Interferon inhibition with viral proteins and fragments and fusions thereof

Abstract

Provided herein is a method of inhibiting a mRNA therapeutic-mediated interferon response in a subject, comprising administering to the subject: 1) at least one mRNA encoding a therapeutic protein; and 2) at least one mRNA encoding a viral protein or fragment thereof capable of inhibiting the mRNA therapeutic-mediated interferon response. Also provided are therapeutic protein – interferon-repressing viral protein fusions.

Description

[0001] INTERFERON INHIBITION WITH VIRAL PROTEINS AND FRAGMENTS AND FUSIONS THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 532,808, filed August 15, 2023. The entire content of the above-referenced patent application is incorporated by reference in its entirety herein.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Messenger RNA (mRNA)-based therapeutics offer a promising new modality for the treatment of numerous diseases and disorders, including cancer, rare genetic disorders, infections. However, mRNA therapeutics are not without their harmful side effects. The administration of mRNA to a subject can trigger a potent negative immune response, such as the activation of Type I interferon that can lead to, among other things, systemic immune response syndrome or a cytokine storm. For mRNA therapeutics to achieve their full potential, strategies must be developed to inhibit these deleterious effects while retaining the therapeutic efficacy of the mRNA.

[0006] SUMMARY OF THE DISCLOSURE

[0007] In one aspect, the disclosure provides a method of inhibiting a mRNA therapeutic- mediated interferon response in a subject, comprising administering to the subject: 1) at least one mRNA encoding a therapeutic protein; and 2) at least one mRNA encoding a viral protein or fragment thereof capable of inhibiting the mRNA therapeutic-mediated interferon response, thereby inhibiting the mRNA therapeutic-mediated interferon response in the subject.

[0008] In certain embodiments, the at least one mRNA encoding the therapeutic protein is produced by in vitro transcription (IVT).

[0009] In certain embodiments, the at least one mRNA encoding the therapeutic protein is not chemically modified.

[0010] In certain embodiments, the at least one mRNA encoding the therapeutic protein comprises at least one chemically modified nucleotide.

[0011] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof is produced by IVT. In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof is not chemically modified.

[0012] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof comprises at least one chemically modified nucleotide.

[0013] In certain embodiments, the at least one chemically modified nucleotide is pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- methylcytosine, 2- thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2- thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5-methyluridine, 5 -methyluridine, 5- methoxyuridine, or 2’-O-methyl uridine, or a combination thereof.

[0014] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof is administered to the subject before the at least one mRNA encoding the therapeutic protein.

[0015] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof and the at least one mRNA encoding the therapeutic protein are administered to the subject simultaneously.

[0016] In certain embodiments, the viral protein or fragment thereof comprises a rhabdovirus phosphoprotein or fragment thereof.

[0017] In certain embodiments, the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof. In certain embodiments, the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUVV) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof. In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45. In certain embodiments, the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD). In certain embodiments, the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

[0018] In certain embodiments, the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 10.

[0019] In certain embodiments, the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 12.

[0020] In certain embodiments, the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 13.

[0021] In certain embodiments, the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 16.

[0022] In certain embodiments, the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 17.

[0023] In certain embodiments, the viral protein or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

[0024] In certain embodiments, the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

[0025] In certain embodiments, the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof.

[0026] In certain embodiments, the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 14.

[0027] In certain embodiments, the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof.

[0028] In certain embodiments, the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 15.

[0029] In certain embodiments, the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

[0030] In certain embodiments, the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 18.

[0031] In certain embodiments, the viral protein or fragment thereof comprises a filoviridae protein or fragment thereof. In certain embodiments, the filoviridae protein or fragment thereof comprises a ebolavirus protein or fragment thereof.

[0032] In certain embodiments, the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

[0033] In certain embodiments, the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 11.

[0034] In certain embodiments, the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 19.

[0035] In certain embodiments, the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 20.

[0036] In certain embodiments, the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 21.

[0037] In certain embodiments, the viral protein or fragment thereof comprises a coronaviridae protein or fragment thereof.

[0038] In certain embodiments, the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

[0039] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP 1 protein or fragment thereof, a betacoronavirus N SP6 protein or fragment thereof, or a betacoronavirus NSP 13 protein or fragment thereof.

[0040] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP 13 protein or fragment thereof.

[0041] In certain embodiments, the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 24.

[0042] In certain embodiments, the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 23.

[0043] In certain embodiments, the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 22.

[0044] In certain embodiments, the viral protein or fragment thereof comprises an orthomyxovidirae protein or fragment thereof. In certain embodiments, the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

[0045] In certain embodiments, the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

[0046] In certain embodiments, the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

[0047] In certain embodiments, the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 27.

[0048] In certain embodiments, the therapeutic protein is selected from the group consisting of an antigen, a nucleic acid editing system or a component thereof, an antigen binding protein or fragment thereof, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and a thrombolytic.

[0049] In certain embodiments, the antigen comprises a viral antigen or a prokaryotic antigen.

[0050] In certain embodiments, the nucleic acid editing system or a component thereof comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, or a Transcription Activator-Like Effector-based Nucleases (TALEN).

[0051] In certain embodiments, the nucleic acid editing system comprises a CRISPR system.

[0052] In certain embodiments, the CRISPR-system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

[0053] In certain embodiments, the nucleobase editing domain comprises an adenosine deaminase, cytidine deaminase, or a functional variant thereof.

[0054] In certain embodiments, the mRNA comprises at least one 5’ untranslated region (5’ UTR), at least one 3’ untranslated region (3’ UTR), and / or at least one polyadenylation (poly A) sequence.

[0055] In certain embodiments, one or both of the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof is encapsulated in a lipid nanoparticle (LNP). In certain embodiments, the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in one LNP.

[0056] In certain embodiments, the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in separate LNPs.

[0057] In certain embodiments, the LNP comprises at least one cationic lipid.

[0058] In certain embodiments, the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

[0059] In certain embodiments, the at least one mRNA encoding a therapeutic protein is present in a composition comprising one or more contaminating RNA species.

[0060] In certain embodiments, the one or more contaminating RNA species are selected from the group consisting of double stranded RNA (dsRNA), uncapped mRNA species, untailed mRNA species, unmodified mRNA species, and prematurely aborted RNA species.

[0061] In certain embodiments, inhibiting the mRNA therapeutic-mediated interferon response in the subject comprises a reduction in an interferon relative to a subject that is not administered at least one mRNA encoding a rhabdovirus phosphoprotein or fragment thereof.

[0062] In certain embodiments, the interferon is interferon beta (IFNP).

[0063] In one aspect, the disclosure provides a composition comprising at least one isolated mRNA encoding a therapeutic protein; and at least one isolated mRNA encoding a viral protein or fragment thereof capable of inhibiting an mRNA therapeutic-mediated interferon response.

[0064] In certain embodiments, the at least one mRNA encoding the therapeutic protein is produced by in vitro transcription (IVT).

[0065] In certain embodiments, the at least one mRNA encoding the therapeutic protein is not chemically modified.

[0066] In certain embodiments, the at least one mRNA encoding the therapeutic protein comprises at least one chemically modified nucleotide.

[0067] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof is produced by IVT.

[0068] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof is not chemically modified.

[0069] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof comprises at least one chemically modified nucleotide.

[0070] In certain embodiments, the at least one chemically modified nucleotide is pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- methylcytosine, 2- thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2- thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5-methyluridine, 5 -methyluridine, 5- methoxyuridine, or 2’-O-methyl uridine, or a combination thereof.

[0071] In certain embodiments, the viral protein or fragment thereof comprises a rhabdovirus phosphoprotein or fragment thereof.

[0072] In certain embodiments, the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof.

[0073] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUW) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof.

[0074] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof.

[0075] In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45.

[0076] In certain embodiments, the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD).

[0077] In certain embodiments, the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

[0078] In certain embodiments, the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 10.

