Trans-amplifying RNAS encoding immunomodulators

Abstract

This application relates to trans amplifying RNAs encoding immune modulating proteins, and methods of use thereof for payload expression in cells and subjects.

Description

[0001] TRANS-AMPLIFYING RNAS ENCODING IMMUNOMODULATORS

[0002] FIELD

[0003] This disclosure relates to polynucleotides encoding gene products which, in some aspects, are useful for modulating gene expression and / or reducing host immune responses in cells and subjects.

[0004] RELATED APPLICATIONS

[0005] This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional Application No. 63 / 631,405, filed April 8, 2024, entitled “TRANS-AMPLIFYING RNAS ENCODING IMMUNOMODULATORS” and US Provisional Application No. 63 / 685,819, filed August 22, 2024, entitled “TRANS-AMPLIFYING RNAS ENCODING IMMUNOMODULATORS”, the contents of each of which are hereby incorporated by reference herein in their entirety for all purposes.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (A141470004WO00-SEQ-KGC.xml; Size: 1,357,902 bytes; and Date of Creation: April 4, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Mammalian host immune responses, such as interferon-mediated responses, degrade viral and synthetic polynucleotides and can result in undesired secondary effects, thus posing several major obstacles for ribonucleic acid (RNA)-based therapies. For example, the rapid clearing of foreign polynucleotides by host immune factors typically necessitates high amounts of and / or multiple doses of RNA to generate an effective dose for expression of payloads, thereby increasing production costs and development time. High dosages of RNA have also been shown to induce adverse secondary effects in subjects, such as inflammation and systemic reactions.

[0010] SUMMARY OF INVENTION

[0011] Described herein are trans amplifying RNAs and self-amplifying RNAs encoding, in some aspects, gene products which reduce inflammatory markers and / or improve expression of pay loads. Provided herein, in some aspects, is a trans-amplifying ribonucleic acid (RNA) (taRNA) comprising: a first RNA polynucleotide (e.g., replicase construct) comprising a nucleic acid encoding a replicase; and a second RNA polynucleotide (e.g., trans replicon construct (trRNA)) comprising a nucleic acid payload or a nucleic acid encoding a payload; and a nucleic acid encoding an immune modulating protein (IMP). In some embodiments, the first RNA polynucleotide (e.g., replicase construct) comprises a nucleic acid encoding a replicase and a nucleic acid encoding an IMP.

[0012] In some embodiments, the IMP is a viral immune evasion protein(s) (VIEP). In some embodiments, the VIEP is from vaccinia virus (VACV), herpes simplex virus (HSV), influenza virus, Toscana Virus (TOSV), or encephalomyocarditis virus (EMCV). In some embodiments, the VIEP is a VACV soluble IFN alpha / beta receptor B 18 (B18R), VACV RNA-binding protein E3 (E3L), VACV F1L, VACV NIL, HSV infected cell protein 34.5 (ICP34.5), HSV unique short 1 (US1), HSV unique short 11 (US 11), influenza virus non- structural protein 1 (NS1), TOSV non-structural (NSs) protein, or EMCV Lpro. In some embodiments, the NS1 is from Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) (PR8 NS1). In some embodiments, the VIEP comprises an amino acid sequence of any one of SEQ ID NOs: 229-238 or 245.

[0013] In some embodiments, the IMP antagonizes a mammalian antiviral factor(s). In some embodiments, the IMP that antagonizes a mammalian antiviral factor is dominant negative mitochondrial antiviral- signaling protein (dnMAVS), microprotein in antiviral immunity 1 (MA VII), dominant negative protein kinase R (dnPKR), suppressor of cytokine signaling (SOCS), or dominant negative Zinc-finger Antiviral Protein (dnZAP). In some embodiments, the dnZAP is rat ZAP with cysteine-to-arginine mutation at position 88 (rZAPC88R). In some embodiments, the SOCS is SOCS1 or SOCS3. In some embodiments, the mammalian antiviral factor comprises an amino acid sequence of any one of SEQ ID NOs: 239-244.

[0014] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises nucleic acid(s) encoding a first IMP and a second IMP. In some embodiments, the first IMP and / or the second IMP is B18, E3L, F1L, PR8 NS1, dnPKR, SOCS1, rZAPCC88r, or Lpro. In some embodiments, the first IMP and the second IMP are joined by a linker. In some embodiments, the linker is a nucleic acid encoding a 2A peptide, a 3 / 4J, a subgenomic promoter (SGP) or an internal ribosome entry site (IRES) element. In some embodiments, the 2A peptide is a 2A peptide selected from thosea asigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), equine rhinitis A virus 2A (E2A), or Furin-2A. In some embodiments, the IRES element is an IRES from Coxsackievirus B3 (CVB3), encephalomyocarditis virus (EMCV), porcine kobuvirus

[0015] (PKV), Manhattan Parechovirus (MPV), or tortoise rafivirus (TraV).

[0016] In some embodiments, the first IMP is E3L, the second IMP is B18R and the 2A peptide is P2A. In some embodiments, the first IMP is E3L, the second IMP is dnPKR, and the 2A peptide is P2A. In some embodiments, the first IMP is E3L, the second IMP is F1L, and the 2A peptide is P2A. In some embodiments, the first IMP is E3L, the second IMP is PR8 NS1, and the 2A peptide is P2A. In some embodiments, the first IMP is E3L, the second IMP is SOCS1, and the 2A peptide is P2A. In some embodiments, the first IMP is E3L, the second IMP is SOCS1, and the 2A peptide is T2A. In some embodiments, the first IMP is SOCS1, the second IMP is dnPKR, and the 2A peptide is P2A. In some embodiments, the first IMP is SOCS1, the second IMP is F1L, and the 2A peptide is P2A. In some embodiments, the first IMP is SOCS1, the second IMP is PR8 NS1, and the 2A peptide is P2A. In some embodiments, the first IMP is SOCS1, the second IMP is rZAPCC88r, and the 2A peptide is P2A.

[0017] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises a nucleic acid(s) encoding a first IMP, a second IMP, and a third IMP. In some embodiments, the third IMP is selected from E3L, SOCS1, and F1L. In some embodiments, the third IMP is joined to the first IMP and / or the second IMP by a linker. In some embodiments, the first IMP is E3L, the second IMP is SOCS1, and the third IMP is F1L.

[0018] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises nucleic acid(s) encoding a first IMP, a second IMP, a third IMP fourth IMP, fifth IMP, and / or sixth IMP. In some embodiments, the adjacent IMPs are joined by a linker. In some embodiments, one or two IMPs are joined to the nucleic acid payload or nucleic acid encoding a payload by a linker.

[0019] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises a 5’ untranslated region (UTR) and a 3’ UTR flanking the nucleic acid encoding the IMP(s) and the nucleic acid encoding the payload or the nucleic acid payload. In some embodiments, the 5’ UTR comprises a conserved sequence element (CSE) which is cognate to the replicase. In some embodiments, the 5’ UTR is a SINV 5’ UTR or SFV 5’ UTR. In some embodiments, the 3’ UTR comprises a CSE which is cognate to the replicase. In some embodiments, the 3’ UTR is a SINV 3’ UTR or SFV 3’ UTR.

[0020] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises a 3’ terminal polyadenylation (poly A) tail. In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises, from 5’ to 3’: i. the nucleic acid encoding the IMP; ii. a linker; and iii. the nucleic acid encoding a payload or nucleic acid payload.

[0021] In some embodiments, the IMP is E3L or SOCS1. In some embodiments, the linker is a CVB3 IRES element or a nucleic acid encoding T2A. In some embodiments, i-iii of the second RNA polynucleotide (e.g., trRNA) are flanked by a 5’ UTR and a 3’ UTR.

[0022] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises, from 5’ to 3’: i. the nucleic acid encoding a payload; ii. a linker; and iii. the nucleic acid encoding the IMP.

[0023] In some embodiments, the IMP is E3L, SOCS1, or PR8 NS1. In some embodiments, the linker is CVB3 IRES element or T2A. In some embodiments, i-iii of the second RNA polynucleotide (e.g., trRNA) are flanked by a 5’ UTR and a 3’ UTR.

[0024] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises a sequence of any one of SEQ ID NOs: 177-220.

[0025] In some embodiments, a payload comprises an antigen.

[0026] In some embodiments, a payload comprises a reporter. In some embodiments, the reporter is SEAP.

[0027] Provided herein, in some aspects, is a taRNA comprising a first RNA polynucleotide (e.g., replicase construct) comprising a nucleic acid encoding a replicase and a nucleic acid encoding an IMP and a second RNA polynucleotide (e.g., trRNA) comprising a nucleic acid payload or a nucleic acid encoding a payload. In some embodiments, a replicase comprises an IMP between a third and fourth domain of the replicase. In some embodiments, the first RNA polynucleotide (e.g., replicase construct) of the taRNA comprises a linker between a nucleic acid encoding a replicase and a nucleic acid encoding an IMP.

[0028] In some embodiments, the IMP is a VIEP. In some embodiments, the VIEP is VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, or TOSV NS. In some embodiments, the IMP antagonizes a mammalian antiviral factor. In some embodiments, the IMP that antagonizes a mammalian antiviral factor is dnMAVS, MA VII, dnPKR, SOCS, dnZAP, or EMCV Lpro. In some embodiments, the linker is a 2A peptide, a 3 / 4J, or an IRES element.

[0029] In some embodiments, the first RNA polynucleotide (e.g., replicase construct) of the taRNA comprises, from 5’ to 3’, a nucleic acid encoding a replicase, a linker, and a nucleic acid encoding an IMP. In some embodiments, the first RNA polynucleotide (e.g., replicase construct) comprises, from 5’ to 3’, a nucleic acid encoding an IMP, a linker, and a nucleic acid encoding a replicase. In some embodiments, the IMP is E3L, dnPKR, or SOCS1. In some embodiments, the linker is a nucleic acid encoding a 2A peptide.

[0030] In some embodiments, the replicase is a Semliki Forest virus (SFV) replicase or a variant thereof.

[0031] In some embodiments, the nucleic acid encoding the replicase comprises the sequence set forth in SEQ ID NO: 129 or SEQ ID NO: 130.

[0032] In some embodiments, the first RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 148-176.

[0033] Provided herein, in some aspects, is a taRNA comprising a first RNA polynucleotide (e.g., replicase construct) comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (e.g., trRNA) comprising a nucleic acid payload or a nucleic acid encoding a payload, and a nucleic acid encoding a second IMP.

[0034] In some embodiments, the first IMP is SOCS1, and the second IMP is E3L. In some embodiments, the first IMP is E3L, and the second IMP is SOCS1. In some embodiments, the first IMP is dnPKR and the second IMP is F1L.

[0035] In some aspects, a trans-amplifying ribonucleic acid (RNA) (taRNA) comprises:

[0036] (a) a first RNA polynucleotide comprising:

[0037] (i) a nucleic acid encoding a replicase; and

[0038] (b) a second RNA polynucleotide comprising:

[0039] (i) a nucleic acid payload or a nucleic acid encoding a payload; and

[0040] (ii) a nucleic acid encoding a first immune modulating protein (IMP), optionally wherein the second RNA polynucleotide comprises: a second IMP, a third IMP, a fourth IMP, a fifth IMP and / or a sixth IMP, optionally wherein adjacent IMPs in the second RNA polynucleotide are joined by a linker.

[0041] In some embodiments, the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, ICPO, PR8 NS1, TOSV NS, dnMAVS, MA VII, dnPKR, SOCS, dnZAP, and EMCV Lpro.

[0042] In some aspects, a trans-amplifying ribonucleic acid (RNA) (taRNA) comprises:

[0043] (a) a first RNA polynucleotide comprising:

[0044] (i) a nucleic acid encoding a replicase; and

[0045] (b) a second RNA polynucleotide comprising, from 5’ to 3’: 5’-CSEl-IMP 1-L1-IMP2-

[0046] L2-IMP3-L3-PCS-L4-IMP4-L5-IMP5-L6-IMP6-CSE2-3’ ; wherein:

[0047] CSE1 and CSE2 are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0048] IMP1, IMP2, IMP3, IMP4, IMP5, and IMP6 are each independently an immune modulating protein (IMP);

[0049] LI, L2, L3, L4, L5, and L6 are each independently a linker;

[0050] PCS is a nucleic acid pay load or a nucleic acid encoding a payload; and

[0051] IMP2-L2, IMP3-L3, L4-IMP4, L5-IMP5, and L6-IMP6 are each independently optionally absent.

[0052] In some aspects, a trans-amplifying ribonucleic acid (RNA) (taRNA) comprises:

[0053] (a) a first RNA polynucleotide comprising:

[0054] (i) a nucleic acid encoding a replicase; and

[0055] (b) a second RNA polynucleotide comprising, from 5’ to 3’: 5’-CSEl-IMPl-Ll-IMP2-

[0056] L2-IMP3-L3-PCS-L4-IMP4-L5-IMP5-L6-IMP6-CSE2-3’ ; wherein:

[0057] CSE1 and CSE2 are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0058] IMP1, IMP2, IMP3, IMP4, IMP5, and IMP6 are each independently an immune modulating protein (IMP);

[0059] LI, L2, L3, L4, L5, and L6 are each independently a linker;

[0060] PCS is a nucleic acid pay load or a nucleic acid encoding a payload; and

[0061] IMP1-L1, IMP2-L2, IMP3-L3, L5-IMP5, and L6-IMP6 are each independently optionally absent.

[0062] In some aspects, a trans-amplifying ribonucleic acid (RNA) (taRNA) comprises:

[0063] (a) a first RNA polynucleotide comprising:

[0064] (i) a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’: 5’-CSEl-IMPl-Ll-IMP2- L2-IMP3-L3-PCS-L4-IMP4-CSE2-3’ ; wherein:

[0065] CSE1 and CSE2 are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0066] IMP1, IMP2, IMP3, and IMP4 are each independently an immune modulating protein (IMP);

[0067] LI, L2, L3, and L4 are each independently a linker;

[0068] PCS is a nucleic acid pay load or a nucleic acid encoding a payload; and IMP2-L2, IMP3-L3, and L4-IMP4 are each independently optionally absent.

[0069] In some aspects, a trans-amplifying ribonucleic acid (RNA) (taRNA) comprises:

[0070] (a) a first RNA polynucleotide comprising:

[0071] (i) a nucleic acid encoding a replicase; and

[0072] (b) a second RNA polynucleotide comprising, from 5’ to 3’: 5’-CSEl-IMPl-Ll-PCS- IMP2-L2-IMP3-L3-L4-IMP4-CSE2-3’ ; wherein:

[0073] CSE1 and CSE2 are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0074] IMP1, IMP2, IMP3, and IMP4 are each independently an immune modulating protein (IMP);

[0075] LI, L2, L3, and L4 are each independently a linker;

[0076] PCS is a nucleic acid pay load or a nucleic acid encoding a payload; and

[0077] IMP1-L1, IMP3-L3, and L4-IMP4 are each independently optionally absent.

[0078] Provided herein, in some aspects, is a composition comprising a deoxyribonucleic acid (DNA) polynucleotide that can be transcribed to produce the second RNA polynucleotide (e.g., a trRNA) of any taRNA described herein. In some embodiments, the composition comprises an additional DNA polynucleotide that can be transcribed to produce the first RNA polynucleotide of any taRNA described herein.

[0079] Provided herein, in some aspects, is a cell comprising any taRNA described herein. In some embodiments, a payload is expressed in the cell via a method comprising transfecting a cell with a taRNA described herein, or a composition comprising a DNA polynucleotide encoding a taRNA described herein.

[0080] In some embodiments, the cell is in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Provided herein, in some aspects, is a fusion protein comprising, from N-terminal to C- Terminal, a replicase and a protein or peptide.

[0081] In some embodiments, the replicase and the protein or peptide are joined by a 2A peptide. In some embodiments, the protein or peptide comprises an IMP. In some embodiments, the protein or peptide comprises a first IMP, a second IMP and / or a third IMP.

[0082] Provided herein, in some aspects, is a fusion protein comprising, from N-terminal to C- terminal, a first protein or peptide, a replicase, and a second protein or peptide. In some embodiments, the first protein or peptide comprises a first immune modulating protein (IMP). In some embodiments, the second protein or peptide comprises a second IMP. In some embodiments, the first protein or peptide comprises a first IMP and the second protein or peptide comprises a second IMP. In some embodiments, the first IMP comprises an Influenza A / Puerto Rico / 8 / 34 virus non- structural protein 1 (PR8 NS1) protein and the second IMP comprises VACV RNA-binding protein E3 (E3L). In some embodiments, the first IMP comprises a VACV RNA-binding protein E3 (E3L), and the second IMP comprises an Influenza A / Puerto Rico / 8 / 34 virus non- structural protein 1 (PR8 NS1) protein.

[0083] In some embodiments, a DNA polynucleotide encodes a fusion protein described herein.

[0084] In some embodiments, an RNA polynucleotide encodes a fusion protein described herein.

[0085] Provided herein, in some aspects, is a self-amplifying RNA (saRNA) encoding an IMP.

[0086] In some embodiments, a saRNA comprises:

[0087] (i) a nucleic acid encoding, from 5’ to 3’, a replicase, a 2A peptide, and a first immune modulating protein (IMP); and

[0088] (ii) a nucleic acid payload or a nucleic acid encoding a payload.

[0089] In some embodiments, a saRNA comprises:

[0090] (i) a nucleic acid encoding, from 5’ to 3’, a first immune modulating protein (IMP), a 2A peptide, and a replicase; and

[0091] (ii) a nucleic acid payload or a nucleic acid encoding a payload.

[0092] In some embodiments, a saRNA encodes a second IMP.

[0093] In some embodiments, the nucleic acid payload or nucleic acid encoding a payload are joined by a nucleic acid sequence encoding a 2A peptide. BRIEF DESCRIPTION OF DRAWINGS

[0094] FIG. 1 shows fold-expression of a taRNA-encoded secreted alkaline phosphatase (SEAP) in type I interferon receptor knockout (IFNAR7') mice relative to wildtype (WT) mice. Adult mice were injected with taRNAs encoding a wildtype replicase, SEAP and serum was collected at 8, 24, and 72 hours. RLU = relative luminometer units.

[0095] FIGs. 2A-2B show expression and ISG54 induction of a taRNA-encoded SEAP in Raw macrophages. Cells were transfected with 5.5ng, 22ng, or 44ng of total RNA consisting of a taRNA including replicase construct encoding a wildtype replicase (WT Rep) or mutated replicase (RLE Rep) and a trRNA encoding SEAP; and an mRNA encoding Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) NS1 or SOCS1. Expression of SEAP in transfected cell supernatant was assessed 24 hours after transfection.

[0096] FIG. 2A shows fold-expression of SEAP 24 hours after transfection, normalized to matched replicase (WT Rep or RLE Rep) and dose.

[0097] FIG. 2B shows fold ISG54 induction 24 hours after transfection, normalized to untreated Raw macrophages.

[0098] FIGS. 3A-3B show expression of taRNA-encoded SEAP in Raw macrophages co-transfected with SOCS1 -encoding mRNA. Cells were transfected with 5ng of taRNA including a taRNA encoding a wildtype replicase and a trRNA encoding SEAP; 5ng of taRNA including a replicase construct encoding a wildtype replicase, a trRNA encoding SEAP, and 0.5ng, 5ng, or lOng SOCS1 mRNA; or 5ng mRNA encoding SEAP.

[0099] FIG. 3A shows expression of SEAP at 6, 24, 48, and 72 hours after transfection.

[0100] FIG. 3B shows IP- 10 chemokine levels vs SOCS1 expression at 24 hours.

[0101] FIG. 4A-4D show a proof-of-concept experiment in which cells were transfected with two proteins encoded by a single trRNA.

[0102] FIG. 4A is a schematic showing an exemplary trRNA including, from 5’ to 3’, a 5’ conserved sequence element (CSE), a first protein coding sequence (PCS 1), a nucleic acid encoding a linker, a second protein coding sequence (PCS 2), a 3’ CSE, and a polyadenylation (poly A) tail. FIG. 4B shows expression of green fluorescent protein (GFP) and / or mCherry in BHK-21 cells transfected with a taRNA including a replicase construct encoding a wildtype Semliki Forest Virus (SFV) replicase, and a trRNA encoding one or two fluorophores: a trRNA encoding green fluorescent protein (GFP) only (GFP trRNA); a trRNA encoding mCherry only (mCherry trRNA); a GFP trRNA and a mCherry trRNA; or a trRNA encoding GFP and mCherry joined by a T2A peptide linker (GFP-T2A-mCherry trRNA). Expression of each fluorophore is shown as fold-expression over cells transfected only with a replicase construct (Naive).

[0103] FIG. 4C shows representative comparative images of GFP, mCherry, or GFP and mCherry expression in the BHK-21 cells of FIG. 4B.

[0104] FIG. 4D shows representative images GFP, mCherry, and GFP and mCherry expression of BHK-21 cells transfected with GFP-T2A-mCherry trRNA only.

[0105] FIGS. 5A-5B show fold-expression of mCherry in BHK-21 cells, where expression is driven by sub-genomic promoters (SGP) or internal ribosome entry site (IRES) elements in a trRNA.

[0106] FIG. 5A shows fold-expression of mCherry in BHK-21 cells transfected with a wildtype replicase encoded by an mRNA or a self-amplifying RNA (saRNA); and a trRNA encoding mCherry downstream of a variable SGP derived from Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEV), Ross River virus (RRV), Semliki Forest virus (SFV), Sindbis virus (SINV), or Western equine encephalitis virus (WEEV). SGPs are referenced by their length (in nt) relative to the transcriptional start site (e.g., -100 / 50 spans 150 nt, and starts 100 nt upstream of a start site and continues to 50 nt downstream of the start site). Cells were transfected with one of the following trRNA: a trRNA encoding mCherry only (STR- mCherry), a trRNA encoding GFP and mCherry (STR-GFP+mCherry), a trRNA encoding GFP-T2A- mCherry (STR-GFP-T2A-mCherry), a trRNA encoding GFP-CHIKV SGP-mCherry (CHIKV- 75 / 69), a trRNA encoding GFP-EEV SGP-mCherry (EEV-84 / 58), a trRNA encoding GFP-RRV SGP-mCherry (RRV-75 / 48), a trRNA encoding GFP-RRV SGP-mCherry with a Kozak sequence in the RRV SGP (RRV-75 / 48_Kozak), a trRNA encoding GFP-SFV SGP-mCherry with variable SFV promoters (SFV-37 / 51; SFV-50 / 0; SFV-50 / 51, SFV- 100 / 0, SFV- 100 / 51, SFV-150 / 0, SFV-150 / 51, SFV 200 / 0, and SFV-200 / 51), a trRNA encoding GFP-SFV SGP- mCherry with a Kozak sequence in the SFV SGP (SFV-66 / 52_Kozak, SFV-200 / 51_Kozak), a trRNA encoding GFP-SFV SGP / El-mCherry (SFV_El-100 / -99 / 45), a trRNA encoding GFP- SFV SGP / El-mCherry with a Kozak sequence in the SFV SGP (SFV_El-100 / -99 / 45_Kozak), a trRNA encoding GFP-SINV SGP-mCherry (SINV-75 / 49), and a trRNA encoding GFP- WEEV SGP-mCherry (WEEV-84 / 37).

[0107] FIG. 5B shows fold-expression of GFP and mCherry in BHK-21 cells transfected with a replicase construct encoding a wildtype SFV replicase; and a trRNA encoding mCherry downstream of a variable IRES element derived from Aichivirus (iAichi), Coxsackievirus B3 (iCVB3), encephalomyocarditis virus (iEMCV), enterovirus 71 (iEV71), or human rhinovirus B3 (iiHRV-B3). Cells were transfected with one of the following trRNA: a trRNA encoding mCherry only (STR-GFP), a trRNA encoding GFP and mCherry (STR-mCherry), a trRNA encoding GFP and mCherry (STR-GFP+mCherry), a trRNA encoding GFP-T2A-mCherry (STR-GFP-T2A-mCherry), a trRNA encoding GFP-iAichi-mCherry (iAichi), a trRNA encoding GFP-iCVB3-mCherry (iCVB3), a trRNA encoding GFP-iEMCV-mCherry (iEMCV), a trRNA encoding GFP-iEV71 (iEV71), a trRNA encoding GFP-iHRV-B3 (iiHRV-B3-#3, iiHRV-B3, iiHRV-B3-#7, iiHRV-B3-#9, iiHRV-B3-Aptv6). As additional controls, cells were transfected with only mRNA encoding GFP (GFP mRNA) or only mRNA encoding mCherry (mCherry mRNA).

