twice the amount of one or more second RNAs. In some embodiments, a subject is administered one or more first RNAs and one or more second RNAs, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the concentration of one or more first RNAs is three times the concentration of one or more second RNAs.

[0260] In some embodiments, a subject is administered two first RNAs, each encoding an antigen derived from an influenza strain or variant, or the composition comprises two first RNAs, each encoding an antigen derived from an influenza strain or variant, wherein the amounts of each RNA are not equal. For example, in some embodiments, 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, two first RNAs are administered to a subject in a 1:3 ratio, or the composition contains two first RNAs in a 1:3 ratio. In some embodiments, two first RNAs are administered to a subject in a 1:2 ratio, or the composition contains two first RNAs in a 1:2 ratio.

[0261] For example, in some embodiments, 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. These are 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, three first RNAs are administered to a subject in a 1:1:3 ratio, or the composition contains three first RNAs in a 1:1:3 ratio. In some embodiments, three first RNAs are administered to the subject in a 1:3:3 ratio, or the composition contains three first RNAs in a 1:3:3 ratio.

[0262] In some embodiments, a subject is administered two or more secondary RNAs, or the composition comprises two or more secondary RNAs, one or more of which encode the HA protein of influenza A virus and one or more of which encode the HA protein of influenza B virus. In some embodiments, one or more secondary RNAs encoding the HA protein of influenza A virus and one or more secondary RNAs encoding the HA protein of influenza B virus are present or administered in the same amount (i.e., in a 1:1 ratio). In some embodiments, one or more secondary RNAs encoding the HA protein of influenza A virus and one or more secondary RNAs encoding the HA protein of influenza B virus are administered in different amounts (for example, in a ratio between 1:10 and 10:1, or in a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (total RNA encoding A antigen: total RNA encoding B antigen)).

[0263] In some embodiments, a subject is administered two second RNAs, each encoding a different influenza virus type 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), or the composition comprises two second RNAs, each encoding a different influenza virus type 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 second RNAs are administered or present in equal amounts (i.e., in a 1:1 ratio). In some embodiments, the second RNAs are administered or present in different amounts (e.g., in a ratio between 1:10 and 10:1, or in a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (A:B)).

[0264] In some embodiments, three second RNAs are administered to the subject, each encoding a different influenza virus subtype HA protein (e.g., HA proteins of A / Wisconsin(H1N1) virus, A / Darwin(H3N2) virus, and B / Austria(Victoria) virus), or the composition contains three second RNAs, each encoding a different influenza virus subtype HA protein (e.g., HA proteins of A / Wisconsin(H1N1) virus, A / Darwin(H3N2) virus, and B / Austria(Victoria) virus). In some embodiments, each of the three second RNAs is administered to the subject in equal amounts (i.e., in a 1:1:1 ratio), or the composition contains each of the three second RNAs in equal amounts (i.e., in a 1:1:1 ratio). In some embodiments, one or more of the three second RNAs are administered to a subject in different amounts (for example, in ratios between 1:1:2 and 1:1:10 (e.g., 1:1:2, 1:1:3, 1:1:4, or 1:1:5), or in ratios between 2:2:1 and 2:2:10 (e.g., 2:2:1, 3:3:1, 4:4:1, or 5:5:1), or the composition contains one or more of the three second RNAs in different amounts (for example, in ratios between 1:1:2 and 1:1:10 (e.g., 1:1:2, 1:1:3, 1:1:4, or 1:1:5), or in ratios between 2:2:1 and 2:2:10 (e.g., 2:2:1, 3:3:1, 4:4:1, or 5:5:1).

[0265] In some embodiments, the RNA administered to the target or composition comprises three second RNAs, two of which encode HA proteins of different influenza A viruses and one of which encodes HA proteins of influenza B viruses. In some such embodiments, the second RNA encoding the HA protein of influenza B viruses is present or administered in a greater amount than any of the second RNAs encoding HA proteins of the A virus (for example, in some embodiments, the ratio of the two second RNAs encoding HA proteins of influenza A viruses to the second RNA encoding HA proteins of influenza B viruses is 1:1:1 to 10, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 (A:A:B)). In some embodiments, three secondary RNAs are administered to the target, two encoding the HA protein of influenza A virus and one encoding the HA protein of influenza B virus, or the composition comprises three secondary RNAs, two encoding the HA protein of influenza A virus and one encoding the HA protein of influenza B virus, where the ratio of the three secondary RNAs is 1:1:4 (A:A:B). In some embodiments, the two secondary RNAs encoding the HA protein of influenza A virus are each present in a larger amount than the secondary RNA encoding the HA protein of influenza B virus, or each is administered in a larger amount than the secondary RNA encoding the HA protein of influenza B virus (for example, in some embodiments, the ratio of the two secondary RNAs encoding the HA protein of influenza A virus to the secondary RNA encoding the HA protein of 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)).

[0266] In some embodiments, four secondary RNAs are administered to the target, each encoding a different influenza virus subtype's HA protein, or the composition contains four secondary RNAs, each encoding a different influenza virus subtype's HA protein. In some such embodiments, the four secondary RNAs include two secondary RNAs encoding HA proteins of different influenza A viruses and two secondary RNAs encoding HA proteins of different influenza B viruses (e.g., HA protein of H1N1 virus, HA protein of H3N2 virus, HA protein of B / Victoria lineage virus, and HA protein of B / Yamagata lineage virus). In some embodiments, each of the two secondary RNAs encoding influenza A virus HA proteins and each of the two secondary RNAs encoding influenza B virus HA proteins are present in equal amounts (i.e., the ratio of the four secondary RNAs is 1:1:1:1). In some embodiments, the second RNAs encoding the HA proteins of two influenza B viruses are administered or present in greater quantities than either of the second RNAs encoding the HA proteins of the A virus (for example, in some embodiments, the ratio of the two second RNAs encoding the HA proteins of the A virus to the second RNAs encoding the HA proteins of the two influenza B viruses 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 embodiments, four secondary RNAs are administered to the target, two encoding the HA protein of influenza A virus and two encoding the HA protein of influenza B virus, or the composition comprises four secondary RNAs, two encoding the HA protein of influenza A virus and two encoding the HA protein of influenza B virus, where the ratio of the four secondary RNAs is 1:1:5:5 (A:A:B:B). In some embodiments, two second RNAs encoding the HA protein of influenza A virus are administered in greater amounts than any of the second RNAs encoding the HA protein of influenza B virus, or are present in greater amounts than any of the second RNAs encoding the HA protein of influenza B virus (for example, in some embodiments, the ratio of two second RNAs encoding the HA protein of influenza A virus to two second RNAs encoding the HA protein of 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)).