[0079] In certain embodiments, the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 12.

[0080] In certain embodiments, the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 16.

[0081] In certain embodiments, the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 17.

[0082] In certain embodiments, the viral protein or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

[0083] In certain embodiments, the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

[0084] In certain embodiments, the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof.

[0085] In certain embodiments, the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 14.

[0086] In certain embodiments, the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof.

[0087] In certain embodiments, the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 15.

[0088] In certain embodiments, the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

[0089] In certain embodiments, the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 18.

[0090] In certain embodiments, the viral protein or fragment thereof comprises a filoviridae protein or fragment thereof.

[0091] In certain embodiments, the filoviridae protein or fragment thereof comprises a ebolavirus protein or fragment thereof.

[0092] In certain embodiments, the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

[0093] In certain embodiments, the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 19.

[0094] In certain embodiments, the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 20.

[0095] In certain embodiments, the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 21.

[0096] In certain embodiments, the viral protein or fragment thereof comprises a coronaviridae protein or fragment thereof.

[0097] In certain embodiments, the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

[0098] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP 1 protein or fragment thereof, a betacoronavirus N SP6 protein or fragment thereof, or a betacoronavirus NSP 13 protein or fragment thereof.

[0099] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP 13 protein or fragment thereof.

[0100] In certain embodiments, the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 24.

[0101] In certain embodiments, the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 23.

[0102] In certain embodiments, the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 22.

[0103] In certain embodiments, the viral protein or fragment thereof comprises an orthomyxovidirae protein or fragment thereof.

[0104] In certain embodiments, the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

[0105] In certain embodiments, the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

[0106] In certain embodiments, the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments, the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 27.

[0107] In certain embodiments, the therapeutic protein is selected from the group consisting of an antigen, a nucleic acid editing system or a component thereof, an antigen binding protein or fragment thereof, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and a thrombolytic.

[0108] In certain embodiments, the antigen comprises a viral antigen or a prokaryotic antigen.

[0109] In certain embodiments, the nucleic acid editing system or a component thereof comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, or a Transcription Activator-Like Effector-based Nucleases (TALEN).

[0110] In certain embodiments, the nucleic acid editing system comprises a CRISPR system.

[0111] In certain embodiments, the CRISPR-system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

[0112] In certain embodiments, the nucleobase editing domain comprises an adenosine deaminase, cytidine deaminase, or a functional variant thereof.

[0113] In certain embodiments, the mRNA comprises at least one 5’ untranslated region (5’ UTR), at least one 3’ untranslated region (3’ UTR), and / or at least one polyadenylation (poly A) sequence.

[0114] In certain embodiments, one or both of the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof is encapsulated in a lipid nanoparticle (LNP).

[0115] In certain embodiments, the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in one LNP.

[0116] In certain embodiments, the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in separate LNPs.

[0117] In certain embodiments, the LNP comprises at least one cationic lipid.

[0118] In certain embodiments, the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

[0119] In certain embodiments, the at least one mRNA encoding a therapeutic protein is present in a composition comprising one or more contaminating RNA species. In certain embodiments, the one or more contaminating RNA species are selected from the group consisting of double stranded RNA (dsRNA), uncapped mRNA species, untailed mRNA species, unmodified mRNA species, and prematurely aborted RNA species.

[0120] In one aspect, the disclosure provides a fusion protein comprising a therapeutic protein domain linked to an interferon-repressing viral protein domain or fragment thereof.

[0121] In certain embodiments, the interferon-repressing viral protein domain or fragment thereof comprises a rhabdovirus phosphoprotein or fragment thereof.

[0122] In certain embodiments, the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof.

[0123] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUW) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof.

[0124] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof.

[0125] In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45.

[0126] In certain embodiments, the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD).

[0127] In certain embodiments, the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

[0128] In certain embodiments, the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 10.

[0129] In certain embodiments, the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 12.

[0130] In certain embodiments, the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 16.

[0131] In certain embodiments, the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 17.

[0132] In certain embodiments, the interferon-repressing viral protein domain or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

[0133] In certain embodiments, the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

[0134] In certain embodiments, the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof.

[0135] In certain embodiments, the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 14.

[0136] In certain embodiments, the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof.

[0137] In certain embodiments, the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 15.

[0138] In certain embodiments, the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

[0139] In certain embodiments, the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 18.

[0140] In certain embodiments, the interferon-repressing viral protein domain or fragment thereof comprises a filoviridae protein or fragment thereof.

[0141] In certain embodiments, the filoviridae protein or fragment thereof comprises an ebolavirus protein or fragment thereof.

[0142] In certain embodiments, the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

[0143] In certain embodiments, the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 19.

[0144] In certain embodiments, the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 20.

[0145] In certain embodiments, the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 21.

[0146] In certain embodiments, the interferon-repressing viral protein domain or fragment thereof comprises a coronaviridae protein or fragment thereof.

[0147] In certain embodiments, the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

[0148] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP 1 protein or fragment thereof, a betacoronavirus N SP6 protein or fragment thereof, or a betacoronavirus NSP 13 protein or fragment thereof.

[0149] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP 13 protein or fragment thereof.

[0150] In certain embodiments, the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 24.

[0151] In certain embodiments, the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 23.

[0152] In certain embodiments, the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 22.

[0153] In certain embodiments, the interferon-repressing viral protein domain or fragment thereof comprises an orthomyxovidirae protein or fragment thereof.

[0154] In certain embodiments, the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

[0155] In certain embodiments, the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

[0156] In certain embodiments, the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments, the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 27.

[0157] In certain embodiments, the therapeutic protein is selected from the group consisting of an antigen, a nucleic acid editing system or a component thereof, an antigen binding protein or fragment thereof, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and a thrombolytic.

[0158] In certain embodiments, the antigen comprises a viral antigen or a prokaryotic antigen.

[0159] In certain embodiments, the nucleic acid editing system or a component thereof comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, or a Transcription Activator-Like Effector-based Nucleases (TALEN).

[0160] In certain embodiments, the nucleic acid editing system comprises a CRISPR system.

[0161] In certain embodiments, the CRISPR-system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

[0162] In certain embodiments, the nucleobase editing domain comprises an adenosine deaminase, cytidine deaminase, or a functional variant thereof.

[0163] In one aspect, the disclosure provides nucleic acid encoding the fusion protein described herein.

[0164] In certain embodiments, the nucleic acid comprises a DNA vector.

[0165] In certain embodiments, the DNA vector comprises a viral vector.

[0166] In certain embodiments, the nucleic acid comprises an mRNA.

[0167] In certain embodiments, the mRNA is produced by in vitro transcription (IVT).

[0168] In certain embodiments, the mRNA is not chemically modified.

[0169] In certain embodiments, the at mRNA comprises at least one chemically modified nucleotide.

[0170] In certain embodiments, the at least one chemically modified nucleotide is pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- methylcytosine, 2- thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2- thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5-methyluridine, 5 -methyluridine, 5- methoxyuridine, or 2’-O-methyl uridine, or a combination thereof.

[0171] In certain embodiments, the mRNA comprises at least one 5’ untranslated region (5’ UTR), at least one 3’ untranslated region (3’ UTR), and / or at least one polyadenylation (poly A) sequence.

[0172] In certain embodiments, the mRNA is encapsulated in a lipid nanoparticle (LNP).

[0173] In certain embodiments, the LNP comprises at least one cationic lipid.

[0174] In certain embodiments, the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

[0175] In one aspect, the disclosure provides a method of inhibiting a mRNA therapeutic- mediated interferon response in a subject, comprising administering to the subject the fusion protein described herein or the nucleic acid described herein, thereby inhibiting the mRNA therapeutic-mediated interferon response in the subject.

[0176] BRIEF DESCRIPTION OF THE DRAWINGS

[0177] FIG. 1 depicts a schematic of RABV phosphoprotein (RABV P).