[0108] FIGs. 6A-6D are schematics of exemplary trRNA constructs with a payload coding nucleic acid sequence (PCS) and one or more immune modulating protein (IMP) coding sequences in different configurations. Dashed lines indicate optional nucleic acid sequences.

[0109] FIG. 6A is a schematic of an exemplary trRNA comprising a PCS and at least one and as many as six IMP-coding nucleic acid sequences (IMP1-IMP6) The pictured trRNA comprises, from 5’ to 3’, a 5’ conserved sequence element (CSE), a first IMP-coding sequence (IMP1), a first linker (El), an optional second IMP-coding sequence (IMP2), an optional second linker (E2), an optional third IMP-coding sequence (IMP3), an optional third linker (E3), a PCS, an optional fourth IMP-coding sequence (IMP4), an optional fourth linker (E4), an optional fifth IMP- coding sequence (IMP5), an optional fifth linker (E5), an optional sixth IMP-coding sequence (IMP6), an optional sixth linker (E6), and a 3’ CSE.

[0110] FIG. 6B is a schematic of another exemplary trRNA comprising a PCS at least one and as many as six IMP-coding nucleic acid sequences. The pictured trRNA comprises, from 5’ to 3’, a 5’ CSE, an optional first IMP-coding sequence (IMP1), an optional first linker (LI), an optional second IMP-coding sequence (IMP2), an optional second linker (L2), an optional third IMP- coding sequence (IMP3), an optional third linker (L3), a PCS, a fourth IMP-coding sequence (IMP4), a fourth linker (L4), an optional fifth IMP-coding sequence (IMP5), an optional fifth linker (L5), an optional sixth IMP-coding sequence (IMP6), an optional sixth linker (L6), and a 3’ CSE.

[0111] FIG. 6C is a schematic of an exemplary trRNA comprising a PCS and at least one and as many as four IMP-coding nucleic acid sequences (IMP1-IMP4). The pictured trRNA comprises, from 5’ to 3’, a 5’ CSE, a first IMP-coding sequence (IMP1), a first linker (LI), an optional second IMP-coding sequence (IMP2), an optional second linker (L2), an optional third IMP-coding sequence (IMP3), an optional third linker (L3), a PCS, an optional fourth IMP-coding sequence (IMP4), an optional fourth linker (L4), and a 3’ CSE.

[0112] FIG. 6D is a schematic of another exemplary trRNA comprising a PCS and at least one and as many as four IMP-coding nucleic acid sequences (IMP1-IMP4). The pictured trRNA comprises, from 5’ to 3’, a 5’ CSE, an optional first IMP-coding sequence (IMP1), an optional first linker (LI), a PCS, a second IMP-coding sequence (IMP2), a second linker (L2), an optional third IMP-coding sequence (IMP3), an optional third linker (L3), a PCS, an optional fourth IMP- coding sequence (IMP4), an optional fourth linker (L4), and a 3’ CSE.

[0113] FIGs. 7A-7B are schematics of exemplary trRNA constructs with a payload coding nucleic acid sequence (PCS) and one immune modulating protein (IMP) coding nucleic acid sequence.

[0114] FIG. 7A is a schematic of an exemplary trRNA as in FIG. 6A or FIG. 6C, without optional IMP-coding sequences; this “IMP-linker-PCS” trRNA would comprise, from 5’ to 3’, a 5’ CSE, an IMP-coding sequence, a linker, a PCS, and a 3’ CSE.

[0115] FIG. 7B is a schematic of an exemplary trRNA as in FIG. 6B (top) or FIG. 6D (bottom), without optional IMP-coding sequences; this “PCS-linker-IMP” trRNA would comprise, from 5’ to 3’, a 5’ CSE, a PCS, a linker, an IMP, and a 3’ CSE.

[0116] FIGs. 8A-8H show in vitro examples of taRNA including trRNA encoding a payload, an IRES linker, and an immune modulating protein (IMP) in a configuration- specific manner.

[0117] FIG. 8A shows ISG54 induction in Raw macrophages transfected with 5.5ng of taRNA including: a replicase construct encoding a wildtype (WT) replicase or RLE replicase and one of three trRNAs according to FIG. 7A (IMP-linker-PCS): a trRNA encoding SEAP (oeSTR) only; or a SOCS1-IRES-SEAP trRNA, a NS1-IRES-SEAP trRNA, or a E3L-IRES-SEAP trRNA. ISG54 induction was measured 24 hours after transfection (normalized to untreated Raw macrophages).

[0118] FIG. 8B shows SEAP expression in 3T3 cells or Raw macrophages transfected with 5ng of taRNA including a replicase construct encoding a wildtype SFV (WT) replicase or RLE replicase, and one of: a trRNA encoding SEAP only (oestrRNA), a trRNA encoding SEAP and a Golden Gate cloning sequence (GGS), a SEAP-EGFP trRNA, or one of six trRNAs according to FIG. 7B (PCS-linker-IMP): SEAP-CVB3-E3L, SEAP-CVB3-SOCS1, SEAP-CVB3-dnPKR, SEAP-CVB3-PR8 NS1, SEAP-CVB3-F1L, or SEAP-CVB3-rZAPC88R. FIG. 8C shows ISG54 fold-induction in the Raw macrophages of FIG. 8B at 24 hours post transfection relative to untreated Raw cells.

[0119] FIGs. 8D shows SEAP expression in 3T3 cells or Raw macrophages transfected with 5ng of taRNA including a replicase construct encoding a wildtype SFV replicase (WT) or RLE replicase, and a trRNA encoding: SEAP only (oestrRNA); E3L-CVB3-SEAP; SEAP-CVB3- E3L; SEAP-CVB3-SOCS1; and SEAP-CVB3-PR8 NS1.

[0120] FIG. 8E shows ISG54 fold-induction in the Raw macrophages of FIG. 8D at 24 hours post transfection relative to untreated Raw cells (cells only).

[0121] FIG. 8F shows SEAP expression in 3T3 cells transfected with 5ng of taRNA including a replicase construct encoding a wildtype SFV replicase (WT) or RLE replicase, and a trRNA encoding: SEAP only (oestrRNA); E3L-CVB3-SEAP; SEAP-CVB3-E3L; SEAP-CVB3-ICP0; SEAP-CVB3-ICP34.5; SEAP-CVB3-US11; and SEAP-CVB3-US1. Expression is shown as fold-change over Naive (cells treated with transfection agents, but no RNA).

[0122] FIG. 8G shows SEAP expression in Raw macrophages transfected with 5ng of taRNA as in FIG. 8F.

[0123] FIG. 8H shows ISG54 fold-induction in the Raw macrophages of FIG. 8G at 24 hours post transfection. Induction is shown as fold-induction over Naive.

[0124] FIGs. 9A-9C show in vivo examples of taRNA including trRNA encoding a payload, an IRES linker, and an immune modulating protein (IMP) in different configurations.

[0125] FIG. 9A shows SEAP expression at different time points in the serum of adult wildtype mice treated with Ipg RNA total of: a taRNA including a replicase construct encoding a wildtype SFV (WT) replicase and a E3L-CVB3-SEAP trRNA (WT / oeE3L-IRES-SEAP); a taRNA including a replicase construct encoding a SFV WT replicase and a trRNA encoding SEAP only (WT / oeSTR); or an mRNA encoding SEAP only (mRNA).

[0126] FIG. 9B shows SEAP expression at different time points in the serum of adult wildtype mice treated with Ipg RNA total of: a taRNA including a replicase construct encoding a wildtype SFV (WT) replicase and a NS1-IRES-SEAP trRNA (WT / oeNSl-CVB3-SEAP); a taRNA consisting of a replicase construct encoding a WT replicase and a E3L-CVB3-SEAP trRNA (WT / oeE3L-CVB3-SEAP); a taRNA including a replicase construct encoding a WT replicase and a trRNA encoding SEAP only (WT / oeSTR); or an mRNA encoding SEAP only (mRNA).

[0127] FIG. 9C shows SEAP expression in sera of wildtype mice treated with: Ipg of taRNA including a replicase construct encoding a wildtype SFV replicase (WT) or RLE replicase (RLE), and an IMP-linker-payload trRNA encoding E3L-CVB3-SEAP (oeE3L-IRES-SEAP); or an mRNA encoding SEAP only (SEAP). Sera was collected at 8, 24, 72, 120, and 168 hours. Expression is shown relative to the serum of untreated mice (RLU).

[0128] FIG. 10 shows expression of SEAP in the sera of adult wildtype mice treated with taRNA including PCS-linker-IMP trRNAs (as in FIG. 7B). Mice were treated with Ipg of taRNA including a replicase construct encoding a wildtype SFV replicase (WT) or RLE replicase (RLE), and a trRNA encoding SEAP-T2A-E3L; or an mRNA encoding SEAP only. Sera was collected at 8, 24, 72, 120, and 168 hours.

[0129] FIGs. 11A-11C show effects of transfection of 3T3 cells or Raw macrophages with: no RNA (Control); Naive; or 5ng of taRNA including a replicase construct encoding a wildtype SFV (WT) replicase or RLE replicase (RLE) and one of six trRNAs: an E3L-CVB3-SEAP trRNA; a SOCS1-CVB3-SEAP trRNA; a SEAP-T2A-SOCS1 trRNA; a SOCS1-T2A-SEAP trRNA; a SEAP-T2A-E3L trRNA; or an E3L-T2A-SEAP trRNA.

[0130] FIG. 11A shows fold-change expression of SEAP in 3T3 cells and Raw cells at 6 hours after transfection.

[0131] FIG. 11B shows fold-change expression of SEAP in Raw cells at 24 hours after transfection. FIG. 11C shows ISG54 induction in Raw cells at 24 hours.

[0132] FIGs. 12A-12B show comparisons of SEAP expression in sera of adult wildtype mice treated with taRNA including configuration- specific trRNA constructs, at 8, 24, 72, 120, and 168 hours after transfection.

[0133] FIG. 12A shows SEAP expression in sera of wildtype mice treated with: Ipg of: taRNA including a replicase construct encoding a wildtype SFV replicase (WT) and an IMP-linker-PCS trRNA (as in FIG. 7A) encoding NS1-CVB3-SEAP (oeNSl-IRES-SEAP) or a PCS-linker-IMP trRNA (as in FIG. 7B) encoding SEAP-CVB3-NS1 (oeSEAP-IRES-NSl); or an mRNA encoding SEAP only.

[0134] FIG. 12B shows SEAP expression in sera of wildtype mice treated with: Ipg of: taRNA including a replicase construct encoding a wildtype replicase (WT) and an IMP-linker-PCS (FIG. 7A) trRNA encoding E3L-CVB3-SEAP (oeE3L-IRES-SEAP) or a PCS-linker-IMP trRNA (FIG. 7B) encoding SEAP-CVB3-E3L (oeSEAP-IRES-E3L); or an mRNA encoding SEAP only. FIGs. 13A-13B are schematics of exemplary trRNA constructs with a payload coding nucleic acid sequence (PCS) and two immune modulating protein (IMP) coding nucleic acid sequence in different configurations.

[0135] FIG. 13A shows an exemplary trRNA as in FIG. 6A or FIG. 6C, with one optional IMP-coding sequence included; this “IMP-linker-IMP-linker-PCS” trRNA comprises, from 5’ to 3’, a 5’ conserved sequence element (CSE), a first IMP coding sequence, a first linker, a second IMP coding sequence, a second linker, a PCS, and a 3’ CSE.

[0136] FIG. 13B shows an exemplary trRNA as in FIG. 6B (top) or FIG. 6D (bottom), with one optional IMP-coding sequence included. These “PCS-linker-IMP-linker-IMP” trRNAs comprise, from 5’ to 3’: a 5’ conserved sequence element (CSE), a PCS, a fourth IMP coding sequence, a fourth linker, a fifth IMP coding sequence, and a 3’ CSE (top); or a 5’ conserved sequence element (CSE), a PCS, a second linker, a second IMP coding sequence, a third linker, a third IMP coding sequence, and a 3’ CSE (bottom).

[0137] FIG. 14 shows expression of SEAP in Raw macrophages transfected with 44ng, 22ng, or 5.5ng of taRNA including: a replicase construct encoding a wildtype SFV (WT) replicase and a trRNA encoding SEAP only; a replicase construct encoding a mutant (RLE) replicase and a trRNA encoding SEAP only; or a replicase construct encoding a WT replicase or RLE replicase and one of three trRNAs: a trRNA encoding SOCS1 (SOCS), a trRNA encoding SEAP and dnPKR, or a trRNA encoding SOCS1 and dnPKR. Expression of SEAP was assessed at 24 hours after transfection and is shown relative to taRNA having a trRNA encoding SEAP only.

[0138] FIG. 15 shows expression of SEAP in 3T3 cells or Raw macrophages with: 1 Ing of taRNA including a replicase construct encoding a wildtype SFV (WT) replicase or RLE replicase and one of five trRNAs: a trRNA encoding SEAP only; a B18-P2A-E3L-CVB3-SEAP trRNA (B18- P2A-E3L-IRES-SEAP); an E3L-CVB3-SEAP trRNA (E3L-IRES-SEAP); an NS1-CVB3-SEAP trRNA (NSl-IREs-SEAP); or a SOCS1-CVB3-SEAP trRNA (SOCS1-IRES-SEAP).

[0139] FIGs. 16A-16B are schematics of exemplary trRNA constructs with a payload coding nucleic acid sequence (PCS) and three immune modulating protein (IMP) coding nucleic acid sequence in different configurations.

[0140] FIG. 16A shows an exemplary trRNA as in FIG. 6A or FIG. 6C, with two optional IMP-coding sequence included; this “IMP-linker-IMP-linker-IMP-linker-PCS” trRNA comprises, from 5’ to 3’, a 5’ conserved sequence element (CSE), a first IMP coding sequence, a first linker, a second IMP coding sequence, a second linker, a third IMP coding sequence, a third linker, a PCS, and a

[0141] 3’ CSE.

[0142] FIG. 16B shows an exemplary trRNA as in FIG. 6B (top) or FIG. 6D (bottom), with two optional IMP-coding sequence included. These “PCS-linker-IMP-linker-IMP-linker-IMP” trRNAs comprise, from 5’ to 3’: a 5’ conserved sequence element (CSE), a PCS, a fourth linker, a fourth IMP coding sequence, a fifth linker, a fifth IMP coding sequence, a sixth linker, a sixth IMP coding sequence, and a 3’ CSE (top); or a 5’ conserved sequence element (CSE), a PCS, a second linker, a second IMP coding sequence, a third linker, a third IMP coding sequence, a fourth linker, a fourth IMP coding sequence, and a 3’ CSE (bottom).

[0143] FIGs. 17A-17H show expression and ISG54 induction of trRNA constructs encoding 1-3 IMPs. FIG. 17A shows expression of SEAP in 3T3 cells transfected with a replicase construct encoding a wildtype SFV replicase (WT replicase) and one of seven trRNAs: a trRNA encoding SEAP only (oeSTR); an E3L-CVB3-SEAP trRNA; a SEAP-T2A-E3L; a SEAP-T2A-E3L-P2A- SOCS1 trRNA; a SEAP-T2A-SOCS1-P2A-E3L trRNA; or a SEAP-T2A-E3L-P2A-SOCS1- E2A-F1L trRNA. Expression was measured at 6, 24, 48, and 72 hours after transfection. FIG. 17B shows expression of SEAP in 3T3 cells transfected with a replicase construct encoding a RLE replicase and one of seven trRNAs: a trRNA encoding SEAP only (oeSTR); an E3L-CVB3-SEAP trRNA; a SEAP-T2A-E3L; a SEAP-T2A-E3L-P2A-SOCS1 trRNA; a SEAP- T2A-SOCS1-P2A-E3L trRNA; or a SEAP-T2A-E3L-P2A-SOCS1-E2A-F1L trRNA. Expression was measured at 6, 24, 48, and 72 hours after transfection.

[0144] FIG. 17C shows expression of SEAP in Raw macrophages transfected with a replicase construct encoding a wildtype SFV replicase (WT replicase) and one of seven trRNAs: a trRNA encoding SEAP only (oeSTR); an E3L-CVB3-SEAP trRNA; a SEAP-T2A-E3L; a SEAP-T2A-E3L-P2A- SOCS1 trRNA; a SEAP-T2A-SOCS1-P2A-E3L trRNA; or a SEAP-T2A-E3L-P2A-SOCS1- E2A-F1L trRNA. Expression was measured at 6, 24, 48, and 72 hours after transfection.

[0145] FIG. 17D shows expression of SEAP in Raw macrophages transfected with a replicase construct encoding a RLE replicase and one of seven trRNAs: a trRNA encoding SEAP only (oeSTR); an E3L-CVB3-SEAP trRNA; a SEAP-T2A-E3L; a SEAP-T2A-E3L-P2A-SOCS1 trRNA; a SEAP- T2A-SOCS1-P2A-E3L trRNA; or a SEAP-T2A-E3L-P2A-SOCS1-E2A-F1L trRNA. Expression was measured at 6, 24, 48, and 72 hours after transfection. FIG. 17E shows induction of ISG54 in the Raw macrophages of FIGs. 17C and 17D at 24 hours. Induction is shown as fold-change over Naive (cells treated with transfection agents, but no RNA).

[0146] FIG. 17F shows expression of SEAP in 3T3 cells transfected with a replicase construct encoding a wildtype SFV replicase (WT) or RLE replicase (RLE) and one of twelve trRNAs: a trRNA encoding SEAP only (oeSTR); a SEAP-CVB3-SOCS1 trRNA; a SEAP-CVBE-SOCS1- P2A-E3L trRNA; a SEAP-CVB3-SOCSl-P2A-dnPKR trRNA; a SEAP-CVB3-SOCS1-P2A- NS1 trRNA; a SOCS1-CVB3-SEAP trRNA; a SOCS1-P2A-E3L-CVB3-SEAP trRNA; a SOCSl-P2A-dnPKR-CVB3-SEAP trRNA; a SOCS-P2A-NS1-CVB3-SEAP trRNA; a SOCS1- P2A-F1L-CVB3-SEAP trRNA; a SEAP-T2A-E3L-PS1A-SOCS1 trRNA; a SEAP-T2A-SOCS1- P2A-E3L trRNA; or a SEAP-T2A-E3L-P2A-SOCS1-E2A-F1L trRNA. Expression was measured 24 hours after transfection.

[0147] FIG. 17G shows expression of SEAP in Raw macrophages transfected with a replicase construct encoding a wildtype SFV replicase (WT) or RLE replicase (RLE) and one of twelve trRNAs: a trRNA encoding SEAP only (oeSTR); a SEAP-CVB3-SOCS1 trRNA; a SEAP- CVBE-SOCS1-P2A-E3L trRNA; a SEAP-CVB3-SOCSl-P2A-dnPKR trRNA; a SEAP-CVB3- SOCS1-P2A-NS1 trRNA; a SOCS1-CVB3-SEAP trRNA; a SOCS1-P2A-E3L-CVB3-SEAP trRNA; a SOCSl-P2A-dnPKR-CVB3-SEAP trRNA; a SOCS-P2A-NS1-CVB3-SEAP trRNA; a SOCS1-P2A-F1L-CVB3-SEAP trRNA; a SEAP-T2A-E3L-PS1A-SOCS1 trRNA; a SEAP-T2A- SOCS1-P2A-E3L trRNA; or a SEAP-T2A-E3L-P2A-SOCS1-E2A-F1L trRNA. Expression was measured 24 hours after transfection.

[0148] FIG. 17H shows induction of ISG54 in the Raw macrophages of FIG. 17G at 24 hours. Induction is shown as fold-change over Naive (cells treated with transfection agents, but no RNA).

[0149] FIGs. 18A-18D are schematics of an alphavirus replicase fusion proteins.

[0150] FIG. 18A is a schematic of a “replicase-3 / 4J-protein” fusion protein, including a protein inserted between a third domain (nsP3) and fourth domain (nsP4) of an alphavirus replicase.

[0151] FIG. 18B is a schematic of a “replicase-linker-protein” fusion protein, including an alphavirus replicase joined at the N-terminus to a different protein by a linker (L).

[0152] FIG. 18C is a schematic of a “protein-linker-replicase” fusion protein, including an alphavirus replicase joined at the C-terminus to a different protein by a linker (L). FIG. 18D is a schematic of a “protein-linker-replicase-linker-protein” fusion protein, including an alphavirus replicase joined at the C-terminus to a first protein (protein 1) by a first linker (LI) and joined at the N-terminus to a second protein (protein 2) by a second linker (L2).

[0153] FIGs. 19A-19D are schematics of polynucleotides encoding alphavirus replicase fusion proteins comprising IMPs.

[0154] FIG. 19A shows an exemplary replicase construct comprising, from 5’ to 3’, a 5’ UTR comprising a conserved sequence element (CSE), a nucleic acid encoding a replicase-3 / 4J- IMP fusion protein (as in FIG. 18A), and a 3’ UTR comprising a CSE.

[0155] FIG. 19B shows an exemplary replicase construct including, from 5’ to 3’, a 5’CSE, a nucleic acid encoding a replicase-linker-IMP fusion protein (as in FIG. 18B), and a 3’ CSE.

[0156] FIG. 19C shows an exemplary replicase construct including, from 5’ to 3’, a 5’CSE, a nucleic acid encoding an IMP-linker-replicase fusion protein (as in FIG. 18C), and a 3’ CSE.

[0157] FIG. 19D shows an exemplary replicase construct including, from 5’ to 3’, a 5’CSE, a nucleic acid encoding an IMP-linker-replicase-linker-IMP fusion protein (as in FIG. 18D), and a 3’ CSE.

[0158] FIGs. 20A-20D show examples of taRNA including replicase constructs encoding replicase- IMP fusion proteins.

[0159] FIG. 20A shows expression of SEAP in 3T3 and Raw macrophages, 24 hours after transfection with a taRNA including: a replicase construct encoding wildtype SFV (WT) replicase, a mutant (RLE) replicase, a replicase-3 / 4J-IMP fusion protein (WT-3 / 4J-dnPKR), or a replicase-linker- IMP fusion protein (WT-T2A-E3L or WT-T2A-S0CS1); and a trRNA encoding SEAP only (oeSTR), an E3L-CVB3-SEAP trRNA, a SEAP-CVB3-E3L trRNA, a SEAP-CVB3-SOCS1 trRNA, a SEAP-CVB3-PR8 NS1 trRNA, or a SEAP-CVB3-F1L trRNA. Expression is shown as fold change over Naive (cells treated with transfection agents, but no RNA). Horizonal dashed lines indicate taRNA control expression in 3T3 or Raw cells treated with a control taRNA including a replicase construct encoding a WT replicase and an oeSTR trRNA (no IMP).

[0160] FIG. 20B shows ISG54 induction in Raw macrophages 24 hours after transfection with taRNA according to FIG 20A. Raw cells were transfected with replicase constructs encoding WT, RLE, WT-T2A-S0CS1, WT-T2A-E3L, or WT-3 / 4J-dnPKR; and oeSTR trRNA, a SEAP-CVB3-F1L trRNA (SEAP-IRES-F1L), a SEAP-CVB3-NS1 trRNA (SEAP-IRES-F1L), an E3L-CVB3- SEAP trRNA (E3L-IRES-SEAP), or a SEAP-CVB3-SOCS1 trRNA (SEAP-IRES-SOCS1). ISG54 induction is shown relative to Naive (cells treated with transfection agents, but no RNA). FIG. 20C shows SEAP expression in 3T3 and Raw macrophages 24 hours after transfection with taRNA including one of five replicase constructs: a replicase construct encoding a wildtype replicase (WT), a replicase construct encoding a wildtype replicase-FIL fusion protein (WT- T2A-F1L), a replicase construct encoding a RLE replicase (RLE), a replicase construct encoding a RLE replicase-SOCSl fusion protein (RLE-T2A-SOCS1), or a replicase construct encoding a RLE replicase-E3L fusion protein (RLE-T2A-E3L); and one of three trRNAs: a trRNA encoding SEAP only (oeSTR), a SEAP-T2A-E3L trRNA, and an E3L-IRES-SEAP trRNA. Horizonal dashed lines indicate taRNA control expression in 3T3 or Raw cells treated with a control taRNA including a replicase construct encoding a WT replicase and an oeSTR trRNA (no IMP).