[0267] In some embodiments, the composition contains or is administered to a subject four secondary RNAs (e.g., A / Wisconsin(H1N1), A / Darwin(H3N2), A / Cambodia(H3N2), and B / Austria(Victoria)) including three secondary RNAs encoding the HA protein of different influenza A viruses and one secondary RNA encoding the HA protein of influenza B virus. In some such embodiments, each of the four secondary RNAs is administered or present in the same amount (i.e., in a ratio of 1:1:1:1). In some embodiments, the amount of the second RNA encoding the HA protein of influenza B virus is higher than any one of the second RNAs encoding the HA protein of influenza A virus (for example, in some embodiments, the ratio of the 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 ratio of the second RNA administered or in the composition is 1:1:1:5 (A:A:A:B). In some embodiments, the amount of the second RNA encoding the HA protein of influenza A virus is greater than the amount of the second RNA encoding the HA protein of influenza B virus (for example, in some embodiments, the ratio of the 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)).

[0268] In some embodiments, one or more secondary RNAs encoding the HA protein of influenza viruses (e.g., two secondary RNAs, three secondary RNAs, or four secondary RNAs, each encoding the HA protein of a different influenza virus) are administered to the target 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). The component or product contains one or more secondary RNAs encoding the HA protein of influenza viruses (for example, two secondary RNAs, three secondary RNAs, or four secondary RNAs, each encoding the HA protein of a different influenza virus) in a total amount of 0.1 to 100 μg (for example, 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, one or more secondary RNAs encoding the HA protein of the influenza virus are administered to a subject 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, or the composition contains one or more secondary RNAs encoding the HA protein of the 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.

[0269] In some embodiments, three or four secondary RNAs, each encoding the HA antigen of a different influenza strain, are administered to the subject in one of the amounts listed in Table C below, or the composition contains three or four secondary RNAs, each encoding the HA antigen of a different influenza strain, in one of the amounts listed in Table C below (each “influenza component” corresponds to a secondary RNA encoding the HA antigen (e.g., the secondary RNAs described herein).

[0270] In some embodiments, the compositions described herein are characterized by producing an influenza neutralizing antibody titer against each influenza virus on which the composition encodes an antigen, within a range of at least twice the influenza neutralizing antibody titer produced by a reference vaccine (for example, the reference vaccine is a quadrivalent influenza RNA vaccine administered alone, or an approved (non-RNA) influenza vaccine).

[0271] In some embodiments, the influenza vaccine is an alpha influenza virus, beta influenza virus, gamma influenza virus, or delta influenza virus vaccine. In some embodiments, the vaccine is an influenza A virus, influenza B virus, influenza C virus, or influenza D virus vaccine. In some embodiments, the influenza A virus vaccine comprises 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 one of them. In some embodiments, the influenza A vaccine comprises or encodes 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 one of them. In some embodiments, the influenza vaccine contains 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 acid protein (PA), polymerase basic proteins PB1, PB1-F2, and / or polymerase It comprises a nucleic acid (e.g., RNA) encoding basic protein 2 (PB2), or an immunogenic fragment or variant thereof, or any one thereof. [Examples]

[0272] (Example 1) Description of the manufacturing process This section describes the manufacturing process and process control of the influenza saRNA vaccine active pharmaceutical ingredient. The manufacturing process includes RNA synthesis via an in vitro transcription (IVT) step and a purification step by ultrafiltration / diafiltration (UFDF-1). Subsequently, the RNA is enzymatically capped, purified by chromatography and final UFDF-2, and then subjected to final filtration and preparation.

[0273] To produce clinical materials, the process was scaled up to a starting IVT volume of 1.5 L. Aside from the requirements for scaling the process up to 1.5 L, there were no significant changes in the non-clinical toxicology / development process.

[0274] RT-ddPCR (identity of encoded RNA sequence) The identity of influenza saRNA is confirmed by performing a one-step reverse transcription (RT)-ddPCR assay on the RNA in the sample. If the tested sample is positive for both the replicase sequence (confirmation of self-amplification of the RNA construct) and the target sequence (confirmation of the encoded influenza sequence), then identity is confirmed. Digital droplet polymerase chain reaction (ddPCR) technology is a digital form of polymerase chain reaction (PCR) that uses a water-oil emulsion system to quantify the target nucleic acid. The RNA sample is diluted to the final theoretical input concentration that falls within the linear range of the ddPCR assay. The reaction mixture, containing reverse transcriptase, DNA polymerase, and sequence-specific primers and probes, is distributed into droplets, and the PCR reaction is performed individually for each distribution. The results are calculated by counting the number of amplified target sequences (positive droplets, measured by fluorescence amplitude above the background) and distributions where no amplification is present (negative droplets). After examining the positive and negative controls and determining them to be valid and acceptable, identity is confirmed if the positive droplet count exceeds an established threshold.

[0275] Reverse-phase HPLC (presence of pseudouridine) The presence of pseudouridine is determined by reverse-phase high-performance liquid chromatography (RP-HPLC) after complete digestion of mRNA. The resulting individual nucleosides exhibit characteristic elution patterns, including the separation of uridine and pseudouridine. The presence of pseudouridine is confirmed by comparison with uridine and pseudouridine reference and limiting standards.