[0178] FIG. 2 depicts interferon beta (IFNP) levels in fibroblasts transfected with mRNA encoding one of several RABV proteins (L, G, M, P, N), an adenosine base editor (ABE-eGFP), or the COCV GP protein.

[0179] FIG. 3 depicts IFNP levels in A549 cells transfected with dirty mRNA, poly IC, dirty mRNA in combination with mRNA encoding the RABV P protein (P mRNA), or poly IC in combination with P mRNA.

[0180] DETAILED DESCRIPTION

[0181] Interferon-Repressing Viral Proteins

[0182] Provided herein are interferon-repressing viral proteins and fragments thereof and fusions proteins thereof. Also provided are nucleic acids (e.g., mRNA) encoding the interferon-repressing viral proteins and fragments thereof and fusions proteins thereof.

[0183] As used herein, an “interferon-repressing viral protein” refers to a viral protein capable of inhibiting the interferon response in a cell or an organism, in particular in an organism that has been administered a therapeutic mRNA encoding a therapeutic protein. A significant antiviral response in mammals is the induction of interferons and other cytokines, which interfere with viral replication. Interferons act via autocrine and paracrine pathways to induce an antiviral state in infected cells and in neighboring cells containing interferon receptors. Accordingly, viruses have evolved mechanisms to evade the host interferon response. Numerous viral proteins have been identified with interferon-repressing activities, through their interaction with one or more intracellular proteins involved in the interferon signaling cascade.

[0184] As used herein, an “interferon-repressing viral protein fragment” refers to a truncated version of the wild-type interferon-repressing viral protein that retains the interferon-repressing activity. Retention of interferon-repressing activity in a viral protein fragment can be readily determined by one of skill in the art using art-recognized interferon assays. Exemplary assays may measure the amount of a protein expressed when the type-I interferon pathway is activated. For example, but in no way limiting, IFNP can be measured following incubation of a cell line with the interferon-repressing viral protein fragment or a nucleic acid encoding the same and a stimulator of the type-I interferon response (such as polylC). An exemplary assay for measuring IFNP is a western blot with an anti-IFNP antibody.

[0185] The viral protein fragment includes amino acid deletions from one or both of the N- terminus or C-terminus of the wild-type viral protein. Any number of amino acid deletions are envisioned, including single amino acid deletions. Amino acid deletions need not be from the N- or C-terminus but may include internal deletions of amino acids. Exemplary interferon- repressing viral protein fragments include the RABV P fragments as recited in any one of amino acid sequences of SEQ ID NOs: 28-33.

[0186] Rhabdovirus Phosphoproteins

[0187] In the interferon signaling cascade, rhabdovirus phosphoproteins (e.g., RABV phosphoprotein) prevent phosphorylation of the IRF3 dimer, preventing its nuclear entry. Rhabdovirus phosphoproteins also bind to phosphorylated STAT1 / STAT1 and STAT1 / STAT2 dimers, preventing their nuclear entry. A schematic of an exemplary rhabdovirus phosphoprotein is provided in FIG. 1, which displays the N-terminal domain (NTD), the L protein binding domain, the dimerization domain (DD), and the C-terminal domain (CTD), which is involved in RNP complex binding, STAT1 / STAT1 binding, and IRF3 dimer.

[0188] Provided herein are rhabdovirus phosphoproteins, including fragments and fusions thereof, useful for repressing an mRNA-mediated interferon response in a subject. The Rhabdoviridae family comprises several genera, including lyssavirus, tibrovirus, vesiculovirus, ephemerovirus, novirhabdovirus, perhabdovirus, sigmavirus, sprivivirus, and tupavirus.

[0189] In certain embodiments, the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof.

[0190] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUW) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof.

[0191] RABV Phosphoprotein

[0192] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof.

[0193] In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45.

[0194] In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 45.

[0195] In certain embodiments, the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD).

[0196] In certain embodiments, the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

[0197] In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 33.

[0198] Non-RABV Rhabdovirus Phosphoproteins

[0199] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a ABLV phosphoprotein or fragment thereof. In certain embodiments, the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 10.

[0200] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a EBLV1 phosphoprotein or fragment thereof. In certain embodiments, the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 12.

[0201] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a LBV phosphoprotein or fragment thereof. In certain embodiments, the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 13.

[0202] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a MOKV phosphoprotein or fragment thereof. In certain embodiments, the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 16.

[0203] In certain embodiments, the lyssavirus phosphoprotein or fragment thereof comprises a VSV phosphoprotein or fragment thereof. In certain embodiments, the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 17.

[0204] Paramyxovirus Proteins

[0205] All paramyxoviruses have genes encoding nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), attachment protein (HN for respiroviruses, rubulaviruses, and avulaviruses, H for morbilliviruses, and G for henipaviruses and members of the Pneumovirinae subfamily), and RNA-dependent RNA polymerase (L). The paramyxovirus P gene has overlapping ORFs that give rise to multiple, distinct gene products. The morbilliviruses, respiroviruses, henipaviruses, and avulaviruses, among others, generate a primary transcript whose mRNA is translated authentically to generate the P protein; however, a process of RNA editing (also known as pseudo-template addition) can result in the insertion of G residues at an editing site in the middle of the transcript to generate mRNAs where there is a frameshift in the coding sequence downstream from this site. The insertion of a single G generates an mRNA that translates into a protein called the “V protein,” which has a common N-terminus to the P protein, but a unique C-terminus. The insertion of a second G residue creates an mRNA that encodes a protein with a different C-terminus called, variously, “W”, “D,” or “I.” Interestingly, in the rubulaviruses it is the V protein that is genomically templated, and production of the P protein requires an insertion of 2Gs into the mRNA by RNA editing; the addition of 1 or 4 Gs creates an mRNA for the I protein. In addition to these products, morbilliviruses, respiroviruses, and henipaviruses translate the P / V / W7D mRNAs using an alternative reading frame(s) to generate poorly conserved “C” protein(s); although only a single C protein is normally made, SeV can utilize 4 distinct start codons to generate C', C, Yl, and Y2 proteins that have a different N-terminus but a common C-terminus. The P genes of viruses within the Pneumovirinae subfamily do not encode more than 1 protein, but rather some of these viruses, including HRSV and BRSV, have 2 extra genes, NS1 and NS2 that, like the V and C proteins, act as interferon antagonists. These P / V / C proteins of paramyxovirus are interferon-repressing viral proteins. The V proteins of most paramyxoviruses are able to antagonize both the NF -Kb and IRF-3 arms of the dsRNA signaling responses, and MDA-5 is the interacting partner for the V proteins (see Goodboum et al. J Interferon Cytokine Res. 2009. 29(9): 539-547, incorporated herein by reference).

[0206] In certain embodiments, the viral protein or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

[0207] In certain embodiments, the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

[0208] In certain embodiments, the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof. In certain embodiments, the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 14.

[0209] In certain embodiments, the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof. In certain embodiments, the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 15.

[0210] In certain embodiments, the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

[0211] In certain embodiments, the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 18.

[0212] Filovirus Protein

[0213] Filoviruses, such as Ebola and Marburg virus, encode viral proteins with the ability to counteract the type I interferon (IFN-I) response. These IFN-I antagonist proteins are crucial to ensure virus replication, prevent an antiviral state in infected and bystander cells, and impair the ability of antigen-presenting cells to initiate adaptive immune responses. Ebola viruses have seven genes coding for eight major viral products, two of which (VP24 and VP35) have been shown to act as IFN-antagonist proteins. VP24 inhibits karyopherin-mediated nuclear translocation of STAT1 (see, Escudero-Perez et al. Vaccines (Basel). 2019. 7(1): 22, incorporated herein by reference).

[0214] In certain embodiments, the viral protein or fragment thereof comprises a filoviridae protein or fragment thereof.