[0161] FIG. 20D shows ISG54 induction in the Raw macrophages of FIG. 20C.

[0162] FIG. 21 shows expression of SEAP in BJ fibroblasts, 24 hours after transfection with a taRNA including: a replicase construct encoding wildtype SFV (WT) replicase, a mutant (RLE) replicase, a PR8 NS1-P2A-WT-T2A-VACV E3L fusion protein (NS1-WT-E3L), WT-T2A-PR8 NS1 fusion protein, PR8 NS1-T2A-WT fusion protein, WT-T2A-T0SV NSs (WT-Tosc NSs) fusion protein, VACV E3L-P2A-WT-T2A-VACV K3L (E3L-WT-K3L), VACV K3L-P2A-WT- -T2A-VACV E3L (K3L-WT-E3L), or WT-T2A-VACV E3L (WT-E3L); and a trRNA encoding SEAP only (oeSTR) or VACV E3L-CVB3-SEAP (E3L-IRES-SEAP) trRNA. Expression is shown as fold change over Naive (cells treated with transfection agents, but no RNA).

[0163] DETAILED DESCRIPTION OF INVENTION

[0164] In some aspects, this disclosure describes a trans-amplifying ribonucleic acid (RNA) (taRNA) comprising: (i) a first RNA polynucleotide comprising a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (ii) a second RNA polynucleotide comprising a nucleic acid payload or a nucleic acid encoding a payload, and a nucleic acid encoding an immune modulating protein (IMP). trans -Amplifying Ribonucleic Acids (taRNAs)

[0165] A “trans-amplifying RNA,” hereinafter referred to as “taRNA,” comprises a first and second RNA polynucleotide, wherein the first RNA polynucleotide (i.e., the replicase construct) encodes a replicase, wherein the second RNA polynucleotide (i.e., the trRNA) comprises a conserved sequence element (CSE) cognate to the replicase, and a nucleic acid payload or nucleic acids encoding a payload, and wherein the second RNA polynucleotide can be replicated by the encoded replicase in trans. taRNAs do not consist of a single polynucleotide that comprises both the first RNA polynucleotide (i.e., the replicase construct) and the second RNA polynucleotide (i.e., the trRNA).

[0166] A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A), uridine (U) / deoxythymidine (T), guanosine / deoxyguanosine (G), cytidine / deoxycytidine (C)) and joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications. In some sequences described herein, T / U is used to denote a particular nucleotide may be a T or U depending on whether the polynucleotide is an RNA polynucleotide (U) or a DNA polynucleotide (T).

[0167] The first and second RNA polynucleotides of a taRNA are separate polynucleotides (i.e., separate molecules which are not a single continuous strand of RNA). The terms “replicase construct” and “trans replicon” (trRNA) construct are used synonymously herein to refer to the first and second RNA polynucleotides of a taRNA, respectively. As used herein, a “construct” refers to an artificial (i.e., not naturally occurring) polynucleotide.

[0168] Replicase Constructs

[0169] In the context of taRNAs, a “replicase construct” (i.e., the first RNA polynucleotide) refers to an mRNA which comprises nucleic acids encoding a replicase (e.g., an alphavirus replicase) and which does not comprise nucleic acids encoding the payload of the taRNA . In some embodiments, the replicase construct is a non-replicating mRNA. As used herein, the term “non-replicating mRNA” refers to an mRNA which is processed for translation into a gene product or else degraded, and which does not self-replicate. Once introduced to an environment comprising translational machinery (such as a cell), replicase constructs can be translated to generate the encoded replicase. In some embodiments, a replicase construct is comprised in a replicating mRNA. As used herein, the term “replicating mRNA” refers to an mRNA molecule which is processed for translation into a gene product, or else degraded, and which is capable of replicating itself; for example, a self- amplifying RNA (saRNA). A saRNA, unlike a taRNA, comprises a CSE, a nucleic acid encoding a payload or a nucleic acid payload, and nucleic acids encoding a replicase (e.g., a replicase construct) in a single polynucleotide. saRNAs are known, e.g., as described in Comes JDG et al., Trends Biotechnol. 2023 Nov;41(ll): 1417-1429.

[0170] A “replicase” is an RNA-dependent RNA polymerase capable of transcribing (i.e., reading) an RNA template to produce an RNA (e.g., trRNA). A replicase construct may encode a modified replicase from an RNA virus, for example, an alphavirus. The term “alphavirus” refers to an RNA virus belonging to the Togaviridae family. Alphaviruses generally comprise a singlestranded RNA genome encoding at least nsPl, nsP2, nsP3, nsP4, El, E2, E3, 6K / TF and capsid proteins. An alphavirus may be any alphavirus known in the art; non-limiting examples include Semliki Forest virus (SFV), Sindbis virus SINV), Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). In some embodiments, a taRNA comprises a replicase construct encoding an alphavirus replicase. Typically, an alphaviral replicase comprises a complex formed by the non-structural proteins nsPl, nsP2, nsP3, and nsP4. Thus, a nucleic acid encoding an alphaviral replicase (also referred to herein as a “replicase-coding sequence”) will be understood to encode at least nsPl, nsP2, nsP3, nsP4, and variants thereof.

[0171] In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Semliki Forest virus (SFV) (SFV replicase). In some embodiments, a wildtype SFV replicase comprises the amino acid sequence set forth in SEQ ID NO: 227. In some embodiments, the SFV replicase is a REE replicase. A RLE replicase is an SFV replicase variant comprising (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 227; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 227; and (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 227. In some embodiments, the RLE replicase comprises an amino acid sequence of SEQ ID NO: 228 (also referred to as an RLE replicase). In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Sindbis virus (SINV) (SINV replicase). Once expressed, an alphaviral replicase may interact with an RNA polynucleotide comprising one or more conserved sequence elements (CSEs) which are cognate to the replicase and generate mirrored copies of the RNA polynucleotide, which can be subsequently translated (e.g., by a host cell).

[0172] A “conserved sequence element (CSE),” refers to a recognition site for an alphavirus replicase. Typically, a CSE functions as a core promoter or enhancer for initiation of replication of a downstream sequence, such that a 5 ’-CSE may initiate synthesis of a plus-strand and a 3’- CSE may initiate synthesis of a minus-strand. A polynucleotide may comprise one or more 5’- CSEs and / or 3’-CSEs. In some embodiments, a CSE forms one or more secondary structure, such as one or more stem-loops. Non-limiting examples of CSEs include CSE1, CSE2, CSE3, CSE4, and variants or derivatives thereof. Replicase constructs of taRNA necessarily do not comprise a CSE; thus, once the replicase of a replicase construct is translated, the encoded replicase cannot replicate the replicase construct.

[0173] In some embodiments, a replicase construct comprises one or more untranslated regions. An “untranslated region,” hereinafter referred to as “UTR,” is a region in a polynucleotide which may be transcribed, but which is not translated into a gene product. UTRs may function as stabilizing elements and / or provide regulation of transcription of a gene or transgene. Typically, UTRs are found upstream and / or downstream of a gene or transgene. A UTR located directly upstream of a start codon operably linked to a gene or transgene is referred to herein as a 5’- UTR. As a skilled artisan will understand, 5 ’-UTRs may comprise sequence elements which play roles in regulation of expression (e.g., Kozak sequences) or structural elements which alter stability of the molecule (e.g., 5’ cap structures). A UTR located directly downstream of a stop codon operably linked to a gene or transgene is referred to herein as a 3 ’-UTR. 3 ’-UTRs may comprise structural elements which alter the stability of a construct and / or provide transcriptional control, including, but not limited to AU-rich elements and polyadenylation (poly A) tails. A variety of 5 ’-UTRs and a 3 ’-UTRs are known to those of ordinary skill in the art. UTRs may be naturally occurring or synthetic. In some embodiments, a polynucleotide comprises a 5 ’-UTR and / or a 3 ’-UTR.

[0174] In some embodiments, a replicase construct comprises a 5 ’-UTR. In some embodiments, a replicase construct comprises a 3’-UTR. In some embodiments, a replicase construct comprises a 5 ’-UTR and a 3 ’-UTR. In some embodiments, a replicase construct comprises a 5’- UTR derived from human alpha-globin (5’-HAG-UTR). An exemplary 5’-HAG-UTR is provided in SEQ ID NO: 111. In some embodiments, a replicase construct comprises a 3’-UTR derived from human alpha-globin (3’-HAG-UTR). An exemplary 3’-HAG-UTR is provided in SEQ ID NO: 115. In some embodiments, a replicase construct comprises a 5’-HAG-UTR and a 3’-HAG-UTR. trRNA

[0175] The term “trans replicon construct” (i.e., second RNA polynucleotide), also known as a “trans replicating RNA,” hereinafter referred to as “trRNA” refers to an RNA polynucleotide capable of being replicated by a replicase of a taRNA. A trRNA comprises at least one (e.g., 1, 2, 3, 4, 5, 6) nucleic acid encoding a payload or a nucleic acid payload (i.e., a payload-encoding sequence”) operably linked to one or more CSEs. The trRNA does not comprise the replicase that amplifies the trRNA. The simultaneous expression of a replicase (e.g., a replicase encoded by a replicase construct) and presence of a trRNA comprising a CSE cognate to the replicase in a cell can thus result in amplification of the trRNA and its cargo (e.g., a nucleic acid encoding a pay load or a nucleic acid pay load).

[0176] As used herein, a “payload” refers to one or more gene products of interest for delivery to or expression by an organism. A payload may be a functional nucleic acid (e.g., RNA), a protein, a peptide or protein fragment, or a fusion protein.

[0177] In some embodiments, a payload is a selectable marker. As used herein, a “selectable marker” is a peptide or protein that can be used to screen cells by artificial selection. Nonlimiting examples of selectable markers include antibiotic resistance proteins (e.g., ampicillin, puromycin); surface markers (e.g., surface proteins, such as low-affinity nerve growth factor receptor) capable of binding with a substrate for visual and / or physical separation (e.g., antibodies, microbeads, magnetic substrates); and negative selection markers (e.g., thymidine kinase).

[0178] In some embodiments, a payload is a reporter. A “reporter” is a peptide or protein which alters the appearance of a cell such that cells can be visually or optically screened for presence or absence of the peptide or protein. In some embodiments, a reporter is an enzyme which alters the appearance of a cell, such as beta-galactosidase. In some embodiments, a reporter is a peptide or peptide fragment (e.g., secreted embryonic alkaline phosphatase (SEAP)) which can be detected in combination with additional reagents (e.g., assay-specific media). In some embodiments, a reporter is a fluorophore, such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or any derivative thereof. In some embodiments, a reporter is a recombinase, such as a Flp recombinase, Cre recombinase, or derivative thereof.

[0179] In some embodiments, a payload is “therapeutic payload,” here referring to a gene product useful for treating or preventing a disease or disorder. In some embodiments, a therapeutic payload knocks down, knocks in, increases, inhibits, or otherwise modulates gene expression. In some embodiments, a therapeutic payload replaces or edits an endogenous gene or gene product. In some embodiments, the payload does not encode a protein. In some embodiments, a therapeutic payload is a functional RNA; non-limiting examples of include short hairpin RNA (shRNA), microRNA (miRNA), artificial microRNA (amiRNA), small interfering RNA (siRNA), or circular RNA (circRNA).

[0180] In some embodiments, a therapeutic payload is a protein. A therapeutic protein may replace or interfere with activity of deficient or absent endogenous proteins; augment activity of existing metabolic or synthetic pathways; provide a novel function or activity; or interfere with the activity of a pathogen or toxic molecule. Non-limiting examples of therapeutic proteins suitable for use as payloads include membrane proteins, membrane- associated proteins, secreted proteins, intracellular proteins, antigens, antibodies or fragments thereof (e.g., an antibody heavy chain and / or an antibody light chain), antigen binding fragments of an antibody (e.g., Fab, single chain Fv (scFv)), fragments thereof. For example, a therapeutic protein may be an antigen of a pathogen, e.g., for use in vaccination. In some embodiments, the antigen is a viral antigen or bacterial antigen. In some embodiments, a payload is a nuclease. In some embodiments, a payload is a genome editing enzyme. In some embodiments, a genome editing enzyme comprises a CRISPR associated enzyme (Cas). In some embodiments, a Cas protein is from a CRISPR type I system, a CRISPR type II system, or a CRISPR type III system. In some embodiments, a Cas protein is an RNA cleaving Cas protein. In some embodiments, a Cas protein is an DNA cleaving Cas protein. In some embodiments, a Cas protein is a nickase. In some embodiments, a Cas protein is a Cas9, Cas 10, Cas 11 or Cas 12 protein. In some embodiments, a genome editing enzyme is a base editor (e.g., a cytosine base editor or an adenosine base editor). In some embodiments, a genome editing enzyme is a prime editor. In some embodiments, a genome editing enzyme is a Talen protein. In some embodiments, a pay load encodes an enzyme and comprises nucleic acids, that when transcribed, produces a corresponding guide RNA (e.g., a CRISPR guide RNA, a prime editing RNA) for a therapeutic target gene or transcript (e.g., targeting an oncogene). trRNA constructs comprise one or more CSEs. In some embodiments, a trRNA comprises one or more CSEs, wherein the CSEs are present in one or more UTRs. In some embodiments, a trRNA comprises a 5’-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 3’-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5’-UTR having one or more CSEs, a nucleic acid pay load or nucleic acids encoding a pay load, and a 3’-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5’-UTR having one or more CSEs, a nucleic acid pay load or nucleic acids encoding a pay load, and a 3’-UTR having one or more CSEs. In some embodiments, a trRNA comprises UTRs having one or more CSEs, wherein the UTRs are derived from one or more alphaviruses. A skilled artisan will appreciate that UTRs derived from alphaviruses comprise one or more CSEs unless stated otherwise. In some embodiments, a trRNA comprises a 5’-UTR derived from a first alphavirus and a 3’-UTR derived from a second alphavirus. In some embodiments, a trRNA comprises a 3’-UTR comprising one or more repeat sequence elements (RSE).

[0181] In some embodiments, a trRNA comprises a 5’-UTR and / or 3’-UTR derived from a Semliki Forest virus (SFV), hereinafter referred to as a “SFV-UTR”. In some embodiments, a trRNA comprises a 5’-UTR and / or 3’-UTR derived from a Sindbis virus, hereinafter referred to as a “SINV-UTR”. In some embodiments, a trRNA comprises a 5’-UTR and 3’-UTR from the same virus. Non-limiting examples include a trRNA comprising a 5’-UTR derived from a SINV (5’ -SINV-UTR), and a 3’-UTR derived from a SINV (3’-SINV-UTR) or a trRNA comprising a 5’-UTR derived from a SFV (5’-SFV-UTR), and a 3’-UTR derived from a SFV (3’-SFV-UTR). In some embodiments, a trRNA comprises a 5’-UTR and 3’-UTR from different viruses. Nonlimiting examples include a trRNA comprising a 5 ’-SINV-UTR and a 3 ’-SFV-UTR. In some embodiments, a 5’-SINV-UTR comprises the sequence set forth in any one of SEQ ID NOs: 112-114. In some embodiments, a 3’-SINV-UTR comprises the sequence set forth in SEQ ID

[0182] 116. In some embodiments, a 3’-SFV-UTR comprises the sequence set forth in SEQ ID NO:

[0183] 117.

[0184] In some embodiments, a trRNA comprises a 5’ UTR having an “oeSTR” extension. An oeSTR extension is a sequence of 9 nucleotides in length (AGAAGAUGG) inserted into the 5’ terminal of a 5’ UTR. The term “5’ terminal of a 5’ UTR” refers to nucleotides at the 5’ end of the 5’ SINV UTR. The 5’ terminal of the wildtype 5’ SINV UTR comprises six conserved nucleotides AUCGGC. The 5’ extension described herein is inserted between the AU and CGGC (e.g., AUAGAAGAUGGCGGC (SEQ ID NO: 322), as shown in SEQ ID NO: 117).

[0185] In some embodiments, a taRNA comprises a compatible or cognate replicase construct and trRNA construct. A replicase construct and trRNA construct are considered “compatible” or “cognate” when a trRNA comprises a CSE to which the replicase encoded by the replicase construct can bind, such that the trans replicon is replicated. In some embodiments, a compatible replicase construct and CSE are derived from the same alphavirus. In some embodiments, a replicase derived from a SFV is capable of binding to (e.g., is capable of replicating) a trRNA comprising a CSE from an SFV. In some embodiments, a replicase derived from a SINV is capable of binding to (e.g., is capable of replicating) a trRNA comprising a CSE from an SINV. In some embodiments, a compatible replicase construct and CSE are derived from different alphaviruses. In some embodiments, a replicase derived from a SFV is capable of binding to (e.g., is capable of replicating) a trRNA comprising a CSE from a SINV. In some embodiments, a replicase derived from a SINV is capable of binding to (e.g., is capable of replicating) a trRNA comprising a CSE from an SFV.

[0186] Immune Modulating Proteins (IMPs)

[0187] In some embodiments, a trRNA construct encodes one or more immune modulating protein. As used herein, the term “immune modulating protein,” referred to hereinafter as IMP, refers to a protein, peptide or protein fragment, or fusion protein, which interferes with a mammalian innate immune response (IIR) pathway. Innate immune response (IIR) pathways comprise numerous signaling pathways by which cells recognize, activate, and ultimately respond to non-specific danger molecules in the cytosol or in extracellular space. IIRs comprise cellular and host organism responses to non-specific danger molecules. Danger molecules, also known as alarmins and danger signals, are molecular warning signals which are not highly specific to a particular antigen, but which generally signal infection by pathogens, cell injury / damage, or cell death (e.g., by necrosis / necroptosis). Danger molecules include, but are not limited to, pathogen-associated molecular patterns (PAMPs), damage- associated molecular patterns (DAMPs), non- self-nucleic acids, and foreign substances. A “non-self” nucleic acid refers to any nucleic acid which is not part of a given host cell, for example, a nucleic acid of a pathogen (e.g., virus, bacteria), a synthetic nucleic acid, or certain nucleic acids of other cells of the same organism.

[0188] A “pathway,” as used herein, is a molecular process by which a cell detects and responds to a signal. A pathway comprises at least a receptor of a signal and an effector of a response to the signal, which may be comprised in one or more molecules. Molecular signals, also referred to herein as “ligands,” bind and activate protein-based receptors. Once a receptor interacts with a signal, one or more molecular events may occur which enable a response to the signal. Nonlimiting examples of responses to a signal include changes to gene expression, changes to cell cycle stage, degradation / sequestering of a molecule, changes to membrane properties (e.g., voltage, permeability), movement of the cell, recruitment of immune cells and / or molecules to a particular location. In some embodiments, IIRs are inflammatory or apoptotic. Inflammatory IIR pathways comprise several mechanisms by which non-specific immune cells quickly damage, destroy, or otherwise neutralize pathogens, molecular danger signals (e.g., non-self-nucleic acids), and injured host tissue. Inflammatory responses are typically rapid and self-limiting, and thus require both pro-inflammatory pathways (i.e., pathways which trigger an inflammatory response) and anti-inflammatory pathways (i.e., pathways which suppress or eliminate an inflammatory response. Notably, inflammatory responses differ from adaptive immune responses, which comprise mechanisms by which pathogen- specific lymphocytes precisely target a particular pathogen. Adaptive immune responses are highly effective and specific (i.e., do not target normal, healthy host tissue), but are a much slower response than inflammatory responses. Apoptosis is a type of programmed cell death which occurs in response to intrinsic or extrinsic signaling pathways. Apoptosis can occur in response to detection of danger molecules (e.g., non- self-nucleic acids). During viral infection, for example, programmed death of an infected cell can greatly reduce release of progeny virus. Apoptosis can also suppress inflammatory responses by releasing anti-inflammatory molecules.

[0189] An IMP “interferes” with an IIR pathway when the presence of the IMP affects the induction or activity of the pathway such that the outcome of the pathway (e.g., inflammatory response, apoptotic response) is reduced, suppressed, prevented, or reversed compared to the outcome of the pathway in the absence of the IMP. Similarly, an IMP “interferes” with a particular molecule when the presence of the IMP reduces the quantity, availability, or function of the molecule relative to the same in the absence of the IMP. For example, an IMP can interfere with the response of an IIR pathway such that the immune response of a cell to a non- self-nucleic acid is reduced, suppressed, prevented, or reversed compared to the response of the cell to the non-self-nucleic acid in the absence of the IMP. The ability of an IMP to interfere with a molecule of pathway may be direct (e.g., by direct contact with a molecule) or indirect (e.g., by contact with a first pathway which regulates a second pathway). For example, an IMP may interfere with an IIR pathway by directly antagonizing a molecule associated with the pathway and / or by competing with a molecule associated with the pathway.

[0190] In some embodiments, an IMP that interferes with an IIR-pathway associated molecule is an antagonist of the IIR pathway-associated molecule. An “antagonist,” as used herein, refers to a molecule which blocks or dampens binding activity of a protein (e.g., enzyme, receptor) ; nonlimiting examples of antagonists include competitive antagonists, allosteric antagonists, partial agonists, and inverse agonists. A “competitive antagonist” is a molecule which can bind to the active site of a target protein, and which competes with endogenous ligands, such that other molecules compete with the competitive antagonist to bind the same target protein. An “allosteric antagonist” is a molecule which binds to an allosteric site of a target protein and indirectly block binding of endogenous ligands to the active site (e.g., by preventing a conformational change required for activation, or by inducing a conformational change of the active site). A “partial agonist” is a molecule which partially activate (i.e., activate to a reduced level compared to an endogenous ligand) a target protein, such that activity of the target protein is reduced. An “inverse agonist” is a molecule which binds to a target protein and alters its natural activity, for example, by triggering an alternative pathway or reversing its activity. Antagonists may be reversible or irreversible.

[0191] In some embodiments, an IMP that interferes with an IIR pathway-associated protein competes with the IIR pathway-associated protein. An IMP can be understood to “compete” with a wildtype protein when it perturbs the normal activity of the protein by interacting with ligands of the wildtype protein, such that the ligands are less available for binding to the wildtype protein. For example, an IMP may be a dominant negative mutant (e.g., variant) of a protein. Dominant negative mutants, also referred to herein as “dn” proteins, are competitive mutants which comprise genetic mutations that render the protein partially functional or non-functional compared to a wildtype version of the same protein. Dominant negative mutants can bind ligands of the wildtype protein but are less effective (or ineffective) in propagating pathways associated with the wildtype protein.

[0192] Mammalian Antiviral Factors

[0193] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) a mammalian antiviral factor. “Antiviral factors” are molecules which mediate IIR pathways related to the suppression of viral infection, elimination of viral proteins, degradation of non- self-nucleic acids, and / or clearance of viral infections from a host. Non-limiting examples of antiviral factors include proteins which recognize viral components (e.g., nucleic acids, viral proteins), molecules involved in antiviral IIR signaling, transcription factors which mediate synthesis of antiviral-associated genes, and proteins which target, sequester, or otherwise interfere with viral reproduction, infection, or stability. Mammalian antiviral factors are those which are natively expressed in mammalian cells.

[0194] In some embodiments, an IMP that interferes with an IIR-associated protein interferes with recognition of danger molecules (e.g., non-self-nucleic acids) by the IIR-associated protein. In some embodiments, recognition of a danger molecules comprises detection of the danger molecule by a pattern recognition receptor (PRR).

[0195] IMPs interfering with Pattern Recognition Receptors (PRRs)

[0196] Pattern recognition receptors (PRRs) are germline-encoded proteins which can detect danger molecules in both the extracellular and intracellular space and which are localized in the cell membrane or in the cytoplasm. In some embodiments, an IMP interferes with a PRR. Nonlimiting examples of PRRs include: RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), absent in melanoma-2 (AIM2)-like receptors (ALRs), and cyclic GMP-AMP synthase (cGAS); some scavenger receptors (SRs) have also been classed as PRRs. Any PRR which can detect a non-self-nucleic acid (such as an RLR, TLR, ALR, NLR, cGAS) can be considered an antiviral factor. Importantly, while non-self-nucleic acids are not always viral nucleic acids, responses to non-self-nucleic acids generally overlap with antiviral IIR responses. Indeed, antiviral IIR responses have been identified as contributing significantly to instability of mRNA therapeutics and regulation immunogenicity and / or inflammation in response to the same (see: Wang, Y. Z. (2021). mRNA vaccine: a potential therapeutic strategy. Molecular Cancer, 20(1), 33).

