Innate immune modulating RNA vaccines

Abstract

Compositions are provided that include a lipid nanoparticle (LNP): a mRNA encoding a gene of interest loaded within the LNP; and an inhibitory nucleic acid, such as a small interfering RNA (siRNA) targeting IFN-α / β receptor 1(Ifnar1) gene loaded within the LNP, and use of the compositions for generating an immune response.

Description

[0001]

[0002] MIT 26293H

[0003] Innate immune modulating RNA vaccines

[0004] Federal Funding Statement

[0005] This invention was made with government support under Al 144462 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] Sequence Listing Statement

[0007] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on December 4, 2024 having the file name “24-2169-US-PRO. xml” and is 121,914 bytes in size.

[0008] Background

[0009] RNA vaccines have emerged as a breakthrough technology. However, the lipid nanoparticles (LNP) commonly used to deliver RNA trigger innate immune recognition, especially via type I interferon (IFN) signaling.

[0010] Summary

[0011] In one aspect, the disclosure provides compositions, comprising:

[0012] (a) a lipid nanoparticle (LNP);

[0013] (b) a mRNA encoding a gene of interest loaded within the LNP; and

[0014] (c) an inhibitory nucleic acid targeting IFN-a / p receptor l(Ifnarl) gene loaded within the LNP. In one embodiment, the inhibitory nucleic acid comprises a short interfering RNA (siRNA). In another embodiment, the inhibitory’ nucleic acid targets a region of the Ifharl gene comprising the nucleotide sequence selected from SEQ ID NO: 1-4, 83, and 89. In a further embodiment, the inhibitory nucleic acid targets a region of the Ifnar1 gene comprising the nucleotide sequence selected from SEQ ID NO: 1-24 and 83-95.

[0015] In one embodiment, the inhibitory' nucleic acid comprises a double stranded siRNA sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs: (a) sense (5’— >3'): GUU GAU CCG UUU AUU CCA UU (SEQ ID NO:25) and anti-sense (5’— >3'): AAU GGA AUA AAC GGA UCA AC (SEQ ID NO:26) (IFNARl.1 mouse minimal sequence);

[0016] (b) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CC (SEQ ID NO:27) and anti-sense (5’-->3’): GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:28) (IFNARl.2 mouse minimal sequence);

[0017] (c) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UC (SEQ ID NO:29) and anti-sense (5'— >3'): anti-sense (5'— >3'): GAG GAC CAA UCU GAG CUU L1G (SEQ ID NO:30) (IFNARl.1 human minimal sequence);

[0018] (d) sense (5'— >3'): GUG CUC CAA AAC AGU CUG GA (SEQ ID NO:31) and anti-sense (5'— >3'): (5’— >3’): UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:32) (IFNARl.2 human minimal sequence);

[0019] (e) sense (5f— >3’): UCA UAG AUG ACA ACU UUA UC (SEQ ID NO:96) and anti-sense (5’— >3’): GAU AAA GUU GUC AUC UAU GA (SEQ ID NO:97) (IFNARl.3 human minimal sequence); and

[0020] (f) sense (5'- >3'): UGU UCA UUC AUC CCG AGA AC (SEQ ID NO: 108) and anti-sense (5 >3 '): GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 109) (IFNARl.4 human minimal sequence).

[0021] In one embodiment, the 5" end of the inhibitory' nucleic acids are phosphorylated. In another embodiment, the inhibitory' nucleic acids comprise a two nucleotide single stranded overhang at the 3’ terminus. In a further embodiment, the two nucleotide single stranded overhang at the 3 ’ terminus comprises dTdT. In various embodiments, the inhibitory nucleic acid comprises a double stranded siRNA sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs:

[0022] (a) sense (5 '->3 '): GUU GAU CCG UUU AUU CCA UUC dTdT (SEQ ID NO: 72) and anti-sense (5'->3’): GAA UGG AAU AAA CGG AUC AAC dTdT (SEQ ID NO:73) (IFNARl.1 mouse 21 nt sequence with overhang);

[0023] (b) sense (5’— >3’): GUA GAA GUA AAG CAC GCG CCU dTdT (SEQ ID NO: 74) and anti-sense (5 '->3'): AGG CGC GUG CUU UAC UUC UAC dTdT (SEQ ID NO:75); (IFNAR1.2 mouse 21 nt sequence with overhang);

[0024] (c) sense (5 '->3'): CAA AGC UCA GAU UGG UCC UCC dTdT (SEQ ID NO:76) and anti-sense (5'— >3’): GGA GGA CCA AUC UGA GCU UUG dTdT (SEQ ID NO:77) (IFNARl.1 human 21 nt sequence with overhang); and (d) sense (5’— >3'): CAA AGC UCA GAU UGG UCC UCC dTdT (SEQ ID NO:78) and anti-sense (5'— >3’): GGA GGA CCA AUG UGA GCU UUG dTdT (SEQ ID NO:79) (IFNAR1 2 human 21 nt sequence with overhang). In one embodiment, the 5’ end of the siRNA is phosphorylated, and the 3’ end is hydroxylated.

[0025] In another embodiment, the siRNA comprises one or more locked nucleic acids (LNA). In a further embodiment, the gene of interest encodes an antigen, including but not limited to an immunogenic portion of a viral, bacterial, parasitic, protozoan, fungal, or tumor antigen. In some embodiments, the antigen comprises a human immunodeficiency virus (HIV) or a severe acute respiratory syndrome (SARS) antigen.

[0026] In one embodiment, wherein the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a polymer-conjugated lipid. In another embodiment, the helper lipid comprises phosphocholines, phosphoethanolamines, or combinations thereof. In a further embodiment, the polymer-conjugated lipid composes polyethylene glycol or polysarcosine conjugated to phosphoethanolamines, 1,2-dimyristoyl-rac-glycero, or other amphiphilic molecules. In one embodiment, the amine-to-phosphate (N: P) ratio in the LNP loaded with RNA ranges from about 1: 1 to about 20:1. In another embodiment, the lipid-to-RNA volume ratio in the LNP may range from about 1: 1 to about 1:20. In a further embodiment, the LNP comprises

[0027] (a) N 1, N 3, N 5 -tris(3 -(didodecylamino)propyl)benzene -1,3,5 -tricarboxamide (TT3),

[0028] (b) (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA),

[0029] (c) 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE),

[0030] (d) cholesterol, and

[0031] (e) 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k);

[0032] In one embodiment, the disclosure provides vaccines comprising a composition of the disclosure where the gene of interest encodes an antigen. Tire disclosure also provides methods for generating an immune response against an antigen, comprising administering to a subject an amount effective to generate an immune response in the subject of the composition or vaccine of any embodiment herein where the gene of interest encodes an antigen. In another embodiment, the methods comprise of treating an infection or limiting development of an infection in a subject in need thereof comprising administering to the subject the composition or vaccine of any embodiment herein where the gene of interest encodes an antigen in an effective amount to induce an immune response against the antigen. In one embodiment, the antigen comprises an HIV antigen, and the subject is at risk of, or has, an HIV infection. In another embodiment, the antigen comprises a SARS-CoV-2 antigen, and the subject is at risk of, or has, an SARS-CoV-2 infection. The subject may be any mammalian subject, including but not limited to a human subject.

[0033] Description of the Figures

[0034] Figure 1. Antigen-specific immunity from modRNA vaccines improve with siRNA co-delivery. Groups of balb / c mice (n=5 animals / group) were immunized i.m. in each leg with 5 pg modRNA loaded in LNPs and were evaluated for vaccine-elicited immune responses. Shown are counts of total GC B cells (a), follicular helper T cells (b), gating for antigen-specific B cells (c), counts for antigen-specific B cells (d), and the frequency of IgG+ antigen-specific B cells (e). Histogram of antigen-specific B cells with equivalent IgG-expression levels is shown in (f), and the mean fluorescence intensity (MFI) of IgG+ antigenspecific B cells are shown in (g). Serum antibody responses were quantified by ELISA assay conducted on mouse sera collected at 14 days post-vaccination and the endpoint titer at 2-weeks post-vaccination are shown in (h). Each column graph shows means ± s.e.m. Statistics represent one-way ANOVA and Tukey's HSD Test (*, p<0.05; **, p<0.01; ***, p<0.001: ****, p<0.0001).

[0035] Figure 2. Cytokine profile in the muscle and draining lymph node from empty LNPs. Groups of balb / c mice (n==5 animals / group) were immunized i.m. in each leg with empty LNPs and were evaluated for cytokine induction. Shown from the muscle are: (a) MCP-1; (b) IFN-y; (C) CXCLl; (d) CXCL10; (e) IL-6; (f) TNF-a; (g) IL-1; (h) IFN- a; and (i) IFN-p. From the popliteal lymph node, we show IFN-p (j) as a representative cytokine. Graphs show means i s.e.m. Statistics represent one-way ANOVA and Tukey’s HSD Test (ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0036] Figure 3. Effects of silencing Ifnarl on modRNA expression in C2C12 mouse myoblasts during in vitro transfection. C2C12 mouse myoblasts were treated with PBS, LNPs loaded with modRNA encoding for GFP (‘(modGFP)’), or LNPs that co-load modGFP and siRNA against Ifnarl (‘(modGFP / siIFNAR1)’). At 24h post-transfection, cells were analyzed by How cytometry, shown as live cell histogram comparing expression of GFP.

[0037] Figure 4. Germinal center response analyses for modRNA vaccines. Groups of balb / c mice (n=5 animals / group) were immunized i.m. in each leg with 5 pg modRNA loaded in LNPs and were evaluated for vaccine-elicited immune responses. Shown are histogram of GC B cells expressing IgG at similar levels (a), and dilution curves serum antibody at 2 -weeks post-vaccination (b). Statistics represent one-way ANOVA and Tukey’s HSD Test (*, ><0.05; **, / ><0.01; ***, ><0.001; ****,£><0.0001).

[0038] Figure 5. LNP formulation used in Moderns’ s COVID-19 vaccine displays the same enhancements in vaccine-elicited immune response when co-delivering silFNARl.

[0039] Groups of balb / c mice (n=5 animals / group) were immunized i.m. in each leg with 1 pg repRNA or 5 pg modRNA loaded in the LNP formulation used in Mode ma’s COVID-19 vaccine (mRNA-1273). Shown are counts of GC B cells (a), follicular helper T cells (b), gating for antigen-specific B cells (c), counts for antigen-specific B cells (d), and the frequency of IgG+ antigen -specific B cells (e). Histogram of antigen -specific B cells with equivalent IgG-expression levels is shown in (f), and the mean fluorescence intensity (MFI) of IgG+ antigen-specific B cells are shown in (g). Serum antibody responses were quantified by ELISA assay conducted on mouse sera collected at 14 days post-vaccination. The dilution curves are shown in (h), AUG of antibody titer are shown in (i), and the endpoint titer are shown in (j). Each column graph shows means ± s.e.m. Statistics represent one-way ANOVA and Tukey’s HSD Test (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.000I).

[0040] Detailed Description

[0041] As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0042] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the w'ords “herein,” “above” and "below'" and ords of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0043] All embodiments of any aspect of tire disclosure can be used in combination, unless the context clearly dictates otherwise.

[0044] As used herein, "about" will mean up to plus or minus 5% of the particular value. In one aspect, the disclosure provides compositions, comprising:

[0045] (a) a lipid nanoparticle (LNP); (b) a mRNA encoding a gene of interest loaded within the LNP; and

[0046] (c) an inhibitory nucleic acid targeting IFN-a / 'P receptor l(Ifnarl) gene loaded within the LNP.

[0047] As shown in the examples that follow, the inventors have demonstrated that the compositions of the invention silence IFN-a / 'P receptor 1 (Ifnarl) to suppress type 1 interferon response upon administration of the compositions, permitting higher levels and more prolonged expression of mRNAs delivered by LNPs, which could be effective for promoting humoral immunity to vaccine immunogens.

[0048] Unlike self-relicating RNA (repRNA), mRNA may be substituted with modified nucleosides to improve RNA stability and limit innate immune recognition. Tirus, one would not expect modfifed mRNA to benefit from combining with an inhibitory nucleic acid targeting Ifnarl. However, the LNPs used to deliver these mRNA induce significant amounts of cytokine production local to the administration site, including type I IFNs.

[0049] The mRNA may comprise unmodified mRNA, or modified mRNA (“modRNA?’). When modified mRN A is used, the percentage of modified residues may be any percentage as appropriate for an intended use. In embodiments using modRNA, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or all 100% of the residues may be modified. The modRNAs may comprise any triphosphate analog that can be incorporated into mRNA. In various non-limiting embodiments, tire analog may comprise one or more of the following (including the base it can substitute for).

[0050] Analog Base

[0051] 5 -Carboxymethylesteruridine Uridine

[0052] Thienouridine Uridine

[0053] 5 -Methoxyuridine Uridine

[0054] 5 -Carboxyuridine Uridine

[0055] 2-Thiouridine Uridine

[0056] 5 -Methyluridine Uridine

[0057] N 1 -Propylpseudouridine Uridine

[0058] N 1 -Methoxymethylpseudouridine Uridine

[0059] N 1 -Ethylpseudouridine Uridine

[0060] N 1 -Methylpseudouridine Uridine

[0061] Pseudouridine Uridine

[0062] 7 -Deazaadenosine Adenosine

[0063] N1 -Methyladenosine Adenosine N6-Methyladenosine Adenosine

[0064] 6-Chloropurineriboside Adenosine

[0065] 2-Amino-6-chloropurineriboside Adenosine

[0066] 2-Aminoadenosine Adenosine

[0067] Isoguanosine Guanosine

[0068] Thienoguanosine Guanosine

[0069] 2-Aminopurine-riboside Guanosine

[0070] 8-Oxoguanosine Guanosine

[0071] Thienocytidine Cytidine

[0072] 5 -Methoxy cytidi n e Cytidine

[0073] 5 -Hydroxymethylcytidine Cytidine

[0074] 5 -Formylcytidine Cytidine

[0075] 5-Aminoallylcytidine Cytidine

[0076] 5-Methylcytidine Cytidine

[0077] 5 -Hydroxy cytidine Cytidine

[0078] In one embodiment using modRNAs, all uridines in the mRNA are substituted with one or more analog. In one such embodiment, all uridines are substituted with Nl-methylpseudouridine.

[0079] The mouse and human Ifnarl gene sequences are provided in Table 1 below.

[0080] The inhibitory nucleic acid may be any inhibitory / nucleic acid that can serve to inhibit Ifnarl expression, including but not limited to antisense nucleic acids and a short interfering RNA (siRNA).

[0081] In some embodiments, the inhibitory' nucleic acid targets a region of the Ifnarl gene comprising the nucleotide sequence selected from SEQ ID NO: 1-4.

[0082] gttgatccgt ttattccatt (SEQ ID NO: 1) (IFNAR1.1 minimal sequence, mouse); gtagaagtaa agcacgcgcc (SEQ ID NO:2) (IFNAR1 2 minimal sequence, mouse); gtgctccaaa acagtctgga (SEQ ID NO:3) (IFNAR1.1 minimal sequence, human); tcatagatga caactttatc (SEQ ID NO:4) (IFNAR1.2 minimal sequence, human); tcatagatga caactttatc (SEQ ID NO:83) (IFNAR1.3 minimal sequence, human); and tgttcattca tcccgagaac (SEQ ID NO:89) (IFNAR1.4 minimal sequence, human).

[0083] The nucleic acid sequence of SEQ ID NO: 1 is in exon 3 in the mouse Ifnarl gene. The nucleic acid sequence of SEQ ID NO:2 is in exon 11 or 12 in the mouse Ifnarl gene. The nucleic acid sequence of SEQ ID NO:3 is in exon3 or 4 in the human Ifnarl gene. The nucleic acid sequence of SEQ ID NO: 4 is in exon 8 in the human Ifnarl gene. The nucleic acid sequence of SEQ ID NO:83 is in exon 2 in the human Ifnarl gene. The nucleic acid sequence of SEQ ID NO:89 is in exon 11 in the human Ifnarl gene.

[0084] In other embodiments, the inhibitory nucleic acid targets a region of the Ifnarl gene comprising the nucleotide sequence selected from SEQ ID NO: 1-24 and 83-95.