[0215] In certain embodiments, the filoviridae protein or fragment thereof comprises a ebolavirus protein or fragment thereof.

[0216] In certain embodiments, the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

[0217] In certain embodiments, the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 11.

[0218] In certain embodiments, the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 19.

[0219] In certain embodiments, the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 21.

[0220] Coronavirus Proteins

[0221] Coronaviruses employ several non- structural proteins (NSPs) to inhibit a hosts interferon response. NSP1, for example, prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSPl-induced depletion of Tyk2 and STAT2 dampens IFN- stimulated gene (ISG) induction. NSP6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation. NSP13 downregulates interferon and NF-KB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBKl and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression (see, Xia et al. Cell Rep. 2020. 33(1): 108234; Kumar et al. J Virol. 2021. 95(13):e0026621; Vazquez et al. PLoS One. 2021. 16(6): e0253089, each of which is incorporated herein by reference).

[0222] In certain embodiments, the viral protein or fragment thereof comprises a coronaviridae protein or fragment thereof.

[0223] In certain embodiments, the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

[0224] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP 1 protein or fragment thereof, a betacoronavirus N SP6 protein or fragment thereof, or a betacoronavirus NSP 13 protein or fragment thereof.

[0225] In certain embodiments, the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP 13 protein or fragment thereof.

[0226] In certain embodiments, the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 24.

[0227] In certain embodiments, the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 23.

[0228] In certain embodiments, the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 22.

[0229] Orthomyxovirus Proteins

[0230] The orthomyxovirus protein NS 1 has been shown to inhibit the interferon response (see Yuan et al. EMBO J. 2001. 20(3): 362-371, incorporated herein by reference).

[0231] In certain embodiments, the viral protein or fragment thereof comprises an orthomyxovidirae protein or fragment thereof.

[0232] In certain embodiments, the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

[0233] In certain embodiments, the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

[0234] In certain embodiments, the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

[0235] In certain embodiments, the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity (i.e., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID NO: 27.

[0236] Any of the interferon-repressing viral proteins, fragments thereof, or fusions thereof may further comprise one or more nuclear localization sequences. Exemplary NLS sequences are described in Plank et al.. PCTZEP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV,

[0237] KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0238] Messenger RNA & Proteins Encoded Therein

[0239] The mRNA described herein comprise at least one open reading frame (ORF) encoding at least one protein (e.g., a therapeutic protein or an interferon-repressing viral protein). In certain embodiments, the mRNA further comprises a 5’ cap, a 5’ UTR, a 3’ UTR, and / or a poly A tail.

[0240] The mRNA 5’cap consists of a guanine nucleotide connected to mRNA via a 5’ to 5’ triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. It is referred to as a 7-methylguanylate cap, abbreviated m7G (also referred to as Cap-0). The m7G cap serves as a molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Moreover, cap 2’0 methylation (referred to as Cap-1) serves as an identifier of self RNA in the innate immune system against foreign RNA. Thus, an uncapped mRNA is capable of triggering the innate immune system. This response is triggered in part by cellular sensors RIG-I and MDA5 and effectors IFIT1 and IFIT5 of the Type I interferon signally pathway, which act by discriminating Cap-1 RNA from others. Cytoplasmic pattern recognition receptors (PRRs) RIG-I and RIG-I-like receptor MDA5 are sensors that trigger cellular type I interferon (IFN) response to virus infections. While MDA5 interacts with long dsRNA, RIG-I interacts strongly with short dsRNA, and less so with 5 ’-ppp and 5’-pp ssRNA. The Cap-1 structure abolishes the interactions of dsRNA with RIG-I and MDA5 and hence does not activate the IFN signaling pathway. Cap-0 and 5 ’-ppp dsRNA bind to RIG-I with similar affinity. Instead, 2’0 methylation of 5'-ppp RNA (5'-ppp(2'OMe)N. . .) significantly diminishes the binding of the dsRNA to RIG-I and IFN signaling. The interaction with RIG-I and downstream INF induction is further attenuated by the complete Cap-1 structure. The mRNA 5’cap and its function are described in further detail in Ramanathan et al. Nucleic Acids Res. 2016. 44(16): 7511-7526, incorporated herein by reference. In some embodiments, the mRNA of the disclosure includes a 5’ and / or 3’ untranslated region (UTR). In mRNA, the 5’ UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3’ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.

[0241] As used herein, the terms “polyA sequence,” “polyA tail,” and “polyA region” refer to a sequence of adenosine nucleotides at the 3’ end of the mRNA molecule. The polyA tail may confer stability to the mRNA and protect it from exonuclease degradation. The polyA tail may enhance translation.

[0242] Messenger RNA Modification

[0243] Unmodified mRNA produced through IVT induces high level of type I interferon (IFN-I) through activation of toll-like receptors (TLRs). Activation of TLRs results in upregulation of proinflammatory cytokines such as IFNP, IFN-I, IL-6, IL-12, TNF-a and chemokines (see, Vlatkovic. Biomedicines. 2021. 9(5):530).

[0244] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA may be synthesized from modified nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g., 1- methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl- adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5- methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl- guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5- carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxy acetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5 ’-m ethoxy carbonylmethyl-uracil, 5-methoxy -uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, P-D-mannosyl-queosine, phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5-methylcytosine, and inosine.

[0245] In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1 -methylpseudouridine, 2- thiouridine, 4’-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2’-O-methyl uridine.

[0246] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof. In some embodiments, the chemical modification comprises Nl- methylpseudouridine.

[0247] Therapeutic Messenger RNA & Proteins Encoded By The Same

[0248] As used herein, a “therapeutic mRNA” refers to an mRNA that encodes a therapeutic protein. As used herein, a “therapeutic protein” refers to protein (including fragments thereof and peptide) that is capable of effecting treatment and / or prophylaxis to a subject in need. In certain embodiments, the therapeutic effect is accomplished by suppression, remission, or eradication of a disease state suffered by the subject.

[0249] The therapeutic protein may encode any therapeutic protein known in the art, for example, without limitation, a therapeutic protein that can replace a deficient or abnormal protein; a therapeutic protein that can augment an existing pathway; a therapeutic protein that can provide a novel function or activity (e.g., a novel function or activity beneficial to a subject suffering from the lack thereof); a therapeutic protein that interferes with a molecule or an organism (e.g., an organism that is different to the organism that hosts the target cell); and / or a therapeutic protein that delivers other compounds or proteins (e.g., a radionuclide, a cytotoxic drug, and / or an effector protein). For example, the therapeutic protein can by, without limitation, a nucleic acid editing protein (e.g., an adenine or cytidine base editor) or system, an antibody or antibody-based drug, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, an antigen, and / or a thrombolytic. Various other types of therapeutic proteins are known to those of ordinary skill in the art.

[0250] In certain embodiments, the therapeutic protein comprises a nucleic acid editing system or components thereof. In some embodiments the therapeutic protein comprises a nucleic acid binding protein (e.g., a zinc finger, a TALE, or a nucleic acid programmable nucleic acid binding protein, such as Cas9). In some embodiments, the nucleic acid editing system component is a nucleic acid programmable nucleic acid binding protein (e.g., Cas9).

[0251] In some embodiments, the therapeutic protein comprises a CRISPR system. In some embodiments, the CRISPR system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain. In some embodiments, the nucleobase editing domain is an adenosine deaminase, cytidine deaminase, cytosine deaminase, or a functional variant thereof (e.g., a functional variant capable of deaminating a nucleobase in a nucleic acid molecule such as DNA or RNA). In some embodiments, the nucleobase editing domain is an adenosine deaminase. In some embodiments, the adenosine deaminase is ABE7.10. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 polypeptide, a Casl2 polypeptide, or a functional variant thereof. In some embodiments, the CRISPR system further comprises a guide RNA (gRNA) or a nucleic acid encoding a gRNA.