[0197] In some embodiments, an IMP interferes with antiviral PRR mediated signaling. An IMP can be considered to interfere with PRR mediated signaling if it interferes with the synthesis, activity, or function of the PRR, adaptor molecules for the PRR, or downstream molecules activated by the PRR or its adaptor molecules. In some embodiments, an IMP that interferes with antiviral PRR mediated signaling interferes with one or more of RLR-mediated signaling, TLR-mediated signaling, ALR-mediated signaling, NLR-mediated signaling, cGAS-mediated signaling, SR-mediated signaling, or a combination thereof.

[0198] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) mediated signaling. Retinoic acidinducible gene I (RIG-I)-like receptors (RLRs) are a family of cytosolic proteins characterized by a central ATPase containing a DExD / H box helicase domain and, optionally, C-terminal repressor domains (RD) and / or N-terminal caspase Recruitment domain (CARD) (see: Dixit, E., & Kagan, J. (2013). Intracellular pathogen detection by RIG-I-like receptors. Advances in Immunology, 117, 99-125. doi: 10.1016 / B978-0- 12-410524-9.00004-9). RLRs are RNA sensors which detect immuno stimulatory (e.g., non-self) nucleic acids to mediate induction of host responses. RLRs are key molecules in IIRs to viral infection and are also thought to mediate responses to synthetic RNAs, such as mRNA vaccines (see: Yong, H., & Luo, D. (2018). RIG-L Like Receptors as Novel Targets for Pan- Antivirals and Vaccine Adjuvants Against Emerging and Re-Emerging Viral Infections. Frontiers in Immunology, 9, 379456). In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an RLR. Non-limiting examples of RLRs include: RIG-1, melanoma differentiation-associated protein (MDA) 5 (MDA5), and laboratory of genetics and physiology (LGP) 2 (LGP2). In some embodiments, an IMP antagonizes an RLR. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) RIG-1. In some embodiments, an IMP antagonizes RIG-1. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) MDA5. In some embodiments, an IMP antagonizes MDA5. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) LGP2. In some embodiments, an IMP antagonizes LGP2.

[0199] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) Toll-like receptor (TLR) mediated signaling. Toll-like receptors (TLRs) are a family of type I transmembrane proteins having N-terminal ectodomains with leucine-rich repeats, a transmembrane domain, and a cytosolic TIR domain. TLRs are activated by various extracellular stimuli, including, but not limited to, viral, bacteria, fungi, and endogenous ligands (see: Fitzgerald, K. A., & Kagan, J. C. (2020). Toll-like Receptors and the Control of Immunity. Cell, 180(6), 1044-1066.). TLRs recognize extracellular ligands (e.g., nucleic acids) which have reached endosomes via endocytosis. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) a TLR. Non-limiting examples of TLRs include: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 in humans, as well as TLR11, TLR12, and TLR13 in other mammals. TLRs localized in the plasma membrane (e.g., TLR1, TLR2, TLR4, TLR 5, and TLR 6) typically detect microbes and extracellular molecules, while TLRs localized in endosomal membranes (e.g., TLR3, TLR7, TLR8, TLR9, TLR13) typically detect nucleic acids (e.g., viral nucleic acids). In particular, TLR3, TR7, and TLR8 are associated with IIRs against non-self RNA. In some embodiments, IMPs interfere with (e.g., antagonizes and / or competes with) TLR3 mediated signaling pathways. In some embodiments, IMPs interfere with (e.g., antagonizes and / or competes with) TLR7 mediated signaling pathways. In some embodiments, IMPs interfere with (e.g., antagonizes and / or competes with) TLR8 mediated signaling pathways.

[0200] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) nucleotide oligomerization domain (NOD)-like receptor (NLR) mediated signaling. Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a family of cytosolic proteins characterized by the presence of a conserved NOD that recognizes danger molecules in the cytosol, as well as a variable amino-terminal effector-binding domains (EBDs) and a carboxyl- terminal ligand-recognition domain (LRD). NLRs are activated by pathogenic ligands and play a key role in initiating pathogen resistance and apoptosis (see: Inohara, N., et al. (2005). NOD- LRR Proteins: Role in Host-Microbial Interactions and Inflammatory Disease. Annual Reviews Biochemistry, 74, 355-383. doi: 10.1146 / annurev.biochem.74.082803.133347). In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an NLR. Nonlimiting examples of NLRs include: PYRIN-containing Apafl-like protein (PYPAL) 7 (PYPAF7; also known as NALP12, NLRP12, Monach 1, and RNO2), NACHT-LRR-and-pyrin domain-containing protein 2 (NALP2; aka PYPAF2, NLRP3), NODI (also known as CARD4), NOD2 (also known as CARD15), NOD3, NOD5 (also known as NALP14), NOD6 (also known as NALP9), NOD8 (NALP10, PYNOD), NOD9, NOD12 (also known as PYPAF3, NALP7), NOD14 (also known as NALP13), NOD16 (also known as NALP8), NOD17 (also known as PYPAL6, NALP11), Caspase- 1 / Interleukin- 1 converting enzyme (ICE) -protease Activating Factor (IPAF; also known as CARD 12, CLAN), Cryopyrin (also known as NALP3, PYPAF1, CIAS1), neuronal apoptosis inhibitor protein (NAIP; also known as BIRC1); nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing (NALP) 1 (NALP1; also known as NAC, DEFCAP, CARD7); Apoptotic protease activating factor (APAF)-l (APAF-1); Pyrin- and-Nacht domain (PAN) 2 (PAN2; also known as PYPAF4, NALP4); Maternal Antigen That Embryos Require (Mater; also known as PYPAF8, NALP5); and Class II Major Histocompatibility Complex Transactivator (CIITA). NLRP3 and NOD2 have been especially recognized for their role in the recognition of RNA. In some embodiments, an IMP interferes with (e.g., antagonizes) NLRP3. In some embodiments, an IMP interferes with (e.g., antagonizes) NOD2.

[0201] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) absent in melanoma-2 (AIM2)-like receptor (ALR) mediated signaling. Absent in melanoma 2 (AIM2)-like receptors (ALRs) are a family of receptors having a pyrin signaling domain and a DNA-binding hematopoietic-expression, interferon-inducible nature, and nuclear-localization (HIN) domain. ALRs. ALRs detect cytosolic DNA, including foreign and genomic DNA, and initiate inflammatory responses (see: Lugrin, J., & F., M. (2017). The AIM2 inflammasome: Sensor of pathogens and cellular perturbations. Immunological Reviews, 281(1), 99-114. doi: 10.1111 / imr.12618). In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an ALR. Non-limiting examples of ALRs include: AIM2, Interferon inducible protein 16 (IFI16); Pyrin and HIN domain Family Member 1 (PYHIN1); and myeloid cell nuclear differentiation antigen (MNDA). AIM2 and IFI16 are known to be particularly important for DNA sensing. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) AIM2. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) IFI16.

[0202] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) cGAS-mediated signaling. cGAS is a cytosolic protein which can bind non-self-nucleic acids and catalyze 2’ -3 ’-cyclic AMP-GMP (cGAMP) for further signaling; though cGAS typically binds DNA, it has been increasingly implicated in RNA binding (see: Ma, Y, et al. (2021). Roles of emerging RNA-binding activity of cGAS in innate antiviral response. Frontiers in Immunology, 12, 741599.). In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) cGAS.

[0203] IMPs interfering with RNA Binding Proteins (RBPs)

[0204] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) RNA binding protein (RBP) mediated signaling. RNA-binding proteins (RBPs) are proteins which are capable of binding double stranded RNA (dsRNA) and / or single stranded RNA (ssRNA); the term “RBP” includes PRRs which directly bind RNA, but also proteins which are otherwise capable of recognizing non-self RNAs. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an RBP. Non-limiting examples of IIR-associated RBPs include: protein kinase R (PKR), 2’-5’ oligoadenylate synthase (OAS), ribonuclease latent (RNaseL), adenosine deaminase acting on RNAs (ADARs), Dicer, Drosha, protein activator of PKR (PACT), transactivation response RBP (TRBP), superkiller viralicidic activity 2-like (SKIV2L), heterogeneous nuclear ribonucleoprotein (hnRNP) M (hnRNP M), hnRNP ULI, tristetraprolin (TTP), human antigen R (HuR), T-cell restricted intracellular antigen- 1 (TIA-1), hnRNP A2B1, GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), AT-rich interactive domain-containing protein 5a (Arid5a), methyltransferase like 3 (Mettl3), serine and arginine-rich splicing factor 1 (SRSF1), zinc finger antiviral proteins (ZAPs), and polypyrimidine tract binding protein 1 (PTBP1).

[0205] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) PKR-mediated signaling. PKR is an antiviral RBP which can bind double-stranded RNA (dsRNA). Binding of dsRNA by PKR triggers dimerization of PKR, after which it can induce various antiviral IIRs. Expression and activation of PKR is mediated by additional activity of IIR pathways. PKR-mediated signaling is associated with inhibition of translation machinery, activation of apoptosis, proinflammatory responses, and cell cycle regulation. In some embodiments, an IMP comprises an antagonist of PKR. In some embodiments, an IMP comprises a mutant PKR that competes with PKR mediated signaling. In some embodiments, an IMP comprises a dominant negative PKR (dnPKR). In some embodiments, dnPKR comprises the amino acid sequence set forth in SEQ ID NO: 242. In some embodiments, dnPKR consists of the amino acid sequence set forth in SEQ ID NO: 242.

[0206] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) ZAP-mediated signaling. ZAPs are CCCH-type zinc finger proteins which function as host restriction factors against non-self RNAs by recognition of CpG motifs. ZAP binds to RNA and recruits additional factors which degrade the RNA. ZAP can also inhibit translation of viral RNA by interactions with eukaryotic initiation factors (elFs). Expression of ZAP can be initiated as part of IIR pathways, but some versions of ZAP also exist constitutively. In some embodiments, an IMP is an antagonist of ZAP. In some embodiments, an IMP is a mutant ZAP that competes with ZAP-mediated signaling. In some embodiments, an IMP is a dominant negative ZAP (dnZAP). In some embodiments, an IMP is dnZAP with an additional mutation. In some embodiments, an IMP is dnZAP with a cysteine to arginine (C to R) mutation at position 88 (ZAPC88R). In some embodiments, an IMP is a rat dnZAPC88R (rZAPC88R). In some embodiments, rZAPC88R comprises the amino acid sequence set forth in SEQ ID NO: 241. In some embodiments, rZAPC88R consists of the amino acid sequence set forth in SEQ ID NO: 241.

[0207] IMPs interfering with Antiviral Adaptor Proteins

[0208] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) antiviral adaptor protein-mediated signaling. Antiviral adaptor proteins are IIR- associated proteins which can integrate signals from antiviral PRRs or RBPs to activate downstream signaling proteins (e.g., via signaling cascades) and, ultimately, contribute to regulation of IIRs. Antiviral adaptor proteins generally mediate protein-protein interactions (e.g., between a PRR and a downstream signaling protein) and lack independent intrinsic enzymatic activity. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an antiviral adaptor protein. Non-limiting examples of antiviral adaptor proteins include myeloid differentiation primary response 88 (MyD88), Toll / IL-1 receptor / resistance (TIR) domain- containing adaptor inducing interferon-P (TRIF, also known as TICAM1), TRIF-related adaptor molecule (TRAM), mitochondrial viral signaling protein (MAVS), microprotein in antiviral immunity 1 (MA VII), apoptosis-associated speck-like protein containing a caspase recruiting domain (CARD) (ASC), stimulator of interferon genes (STING), inflammasomes, and PANoptosomes. In some embodiments, an IMP interferes with synthesis or activity of an antiviral adaptor protein.

[0209] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) MyD88-mediated signaling. MyD88 is an adaptor protein for TLR and interleukin-1 (IL-1) receptors (IL-1R). MyD88 links signals from TLRs and / or IL-lRs to IL-lR-associated kinase (IRAK) family members, which include IRAKI, IRAK4, IRAK2, and IRAKM. MyD88- mediated signaling is associated with activation of inflammatory responses, such as production of pro-inflammatory cytokines (e.g., tumor necrosis factor (TNF), IL-6, IL-1), and type I interferons (IFN). In some embodiments, an IMP antagonizes MyD88.

[0210] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) TRIF-mediated signaling. TRIF is an adaptor protein for TLRs and contributes to both MyD88- dependent and MyD88-independent pathways. TRIF pathways link TLRs to IRAK family members, tumor necrosis factor receptor (TNFR) associated factor (TRAF) family members, receptor-interacting proteins (RIPs), and inflammasomes. TRIF-mediated signaling is associated with activation of inflammatory responses, such as production of type-I IFN-P and analogs thereof, and initiation of apoptosis and necroptosis. In some embodiments, an IMP antagonizes TRIF.

[0211] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) TRAM-mediated signaling. TRAM is an adaptor protein for TLR4 and contributes to both TRIF-dependent and MyD88-dependent signaling pathways. TRAM-mediated signaling is associated with pro-inflammatory responses, such as production of pro -inflammatory cytokines and type I IFN. In some embodiments, an IMP antagonizes TRAM.

[0212] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) mitochondrial antiviral- signaling (MAVS) -mediated signaling. MAVS is a mitochondria- associated adaptor molecule for RLRs. Once activated, MAVS forms prion-like aggregates on the outer membrane of mitochondria, through which downstream signals are propagated. MAVS-mediated signaling is associated with immune and inflammatory responses, such as production of type I IFNs and cytokines (e.g., TNF-a and interleukins). In some embodiments, an IMP antagonizes MAVS. In some embodiments, an IMP is a mutant MAVS that competes with MAVS-mediated signaling. In some embodiments, an IMP is a dominant negative MAVS (dnMAVS). In some embodiments, dnMAVS comprises the amino acid sequence set forth in SEQ ID NO: 243. In some embodiments, dnMAVS consists of the amino acid sequence set forth in SEQ ID NO: 243.

[0213] In some embodiments, an IMP facilitates signaling by inhibitors of MAVS. In some embodiments, an IMP facilitates MAVI1 -mediated signaling. MAVI1 is an endoplasmic- reticulum associated negative regulator of RLR-MAVS signaling, which attenuates MAVS activity. MAVI1 inhibits MAVS aggregation and reduces type I IFN signaling activation. MAVI1 is down-regulated during inflammatory responses (e.g., in response to viral infection), and is up- regulated to deactivate immune responses. In some embodiments, an IMP facilitates overexpression of MAVI1. In some embodiments, an IMP is MAVIl.In some embodiments, MAVI1 comprises the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, MAVI1 consists of the amino acid sequence set forth in SEQ ID NO: 244.

[0214] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) STING-mediated signaling. STING is an endoplasmic reticulum-associated adaptor molecule for cGAS. Once activated, STING forms a complex with TANK-binding kinase 1 (TBK1), which initiates pathways for production of type I interferon and cytokines. In some embodiments, an IMP antagonizes STING. In some embodiments, an IMP antagonizes STING.

[0215] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) inflammasome-mediated signaling. Inflammasomes are multi-protein complexes formed by PRRs in the cytoplasm and which, when activated, trigger activation of caspase- 1. Inflammasomes are comprised of NLRs or ALRs, and adapter protein apoptosis-associated speck- like protein containing a CARD (ASC). In some embodiments, an IMP antagonizes an inflammasome. Non-limiting examples of inflammasomes include NLRP1 inflammasome, NLRP3 inflammasome, Caspase- 11 inflammasome, NAIP-NLRC4 inflammasome, and AIM2 inflammasome.

[0216] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) PANoptosome-mediated signaling. PANoptosomes are complexes formed by numerous pyroptotic, apoptotic, and necroptotic (PANoptotic molecules), and are mainly composed of PRRs, sensory proteins (e.g., Z-DNA binding proteins), adapter proteins with caspase recruiting domains (e.g., apoptosis-associated speck-like protein), and proteins with catalytic effects (e.g., receptor interacting protein kinases, caspases). In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) a PANoptosome or formation thereof. Non-limiting examples of PANoptosomes include: ZBPl-PANoptosome (ZBP1, NLRP3, ASC, caspase-1, caspase-6, caspase-8, RIPK1, RIPK3), AIM2-PANoptosome (AIM2, Pyrin, ZBP1, ASC, caspase-1, caspase-8, FADD, RIPK1, and RIPK3), RIPKl-PANoptosome (RIPK1, RIPK3, NLRP3, ASC, caspase-1, and caspase-8), and NLRP12- PANoptosome (NLRP12, ASC, caspase- 8, and RIPK3).

[0217] IMPs interfering with IIR-associated Transcription Factors

[0218] Mammalian antiviral factors also comprise transcription factors activated as part of antiviral IIR pathways. Transcription factors are sequence- specific proteins which regulate the rate of transcription of endogenous genes by binding to regions of DNA, such as enhancers or promoter regions. An IMP may interfere with transcription factors by for example, modulating the binding of transcription factors to DNA, reducing synthesis of transcription factors, suppressing nuclear localization of transcription factors, or downregulating their activation. Though the precise mechanisms through which IIR pathways ultimately produce immune and / or inflammatory responses vary, IIR pathways generally converge on the transcription factors nuclear factor kappa-light chain-enhancer of activated B cells (NF-KB) and interferon regulatory factors (IRFs).

[0219] In some embodiments, an IMP interferes with regulation of NF-KB and / or transcription of NF-KB target genes. NF-KB is a rapid-acting, inducible transcription factor involved in producing inflammatory cytokine responses to danger signals. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an NF-KB. Non-limiting examples of NF-KBS include NF-KB 1, NF-KB2, RelA, RelB, and c-Rel. In the absence of danger signals, NF- KBS are sequestered in the cytosol by inhibitor of NF-KB (IKB). Detection of certain danger signals by IIR-associated proteins, such as TLRs, can lead to the phosphorylation of IKB inhibitors, which release NF-KB into the cytosol. Once translocated to the nucleus, NF-KB can bind to DNA to regulate transcription of NF-KB target genes, which include several chemokines, cytokines, cell cycle regulators, anti-apoptotic factors, and adhesion molecules. Importantly, NF- KB can induce transcription of genes which promote inflammatory responses, as well as genes which limit the duration and magnitude of inflammatory responses; thus, NF-KB activity can regulate the onset, duration, and offset of inflammation by differential induction of NF-KB target genes. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an NF-KB target gene. Non-limiting examples of NF-KB target genes include first apoptosis signal (Fas), B-cell lymphoma 2 (BCL-2), cellular FLICE-like inhibitory protein (c-FLIP), caspases, inhibitors of apoptosis (IAPS), BCL-2 related gene expressed in fetal liver (BFL-1), Survivin, BCL-2-like protein 1 (BCL-2L1), plasminogen activator inhibitor 2 (PAI2), Cyclin, interleukin (IL)-l, IL-2, IL-6, IL-8, IL- 12, IL- 18, tumor necrosis factor alpha (TNFa), monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand (CCL) 5 (CCL5; also known as RANTES), macrophage inflammatory protein 2 (MIP-2), chemokine (C-X-C motif) ligand (CXCL) 1 (CXCL1), CXCL10, intercellular adhesion molecule (ICAM) 1 (ICAM-1), vascular cell adhesion molecule (VCAM) 1 (VCAM-1), endothelial cell adhesion molecule (ECAM) 1 (ECAM-1), matrix metalloproteinase (MMPs) and Selectin.

[0220] In some embodiments, IMPs interfere with regulation of IRFs and / or transcription of IRF target genes. IRFs are a family of transcription factors that regulate expression of pro- inflammatory responses, apoptosis, and immune responses to danger signals. In the absence of danger signals, IRFs are localized in the cytoplasm in an inactive, monomeric form. Detection of certain danger signals by IIR-associated proteins, such as RLRs, lead to post-translational modifications of IRFs which allow for protein -protein interactions. IRFs can then homodimerize with other IRFs or heterodimerize with other molecules, after which IRFs can translocate to the nucleus and bind to IRF-target genes. In some embodiments, an IMP interferes with IIR- associated IRF-mediated signaling (e.g., IRF homodimerization with other molecules). In some embodiments, an IMP antagonizes an IIR-associated IRF. Non-limiting examples of IIR- associated IRFs include IRF1, IRF2, IRF3, IRF4 (also known as PIP and ICS AT), IRF5, IRF6, IRF7, IRF8 (also known as ISCBP) and IRF9 (also known as p48 and ISG3y). Several IRFs function as transcription factors for type I interferons (e.g., IFNa, IFNP) and interferon stimulated genes (ISGs). Some IRFs are activated by MAVS, STING, and TRIF; several IRFs can also indirectly activate NF-KB, for example, via TNFa signaling. In some embodiments, IMP-mediated interference with regulation of IRFs and / or transcription of IRF target genes comprises interference with MAVS, STING, and / or TRIF. In some embodiments, IMP-mediated interference with regulation of IRFs and / or transcription of IRF target genes comprises interference with TNFa signaling. In some embodiments, IMP-mediated interference with regulation of IRFs and / or transcription of IRF target genes comprises interference with NF-KB- mediated signaling. As used herein, the term “and / or” means “one or more of’ a plurality of articles in any quantity or combination. IMPs interfering with Immune and / or Inflammatory Response Molecules

[0221] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) one or more immune and / or inflammatory response molecules. As used herein, the term “immune and / or inflammatory response molecules” refers to molecules (e.g., proteins, receptors) that are activated by an IIR pathway and that, once activated, mediate (e.g., enact, trigger) one or more immune responses, inflammatory responses, and / or apoptosis. In some embodiments, an immune and / or inflammatory response molecule is a cytokine (e.g., an interferon, an interleukin, a tumor necrosis factor) or an apoptotic factor (e.g., a caspase, Bcl-2).

[0222] IMPs Interfering with Cytokines

[0223] In some embodiments, an IMP interferes with(e.g., antagonizes and / or competes with) cytokine-mediated signaling. Cytokines are cell- signaling, secreted proteins which promote immune responses (e.g., to infection, inflammation, trauma, and disease), for example, by promoting expression of pro-inflammatory genes. In some embodiments, an IMP (e.g., antagonizes and / or competes with) a cytokine. Non-limiting examples of cytokines include interferons (IFNs), interleukins (IL), and tumor necrosis factors (TNFs). In some embodiments, IMPs interfere with transcription of pro-inflammatory genes. In some embodiments, IMPs induce transcription of anti-inflammatory genes. In some embodiments, IMPs interfere with cytokine production.

[0224] In some embodiments, IMPs interfere with (e.g., antagonize, compete with) interferon (IFN) transcription, synthesis, and / or signaling. Interferons (IFNs) are molecules which interfere with viral replication. IFN production in cells can be initiated by PRR activity, after which IFN can act as a signal for further gene transcription (e.g., of interferon stimulated genes (ISGs)) and for responses by neighboring cells. In some embodiments, the IFN is a type I IFN (e.g., IFNa, IFNP). In some embodiments, the IFN is a type III IFN (e.g., IFNX).

[0225] In some embodiments, an IMP interferes with IFNa production or activity. In some embodiments, an IMP interferes with IFNP production or activity. In some embodiments, an IMP interferes with IFNZ. production or activity. Type I IFN (e.g., IFNa, IFNP) synthesis and activity can be promoted by various IIR pathways, for example, via the activity of RLRs, TLRs, MyD88, TRIF, TRAM, MAVS, and PKR. Accordingly, in some embodiments, an IMP that interferes with a type I IFN interferes with (e.g., antagonizes and / or competes with) an RLR, a TLR, MyD88, TRIF, TRAM, MAVS, PKR, or a combination thereof. Synthesis and activity of type I IFNs can also be downregulated by various cytokine signaling inhibitors, such as suppressors of cytokine signaling (SOCS) family members and IKBS. Accordingly, in some embodiments, an IMP that interferes with a type I IFN increases (e.g., provides, promotes overexpression of) a level or activity of a cytokine signaling inhibitor (e.g., SOCS, IKB).