[0276] Capillary gel electrophoresis (RNA integrity) RNA integrity is determined by capillary gel electrophoresis (CGE) based on the discriminative migration of RNAs with different molecular weights within an applied electric field. RNA is subjected to a denaturing agent that unfolds the RNA and dissociates non-covalently bound complexes. The denatured RNA species, upon exposure to the electric field, migrate through the gel matrix toward the anode according to their length and size. During migration, intercalating dyes bind to the RNA and associated fragments, enabling fluorescence detection. Intact RNA is separated from all fragmented species, allowing for the quantification of RNA integrity by determining the relative percentage time-corrected area for the intact (major) peak.

[0277] qPCR (residual DNA template) The level of residual DNA template is determined by quantitative polymerase chain reaction (qPCR) using fluorescence technology. A qPCR master mix containing target-specific primers and a fluorescent qPCR quantification reagent is added to all sample wells. Samples are prepared in a series of dilutions and analyzed in real time by qPCR. The measured fluorescence signal is proportional to the amount of PCR product. DNA quantification is performed at the cycle threshold (CT), which is the point at which amplification of the target sequence is first detected above the established signal threshold during the logarithmic phase of the reaction. This Ct point depends on the amount of DNA originally present in the sample. The concentration of DNA in the test sample is interpolated from a linear regression of the calibration curve, taking the dilution factor into account. The results are reported as ng of DNA per 1 mg of RNA.

[0278] Table 1 presents batch details and batch analysis summary data for one batch of material used for regulatory toxicity assessment and one GMP batch of active pharmaceutical ingredients used in clinical trials.

[0279] [Table 1]

[0280] (Example 2) S.4.1 Description and composition of pharmaceutical products The PF-07867246 (Construction 6 (TC83-delkozak-HA-SGP-NA-80A) (SEQ ID NO: 1)) drug product is a sterile dispersion of liquid nanoparticles (LNPs) in a preservative-free aqueous cryoprotective buffer for intramuscular administration. This drug product is formulated with 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamic acid, at pH 7.4, containing 0.06 mg / mL of RNA. The drug product is supplied in a 2 mL glass vial sealed with an aluminum seal, a chlorobutyl elastomer stopper, and a flip-off plastic cap (nominal volume 0.5 mL). Table 2 shows the composition of the drug product, including applicable unit amounts, volume per vial, and functional and quality standards for each component.

[0281] [Table 2]

[0282] [Table 3-1]

[0283] [Table 3-2]

[0284] [Table 3-3]

[0285] [Table 3-4]

[0286] [Table 3-5]

[0287] [Table 3-6]

[0288] (Example 3) S.4.1. Description and composition of pharmaceutical products The PF-07871987 (Construction 7 TC83-HA-40A 50U-50pU (SEQ ID NO: 2)) drug product is a sterile dispersion of liquid nanoparticles (LNPs) in a preservative-free aqueous cryoprotective buffer for intramuscular administration. This drug product is formulated with 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamic acid, at pH 7.4, containing 0.06 mg / mL of RNA.

[0289] This drug product is supplied in a 2 mL glass vial sealed with an aluminum seal, featuring a chlorobutyl elastomer stopper and a flip-off plastic cap (nominal volume 0.5 mL).

[0290] Table 4 shows the composition of the drug product, including the applicable unit dosage, amount per vial, and functional and quality standards for each component.

[0291] [Table 4]

[0292] [Table 5]

[0293] [Table 6-1]

[0294] [Table 6-2]

[0295] [Table 6-3]

[0296] [Table 6-4]

[0297] [Table 6-5]

[0298] [Table 6-6]

[0299] (Example 4) S.4.4.1 Construct 7: TC83-HA-40A 50U-50pU (PF-07871987) Table 7 presents detailed batch information and batch analysis summary data for one batch of material used for regulatory toxicity assessment and one GMP batch of active pharmaceutical ingredients used in clinical trials.

[0300] [Table 7]

[0301] (Example 5) Assay Hemagglutination inhibition assay The primary serological assay used to measure the immune response induced by influenza vaccines is the hemagglutinin inhibition assay (HAI). HAI quantitatively measures functional antibodies in serum that prevent HA-mediated agglutination of red blood cells in a reaction containing a serum sample pretreated with a receptor-destroying enzyme, the influenza virus, and red blood cells derived from turkey or guinea pig. The HAI titer is the reciprocal of the highest serum dilution that results in a loss of HA activity, visualized as a teardrop shape when the microtiter plate is tilted. The titer, obtained from multiple determinations per sample, is reported as the geometric mean titer (GMT). An HAI titer of 1:40 or higher is generally considered protective in humans.

[0302] Influenza microneutralization assay The influenza virus microneutralization assay (MNT) quantitatively measures functional antibodies in serum that neutralize influenza virus activity, thereby preventing proliferative infection of a host cell monolayer. A neutralization reaction occurs when influenza virus is incubated with a serum sample. The reaction mixture is then applied to a monolayer of Maidin-Derby canine kidney (MDCK) cells to measure the degree of neutralization. MNT titers are reported as the reciprocal of the dilution, resulting in a 50% or 90% reduction in infection compared to a serum-free control. 1-Day MNT measures anti-HA neutralizing antibodies, while 3-Day MNT measures both anti-HA and anti-NA neutralizing antibodies.

[0303] Neuraminidase Inhibition Assay The neuraminidase inhibition assay (NAI) quantitatively measures functional antibodies in serum that prevent NA-mediated cleavage of sialic acid in enzyme-conjugated lectin assays. Briefly, serum containing antibodies is incubated with influenza virus, and the mixture is transferred to a plate coated with fetuin-lectin. Cleavage of sialic acid from fetuin is monitored by a colorimetric quantitative reaction after adding a substrate to a peanut agglutinin conjugated with horseradish peroxidase attached to the exposed galactose portion. The NAI titer is the reciprocal of the highest serum dilution that yields a 50% decrease in NA activity compared to a serum-free control. Titers obtained from multiple determinations per sample are reported as geometric mean titers (GMT).