[0252] In some embodiments the therapeutic protein comprises a nucleobase modifying protein (e.g., a base editor protein). In some embodiments the therapeutic protein comprises an adenosine base editor (e.g., ABE7.10). In some embodiments the therapeutic protein comprises a cytidine base editor. In some embodiments the therapeutic protein comprises a cytosine base editor capable of deaminating a cytosine in DNA or RNA.

[0253] In certain embodiments, the therapeutic protein comprises a nucleic acid editing system, e.g., a base editor system further described herein.

[0254] In certain embodiments, the therapeutic protein comprises an antigen. As used herein, an “antigen” refers to a protein (e.g., peptide or polypeptide) that is derived from a pathogen that is capable of stimulating a protective immune response against the pathogen. In certain embodiments, the antigen is a viral antigen or a prokaryotic antigen.

[0255] Interferon-Repressing Viral Protein Fusions

[0256] Provided herein are interferon-repressing viral protein fusions comprising the interferon-repressing viral protein or fragment thereof described herein linked to a therapeutic protein described herein. The interferon-repressing viral protein fusions may advantageously provide the therapeutic effect of the therapeutic protein while simultaneously repressing the interferon response in a subject administered the fusion protein. Interferon-repressing viral protein fusions may thus be encoded in a single nucleic acid molecule (e.g., a single mRNA molecule).

[0257] The fusions described herein may comprise the full-length interferon-repressing viral protein. Alternatively, the fusions described herein may comprise the interferon-repressing viral protein fragment that retains the interferon repressing activity.

[0258] The interferon-repressing viral protein or fragment thereof may be linked to the therapeutic protein via an amino acid linker. Alternatively, the interferon-repressing viral protein or fragment thereof may be directly linked to the therapeutic protein without an amino acid linker. Typically, a linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, a linker is an amino acid or a plurality of amino acids e.g., a peptide or protein). In some embodiments, a linker is 1-100 amino acids in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0259] Various linker lengths and flexibilities between the interferon-repressing viral protein or fragment thereof and the therapeutic protein can be employed (e.g., ranging from very flexible linkers of the form (GGGS)n, (GGGGS)n, and (G)n to more rigid linkers of the form (EAAAK)n, (SGGS)n, SGSETPGTSESATPES (see, e g., Guilinger JP, et al. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, e.g., PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP)4, P(AP)7, P(AP)io (see, e.g. Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan 25; 10(l):439; the entire contents are incorporated herein by reference). Such proline-rich linkers are also termed “rigid” linkers.

[0260] Methods Of Inhibiting An Interferon Response

[0261] Provided herein are methods of inhibiting a mRNA therapeutic-mediated interferon response in a subject. Innate immune mechanisms in mammals are designed to recognize foreign RNA, thereby mounting an immune response. The type-I interferon response, and the associated proteins in the pathway, serves as a significant mediator of the foreign RNA detection and response system. Double-stranded RNA (dsRNA) and uncapped single-stranded RNA (ssRNA) are recognized, at least, by RIG-I (for RNAs with 5'-triphosphate) and MDA-5 (for dsRNAs), which activate IRFs andNF-KB transcription factors. Endosomally encapsulated ss / dsRNAs are detected by TLR3, 7 and 8, which also activate IRFs and NF-KB transcription factors. Activation of the pathway leads to expression of numerous anti-viral interferons and other cytokines, including IFNP (see Gantier et al. Cytokine Growth Factor Rev. 2007. 18(5- 6): 363-371, incorporated herein by reference).

[0262] Preparations of mRNA therapeutics often posses contaminating RNA species which can trigger the above described interferon response. Uncapped and untailed RNAs will trigger the response, as will contaminating dsRNA in the preparation. Unmodified mRNA produced through IVT may also trigger the response. The activation of the interferon response in a subject administered these therapeutic mRNA preparations can have a deleterious effect on the subject.

[0263] As used herein, a “contaminating RNA species” refers to an RNA molecule that is an undesired product of an in vitro transcription (IVT) reaction.

[0264] In certain embodiments, the at least one mRNA encoding a therapeutic protein is present in a composition comprising one or more contaminating RNA species.

[0265] In certain embodiments, the one or more contaminating RNA species are selected from the group consisting of double stranded RNA (dsRNA), uncapped mRNA species, untailed mRNA species, unmodified mRNA species, and prematurely aborted RNA species.

[0266] As used herein, a “prematurely aborted RNA species” refers to an incomplete product of an mRNA synthesis reaction (e.g., an in vitro synthesis reaction). For a variety of reasons, RNA polymerases do not always complete transcription of a DNA template; i.e., RNA synthesis terminates prematurely. Possible causes of premature termination of RNA synthesis include quality of the DNA template, polymerase terminator sequences for a particular polymerase present in the template, degraded buffers, temperature, depletion of ribonucleotides, and mRNA secondary structures. Prematurely aborted RNA species may be any length that is less than the intended length of the desired transcriptional product. For example, prematurely aborted mRNA species may be less than 1000 bases, less than 500 bases, less than 100 bases, less than 50 bases, less than 40 bases, less than 30 bases, less than 20 bases, less than 15 bases, less than 10 bases or fewer. In certain embodiments, the compositions comprising the mRNA encoding a therapeutic protein comprise about 5% to about 50% contaminating RNA species. In certain embodiments, the compositions comprising the mRNA encoding a therapeutic protein comprise about 50% contaminating RNA species, about 45% contaminating RNA species, about 40% contaminating RNA species, about 35% contaminating RNA species, about 30% contaminating RNA species, about 25% contaminating RNA species, about 20% contaminating RNA species, about 15% contaminating RNA species, about 10% contaminating RNA species, about 5% contaminating RNA species, about 4% contaminating RNA species, about 3% contaminating RNA species, about 2% contaminating RNA species, or about 1% contaminating RNA species. In certain embodiments, the % contaminating RNA species is a w / w %.

[0267] Accordingly, in one aspect of the disclosure, provided herein is a method of inhibiting a mRNA therapeutic-mediated interferon response in a subject, comprising administering to the subject: 1) at least one mRNA encoding a therapeutic protein described herein; and 2) at least one mRNA encoding a viral protein or fragment thereof capable of inhibiting the mRNA therapeutic-mediated interferon response (i.e., an interferon-repressing viral protein or fragment thereof), thereby inhibiting the mRNA therapeutic-mediated interferon response in the subject.

[0268] In certain embodiments, the at least one mRNA encoding the viral protein or fragment thereof and the at least one mRNA encoding the therapeutic protein are administered to the subject simultaneously.

[0269] In other embodiments, the at least one mRNA encoding the viral protein or fragment thereof and the at least one mRNA encoding the therapeutic protein are administered to the subject sequentially. For example, but in no way limiting, the mRNA encoding the viral protein or fragment thereof may be administered to the subject first for a period of time to allow adequate expression of the viral protein or fragment thereof to suppress the interferon response. The mRNA encoding the therapeutic protein may then be administered to the subject.

[0270] In certain embodiments, the inhibiting the mRNA therapeutic-mediated interferon response in the subject comprises a reduction in an interferon relative to a subject that is not administered at least one mRNA encoding the viral protein or fragment thereof. In certain embodiments, the interferon is a type-I interferon. In certain embodiments, the interferon is interferon beta (IFNP). In certain embodiments, the interferon is interferon alpha (IFN-a). In another aspect of the disclosure, provided herein is a method of inhibiting a mRNA therapeutic-mediated interferon response in a subject, comprising administering to the subject at least one mRNA encoding the fusion protein described herein (i.e., a therapeutic protein domain linked to an interferon-repressing viral protein domain or fragment thereof), thereby inhibiting the mRNA therapeutic-mediated interferon response in the subject.