[0226] In some embodiments, an IMP increases (e.g., provides, promotes overexpression of) a level or activity of a suppressor of cytokine signaling (SOCS) protein. Suppressor of cytokine signaling (SOCS) proteins are negative-feedback inhibitors of cytokine signaling pathways and are activated via JAK / STAT pathways. Non-limiting examples of SOCS proteins include cytokine inducible SH2-containing protein (CISH), SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7. In some embodiments, an IMP comprises a SOCS protein. In some embodiments, an IMP is SOCS1. In some embodiments, SOCS1 comprises the amino acid sequence set forth in SEQ ID NO: 239. In some embodiments, SOCS1 consists of the sequence set forth in SEQ ID NO: 239. In some embodiments, an IMP is SOCS3. In some embodiments, SOCS3 comprises the amino acid sequence set forth in SEQ ID NO: 240. In some embodiments, SOCS3 consists of the sequence set forth in SEQ ID NO: 240.

[0227] In some embodiments, IMPs interfere with (e.g., antagonize, compete with) interleukin (IL) transcription, synthesis, or signaling. Interleukins (ILs) are molecules which are secreted by white blood cells and are critical for immune function. Several interleukins are known (e.g., IL- 1, IL-2, IL-3, IL-4, etc.), each having regulatory and / or signaling functions in immune responses and / or inflammatory responses. In some embodiments, an IMP is an antagonist of one or more ILs.

[0228] In some embodiments, an IMP interferes with a tumor necrosis factors (TNF) transcription, synthesis, or signaling. Tumor necrosis factors (TNLs) are molecules mediate inflammatory and apoptotic responses. TNL signaling is mediated by two receptors: TNL receptor 1 (TNLR1) and TNLR2. TNER1 -mediated signaling is generally pro-inflammatory and apoptotic. TNLR2-mediated signaling is generally anti-inflammatory and pro-cell growth. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an TNER1- mediated immune and / or inflammatory response. In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an TNLR2-mediated immune and / or inflammatory response. In some embodiments, an IMP is a TNLR (e.g., TNLR1, TNLR2) antagonist.

[0229] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) TNL-a. TNL-a is a TNL produced by several IIR pathways that can bind to both TNLR1 and TNLR2 to differentially regulate immune responses. In some embodiments, IMPs interfere with TNL-a transcription, synthesis, or signaling. IMPs Interfering with Apoptotic Factors

[0230] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) an apoptotic factor. Apoptotic factors are proteins which promote or inhibit apoptosis. Nonlimiting examples of apoptotic factors include caspases, Bcl-2 proteins, and TNFs. In some embodiments, an IMP interferes with a pro-apoptotic signaling pathway. In some embodiments, an IMP promotes an anti- apoptotic signaling pathway.

[0231] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) Fas ligand transcription, synthesis, or signaling. First apoptosis signal (Fas) ligand is a member of the TNF family which can interact with Fas receptors. Binding of Fas ligand to Fas receptors initiates an apoptosis pathway. In some embodiments, an IMP is a Fas receptor antagonist.

[0232] In some embodiments, an IMP interferes with (e.g., antagonizes and / or competes with) caspase transcription, synthesis, or signaling. Caspases are cysteine-aspartic proteases which can initiate apoptosis, execute apoptosis, and / or promote inflammation. Caspase synthesis is tightly regulated and can occur as a result of IIR pathway initiation. Caspases enter an active form when they dimerize and form multi-protein complexes with various functions. In some embodiments, an IMP interferes with caspase synthesis. In some embodiments, an IMP interferes with caspase dimerization. In some embodiments, an IMP interferes with caspase complex functions. In some embodiments, an IMP is a caspase antagonist.

[0233] In some embodiments, an IMP interferes with Bcl-2 protein transcription, synthesis, or signaling. B-cell lymphoma 2 (Bcl-2) proteins are regulatory proteins which regulate apoptosis. Bcl-2 is a receptor localized on the outer membrane of mitochondria and can promote or inhibit apoptosis. In some embodiments, an IMP interferes with Bcl-2 synthesis or function. In some embodiments, an IMP is a Bcl-2 antagonist.

[0234] Viral Immune Evasion Proteins

[0235] In some embodiments, an IMP is a viral immune evasion protein (VIEP). Viral immune evasion proteins (VIEPs) are viral proteins which interfere with one or more antiviral IIR pathways and ultimately reduce the magnitude of a host’s response (e.g., reduce magnitude of an inflammatory response) against the virus or extend the amount of time before which an IIR against the virus is initiated. VIEPs are expressed by numerous viruses, for example, vaccinia virus (VACV), Orf virus, herpes simplex virus (HSV), influenza A viruses (IAV), and Toscana virus (TOSV). VIEPs may exert their effect through various mechanisms. For example, a VIEP may interfere with (e.g., antagonize) PRRs, RBPs, adaptor molecules, transcription factors, cytokines, and regulators thereof which are associated with antiviral IIR pathways.

[0236] Vaccinia Virus (VACV) VIEPs

[0237] In some embodiments, an IMP is a VIEP from vaccinia virus (VACV). VACV is an enveloped virus of the Poxviridae family and comprises a linear, double- stranded DNA genome. Because VACV replicates in the cytoplasm of a host cell and not the nucleus, it possesses a large genome which encodes enzymes and proteins related to DNA replication and transcription, as well as VIEPs which endow the virus with resistance to IIRs, for example, F1L, K3L, E3L, and B18R (see: Perdiguero, B., & Esteban, M. (2009). The Interferon System and Vaccinia Virus Evasion Mechanisms. Journal of Interferon and Cytokine Research, 29(9), 581-598.).

[0238] In some embodiments, an IMP is F1L. F1L is a VACV protein which has been shown to interfere with NLR-mediated signaling. VACV is a homolog of Bcl-2, and can inhibit NLRP1 by directly binding to it. In some embodiments, F1L comprises the sequence set forth in SEQ ID NO: 231. In some embodiments, F1L consists of the sequence set forth in SEQ ID NO: 231.

[0239] In some embodiments, an IMP is K3L. K3L is a VACV protein with homology to an elF, eIF2a. eIF2a is a substrate of PKR and is important for initiation of translation of many proteins, including cytokines. K3L is a partial agonist of PKR, and competitively inhibits activation of endogenous eIF2a.

[0240] In some embodiments, an IMP is RNA-binding protein E3 (E3L). E3L is a VACV dsRNA binding protein which has been shown to interfere with PKR, OAS, ADAR, IRF3, IRF7, and NF-KB-mediated signaling, as well as the expression of IFNs and ISGs. E3L is also capable of binding directly to PKR to prevent its activation of elFs. In some embodiments, E3L comprises the amino acid sequence set forth in SEQ ID NO: 230. In some embodiments, E3L consists of the amino acid sequence set forth in SEQ ID NO: 230.

[0241] In some embodiments, an IMP is soluble IFN-a / p receptor B 18 (B18R). B18R is a VACV glycoprotein with some similarity to cellular IFNa and IFNP receptors; thus, B18R competes with IFNa and IFNP receptors to bind IFNa and IFNP, and thereby interferes with IFN related signaling. In some embodiments, B18R comprises the amino acid sequence set forth in SEQ ID NO: 229. In some embodiments, B18R consists of the amino acid sequence set forth in SEQ ID NO: 229.

[0242] In some embodiments, an IMP is NIL. NIL is a VACV protein predicted to contain BH3-like binding domains; though the host factors with which NIL interferes are not fully understood, NIL may be able to bind BH3 to exert control over induced apoptosis in infected cells. In some embodiments, NIL comprises the amino acid sequence set forth in SEQ ID NO: 232. In some embodiments, NIL consists of the amino acid sequence set forth in SEQ ID NO: 232.

[0243] Herpes Simplex Virus (HSV) VIEPs

[0244] In some embodiments, an IMP is a VIEP from herpes simplex virus (HSV). HSV is an enveloped virus comprising a large, double- stranded linear DNA genome. HSVs include HSV 1 and HSV2. Several DNA sequences, dsRNA intermediates, and viral proteins of HSV are recognized by PRRs (including TLRs, RLRs, NLRs, ALRs, and cGAS). IIR pathways which respond to HSV involve several molecules, such as MyD88, STING, and LGP2. HSV encodes several VIEPs, including US1, US11, and ICP34.5 (see: Zhang, M. L., & Yan, Y. (2015). HSV- 2 immediate-early protein US1 inhibits IFN-P production by suppressing association of IRF-3 with IFN-P promoter. The Journal of Immunology, 194(7), 3102-3115; Zhu, H., & Zheng, C. (2020). The race between host antiviral innate immunity and the immune evasion strategies of herpes simplex virus 1. Microbiology and Molecular Biology Reviews, 84(4), 10-1128. doi: 10.1128 / MMBR.00099-20).

[0245] In some embodiments, an IMP is US1. Unique short 1 (US1) is an HSV immediate early protein which has been found to suppress production of IFN-P through inhibition of IRF3. Specifically, US1 binds to DNA binding domains of IRF3 to prevent its association with the IFN-P promoter. In some embodiments, US1 comprises the amino acid sequence set forth in SEQ ID NO: 235. In some embodiments, US1 consists of the amino acid sequence set forth in SEQ ID NO: 235.

[0246] In some embodiments, an IMP is US11. Unique short 11 (US11) is an HSV RNA- binding protein which interrupts RLR-mediated antiviral IIR pathways. US 11 is an antagonist of RIG-I and MDA5 and binds directly to the C-terminal domains of both receptors. In some embodiments, US 11 comprises the amino acid sequence set forth in SEQ ID NO: 236. In some embodiments, US 11 consists of the amino acid sequence set forth in SEQ ID NO: 236.

[0247] In some embodiments, an IMP is infected cell protein 34.5 (ICP34.5, also known as ISC34.5). ICP34.5is an HSV protein which acts on eIF-2A, and which has been shown to target STING by preventing its translocation from the endoplasmic reticulum, thereby inhibiting downstream production of cytokines and propagation of antiviral IIRs. In some embodiments, ICP34.5 comprises the amino acid sequence set forth in SEQ ID NO: 234. In some embodiments, ICP34.5 consists of the amino acid sequence set forth in SEQ ID NO: 234.

[0248] In some embodiments, an IMP is infected cell protein 0 (ICPO). ICPO is an HSV protein and ubiquitination (Ub) ligase which targets proteins in numerous cell pathways, such as SUMOylation pathways, cell cycle checkpoint factors, DNA repair pathways, TLR-associated proteins, and cytokine signaling pathways to promote HSV lytic replication and propagation. In some embodiments, ICPO comprises the amino acid sequence set forth in SEQ ID NO: 233. In some embodiments, ICPO consists of the amino acid sequence set forth in SEQ ID NO: 233.

[0249] Influenza A Virus (IAV) VIEPs

[0250] In some embodiments, an IMP is a VIEP from influenza A (IAV). IAV is a negativesense, single- stranded segmented, enveloped RNA virus of the family Orthomyxoviridae . IAVS subtypes are categorized by the type of surface protein in its envelope: hemagglutinin (H) and neuraminidase (N). IAVs are recognized by TLRs, RLRs, and NLRs. One critical IAV VIEP is non-structural protein 1 (NS1) ( see: Van de Sandt, C., Kreijtz, J., & Rimmelzwaan, G. (2012). Evasion of influenza A viruses from innate and adaptive immune responses. Viruses, 4(9), 1438- 1476.).

[0251] In some embodiments, an IAP is an IAV NS1. IAV NS1 blocks recognition of RIG-I mediated signaling through various means, such as antagonizing RIG-I, inhibiting oligomerization of TRIM25, inhibiting IRF-3, inhibiting NF-KB, and interfering with mRNA export machinery. In some embodiments, an IAP is NS1 from influenza A / Puerto Rico / 8 / 34 (PR8 NS1). In some embodiments, PR8 NS1 comprises the amino acid sequence set forth in SEQ ID NO: 237. In some embodiments, PR8 NS1 consists of the amino acid sequence set forth in SEQ ID NO: 237.

[0252] IMPs from Toscana Virus (TOSV) VIEPs

[0253] In some embodiments, an IMP is a VIEP from Toscana virus (TOSV). Toscana virus an enveloped RNA arbovirus of the family Phenuiviridae . In some embodiments, an IMP is a non- structural (NSs) protein of TOSV. The NSs proteins of TOSV have been associated with inhibition of IIRs in cells (see: Gori-Savellini, G. V. (2013). Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I. Journal of Virology, 87(12), 6660- 6667; Kalveram, B., & I., T. (2013). Toscana virus NSs protein promotes degradation of doublestranded RNA-dependent protein kinase. Journal of Virology, 87(7), 3710-3718.). TOSV NSs have been shown to downregulate PKR and inhibit the induction of IFN production by RLR- mediated IIR pathways. In some embodiments, TOSV NSs comprises the amino acid sequence set forth in SEQ ID NO: 238. In some embodiments, TOSV NSs consists of the amino acid sequence set forth in SEQ ID NO: 238.

[0254] IMPs from Encephalomyocarditis Virus (EMCV)

[0255] In some embodiments, an IMP is a VIEP from encephalomyocarditis virus (EMCV). EMCV is a single-stranded, non-enveloped virus of the family Picornaviridae. In some embodiments, an IMP is an Lpro protein of EMCV. The leader protease (Lpro) protein of EMCV has been associated with interfering with IRF pathways and ultimately inhibiting the induction of IFN production. In some embodiments, EMCV Lpro comprises the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, EMCV Lpro consists of the amino acid sequence set forth in SEQ ID NO: 245. trRNAs encoding IMPs

[0256] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA and / or a replicase construct) comprising a nucleic acid encoding an IMP. In some embodiments, a taRNA comprises an RNA polynucleotide comprising a nucleic acid encoding multiple IMPs, for example, a first IMP and a second IMP. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding two or more IMPs (e.g., 2, 3, 4, 5, 6, or more IMPs). In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding three or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding four or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding five or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding six or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding one, two, three, four, five, or six IMPs. In some embodiments, the multiple IMPs are different IMPs. In some embodiments, multiple IMPs are joined by a linker.

[0257] Linkers

[0258] As used herein, the term “linker” refers to sequences which allow for the concatenation of one or more nucleic acids encoding a gene product, for example, in a multicistronic vector. Linkers may be nucleic acid linkers, for example, nucleic acid elements which can induce cap- independent translation and internal promoters, or peptide linkers, such as ribosomal skip proteins. Proteins (e.g., IMPs) are considered to be “joined” by a linker if the nucleic acids encoding the proteins and the nucleic acid linkers or nucleic acids encoding a linker are comprised within a continuous polynucleotide.

[0259] IRES Elements

[0260] In some embodiments, a linker is an internal ribosome entry site (IRES) element. As used herein, the term “IRES element” refers to a cis RNA sequence region which allows for the initiation of translation of a sequence in a cap-independent manner. The terms “IRES element,” “IRES,” “IRES sequence,” and “IRES region” may be used interchangeably. The term “capindependent” refers to a mechanism for initiation of translation of an RNA polynucleotide which does not require a 5’ cap (e.g., Cap-0, Cap-1, Cap-2); cap-independent mechanisms for initiating translation are found in many viral RNA and in some eukaryotic mRNA. In contrast, “capdependent” mechanisms require a 5’ cap (e.g., Cap-0) which recruits a series of initiation factors and catalyzes the formation of a functional ribosome to initiate translation by a ribosome; capdependent mechanisms for initiating translation are found in most eukaryotic mRNA. When used as linkers in an RNA polynucleotide, IRES elements can initiate the translation of downstream nucleic acids (i.e., coding sequence). Though rates of translation of a downstream nucleic acid can vary between different IRES elements and / or promoters, IRES elements may be useful for expressing multiple gene products off of a single polynucleotide, as translation can be reinitiated at each IRES element. IRES elements also allow for expression of multiple proteins without further alteration of their amino acid sequence (e.g., as with some peptide-based linkers). IRES elements are often observed in viruses, though virus-like endogenous IRES elements in eukaryotes have been observed. Exemplary viral and eukaryotic IRES sequences can be found on IRESbase (available on the worldwide web at reprod.njmu.edu.cn / cgi- bin / iresbase / index.php). In some embodiments, a linker is a viral IRES element. Non-limiting examples of viral IRES elements include IRES elements of Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitis virus, simian Virus 40 (SV40), Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Manhattan Parechovirus (MPV), Human rhinovirus (iHRV), Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 (EV71), Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Porcine kobuvirus (PKV), Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), tortoise rafivirus (TraV), and Poliovirus (PV). In some embodiments, a linker is an IRES from CVB3. In some embodiments, an IRES from CVB3 comprises the sequence set forth in SEQ ID NO: 118. In some embodiments, a linker is an IRES from EMCV In some embodiments, an IRES from EMCV comprises the sequence set forth in SEQ ID NO: 119. In some embodiments, a linker is an IRES from PKV. In some embodiments, an IRES from PKV comprises the sequence set forth in SEQ ID NO: 120. In some embodiments, a linker is an IRES from MPV. In some embodiments, an IRES from MPV comprises the sequence set forth in SEQ ID NO: 121. In some embodiments, a linker is an IRES from TraV. In some embodiments, an IRES from TRAV comprises the sequence set forth in SEQ ID NO: 122.

[0261] Ribosomal Skip Peptides

[0262] In some embodiments, a linker is a ribosomal skip peptide. Ribosomal skip peptides, also known as “self-cleaving peptides,” are peptides which induce ribosomal skipping during translation of an RNA polynucleotide, such that the ribosome fails to covalently bond an amino acid to the nascent chain, then resumes translation downstream, such that multiple, separate proteins are produced. Sequences encoding ribosomal skip peptides tend to be significantly shorter than most IRES elements, and so may be particularly useful when constraints on polynucleotide length are important. Ribosomal skip peptides are typically viral peptides such as 2A peptides. 2A peptides are peptides of about 18-22 amino acids in length which can induce ribosomal skipping. In some embodiments, a linker is a 2A peptide. Non-limiting examples of 2A peptides include 2A peptides from: thosea asigna virus 2A (T2A), porcine teschovirus- 1 2A (P2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 18 (F2A). In some embodiments, a linker is a 2A peptide. In some embodiments, a linker is a T2A peptide. In some embodiments, a T2A peptide comprises the sequence set forth in SEQ ID NO: 221. In some embodiments, a T2A peptide is encoded by the sequence set forth in SEQ ID NO: 123. In some embodiments, a linker is a P2A peptide. In some embodiments, a P2A peptide comprises the sequence set forth in SEQ ID NO: 222. In some embodiments, a P2A peptide is encoded by the sequence set forth in SEQ ID NO: 124. In some embodiments, a linker is an E2A peptide. In some embodiments, an E2A peptide comprises the sequence set forth in SEQ ID NO: 223. In some embodiments, an E2A peptide is encoded by the sequence set forth in SEQ ID NO: 125.

[0263] Though expression of multiple proteins linked by 2A peptides is generally even, inclusion of a 2A peptide results in additional amino acids being appended onto each protein flanking the 2A peptide, which may be undesirable for certain proteins which poorly (e.g., do not) tolerate terminal modifications. In some embodiments, a 2A peptide is combined with one or more furin recognition sites to allow for removal of these 2A residues from a translated protein. Furin, also known as Paired basic Amino acid Cleaving Enzyme (PACE), is a protease capable of cleaving proteins at furin recognition sites. As used herein, the term “Furin-2A” refers to a 2A peptide (e.g., T2A, P2A) comprising a furin cleavage site (FCS) upstream of said 2A peptide . In some embodiments, a linker is a Furin-2A peptide. In some embodiments, a linker is a Furin-T2A peptide. In some embodiments, a Furin-T2A peptide comprises the sequence set forth in SEQ ID NO: 224. In some embodiments, a Furin-T2A peptide is encoded by the sequence set forth in SEQ ID NO: 126. In some embodiments, a linker is a Furin-P2A peptide. In some embodiments, a Furin-P2A peptide comprises the sequence set forth in SEQ ID NO: 225. In some embodiments, a Furin-P2A peptide is encoded by the sequence set forth in SEQ ID NO: 127.

[0264] Subgenomic Promoters (SGPs)

[0265] In some embodiments, a linker is an alphaviral subgenomic promoter (SGP). Subgenomic promoters (SGP) comprise recognition and binding sites for RNA polymerase (e.g., RNA-dependent RNA polymerase), and may also comprise recognition sites or binding sites for other transcription factors. An alphaviral SGP is a promoter which is native to an alphavirus, and which can regulate transcription of nucleic acids to which it is operably linked. In some embodiments, wildtype alphavirus SGPs regulate transcription of a structural protein and / or a non-structural protein (e.g., of a replicase protein). Wildtype alphavirus SGPs are typically found at a junction between nsP4 and structural (STR) coding regions of the alphaviral genome. These alphavirus SGPs are named according to the virus from which they are derived and the nucleotide positions spanned by the SGP, relative to the transcription start site. For example, an SFV SGP spanning 151 nucleotides, starting lOOnt upstream of the start site and ending 51nt downstream of the start site would be described as “SFV (-100 / 51).” An alphavirus SGP may be any alphavirus SGP known in the art.

[0266] In some embodiments, a linker is an SGP (e.g., an alphavirus SGP). In some embodiments, an SGP (e.g., an alphavirus SGP) is positioned upstream (i.e., 5’) of a nucleic acid encoding a payload, to which it is operably linked. In some embodiments, an SGP is a Chikungunya virus (CHIKV) SGP. In some embodiments, a CHIKV SGP is CHIKV (-75 / 69). In some embodiments, an SGP is an Eastern equine encephalitis virus (EEV) SGP. In some embodiments, a EEV SGP is EEV (-84 / 58). In some embodiments, an SGP is a Ross River virus (RRV) SGP. In some embodiments, an RRV SGP is RRV (-75 / 48). In some embodiments, an SGP is an SFV SGP. In some embodiments, a SFV SGP is SFV (-37 / 51); SFV (-50 / 0); SFV (- 50 / 51), SFV (-100 / 0), SFV (-100 / 51), SFV (-150 / 0), SFV (-150 / 51), SFV (-200 / 0), or SFV (- 200 / 51). In some embodiments, an SGP is an SINV SGP. In some embodiments, an SINV SGP is SINV (SINV-75 / 49). In some embodiments, an SGP is a western equine encephalitis virus (WEEV) SGP. In some embodiments, an WEEV SGP is WEEV (-84 / 37).

[0267] Alphavirus Replicase Junctions

[0268] In some embodiments, a linker is an alphaviral replicase junction region. The term “junction,” as used herein, refers to a region between the C-terminal amino acid of the first domain of a protein and the N-terminal amino acid of the second domain of a protein. A protein “domain” refers to a distinct functional or structural subunit of a protein or protein complex. For example, a replicase protein is a protein complex comprising nsPl, nsP2, nsP3, and nsP4, each of which may be referred to herein as a domain of a replicase; the skilled artisan will understand that a junction thus exists between nsPl and nsP2, between nsP2 and nsP3, and between nsP3 and nsP4. The junction between nsP3 and nsP4 domains is referred to herein as “3 / 4J”; similar nomenclature may be adopted for other junctions. In some embodiments, nsPl and nsP2 are in a fusion protein that comprises an amino acid sequence having 95% similarity to the sequence set forth in SEQ ID NO: 319. In some embodiments, nsPl and nsP2 are in a fusion protein that comprises the amino acid sequence set forth in SEQ ID NO: 319. In some embodiments, nsP3 comprises an amino acid sequence having 95% similarity to the sequence set forth in SEQ ID NO: 320. In some embodiments, nsP3 comprises the amino acid sequence set forth in SEQ ID NO: 320. In some embodiments, nsP4 comprises an amino acid sequence having 95% similarity to the sequence set forth in SEQ ID NO: 321. In some embodiments, nsP4 comprises the amino acid sequence set forth in SEQ ID NO: 321. In some embodiments, a 3 / 4J comprises the amino acid sequence set forth in SEQ ID NO: 226. In some embodiments, a 3 / 4J is encoded by the sequence set forth in SEQ ID NO: 128. In some embodiments, a 3 / 4J linker is joined to an FCS.

[0269] Illustrative taRNAs encoding IMP

[0270] In some embodiments, a taRNA comprises an RNA polynucleotide encoding one, two, three, four, five, six, or more IMPs. In some embodiments, IMPs encoded by an RNA polynucleotide may each be different. In some embodiments, a linker is between adjacent IMPs in the RNA polynucleotide; for example, a second IMP may be joined to a first IMP and / or a third IMP by a linker. In some embodiments, a taRNA comprises a nucleic acid encoding a first IMP and, optionally, a second IMP, a third IMP, a fourth IMP, a fifth IMP, and / or a sixth IMP.