[0304] (Example 6) Evaluation of bicistronic HA-NA saRNA vaccine design for influenza in mice. This study was conducted 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 the effects of immunogenicity and polyA tail length (40A or 80A) with and without an exogenous Kozak sequence upstream of the primary target gene.

[0305] The key bicistronic design elements evaluated in this study include the regulatory elements used to drive the expression of the second target gene (subgenome promoter (SGP) and its internal ribosomal entry site (IRES)), and the sequence of antigen placement on the vector (HA-NA or NA-HA).

[0306] Intramuscular immunization of Balb / c mice with LNP-formulated saRNA vaccines encoding A / Wisconsin / 588 / 2019(H1N1)HA and / or NA antigens induced functional and neutralizing antibody responses.

[0307] Overall, similar titers were obtained from all the bicistronic saRNA vaccines tested, and these titers were also similar to those of individually formulated saRNA vaccines consisting of saRNA-HA + saRNA-NA. These results confirm that the bicistronic saRNA approach is viable and capable of extracting titers.

[0308] All saRNA vectors used in this study were based on the TC-83 backbone. The study was designed to evaluate the effects of immunogenicity and poly(A) tail length (40A or 80A) with and without an exogenous Kozak sequence upstream of the primary target gene. Key bicistronic design elements evaluated in this study included regulatory elements necessary to drive the expression of the secondary target gene, and the order of antigen placement on the vector (HA-NA or NA-HA). For comparison, the selected regulatory elements were the native VEEV subgenome promoter (SGP; 61 nucleotides) and the internal ribosomal entry site (IRES, 587 nucleotides) derived from encephalomyocarditis virus. ModRNA vaccines encoding influenza HA or NA were also included in the study for comparison.

[0309] Mice were immunized with saRNA or modRNA LNP preparations on day 0 and day 28, and serum was collected 21 days after the prime and 14 days after the boost. Immunogenicity was determined by measuring neutralizing and functional antibodies on days 21 and 42.

[0310] This study was designed using 15 groups, as shown in Table 8, with a total of 10 female mice in each group (mouse strain: BALB / c). mRNA drug products were evaluated in 0.05 mL dose volumes.

[0311] [Table 8]

[0312] [Table 9]

[0313] [Table 10-1]

[0314] [Table 10-2]

[0315] M. Analysis results of the test specimen

[0316] [Table 11]

[0317] 2. Results and Discussion As measured by HAI, 1-Day MNT, 3-Day MNT, and NAI (Figures 1, 2, 3, and 4, respectively), intramuscular immunization of Balb / c mice with LNP-formulated saRNA vaccines encoding A / Wisconsin / 588 / 2019(H1N1)HA and / or NA antigens induced functional and neutralizing antibody responses, accompanied by a clear boost effect two weeks after the second immunization. Results from 3-Day MNT at day 42 indicated that the contribution of NA to neutralization was minimal compared to HA in this assay. Overall, similar titers were achieved for all bicistronic saRNA vaccine designs evaluated, and these titers were also similar to those of saRNA vaccines composed of individually formulated HA and NA monocistronic saRNAs (HA / NA Post-Mix) as well as modRNA. The titers of saRNA vaccines expressing both antigens were similar to or only slightly lower than those of the saRNA-HA or saRNA-NA-only controls. These data confirmed that the bicistronic saRNA approach is feasible.

[0318] Neither deletion of the Kozak sequence, the length of the polyA tail, nor the regulatory element used to drive the secondary target gene (whether IRES or SGP), nor the order of antigen positioning (HA-NA or NA-HA) had any apparent effect on the titer drawn.

[0319] Regarding Figure 1, female Balb / c mice were immunized with various LNP-formulated influenza saRNA vaccine constructs on days 0 and 28, as well as with influenza modRNA comparators encoding A / Wisconsin / 588 / 2019(H1N1)HA and / or NA. The HA / NA post-compound preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by HAI on day 21 (3 weeks after prime) and day 42 (2 weeks after boost).

[0320] Regarding Figure 2, female Balb / c mice were immunized with various LNP-formulated influenza saRNA vaccine constructs on days 0 and 28, as well as with influenza modRNA comparators encoding A / Wisconsin / 588 / 2019(H1N1)HA and / or NA. The HA / NA post-compound preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by a 1-Day MNT assay on days 21 (3 weeks after prime) and 42 (2 weeks after boost). 50% neutralizing titers were reported.

[0321] Regarding Figure 3, female Balb / c mice were immunized with various LNP-formulated influenza saRNA vaccine constructs on days 0 and 28, as well as with influenza modRNA comparators encoding A / Wisconsin / 588 / 2019(H1N1)HA and / or NA. The HA / NA post-compound preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by a 3-Day MNT assay on day 42 (two weeks after boost). 50% neutralizing titers were reported.

[0322] Regarding Figure 4, female Balb / c mice were immunized with various LNP-formulated influenza saRNA vaccine constructs on days 0 and 28, as well as with influenza modRNA comparators encoding A / Wisconsin / 588 / 2019(H1N1)HA and / or NA. The HA / NA post-compound preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by NAI on days 21 (3 weeks after prime) and 42 (2 weeks after boost).

[0323] 3. Conclusion Seasonal influenza saRNA vaccines are intended to express four different HA proteins and four different NA proteins to match the influenza strains that are primarily prevalent in each season. This has the potential to be achieved using eight individual saRNA components or four bicistronic saRNA components. To that end, the feasibility of the bicistronic saRNA approach was evaluated in mouse immunogenicity studies. Bicistronic saRNA vaccine candidates encoding both HA and NA antigens (TC83-delkozak-HA-SGP-NA-80A) were compared to monocistronic saRNA-HA or saRNA-NA controls encoding a single antigen (TC83-HA-40A or TC83-NA-40A), as well as to individually formulated 1:1 mixtures of 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. In Balb / c mice, IM immunization was performed on day 0 using 20 ng of a bicistronic saRNA vaccine preparation, 20 ng of a monocistronic saRNA vaccine preparation, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA and saRNA-NA, and 200 ng of modRNA control. All saRNA LNPs were placed in a 10 mM Tris / 10% sucrose + 20 mM glutamate, pH 7.4 base selected for clinical use. On day 21 (3 weeks after initial immunization), an anti-HA antibody response was induced as measured by HAI and MNT (Figures 1 and 2), and an anti-NA antibody response was induced as measured by NAI (Figure 4). The bicistronic saRNA vaccine candidate achieved titers similar to those of saRNA vaccines composed of individually formulated monocistronic saRNA HA + saRNA-NA 1:1 mixtures. The titers of saRNA vaccine formulations expressing two antigens were similar to, or only slightly lower than, those obtained by saRNA controls expressing only a single antigen.These results confirm the feasibility of the bicistronic saRNA approach, and demonstrate that in Balb / c mice, using a saRNA vaccine based on antibody titers after a single immunization resulted in dose savings compared to modRNA.