[0271] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0272] EXAMPLES

[0273] EXAMPLE 1 - RABV Phosphoprotein mRNA Inhibits IFNB Induction

[0274] In the process of analyzing in vitro transcribed (IVT) mRNA for quality via an IFNP expression assay, it was surprisingly observed that the mRNA encoding RABV phosphoprotein (RABV P) did not induce detectable IFNP expression. This effect was not observed with mRNA encoding other RABV proteins (i.e., L, G, M, or N), an mRNA encoding an adenosine base editor-eGFP fusion protein (ABE-eGFP), the cocal virus (COCV) glycoprotein protein (GP), or the full length RABV replicon (see FIG. 2). To perform the assay, about 1.44xl04fibroblasts were transfected with 100 ng of one of the recited mRNA molecules. Twenty -four hours after transfection, a Meso Scale Discovery® (MSD) human IFNP S-PLEX® assay was performed to detect IFNP levels.

[0275] Based on this observation, it was hypothesized that viral proteins or fragments thereof (and mRNA encoding the same) that reduce the interferon response in cells could be used to ablate that response in the context of therapeutic mRNA administration to a subject. Such viral proteins include, but are not limited to, rhabdovirus phosphoproteins (e.g., rabies phosphoprotein), paramyxovirus phosphoproteins (also known as P or V proteins), filovirus VP24 or VP35 proteins, or coronavirus NSP1, NSP6, or NSP13 proteins.

[0276] To test this hypothesis, cells were transfected with mRNA encoding an exemplary interferon-repressing viral protein, RABV P. A549 cells were co-transfected with RABV P mRNA and one of either polylC (a known immunostimulant) or dirty mRNA. Dirty mRNA refers to mRNA that is not capped or polyA-tailed. Both polylC and the dirty mRNA induce a foreign RNA sensing response that leads to interferon induction. As shown in FIG. 3, increasing amounts of either polylC or dirty mRNA increased IFNp. When the cells were cotransfected with RABV P mRNA, it completely eliminated IFNP expression.

[0277] This data demonstrates that co-admini strati on of an interferon-repressing viral protein or fragment thereof or a nucleic acid encoding the same can be used in a therapeutic mRNA context. Therapeutic mRNA molecules may have residual uncapped and / or untailed mRNA molecules that may lead to the deleterious effects of an interferon response when administered to a subject. Other components of the IVT reaction (i.e., enzymes, buffer components, etc.) to produce the therapeutic mRNA may remain after purification which can also trigger an interferon response in a subject. Finally, mRNA lacking chemical modifications, such a pseudouridine modification, can also trigger an interferon response in a subject. Coadministration of an interferon-repressing viral protein in the context of unmodified mRNA can also repress the interferon response.

[0278] EXAMPLE 2 - RABV Phosphoprotein Fusions

[0279] Fusion proteins comprising a rhabdovirus phosphoprotein linked to therapeutic protein may be employed to attenuate the mRNA-mediated interferon response. Said fusion proteins are encoded in the mRNA therapeutic and thus in a single mRNA molecule, the therapeutic effect is achieved while simultaneously inhibiting the deleterious effects of the interferon response on the subject.

[0280] As it is the CTD of RABV P that mediates interferon repression, CTD fragments will be linked to other proteins to form exemplary fusion proteins described herein. The CTD fragments include any one of the amino acid sequences set forth in SEQ ID NOs: 28-33.

[0281] Other Embodiments

[0282] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0283] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

Claims

CLAIMSWhat is claimed:

1. A method of inhibiting a mRNA therapeutic-mediated interferon response in a subject, comprising administering to the subject:1) at least one mRNA encoding a therapeutic protein; and2) at least one mRNA encoding a viral protein or fragment thereof capable of inhibiting the mRNA therapeutic-mediated interferon response, thereby inhibiting the mRNA therapeutic-mediated interferon response in the subject.

2. The method of claim 1, wherein the at least one mRNA encoding the therapeutic protein is produced by in vitro transcription (IVT).

3. The method of claim 1 or claim 2, wherein the at least one mRNA encoding the therapeutic protein is not chemically modified.

4. The method of claim 1 or claim 2, wherein the at least one mRNA encoding the therapeutic protein comprises at least one chemically modified nucleotide.

5. The method of any one of claims 1-4, wherein the at least one mRNA encoding the viral protein or fragment thereof is produced by IVT.

6. The method of claim 5, wherein the at least one mRNA encoding the viral protein or fragment thereof is not chemically modified.

7. The method of claim 5, wherein the at least one mRNA encoding the viral protein or fragment thereof comprises at least one chemically modified nucleotide.

8. The method of any one of claims 4-7, wherein the at least one chemically modified nucleotide is pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- m ethylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy -pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, or 2’-O-methyl uridine, or a combination thereof.

9. The method of any one of claims 1-8, wherein the at least one mRNA encoding the viral protein or fragment thereof is administered to the subject before the at least one mRNA encoding the therapeutic protein.

10. The method of any one of claims 1-8, wherein the at least one mRNA encoding the viral protein or fragment thereof and the at least one mRNA encoding the therapeutic protein are administered to the subject simultaneously.

11. The method of any one of claims 1-10, wherein the viral protein or fragment thereof comprises a rhabdovirus phosphoprotein or fragment thereof.

12. The method of claim 11, wherein the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof.

13. The method of claim 12, wherein the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUVV) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof.

14. The method of claim 12 or claim 13, wherein the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof.

15. The method of claim 13 or claim 14, wherein the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45.

16. The method of claim 14, wherein the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD).

17. The method of claim 16, wherein the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

18. The method of claim 12 or claim 13, wherein the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 10.

19. The method of claim 12 or claim 13, wherein the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 12.

20. The method of claim 12 or claim 13, wherein the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 13.

21. The method of claim 12 or claim 13, wherein the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 16.

22. The method of claim 12 or claim 13, wherein the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 17.

23. The method of any one of claims 1-10, wherein the viral protein or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

24. The method of claim 23, wherein the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

25. The method of claim 24, wherein the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof.

26. The method of claim 25, wherein the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO:14.

27. The method of claim 24, wherein the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof.

28. The method of claim 27, wherein the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO:15.

29. The method of claim 24, wherein the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

30. The method of claim 29, wherein the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO:18.

31. The method of any one of claims 1-10, wherein the viral protein or fragment thereof comprises a filoviridae protein or fragment thereof.

32. The method of claim 31, wherein the filoviridae protein or fragment thereof comprises a ebolavirus protein or fragment thereof.

33. The method of claim 32, wherein the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

34. The method of claim 33, wherein the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 11.

35. The method of claim 33, wherein the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO:19.

36. The method of claim 33, wherein the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO:20.

37. The method of claim 33, wherein the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO:21.

38. The method of any one of claims 1-10, wherein the viral protein or fragment thereof comprises a coronaviridae protein or fragment thereof.

39. The method of claim 38, wherein the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

40. The method of claim 39, wherein the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP1 protein or fragment thereof, a betacoronavirus NSP6 protein or fragment thereof, or a betacoronavirus NSP13 protein or fragment thereof.

41. The method of claim 39 or 40, wherein the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP13 protein or fragment thereof.

42. The method of claim 41, wherein the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 24.

43. The method of claim 41, wherein the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 23.

44. The method of claim 41, wherein the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 22.

45. The method of any one of claims 1-10, wherein the viral protein or fragment thereof comprises an orthomyxovidirae protein or fragment thereof.

46. The method of claim 45, wherein the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

47. The method of claim 45, wherein the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

48. The method of claim 47, wherein the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

49. The method of claim 47, wherein the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 27.

50. The method of any one of claims 1-49, wherein the therapeutic protein is selected from the group consisting of an antigen, a nucleic acid editing system or a component thereof, an antigen binding protein or fragment thereof, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and a thrombolytic.

51. The method of claim 50, wherein the antigen comprises a viral antigen or a prokaryotic antigen.

52. The method of claim 50, wherein the nucleic acid editing system or a component thereof comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, or a Transcription Activator-Like Effector-based Nucleases (TALEN).