[0271] In some embodiments, an RNA polynucleotide (e.g., trRNA) encodes one IMP. In some embodiments, an RNA polynucleotide encoding one IMP is a trRNA. In some embodiments, an RNA polynucleotide encoding two or more IMPs is a trRNA. It should be understood that unless a 5’ to 3’ configuration is specifically noted, the terms “first,” “second,” “third,” etc. do not denote a particular order.

[0272] In some embodiments, a taRNA comprises an RNA polynucleotide encoding a first IMP and a second IMP. In some embodiments, the first IMP and second IMP are different IMPs. In some embodiments, the first IMP and the second IMP are joined by a linker.

[0273] In some embodiments, the first IMP is E3L, and the second IMP is a different IMP. In some embodiments, the first IMP is E3L, and the second IMP is B18R. In some embodiments, the first IMP is E3L, and the second IMP is dnPKR. In some embodiments, the first IMP is E3L, and the second IMP is F1L. In some embodiments, the first IMP is E3L, and the second IMP is PR8 NS1. In some embodiments, the first IMP is E3L, and the second IMP is SOCS1. In some embodiments, the first IMP is E3L, the second IMP is a different IMP, and the two IMPs are joined by a linker. In some embodiments, the first IMP is E3L, the second IMP is B18R, and the linker is P2A. In some embodiments, the first IMP is E3L, the second IMP is dnPKR, and the linker is P2A. In some embodiments, the first IMP is E3L, the second IMP is F1L, and the linker is P2A. In some embodiments, the first IMP is E3L, the second IMP is PR8 NS1, and the linker is P2A. In some embodiments, the first IMP is E3L, the second IMP is SOCS1, and the linker is P2A. In some embodiments, the first IMP is E3L, the second IMP is S0CS1, and the linker is T2A.

[0274] In some embodiments, the first IMP is S0CS1, and the second IMP is a different IMP In some embodiments, the first IMP is SOCS1, and the second IMP is dnPKR. In some embodiments, the first IMP is SOCS1, and the second IMP is F1L. In some embodiments, the first IMP is SOCS1, and the second IMP is PR8 NS1. In some embodiments, the first IMP is SOCS1, and the second IMP is rZAPCC88R. In some embodiments, the first IMP is SOCS1, the second IMP is a different IMP, and the two IMPs are joined by a linker. In some embodiments, the first IMP is SOCS1, the second IMP is dnPKR, and the linker is P2A. In some embodiments, the first IMP is SOCS1, the second IMP is F1L, and the linker is P2A. In some embodiments, the first IMP is SOCS1, the second IMP is PR8 NS1, and the linker is P2A. In some embodiments, the first IMP is SOCS1, the second IMP is PR8 NS1, and the linker is P2A.

[0275] In some embodiments, the first IMP is PR8 NS1 and the second IMP is a different IMP In some embodiments, the first IMP is PR8 NS1 and the second IMP is TOSV NSs. In some embodiments, the first IMP is PR8 NS1, the second IMP is a different IMP, and the two IMPs are joined by a linker.

[0276] In some embodiments, a taRNA comprises a polynucleotide encoding a first IMP, a second IMP, and a third IMP. In some embodiments, the first IMP, second IMP, and third IMP are different IMPs. In some embodiments, the third IMP is E3L. In some embodiments, the third IMP is SOCS1. In some embodiments, the third IMP is F1L. In some embodiments, the third IMP is joined to the first IMP by a linker. In some embodiments, the third IMP is joined to the second IMP by a linker. In some embodiments, the first IMP is E3L, the second IMP is SOCS1, and the third IMP is F1L.

[0277] In some embodiments, a taRNA comprises a polynucleotide encoding a first IMP, a second IMP, a third IMP, and a fourth IMP. In some embodiments, the first IMP, the second IMP, the third IMP, and the fourth IMP are different IMPs. In some embodiments, a taRNA comprises a polynucleotide encoding a first IMP, a second IMP, a third IMP, a fourth IMP, and a fifth IMP. In some embodiments, the first IMP, the second IMP, the third IMP, the fourth IMP, and the fifth IMP are different IMPs. In some embodiments, a taRNA comprises a polynucleotide encoding a first IMP, a second IMP, a third IMP, a fourth IMP, a fifth IMP, and a sixth IMP. In some embodiments, the first IMP, the second IMP, the third IMP, the fourth IMP, the fifth IMP, and the sixth IMP are different IMPs. In some embodiments, the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B 18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and EMCV Lpro.

[0278] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid encoding an IMP and a nucleic acid encoding a payload or a nucleic acid payload, wherein the IMP and the nucleic acid encoding a payload or the nucleic acid payload are joined by a linker. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising nucleic acids encoding two or more IMPs and a nucleic acid encoding a payload or a nucleic acid payload, wherein one or two of the IMPs are joined to the nucleic acid pay load or nucleic acid encoding a pay load by one or more linkers.

[0279] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid encoding an IMP, and two or more (e.g., 2, 3, 4, 5, 6 or more) nucleic acids encoding payloads or nucleic acid payloads, optionally further comprising one or more linkers. In some embodiments, a trRNA comprises 2 nucleic acids encoding pay loads or nucleic acid pay loads. In some embodiments, the skilled artisan will appreciate that each nucleic acid in such an RNA polynucleotide may be arranged in any desired configuration. For example, a taRNA may comprise an RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid encoding an IMP, a first nucleic acid encoding a payload or a nucleic acid payload, and second a nucleic acid encoding a payload or a nucleic acid payload, wherein the IMP and the first nucleic acid encoding a payload or a nucleic acid payload are joined by a first linker and the IMP and the second the nucleic acid encoding a pay load or the nucleic acid pay load are joined by linkers. In another example, a taRNA may comprise an RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid encoding an IMP, a first nucleic acid encoding a payload or a nucleic acid payload, and second a nucleic acid encoding a payload or a nucleic acid payload, wherein the first nucleic acid encoding a payload or a nucleic acid payload and the second the nucleic acid encoding a payload or the nucleic acid payload are joined by linkers, and the nucleic acid encoding the IMP is joined to the first or second nucleic acid encoding a payload or a nucleic acid payload.

[0280] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP and (ii) a nucleic acid encoding a payload or a nucleic acid pay load, wherein the nucleic acids of (i) and (ii) are flanked by a 5’ CSE and a 3’ CSE. In some embodiments, a taRNA comprises an RNA polynucleotide comprising (i) a nucleic acid encoding an IMP and (ii) a nucleic acid encoding a payload or a nucleic acid pay load, wherein the nucleic acids of (i) and (ii) are flanked by a 5’ SFV UTR comprising a CSE and a 3’ SFV UTR comprising CSE. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP and (ii) a nucleic acid encoding a payload or a nucleic acid payload, wherein the nucleic acids of (i) and (ii) are flanked by a 5’ SINV UTR comprising a CSE UTR and a 3’ SINV UTR comprising a CSE. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP and (ii) a nucleic acid encoding a payload or a nucleic acid pay load, wherein the nucleic acids of (i) and (ii) are flanked by a 5’ SINV UTR comprising a CSE and a 3’ SFV UTR comprising a CSE.

[0281] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ CSE and a 3’ CSE. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ SINV UTR comprising a CSE and a 3’ SINV UTR comprising a CSE. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ SFV UTR comprising a CSE and a 3’ SFV UTR comprising a CSE. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ SINV UTR comprising a CSE and a 3’ SFV UTR comprising a CSE.

[0282] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ UTR and a 3’ UTR. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ UTR and a 3’ UTR, wherein the 5’ UTR comprises a CSE cognate to the replicase of the taRNA. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ UTR and a 3’ UTR, wherein the 3’ UTR comprises a CSE cognate to the replicase of the taRNA. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ SINV UTR and a 3’ SINV UTR. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ SFV UTR and a 3’ SFV UTR. In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a trRNA) comprising (i) a nucleic acid encoding an IMP, (ii) a nucleic acid encoding a payload or a nucleic acid payload, and (iii) a linker, wherein the nucleic acids of (i) and (ii) are joined by the linker, and are flanked by a 5’ SINV UTR and a 3’ SFV UTR.

[0283] Configuration-specific trRNAs encoding IMPs

[0284] Described hereinabove are various embodiments of RNA polynucleotides comprising IMPs. As evidenced herein, such as by the Examples, these various designs are suitable for numerous applications. Further described herein are RNA polynucleotides (e.g., trRNAs) having specific configurations of nucleic acids encoding IMP(s) and nucleic acids encoding payloads or nucleic acid pay loads.

[0285] In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding an IMP; a linker; and nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP or EMCV Lpro; a linker; and nucleic acid encoding a payload or a nucleic acid payload.

[0286] In some embodiments, a trRNA comprises, from 5’ to 3’ : a nucleic acid encoding E3L; a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide); and nucleic acid encoding a payload or a nucleic acid pay load. In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding SOCS1; a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide); and nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, a trRNA comprises, from 5’ to 3’ : a nucleic acid encoding PR8 NS1; a linker (e.g., a CVB3 IRES, a T2A peptide, a

[0287] P2A peptide); and nucleic acid encoding a payload or a nucleic acid payload.

[0288] In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVH, dnPKR, SOCS, dnZAP, and / or EMCV Lpro); a CVB3 IRES; and nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVH, dnPKR, SOCS, dnZAP, and / or EMCV Lpro); a T2A peptide; and a nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVH, dnPKR, SOCS, dnZAP, and / or EMCV Lpro); a P2A peptide; and a nucleic acid encoding a payload or a nucleic acid payload.

[0289] In some embodiments, a trRNA comprises, from 5’ to 3’: a 5’ CSE, a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US 11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVH, dnPKR, SOCS, dnZAP, and / or EMCV Lpro), a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide), a nucleic acid encoding a pay load or a nucleic acid pay load, and a 3’ CSE. In some embodiments, a trRNA comprises, from 5’ to 3’: a 5’ UTR, a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVH, dnPKR, SOCS, dnZAP, and / or EMCV Lpro), a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide), a nucleic acid encoding a pay load or a nucleic acid pay load, and a 3’ UTR.

[0290] In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding a payload or a nucleic acid payload; a linker; and a nucleic acid encoding an IMP. In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding a pay load or a nucleic acid payload; a linker; and a nucleic acid encoding an IMP independently selected from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVH, dnPKR, SOCS, dnZAP or Lpro.

[0291] In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding a payload or a nucleic acid payload; a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide); and a nucleic acid encoding E3L. In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding a payload or a nucleic acid payload; a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide); and a nucleic acid encoding SOCS 1. In some embodiments, a trRNA comprises, from 5’ to 3’ : a nucleic acid encoding a payload or a nucleic acid payload; a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide); and a nucleic acid encoding PR8 NS1.

[0292] In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding a payload or a nucleic acid payload; a CVB3 IRES; and a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and / or EMCV Lpro). In some embodiments, a trRNA comprises, from 5’ to 3’: a nucleic acid encoding a pay load or a nucleic acid payload; T2A peptide; and a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and / or EMCV Lpro).

[0293] In some embodiments, a trRNA comprises, from 5’ to 3’: a 5’ CSE, a nucleic acid encoding a payload or a nucleic acid payload, a linker (e.g., a CVB3 IRES, a T2A peptide, a P2A peptide), a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and / or EMCV Lpro), and a 3’ CSE. In some embodiments, a trRNA comprises, from 5’ to 3’: a 5’ UTR, a nucleic acid encoding a pay load or a nucleic acid payload, a linker, a nucleic acid encoding an IMP (e.g., VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and / or EMCV Lpro), and a 3’ UTR.

[0294] In some embodiments, a taRNA comprises a trRNA comprising, from 5’ to 3’: S’-CSE1- IMP1-L1-IMP2-L2-IMP3-L3-PCS-L4-IMP4-L5-IMP5-L6-IMP6-CSE2-3’, wherein:

[0295] CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0296] IMP1, IMP2, IMP3, IMP4, IMP5, and IMP6are each independently an IMP;

[0297] L1, L2, L3, L4, L5, and L6are each independently a linker;

[0298] PCS is a nucleic acid payload or a nucleic acid encoding a payload; and

[0299] IMP2-L2, IMP3-L3, L4-IMP4, L5-IMP5, and L6-IMP6are each independently optionally absent. In some embodiments, the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and EMCV Lpro. An exemplary RNA polynucleotide according to this construction is depicted in FIG. 6A.

[0300] In some embodiments, a taRNA comprises a trRNA comprising, from 5’ to 3’: 5’-CSEl- IMP1-L1-IMP2-L2-IMP3-L3-PCS-L4-IMP4-L5-IMP5-L6-IMP6-CSE2-3’; wherein:

[0301] CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0302] IMP1, IMP2, IMP3, IMP4, IMP5, and IMP6are each independently an immune modulating protein (IMP);

[0303] L1, L2, L3, L4, L5, and L6are each independently a linker;

[0304] PCS is a nucleic acid payload or a nucleic acid encoding a payload; and

[0305] IMP^L1, IMP2-L2, IMP3-L3, L5-IMP5, and L6-IMP6are each independently optionally absent. In some embodiments, the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and EMCV Lpro. An exemplary RNA polynucleotide according to this construction is depicted in FIG. 6B.

[0306] In some embodiments, a taRNA comprises a trRNA comprising, from 5’ to 3’: S’-CSE1- IMP1-L1-IMP2-L2-IMP3-L3-PCS-L4-IMP4-CSE2-3’ ; wherein:

[0307] CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0308] IMP1, IMP2, IMP3, and IMP4are each independently an immune modulating protein (IMP);

[0309] L1, L2, L3, and L4are each independently a linker;

[0310] PCS is a nucleic acid payload or a nucleic acid encoding a payload; and

[0311] IMP2-L2, IMP3-L3, and L4-IMP4are each independently optionally absent. In some embodiments, the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and EMCV Lpro. An exemplary RNA polynucleotide according to this construction is depicted in FIG. 6C.

[0312] In some embodiments, a taRNA comprises a trRNA comprising, from 5’ to 3’: S’-CSE1- IMP1-L1-PCS-IMP2-L2-IMP3-L3-L4-IMP4-CSE2-3’; wherein: CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;

[0313] IMP1, IMP2, IMP3, and IMP4are each independently an immune modulating protein (IMP);

[0314] L1, L2, L3, and L4are each independently a linker;

[0315] PCS is a nucleic acid payload or a nucleic acid encoding a payload; and

[0316] IMP^L1, IMP3-L3, and L4-IMP4are each independently optionally absent. In some embodiments, the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and EMCV Lpro. An exemplary RNA polynucleotide according to this construction is depicted in FIG. 6D.

[0317] Additional examples of RNA polynucleotides (e.g., trRNAs) having specific configurations of nucleic acids encoding IMP(s) and nucleic acids encoding payloads or nucleic acid pay loads are provided in Table 7.

[0318] Replicase-IMP Fusion Proteins

[0319] Described herein, in some aspects, are RNA polynucleotides encoding a replicase and an IMP. In some embodiments, an RNA polynucleotide encoding a replicase and an IMP encodes a replicase-IMP fusion protein. The term “fusion protein,” also known as a chimeric protein, refers to any protein comprising at least two domains wherein each domain is derived from a different protein, and wherein the at least two domains are joined at a fusion junction. Domains of a fusion protein may be joined directly by a covalent bond, or by a linker. Though domains of a fusion protein are encoded by the same polynucleotide, these domains may be cleaved or otherwise separated, for example, during translation or via post-translational modification.

[0320] In some aspects, a replicase-IMP fusion protein comprises a replicase protein wherein an IMP is inserted between domains of the replicase, for example, after the 3 / 4J between the nsP3 and nsP4 domains, but before the nsP4 (as in FIG. 18A), such that the replicase-IMP fusion protein comprises nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4. In some embodiments, a nsPl-nsP2-nsP3- 3 / 4J-IMP-nsP4 replicase-IMP fusion protein is encoded by a replicase construct comprising, from 5’ to 3’: a 5’UTR, a nucleic acid encoding nsPl, a nucleic acid encoding nSP2, a nucleic acid encoding nsP3, a nucleic acid encoding an IMP, a nucleic acid encoding nsP4, and a 3’UTR (e.g., as shown in FIG. 19A). In some embodiments, a nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4 replicase-IMP fusion protein further comprises a 3 / 4J duplicate. A “3 / 4J duplicate”, as referred to herein, comprises a 3 / 4J which is inserted into a replicase protein, such that the replicase protein comprises two 3 / 4Js. In some embodiments, an IMP is flanked between a 3 / 4J and a 3 / 4J duplicate and comprises: nsPl-nsP2-nsP3-3 / 4J-IMP-3 / 4J-nsP4. In some embodiments, a nsPl- nsP2-nsP3-3 / 4J-IMP-nsP4 (e.g., nsPl-nsP2-nsP3-3 / 4J-IMP-3 / 4J-nsP4) replicase-IMP fusion protein is made using a wild-type replicase (e.g., a wildtype SFV replicase), and is referred to herein as a WT SFV a nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4 (e.g., WT SFV nsPl-nsP2-nsP3-3 / 4J- IMP-3 / 4J-nsP4). In some embodiments, the 3 / 4J replicase-IMP fusion protein is made using an RLE replicase. and is referred to herein as a RLE a nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4 (e.g., RLE nsP 1 -nsP2-nsP3-3 / 4J-IMP-3 / 4J-nsP4).

[0321] In some embodiments, a replicase-IMP fusion protein comprises dnPKR inserted between two domains of a replicase. In some embodiments, a replicase-IMP fusion protein comprises dnPKR inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4). In some embodiments, a replicase-IMP fusion protein comprises dnPKR inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4) and a 3 / 4J duplicate. In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2- nsP3-3 / 4J-dnPKR-3 / 4J-nsP4 replicase-IMP fusion protein. In some embodiments, an RLE nsPl- nsP2-nsP3-3 / 4J-dnPKR-3 / 4J-nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, an RLE nsPl-nsP2-nsP3-3 / 4J- dnPKR-3 / 4J-nsP4 replicase-IMP fusion protein is encoded by the nucleic acid sequence set forth in SEQ ID NO: 148. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl- nsP2-nsP3-3 / 4J-dnPKR-3 / 4J-nsP4 replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J-dnPKR-3 / 4J-nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, an SFV WT nsPl-nsP2- nsP3-3 / 4J-dnPKR-3 / 4J-nsP4 replicase-IMP fusion protein is encoded by the nucleic acid sequence set forth in SEQ ID NO: 149.

[0322] In some embodiments, a replicase-IMP fusion protein comprises E3L inserted between two domains of a replicase. In some embodiments, a replicase-IMP fusion protein comprises E3L inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4). In some embodiments, a replicase-IMP fusion protein comprises E3L inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4) and a 3 / 4J duplicate. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-3 / 4J-E3L-3 / 4J- nsP4 replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J- E3L-3 / 4J-nsP4 replicase-IMP fusion protein further comprises an FCS between the E3L and 3 / J duplicate, such that the replicase-IMP fusion protein comprises SFV WT nsPl-nsP2-nsP3-3 / 4J- E3L-FCS-3 / 4J-nsP4. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J-E3L-FCS-3 / 4J- nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J-E3L-FCS-3 / 4J-nsP4 replicase- IMP fusion protein is encoded by the nucleic acid sequence set forth in SEQ ID NO: 150.

[0323] Subgenomic promoters within the nsP4 domain of the replicase protein are considered essential for expression of downstream sequences and are thought to function as a conserved sequence element (CSE) (see: Schmidt, C., & Schnierle, B. S. (2023). Self-amplifying RNA Vaccine Candidates: Alternative Platforms for mRNA Vaccine Development. Pathogens, 12, 138. doi: 10.3390 / pathogens 12010138). Accordingly, functional replicase constructs encoding a replicase protein fused to another protein at the C-terminus are considered to render replicases unable to mediate expression of downstream sequences. However, the disclosure describes the surprising discovery that such fusion proteins are not only functional as replicase proteins, but may be useful for IMP expression (e.g., see Example 12).

[0324] In some embodiments, a replicase-IMP fusion protein comprises a replicase which is fused to an IMP on either or both terminals (i.e., not between domains of the replicase). In some embodiments, a replicase-IMP fusion protein comprises an IMP fused to the C-terminal of a replicase protein. In some embodiments, a replicase-IMP fusion protein comprises an IMP fused to the N-terminal of a replicase protein. In some embodiments, a replicase-IMP fusion protein comprises a first IMP fused to the C-terminal of a replicase protein, and a second IMP fused to the N-terminal of a replicase protein. In some embodiments, the first IMP and the second IMP are the same IMP. In some embodiments, the first IMP and the second IMP are different IMPs. In some embodiments, an IMP is fused to the replicase using a linker (e.g., linked to the C- terminal and / or N-terminal of the replicase).

[0325] In some embodiments, a replicase-IMP fusion protein comprises a replicase which is fused to an VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, HSV ICP0, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and / or Lpro. In some embodiments, a replicase-IMP fusion protein comprises nsPl-nsP2-nsP3-nsP4- IMP. In some embodiments, a replicase-IMP fusion protein comprises nsPl-nsP2-nsP3-nsP4- 3 / 4J-IMP. In some embodiments, a replicase-IMP fusion protein comprises nsPl-nsP2-nsP3- nsP4-linker-IMP (e.g., as shown in FIG. 18B). In some embodiments, a nsPl-nsP2-nsP3-nsP4- linker-IMP replicase-IMP fusion protein is encoded by a replicase construct comprising, from 5’ to 3’: a 5’ UTR, a nucleic acid encoding nsPl, a nucleic acid encoding nsP2, a nucleic acid encoding nsP3-nsP4, a nucleic acid encoding a linker, a nucleic acid encoding an IMP, and a 3’ UTR (e.g., as shown in FIG. 19B). In some embodiments, a replicase-IMP fusion protein comprises IMP-nsPl-nsP2-nsP3-nsP4. In some embodiments, a replicase-IMP fusion protein comprises IMP-3 / 4J-nsPl-nsP2-nsP3-nsP4. In some embodiments, a replicase-IMP fusion protein comprises IMP-linker-nsPl-nsP2-nsP3-nsP4 (e.g., as shown in FIG. 18C). In some embodiments, an IMP-linker-nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein is encoded by a replicase construct comprising, from 5’ to 3’: a 5’ UTR, a nucleic acid encoding an IMP, a nucleic acid encoding a linker, a nucleic acid encoding nsPl, a nucleic acid encoding nsP2, a nucleic acid encoding nsP3-nsP4, and a 3’ UTR (e.g., as shown in FIG. 19C). In some embodiments, a replicase-IMP fusion protein comprises IMP-nsPl-nsP2-nsP3-nsP4-IMP. In some embodiments, a replicase-IMP fusion protein comprises IMP-nsPl-nsP2-nsP3-nsP4-3 / 4J- IMP. In some embodiments, a replicase-IMP fusion protein comprises IMP 1 -linker l-nsPl-nsP2- nsP3-nsP4-linker2-IMP2 (e.g., as shown in FIG. 18D), wherein IMP1 and IMP2 are both IMPs and linkerl and linker2 are both linkers; IMP1 and IMP2 may be the same IMP, or may be different IMPs, and linkerl and linker2 may be the same linker, or may be different linkers. In some embodiments, an IMP-linker-nsPl-nsP2-nsP3-nsP4-linker-IMP replicase-IMP fusion protein is encoded by a replicase construct comprising, from 5’ to 3’: a 5’ UTR, a nucleic acid encoding a first IMP, a nucleic acid encoding a first linker, a nucleic acid encoding nsPl, a nucleic acid encoding nsP2, a nucleic acid encoding nsP3-nsP4, a nucleic acid encoding a second linker, a nucleic acid encoding a second IMP, and a 3’ UTR (e.g., as shown in FIG.

[0326] 19D). Generally, replicase-IMP fusion proteins comprising a linker comprise a 2A peptide and do not comprise an IRES.

[0327] Exemplary Replicase-IMP Fusion Proteins

[0328] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to dnPKR. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to dnPKR via a 3 / 4 J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to dnPKR via a linker. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to dnPKR and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-T2A- dnPKR replicase-IMP fusion protein. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4- T2A-dnPKR replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-dnPKR replicase-IMP fusion protein is encoded by SEQ ID NO: 151. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-dnPKR replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-dnPKR replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-dnPKR replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 152. In some embodiments, a replicase-IMP fusion protein is an REE nsPl-nsP2-nsP3-nsP4-T2A-dnPKR replicase-IMP fusion protein. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-dnPKR replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4- T2A-dnPKR replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 159. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3- nsP4-T2A-dnPKR replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2- nsP3-nsP4-T2A-dnPKR replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 258. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A- dnPKR replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 160.