[0324] Overall, the evaluated bicistronic saRNA constructs yielded functional and neutralizing antibody titers similar to those induced by saRNA vaccines composed of individually formulated HA and NA monocistronic saRNAs. The titers of saRNA vaccines expressing both antigens were similar to or only slightly lower than those of the saRNA-HA or saRNA-NA-only controls. These preliminary test results confirm that the bicistronic saRNA approach is viable regardless of the regulatory element used to drive the second target gene (IRES or SGP), the order of antigen repositioning (HA-NA or NA-HA), Kozak sequence deletions, and polyA tail length (40A or 80A).

[0325] (Example 7) Immunogenicity of saRNA influenza vaccines containing modified nucleosides To determine whether incorporating modified bases could produce saRNA vaccines that were more tolerable and potent, saRNA preparations expressing influenza HA were generated by replacing uridine with varying amounts of N1-methylpseudridine, ranging from 0% to 100%. The effect of increasing the percentage of modified bases on in vitro antigen expression was cell type-dependent. Specifically, in immunocompetent human cell lines, such as HeLa, saRNAs containing 25–75% N1-methylpseudridine resulted in higher in vitro antigen expression than unmodified (0%) saRNA controls. However, saRNAs with 100% modified bases consistently produced low levels of antigen, regardless of cell type. This may be due to impaired replicase function. Increasing the percentage of modified nucleosides incorporated into saRNA also correlated with decreased activation levels of various PRR or RNA sensors in reporter cell lines, such as TLR3, TLR7, and RIG-1 (data not shown).

[0326] To assess immunogenicity, Balb / c mice were immunized on day 0 with 200 ng of saRNA vaccine preparations containing varying amounts of N1-methylpseudridine. As measured by serum cytokine and chemokine secretion on day 1 postvaccination, higher levels of modified nucleosides correlated with lower innate immune activation (Figure 5), potentially improving vaccine tolerability. However, higher levels of modified nucleosides in the saRNA also correlated with a decrease in neutralizing antibody titers three weeks postvaccination (Figure 6), which may potentially reflect an effect on replicase activity. These results were confirmed in different C57BL6 / J mouse species (Figures 7 and 8). Based on mouse data, saRNA constructs incorporating 50% modified nucleosides substantially reduced cytokine and chemokine secretion compared to unmodified controls, and the decrease in antibody titers was more moderate, reaching levels similar to or higher than the modRNA-HA benchmark. Overall, the data suggest that partial incorporation of modified bases may be tolerated by saRNAs to reduce initial innate immune stimulation while still eliciting a robust adaptive humoral response.

[0327] While the use of 50% modified bases may partially affect replicase function, in humans, enhanced tolerability could potentially lead to improved saRNA vaccines.

[0328] Regarding Figure 5, female Balb / c mice were immunized on day 0 with either 200 ng of an LNP-formulated influenza saRNA vaccine preparation containing different amounts (0% to 100%) of N1-methylpseudridine, or with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. Serum cytokines and chemokines were measured 24 hours after the first immunization using the Mouse Anti-Virus Response panel LEGENDplex assay. Data are reported as median values ​​along with interquartile ranges.

[0329] Regarding Figure 6, female Balb / c mice were immunized on day 0 with either 200 LNP-formulated influenza saRNA vaccine preparations containing different amounts (0% to 100%) of N1-methylpseudridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. On day 21 (3 weeks after immunization), the antibody response to A / Wisconsin / 588 / 2019 was measured by HAI or 1-Day MNT assay. HAI and 50% neutralizing titers are reported (geometric mean and geometric SD).

[0330] Regarding Figure 7, female C57BL6 / J mice were immunized on day 0 with either 200 ng of an LNP-formulated influenza saRNA vaccine preparation containing different amounts (0% or 50%) of N1-methylpseudridine, or with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. Serum cytokines and chemokines were measured 24 hours after the first immunization using the Mouse Anti-Virus Response panel LEGENDplex assay. Data are reported as median values ​​along with interquartile ranges.

[0331] Regarding Figure 8, female C57BL6 / J mice were immunized on day 0 with 200 LNP-formulated influenza saRNA vaccine preparations containing different amounts (0% or 50%) of N1-methylpseudridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019(H1N1)HA. Antibody response to A / Wisconsin / 588 / 2019 was measured on day 21 (3 weeks after immunization) by HAI or 1-Day MNT assay. HAI and 50% neutralizing titers are reported (geometric mean and geometric SD).