[0085] IFNAR1.1 (mouse) Exon 3

[0086] RefSeq: NM_010508.2

[0087] SEQ ID NO Sequence

[0088] 1 gttgatccgt ttattccatt

[0089] 5 gttgatccgt ttattccatt c

[0090] 6 gttgatccgt ttattccatt ct

[0091] gttgatccgt ttattccatt eta

[0092] 8 gttgatccgt ttattccatt ctac

[0093]

[0094] 9 gttgatccgt ttattccatt ctaca

[0095] IFNAR1.2 (mouse) Exon 11

[0096] RefSeq: NM_010508.2

[0097] SEQ ID NO Sequence

[0098] 2 gtagaagtaa agcacgcgcc

[0099] 10 gtagaagtaa agcacgcgcc t

[0100] 11 gtagaagtaa agcacgcgcc tg

[0101] 12 gtagaagtaa agcacgcgcc tga

[0102] 13 gtagaagtaa agcacgcgcc tgag

[0103]

[0104] 14 gtagaagtaa agcacgcgcc tgagg

[0105] IFNAR1.1 (human) Exon 3 or 4

[0106] RefSeq: NP_000620.2; XP_005261021. 1; XP_011527854. 1- SEQ ID NO Sequence

[0107] 3 caaagctcag attggtcctc

[0108] 15 caaagctcag attggtcctc c

[0109] 16 caaagctcag attggtcctc ca

[0110] 17 caaagctcag attggtcctc cag

[0111] 18 caaagctcag attggtcctc caga

[0112]

[0113] 19 caaagctcag attggtcctc cagaa

[0114] IFNAR1.2 (human) Exon 8

[0115] RefSeq: NP_000620.2; XP_005261021. 1

[0116] SEQ ID NO Sequence

[0117]

[0118] 4 gtgctccaaa acagtctgga 20 gtgctccaaa acagtctgga a

[0119] 21 gtgctccaaa. acagtctgga aa.

[0120] 22 gtgctccaaa acagtctgga aac

[0121] 23 gtgctccaaa acagtctgga aaca

[0122]

[0123] 24 gtgctccaaa. acagtctgga aa.cac

[0124] IFNAR1.3 (human) Exon 2

[0125] RefSeq: NM_000629.2, XM_005260964. 2, XM_011529552. 1

[0126] SEQ ID NO Sequence

[0127] 83 tca.tagatga. caactttatc

[0128] 84 tcatagatga caactttatc c

[0129] 85 tcatagatga caacttt.atc at

[0130] 86 tca.tagatga. caactttatc ctg

[0131] 87 tcatagatga caactttatc ctga

[0132]

[0133] 88 tcatagatga caactttatc ctgag

[0134] IFNAR1.4 (human) Exon 11

[0135] RefSeq: NM_000629. 2; XM 005260964. 2

[0136] SEQ ID NO Sequence

[0137] 89 tgttcattca tcccgagaac

[0138] 90 tgttcattca tcccgagaac a

[0139] 91 tgttcattca tcccgagaac at

[0140] 92 tgttcattca tcccgagaac att

[0141] 93 tgttcattca tcccgagaac attg

[0142]

[0143] 95 tgttcattca tcccgagaac attgg

[0144] In one specific embodiment, the inhibitory nucleic acid comprises a short interfering RNA (siRNA).

[0145] siRNA is a class of double -stranded RNA at first non-coding RNA molecules, typically 20-25 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation, siRNAs have a well-defined structure that is a short (usually 20 to 25-bp) doublestranded RNA (dsRNA), and may have phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides.

[0146] In some embodiments, the inhibitory nucleic acid comprises a double stranded siRNA sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs:

[0147] (a) sense (5'-->3’): GUU GAU CCG UUU AUU CCA UU (SEQ ID NO:25) and anti-sense (5'-->3'): AAU GGA AUA AAC GGA UCA AC (SEQ ID NO:26) (IFNAR1.1 mouse minimal sequence); (b) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CC (SEQ ID NO:27) and anti-sense (5’— >3'): GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:28) (IFNAR1.2 mouse minimal sequence);

[0148] (c) sense (5’— >3'): CAA AGC UCA GAU UGG UCC UC (SEQ ID NO:29) and anti-sense (5'— >3'): anti-sense (5'— >3'): GAG GAC CAA UCU GAG CUU UG (SEQ ID NO:30) (IFNAR1.1 human minimal sequence);

[0149] (d) sense (5 >3 ’): GUG CUC CAA AAC AGU CUG GA (SEQ ID NO:31) and anti-sense (5’— >3’): (5'— >3’): UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:32) (IFNARI.2 human minimal sequence);

[0150] (e) sense (5'->3'): UCA UAG AUG ACA ACU UUA UC (SEQ ID NO:96) and anti-sense (5'— >3'): GAU AAA GUU GUC AUC UAU GA (SEQ ID NO:97) (IFNAR1.3 human minimal sequence); and

[0151] (f) sense (5'->3'): UGU UCA UUC AUC CCG AGA AC (SEQ ID NO: 108) and anti-sense (5'->3’): GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 109) (IFNAR1.4 human minimal sequence).

[0152] In various other embodiments, the inhibitory nucleic acid comprises a double stranded siRNA sequence composing the nucleotide sequence of a pair of sequences selected from the following pairs:

[0153] (a) sense (5'— >3’): GUU GAU CCG UUU AUU CCA UU (SEQ ID NO:25) and anti-sense 5'->3r) AAU GGA AU A AAC GGA UCA AC (SEQ ID NO:26) (IFNAR1.1 mouse minimal sequence);

[0154] (b) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CC (SEQ ID NO:27) and anti-sense (5'~~ >3'): GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:28) (IFNARI.2 mouse minimal sequence);

[0155] (c) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UC (SEQ ID NO:29) and anti-sense (5'— >3'): anti-sense (5'— >3'): GAG GAC CAA UCU GAG CUU UG (SEQ ID NO:30) (IFNAR1.1 human minimal sequence);

[0156] (d) sense (5 >3 '): GUG CUC CAA AAC AGU CUG GA (SEQ ID NO:31 ) and anti-sense (5'-->3'): (5'-->3'): UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:32) (IFNAR1.2 human minimal sequence);

[0157] (e) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UUC (SEQ ID NO: 80) and anti-sense (5’— >3'): GAA UGG AAU AAA CGG AUC AAC (SEQ ID NO:33) (IFNARI. I mouse 21 nt sequence); (f) sense (5’— >3'): GUU GAU CCG UUU AUU CCA UUC U (SEQ ID NO:34) and anti-sense (5'— >3'): AGA AUG GAA UAA ACG GAU CAA C (SEQ ID NO:35) (IFNARl.l mouse 22 nt sequence);

[0158] (g) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UUC UA (SEQ ID NO: 36) and anti-sense (5'->3'): UAG AAU GGA ALTA AAC GGA UCA AC (SEQ ID NO:37) (IFNAR1.1 mouse 23 nt sequence);

[0159] (h) sense (5'— >3’): GUU GAU CCG UUU AUU CCA UUC UAC (SEQ ID NO:38) and anti-sense (5'— >3'): GUA GAA UGG AAU AAA CGG AUC AAC (SEQ ID NO:39) (IFNARl.l mouse 24 nt sequence);

[0160] (i) sense (5 '->3'): GUU GAU CCG UUU AUU CCA UUC UAC A (SEQ ID NO:40) and anti-sense (5'— >3’): UGU AGA AUG GAA UAA ACG GAU CAA C (SEQ ID NO:41) (IFNARl.l mouse 25 nt sequence);

[0161] (j) sense (5'— >3’): GUA GAA GUA AAG CAC GCG CCU (SEQ ID NO:42) and anti-sense (5’— >3'): AGG CGC GUG CUU UAC UUC UAC (SEQ ID NO:43);

[0162] (IFNAR1.2 mouse 21 nt sequence);

[0163] (k) sense (5'->3'): GUA GAA GUA AAG CAC GCG CCU G (SEQ ID NO: 44) and anti-sense (5'— >3’): CAG GCG CGU GCU UUA CUU CUA C (SEQ ID NO:45) (IFNARI.2 mouse 22 nt sequence);

[0164] (l) sense (5'— >3’): GUA GAA GUA AAG CAC GCG CCU GA (SEQ ID NO:46) and anti-sense (5'->3'): UCA GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:47) (IFNAR1.2 mouse 23 nt sequence);

[0165] (m) sense (5’— >3'): GUA GAA GUA AAG CAC GCG CCU GAG (SEQ ID NO:48) and anti-sense (5'— >3'): CUC AGG CGC GUG CUU UAC UUC UAC (SEQ ID NO:49) (IFNAR1.2 mouse 24 nt sequence);

[0166] (n) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU GAG G (SEQ ID NO:50) and anti-sense (5'— >3'): CCU CAG GCG CGU GCU UUA CUU CUA C (SEQ ID NO:51) (IFNAR1.2 mouse 25 nt sequence);

[0167] (o) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UCC (SEQ ID NO:52) and anti-sense (5’— >3’): GGA GGA CCA AUC UGA GCU UUG (SEQ ID NO:53) (IFNARl.l human 21 nt sequence);

[0168] (p) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UCC A (SEQ ID NO:54) and anti-sense (5'— >3'): UGG AGG ACC AAU CUG AGC UUU G (SEQ ID NO:55) (IFNARl.l human 22 nt sequence); (q) sense (5 >3 ' ): CAA AGC UCA GAU UGG UCC UCC AG (SEQ ID NO: 56) and anti-sense (5'->3’): CUG GAG GAC CAA UCU GAG CUU UG (SEQ ID NO:57) (IFNAR1.1 human 23 nt sequence);

[0169] (r) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UCC AGA (SEQ ID NO:58) and anti-sense (5’— >3’): UCU GGA GGA CCA AUC UGA GCLT UUG (SEQ ID NO:59) (IFNAR1.1 human 24 nt sequence);

[0170] (s) sense (5'— >3’): CAA AGC UCA GAU UGG UCC UCC AGA A (SEQ ID NO:60) and anti-sense (5 >3 ’): UUC UGG AGG ACC AAU CUG AGC UUU G (SEQ ID NO:61) (IFNAR1 1 human 25 nt sequence);

[0171] (t) sense (5 '->3'): GUG CUC CAA A AC AGU CUG GAA (SEQ ID NO: 62) and anti-sense (5'— >3’): UUC CAG ACU GUU UUG GAG CAC (SEQ ID NO:63) (IFNAR1.2 human 21 nt sequence);

[0172] (u) sense (5'->3'): GUG CUC CAA AAC AGU CUG GAA A (SEQ ID NO: 64) and anti-sense (5’— >3’): UUU CCA GAC UGU UUU GGA GCA C (SEQ ID NO:65) (IFNAR1.2 human 22 nt sequence);

[0173] (v) sense (5'— >3'): GUG CUC CAA AAC AGU CUG GAA AC (SEQ ID NO:66) and anti-sense (5'— >3’): GUU UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:67) (IFNAR1.2 human 23 nt sequence);

[0174] (w) sense (5'— >3’): GUG CUC CAA AAC AGU CUG GAA ACA SEQ ID NO:68) and anti-sense (5'->3'): UGU UUC CAG ACU GUU UUG GAG CAC (SEQ ID NO:69) (IFNAR1.2 human 24 nt sequence);

[0175] (x) sense (5’— >3'): GUG CUC CAA AAC AGU CUG GAA ACA C (SEQ ID NO:70) and anti-sense (5'— >3’): GUG UU U CCA GAC UGU UUU GGA GCA C (SEQ ID NO:71) (IFNAR1.2 human 25 nt sequence);

[0176] (y) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UC (SEQ ID NO:96) and anti-sense (5’— >3’): GAU AAA GUU GUC AUC UAU GA (SEQ ID NO:97) (IFNAR1.3 human minimal sequence);

[0177] (z) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UCC (SEQ ID NO:98) and anti-sense (5’— >3'): GGA UAA AGU UGU CAU CUA UGA (SEQ ID NO:99) (IFNAR1.3 human 21 nt sequence);

[0178] (aa) sense (5'->3'): UCA UAG AUG ACA ACU UUA UCC U (SEQ ID NO: 100) and anti-sense (5'— >3'): AGG AUA AAG UUG UCA UCU AUG A (SEQ ID NO: 101) (IFNAR1.3 human 22 nt sequence); (bb) sense (5 '->3'): UCA UAG AUG ACA ACU UUA UCC UG (SEQ ID NO: 102) and anti-sense (5'-->3'): CAG GAU AAA GUU GUC AUC UAU GA (SEQ ID NO: 103) (IFNAR1.3 human 23 nt sequence);

[0179] (cc) sense (5'-->3'): UCA UAG AUG ACA ACU UUA UCC UGA (SEQ ID NO: 104) and anti-sense (5’— >3’): UCA GGA UAA AGU UGU CAU CUA UGA (SEQ ID NO: 105) (IFNAR1.3 human 24 nt sequence);

[0180] (dd) sense (5’— >3’): UCA UAG AUG ACA ACU UUA UCC UGA G (SEQ ID NO: 106) and anti-sense (5 >3 '): CUC AGG AUA AAG UUG UCA UCU AUG A (SEQ ID NO: 107) (IFNAR1.3 human 25 nt sequence);

[0181] (ee) sense (5'->3'): UGU UCA UUC AUC CCG AGA AC (SEQ ID NO: 108) and anti-sense (5 >3 '): GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 109) (IFNAR1.4 human minimal sequence);

[0182] (ff) sense (5f— >3’): UGU UCA UUC AUC CCG AGA ACA (SEQ ID NO: 110) and anti-sense (5 >3'): UGU UCU CGG GAU GAA UGA ACA (SEQ ID NO: 111) (IFNAR1.4 human 21 nt sequence);

[0183] (gg) sense (5'->3'): UGU UCA UUC AUC CCG AGA ACA U (SEQ ID NO: 112) and anti-sense (5'— >3'): AUG UUC UCG GGA UGA AUG AAC A (SEQ ID NO: 113) (IFNAR1.4 human 22 nt sequence);

[0184] (hh) sense (5’->3'): UGU UCA UUC AUC CCG AGA ACA UU (SEQ ID NO: 114) and anti-sense (5f— >3'): AAU GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 115) (IFNAR1.4 human 23 nt sequence);

[0185] (li) sense (5’— >3'): UGU UCA UUC AUC CCG AGA ACA UUG (SEQ ID NO: 116) and anti-sense (5 '->3'): CAA UGU UCU CGG GAU GAA UGA ACA (SEQ ID NO: 117) (IFNAR1.4 human 24 nt sequence); and

[0186] (jj ) sense (5'— >3'): UGU UCA UUC AUC CCG AGA ACA UUG G (SEQ ID NO: 118) and anti-sense (5'->3'): CCA AUG UUC UCG GGA UGA AUG AAC A (SEQ ID NO: 119) (IFNAR 1,4 human 25 nt sequence).

[0187] In one embodiment, the 5’ end of the sequences are phosphorylated. In another embodiment, the nucleic acids comprise a two nucleotide single stranded overhang at tire 3 ’ terminus. In one such embodiment, the two nucleotide single stranded overhang at the 3’ terminus comprises dTdT. In other embodiments, the two nucleotide single stranded overhang at the 3 ’ terminus may comprise LTU, or may be complementary to the target mRNA. In further embodiments, the inhibitory nucleic acid comprises a double stranded siRNA sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs:

[0188] (a) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UUC dTdT (SEQ ID NO: 72) and anti-sense (5'->3'): GAA UGG AAU AAA CGG AUC AAC dTdT (SEQ ID NO:73) (IFNAR1.1 mouse 21 nt sequence with overhang);

[0189] (b) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU dTdT (SEQ ID NO: 74) and anti-sense (5’— >3’): AGG CGC GUG CUU UAC UUC UAC dTdT (SEQ ID NO:75); (IFNAR1.2 mouse 21 nt sequence with overhang);

[0190] (c) sense (5 '->3'): CAA AGC UCA GAU UGG UCC UCC dTdT (SEQ ID NO:76) and anti-sense (5'— >3’): GGA GGA CCA AUC UGA GCU UUG dTdT (SEQ ID NO:77) (IFNAR1.1 human 21 nt sequence with overhang);

[0191] (d) sense (5f-->3’): CAA AGC UCA GAU UGG UCC UCC dTdT (SEQ ID NO:78) and anti-sense (5'->3'): GGA GGA CCA AUC UGA GCU UUG dTdT (SEQ ID NO:79) (IFNAR1.2 human 21 nt sequence with overhang);

[0192] (e) sense (5 ’->3'): UCA UAG AUG ACA ACU UUA UCC dTdT (SEQ ID NO: 120) and anti-sense (5'— >3’): GGA UAA AGU UGU CAU CUA UGA dTdT (SEQ ID NO: 121) (IFNAR1.3 human 21 nt sequence with overhang); and

[0193] (f) sense (5'-->3'): UGU UCA UUC AUC CCG AGA ACA (SEQ ID NO: 122) and anti-sense (5'->3'): UGU UCU CGG GAU GAA UGA ACA (SEQ ID NO: 123) (IFNAR1.4 human 21 nt sequence with overhang).

[0194] In some embodiments, the 5’ end of the siRNA strands may be phosphorylated. In other embodiments, the 5’ end of the siRNA strands are not phosphorylated. In another embodiment, the 3' end of the siRNA strands are hydroxylated; in other embodiments they are not hydroxylated. In one embodiment, the 5’ end of the siRNA strands are phosphorylated and the 3' end of the siRNA strands are hydroxylated.

[0195] The siRNAs may comprise chemically modified residues to enhance stability or other beneficial characteristics. Modifications may include, for example, end modifications, e.g., 5 '-end modifications (phosphorylation, conjugation, inverted linkages) or 3 '-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of the sugar; and / or backbone modifications, including modification or replacement of the phosphodiester linkages. In one embodiment, the siRNA may be modified to comprise one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively “locks” the ribose in the 3'-endo structural conformation.