[0329] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to E3L. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to E3L via a 3 / 4J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to E3L via a linker. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to E3L and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-E3L. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-E3L comprises the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-E3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 153. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-E3L. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-E3L comprises the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, an SFV WT nsPl-nsP2-nsP3- nsP4-3 / 4J-E3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 154. In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2-nsP3- nsP4-T2A-E3L. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-E3L comprises the amino acid sequence set forth in SEQ ID NO: 259. In some embodiments, an RLE nsPl-nsP2- nsP3-nsP4-T2A-E3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 161. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2- nsP3-nsP4-T2A-E3L. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-E3L comprises the amino acid sequence set forth in SEQ ID NO: 260. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-E3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 162.

[0330] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to Lpro. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to Lpro via a 3 / 4J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to Lpro via a linker. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to Lpro and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-Lpro replicase-IMP fusion protein. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-Lpro replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-Lpro replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 155. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-Lpro replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-Lpro replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-Lpro replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 156. In some embodiments, a replicase-IMP fusion protein is RLE nsPl-nsP2-nsP3-nsP4-T2A-Lpro replicase-IMP fusion protein. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-Lpro replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 262. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4- T2A-Lpro replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 164. In some embodiments, a replicase-IMP fusion protein is SFV WT nsPl-nsP2-nsP3-nsP4- T2A-Lpro replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3- nsP4-T2A-Lpro replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 263. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-Lpro replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 165.

[0331] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to F1L. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to F1L via a 3 / 4J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to F1L via a linker. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to F1L and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-FlL replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A- F1L replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 261. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-FlL replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 163.

[0332] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to SOCS1. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to SOCS1 via a 3 / 4J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to SOCS1 via a linker. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to SOCS1 and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J- SOCS1 replicase-IMP fusion protein. In some embodiments, an SFV RLE nsPl-nsP2-nsP3- nsP4-3 / 4J-SOCSl replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-SOCSl replicase- IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 157. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-SOCSl replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J- SOCS1 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-SOCSl replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 158. In some embodiments, a replicase-IMP fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-T2A-SOCSl replicase-IMP fusion protein. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-SOCSl replicase- IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 264. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-SOCSl replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 166. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-SOCSl replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-SOCSl replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 265. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-SOCSl replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 167. In some embodiments, a replicase-IMP fusion protein is an RLE SOCSl-3 / 4J-nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, an RLE SOCSl-3 / 4J-nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 266. In some embodiments, an RLE SOCSl-3 / 4J-nsPl- nsP2-nsP3-nsP4 replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 168. In some embodiments, a replicase-IMP fusion protein is an RLE SOCSl-T2A-nsPl- nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, an RLE SOCS1-T2A- nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 267. In some embodiments, an RLE SOCSl-T2A-nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 169.

[0333] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to PR8 NS1. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to PR8 NS1 via a 3 / 4J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to PR8 NS1 via a linker. In some embodiments, a replicase-IMP fusion protein is a SFV WT nsPl-nsP2-nsP3-nsP4-T2A-PR8 NS1 replicase-IMP fusion protein. In some embodiments, a SFV WT nsPl-nsP2-nsP3-nsP4-T2A-PR8 NS1 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 268. In some embodiments, a SFV WT nsPl- nsP2-nsP3-nsP4-T2A-PR8 NS1 replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 170. In some embodiments, a replicase-IMP fusion protein is a PR8 NS1- T2A-SFV WT nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, a PR8 NS1-T2A-SFV WT nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 269. In some embodiments, a PR8 NS1-T2A-SFV WT nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 171.

[0334] In some embodiments, a replicase-IMP fusion protein comprises a replicase fused to TOSV NSs. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to TOSV NSs via a 3 / 4J. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to TOSV NSs via a linker. In some embodiments, a replicase-IMP fusion protein is a TOSV NSs-T2A-SFV WT nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, a TOSV NSs-T2A-SFV WT nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 271. In some embodiments, a TOSV NSs-T2A-SFV WT nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 173. In some embodiments, a replicase-IMP fusion protein is a SFV WT nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein. In some embodiments, a SFV WT nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 270. In some embodiments, a SFV WT nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 172.

[0335] In some embodiments, a replicase-IMP fusion protein comprises a replicase which is fused to a first IMP fused to the C-terminal of a replicase protein and an IMP fused to the N- terminal of the replicase protein. In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to PR8 NS1 and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to VACV E3L and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to K3L and another IMP (e.g., directly fused, via a 3 / 4J, via a linker). In some embodiments, a replicase-IMP fusion protein comprises a replicase joined to TOSV NSs and another IMP (e.g., directly fused, via a 3 / 4 J, via a linker).

[0336] In some embodiments, a replicase-IMP fusion protein is a PR8 NS1-T2A-SFV WT nsPl- nsP2-nsP3-nsP4-VACV E3L replicase-IMP fusion protein. In some embodiments, a PR8 NS1- T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A-VACV E3L replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 272. In some embodiments, a PR8 NS1-T2A- SFV WT nsPl-nsP2-nsP3-nsP4-P2A-VACV E3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 174.

[0337] In some embodiments, a replicase-IMP fusion protein is a VACV E3L-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-VACV K3L replicase-IMP fusion protein. In some embodiments, a VACV E3L-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A-VACV K3L replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 273. In some embodiments, a VACV E3L-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A-VACV K3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 175. In some embodiments, a replicase-IMP fusion protein is a VACV K3L-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A-VACV E3L replicase-IMP fusion protein. In some embodiments, a VACV K3L-T2A-SFV WT nsPl- nsP2-nsP3-nsP4-P2A-VACV E3L replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 274. In some embodiments, a VACV K3L-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A-VACV E3L replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 176.

[0338] In some embodiments, a replicase-IMP fusion protein is a PR8 NS1-P2A-SFV WT nsPl- nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein. In some embodiments, a PR8 NS1-P2A-SFV WT nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 324. In some embodiments, a PR8 NS1-P2A-SFV WT nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 323. In some embodiments, a replicase-IMP fusion protein is a TOSV NSs-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A-PR8 NS1 replicase- IMP fusion protein. In some embodiments, a TOSV NSs-T2A-SFV WT nsPl-nsP2-nsP3-nsP4- P2A-PR8 NS1 replicase-IMP fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 327. In some embodiments, a TOSV NSs-T2A-SFV WT nsPl-nsP2-nsP3-nsP4-P2A- PR8 NS1 replicase-IMP fusion protein is encoded by the sequence set forth in SEQ ID NO: 326.

[0339] In some embodiments, a replicase-IMP fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 246-274, 324, or 327. In some embodiments, a replicase- IMP fusion protein is encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs: 148-176, 323, or 326. taRNAs encoding IMPs

[0340] Provided herein are taRNAs comprising a first RNA polynucleotide (i.e., a replicase construct) and a second RNA polynucleotide (i.e., a trRNA), wherein one or both RNA polynucleotide encodes an IMP. In some embodiments, a taRNA comprises: a first RNA polynucleotide (i.e., a replicase construct) comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a payload and a nucleic acid encoding a second IMP. For example, a taRNA may comprise a replicase construct encoding a replicase-IMP fusion protein and a trRNA encoding a payload and an IMP.

[0341] In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a pay load and a nucleic acid encoding a second IMP, wherein the first IMP is SOCS1. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a payload and a nucleic acid encoding a second IMP, wherein the first IMP is E3L. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a payload and a nucleic acid encoding a second IMP, wherein the first IMP is dnPKR. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a pay load and a nucleic acid encoding a second IMP, wherein the second IMP is E3L. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a payload and a nucleic acid encoding a second IMP, wherein the second IMP is SOCS1. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a payload and a nucleic acid encoding a second IMP, wherein the second IMP is F1L.

[0342] In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a SOCS1; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a pay load and a nucleic acid encoding E3L. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a E3L; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a pay load and a nucleic acid encoding SOCS1. In some embodiments, a taRNA comprises: a first RNA polynucleotide comprising a nucleic acid encoding a replicase and a nucleic acid encoding a dnPKR; and a second RNA polynucleotide (i.e., a trRNA) comprising a nucleic acid sequence encoding a payload and a nucleic acid encoding F1L.

[0343] In some embodiments, a taRNA comprises a replicase construct comprising the sequence set forth in any one of SEQ ID NO: 148-176. In some embodiments, a replicase construct is encoded by a DNA polynucleotide comprising the sequence set forth in any one of SEQ ID NO: 38-66.

[0344] In some embodiments, a taRNA comprises a trRNA comprising the sequence set forth in any one of SEQ ID NO: 177-220. In some embodiments, a trRNA is encoded by a DNA polynucleotide comprising the sequence set forth in any one of SEQ ID NO: 67-110.

[0345] In some embodiments, a taRNA comprises replicase constructs and trRNA constructs in about equal amounts (e.g., a molar ratio of replicase construct:trRNA of about 1:1). In some embodiments, a taRNA comprises more replicase constructs than trRNA (e.g., a molar ratio of replicase construct:trRNA construct of about 5:1, 10:1, 100:1, 1000:1, or more). saRNA encoding IMPs

[0346] Provided herein, in some aspects, are self-amplifying ribonucleic acids (saRNAs) comprising a nucleic acid encoding a nucleic acid encoding an IMP, a linker, and a replicase; and a nucleic acid encoding a payload or a nucleic acid payload. saRNAs are a single polynucleotide which comprises, from 5’ to 3’, an alphavirus UTR, a nucleic acid encoding a replicase, a nucleic acid comprising an alphaviral gene or pay load encoding sequence, and a 3’ alphavirus UTR. saRNAs are known, e.g., as described in Comes JDG et al., Trends Biotechnol. 2023 Nov;41(l l):1417-1429.

[0347] In some embodiments, an saRNA comprises a nucleic acid encoding a replicase-IMP fusion protein. In some embodiments, an saRNA comprises a nucleic acid encoding a replicase- IMP fusion protein and a nucleic acid encoding an IMP. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, an IMP, a linker, and a replicase; and a nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, an IMP, a 2A peptide, and a replicase; and a nucleic acid encoding a payload or a nucleic acid payload.

[0348] In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a first linker, and a replicase; a nucleic acid encoding a payload or a nucleic acid payload, and a second IMP. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a 2A peptide, and a replicase; a nucleic acid encoding a payload or a nucleic acid payload, and a second IMP. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a first linker, and a replicase; a nucleic acid encoding a payload or a nucleic acid payload, a second linker and a second IMP. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a 2A peptide, and a replicase; a nucleic acid encoding a payload or a nucleic acid payload, a linker and a second IMP.

[0349] In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a first linker, and a replicase; a nucleic acid encoding a second IMP, and a nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a 2A peptide, and a replicase; a nucleic acid encoding a second IMP, and a nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a linker, and a replicase; a nucleic acid encoding a second IMP, a linker, and a nucleic acid encoding a payload or a nucleic acid payload. In some embodiments, an saRNA comprises a nucleic acid encoding, from 5’ to 3’, a first IMP, a 2A peptide, and a replicase; a nucleic acid encoding a second IMP, a linker, and a nucleic acid encoding a payload or a nucleic acid payload.

[0350] Methods of Use

[0351] In some embodiments, this disclosure describes a cell comprising one or more of any of the taRNAs described herein. In some aspects, the taRNAs described herein may be used to express a payload in a mammalian cell. Mammalian cells may be derived from any mammal, including, but not limited to, mice, hamsters, pigs, cows, sheep, goats, horses, and primates, including humans. In some embodiments, the taRNA described herein are used to express pay loads in isolated cells from established cell lines generally known in the art, such as 3T3, A549, BHK21, C127, CHO, HeLa, HEK, HT-1080, Huh7, Jurkat, NSO, PER.C6, Sp2 / 0, Vero, and derivatives thereof. In some embodiments, the taRNA described herein are used to express payloads in cells isolated from a subject (e.g., immune cells).

[0352] In some embodiments, the taRNAs described herein are transfected into a cell. The term “transfection” refers to the process by which cells uptake foreign polynucleotides into the cytoplasm, in the absence of viral vectors. Non-limiting examples of transfection include electroporation, heat shock, liposome-mediated delivery, nanoparticle-mediated delivery, microinjections, sonoporation, photoporation, magnetofection, hydroporation, biolistics, continuous infusion, impalefection, and any technique known to those of ordinary skill in the art. In some embodiments, a cell transfected with a taRNA is transfected with the replicase construct and trans replicon construct concurrently (i.e., at the same time). In some embodiments, a cell transfected with a taRNA is transfected with the replicase construct and trans replicon construct sequentially; for example, the replicase construct may be transfected at a first time point and a trans replicon construct transfected at a second, later, time point. Once one or more foreign polynucleotides have entered the cytoplasm of a cell, the polynucleotides may be expressed by the cell. In some embodiments, a cell transfected with a taRNA expresses the replicase encoded by the replicase construct. In some embodiments, a cell transfected with a taRNA expresses (i.e., translates) the pay load encoded by the trans replicon construct or its replicants.

[0353] In some aspects, the taRNAs described herein may be used to express a payload in a cell (e.g., a cell of subject). taRNAs described herein may be administered to a subject using any method known to those of ordinary skill in the art, such as injection (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). In some embodiments, the taRNAs described herein are formulated as a pharmaceutical composition. A “pharmaceutical composition” refers to a composition comprising taRNAs formulated with one or more pharmaceutically acceptable excipients. In some embodiments, taRNAs are formulated in a carrier (e.g., lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, ligand). In some embodiments, one or more cells transfected with taRNAs may be administered to the subject.

[0354] In some embodiments, taRNAs are administered to mammalian subjects. Non-limiting examples of mammalian subjects include mice, hamsters, pigs, cows, sheep, goats, horses, and primates, including humans. In some embodiments, taRNAs are administered to human subjects.

[0355] In some embodiments, taRNAs are expressed in a cell-free system, for example, using in vitro transcription.

[0356] Exemplary Sequences

[0357] Table 1- 5’ UTRs

[0358] Table 2- 3’ UTRs

[0359] Table 3- Exemplary Linker Sequences

[0360] Table 4- Exemplary Replicase Sequences Table 5- Exemplary IMP Sequences

[0361] VACV E3L

[0362] TOSV NS

[0363] Table 6- Exemplary Replicase Fusion Protein Sequences -2T2- -Zll -

[0364] Table 7- Exemplary trRNA Constructs

[0365] Table 8- SFV nSP sequences

[0366] EXAMPLES

[0367] Example 1: Interferon Signaling Limits Expression of taRNA in vivo

[0368] Host anti-viral immune responses have been suggested to contribute to instability and low expression RNA therapeutics. To evaluate the effects of interferon (IFN) signaling on trans amplifying RNA (taRNA) expression in vivo, adult (2-6 months) wildtype mice and Type 1 IFN receptor knockout (IFNAR7') mice were injected with secreted embryonic alkaline phosphatase (SEAP)-encoding taRNA. SEAP-encoding taRNA included a replicase construct encoding a wildtype replicase, and a trans replicating RNA (trRNA) encoding SEAP; and were produced by a standard in vitro transcription reaction using a linearized DNA template. taRNA were prepared for injection and mice were injected with Ipg taRNA (1:1 molar ratio of replicase construct:trRNA) total via intramuscular (IM) administration. Mice were bled and sera was collected at 8 hours, 24 hours, and 72 hours and SEAP expression was determined using a standard SEAP reporter gene assay.

[0369] As shown in FIG. 1, taRNA-mediated SEAP expression was 13.1 -fold (AUC) higher in IFNAR7' mice than in wildtype mice; notably, this effect persisted through 72 hours. These results suggest that antagonism of IFN responses may improve expression of taRNA-encoded pay loads in vivo. Example 2: Co-transfection oftaRNA and. Immune Modulating Protein (IMP fencoding mRNAs

[0370] In these experiments, Raw macrophages were co-transfected with payload-encoding taRNA and mRNAs encoding an immune modulating protein (IMP). SEAP-encoding taRNAs, including: a replicase construct encoding a wildtype Semliki Forest Virus (SFV) replicase or RLE replicase; and a trRNA encoding SEAP, and mRNA encoding Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) NS1 or SOCS1 were produced as described in Example 1. Raw macrophages were transfected with 5.5ng, 22ng, or 44ng of total RNA.

[0371] Raw macrophages transfected with SEAP-encoding taRNA, and SOCS1 -encoding mRNA exhibited 2-4x expression with wildtype SFV replicase and more than 8x expression with RLE replicase at 24 hours post-transfection (FIG. 2A), supporting antagonism of IFN to maximize expression of taRNA-encoded pay loads. Importantly, though higher doses of RNA increased ISG54 induction overall, combinations of taRNAs with IMP-encoding mRNA reduced ISG54 induction by as much as 4x, compared to equivalent doses of taRNA only (FIG. 2B).

[0372] In a related experiment, SEAP expression and IP- 10 chemokine levels was measured from supernatant of the Raw cells at 6, 24, 48, and 72 hours after transfection with SEAP- encoding taRNA, SEAP-encoding taRNA and SOCS1 -encoding mRNA, or SEAP-encoding mRNA. Early expression (at 6 hours and 24 hours) was enhanced in cells co-transfected with SEAP-encoding taRNA and SOCS1 -encoding mRNA, with peak expression enhancement occurring at 24 hours (FIG. 3A). IP- 10 chemokine levels at 24 hours were compared to SEAP expression at the same time (FIG. 3B). Reduced IP- 10 chemokine levels were found to generally correlate with increased SEAP expressions, further suggesting that antagonism of immune pathways may increase RNA-mediated payload expression.

[0373] Example 3: Co-Expression of Multiple 2A Peptide-Linked Proteins on a Single trRNA

[0374] While co-transfection of cells with taRNA and IMP-encoding mRNAs improved expression of taRNA-mediated pay loads in vitro, co-localization of multiple different mRNAs in vivo can introduce additional complexities to heterologous RNA expression. Accordingly, it may be desirable to express multiple proteins (e.g., an IMP), on a single RNA polynucleotide. In these proof-of-concept experiments, nucleic acids encoding multiple proteins joined by a linker were embedded into trRNA constructs (FIG. 4A). BHK-21 cells were transfected with a taRNA including a replicase construct encoding a wildtype SFV replicase; and one of the following trRNAs encoding one or two fluorophores separated by a 2A ribosomal skip peptide (2A peptide): a trRNA encoding green fluorescent protein (GFP) only (GFP trRNA); a trRNA encoding mCherry only (mCherry trRNA); a GFP trRNA and a mCherry trRNA; or a trRNA encoding GFP and mCherry joined by a T2A peptide linker (GFP-T2A-mCherry trRNA).

[0375] Expression of each fluorophore is shown as fold-expression over cells transfected only with a replicase construct (FIG. 4B). All cells transfected with a trRNA encoding a fluorophore expressed mCherry and / or GFP; however, cells transfected with two different trRNAs (GFP trRNA and mCherry trRNA) expressed lower levels of GFP and mCherry relative to cells transfected with only one type of trRNA (FIG. 4C). Surprisingly, cells transfected with GFP- T2A-mCherry trRNA expressed both GFP and mCherry at similar levels to cells transfected with only one type of trRNA; GFP and mCherry expression in these cells is shown separately in (FIG. 4D). These results indicate that expression of two proteins on a single trRNA is not only possible but occurs at comparable efficiency as traditional trRNA.

[0376] Example 4: Co-Expression of Multiple Subgenomic Promoter (SGP)- or IRES- Linked Proteins on a Single trRNA

[0377] Though 2A peptides proved effective as linkers of multiple proteins for taRNA-mediated expression, 2A peptides require addition of multiple amino acids on the C-terminus of a first protein (e.g., “protein 1” of FIG. 4A) and an amino acid on the N-terminal of a second protein (e.g., “protein 2” of FIG. 4A). Because certain proteins do not tolerate modification to the C- terminus and / or N-terminus, additional linker types which do not require modification to a protein were tested, including alphaviral sub-genomic promoters (SGPs) and internal ribosomal entry site (IRES) elements. In this Example, taRNA-mediated expression of multiple protein encoded by a single trRNA having an SGP or IRES linker was assessed.

[0378] SGP linkers were tested by transfection of BHK-21 cells with a wildtype replicase encoded by an mRNA or a self-amplifying RNA (saRNA); and a trRNA encoding GFP-SGP- mCherry, with a variable SGP derived from Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEV), Ross River virus (RRV), Semliki Forest virus (SFV), Sindbis virus (SINV), or Western equine encephalitis virus (WEEV). Expression of mCherry was assessed at 24 hours as fold-expression compared to Naive.

[0379] Surprisingly, though BHK-21 cells transfected with trRNA including an SGP expressed mCherry, expression levels were relatively low and significantly less than with traditional trRNA (FIG. 5A). Of these constructs, cells transfected with saRNAs encoding replicases expressed higher levels of mCherry compared to cells transfected with mRNAs encoding replicases. These surprising findings indicate that while SGPs still allow for replication of multiple proteins, the kinetics of mRNA-encoded replicases are incompatible with SGP-driven expression.

[0380] IRES linkers were evaluated by transfection of BHK-21 cells with a wildtype taRNA; and a trRNA encoding GFP-IRES-mCherry, with a variable IRES selected from Aichivirus (iAichi), Coxsackievirus B3 (iCVB3), encephalomyocarditis virus (iEMCV), enterovirus 71 (iEV71), or human rhinovirus B3 (iiHRV-B3) IRES. Expression of GFP and mCherry was assessed at 24 hours as fold-expression compared to Naive.

[0381] All tested trRNA with IRES elements enabled detectable expression of mCherry and GFP at significant levels relative to Naive conditions. Thus, though IRES elements resulted in lower expression than T2A, IRES elements are effective for expression of multiple proteins on a single trRNA.

[0382] Example 5: trRNAs Encoding a Payload, an IRES linker, and an IMP Improve Payload Expression and Reduce ISG54 Induction in a Configuration-Specific Manner in vitro

[0383] While co-transfection of cells with taRNA and IMP-encoding mRNAs improved expression of taRNA-mediated pay loads in vitro, co-localization of multiple different mRNAs in vivo can introduce additional complexities to heterologous RNA expression. In these proof-of- concept experiments, nucleic acids encoding IMPs and IRES linkers were “embedded” into trRNAs for transfection into Raw macrophages and 3T3 cells. Cells were co-transfected with a replicase construct encoding wildtype SFV replicase or a mutant (RLE) replicase and a trRNA. Several trRNA in either IMP-linker-payload coding sequence (PCS) configuration or PCS- linker-IMP configuration (see: FIG. 7A-4C) were designed with one of various IMPs: SOCS1- CVB3-SEAP, NS1-CVB3-SEAP, E3L-CVB3-SEAP, SEAP-CVB3-E3L, SEAP-CVB3-SOCS1, SEAP-CVB3-dnPKR, SEAP-CVB3-PR8 NS1, SEAP-CVB3-F1L, SEAP-CVB3-rZAPC88R, SEAP-CVB3-ICP0, SEAP-CVB3-ICP34.5, SEAP-CVB3-US11, and SEAP-CVB3-US1. In control experiments, cells were transfected with trRNA encoding SEAP only (oestrRNA;). taRNA with WT replicase or RLE replicase were prepared as described in Examples 1-2 and cells were transfected with 5 or 5.5ng total RNA.

[0384] Several IMPs improved expression of SEAP in 3T3 and / or Raw cells. E3L, a vaccinia virus viral immune evasion protein (VIEP), was found to improve expression regardless of SEAP when embedded in either IMP-linker-payload or payload-linker- IMP trRNAs (FIG. 8B, 8D, 8F, 8G). For example, PR8 NS1, an influenza A virus VIEP, and US11, a herpes simplex virus VIEP, were both found to particularly improve expression of trRNA when embedded in payload-linker- IMP trRNAs (FIG. 8B, 8D). Both E3L (FIG. 8C, 8E) and PR8 NS1 (FIG. 8A) were also found to reduce ISG54 in IMP-IRES-SEAP configurations. However, improvements to payload expression were not always correlated with reductions in ISG54 induction. While SOCS1, an antagonist of human antiviral factors, showed only low-to-moderate improvements to expression of a payload in either trRNA configuration (FIG. 8B, 8D), ISG54 induction was significantly reduced in both (FIG. 8A, 8C, 8E).