[0332] (Example 8) Immunogenicity of a quadrivalent bicistronic saRNA influenza vaccine encoding HA and NA derived from four seasonal influenza strains. The key pharmacological properties of influenza saRNA vaccines were evaluated in in vitro and in vivo non-clinical studies. In vitro and in vivo studies demonstrated that influenza saRNA vaccines, encoding influenza HA and / or NA proteins, induced potent functional and neutralizing antibody responses as well as robust CD4+ and CD8+ T cell responses in mice, enabling significantly lower doses compared to modRNA. SaRNA replication also leads to innate immune activation, potentially enhancing the adaptive immune response to the expressed antigen(s). Efficient in vitro expression of HA and NA glycoproteins from influenza saRNA vaccines was demonstrated in cultured cells. Immunogenicity studies in mice, rats, and ferrets demonstrated that various influenza saRNA vaccine preparations elicited potent functional and neutralizing antibody responses as well as T cell responses. Innate immune activation, measured by serum cytokine and chemokine release 24 hours after immunization, was also demonstrated in mice and rats. Immunogenicity studies in mice, benchmarked against influenza modRNA vaccines, support the use of bicistronic influenza saRNA constructs expressing two distinct influenza antigens (HA and NA) from the same saRNA vector. Immunogenicity studies in mice also demonstrate that saRNAs may tolerate partial incorporation of modified nucleosides to elicit a potent adaptive humoral response while reducing initial innate immune stimulation. Finally, immunogenicity studies in mice support combinations of four bicistronic influenza saRNA constructs, each encoding a different HA and NA, for targeting four seasonal influenza strains.

[0333] The influenza saRNA vaccine candidate selected for the initial proof-of-concept trial will contain a codon-optimized coding sequence of the full-length HA or NA glycoprotein derived from a viral strain based on A / Wisconsin / 588 / 2019(H1N1) cells, which is recommended for use in the Northern Hemisphere during the 2021, 2022, and 2022-2023 influenza seasons and in the Southern Hemisphere during the 2022 season.

[0334] [Table 12]

[0335] A seasonal influenza saRNA vaccine expressing four different HA proteins and four different NA proteins to adapt to the influenza strains prevalent in each season can be realized using four bicistronic saRNA components. The feasibility of the quadrivalent bicistronic saRNA approach was evaluated in a mouse immunogenicity study benchmarking the approved adjuvant-added seasonal quadrivalent influenza vaccine (QIV; FluAd) for the Northern Hemisphere 2021-22 season. BALB / c mice were immunized with either a total dose of 0.8 μg of the quadrivalent saRNA vaccine (0.2 μg per component) or 2.4 μg of the approved QIV comparator on days 0 and 28. On day 42 (two weeks after the second immunization), anti-HA antibody responses were induced for each of the four components as measured by HAI and MNT (Figure 9), and anti-NA antibody responses were also induced as measured by NAI (Figure 10). The quadrivalent bicistronic saRNA vaccine candidate achieved HA and NA titers similar to or higher than those of the QIV control group.

[0336] Regarding Figure 9, female Balb / c mice were immunized on day 0 with either 20 ng of LNP-formulated tetravalent saRNA, 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 strain), and B / Phuket / 3073 / 2013 (B / Yamagata strain), or 2.4 μg of an approved adjuvant-added tetravalent inactivated vaccine (QIV; FluAd). On day 42 (two weeks after the second dose), the antibody response to each vaccine component was measured by HAI or 1-Day MNT assay. HAI and 50% neutralizing titers are reported (geometric mean and geometric SD).

[0337] Regarding Figure 10, female Balb / c mice were immunized on day 0 with either 20 ng of LNP-formulated quadrivalent saRNA, 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 strain), and B / Phuket / 3073 / 2013 (B / Yamagata strain), or 2.4 μg of an approved adjuvant-added quadrivalent inactivated vaccine (QIV; FluAd). On day 42 (two weeks after the second dose), the antibody response to each vaccine component was measured by NAI. NAI titers have been reported for three of the four strains (geometric mean and geometric standard deviation). Due to technical issues associated with the NAI assay for this strain, the H3N2 NAI titer could not be reported for either saRNA or QIV.

[0338] (Example 9) Effects in humans C4861001 is an ongoing Phase 1 First-In, First-Hear trial evaluating the safety, tolerability, and immunogenicity of PF-07845104. As of the data cutoff date of November 15, 2022, 253 participants had been randomized, and 248 had received the vaccine. A total of 5 participants did not receive the vaccine (1 participant in vaccine preparation 4 of the 2.5 μg group, 2 participants in vaccine preparation 5 of the 2.5 μg group, 1 participant in vaccine preparation 5 of the 10 μg group, and 1 participant in vaccine preparation 6 of the 2.5 μg group). The approved QIV was used as the comparator.

[0339] Safety and efficacy - C4861001 test C4861001 is an ongoing, FIH Phase 1, randomized, placebo-controlled, evaluator-blinded, sponsor-open-label, dose-finding, vaccine composition / formulation selection study in healthy adults. This study will evaluate the safety, tolerability, and immunogenicity of single doses of various monocistronic and ultimately bicistronic saRNA vaccine preparations for influenza. Participants aged 18–49 years will be randomly assigned in a 4:1 ratio to receive either a saRNA vaccine preparation or placebo. An additional group of participants receiving approved QIV will be independently enrolled as a control.

[0340] Table 13 shows the details of the saRNA vaccine preparations.

[0341] [Table 13]

[0342] Vaccine preparations 1, 2, and control group - randomly assigned participants A total of 36 participants were randomly assigned to either vaccine preparation 1 or vaccine preparation 2, 63 participants to the placebo group, and 33 participants to the control group.

[0343] Vaccine preparation 1 In this group, 11 participants received a 1 μg dose, 13 participants received a 2.5 μg dose, and 12 participants received a 10 μg dose. One participant was randomly assigned to the 2.5 μg group but mistakenly received a 1 μg dose and was therefore included in the 1 μg group for safety analysis purposes.

[0344] Five participants (one from the 1 μg group and two each from the 2.5 μg and 10 μg groups) discontinued participation in the trial after vaccination.

[0345] Vaccine preparation 2 In this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg of vaccine. One participant in the 1 μg group lost track of time after vaccination.

[0346] Vaccine preparations 3, 4, and 7 and control group - randomly assigned participants A total of 36 participants were randomly assigned to receive vaccine preparation 3, vaccine preparation 4, and vaccine preparation 7, while 63 participants were randomly assigned to receive a placebo. One participant in the vaccine preparation 4 2.5 μg group did not receive the vaccine.

[0347] Vaccine preparation 3 In this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg of vaccine.

[0348] Two participants in the 1 μg group discontinued participation in the trial after vaccination.