[0196] The mRNA may encode any gene of interest, including but not limited to a gene encoding a therapeutic protein, antigen, immunogen, reporter protein, cytokine, etc. In one embodiment, the gene of interest encodes an antigen, The antigen may be any antigen suitable for an intended purpose. In some embodiments, the antigen comprises an immunogenic portion of a viral, bacterial, parasitic, protozoan, fungal, or tumor antigen.

[0197] Exemplary viruses comprising suitable antigens include, but are not limited to, e.g., respiratory syncytial virus (RSV), hepatitis B virus (HBV), hepatitis C virus (HCV), Dengue virus, herpes simplex virus (HSV; e.g., HSV-I, HSV-II), molluscum contagiosum virus, vaccinia virus, variola virus, lentivirus, human immunodeficiency virus (HIV), human papilloma virus (HPV), cytomegalovirus (CMV), varicella zoster virus (VZV), rhinovirus, enterovirus, adenovirus, coronavirus (e.g., SARS), influenza virus (flu), para-influenza virus, mumps virus, measles virus, papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus (e.g., Lymphocytic Choriomeningitis Virus, Junin virus, Machupo virus, Guanarito virus, or Lassa vims), norovirus, yellow fever virus, rabies virus, Filovirus (e.g., Ebola virus or marbug virus), hepatitis C virus, hepatitis B virus, hepatitis A virus, Morbilliviruses (e.g., measles virus), Rubulaviruses (e.g., mumps virus), Rubiviruses (e.g., rubella virus), bovine viral diarrhea virus. For example, the antigen can be CMV glycoprotein gH, or gL;

[0198] Parvovirus; HIV glycoprotein gp!20 or gp!40, HIV p55 gag, pol; or RSV-F antigen,. In some embodiments, the antigen is a viral antigen. In some embodiments, the viral antigen is an HIV antigen comprising gpl20 or gp!40. In some engineered HIV antigen is an engineered variant of gpl20 (engineered Outer Domain, eOD). In some embodiments, the engineered HIV antigen is an engineered variant of gp!40 (SOSIP). Further description of SOSIP is provided by WO201605704 A3, US20160185825A1, Georgiev et al., (2015) J Virol 89( 10): 5318-5329, all of which are incorporated herein by reference in their entirety.

[0199] In some embodiments, the antigen is from a parasite. In some embodiments, the antigen is derived from a species from -within the Plasmodium genus, such as P. falciparum, P. vivax, P. malariae or P. ovale. In this embodiment, the immunogenic composition may be used for preparation of a vaccine for immunizing against malaria.

[0200] In some embodiments, the antigen is from a bacterial pathogen. Exemplary bacterial pathogens include, e.g., Neisseria spp, including N. gonorrhea and N. meningitides;

[0201] Streptococcus spp, including S. pneumoniae, S. pyogenes, S. agalactiae, S. mutans;

[0202] Haemophilus spp, including H. influenzae type B, non typeable H. influenzae, H. ducreyi; Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis;

[0203] Bordetella spp, including B pertussis, B. parapertussis and B. bronchiseptica;

[0204] Mycobacterium spp., including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli, enterohemorragic E. coli, enteropathogenic E. coli; Vibrio spp, including V, cholera, Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii;

[0205] Yersinia spp, including Y. enterocolitica, Y. pestis, Y. pseudotuberculosis, Campylobacter spp, including C. jejuni and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes;

[0206] Helicobacter spp, including H pylori; Pseudomonas spp, including P. aeruginosa, Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani, C. botulinum, C. difficile;

[0207] Bacillus spp., including B, anthracis; Corynebacterium spp., including C. diphtheriae;

[0208] Borrelia spp., including B. burgdorferi, B. garinii, B afzelii, B. andersonii, B. hermsii;

[0209] Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp., including C trachomatis, C. neumoniae, C. psittaci; Leptsira spp,, including L, interrogans; Treponema spp,, including T. pallidum, T. denticola, T. hyodysenteriae

[0210] In certain embodiments, the antigen is from a fungal pathogen. Exemplary fungal pathogens include, e.g., Aspergillus fumigatus, A. flavus, A. niger, A. terreus, A. nidulans, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, and Pneumocystis jirovecii.

[0211] In certain embodiments, the antigen is from a protozoan pathogen. Exemplary protozoan pathogens include, e.g., Toxoplasma gondii and Strongyloides stercoralis.

[0212] In certain embodiments, the antigen is from a multicellular parasitic pathogen.

[0213] Exemplary multicellular parasitic pathogens include, e.g., trematodes (flukes), cestodes (tapeworms), nematodes (roundworms), and arthropods.

[0214] In some embodiments, the antigen is derived from a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors); (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21 / Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2 / neu (associated with, e.g., breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-l / Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein- 1 / TRP I and tyrosinase related protein-2 / TRP2 (associated with, e.g., melanoma); (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer; (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, pl 5, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p!85erbB2, p!80erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p!6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29VBCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCASI, SDCCAGI6, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

[0215] In some embodiments, the antigen comprises a human immunodeficiency virus (HIV) or a severe acute respiratory syndrome (SARS) antigen. The compositions may comprise any lipid nanoparticle (LNP). In one embodiment, the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a polymer-conjugated lipid. In this embodiment, helper lipid may comprise, for example phosphocholines, phosphoethanolamines, or combinations thereof. In another embodiment, the polymer- conjugated lipid may comprise, for example, polyethylene glycol or polysarcosine conjugated to phosphoethanolamines, 1,2-dimyristoyl-rac-glycero, or other amphiphilic or lipid-like molecules.

[0216] In some embodiments, the amine-to-phosphate (N: P) ratio in the LNP loaded with RNA may range from about 1: 1 to about 20: 1. In other embodiments, the lipid-to-RNA volume ratio in the LNP may range from about 1: 1 to about 1:20 (i.e,, the ratio between the volume of lipids (all components mixed at predetermined molar ratio) in ethanol, and the volume of the RNA (mRNA + siRNA) in water or acidic buffer).

[0217] In another embodiment, the LNP comprises

[0218] (a) Nl, N3, NT5-tris(3-(didodecylamino)propyl)benzene-l,3,5-tricarboxamide (TT3),

[0219] (b) (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA),

[0220] (c) 1,2-dioIeoyl-sn-glycero-3-phosphoethanolamine (DOPE),

[0221] (d) cholesterol, and

[0222] (e) l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol -2000 (DMG-PEG2k);

[0223] In one exemplary embodiment, the molar ratio of LNP components is about 10 (TT3):25 (DLin-MC3-DMA):20 (DOPE):40 (cholesterol): 5 (DMG-PEG2k).

[0224] The compositions may comprise any other components as appropriate for an intended purpose. In one embodiment, the composition may further comprise an adjuvant.

[0225] As used herein, the term "adjuvant" refers to any substance that acts to augment and / or direct antigen -specific immune responses when used in combination with specific antigens. When combined with a vaccine antigen, adjuvant increases the immune response to the vaccine antigen as compared to the response induced by the vaccine antigen alone.

[0226] Adjuvants help drive immunological mechanisms and shape the output immune response to vaccine antigens. Exemplary antigens that might be used with the compositions of the disclosure may comprise aluminum salts (‘"alum”; aluminum hydroxide, aluminum phosphate), “Adjuvant System 04” (AS04), trehalose-6,6’-dimycolate (TDM), muramyl dipeptide (MDP), pluronic block copolymers, alum solution, aluminum hydroxide, ADJ UMER® (polyphosphazene); aluminum phosphate gel; glucans from algae; algammulin; aluminum hydroxide gel (alum); highly protein-adsorbing aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVR1DINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOL™ (l-alpha,2S-dihydroxy-vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-Al-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC / alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i) N-acetylglucosaminyl-(Pl-4)-N- acety Imuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(bl-4)-N-acetylmuraniyl-L-alanyl-D-isoglutamine); imiquimod (l-(2-methylpropyl)-lH-imidazol-4,5-c)quinoline-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-gamma; interleukin-1 beta; interleukin-2; interleukin-7; interleukin- 12; ISCOMS™; ISCOPREP 7.0.3.™; liposomes; LOXORIB1NE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile enterotoxin- protoxin); microspheres and microparticles of any composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 1™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ( (N-acetyl-L -alanyl -D-isoghitaminyl-L-alanine-2-( 1,2-dipalniitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt);

[0227] MURAMETIDE™ (Nac-Mur-L-Ala-D-GCn-OCH3); MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoCln-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN™ (P-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres / nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly -rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyI-2-ethoxymethyl-lH-imidazo[4,5-c]quinoline-l -ethanol); SAF-l™(“Syntex adjuvant formulation”); Sendai proteoliposomes and Sendai-containing lipid matrices; Span- 85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and 2,6, 10, 15,19, 23-hexamethyl-2,6, 10,14,18,22-tetracosahexane); stearoyltyrosine (octadecyltyrosine hydrochloride);

[0228] Theramid® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide™ or [thr 1J-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminum hydroxide), and lipopeptides, including Pam3Cys, in particular aluminum salts, such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IF A, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, ISCOMATRIX®, 1SCOM; adjuvants suitable for co-stimulation including Tomatine, biopolymers, including PLG, PMM, Inulin; microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII / Lewis X, antimicrobial peptides, UC- 1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide

[0229] The compositions may be used for any suitable purpose, including but not limited for delivery of therapeutics. In another embodiment, the gene of interest encodes an antigen, and the compositions may comprise vaccines.

[0230] In another embodiment, the disclosure provides pharmaceutical compositions comprising the composition or vaccine of any embodimen t herein and a pharmaceutically acceptable carrier.

[0231] The disclosure further comprises methods for generating an immune response against an antigen, comprising administering to a subject an amount effective to generate an immune response in the subject of the composition or vaccine of any embodiment herein, wherein the gene of interest encodes an antigen. The disclosure also provides methods of treating an infection or limiting development of an infection comprising administering to a subject in need thereof the composition or vaccine of any embodiment herein in an effective amount to induce an immune response against the antigen.

[0232] In one embodiment, the antigen comprises an HIV antigen, and the subject is at risk of, or has, an HIV infection. In another embodiment, the antigen comprises a SARS-CoV-2 antigen, and the subject is at risk of, or has, an SARS-CoV-2 infection.

[0233] In embodiments where the antigen is an HIV or SARS-CoV-2 antigen, "limiting development" includes, but is not limited to accomplishing one or more of the following: (a) generating an immune response (antibody and / or cell-based) HIV or SARS- CoV-2 in the subject; (b) generating neutralizing antibodies against HIV or SARS- CoV-2 in the subject (b) limiting build-up of HIV or SARS- CoV-2 titer in the subject after exposure to HIV or SARS- CoV-2; and / or (c) limiting or preventing development of HIV or SARS- CoV-2 symptoms after infection. Exemplary symptoms of HIV infection include, but are not limited to, fever, fatigue, swollen lymph nodes, diarrhea, weight loss, oral yeast infection, shingles, and / or pneumonia. Exemplary symptoms of SARS-CoV-2 infection include, but are not limited to, fever, fatigue, cough, shortness of breath, chest pressure and / or pain, loss or diminution of the sense of smell, loss or diminution of the sense of taste, and respiratory- issues including but not limited to pneumonia, bronchitis, severe acute respiratory syndrome (SARS), and upper and lower respiratory' tract infections.

[0234] In embodiments where the antigen is an HIV or SARS- CoV-2 antigen, "treat" or "treating" includes, but is not limited to accomplishing one or more of the following: (a) reducing HIV titer in the subject; (b) limiting any increase of HIV titer in the subject; (c) reducing the severity of HIV symptoms; (d) limiting or preventing development of HIV symptoms after infection; (e) inhibiting worsening of HIV symptoms; (f) limiting or preventing recurrence of HIV symptoms in subjects that were previously symptomatic for HIV infection; and / or (e) improving survival,

[0235] In some embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for s.c. and / or I V. administration. In some embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery’ vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, cd., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% Sucrose. In some embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the immunogenic composition.

[0236] The subject may be any mammalian subject that may benefit from the methods of the disclosure, including but not limited to humans, dogs, cats, horses, cattle, chickens, goats, etc.

[0237] Table 1

[0238] Nam Sequence

[0239] e

[0240] Ifnar 1 ggaaggggcg gggcgc tgga gggggcgggg cgtcgcggag ggcctagctg cccagaggta (moo 61 gtctccagct ccgcggtgct gctgaggaga aggaggagaa tgtgagccgc cgcccggcct se) 121 cccaagacga tgctcgctgt cgtgggcgcg gcggccctgg tgctggtggc cggggcgcct 181 tgggtgctac cctcagctgc aggtggagaa aatc tgaaac ctcctgagaa tatagacgtc

[0241]

[0242] 241 tacattatag atgacaacta caccctaaag tggagcagcc acggagagtc aatgggcagt NCB 301 g tgacctt tt cagcagaata tcgaacaaaa gacgaggcga agtggtt aaa agtgcctgaa I 361 tgtcaacata utacaacgac caagtgtgaa ttctctttac tggacacaaa tgtgtatatc Refer 421 aaaacacagt ttcgtgtcag agcagstggaa gggaacagca catcttcgtg gaatgaggtt ence 481 gatccgttta ttccattcta cacagcc cac atgagccccc cagaagtacg tttagaagct Sequ 541 gaacataaag ccatactagt ccacatctct cctcccggac aagacgggaa catgtgggca CHOC: 601 ctggagaaac cttccttcag ttacaccata cgaatctggc agaagtette c a g t Q a c a a a NM aaaactatta actctacgta ttatgtagaa aagataccag aactcttgcc agagactact 0105 tac tgtttag aagttaaagc a a t ac a t c eg tcacttaaga aacacagcaa 11 acagcac t 08.2 781 gtgcagtgta taagcaccac agtggcaaat aaaatguc i~g tgccaggaaa tctccaagtg 841 gatgcccaag gcaagagctct tgtcctgaaa tgggactaca ttgcgtctgc agacgtgctc SEQ 901 ttcagggcac 5-gtggcttcc tggetattea aaaagcagtt etggaageeg ttcagataaa ID tggaaaccaa taccaacctg tgcaaatgtc cagactacgc actgtgtctt t t c t c a. ag a t NO:8 1021 actgtctaca caggaacgtt ctttctccat gtacaagcct cagagggaaa tcacacatcc 1 1081 ttttggtctg aagagaagtt tattgattet caaaaacaca ttctccctcc tcctccggtc 1141 attaetgtea ccgccatgag tgacaccttg cttgtttatg teaaetgtea ggacagcaca 1201 tgtgatggac t c a a 11 a eg a aatcatcttt tgggaaaaca cttccaatac taagataagc 1261 atggagaagg atggcccagat gttcaccctc aagaacctgc agccgctgac tgtgtactgt 1321 gtccaggcca gagtgctctt cagggccctg ctgaataaga ccagcaactt cagtgaaaag 1381 ctgtgtgaga aaacacgtcc aggaagtt tt 0- c* c <a 0- c* t gc? at t a t aac tggattaggt 1441 gttgtgttct tctctgtcat ggtcctttat gctttgagga gcgtctggaa atacctgtgt 1501 catgtgtgct tcccaccact caagcctccc cgcagtattg atgagttttt ctctgagccg 1561 - C 0- acct, tg tact tc tga egg ct gaggagcaca eggaaagatg c 11 c a t c a 11 1621 gagaatacag acacggtcgc tgtagaagta aagcacgcgc etgaggagga cctcaggaag 1681 tacagctcac ag ac c ag c c a ggactcgggc aactattcca aegaagagga ggagagtgtg 1741 ggcaccgaga gcggccaagc tgtgctctcc aaagctccct gcggggggcc atgcagcgtg 1801 cctagccctc ctgggacctt ggaagacggg acctgcttcc Lc?ggaaa tea ci <a v. C* t 0- 1861 cagagcccag ccctgaggac agagccagct cttctctgct gacaggtgct cggcccaggc 1921 tgggggccgt gtggaggaga cagacttatc tgaagtggct gccagcttct gctgccagct 1981 ggaagccctg ggtgctgttt gtgccacaga tgaggctcac tgatgtcagg catctggtgg 2041 ttctgtctcg gtgtttgtcc tctaggtgac atttagaaca ttccgatttt taatcctgcc 2101 gtagcccctc agtgtgtcac aggtaccatt gtctacagcc gcgcttccta acccatacct 2161 caccacagcc gtggcttctg accagaagtc actgaaacca tggagaaagg ggacactaac 2221 tggaggaget ggcgt tgttt aagatctcag tgtggcctgg gtgtgcccgt tgtactctag 2281 taettgeaga ac cagaaaac gccagggaca aaaagcaggc atgaaccatt cagtaagaga 2341 gaagetagtg aacctgatgg tgaaagaaga ccgtgtccag tcagtgtttg tgtgaaggcc 2401 etgtgetget ctctgtagcc ttcccgtggt s.ct t ataaaa ccaggccgaa ccctgtcctc 2461 tgtccggcac tattcaggtc ccttcccagc tccaggtact taaaa aacaa agtgttgggt 2521 cctggggtga gggaacagct tcgacggcac agtgctgcct gcaggcgcct ataagtgacc 2581 ttagatgaat gctccatgag catcttcggt aggcaggaca tccagcagtg ctggtggctc 2641 cagggcagac tcagt tgcag gccaaaaagg aagagagagg agteatagee c tgccccaat 2701 cgagcttgtt gctcgctgca ctgcgccttt gtaatcaatc ttggtttttt tgttttgttt tgtttttttt aacttttagg tgataacata ttctcagata tagcacacaa gagacactac 2821 acaaattacg tc tg aaag t t ttgatagaaa agagcttttg actggggtag acagcttgct 2881 cctcactact accttgtatc tcagtctcct caacagagag agaatgeaga ttgattttca 2941 tcctgagttt tattttcttg cccggtttct gggttcgggt gtgctttcaa ggctctgaac 3001 agcttgtctg cccacagggg agcccggaga cccagccttt tggctaaatg ttgcaggttc 3061 ctgtacttga aggac tccag c tcctctgta gagggcatgg teategeggt c tgcaatcac 3121 gctgataccc agaaaaatgg ccacacaact caaatctggt gccacgcttt aat aa t i»c t a 3181 cccagcacgg cccagccctg ttcacggcgg aaaagctcct tctgccagga cagcctgtga 3241 agaaacaaga cctccgagga cgtggccaag gaaggcccag atgetagegg gaagttgtta 3301 gttggcactc tttcatctct caacaactgt gacacagg i»c ttcagggtga agcccaaagc 3361 agetateggt tgcagcagcg ggtaaggagg gggcttcacc tgagcctggc cttctgggcg 3421 ageagtteta cctgcacact tcaagacagc ctccatgacc cttttcacgg cacaccgcct 3481 gcttttggtg tgaccggggt t teetgtgea ggcacatgt t ettaaggaat gccatgcaca 3541 caggcctgaa gagctggctc agegggtaga gcacaggttg ctcttgtctg ggatctgagc 3601 tctgtcccca gcacccacag agtgcttctg gttgcctcag actccagggg tgcacaaggc 3661 c a t g t a ag c a aagacatgta ac?c aaag aca g 11 g t a c a c a gagaagteaa acctttttga 3721 aaaaaagaaa caccacgggc tggagagatg gctcagtggt taagaacact gaccactctt 3781 ccagaggtcc tgcigttcaat tcccagcaac cacatggtgg ctcacaacca tctgtaatag 3841 gatetgatge ccccttctgg tgtgtctgaa gatagctaca gtgtattcat atataaaata 3901 ataaatacat cctttttttt c tttttttac t at tgcfa.t a t tttctttatt t ac a 1 t 1 c aa 3961 atgttatccc ctttcctgat ttacccccct cccagaagcc ccctatccca tcccccctcc 4021 cctgcttcta tgagggtgtt cacccaccca cccactccca cttccctgcc cttgattccc 4081 ctacactggg ttatttatca a c? c c a t c a t a ggaccaaaga cctctccttc cattgatcca 4141 tgacaaagcc atcctctgca acatatgcag c utgaguta t gtgtacttct tggttgatgg 4201 catagtccct gggagttctg gggcagtggg gggttggggg gagtctggtt agt tga ta 11 4261 attcttccta ttggggttgc aaaccccttc aatgccttca gtcccttctc taactcctct