53. The method of claim 52, wherein the nucleic acid editing system comprises a CRISPR system.

54. The method of claim 53, wherein the CRISPR-system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

55. The method of claim 54, wherein the nucleobase editing domain comprises an adenosine deaminase, cytidine deaminase, or a functional variant thereof.

56. The method of any one of claims 1-55, wherein the mRNA comprises at least one 5’ untranslated region (5’ UTR), at least one 3’ untranslated region (3’ UTR), and / or at least one polyadenylation (poly A) sequence.

57. The method of any one of claims 1-56, wherein one or both of the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof is encapsulated in a lipid nanoparticle (LNP).

58. The method of claim 57, wherein the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in one LNP.

60. The method of claim 57, wherein the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in separate LNPs.

61. The method of any one of claims 57-60, wherein the LNP comprises at least one cationic lipid.

62. The method of claim 61, wherein the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

63. The method of any one of claims 1-62, wherein the at least one mRNA encoding a therapeutic protein is present in a composition comprising one or more contaminating RNA species.

64. The method of claim 63, wherein the one or more contaminating RNA species are selected from the group consisting of double stranded RNA (dsRNA), uncapped mRNA species, untailed mRNA species, unmodified mRNA species, and prematurely aborted RNA species.

65. The method of any one of claims 1-64, wherein inhibiting the mRNA therapeutic- mediated interferon response in the subject comprises a reduction in an interferon relative to a subject that is not administered at least one mRNA encoding a rhabdovirus phosphoprotein or fragment thereof.

66. The method of claim 65, wherein the interferon is interferon beta (IFNP).

67. A composition comprising at least one isolated mRNA encoding a therapeutic protein; and at least one isolated mRNA encoding a viral protein or fragment thereof capable of inhibiting an mRNA therapeutic-mediated interferon response.

68. The composition of claim 67, wherein the at least one mRNA encoding the therapeutic protein is produced by in vitro transcription (IVT).

69. The composition of claim 67 or claim 68, wherein the at least one mRNA encoding the therapeutic protein is not chemically modified.

70. The composition of claim 67 or claim 68, wherein the at least one mRNA encoding the therapeutic protein comprises at least one chemically modified nucleotide.

71. The composition of any one of claims 67-70, wherein the at least one mRNA encoding the viral protein or fragment thereof is produced by IVT.

72. The composition of claim 71, wherein the at least one mRNA encoding the viral protein or fragment thereof is not chemically modified.

73. The composition of claim 71, wherein the at least one mRNA encoding the viral protein or fragment thereof comprises at least one chemically modified nucleotide.

74. The composition of any one of claims 70-73, wherein the at least one chemically modified nucleotide is pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- methylcytosine, 2-thio-l-m ethyl- 1-deaza-pseudouri dine, 2-thio-l-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy -pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, or 2’-O-methyl uridine, or a combination thereof.

75. The composition of any one of claims 67-74, wherein the viral protein or fragment thereof comprises a rhabdovirus phosphoprotein or fragment thereof.

76. The composition of claim 75, wherein the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof.

77. The composition of claim 76, wherein the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUVV) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof.

78. The composition of claim 75 or claim 76, wherein the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof.

79. The composition of claim 77 or claim 78, wherein the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45.

80. The composition of claim 79, wherein the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD).

81. The composition of claim 80, wherein the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

82. The composition of claim 75 or claim 76, wherein the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 10.

83. The composition of claim 75 or claim 76, wherein the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 12.

84. The composition of claim 75 or claim 76, wherein the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 13.

85. The composition of claim 75 or claim 76, wherein the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 16.

86. The composition of claim 75 or claim 76, wherein the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 17.

87. The composition of any one of claims 67-74, wherein the viral protein or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

88. The composition of claim 87, wherein the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

89. The composition of claim 88, wherein the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof.

90. The composition of claim 89, wherein the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 14.

91. The composition of claim 88, wherein the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof.

92. The composition of claim 91, wherein the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 15.

93. The composition of claim 88, wherein the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

94. The composition of claim 93, wherein the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 18.

95. The composition of any one of claims 67-74, wherein the viral protein or fragment thereof comprises a filoviridae protein or fragment thereof.

96. The composition of claim 95, wherein the filoviridae protein or fragment thereof comprises a ebolavirus protein or fragment thereof.

97. The composition of claim 96, wherein the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

98. The composition of claim 97, wherein the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 11.

99. The composition of claim 97, wherein the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 19.

100. The composition of claim 97, wherein the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 20.

101. The composition of claim 97, wherein the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 21.

102. The composition of any one of claims 67-74, wherein the viral protein or fragment thereof comprises a coronaviridae protein or fragment thereof.

103. The composition of claim 102, wherein the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

104. The composition of claim 103, wherein the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP1 protein or fragment thereof, a betacoronavirus NSP6 protein or fragment thereof, or a betacoronavirus NSP13 protein or fragment thereof.

105. The composition of claim 103 or 104, wherein the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP13 protein or fragment thereof.

106. The composition of claim 105, wherein the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 24.

107. The composition of claim 105, wherein the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 23.

108. The composition of claim 105, wherein the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 22.

109. The composition of any one of claims 67-74, wherein the viral protein or fragment thereof comprises an orthomyxovidirae protein or fragment thereof.

110. The composition of claim 109, wherein the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

111. The composition of claim 109, wherein the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

112. The composition of claim 109, wherein the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

113. The composition of claim 109, wherein the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 27.

114. The composition of any one of claims 67-113, wherein the therapeutic protein is selected from the group consisting of an antigen, a nucleic acid editing system or a component thereof, an antigen binding protein or fragment thereof, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and a thrombolytic.

115. The composition of claim 114, wherein the antigen comprises a viral antigen or a prokaryotic antigen.

116. The composition of claim 114, wherein the nucleic acid editing system or a component thereof comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, or a Transcription Activator-Like Effector-based Nucleases (TALEN).

117. The composition of claim 114, wherein the nucleic acid editing system comprises a CRISPR system.

118. The composition of claim 117, wherein the CRISPR-system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

119. The composition of claim 118, wherein the nucleobase editing domain comprises an adenosine deaminase, cytidine deaminase, or a functional variant thereof.

120. The composition of any one of claims 65-117, wherein the mRNA comprises at least one 5’ untranslated region (5’ UTR), at least one 3’ untranslated region (3’ UTR), and / or at least one polyadenylation (poly A) sequence.

121. The composition of any one of claims 67-120, wherein one or both of the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof is encapsulated in a lipid nanoparticle (LNP).

122. The composition of claim 121, wherein the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in one LNP.

123. The composition of claim 121, wherein the mRNA encoding a therapeutic protein and the mRNA encoding a viral protein or fragment thereof are encapsulated in separate LNPs.

124. The composition of any one of claims 121-123, wherein the LNP comprises at least one cationic lipid.

125. The composition of claim 124, wherein the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

126. The composition of any one of claims 67-125, wherein the composition further comprises one or more contaminating RNA species.

127. The composition of claim 126, wherein the one or more contaminating RNA species are selected from the group consisting of double stranded RNA (dsRNA), uncapped mRNAspecies, untailed mRNA species, unmodified mRNA species, and prematurely aborted RNA species.

128. A fusion protein comprising a therapeutic protein domain linked to an interferon- repressing viral protein domain or fragment thereof.

129. The fusion protein of claim 128, wherein the interferon-repressing viral protein domain or fragment thereof comprises a rhabdovirus phosphoprotein or fragment thereof.

130. The fusion protein of claim 129, wherein the rhabdovirus phosphoprotein or fragment thereof comprises a lyssavirus phosphoprotein or fragment thereof.