[0349] Vaccine preparation 4 In this group, 12 participants received 1 μg and 10 μg of vaccine, and 11 participants received 2.5 μg of vaccine.

[0350] Two participants (one from the 1 μg group and one from the 10 μg group) discontinued participation in the trial after vaccination.

[0351] Vaccine preparation 7 In this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg of vaccine.

[0352] Vaccine preparations 5, 6 and control group - randomly assigned participants A total of 38 participants were randomly assigned to vaccine preparation 5, 35 to vaccine preparation 6, 63 to placebo, and 33 to the control group. Three participants in vaccine preparation 5 (two in the 2.5 μg group and one in the 10 μg group) and one participant in vaccine preparation 6 (2.5 μg) did not receive the vaccine.

[0353] Vaccine preparation 5 In this group, 12 participants received 1 μg and 2.5 μg of vaccine, and 11 participants received 10 μg of vaccine. Two participants in the 2.5 μg group and one participant in the 10 μg group did not receive the vaccine.

[0354] Two participants in the 1 μg group discontinued participation in the trial after vaccination.

[0355] Vaccine preparation 6 In this group, 11 participants received 1 μg and 2.5 μg of vaccine, and 12 participants received 10 μg of vaccine. One participant in the 2.5 μg group did not receive the vaccine.

[0356] Five participants (one from the 1 μg group and four from the 2.5 μg group) discontinued participation in the trial after vaccination.

[0357] immunogenicity A total of 253 participants were randomly assigned to receive vaccination, and 196 of them were eligible for immunogenicity assessment.

[0358] A dose-dependent increase in HAI GMT was observed 4 weeks after vaccine administration. HAI GMT on day 1 (before vaccination), and 1, 2, and 4 weeks after vaccine administration are shown in Figures 11, 12, and 13.

[0359] Vaccine preparations 1 and 2 The proportion of participants achieving seroconversion after 4 weeks with 10 μg was higher than the proportion of participants achieving seroconversion after 1 μg and 2.5 μg (Table 14).

[0360] Vaccine preparations 3, 4, and 7 Regarding vaccine preparation 3, the proportion of participants achieving seroconversion 4 weeks after 10 μg was higher than the proportion of participants achieving seroconversion after 1 μg and 2.5 μg (Table 15).

[0361] [Table 14-1]

[0362] [Table 14-2]

[0363] [Table 15]

[0364] [Table 16]

[0365] Regarding vaccine preparation 4, the proportion of participants achieving seroconversion 4 weeks after 1 μg was higher than the proportion of participants achieving seroconversion after 2.5 μg and 10 μg (Table 12).

[0366] For vaccine preparation 7, the proportion of participants who achieved seroconversion 4 weeks after 1 μg was equal to the proportion of participants who achieved seroconversion after 2.5 μg (Table 12).

[0367] Vaccine preparations 5 and 6 Regarding vaccine preparation 5, no participants achieved seroconversion after 4 weeks. Regarding vaccine preparation 6, the proportion of participants achieving seroconversion after 4 weeks with 10 μg was higher than the proportion of participants achieving seroconversion after 1 μg and 2.5 μg (Table 13).

[0368] (Example 10) Enhancement of saRNA flu efficacy by co-delivering modRNA-NS1 in trans. We used a saRNA platform based on the genome of Venezuelan encephalitis alphavirus, in which structural genes under the subgenome promoter (SGP) were replaced with vaccine antigens. Influenza hemagglutinin (HA) derived from A / Wisconsin / 588 / 2019(H1N1) was used as the target vaccine antigen. Upon delivery into the cell, the saRNA expresses RNA-dependent RNA polymerase (RDRP), which replicates the RNA and produces multiple copies of the gene(s) expressed under the SGP. One potential strategy to evade innate immune sensing, which is potentially induced during saRNA delivery, is based on the use of proteins that can efficiently inhibit interferon (IFN) induction and / or signaling cascades, similar to the mechanisms used by various RNA viruses to evade innate immune recognition. In this study, unless otherwise explicitly disclosed herein, the influenza NS1 antigen (AF389122 from Genbank (NCBI)), a well-known potent IFN antagonist derived from A / Puerto Rico / 08 / 1934 (H1N1), was used to enhance saRNA expression in vitro in HeLa cells, a cell line sensitive to saRNA. Codon optimization of the NS1 sequence was performed by a commercial vendor. Our data show that co-delivery of small amounts of NS1 as modRNA in trans significantly increases saRNA-HA expression levels and promotes the preservation of overall cell viability. These results provide evidence that IFN antagonist proteins can effectively block the naturally induced innate immune response by saRNA and may be used to enhance the immunogenicity and efficacy of saRNA vaccines. Figure 14 shows that trans addition of modNS1 increases saRNA-HA expression and cell viability in HeLa cells. As used herein, "modNS1" refers to a polynucleotide encoding the NS1 protein, wherein the polynucleotide comprises N1-methylpseudridine (m1ψ).Figure 14 shows the increased antigen expression and cell viability mediated by saRNA when modNS1 was delivered trans-co-delivered. HeLa cells were transfected with gradually increasing doses of modNS1 or modGFP (as a control) in combination with 25 ng of saRNA-HA-Wisconsin. The total number of cells for which % HA-positive cells, protein expression levels (MFI), and cell viability were assessed was measured at 24 hours. C. Confocal images (10x) of HeLa cells transfected with 25 ng of saRNA-HA-Wisconsin alone (left) or with 25 ng of modNS1 (right). Cells were fixed at 24 hours and stained with FI6 HA human monoclonal antibody and goat anti-human A647 secondary antibody. It is also shown that low doses of modNS1 are sufficient to increase HA expression mediated by saRNA containing unmodified or modified nucleosides. See Figures 15, 16, and 17, which illustrate how low doses of modNS1 increase antigen expression in both normal saRNA and saRNA containing 50% m1ψA. A total of 25 ng of saRNA-HA-Wisconsin was transfected into HeLa cells in combination with low-escalation modNS1 (1 ng to 25 ng) for blue (i.e., "RMM59 + NS1"), or with saRNA (dose-balanced control) for red (i.e., "RMM59 + (X)RMM59"). Cells were fixed at 24 hours and stained with FI6 HA human monoclonal antibody and goat anti-human A488 secondary antibody. (%) HA-positive cells and cell counts were measured. Confocal images (10x) of HeLa cells transfected with 25 ng of saRNA-HA-Wisconsin alone or with 25 ng of modNS1 were observed (data not shown). Table 17 summarizes the transfected amount, %HA(+) cells, and absolute number of cells obtained for each of the tested conditions.