[0243]

[0244] 4321 attagggacc ccgagctcag tccagtggtt ggctgctaac atccacc tct gtatttgtaa 4381 ggc tctggca gggectctca agagacagcc a t a t cagg c t cctttcagca tgt actt.ctt 4441 ggcacccaca acagtgtctg gtttggtaac getgtaegge atgaatteeg aggtgggaca 4501 atctatgggt ggcctttcat teagtetetg ctctacactt tatctccata tttgctccag 4561 tgagtatttt gttctccttc taagaaggac agaagcaccc acactttgct cgtccttctt 4621 attgagcttc atgtggtctg tgaattgtat cet ggttatt tggaget ttg gggctagt.at 4681 c c a c 11 a t c a gtgagtacat attgtgtgtg ttcttttgtg attgggttac ctcactcagg 4741 atgatatcct ccagatccat ccatttgcct aagaatttca tgaattcatc gtttttaata 4801 gctgtgcagt actctgttgt ggaaatgcac cacgttttct gtatccattc ctctgtt.gag 4861 gggcatctgg gttctttcca gettetgget a utaqaaa i_a aggetgetat gaacatagtg 4921 gagcatgtgt ccttcttacc agttggagca tcttctgggt atatgcccag gagtagcata 4981 gctgggtcct caggtaatac tatgtccaat tttetgagga ccggccaaac tgatttccag 5041 agtggttgta cgagct tgta t tcccaccag c aa t gc? agg a gtgttcc tct t-tc tccacat 5101 cctcaccaac atetgctgte aacagagttt ttaattttag ccattctgac tggtgtgaaa 5161 tggaatctca gggtggtttt gatttgtaat tcaccaatga ctaaggatgt tgaacatttc 5221 t 1 t agg tg c t tcttggccat t tgatat t cc tcagttgaaa attetttgtt tagctct.cta 5281 ccccattttt taatagggtt a tttggttct etggagteta aettettgag ttctttgtat 5341 atattggata ttoigccctct atcggatgta ggattggtaa agatcttttc ccaatctgtt 5401 ggtattgttt tgtccttttg tcagtgtcct ttgccttaca gaagctttgc aattttatga 5461 ggtcccactt g teg at tett. t ttttttt tt tttt tttttt ttttt.cgaga cagggttt.ct 5521 ctgtatagcc ctggctgtcc tggagctcac tttgtagacc aggctggcct cgaactcaga 5581 aatccgcctg cctctgcctc ccgagtgctg ggattaaagg cgtgcaccac cacacccggc 5641 t coat t t g t c gattet tgat cttacagcat tagccattgg tgttctatta aggaattttt 5701 t c c c c t g t ac ccgtatgttt gaggetettt ccaaattgct cctctataaa gagagagagg 5761 gggagaggga gaaggagaga gcaaggaagg aagaaaagag aagagacaag acaagaagga 5821 aaaggagaga aaagaaaaga gaaaaaagaa atgaaaagaa gagaagagaa aagagaaaaa 5881 agaaaagaaa gaccatgcac t tgagggcac tttgtttat.g ttgatagttc ttggagaagc 5941 tgagcgccgt c t a a ga t 1 u a tgtaaaagee tcatgagaaa teagtgettt cacagacaca 6001 tgtaaatagc aacaggcctg tgagcacagc tacacagggg agctggctgc actccactag 6061 aat tgccacg a c g t a c c tat cacag tg acg tctacagtga gaaatgetgt t c t ag a c tea 6121 ttacatatgt a a c a 111 a a a tg taa t cat ttagagcctg ggcagaaatc tataacctat 6181 gaaaatgttc atttaaaatc aggtcctggca caagtgtgcc tggcttgttc tcatcccagc 6241 tgcctaggaa tgtgggccaa cctcagccac atagcaagag cctgtcgtac acatatgtac 6301 aggtgtagag gtatgtgtgc atgtatatag gt.ac tatat.a aaat acagag eceagagect 6361 gtgatgcttc agatggcctc ggaettetet ggagacctgg ggaacgtgtt aacaaagcct 6421 tccctaaccc catggaagcc gtgtgtccct gtgtgcattg gtcctttggt gtttgcetcc 6481 c t c t g t a t t g cagaagctct t tgagcaggg tctcagttcc tccctgtggc cccccagcec 6541 ctgtgacagt gcagcgctgg cacagaca c?t gcccacagct ac uga a t c?aa tgagatetga 6601 aattgaagca gatgactgtt ctggctgttt gtgtaatgaa ataaccacgt ttgtgtgttt 6661 ctatgcatca cctttgtgtc tgcatccttt tatatttttg tctggagccc eactgttgta 6721 tatcgacaaa cctaacacac accggaacca ggac tctgga tctcaggtgc t.ge tcettta 6781 aactggaatc cgtgaggcag aggtgeegte tacaacccgt tcctgacagc aggaettgea 6841 ttctggcatt ctggatccac actgctgtcc cttccaagtg ttactgtctc tcattcetct 6901 gtc tctgaga tccettctgc tctacctcaa gagactaaga gggtgaacac agt ttg tact 6961 111 c a t c 11 c cttacagaga acfgaaa t a a t a a a 11 c a t a t ttgttcccaa g aaaaa aa aa 7021 aaaaa

[0245] Ifhar ttaggacggg gegatggegg etgagaggag ctgcgcgtgc gcgaacatgt aactggtggg (hum 61 atetgeggeg gctcccagat gatggtegte ctcctgggcg cgacgaccct agtgetegte an) 121 gccgtgggcc catgggtgtt. gtccgcagcc gcaggtggaa aaaatctaaa atc tcctcaa 181 aaagtagagg tcgacatcat agatgacaac tttatcctga ggtggaacag gagcgatgag SEQ 241 tctgtcggga atgtgacttt ttcattcgat tatcaaaaaa ctgggatgga taattggata ID 301 aaattgtctg ggtgtcagaa tattactagt accaaatgca acttttcttc actcaagctg NO:8 361 aatgtttatg aagaaattaa attgegtata agagcagaaa aagaaaacac ttcttcatgg 2 421 tatgaggttg actcatttac accatttcgc aaagctcaga ttggtcctcc agaagtacat 481 ttagaagctg aagataaggc aatagtgata cacatctctc ctggaacaaa agatagtgtt 541 atgtgggctt tggatggttt. aagctttaca tatagcttac ttatctggaa aaactcttca 601 ggtgtagaag aaaggattga aaatatttat tccagacata aaatttataa actctcacca 661 gagactactt attgtctaaa agttaaagca gcactactta cgtcatggaa aattggtgtc 721 t.atagtccag tacattgtat aaagaccaca gttgaaaatg aactacctcc accagaaaat 781 atagaagtea gtgtccaaaa tcagaactat gttettaaat gggattatac atatgcaaac 841 atgacctttc aagttcagtg gctccacgcc tttttaaacia ggaatcctgg aaaccatttg 901 tataaatgga aacaaatacc tgactgtgaa aatgtcaaaa ctacccagtg tgtctttcct 961 caaaacgttt tccaaaaagg aatttacc tt ctccgcgtac aagcatc tca tggaaataac 1021 acatcttttt ggtetgaaga gataaagttt gataetgaaa tacaagcttt cctacttcct 1081 ccagtcttta acattagatc ccttagtgat tcsittccata tetatategg tgctccaaaa 1141 cagtctggaa acacgcctgt gatccaggat tatccactga tttatgaaat tattttttgg 1201 gaaaacactt caaatgctga gagaaaaatt ategagaaaa aaactgatgt tacagttcct

[0246]

[0247] 1261 aatttgaaac cactgactgt atattgtgtg aaagccagag cacacaccat ggatgaaaag 1321 ctgaataaaa gcagtg tttt tagtgacgct gtatgtgaga aaacaaaacc aggaaatacc 1381 tctaaaattt ggcttatagt tggaatttgt a i- u g c a 11 a 1. ttgctctccc gtttgtcatt 1441 tatgctgcga aagtcttctt gagatgcatc aattatgtct tctttccata acttaaacct 1501 tcttccagta tagatgagta tttctctgaa cagccattga agaatcttct gctttcaact 1561 tctgaggaac aaat cgaaaa atgtttcata a t tg aaaa t a t aag c ac aa t t’.gc tacag ta 1621 g aaqa.aac t a atcaaactga tgaagatcat aaaaaataca gttcccaaa u a g c c a a g a 1.

[0248] 1681 tcaggaaatt attctaatga agatgaaagc gaaagtaaaa caagtgaag actacagcag 1741 gac tttgtat gacc ag aaat gaactgtgtc aagtataagg tttttcagca ggagt tacac 1801 tgggagcctg aggtcctcac cttcctctca g u a a c t a c a g agaggacgtt tcctgtttag 1861 ggaaagaaaa aacatcttcci gatcataggt cctaaaaata cgggcaagct cttaactatt 1921 taaaaatgaa attacaggcc cgggcacggt ggctcacacc tgtaatccca gcactttggg 1981a99c t9a99caggcagatca tgaggtcaag agatcgagac cagcctggcc aacgtggtga 2041 aaccccatct c t ac taaaa a taaaa aaatt agccgggtag taggtaggcg cgcgcctgtt 2101 gtcttagcta ctcaggaggc tgaggcagga gaatcgcttg aaaacaggag gtggaggttg 2161 cag tgagccg agatcacgcc actgcactcc agcctggtga cagcgtgaga c tc t L taaaa 2221 aaagaaa t ta aaagagttga gacaaacgt t tcctacattc ttttcca tgt gtaaaatcat 2281 gaaaaagcct gtcaccggac ttgcattgga tgagatgagt cagaccaaaa cagtggccac 2341 ccgtcttcct cctgtgagcc taagtgcagc cgtgctagct gcgcaccgtg gctaaggatg 2401 acgtctgtgt tcctgtccat cactgatgct gctggctact gcatgtgcca cacctgtctg 2461 ttcgccattc ctaaca ttct gtttcattct tcctcgggag atatttcaaa catttggtct 2521 tttcttttaa cactgagggt aggcccttag gaaatttatt taggaaagtc tgaacacgtt 2581 atcacttggt tttctggaaa g tagc t t. acc c tagaaaac a gctgcaaatg ccagaaagat 2641 gatccc i,aaa aatgttgagg gacttctgtt cattcatccc gagaaca ttg gcttccacat 2701 cacagtatct acccttacat ggtttciggat taaagccagg caatctttta ctatg

[0249]

[0250] Examples

[0251] Effective delivery of antigen-encoding mRNA in humans using lipid nanoparticles (LNPs) represents a significant advancement in vaccinology. In addition to the RNA itself, LNPs commonly used to deliver both mRNA and self-replicating (repRNA) vaccines themselves trigger NF-KB activation and type 1 interferon (IFN) responses. A o!>

[0252] A key output of these RNA recognition pathways is the production of type 1 IFNs, which can act in both an autocrine and paracrine manner. The effect of global IFN signaling deficiency on the immunogenicity of RNA vaccines is unclear.

[0253] Here, we developed an approach to focus modulation of type I IFN signaling on transfected cells in vivo, by co-delivering siRNA targeting IFNAR1 together with repRNA or modRNA in the same LNP. Using RNA vaccines encoding a clinically-relevant HIV immunogen- a transmembrane form of a germline targeting HIV Env SOSIP trimer- we assessed the impact of in vivo Ifnar1 silencing on repRNA expression, changes in the composition of immune cells recruited at the injection site and draining lymph nodes, antigen presentin cell (APC) activation, and downstream cellular and humoral immune responses. These studies revealed that co-delivery of RNA vaccines with siRNA against Ifriarl enhances antigen expression, immune cell infiltration and stimulation, and diverse downstream antigen-specific vaccine responses.

[0254] RESULTS Designing RNA vaccines to query the role of IFNAR1 in vaccine-elicited immunity As a model system, we synthesized repRNA encoding a transmembrane form of a germline targeting HIV Env trimer termed N332-GT2.35This Env trimer immunogen is designed to prime B cells in humans capable of evolving to produce broadly neutralizing antibodies targeting the V2-Apex neutralizing site on HIV; a closely related trimer is currently in phase I human testing.36To determine whether type I IFN signaling impacts the development of antigen-specific B cells in germinal centers (GCs) following RNA immunization, we vaccinated wildtype and ifnar'' mice with LNPs carrying repRNA encoding N332-GT2. Two weeks post-vaccination, we observed that while there were no significant differences in the total frequency of germinal center (GC) B cell or follicular helper T (Tih) cell populations, antigen-specific GC B cells that were present at a frequency of less than 1% in WT animals were increased 9.4-fold in ifnar'1' mice (data not shown). Further, 60% of these animals had also already seroconverted, while antibody titers in WT animals were still at baseline at this timepoint (data not shown). Thus, the type 1 IFN pathway appears to play an important role in restraining humoral responses to repRNA.

[0255] We hypothesized that co-delivery of repRNA together with siRNA targeting IFN sensing might provide a means to pharmacologically modulate the interferon response in a manner focused on RNA-transfected cells without global IFN suppression, and over a time window that would be relevant for amplifying repRNA vaccine responses. To test this idea, we prepared (1) LNPs carrying repRNA alone, (2) LNPs co-loaded with repRNA and one of two different siRNAs targeting ifnar 1 or a control scrambled siRNA sequence (siScramble), and (3) a “cocktail” case consisting of LNPs carrying repRNA alone mixed with LNPs carrying siRNA. LNPs were synthesized using microfluidic mixing of lipids in ethanol with aqueous phase repRNA in water. For these initial studies, we utilized an LNP formulation containing two ionizable lipids, TT337and DLin-MC3-DMA38, which provided more efficient transfection of mouse muscles with repRN A compared to LNP compositions employed in the Moderna (MDR) or Pfizer (PFZ) COVID- 19 mRNA vaccines.