131. The fusion protein of claim 130, wherein the lyssavirus phosphoprotein or fragment thereof is selected from the group consisting of a rabies virus (RABV) phosphoprotein or fragment thereof, a Lagos bat virus (LBV) phosphoprotein or fragment thereof, a Mokola virus (MOKV) phosphoprotein or fragment thereof, a Duvenhage virus (DUVV) phosphoprotein or fragment thereof, a European bat lyssavirus type 1 (EBLV1) phosphoprotein or fragment thereof, a European bat lyssavirus type 2 (EBLV2) phosphoprotein or fragment thereof, an Australian bat lyssavirus (ABLV) phosphoprotein or fragment thereof, and a Vesicular stomatitis virus (VSV) phosphoprotein or fragment thereof.

132. The fusion protein of claim 130 or claim 131, wherein the lyssavirus phosphoprotein or fragment thereof comprises a RABV phosphoprotein or fragment thereof.

133. The fusion protein of claim 131 or claim 132, wherein the RABV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9 and 28-45.

134. The fusion protein of claim 133, wherein the RABV phosphoprotein fragment comprises a RABV phosphoprotein C terminal domain (CTD).

135. The fusion protein of claim 134, wherein the RABV phosphoprotein CTD comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 28-33.

136. The fusion protein of claim 130 or claim 131, wherein the ABLV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 10.

137. The fusion protein of claim 130 or claim 131, wherein the EBLV1 phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 12.

138. The fusion protein of claim 130 or claim 131, wherein the LBV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 13.

139. The fusion protein of claim 130 or claim 131, wherein the MOKV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 16.

140. The fusion protein of claim 130 or claim 131, wherein the VSV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 17.

141. The fusion protein of claim 128, wherein the interferon-repressing viral protein domain or fragment thereof comprises a paramyxoviridae protein or fragment thereof.

142. The fusion protein of claim 141, wherein the paramyxoviridae protein or fragment thereof comprises a rubulavirus protein or fragment thereof, a morbillivirus protein or fragment thereof, or a respirovirus protein or fragment thereof.

143. The fusion protein of claim 142, wherein the rubulavirus protein or fragment thereof comprises a mapura virus (MPRV) phosphoprotein or fragment thereof.

144. The fusion protein of claim 143, wherein the MPRV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 14.

145. The fusion protein of claim 142, wherein the morbillivirus protein or fragment thereof comprises a measles virus (MV) phosphoprotein or fragment thereof.

146. The fusion protein of claim 145, wherein the MV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 15.

147. The fusion protein of claim 142, wherein the respirovirus protein or fragment thereof comprises a sendai virus (SEV) phosphoprotein or fragment thereof.

148. The fusion protein of claim 147, wherein the SEV phosphoprotein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 18.

149. The fusion protein of claim 128, wherein the interferon-repressing viral protein domain or fragment thereof comprises a filoviridae protein or fragment thereof.

150. The fusion protein of claim 149, wherein the filoviridae protein or fragment thereof comprises an ebolavirus protein or fragment thereof.

151. The fusion protein of claim 150, wherein the ebolavirus protein or fragment thereof comprises a Zaire ebolavirus (ZEBV) VP24 or VP35 protein or fragment thereof, a Sudan ebolavirus (SEBV) VP24 or VP35 protein or fragment thereof, a Tai Forest ebolavirus (TFEBV) VP24 or VP35 protein or fragment thereof, or a Bundibugyo ebolavirus (BEBV) VP24 or VP35 protein or fragment thereof.

152. The fusion protein of claim 151, wherein the ZEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 11.

153. The fusion protein of claim 151, wherein the SEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 19.

154. The fusion protein of claim 151, wherein the TFEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 20.

155. The fusion protein of claim 151, wherein the BEBV VP24 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 21.

156. The fusion protein of claim 128, wherein the interferon-repressing viral protein domain or fragment thereof comprises a coronaviridae protein or fragment thereof.

157. The fusion protein of claim 156, wherein the coronaviridae protein or fragment thereof comprises a betacoronavirus protein or fragment thereof.

158. The fusion protein of claim 157, wherein the betacoronavirus protein or fragment thereof comprises a betacoronavirus NSP1 protein or fragment thereof, a betacoronavirus NSP6 protein or fragment thereof, or a betacoronavirus NSP13 protein or fragment thereof.

159. The fusion protein of claim 157 or 158, wherein the betacoronavirus protein or fragment thereof comprises a SARS-CoV-2 (SCV2) NSP1 protein or fragment thereof, a SCV2 NSP6 protein or fragment thereof, or a SCV2 NSP13 protein or fragment thereof.

160. The fusion protein of claim 159, wherein the SCV2 NSP1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 24.

161. The fusion protein of claim 159, wherein the SCV2 NSP6 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 23.

162. The fusion protein of claim 159, wherein the SCV2 NSP13 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 22.

163. The fusion protein of claim 128, wherein the interferon-repressing viral protein domain or fragment thereof comprises an orthomyxovidirae protein or fragment thereof.

164. The fusion protein of claim 163, wherein the orthomyxovidirae protein or fragment thereof comprises an alphainfluenzavirus protein or fragment thereof or an betainfluenzavirus protein or fragment thereof.

165. The fusion protein of claim 164, wherein the alphainfluenzavirus protein or fragment thereof or the betainfluenzavirus protein or fragment thereof comprises an NS1 protein or fragment thereof.

166. The fusion protein of claim 165, wherein the alphainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

167. The fusion protein of claim 165, wherein the betainfluenzavirus NS1 protein comprises an amino acid sequence with at least about 80% identity to an amino acid sequence set forth in SEQ ID NO: 27.

168. The fusion protein of any one of claims 128-167, wherein the therapeutic protein is selected from the group consisting of an antigen, a nucleic acid editing system or a component thereof, an antigen binding protein or fragment thereof, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and a thrombolytic.

169. The fusion protein of claim 168, wherein the antigen comprises a viral antigen or a prokaryotic antigen.

170. The fusion protein of claim 168, wherein the nucleic acid editing system or a component thereof comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, or a Transcription Activator-Like Effector-based Nucleases (TALEN).

171. The fusion protein of claim 170, wherein the nucleic acid editing system comprises a CRISPR system.

172. The fusion protein of claim 171, wherein the CRISPR-system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

173. The fusion protein of claim 172, wherein the nucleobase editing domain comprises an adenosine deaminase, cytidine deaminase, or a functional variant thereof.

174. A nucleic acid encoding the fusion protein of any one of claims 128-173.

175. The nucleic acid of claim 174, comprising a DNA vector.

176. The nucleic acid of claim 175, wherein the DNA vector comprises a viral vector.

177. The nucleic acid of claim 174, comprising an mRNA.

178. The nucleic acid of claim 177, wherein the mRNA is produced by in vitro transcription (IVT).

179. The nucleic acid of claim 177 or claim 178, wherein the mRNA is not chemically modified.

180. The nucleic acid of claim 177, wherein the at mRNA comprises at least one chemically modified nucleotide.

181. The nucleic acid of claim 180, wherein the at least one chemically modified nucleotide is pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- m ethylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy -pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, or 2’-O-methyl uridine, or a combination thereof.

182. The nucleic acid of any one of claims 177-181, wherein the mRNA comprises at least one 5’ untranslated region (5’ UTR), at least one 3’ untranslated region (3’ UTR), and / or at least one polyadenylation (poly A) sequence.

183. The nucleic acid of any one of claims 177-182, wherein the mRNA is encapsulated in a lipid nanoparticle (LNP).

184. The nucleic acid of claim 183, wherein the LNP comprises at least one cationic lipid.

185. The nucleic acid of claim 184, wherein the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

186. A method of inhibiting a mRNA therapeutic-mediated interferon response in a subject, comprising administering to the subject the fusion protein of any one of claims 128- 173 or the nucleic acid of any one of claims 174-185, thereby inhibiting the mRNA therapeutic-mediated interferon response in the subject.

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