[0369] [Table 17]

[0370] Figure 16. We tested saRNA encoding HA Wisconsin but containing 50% m1ψ under the same conditions as described for Figure 15 (and related Table 17), and the results are shown in Figure 16 and related Table 18.

[0371] [Table 18]

[0372] (Example 11) The presence of modNS1 in human monocytes and antigen expression via saRNA. Figure 17 shows data demonstrating the ability to improve saRNA replication in human monocytes. Human CD14+ cells (stem cells) were transfected with GFP-expressing saRNA at varying doses (5 ng to 100 ng) in combination with modNS1 (0 to 50 ng) in increasing doses, and modRNA-GFP was used as a control. The total percentage of positive cells was measured by FACS at 24 hours, and cell viability was also measured. Figure 18 shows the total percentage of positive cells measured using aqua live / dead staining.

[0373] (Example 12) Trans-delivery of modNS1 is an advantage compared to the cis (bisistronic) method. Figure 19A supports the idea that the combination of monocistronic saRNA and modNS1 contributes to higher HA levels compared to the bicistronic method. A. HeLa cells were transfected 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. (%) HA-positive cells and protein expression levels (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 19B: Diagrams of different saRNA configurations (monocistronic and bicistronic) alone or in combination with modNS1 as shown in Figure 19A.

[0374] (Example 13) modNS1 increases antigen expression levels from uRNA but does not increase antigen expression levels from modRNA. Figures 20A-B show that NS1 can increase HA antigen expression using conventional uridine-containing RNA. A fixed total amount of RNA (100 ng) was added to HeLa cells for each condition. Combinations of u RNA (i.e., unmodified RNA) or conventional modRNA with modNS1 or modGFP (control) were added in different ratios, and %HA(+) cells (Figure 20A) and cell count (Figure 20B) were assessed at 24 hours. Confocal images in HeLa cells were observed comparing those with 100 ng of RMM71 (uRNA HA Wisconsin) and those with 50 ng of uRNA and 50 ng of modNS1 (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 20C: Comparison of different configurations tested.

[0375] (Example 14) Discharge via the modNS1 transformer Trans-delivery of modNS1 significantly increases saRNA-mediated antigen expression, promoting the preservation of cell viability. See Figure 21.

[0376] Further findings based on this disclosure include the fact that combining modNS1 with saRNA containing 50% modified nucleosides promotes more efficient antigen expression and enhances the preservation of cell viability. ModNS1 increases antigen expression by uRNA but not by modRNA, thus suggesting a role for NS1 in preventing the activation of the uridine-induced innate immune response in cells. Furthermore, modNS1 does not improve antigen expression by saRNA in human monocytes.

[0377] (Example 15) Immunogenicity of 8-valent influenza HA / NA saRNA vaccine in mice This study was conducted to test the immunogenicity of an octvalent saRNA-LNP vaccine encoding HA and NA antigens derived from four influenza virus strains in a mouse model. The saRNA constructs used in this study encoded 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 control FluAd, the octvalent saRNA-LNP vaccine elicited equivalent or higher levels of functional anti-HA, anti-NA, and virus-neutralizing antibodies.

[0378] Eighteen-valent vaccine formulations consisting of either a monocistronic or bicistronic saRNA construct exhibited comparable immunogenicity in mice after two doses. Polyvalent saRNA vaccines, either pre-mixed ("pre-mixed") or post-mixed ("post-mixed") with LNPs, exhibited comparable immunogenicity in mice. Eighteen-valent saRNA-LNP vaccines showed moderate interference with the immunogenicity of some viral strains, particularly influenza B virus strains, compared to mice vaccinated with a monocistronic single-antigen control. Overall, these data support continued evaluation of polyvalent saRNA vaccines encoding influenza virus antigens.

[0379] The primary objective of this study was to evaluate the immunogenicity of an octavalent saRNA vaccine encoding hemagglutinin (HA) or neuraminidase (NA) proteins derived from influenza virus in mice, compared with monocistronic and bicistronic saRNA vaccine controls. The octavalent vaccine consisted of either eight monocistronic HA or NA saRNAs or four bicistronic HA-NA saRNA constructs. The octavalent saRNA vaccine was also compared with an approved quadrivalent inactivated influenza virus vaccine control (containing eight HA / NA antigens derived from four strains) and a quadrivalent modified RNA...

Claims

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

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

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

4. The composition according to claim 3, wherein the self-amplified RNA comprises unmodified nucleotides.

5. The composition according to claim 3, wherein the self-amplified RNA comprises modified nucleotides.

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

7. The composition according to claim 1, 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 an alphavirus; a first subgenome promoter derived from an alphavirus; a first open reading frame encoding a first target gene derived from influenza virus hemagglutinin (HA); a second subgenome promoter derived from an alphavirus; a second open reading frame encoding a second target gene derived from an influenza virus; a 3' untranslated region (3'UTR); and a 3' polyA sequence, wherein the second target gene derived from the influenza virus is an influenza 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 to 8, wherein the first RNA molecule and the second RNA molecule are not linked.

10. The composition according to any one of claims 1 to 9, wherein the influenza 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 to 10, wherein the influenza 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 to 11, wherein the influenza NS1 is encoded by an amino acid sequence having at least about 80% sequence identity with the amino acid sequence shown in Sequence ID No.

33.

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

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

15. The composition according to any one of claims 1 to 14, wherein the increased expression of the target mRNA is sustained for at least about 6 hours.

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

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

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

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

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