[0256] We characterized LNPs loaded with repRNA alone, repRNA together with an siRNA targeting If ar 1 (silFNARl), or LNPs loaded with only silFNARl. For the co-loaded formulation, we selected a repRNA:siRNA mass ratio of 1:2 based on enhanced early replicon expression observed when delivering repRNA expressing luciferase as a reporter gene. Cryo-EM and dynamic light scattering showed that all three formulations formed relatively monodisperse populations of nanoparticles approximately 40 run in diameter, with near-neutral zeta potentials and high RNA encapsulation efficiencies (data not shown). Silencing Ifnarl augments repRNA expression in vitro

[0257] C2C12 mouse myoblast cells transfected with LNPs carry ing GFP-encoding repRNA alone, repRNA + siRNA, or mixtures of LNPs carrying only repRNA and only siRNA all showed similar high levels of cell viability and LNP uptake (data not shown). We compared knockdown of Ifnarl using two different co-delivered siRNA sequences (silFNARl.1 and siIFNAR1.2), and characterized replication of the RNA by qRT-PCR using primers targeting the nsP3 domain of the backbone and the subgenomic Gfp payload. LNPs co-loaded with replicon and silFNARl triggered over 95% knockdown of Ifnarl, while LNPs carrying repRNA and control scrambled siRNA led to no significant change, and co-delivery of separate LNPs carrying repRNA and siRNA showed Ifnar knockdown but -with lower efficiency (77%, data not shown). Knockdown of Ifnarl in cells receiving siRNA / repRNA co-delivery' was accompanied by a significant increase in copies of the repRNA backbone (over 6-fold increase in nsP3 transcripts for siIFNAR1.2,) and up to 17-fold increases in copies of the replicon payload Gfp (data not sho n ) compared to cells transfected with replicon alone or co-delivered with siScramble. At the protein level, the mean fluorescence intensity (MFI) of GFP -expressing cells also significantly increased when cells were transfected with LNPs co-delivering repGFP and silFNARl, up to 1.7-fold over repRNA alone for the optimal silFNARl.2 sequence (data not shown). These results indicate that replicon / siRNA co-delivery can be used to substantially enhance both repRNA genome replication as well as copies of payload gene transcripts without compromising cell viability.

[0258] siRNA / repRNA co-delivery enhances replicon expression in vivo

[0259] Next, we evaluated effects of delivering 7 / har / -targeting siRNA on replicon expression in vivo. Balb / c mice were vaccinated intramuscularly (i.m.) in the gastrocnemius with LNPs carrying repRNA encoding GFP (“(repRNA)”), silFNARl.2 co-loaded LNPs (“(repRNA / silFNAR I)”), or a mixture of the repGFP-loaded and silFNAR l, 2 -loaded LNPs (“(repRNA) + (silFNARl)”). qRT-PCR analysis of injected muscles showed that while administration of replicon alone led to a 6.7-fold increase in Ifnarl expression 1-day postadministration, this upregulation was completely blocked for at least 3 days following replicon / siRNA co-delivery’ (data not shown). Interestingly, Ifnarl expression then rebounded at day 7 significantly to ~3-fold higher expression than replicon-only treatment, which had dropped substantially by this timepoint. This suppression of Ifnarl correlated w ith > 10-fold increased transcripts for the repRNA backbone and its subgenome payload, which were maintained over time. Although co-treatment with repRNA and siRNA delivered by separate LNPs also blocked Ifnar1 upregulation at early times, this treatment failed to elicit the same increases in replicon backbone and Gfp transcript levels (data not shown). In draining popliteal lymph nodes (dLNs), Ifnarl expression was transiently upregulated at day 1 post-RNA administration; siRN A co-delivery reduced this upregulation compared to replicon alone by 2-fold, accompanied by 4.4-fold and 5.5-fold increased peak levels of the replicon backbone and subgenome transcripts, respectively, in the dLN at day 3 (data not shown).

[0260] We next used cryofluorescence tomography (CFT)- where frozen tissues are serially sectioned and imaged using fluorescence microscopy, followed by digital reconstruction to enable whole-tissue 3D fluorescence imaging- to visualize the muscle injection site following delivery of fluorescent LNPs carrying replicons encoding the reporter gene mCherry'™. As shown in 2D maximum -intensity projections of the 3D imaging data and in volumetric quantification of mCherry™ signal, replicon / siRNA co-administration led to a 10- fold higher integrated intensity of mCherry™ expression in an 11-fold greater volume of the muscle tissue at day 3 compared to immunization with repRNA alone (data not shown). Reporter gene expression increased over time for repRNA alone, but repRNA / siRNA co-delivery' maintained a 3.8-fold higher integrated intensity of mCherry'™ over the repRNA-alone group 7 days post-vaccination. Notably, delivery' of repRNA LNPs mixed with siRNA LNPs led to lower mCherry™ expression signal and transfected volume compared to delivery' of the replicon and siRNA together in the same LNP (data not shown). As expected, the total LNP signal in the tissues was not statistically different between groups. We next quantified mCherry'™ expression in the muscles overtime by ELISA analysis of tissue lysates. Similar to the findings from CFT, we saw' found 3.4-fold higher levels of mCherry™ expressed for repRNA / silFNARl co-loaded LNPs compared to repRNA alone at day 7 post-administration (data not shown). Altogether, silencing of the type 11FN responsiveness through Ifnar1 knockdown substantially amplified expression of replicon-encoded payload genes over at least the first week post administration.

[0261] Suppression of Ifnarl enhances immune cell recruitment and stimulation in muscle tissue and draining lymph nodes

[0262] We next characterized the impact of Ifnarl silencing on immune cell recruitment / phenotypes in the muscle and dLNs by flow' cytometry. As LNPs induce a variety of cytokines, we expected that IFNAR1 silencing would not block cytokine / chemokine expression in injected muscles. This was confirmed by multiplex ELISA analysis of muscle lysates following repRNA administration, where we saw that IL- 1 p, TNF'-a, and IL-6 were all induced for both repRNA alone and repRNA / siRNA co-delivery, and some chemokines were actually induced in the muscle by repRNA / siRNA co-delivery at higher levels than repRNA delivery alone (data not shown). Immune cell infiltration in the muscle was detected by 3 days post-vaccination, and interestingly, repRNA / siRNA co-delivery amplified immune cell recruitment, reaching 7-fold higher levels vs. repRNA alone at day 7 (data not shown). By day 7, repRNA / silFNARl co-delivery had increased the recruitment of cDCl dendritic cells (~8-fold), cDC2 dendritic cells (~3-fold,), and monocytes (~ 13-fold), as well as neutrophils and macrophages (data not shown). Interestingly, adaptive immune cells were also enriched at the injected muscle, with B cells increased 20-fold over repRNA alone at day 7. By contrast, control immunization with repRNA / scrambled siRNA co-delivery or repRNA LNPs mixed with siRNA LNPs generally elicited immune cell infiltrates very similar to repRNA alone (data now' shown). Among these recruited cells, mCherryIMexpression was primarily detected in myeloid cells, and most prominently monocytes (data not shown). In addition to more transfected cells, repRNA / siRNA co-delivery vaccination yielded higher mean fluorescence intensities of mCherryIMexpression on a per-cell basis (1,8-fold and 1.4-fold on days 3 and 7, respectively.

[0263] Replicon vaccination induced expansion of draining lymph nodes, with Ifnarl silencing via siRNA co-delivery modestly increasing total cellularity vs. repRNA alone ( i.6-1.8-fold higher data not shown). Among various cell subsets, siRNA co-deliver ' most impacted cDCls, cDC2s, and monocytes, increasing each of these populations by ~2-fold by day 7.

[0264] Next, we evaluated activation of antigen presenting cells in the muscle and dLN via expression of tire key costimulatory receptors CD80 and CD86. Interestingly, immunization with replicon alone did not result in substantial increases in APC activation over naive animals in either the muscle or dLN in the first week post-vaccination. How ever, vaccination with tire replicon in conjunction with siRNA against Ifnarl yielded notable upregulation of CD80 and CD86 in monocytes and cDC2s in the muscle at day 7 (data not shown). In the draining lymph node, costimulatory receptor expression was significantly upregulated by day 7 in cDCls, and cDC2s (data not shown). Overall, we found that despite suppressing one inflammatory'- cue, co-delivery' of replicon with siRNA against Ifnar1 stimulated significantly higher infiltration of immune cells to the administration site and amplified APC activation in the muscle and dLN s. Ifnarl silencing enhances repRNA vaccine-elicited immune responses We next assessed how these changes in gene expression and local inflammation relate to the immunogenicity of LNP-repRNA vaccines. We first immunized BALB / c mice with LNPs loaded with repRNA encoding N332-GT2 trimer with or without silFNARl.2, siScramble, or with the co-treatment vaccine. Flow cytometric analyses of cells recovered from the draining popliteal lymph nodes at day 14 revealed that vaccines co-delivering antigen-encoding repRNA and silFNARl significantly increased expansion of total GC B cells and follicular helper T cells (data not shown). Most strikingly, staining with antigen tetramers revealed that siRNA co-delivery substantially amplified the antigen-specific GC B cell response, increasing both tire total number of GC B cells capable of recognizing the intact antigen and the proportion of these cells that had class switched to IgG (data not shown). Gating on GC B cells with similar IgG expression levels, we found that antigenspecific B cells elicited by repRNA / silFNARl co-delivery had an -8-fold higher mean fluorescence intensity of antigen binding (data not shown), suggesting enhanced affinity¬ maturation induced by Ifnar1 silencing.

[0265] A single immunization with N332-GT2 trimer-encoding repRNA co-delivered with Ifnarl -targeting siRNA led to rapid seroconversion, with all animals expressing high titers of trimer-specific IgG by day 14, while mean titers of repRNA alone were -2 logs lower at this time point (data not shown). Antibody titers elicited by repRNA alone slowly rose but even following their plateau at 60 days, they remained 1.4 logs lower than titers in the repRNA / siRNA co-delivery group. repRNA / silFNARl vaccination induced higher levels of IgG2b and a trend toward increased IgG I compared to repRN A, but other isotypes were elicited at similar levels (data not shown). ELISPOT analysis of trimer-specific and total IgG-producing cells from bone marrow at 6 weeks post-vaccination revealed a trend toward increased antigen-specific plasma cell responses elicited by co-delivery of silFNAR1 with repRNA in the same LNPs (data not shown). Finally, antigen-specific T cell responses in the spleen were also amplified by repRNA / siRNA co-delivery compared to the baseline repAg vaccine or with a scramble siRNA or the co-treatment group, as read out by IFN-y ELISPOT (data not shown). Altogether, these results indicate that Ifnarl silencing during replicon vaccination can augment diverse elements of the innate and adaptive immune response,

[0266] Ifnarl silencing enhances modRNA vaccine-elicited immune responses We found that empty LNPs used in this study (‘LNP ), as well as clinical formulations used in COVID-19 vaccines from Moderna (mRNA-1273; MDR ) and Pfizer-BioNTech (BNT162b2; ‘PFZ’) induce significant amounts of cytokine production local to the administration site, including type I IFNs (Fig. 2). To assess whether Ifnarl silencing could also benefit non-replicating mRNA vaccines, we first co-transfected siRNA against Ifnar1 with modRNA (all uridine residues substituted with N1-methylpseudouridine) encoding for GFP in C2C12 murine myoblasts in vitro (Fig. 3). At 24h post-LNP transfection, we observed that co-delivery of siRNA against Ifnarl with modRNA (modGFP / siIFNAR1) increased the expression of GFP over the delivery of modGFP alone (modGFP).

[0267] Next, we tested the impact of co-delivering modRNA vaccines with Ifnar1-targeted siRNA on vaccine-elicited immune responses in vivo. Mice were immunized with LNPs loaded with modRNA encoding N332-GT2 trimer (all uridine residues substituted with N1-methylpseudouridine; see Steichen, J. M. et al. Science 366, eaax4380 (2019)), alone (‘(modRNA) ), modRNA co-delivered with silFNARl.2 (‘(modRNA / silFNARl)’), modRNA co-delivered with scrambled-sequence control siRNA (‘(modRNA / siScramble)’), or with a co-treatment vaccine putting modRNA and siRNA in separate LNPs (‘(modRN A) + (silFNARl)’). At 2 -weeks post-vaccination, we observed approximately 2-fold increases in the number of GC B cells (Fig. la) and T follicular helper cells (Fig. lb) in mice vaccinated with the co-delivery vaccine compared to modRNA alone. Antigen-specific GC B cell responses were also significantly increased with siRNA co-delivery, with the total number of antigen-specific GC B cells increasing by 3.6-fold, and the proportion of IgG class-switched cells by 8.6-fold compared to replicon-only group (Fig. Ic-e). In GC B cells with similar IgG expression (Fig. 4a), these antigen-specific B cells also displayed 1.7-fold higher mean fluorescence intensity in binding to antigen tetramers (Fig. If-g), again suggesting that the B cells generated by the modRNA / silFNARl co-delivery vaccine have higher binding affinity. Lastly, co-delivery of modRNA encoding for the N332-GT2 trimer with siRNA against Ifnar1 induced rapid seroconversion, such that by week 2 post-vaccination, mice vaccinated with the co-delivery vaccine showed a trend toward ~1.3 log-fold higher antibody titer (Fig. Ih and Fig. 4b). Thus, Ifnar1 silencing can also substantially augment non-replicating mRNA vaccines.

[0268] Impact of the LNP composition on siIFNAR1-mediated enhancement of vaccine-elicited immune response To discern the impact of LNP formulation on the observed improvements in vaccine efficacy with siRNA co-delivery, we finally carried out a vaccination study using the clinical LNP formulation used in Modema’s COVID-19 vaccine (mRNA-1273). We immunized balb / c mice with either repRNA or modRNA encoding N332-GT2 HIV Env trimer, with or without Ifnarl -targeting siRNA (Fig. 5). As we saw with our replicon-tailored LNPs, the Modema formulations co-loaded with the immunogen-encoding RNA and silFNARl also yielded significant increases in the vaccine-elicited immune response over the modRNA or repRNA vaccine groups without silFNARl, including increased total GC B cells (Fig. 5a), increased Tfh cells (Fig. 5b), and increased antigen-specific B cells- by 8.7-fold for the modRNA / silFNARl vaccine and 11.3-fold for repRNA / silFNARl (Fig. 5c-d) The proportion of IgG class-switched antigen-binding GC B cells also increased by 10-fold for modRNA / silFNARl vaccine and -5-fold for repRNA / silFNARl (Fig. 5e). As seen with our replicon-tailored LNPs, the mean fluorescence intensity of trimer-binding IgG+B cells also increased by ~2.5-fold for both modRNA / silFNARl and repRNA / silFNARl vaccine groups (Fig. 5f-g). Finally, we observed significant increases in the serum antibody titer in the silFNARl co-delivery vaccine over the modRNA or repRNA vaccine alone (Fig. 5i-j) These results suggest that silFNARl co-delivery may be a generalizable strategy to improve vaccine-elicited immune response of repRNA and modRNA vaccines.

[0269] DISCUSSION

[0270] We hypothesized that formulation of LNPs carrying RNA together with siRNA against Ifnarl could focus type 1 IFN signaling inhibition on transfected cells and limit the duration of IFN modulation, to avoid blocking potentially beneficial IFN signaling in responding immune and stromal cells in the injection site and / or draining lymph nodes. In mice, we found that 3 days of robust Ifnarl silencing led to drastically enhanced replication and expression of repRNA by over 10-fold at both transcriptomic and proteomic levels, correlating with increased antigen-specific B (~8-fold higher antigen-specific GC B cells) and T (4.4-fold) cell responses, as well as >10-fold increased serum antibody titers compared to vaccines delivered without any siRNA or with scramble sequence of siRNA against Ifnarl. We found similar enhancements in the immunogenicity of non-replicating RNA vaccines: While modRNA is designed to evade innate recognition, emerging evidence suggests that modRNA is not completely immune-silent in vivo, and the LNP delivery vehicle is known to stimulate type I IFN production.18-20Thus, this approach is generally useful for RNA-based vaccines. Notably, we found that it is key to encapsulate antigen-encoding RNAs and siRNA against Ifnarl in the same LNP for co-delivery into the same cells, as opposed to independent delivery of the two RNA payloads in separate LNPs to the same tissue, but not necessarily the same cell.

[0271] Overall, our findings demonstrate that while innate recognition of RNA and LNPs can inhibit RNA vaccine function, transiently suppressing the type 1 IFN response both augments antigen expression and enhances early injection site and lymph node innate immune activation, leading to dramatically improved downstream vaccine -elicited immune responses. Ifnar1 silencing through siRNA co-delivery is a simple approach to augment many elements of vaccine-elicited immunity for both replicating and non-replicating RNA vaccines, which could be readily translated to humans.

[0272] ONLINE METHODS

[0273] Materials

[0274] N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3) was synthesized as previously described3'; (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA) was purchased from MedChemExpress (CAT#HY-112251); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE;

[0275] CAT#850725), Cholesterol (CAT#700100), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k; CAT#88015) were purchased from Avanti Polar Lipids, Soluble HIV Env trimer protein N332-GT2 was prepared as previously described.35Citrate buffer (pH 3; CAT#J61391-AK) was purchased from Alfa Aesar. For dialysis, 20K MWCO Slide-A-Lyzer™ MINI Dialysis Device (ThermoFisher Scientific), RNAse-free PBS (AM9625; ThermoFisher Scientific) was used. Quant-iT RiboGreen™ RNA kit (CAT#R11490, Invitrogen) was used to evaluate the RNA encapsulation efficiency in LNPs. For bioluminescence studies, luciferin was purchased from GoldBio (CAT# LUCK- 1G).

[0276] RNA synthesis

[0277] Development of a backbone for self-amplifying RNAs (repRNA) was described previously.6,33In brief, structural genes from the Venezuelan Equine Encephalitis Virus genome were replaced with a reporter gene and favorable mutations were introduced via in vitro evolution. In this study, we replaced the reporter gene of this backbone with green fluorescent protein (GFP), mCherry™, firefly luciferase, or HIV immunogen N332-GT2 (a transmembrane form of a stabilized SOSIP HIV envelope trimer35) using traditional cloning methods. For modRNA Synthesis, template DNA plasmids used in the production of modRNA were created using a commercially available Cloning Kit for mRNA Templates (Takara #6143) according to manufacturer’s instructions. Resultant plasmid DNA was linearized via endonuclease digestion and purified with PureLink™ PCR Purification columns (ThermoFisher #K310002) following the manufacturer’s instructions. To synthesize RNA, 20 pL in vitro transcription (IVT) reactions were performed using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB #E2040S) and 1-2 μg of linear DNA template (scaled as needed). For modRNA, modified base Ml -methylpseudouridine triphosphate (TriLink #N-1081) was added to the reaction mixture instead of canonical uridine triphosphate, and CleanCapTMReagent AG (TriLink #N-7113) was utilized to co-enzymatically add 5’ Cap-1 structures to synthesized RNA. The IVT product was purified using PureLink™ RNA Mini columns (ThermoFisher #12183018A) following tire manufacturer’s instructions. Replicon RNA was capped and methylated using the ScriptCap™ Cap 1 Capping System (CellScript #C-SCCS2250) following the manufacturer’s instructions, after which RNA was purified a final time using PureLink™ RNA Mini columns. Quality of the resulting repRNA was assessed using UV-Vis spectrophotometry and gel electrophoresis.

[0278] Lipid nanoparticle synthesis

[0279] Lipids were stored in ethanol at -20°C, and RNA constructs were stored in RNAse-free water at -80°C and were thawed on ice before use. Lipid nanoparticles (LNPs) were synthesized using a microfluidic organic-aqueous nanoprecipitation method, as previously described.64The organic phase was prepared by solubilizing the lipids TT337, DLin-MC3-DMA, DOPE, Cholesterol, and DMG-PEG2k in ethanol at a molar ratio of 10:25:20:40:5. Tire aqueous phase of RNA was prepared by diluting the RNA (stored in RNAse-free water) with 10 mM citrate buffer, pH 3.0 (CAT#J61391-AK; Alfa Aesar). For empty LNP synthesis, RNA was replaced with additional citrate buffer. The two phases were prepared at an ethanol:aqueous volume ratio of 1:2, and RNA and lipids combined at an N: P (number of amines on TT3 and MC3 to number of phosphates on repRNA) ratio of 3: 1 (for mRNA, N: P ratio was set to 8: 1). Each phase was loaded into a syringe (BD), and locked onto the NxGen™ microfluidic cartridge for mixing using a NanoAssemblr™ Ignite instrument (Precision Nanosystems). The Ignite was set to operate with the following settings: volume ratio- 2:1; flow rate- 12 ml / min; waste volume- 0 mL. The resulting LNPs were dialyzed against PBS using 20K MWCO Slide-A-Lyzer™ MINI Dialysis cassettes (ThermoFisher Scientific) at 25°C for 90 min, with an exchange of the buffer reservoir after 45 min. Particle characterization

[0280] Hydrodynamic size, polydispersity index, and zeta potential of the LNPs was determined by dynamic light scattering (DLS; Malvern Panalytical). To measure hydrodynamic size, 10 uL of particles were diluted in 800 uL of deionized water and placed into a 1.5 mL cuvette (Fisher Scientific) and measured. Same sample preparation was used to measure zeta-potential using the folded capillary zeta cell (Malvern Panalytical). Cryogenic electron microscopy (cryo-EM) was used to qualitatively assess the LNP size and polydispersity as previously described.64Briefly, 3 uL of the particles in deionized water was dropped on a lacey copper grid coated with a continuous carbon film. Excess liquid was blotted and the grid was frozen in liquid ethane cooled by liquid nitrogen in the Gatan Cryo Plunge™ III. Then the grid was mounted on a Gatan 626 single tilt cryo-holder of the TEM column while being kept chill with liquid nitrogen. Imaging on a JEOL 2100 FEG microscope was operated at 200 kV with a magnification range of 10,000-60,000. Gatan 2kx2k UltraScan CCD camera was used to record all images.

[0281] Quant-iT RiboGreen™1RNA kit (Invitrogen) was used to evaluate the RNA encapsulation efficiency in LNPs according to the manufacturer instructions. In a black 96 well plate, control RNA provided in the kit was used to make two sets of standard curves with serial dilution in either RNAse-free water or Triton-X-100. Samples were 100-fold diluted in either RNAse-free water (to quantify RNA present outside of LNPs) or Triton-X- 100 (to quantify total RNA present inside and outside of LNPs). Fluorescence was measured with excitation wavelength = 480 nm, and emission wavelength = 520 nm.

[0282] In vitro cell culture and transfection

[0283] Cell lines (C2C12 mouse myoblasts and LLC-MK2 macaque kidney epithelial cells) were cultured in growth media (DMEM supplemented with 10% FBS and 1% penicillin / streptomycin, and were incubated at 37°C in 5% CO2). For transfection, 25,000 cells were seeded in 96 well plates for overnight attachment, and 1 μg of GFP-encoding repRNA loaded in DiI-labeled LNPs were added in 10 uL of PBS with 20 uL Opti-MEM (Gibco) and 70 uL of growth media. At 24h post-transfection, cells stained with Zombie Aqua™ (BioLegend) for cell viability, and were transferred to a V-bottom 96 well plate for quantification of GFP expression on the BD LSRFortessa™ Cell Analyzer (BD Biosciences).

[0284] Mice All animal studies and procedures were carried out following federal, state, and local guidelines under an IACUC-approved animal protocol. Female BALB / c (JAX Stock No. 000651) mice, C57BL / 6 (JAX Stock No. 000664), and B6(Cg)-Ifnar1tm1.2Ees / J (JAX Stock No. 028288) at 6-8 weeks of age were maintained in the animal facility at the Massachusetts Institute of Technology (MIT). For ail LNP administrations, mice were intramuscularly injected in the left and right gastrocnemius muscles at dose of 1 μg repRNA or 5 μg mRNA in 20 pL of PBS.

[0285] In vivo mouse imaging

[0286] To assess the efficacy of different LNP formulations using repRNA encoding for luciferase (repLuc) as a reporter, we immunized BALB / c mice intramuscularly (i.m.) with 1 ug RNA doses of LNPs carrying repLuc in each of the left and right gastrocnemius muscles. At days 1, 3, 7, 10, and 15 post-i.m. injection, the mice were intraperitoneally (i.p.) administered 200 uL of luciferin (50 mg / ml in PBS), and imaged using the In Vivo Imaging System (Xenogen IVIS 200; PerkinElmer) 10 min post-i.p. injection.

[0287] qRT-PCR for relative quantification of RNA expression

[0288] For in vitro quantification of RNA expression, C2C12 mouse myoblasts were seeded at 300,000 cells / well in a 6 well-plate and incubated overnight for attachment. repRNA encoding for GFP was loaded in LNPs and treated at 5 ug / well in 100 uL of PBS, 200 uL of Opti-MEM and 700 uL of growth media for overnight transfection. Total RNA was purified from the cells using a PureLink™ RNA Mini Kit (Invitrogen). Briefly, cells were lysed and the lysates underwent a series of fast spin-column washes. The final RNA concentration was measured using a NanoDrop™ UV-VIS spectrometer (ThermoFisher Scientific), The relative quantification of RNA expression was assessed using a two-step quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR, QuantStudio, ThermoFisher). cDNA was synthesized from RNA using the iScript™ Reverse Transcription Supermix (BioRad), and PCR was run with the iTaq™ Universal SYBR® Green Supermix (Bio-Rad).

[0289] For in vivo quantification of RNA expression, mice were intramuscularly administered 1 ug of repRNA encoding for GFP loaded in LNPs. At days 1, 3, 7, and 14 postadministration, mice were sacrificed and the gastrocnemius and popliteal lymph nodes were harvested. Tissues were kept in RNAlater™ Stabilization Solution (Invitrogen) at -20°C until thawed for RNA purification. Tissues were placed in 1 mL of Trizol™ (Invitrogen) with β-mercaptoethanol (Gibco), and were homogenized in gentleMACS™ M tubes using gentleMACS ™ Octo Dissociator (Miltenyi Biotec) and its built-in muscle protocol at least 5 times or until no large tissue fragments was visible. The M tubes were centrifuged at 600 x g for 5 min at 4°C. Supernatants were collected and gently mixed with chloroform was added at 20% by volume and incubated on ice for 2-3 min. Samples were then centrifuged for 15 minutes at 12,000 x g at 4°C to phase-separate the mixture. The top aqueous phase was transferred to new RNAse-free tubes, and 500 uL of isopropanol was added for 10 min incubation at 4°C. RNA was pelleted by centrifugation for 10 minutes at 12,000 x g at 4°C. The pellet was resuspended and washed in 1 mL of 70% ethanol by centrifugation for 5 minutes at 7500 x g at 4°C. The pellet was dried briefly in air for 5 min before being resuspended in 50 uL of RNAse-free water, and placed on a heat block for 10 min at 60°C. RNA concentration was measured by NanoDrop and were stored in -80°C until cDNA synthesis using iScript™ Reverse Transcription Supermix and qRT-PCR with iTaq™ Universal SYBR® Green Supermix.

[0290] Cryofluorescence Tomography

[0291] Mice were placed on alfalfa-free diet to minimize autofluorescence from the gut for a week prior to repRNA administration. Mice were intramuscularly administered 1 ug of repRNA encoding for mCherry ™ loaded in LNPs in each leg, with 10% of the LNPs labeled with DiR (ThermoFisher). At days 1, 3, and 7 post-repRNA administration, mice were euthanized by CO2, and skin was removed to eliminate autofluorescence. A holder made of polylactic acid (PLA) was 3D-printed to control orientation of the mouse body during freezing in a hexane bath at dry ice temperature for 5 minutes. Afterwards the frozen samples were placed in a -20°C freezer overnight to degas the hexane. Mouse lower limbs were isolated from the holder and were mounted in an OCT “C” block (14x10.5x7cm) and sliced and imaged using an EMIT (Baltimore, MD) Xerra™ CFT system. Slices were cut at 35 pm thicknesses and images were acquired using excitation wavelength at 555 nm and detection filter of 620 nm. Images were analyzed using Fiji with Bio-formats.

[0292] mCherry™ quantification from muscles

[0293] Mice were intramuscularly administered 1 μg of repRNA encoding for mCherry™ loaded in LNPs. At days 1, 3, and 7 post-administration, mice were sacrificed and the left and right gastrocnemius muscles were harvested. Muscles were placed in gentleMACS™ M tubes (Miltenyi Biotec) with 300 μL of lysis buffer composed of RIPA buffer (ThermoFisher Scientific) supplemented with 1% EDTA (Invitrogen) and 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Muscles were lysed using gentleMACS™ Octo Dissociator (Miltenyi Biotec) and its built-in muscle protocol at least 5 times or until no large tissue fragments was visible. The M tubes were centrifuged at 600 x g for 5 min at 4°C. Supernatants were collected and stored in -20°C until use. For protein quantification, mCherry™ ELISA kit (CAT# ab221829; Abeam) was used according to manufacturer instructions. Briefly, a standard curve was set using the provided mCherry™ recombinant protein with serial dilution from 4,000 pg / mL with dilution factor of 2x. Muscle lysates were thawed and 200-fold diluted in sample diluent buffer in each well. In each well, 50 uL of standard or sample and 50 uL of the provided antibody cocktail were placed. The plate was covered and incubated for 1h at 25°C. After three consecutive washes with the provided wash buffer, 100 uL of TMB development solution was added to each well and incubated for 1 min. Finally, 100 uL of the provided stop solution was added and the absorbance was measured at 450 nm using a UV-Vis plate reader.

[0294] Mouse muscle and lymph node immunophenotyping

[0295] Mice were intramuscularly administered 1 ug of repRNA encoding for mCherry™ loaded in LNPs. At days 3, and 7 post-administration, mice were sacrificed and the left and right gastrocnemius muscles and popliteal lymph nodes were harvested. Tissues were placed in gentleMACS™ C tubes (Miltenyi Biotec) with 5 uL of digestion buffer composed of complete RPMI + 0.1% w / v Collagenase I + 0.1% w / v Collagenase IV. Muscles were lysed using gentleMACS™ Octo Dissociator (Miltenyi Biotec) and its built-in muscle protocol at least 2 times or until no large tissue fragments was visible. The samples were incubated at 37°C for Ih on shaker for further digestion. Post-digestion, samples were filtered through 50 pm pores twice and centrifuged at 600 x g for 5 min at 4°C to isolate pellets composed of single cells. Zombie Aqua™ (BioLegend) in PBS was used to stain for cell viability, and antibody staining was performed in fluorescence-activated cell sorting (FACS) buffer (PBS, 1 % bovine serum albumin (BSA), 0.02% NaN3, and 2 mM EDTA). Fc-mediated binding was blocked using purified anti-CD16 / 32 (2.5 pg / ml; 93; BioLegend) at 4°C for 15 min, on top of which we added primary antibodies for cell surface staining at 4°C for 30 min. Immune cells were identified with anti-CD45 PerCP-Cy5.5 (30-F11; BioLegend), B cells were stained with anti-B220 BV421 (RA3-6B2; BioLegend) and anti-CD19 PE-Cy7 (6D5; BioLegend), T cells were marked by anti-CD3 BUV661 (145-2C11; BD Biosciences), macrophages were stained with anti-F4 / 80 APC-Cy7 (BM8; BioLegend) and anti-CD11b BUV396 (MI / 70;

[0296] BioLegend), monocytes were stained with anti-Ly6C BV650 (HK1.4; BioLegend), neutrophils with anti-Ly6G APC (1A8; BioLegend), and anti-CD11b. Dendritic cells were stained with anti-CD11c AF488 (N418; BioLegend), anti-MHC-II AF700 (M5 / 114.15.2; BioLegend), and anti-CD103 BV786 (M290; BD Biosciences). Activation states of myeloid cells were assessed by staining for anti-CD80 BUV737 (16-10A1; BD Biosciences) and anti-CD86 BV605. Stained cells were fixed with 4% paraformaldehyde (ThermoFisher Scientific) for 10 min at 25°C, washed and resuspended in FACS buffer for flow cytometric analysis on a BD FACSymphony™ A3 Cell Analyzer (BD Biosciences). CountBright™ Absolute Counting Beads (ThermoFisher Scientific) were added to each well immediately before running flow cytometry.

[0297] Germinal center (GC) analysis

[0298] Balb / C mice were immunized as described above, and at 2 weeks post-vaccination, popliteal lymph nodes were collected and mechanically dissociated to obtain single cell suspensions. Zombie Aqua™ (BioLegend) in PBS was used to stain for cell viability, and antibody staining was performed in fluorescence-activated cell sorting (FACS) buffer (PBS, 1% bovine serum albumin (BSA), 0.02% NaN3, and 2 mM EDTA). Fc-mediated binding was blocked using purified anti-CD16 / 32 (2.5 μg / ml; 93; BioLegend) at 4°C for 15 min, on top of which we added primary antibodies for cell surface staining at 4°C for 30 min. GC B cells were stained using anti-GL7 PerCPCy5.5 (GL7; BioLegend), anti-CD38 AF488 (90;

[0299] BioLegend), anti-B220 PECy7 (RA3-6B2; BioLegend), and anti-CD4 BV711(GK1.5;

[0300] BioLegend). IgG-expression was characterized by anti-IgG BV786 (X56; BD Biosciences), Follicular helper T cells were assessed using anti-CD4-BV711 (GK1.5; BioLegend), anti- B220-PECy7 (RA3-6B2; BioLegend), anti-CXCR5-PE (phycoerythrin) (L138D7;

[0301] BioLegend), and anti-PDl-BV421 (29F.1A12; BioLegend), The HIV env trimer used as the antigen was conjugated to either BV605 (streptavidin-conjugated; BioLegend) or APC-Cy7 (streptavidin-conjugated; BioLegend), and both probes were used to detect antigen-specific B cells. Stained cells were fixed with 4% paraformaldehyde (ThermoFisher Scientific) for 10 min at 25°C, washed and resuspended in FACS buffer for flow cytometric analysis on a BD FACSymphony™ A3 Cell Analyzer (BD Biosciences). CountBright™ Absolute Counting Beads (ThermoFisher Scientific) were added to each well immediately before running flow cytometry.

[0302] Serum antibody titer quantification To quantitatively assess the efficacy of thawed LNPs compared with freshly prepared samples using repRNA encoding for the HIV env trimer, we vaccinated healthy Balb / C mice by injecting 1 ug RNA doses of the LNP-repRNA i.m. in each of the left and right gastrocnemius muscle. At weeks 2 and 4 post-i.m. injection, the mice underwent retro-orbital bleeding; blood was collected in Z-gel PP tubes for blood serum collection

[0303] (CAT 41.1500.005; Sarstedt), Serum was collected by centrifuging blood at 10,000xg for 4 minutes, and stored at -80°C prior to use. To conduct ELISAs, NUNC MaxiSorp ™ plates were coated overnight with 1 pg / mL purified HIV antigen in PBS, then blocked for 2 h with 10% BSA in PBS. Mouse sera were initially diluted 50x in blocking buffer, followed by 3x serial dilutions. Diluted sera were transferred to blocked plates and incubated for 2 h. HRP-conjugated immunoglobulins (e.g., IgG, IgGl, IgG2a, IgG2b, IgG3, IgM; Bio-Rad) were used as detection antibodies at 1:5,000 for endpoint titer assessments, with gpl20-specific monoclonal antibody VRC01 used as a positive control. 3,3',5,5'-Tetramethylbenzidine (TMB) signal w as read using a microplate reader by subtracting the absorbance at 450 nm by that at 550 nm.

[0304] Enzyme-linked immunosorbent spot (ELISpot) assay of splenocytes

[0305] Spleens were harvested 2 weeks after mice had been vaccinated i.m. with LNPs carrying repRNA encoding for the HIV immunogen in the left and right gastrocnemius muscles of mice, Splenocytes were isolated by mechanical dissociation of the spleen and erythrocytes were removed using the Gibco Ammonium-Chloride-Potassium lysing buffer (Thermo Fisher Scientific). ELISpot™ assays were conducted using the mouse IFN-y ELISPOT™ Kit (BD Biosciences). Cells were seeded on IFN-γ-coated wells at 106cells / well in triplicate for pools of overlapping peptides covering the entire sequence of the HIV env trimer (15-mer peptides overlapping by 11 amino acids), which were added to the cells at 2 μg / mL. Cells were stimulated by wrapping the plate in foil and incubating overnight at 37°C. Plates were developed and detected according to manufacturer’s instructions. Plates were scanned using a CTL-ImmunoSpot™ Plate Reader, and data were analyzed using CTL ImmunoSpot™ Software.

[0306] Enzyme-linked immunosorbent spot (ELISpot) assay of bone-marrow plasma cells Balb / C mice were immunized as described above, and at 6 weeks post-vaccination, bone-marrow plasma cells (BMPCs) were isolated from the femur and tibia. ELISpot™ Flex: Mouse IgG (HRP) kit (CAT# 3825-2H; MABTECH) was used to evaluate antigen-specificity of BMPCs according to manufacturer's instructions. Briefly, a MSIP PVDF-membrane plate was activated by ethanol washing, then coated with the provided anti-IgG antibody overnight at 4°C. Coated plates were seeded with 106BMPCs / well for total IgG quantification and 5x106BMPCs / well for antigen-specific IgG quantification for overnight incubation at 37°C in 5% CO2. Cells were removed by washing before target analytes were added to wells; anti-IgG-biotin was added for total IgG quantification, and biotinylated HIV env trimer was added for antigen-specific IgG quantification. Plates were developed and detected according to manufacturer’s instructions. Plates were scanned using the CTL-ImmunoSpot™ Plate Reader, and data were analyzed using CTL ImmunoSpot™ Software.

[0307] Bead-based ELISA cytokine quantification

[0308] At 24 h post-vaccination with empty LNPs, gastrocnemius muscles and popliteal lymph nodes were collected from mice. Tissues were placed in 0,3 mL of lysis buffer, composed of RIPATMbuffer (ThermoFisher Scientific) supplemented with 1 % EDTA (Invitrogen) and 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Tissues were lysed using gentleMACS™ Octo Dissociator (Miltenyi Biotec) and its built-in muscle protocol at least 2 times or until no large tissue fragments was visible. The M tubes were centrifuged at 600 x g for 5 min at 4°C. Supernatants were collected and stored in -20°C until use. The lysates from mouse gastrocnemius muscles and popliteal lymph nodes were analyzed using the Legendplex mouse antivirus response panel (Biolegend) following the manufacturer's suggested protocol and analyzed using the LEGENDplexTMData Analysis Software Suite.

[0309] REFERENCES

[0310] 1. Pardi, N., Hogan, M. J., Porter, F. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nature Reviews Drug Discovery 17, 261-279 (2018).

[0311] 2. Chaudhary, N., Weissman, D. & Whitehead, K. A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev. Drug Discov. 20, 817-838 (2021). 3. Blakney, A. K., Ip, S. & Geall, A. J. An Update on Self-Amplifying mRNA Vaccine Development. Nato Adv Sci Inst Se 9, 97 (2021).

[0312] 4. Maruggi, G. et al. Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens. Vaccine 352, 361-368 (2017). 5. Brazzoli, M. et al. Induction of Broad-Based Immunity and Protective Efficacy by Selfamplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin. J Virol. 90, 332-344 (2015).

[0313] 6. Pollock, K. M. et al. Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial. Eclinicalmedicine 44, 101262 (2022).

[0314] 7. Melo, M. et al. Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for Self-Assembly into Nanoparticles. Mol Ther 27, 2080-2090 (2019).

[0315] 8. Blakney, A., McKay, P. & Shattock, R. Structural Components for Amplification of Positive and Negative Strand VEEV Splitzicons. Frontiers in Molecular Biosciences 5, null (2018).

[0316] 9. McKay, P. F. et al. Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nat. Commun. 11, 3523 (2020).

[0317] 10. Lundstrom, K. Self-Amplifying RNA Viruses as RNA Vaccines. Int. J. Mol. Sci. 21, 5130 (2020).

[0318] 11. Pepini, T. et al. Induction of an IFN-Mediated Antiviral Response by a Self- Amplifying RNA Vaccine: Implications for Vaccine Design. J Immunol 198, 4012-4024 (2017).

[0319] 12. Blakney, A. et al. Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self- Amplifying RNA. Mol Ther 29, 1174-1185 (2020).

[0320] 13. Lin, G., Yan, H., Sun, J., Zhao, J. & Zhang, Y. Self-replicating RNA nanoparticle vaccine elicits protective immune responses against SARS-CoV-2. Mol. Ther. Nucleic Acids 32, 650-666 (2023).

[0321] 14. Lund, J. M. et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. 101, 5598-5603 (2004).

[0322] 15. Huysmans, H. et al. Expression Kinetics and Innate Immune Response after Electroporation and LNP -Mediated Delivery of a Self-Amplifying mRNA in tire Skin. Mol. Ther. - Nucleic Acids 17, 867-878 (2019).

[0323] 16. Zelkoski, A. E., Ennis, K. H. E., Lu, Z. & Malloy, A. M. W. The BNT162b2 vaccine’s empty lipid nanoparticle is able to induce an NF-KB response. J. Immunol. 210, 160.18- 160.18 (2023).

[0324] 17. Chaudhary, N. et al. Amine headgroups in ionizable lipids drive immune responses to lipid nanoparticles by binding to the receptors TLR4 and CD1d, Nat. Biomed. Eng. 1–16 (2024) doi:10.1038 / s41551-024-01256-w.

[0325] 18. Connors, J. et al. Lipid nanoparticles (LNP) induce activation and maturation of antigen presenting cells in young and aged individuals. Commun. Biol. 6, 188 (2023). 19. Ndeupen, S., Qin, Z., Jacobsen, S. & Igyarto, B. Z. The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory'. J.

[0326] Immunol. 206, 30.01-30.01 (2021).

[0327] 20. Connors, J. R. et al. The impact of lipid nanoparticles (LNP) on anti-viral pathways and immune function in aged individuals. J Immunol. 208, 163.26-163.26 (2022).

[0328] 21. Omo-Lamai, S. et al. Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects. bioRxiv 2024.04.16.589801 (2024) doi: 10.1101 / 2024.04.16.589801.

[0329] 22. Tahtinen, S. et al. IL-1 and IL-lra are key regulators of the inflammatory response to RNA vaccines. Nat Immunol 23, 532-542 (2022).

[0330] 23. Vanluchene, H. et al. Less is more: Self-amplifying mRNA becomes self-killing upon dose escalation in immune-competent retinal cells. Eur. J. Pharm. Biopharm. 196, 114204 (2024).

[0331] 24. Beuckelaer, A. D. et al. Type I Interferons Interfere with the Capacity of mRNA Lipoplex Vaccines to Elicit Cytolytic T Cell Responses. Mol Ther 24 11, 2012-2020 (2016).

[0332] 25. Pollard, C. et al. Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines. Mol Ther J Am Soc Gene Ther 21 1, 251-9 (2013).

[0333] 26. Bon, A. L. et al. Type I Interferons Potently Enhance Humoral Immunity and Can Promote Isotype Switching by Stimulating Dendritic Cells In Vivo. Immunity 14, 461-470 (2001).

[0334] 27. Toporovski, R., Morrow, M. P. & Weiner, D. B. Interferons as potential adjuvants in prophylactic vaccines. Expert Opin. Biol. Ther. 10, 1489-1500 (2010).

[0335] 28. Liu, F., Niu, Q., Liang, Y., Sun, J. & Hu, H. Role of Type I IFN signaling in induction of humoral response induced by viral vectored vaccines. J. Immunol. 200, 126.35-126.35 (2018).

[0336] 29. Zhong, C. et al. Type I Interferon Promotes Humoral Immunity in Viral Vector Vaccination. J. Virol. 95, e00925-21 (2021).

[0337] 30. Zhong, Z. et al. Immunogenicity and Protection Efficacy of a Naked Self-Replicating mRNA-Based Zika Virus Vaccine. Vaccines 7, null (2019).

[0338] 31. Vogel, A. B. et al. Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses. Molecular Therapy 26, 446-455 (2017). 32. Tregoning, J. S. et al. Formulation, inflammation, and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines. Mol. Ther. - Nucleic Acids 31, 29-42 (2023).

[0339] 33. Hoecke, L. V. et al. The Opposing Effect of Type I IFN on the T Cell Response by Nonmodified mRNA-Lipoplex Vaccines Is Determined by the Route of Administration. Mol Ther - Nucleic Acids 22, 373-381 (2020).

[0340] 34. Broos, K. et al. Particle-mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-specific T-cell Responses Despite the Induction of Type I Interferon. Mol Ther Nucleic Acids 5, null (2016).

[0341] 35. Steichen, J. M. et al. A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses. Science 366, (2019).

[0342] 36. Sanders, N. I. of H. (NIH)|Department of H. and H. A Trial to Evaluate an HIV Envelope Trimer, N332-GT5 gp140, Adjuvanted With SMNP in Adult Participants Without HIV, NCT06033209, web site: clinicaltrials.gov / study / NCT06033209#more-information.

[0343] 37. B., L. et al. An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery' in Vivo. Nano Lett 15, 8099-107 (2015).

[0344] 38. Jayaraman, M. et al. Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo**. Angew. Chem. (Int. Ed Engl.) 51, 8529-8533 (2012).

[0345] 39. Iijima, M. C., Fernandez, S. & French, M. A. Characterising the Phenotypic Diversity of Antigen -Specific Memory B Cells Before and After Vaccination. Front. Immunol. 12, 738123 (2021).

[0346] 40. Bloom, K., Berg, F. van den & Arbuthnot, P. Self-amplifying RNA vaccines for infectious diseases. Gene Therapy 28, 117-129 (2020).

[0347] 41. Comes, J. D. G., Pijlman, G. P., & Hick, T. A. H. Rise of the RNA machines - selfamplification in mRNA vaccine design. Trends Biotechnol. 41, 1417-1429 (2023).

[0348] 42. Tam, H. H. et al. Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination. Proc. Natl. Acad. Sci. 113, E6639-E6648 (2016). 43. Cirelli, K. M. et al. Slow Delivery' Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance. Cell 177, 1153- 1171. e28 (2019).

[0349] 44. Johansen, P. et al. Antigen kinetics determines immune reactivity. Proc. Natl. Acad. Sci.

[0350] 105, 5189-5194 (2008).

[0351] 45. Morse, M. A. et al. Clinical trials of self-replicating RNA-based cancer vaccines. Cancer Gene Ther. 30, 803-811 (2023). 46. Oda. Y. et al. Immunogenicity and safety of a booster dose of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2 mRNA COVID-19 vaccine: a doubleblind, multicentre, randomised, controlled, phase 3, non-inferiority trial. Lancet Infect. Dis. (2023) doi: 10.1016 / S1473-3099(23)00650-3.

[0352] 47. Palmer, C. D. et al. GRT-R910: a self-amplifying mRNA SARS-CoV-2 vaccine boosts immunity for >6 months in previously-vaccinated older adults. Nat. Commun. 14, 3274 (2023).

[0353] 48. Ong, E. Z. et al. Immune gene expression analysis indicates the potential of a self-amplifying Covid-19 mRNA vaccine, npj Vaccines 7, 154 (2022).

[0354] 49. Giovanni, M. D. et al. Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells. Nat. Immunol. 21, 321-330 (2020). 50. Teijaro, J. R. & Farber, D. L. COVID- 19 vaccines: modes of immune activation and future challenges. Nat. Rev. Immunol. 21, 195-197 (2021),

[0355] 51. Beissert, T. et al. Improvement of In Vivo Expression of Genes Delivered by Self- Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins. Human Gene Therapy 28, 1138-1146 (2017).

[0356] 52. McGee, J. E. et al. Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA. bioRxiv 2023.09.15.557994 (2023) doi: 10.1101 / 2023.09.15.557994.

[0357] 53. Erasmus, J. H. et al. Intramuscular Delivery’ of Replicon RNA Encoding ZIKV-117 Human Monoclonal Antibody Protects against Zika Virus Infection. Mol. Ther. - Methods Clin. Dev. 18, 402-414 (2020).

[0358] 54. Kairuz, D., Samudh, N., Ely, A., Arbuthnot, P. & Bloom, K. Production, Characterization, and Assessment of Permanently Cationic and Ionizable Lipid Nanoparticles for Use in the Delivery' of Self-Amplifying RNA Vaccines. Pharmaceutics 15, 1173 (2023).

[0359] 55. Beissert, T. et al. A Trans-amplifying RNA Vaccine Strategy for Induction of Potent Protective Immunity, Molecular therapy: the journal of the American Society of Gene Therapy null, null (2020).

[0360] 56. Minnaert, A.-K. et al. Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across. Adv Drug Deliver Rev 176, 113900-113900 (2021).

[0361] 57. Papukashvili, D. et al. Self-Amplifying RNA Approach for Protein Replacement Therapy. Int. J. Mol. Sci. 23, 12884 (2022). 58. Blakney, A. K. et al. Effects of cationic adjuvant formulation particle type, fluidity and immunomodulators on delivery and immunogenicity of saRNA. J. Control. Release 304, 65- 74 (2019).

[0362] 59. Beuckelaer, A. D., Grooten, J. & Koker, S. D. Type I Interferons Modulate CD8-t- T Cell Immunity to mRNA Vaccines. Trends in molecular medicine 23, 216-226 (2017).

[0363] 60. Kranz, L. M. et al. Systemic RNA delivery’ to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396-401 (2016).

[0364] 61. Li, Y. et al. Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nat Cancer 1, 882-893 (2020).

[0365] 62, Li, Y, et al. In vitro evolution of enhanced RNA replicons for immunotherapy. Sci Rep-uk 9, 6932 (2019).

[0366] 63. Wroblewska, L. et al. Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery’. Nat Biotechnol 33, 839-41 (2015),

[0367] 64. Kim, B. et al. Optimization of storage conditions for lipid nanoparticle-formulated self- replicating RNA vaccines. J. Control. Release 353, 241-253 (2023).

Claims

We claim:

1. A composition, comprising:(a) a lipid nanoparticle (LNP);(b) a mRNA encoding a gene of interest loaded within the LNP; and(c) an inhibitory nucleic acid targeting IFN-α / β receptor 1 (Ifnar1) gene loaded within the LNP.

2. The composition of claim 1, wherein the inhibitory nucleic acid comprises a short interfering RNA (siRNA).

3. Tire composition of claim 1 or 2, wherein the inhibitory nucleic acid targets a region of the Ifnar1 gene comprising the nucleotide sequence selected from SEQ ID NO: 1-4, 83, and 89.

4. The composition of claim 3, wherein the inhibitory nucleic acid targets a region of the Ifnar1 gene comprising the nucleotide sequence selected from SEQ ID NO: 1-24 and 83-95.

5. The composition of any one of claims 1-4, wherein the inhibitory nucleic acid comprises a double stranded siRNA sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs:(a) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UU (SEQ ID NO:25) and anti-sense (5’— >3’): AAU GGA AUA AAC GGA UCA AC (SEQ ID NO:26) (1FNAR1.1 mouse minimal sequence);(b) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CC (SEQ ID NO:27) and anti-sense (5'— >3'): GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:28) (IFNAR1.2 mouse minimal sequence);(c) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UC (SEQ ID NO:29) and anti-sense (5'— >3'): anti-sense (5'— >3'): GAG GAC CAA UCU GAG CUU UG (SEQ ID NO:30) (IFNAR1.1 human minimal sequence);(d) sense (5'— >3’): GUG CUC CAA AAC AGU CUG GA (SEQ ID NO:31) and anti-sense (5'->3'): (5’— >3’): UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:32) (IFNAR1.2 human minimal sequence);(e) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UC (SEQ ID NO:96) and anti-sense (5'— >3'): GAU AAA GUU GUC AUC UAU GA (SEQ ID NO:97) (IFNAR1.3 human minimal sequence); and(f) sense (5’— >3'): UGU UCA UUC AUC CCG AGA AC (SEQ ID NO: 108) and anti-sense (5'— >3'): GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 109) (IFNAR1.4 human minimal sequence).

6. The composition of claim 5, wherein the inhibitory nucleic acid comprises a double stranded siRN A sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs:(a) sense (5'-->3'): GUU GALT CCG UUU AUU CCA UU (SEQ ID NO:25) and anti-sense (5’— >3’): AAU GGA AUA AAC GGA UCA AC (SEQ ID NO:26) (IFNAR1.1 mouse minimal sequence);(b) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CC (SEQ ID NO:27) and anti-sense (5'— >3’): GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:28) (IFNAR1.2 mouse minimal sequence);(c) sense (5’— >3’): CAA AGC UCA GAU UGG UCC UC (SEQ ID NO:29) and anti-sense (5'— >3'): anti-sense (5'— >3'): GAG GAC CAA UCU GAG CUU UG (SEQ ID NO:30) (IFNAR1.1 human minimal sequence);(d) sense (5'->3'): GUG CUC CAA AAC AGU CUG GA (SEQ ID NO:31) and anti-sense (5'— >3'): (5'— >3'): UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:32) (IFNAR1.2 human minimal sequence);(e) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UUC (SEQ ID NO:80) and anti-sense (5'->3'): GAA UGG AAU AAA CGG AUC AAC (SEQ ID NO:33) (IFNAR1.1 mouse 21 nt sequence);(f) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UUC U (SEQ ID NO:34) and anti-sense (5'->3'): AGA AUG GAA UAA ACG GAU CAA C (SEQ ID NO:35) (IFNAR1.1 mouse 22 nt sequence);(g) sense (5’— >3'): GUU GAU CCG UUU AUU CCA UUC UA (SEQ ID NO:36) and anti-sense (5'->3’): UAG AAU GGA AUA AAC GGA UCA AC (SEQ ID NO:37) (IFNARI.l mouse 23 nt sequence);(h) sense (5'— >3'): GUU GAU CCG UUU AUU CCA UUC UAC (SEQ ID NO:38) and anti-sense (5'— >3'): GUA GAA UGG AAU AAA CGG AUC AAC (SEQ ID NO:39) (IFNAR1.1 mouse 24 nt sequence);(i) sense (5’— >3'): GUU GAU CCG UUU AUU CCA UUC UAC A (SEQ ID NO:40) and anti-sense (5'— >3’): UGU AGA AUG GAA UAA ACG GAU CAA C (SEQ ID N0:41) (IFNAR1.1 mouse 25 nt sequence);(j) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU (SEQ ID NO:42) and anti-sense (5’— >3'): AGG CGC GUG CUU UAC UUC UAC (SEQ ID NO:43);(IFNAR1.2 mouse 21 nt sequence);(k) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU G (SEQ ID NO:44) and anti-sense (5'— >3'): CAG GCG CGU GCU UUA CUU CUA C (SEQ ID NO:45) (IFNAR1.2 mouse 22 nt sequence);(l) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU GA (SEQ ID NO:46) and anti-sense (5'— >3'): UCA GGC GCG UGC UUU ACU UCU AC (SEQ ID NO:47) (IFNAR1.2 mouse 23 nt sequence);(m) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU GAG (SEQ ID NO:48) and anti-sense (5'-->3'): CUC AGG CGC GUG CUU UAC UUC UAC (SEQ ID NO:49) (IFNAR1.2 mouse 24 nt sequence);(n) sense (5'— >3'): GUA GAA GUA AAG CAC GCG CCU GAG G (SEQ ID NO:50) and anti-sense (5’— >3’): CCU CAG GCG CGU GCU UUA CUU CUA C (SEQ ID NO:51) (IFNAR1.2 mouse 25 nt sequence);(o) sense (5'->3’): CAA AGC UCA GAU UGG UCC UCC (SEQ ID NO:52) and anti-sense (5’— >3’): GGA GGA CCA AUC UGA GCU UUG (SEQ ID NO:53) (IFNAR1.1 human 21 nt sequence);(p) sense (5’— >3'): CAA AGC UCA GAU UGG UCC UCC A (SEQ ID NO:54) and anti-sense (5'->3’): UGG AGG ACC AAU CUG AGC UUU G (SEQ ID NO:55) (IFNAR1.1 human 22 nt sequence);(q) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UCC AG (SEQ ID NO:56) and anti-sense (5'->3’): CUG GAG GAC CAA UCU GAG CUU UG (SEQ ID NO:57) (IFNAR1.1 human 23 nt sequence);(r) sense (5’— >3'): CAA AGC UCA GAU UGG UCC UCC AGA (SEQ ID NO: 58) and anti-sense (5'->3'): UCU GGA GGA CCA AUC UGA GCU UUG (SEQ ID NO:59) (IFNAR1.1 human 24 nt sequence);(s) sense (5 '->3'): CAA AGC UCA GAU UGG UCC UCC AGA A (SEQ ID NO:60) and anti-sense (5'— >3’): UUC UGG AGG ACC AAU CUG AGC UUU G (SEQ ID NO:61) (IFNAR1.1 human 25 nt sequence);(t) sense (5'— >3'): GUG CUC CAA AAC AGU CUG GAA (SEQ ID NO:62) and anti-sense (5'->3'): UUC CAG ACU GUU UUG GAG CAC (SEQ ID NO:63) (IFNAR1.2 human 21 nt sequence);(u) sense (5’— >3'): GUG CUC CAA AAC AGU CUG GAA A SEQ ID NO:64) and anti-sense (5'— >3'): UUU CCA GAC UGU UUU GGA GCA C (SEQ ID NO:65) (IFNAR1.2 human 22 nt sequence);(v) sense (5'— >3'): GUG CUC CAA AAC AGU CUG GAA AC (SEQ ID NO:66) and anti-sense (5'->3'): GUU UCC AGA CUG UUU UGG AGC AC (SEQ ID NO:67) (IFNAR1.2 human 23 nt sequence);(w) sense (5'->3'): GUG CUC CAA AAC AGU CUG GAA ACA SEQ ID NO:68) and anti-sense (5’— >3’): UGU UUC CAG ACU GUU UUG GAG CAC (SEQ ID NO:69) (IFNAR1.2 human 24 nt sequence);(x) sense (5'— >3’): GUG CUC CAA AAC AGU CUG GAA ACA C (SEQ ID NO:70) and anti-sense (5’->3!): GUG UUU CCA GAC UGU UUU GGA GCA C (SEQ ID N0:71) (IFNAR1.2 human 25 nt sequence);(y) sense (5'->3'): UCA UAG AUG ACA ACU UUA UC (SEQ ID NO:96) and anti-sense (5'— >3'): GAU AAA GUU GUC AUC UAU GA (SEQ ID NO:97) (IFNAR1.3 human minimal sequence);(z) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UCC (SEQ ID NO:98) and anti-sense (5'->3'): GGA UAA AGU UGU CAU CUA UGA (SEQ ID NO:99) (IFNAR1.3 human 21 nt sequence);(aa) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UCC U (SEQ ID NO:100) and anti-sense (5'— >3'): AGG AUA AAG UUG UCA UCU AUG A (SEQ ID NO:101) (IFNAR1.3 human 22 nt sequence);(bb) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UCC UG (SEQ ID NO:102) and anti-sense (5’— >3’): CAG GAU AAA GUU GUC AUC UAU GA (SEQ ID NO: 103) (IFNAR1.3 human 23 nt sequence);(cc) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UCC UGA (SEQ ID NO: 104) and anti-sense (5'->3'): UCA GGA UAA AGU UGU CAU CUA UGA (SEQ ID NO: 105) (IFNAR1.3 human 24 nt sequence);(dd) sense (5'— >3'): UCA UAG AUG ACA ACU UUA UCC UGA G (SEQ ID NO: 106) and anti-sense (5'->3'): CUC AGG AUA AAG UUG UCA UCU AUG A (SEQ ID NO: 107) (IFNAR1.3 human 25 nt sequence);(ee) sense (5’— >3'): UGU UCA UUC AUG CCG AGA AC (SEQ ID NO: 108) and anti-sense (5’— >3'): GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 109) (IFNAR1.4 human minimal sequence);(fl) sense (5'— >3'): UGU UCA UUC AUC CCG AGA ACA (SEQ ID NO: 110) and anti-sense (5 >3'): UGU UCU CGG GAU GAA UGA ACA (SEQ ID NO: 111) (IFNAR1.4 human 21 nt sequence);(gg) sense (5 >3 '): UGU UCA UUC AUC CCG AGA ACA U (SEQ ID NO: 112) and anti-sense (5’— >3'): AUG UUC UCG GGA UGA AUG AAC A (SEQ ID NO: 113) (IFNAR1.4 human 22 nt sequence);(hh) sense (5 '->3'): UGU UCA UUC AUC CCG AGA ACA UU (SEQ ID NO: 114) and anti-sense (5 >3 '): AAU GUU CUC GGG AUG AAU GAA CA (SEQ ID NO: 115) (IFNAR1.4 human 23 nt sequence);(ii) sense (5f— >3’): UGU UCA UUC AUC CCG AGA ACA UUG (SEQ ID NO: 116) and anti -sense (5 '->3'): CAA UGU UCU CGG GAU GAA UGA ACA (SEQ ID NO: 117) (IFNAR1.4 human 24 nt sequence); and(jj) sense (5 >3'): UGU UCA UUC AUC CCG AGA ACA UUG G (SEQ ID NO: 118) and anti-sense (5'— >3'): CCA AUG UUC UCG GGA UGA AUG AAC A (SEQ ID NO: 119) (IFNAR1.4 human 25 nt sequence).

7. The composition of claim 5 or 6, wherein the 5’ end of the inhibitor}' nucleic acid sequences are phosphorylated.

8. The composition of any one of claims 5-7, wherein the inhibitor ' nucleic acids comprise a two nucleotide single stranded overhang at the 3’ terminus.

9. Tlie composition of claim 8, wherein the two nucleotide single stranded overhang at the 3‘ terminus comprises dTdT,10. The composition of any one of claims 5-9, wherein the inhibitory nucleic acid comprises a double stranded siRNA sequence comprising the nucleotide sequence of a pair of sequences selected from the following pairs:(a) sense (5 '->3 '): GUU GAU CCG UUU AUU CCA UUC dTdT (SEQ ID NO: 72) and anti-sense (5’— >3’): GAA UGG AAU AAA CGG AUC AAC dTdT (SEQ ID NO:73) (1FNAR1.1 mouse 21 nt sequence with overhang);(b) sense (5’— >3'): GUA GAA GUA AAG CAC GCG CCU dTdT (SEQ ID NO:74) and anti-sense (5!->3'): AGG CGC GUG CUU UAC UUC UAC dTdT (SEQ ID NO:75); (IFNAR1.2 mouse 21 nt sequence with overhang);(c) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UCC dTdT (SEQ ID NO:76) and anti-sense (5’— >3'): GGA GGA CCA AUC UGA GCU UUG dTdT (SEQ ID NO:77) (IFNAR1.1 human 21 nt sequence with overhang); and(d) sense (5'— >3'): CAA AGC UCA GAU UGG UCC UCC dTdT (SEQ ID NO:78) and anti-sense (5'— >3'): GGA GGA CCA AUC UGA GCU UUG dTdT (SEQ ID NO: 79) (IFNAR1 2 human 21 nt sequence with overhang).

11. Tire composition of claim 10, wherein the 5’ end of the siRNA is phosphorylated, and the 3’ end is hydroxylated.

12. The composition of any one of claims 3-11, wherein the siRNA comprises one or more locked nucleic acids (LNA).

13. The composition of any one of claims 1-12, wherein the mRNA is unmodified.

14. The composition of any one of claims 1-12, wherein the mRN A is modified (“modRNA”),15. The composition of claim 14, wherein the modRNA comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% modified residues.

16. Tlie composition of claim 15, wherein the modified residues are selected from the group consisting of 5-carboxymethylesteruridine, thienouridine, 5-methoxyuridine, 5-carboxyuridine, 2-thiouridine, 5 -methyluridine, N1 -propylpseudouridine, N 1 -methoxymethylpseudouridine, N 1 -ethylpseudouridine, N1 -methylpseudouridine, pseudouridine, 7 -deazaadenosine, N1 -methyladenosine, N6-methyladenosine, 6-chloropurineriboside, 2-amino-6-chloropurineriboside, 2-aminoadenosine, isoguanosine, thienoguanosine, 2-aminopurine-riboside, 8-oxoguanosine, thienocytidine, 5- methoxy cytidine, 5 -hydroxymethylcytidine, 5 -formylcytidine, 5 -aminoallylcytidine, 5- methylcytidine, and 5 -hydroxy cytidine.

17. The composition of claim 14 or 15, wherein all uridines in the mRNA are substituted with one or more analog.

18. Tlie compoisition of claim 17, wherin all uridines are substituted with Nl-methylpseudouridine.

19. The composition of any one of claims 1-18, wherein the gene of interest encodes an antigen.

20. The composition of claim 19, wherein the antigen comprises an immunogenic portion of a viral, bacterial, parasitic, protozoan, fungal, or tumor antigen.

21. The composition of claim 19 or 20, wherein the antigen comprises a human immunodeficiciency virus (HIV) or a severe acute respirator}' syndrome (SARS) antigen.

22. The composition of any one of claims 1-21, wherein the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a polymer-conjugated lipid,23. The composition of claim 22, wherein the helper lipid comprises phosphocholines, phosphoethanolamines, or combinations thereof.

24. The composition of claim 22 or 23, wherein the polymer-conjugated lipid comprises polyethylene glycol or polysarcosine conjugated to phosphoethanolamines, 1,2-dimyristoyl-rac-glycero, or other amphiphilic molecules.

25. The composition of any one of claims 22-24, wherein the amine -to-phosphate (N: P) ratio in the LNP loaded with RNA ranges from about 1: 1 to about 20: 1,26. The composition of any one of claims 1-25, wherein the lipid-to-RNA volume ratio in the LNP may range from about 1: 1 to about 1:20.

27. Tire composition of any one of claims 1-26, wherein the LNP comprises(a) N 1, N 3, N 5 -tri s (3 -(didodecylamino)propyl)benzene -1,3,5 -tricarboxamide (TT3),(b) (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA),(c) 1,2-dioIeoy l-sn-glycero-3-phosphoethanolamine (DOPE),(d) cholesterol, and(e) l,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000 (DMG-PEG2k)28. The composition of claim 27, wherein a molar ratio of LNP components is about 10 (TT3):25 (DLin-MC3-DMA):20 (DOPE):40 (cholesterol):5(DMG-PEG2k).

29. Tire composition of any one of claims 1-8, further comprising an adjuvant.

30. A vaccine comprising the composition of any one of claims 19-28.

31. A pharmaceutical composition comprising the composition or vaccine of any one of claims 1-30, and a pharmaceutically acceptable carrier.

32. A method for generating an immune response against an antigen, comprising administering to a subject an amount effective to generate an immune response in the subject of the composition or vaccine of any one of claims 19-31.

33. A method of treating an infection or limiting development of an infection in a subject in need thereof comprising administering to the subject the composition or vaccine of any one of claims 19-31 in an effective amount to induce an immune response against the antigen,34. Tlie method of claim 33 or 34, wherein the antigen comprises an HIV antigen, and the subject is at risk of, or has, an HIV infection,35. The method of claim 33 or 34, wherein the antigen comprises a SARS-CoV-2 antigen, and the subject is at risk of, or has, an SARS-CoV-2 infection.

Images