[0385] Overall, several IMPs functioned well in particular configurations, and both E3L-IRES- SEAP trRNA and SEAP-IRES-E3L trRNA improved expression of SEAP while reducing ISG54 induction.

[0386] Example 6: trRNAs Encoding a Payload, an IRES linker, and an IMP Improve Payload Expression and Reduce ISG54 Induction in a Configuration-Specific Manner in vivo

[0387] In this Example, the efficacy of two taRNAs with trRNA with embedded IRES linkers and IMPs in vivo was assessed. Adult (3-6 months) wildtype mice (N = X) were injected intramuscularly with Ipg total RNA for SEAP expression. Mice were injected with taRNA including: a replicase construct encoding a wildtype replicase or a mutant (RLE) replicase; and a trRNA encoding SEAP only; E3L-CVB3-SEAP trRNA or NS1-CVB3-SEAP trRNA; or an mRNA encoding SEAP only. Mice were bled and sera was collected at 8 hours, 24 hours, 72 hours, 120 hours, and 168 hours, and SEAP expression was determined using a standard SEAP reporter gene assay.

[0388] Mice injected with trRNA constructs, including controls, expressed SEAP at significantly higher levels than mice injected with mRNA only. SEAP expression in mice injected with E3L-CVB3-SEAP trRNA was nearly 3x that of mice injected with trRNA encoding SEAP only (FIG. 9A) during peak expression at 24 hours, indicating that IMP- encoding trRNAs may be more potent than traditional trRNAs. Indeed, both NS1-CVB3-SEAP trRNA and E3L-CVB3-SEAP trRNA exhibited comparable increases in expression of SEAP in vivo compared to traditional trRNA at 24 hours, an effect which persisted through 168 hours (FIG. 9B). For E3L-CVB3-SEAP trRNAs, this effect persisted regardless of replicase construct (FIG. 9C). Overall, trRNAs with embedded IMPs functioned well to increase expression of payloads in vivo for as long as 168 hours, with both wildtype and RLE replicases.

[0389] Example 7: trRNAs Encoding a Payload, a T2A linker, and an IMP Also Improve Payload Expression and Reduce ISG54 Induction in a Configuration-Specific Manner

[0390] In this proof-of-concept experiment, nucleic acids encoding IMPs and T2A peptide linkers were embedded into trRNAs for testing in vivo. Adult (3-6 months) wildtype mice (N = X) were injected intramuscularly with Ipg total RNA for SEAP expression. Mice were injected with taRNA including: a replicase construct encoding a wildtype replicase or a mutant (RLE) replicase; and a trRNA encoding SEAP-T2A-E3L, or an mRNA encoding SEAP only. Mice were bled and sera was collected at 8 hours, 24 hours, 72 hours, 120 hours, and 168 hours, and SEAP expression was determined using a standard SEAP reporter gene assay.

[0391] Like trRNAs with IRES linkers, trRNAs with T2A linkers, regardless of replicase, exhibited increased payload expression relative to traditional trRNAs, an effect which persisted through 168 hours (FIG. 10). These experiments indicate that T2A peptides are suitable linkers for payloads and IMPs encoded by trRNAs; importantly, these results also indicate that both IMPs and pay loads can be expressed off of the endogenous alphaviral promoters of trRNA in the absence of IRES-mediated initiation of translation.

[0392] Example 8: Payload, linker, and IMP trRNAs Exhibit Configuration-Specific and Linker- Specific Effects on Expression in vitro

[0393] In this Example, expression of payloads from trRNAs having IRES or 2A peptide linkers and payload- linker- IMP or IMP-linker-payload configurations was compared. 3T3 cells and Raw macrophages were co-transfected with a replicase construct encoding wildtype SFV replicase or a mutant (RLE) replicase and a trRNA. Several trRNA in either IMP-linker-payload coding sequence (PCS) configuration or PCS-linker-IMP configuration (see: FIG. 7A-7C) were designed: E3L-CVB3-SEAP trRNA, a SOCS1-CVB3-SEAP trRNA, a SEAP-T2A-SOCS1 trRNA, a SOCS1-T2A-SEAP trRNA, a SEAP-T2A-E3L trRNA, and an E3L-T2A-SEAP trRNA. In control experiments, cells were transfected with trRNA encoding SEAP only (oestrRNA;). taRNA were prepared as described in Examples 1-2 and cells were transfected with 5ng total RNA.

[0394] Generally, cells transfected with trRNAs containing 2A peptide linkers exhibited higher expression at 6 hours than trRNAs containing IRES linkers (FIG. 11A). However, cells transfected with trRNAs containing IRES linkers exhibited significantly increased expression of SEAP at 24 hours; at the same time, trRNAs encoding E3L increased expression relative to trRNAs encoding SOCS1 (FIG. 11B). ISG54 induction was similar for both linker types but was generally reduced in cells transfected with trRNA encoding SOCS1 (FIG. 11C).

[0395] Together, these results indicate that while both linker types are effective in increasing expression of payloads, the two linker types exhibit distinct kinetics in the cellular environment. Thus, pairings of specific linker and IMPs may be useful for particular applications; for example, E3L with an IRES may serve as a useful IMP for applications in which long-lasting expression is required, whereas SOCS1 may be useful when immediate expression and / or reduction of inflammatory effects is desired.

[0396] Example 9: Payload, linker, and IMP trRNAs Exhibit Configuration-Specific and Linker- Specific Effects on Expression in vivo

[0397] In this Example, the efficacy of taRNAs with trRNA with embedded IRES or T2A linkers and IMPs was evaluated in different configurations in vivo. Adult (3-6 months) wildtype mice (N = X) were injected intramuscularly with Ipg total RNA for SEAP expression. Mice were injected with taRNA including: a replicase construct encoding a wildtype replicase; and a NS1-CVB3-SEAP trRNA; a SEAP-CVB3-SEAP trRNA; an E3L-CVB3-SEAP trRNA or a SEAP-T2A-E3L trRNA; or an mRNA encoding SEAP only. Mice were bled and sera was collected at 8 hours, 24 hours, 72 hours, 120 hours, and 168 hours, and SEAP expression was determined using a standard SEAP reporter gene assay.

[0398] All tested trRNAs exhibited increased expression relative to mRNA for the duration of sampling (FIG. 12A, 12B). As suggested by the results of Example 6, while the tested IMPs and linkers generally improve expression compared to traditional trRNAs, specific trRNA configurations exhibit distinct kinetics which may be useful for particular uses. For example, SEAP-T2A-E3L exhibited a higher Cmax and reduced durability relative to E3L-IRES-SEAP (FIG. 12A), and NS1-IRES-SEAP was found to be more potent than SEAP-IRES-NS1 (FIG. 12B). Overall, these results indicate that trRNAs are highly expressive in vivo and can be used for a variety of applications.

[0399] Example 10: mRNAs Encoding Two IMPs Can Improve Expression of trRNA-mediated Payloads Synergistically

[0400] In this proof-of-concept experiments, the effects of multiple mRNAs were tested on trRNA expression. In these experiments, Raw macrophages were co-transfected with SEAP- encoding taRNA and mRNAs encoding one or two IMPs (as in FIGs. 7A-7B and FIGs. 13 A- 13B). SEAP-encoding taRNAs, including a replicase construct encoding a wildtype SFV replicase or RLE replicase and a trRNA encoding SEAP were co-transfected into Raw macrophages with mRNAs encoding SOCS1 or dnPKR. RNAs were prepared as described in Examples 1-2. Raw macrophages were transfected with 5.5ng, 22ng, or 44ng of total RNA (1:1:1 molar ratio of replicase construct:trRNA:mRNA).

[0401] Though cells transfected with SEAP-encoding taRNA and only a SOCS1 -encoding mRNA exhibited 4-8x expression with either replicase at 24 hours post-transfection, cells transfected with SEAP-encoding taRNA and only a dnPKR-encoding mRNA exhibited modest increases in expression (FIG. 14). Surprisingly, mRNAs encoding SOCS1 and dnPKR increased expression of SEAP as much as 16x, indicating synergistic effects, rather than additive effects, of combined IMPs.

[0402] Example 11: trRNAs Encoding Two or More IMPs Improves Expression of Pay loads Synergistically

[0403] In this Example, taRNA-mediated SEAP expression with trRNAs encoding one, two, or three IMPs, and a linker was compared (as in FIGs. 7A-7B, FIGs. 13A-13B, FIGs. 16A-16B). 3T3 cells and Raw macrophages were transfected with taRNAs including a replicase construct encoding a wildtype replicase or RLE replicase; and a trRNA encoding SEAP-only, a B18-P2A- E3L-CVB3-SEAP trRNA, a E3L-CVB3-SEAP trRNA, a NS1-CVB3-SEAP trRNA, a SOCS1- CVB3-SEAP trRNA, a SEAP-T2A-E3L trRNA, a SEAP-T2A-E3L-P2A-F1L trRNA, a SEAP- T2A-E3L-P2A-SOCS1 trRNA, a SEAP-T2A-SOCS1-P2A-E3L trRNA, a SEAP-T2A-E3L- P2A-SOCS1-E2A-F1L trRNA, a SEAP-CVB3-SOCS1-P2A-E3L trRNA, a SEAP-CVB3- SOCSl-P2A-dnPKR trRNA, a SEAP-CVB3-SOCS1-P2A-NS1 trRNA, a SOCS1-P2A-E3L- CVB3-SEAP trRNA, a SOCSl-P2A-dnPKR-CVB3-SEAP trRNA, a SOCS1-P2A-NS1-CVB3- SEAP trRNA, a SOCS1-P2A-F1L-CVB3-SEAP trRNA, a SEAP-T2A-E3L-P2A-SOCS1 trRNA, a SEAP-T2A-SOCS1-P2A-E3L trRNA, or a SEAP-T2A-E3L-P2A-SOCS1-E2A-F1L trRNA.

[0404] E3L-CVB3-SEAP trRNA increased expression significantly (3.4x expression in 3T3 cells and 9.4x expression in Raw cells; FIG. 15). B18-P2A-E3L-CVB3-SEAP trRNA improved expression in Raw cells with a RLE replicase, and otherwise performed similarly to other trRNA candidates. In both 3T3s and Raw cells, trRNAs with embedded IMPs exhibited increased expression compared to taRNAs without IMPs. Surprisingly, trRNAs containing E3L, SOCS1, or both E3L and SOCS1 were associated with higher levels of SEAP expression in 3T3 cells compared to other IMP-containing trRNAs, regardless of linker type (FIG. 17A, 17B). In Raw macrophages, trRNAs containing F1L allowed the highest levels of expression when paired with wildtype replicases, while trRNAs with E3L and SOCS1, for example, SEAP-T2A-E3L-P2A- SOCS1, performed well with mutant (RLE) replicase (FIG. 17C, 17D).

[0405] ISG54 induction in Raws was also most reduced for trRNAs containing SOCS1 overall, affirming an important role for SOCS1 in mitigating inflammatory responses in cells transfected with trRNAs (FIG. 17E). Indeed, as shown in FIGs. 17F-17H, SOCS1 -containing trRNAs generally improved expression of trRNAs while suppressing ISG54 induction; of the tested trRNAs, SEAP-T2A-ERL-P2A-SOCS1 and SEAP-T2A-E3L-P2A-SOCS1-E2A-F1L both allowed for higher levels of SEAP expression.

[0406] Example 12: Replicase Proteins Can be Fused, to Other Proteins with a Variable Linker

[0407] Though the results have shown trRNAs encoding IMPs provide improve payload expression and reduce induction of inflammatory markers in transfected cells, it may also be desirable to express IMPs on replicase constructs. For example, taRNAs require the presence of a replicase for expression of trRNAs; thus, replicase construct expression typically occurs before trRNA expression. Co-expression of a replicase and an IMP which are encoded by the same construct may influence overall expression kinetics (e.g., by reducing immune responses prior to trRNA expression, thereby allowing increased expression of the trRNA). In this Example, the pay load expression of taRNA constructs comprising replicase constructs encoding wildtype (WT) replicases, mutant (RLE) replicases, or various replicase fusion proteins was assessed; and trRNAs comprising payload coding sequences (PCS) with or without immune modulating protein (IMP) coding sequences.

[0408] 3T3 and Raw cells were transfected with 5ng total RNA including: a replicase construct encoding WT replicase, RLE replicase, a replicase-3 / 4J-IMP fusion protein (WT-3 / 4J-dnPKR;), or a replicase-linker-IMP fusion protein (WT-T2A-E3L, WT-T2A-SOCS1, WT-T2A-F1L, RLE- T2A-SOCS1, or RLE-T2A-E3L); and a trRNA encoding SEAP only (oeSTR), an E3L-CVB3- SEAP trRNA, a SEAP-CVB3-E3L trRNA, a SEAP-CVB3-SOCS1 trRNA, a SEAP-CVB3-PR8 NS1 trRNA, a SEAP-CVB3-F1L trRNA, or a SEAP-T2A-E3L trRNA.

[0409] Surprisingly, all tested replicase constructs, including those encoding replicase fusion proteins, were expressive in Raw and 3T3 cells. Cells transfected with replicase constructs encoding WT-T2A-SOCS1 and WT-T2A-E3L expressed similar levels of SEAP as cells transfected with replicase constructs encoding non-fusion WT replicases (FIG. 20A). Similarly, cells transfected with replicase constructs encoding RLE-T2A-SOCS1 expressed only slightly lower levels of SEAP compared to cells transfected with replicase constructs encoding RLE only (FIG. 20C). Notably, Raw cells transfected with WT-T2A-S0CS1, WT-T2A-E3L, or RLE- T2A-SOCS1 replicase constructs also experienced significantly reduced ISG54 induction compared to cells transfected with replicase constructs encoding respective non-fusion replicases (FIG. 20B, 20D).

[0410] Example 13: Replicase-IMP Fusion Proteins with C-terminal and. N-terminal IMPs Increase Expression

[0411] In this Example, taRNAs having replicase constructs encoding IMP and replicase fusion proteins in various configurations were assessed for effects on SEAP payload expression. BJ fibroblast cells were transfected with 5ng total RNA including: a replicase construct encoding WT SFV replicase (WT), PR8 NS1-P2A-WT-T2A-VACV E3L (NS1-WT-E3L), WT-T2A-PR8 NS1 (WT-NS1), PR8-NS1-T2A-WT (NS1-WT), WT-T2A-T0SV NSs (WT-Tosc NSs), VACV E3L-P2A-WT-T2A-VACV K3L, or WT-T2A- VACV E3L; and a trRNA encoding SEAP only (oeSTR) or a trRNA encoding VACV E3L-CVB3-SEAP (E3L-IRES-SEAP).

[0412] Several taRNAs having replicase constructs encoding replicase and IMP fusion proteins outperformed (i.e., expressed more trRNA SEAP) than taRNAs having replicase constructs encoding wildtype SFV replicases (FIG. 21). Notably, taRNAs with replicase constructs encoding replicase PR8 NS1 fusion proteins or replicase TOSV NSs fusion proteins increased expression of oeSTR trRNA by more than lOx compared to taRNAs with replicase constructs encoding wildtype SFV replicases only. Not only did taRNAs with replicase constructs encoding replicase fusion proteins with terminal IMPs express SEAP payloads, but taRNAs with replicase constructs encoding NS1-WT-E3L expressed SEAP payloads at similar levels regardless of the trRNA.

Claims

CLAIMSWhat is claimed is:

1. A / / wrs-ampli lying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(b) a second RNA polynucleotide comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a nucleic acid encoding an immune modulating protein (IMP).

2. A trans-amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(ii) a nucleic acid encoding an immune modulating protein (IMP); and(b) a second RNA polynucleotide comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload.

3. The taRNA of claim 1, wherein the first RNA polynucleotide of (a) further comprises:(ii) a nucleic acid encoding an IMP.

4. The taRNA of claim 2, wherein the second RNA polynucleotide of (b) further comprises:(ii) a nucleic acid encoding an IMP.

5. The taRNA of claim 2 or claim 3, wherein the nucleic acid encoding the replicase of (a)(i) encodes a replicase comprising an IMP between a third and fourth domain of the replicase.

6. The taRNA of claim 3, wherein the first RNA polynucleotide of (a) comprises a linker between the nucleic acid of (a)(i) and the nucleic acid of (a)(ii) .

7. The taRNA of claim 1 or claim 2, wherein the IMP is a viral immune evasion protein(s) (VIEP).

8. The taRNA of claim 7, wherein the VIEP is from vaccinia virus (VACV), herpes simplex virus (HSV), influenza virus, Toscana Virus (TOSV), or encephalomyocarditis virus (EMCV).

9. The taRNA of claim 8, wherein the VIEP is a VACV soluble IFN a / p receptor B 18 (B18R), VACV RNA-binding protein E3 (E3L), VACV F1L, VACV NIL, HSV infected cell protein 34.5 (ICP34.5), HSV unique short 1 (US1), HSV unique short 11 (US 11), influenza virus non-structural protein 1 (NS1), TOSV non-structural (NSs) protein, or EMCV Lpro.

10. The taRNA of claim 9, wherein the NS1 is from Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) (PR8 NS1).

11. The taRNA of claim 9, wherein the VIEP is VACV E3L.

12. The taRNA of claim 9, wherein the VIEP is PR8 NS 1.

13. The taRNA of claim 9, wherein the VIEP is TOSV NSs.

14. The taRNA of claim 9, wherein the VIEP comprises an amino acid sequence of any one of SEQ ID NOs: 229-238 or 245.

15. The taRNA of claim 1 or claim 2, wherein the IMP antagonizes a mammalian antiviral factor(s).

16. The taRNA of claim 15, wherein the IMP that antagonizes a mammalian antiviral factor is dominant negative mitochondrial antiviral- signaling protein (dnMAVS), microprotein in antiviral immunity 1 (MAVH), dominant negative protein kinase R (dnPKR), suppressor of cytokine signaling (SOCS), or dominant negative Zinc-finger Antiviral Protein (dnZAP).

17. The taRNA of claim 16, wherein the dnZAP is rat ZAP with cysteine-to-arginine mutation at position 88 (rZAPC88R).

18. The taRNA of any one of claim 16, wherein the SOCS is SOCS1 or SOCS3.

19. The taRNA of claim 16, wherein the mammalian antiviral factor comprises an amino acid sequence of any one of SEQ ID NOs: 239-244.

20. The taRNA of claim 1 or claim 2, wherein the nucleic acid(s) encoding an IMP encode a first IMP and a second IMP.

21. The taRNA of claim 20, wherein the first RNA polynucleotide of (a) encodes the first IMP and the second IMP.

22. The taRNA of claim 20, wherein the second RNA polynucleotide of (b) encodes the first IMP and the second IMP.

23. The taRNA of claim 20, wherein the first IMP and second IMP are different IMPs.

24. The taRNA of claim 20, wherein the first IMP and / or the second IMP is VACV Bl 8,VACV E3L, VACV F1L, PR8 NS1, dnPKR, SOCS1, rZAPCC88r, Lpro, or TOSV NSs.

25. The taRNA of claim 20, wherein the first IMP and the second IMP are joined by a linker.

26. The taRNA of claim 25, wherein the linker is a nucleic acid encoding a 2A peptide, or an internal ribosome entry site (IRES) element.

27. The taRNA of claim 26, wherein the 2A peptide is selected from thosea asigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), equine rhinitis A virus 2A (E2A), or Furin-2A.

28. The taRNA of claim 26, wherein the IRES element is an IRES from Coxsackievirus B3 (CVB3), encephalomyocarditis virus (EMCV), porcine kobuvirus (PKV), Manhattan Parechovirus (MPV), or tortoise rafivirus (TraV).

29. The taRNA of claim 26, wherein the first IMP is E3L, the second IMP is B 18R and the 2A peptide is P2A.

30. The taRNA of claim 26, wherein the first IMP is E3L, the second IMP is dnPKR, and the 2A peptide is P2A.

31. The taRNA of claim 26, wherein the first IMP is E3L, the second IMP is F1L, and the 2A peptide is P2A.

32. The taRNA of claim 26, wherein the first IMP is E3L, the second IMP is PR8 NS1, and the 2A peptide is P2A.

33. The taRNA of claim 26, wherein the first IMP is E3L, the second IMP is SOCS1, and the 2A peptide is P2A.

34. The taRNA of claim 26, wherein the first IMP is E3L, the second IMP is SOCS1, and the 2A peptide is T2A.

35. The taRNA of claim 26, wherein the first IMP is SOCS1, the second IMP is dnPKR, and the 2A peptide is P2A.

36. The taRNA of claim 26, wherein the first IMP is SOCS1, the second IMP is F1L, and the 2A peptide is P2A.

37. The taRNA of claim 26, wherein the first IMP is SOCS1, the second IMP is PR8 NS1, and the 2A peptide is P2A.

38. The taRNA of claim 26, wherein the first IMP is SOCS1, the second IMP is rZAPCC88r, and the 2A peptide is P2A.

39. The taRNA of claim 20, wherein the nucleic acid(s) encoding an IMP further encodes a third IMP.

40. The taRNA of claim 39, wherein the third IMP is selected from E3L, SOCS1, and F1L.

41. The taRNA of claim 40, wherein the third IMP is joined to the first IMP and / or the second IMP by a linker.

42. The taRNA of claim 39, wherein the first IMP is E3L, the second IMP is SOCS1, and the third IMP is F1L.

43. The taRNA of claim 39, wherein the nucleic acid(s) encoding an IMP further encodes a fourth IMP, fifth IMP, and / or sixth IMP.

44. The taRNA of claim 43, wherein adjacent IMPs are joined by a linker.

45. The taRNA of claim 43, wherein one or two IMPs are joined to the nucleic acid payload or nucleic acid encoding a payload by a linker.

46. The taRNA of claim 20, wherein the first RNA polynucleotide of (a) encodes the first IMP, and the second RNA polynucleotide of (b) encodes the second IMP.

47. The taRNA of claim 46, wherein the first IMP is SOCS1, and the second IMP is E3L.

48. The taRNA of claim 46, wherein the first IMP is E3L, and the second IMP is SOCS1.

49. The taRNA of claim 46, wherein the first IMP is dnPKR and the second IMP is F1L.

50. The taRNA of claim 1, wherein the first RNA polynucleotide of (a) comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii).

51. The taRNA of claim 50, wherein the first RNA polynucleotide of (a) comprises, from 5’ to 3’, the nucleic acid of (i), a linker, and the nucleic acid of (ii).

52. The taRNA of claim 50, wherein the first RNA polynucleotide of (a) comprises, from 5’ to 3’, the nucleic acid of (ii), a linker, and the nucleic acid of (i).

53. The taRNA of claim 2, wherein the second RNA polynucleotide of (b) comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii).

54. The taRNA of claim 53, wherein the linker is an IRES element, a % junction amino acid sequence, or a nucleic acid encoding a 2A peptide.

55. The taRNA of claim 54, wherein the linker is a sub-genomic promoter.

56. The taRNA of claim 54, wherein the IRES element is an IRES from CVB3, EMCV, PKV,MPV, or TraV.

57. The taRNA of claim 56, wherein the 2A peptide is T2A, P2A, E2A, or Furin-2A.

58. The taRNA of claim 1 or claim 2, wherein the replicase is an alphavirus replicase.

59. The taRNA of claim 58, wherein the replicase is a Semliki Forest virus (SFV) replicase or a variant thereof.

60. The taRNA of any claim 1 or claim 2, wherein the nucleic acid encoding the replicase comprises the sequence set forth in SEQ ID NO: 129 or SEQ ID NO: 130.

61. The taRNA of claim 1, wherein the second RNA polynucleotide comprises a 5’ untranslated region (UTR) and a 3’ UTR flanking the nucleic acid of (i) and the nucleic acid of (ii).

62. The taRNA of claim 61, wherein the 5’ UTR comprises a conserved sequence element (CSE) which is cognate to the replicase.

63. The taRNA of claim 61 , wherein the 5 ’ UTR is a SINV 5 ’ UTR or SFV 5 ’ UTR.

64. The taRNA of claim 61, wherein the 3’ UTR comprises a CSE which is cognate to the replicase.

65. The taRNA of claim 61 , wherein the 3 ’ UTR is a SINV 3 ’ UTR or SFV 3 ’ UTR.

66. The taRNA of claim 1 or claim 2, wherein the second RNA polynucleotide of (b) further comprises a 3’ terminal polyadenylation (poly A) tail.

67. The taRNA of claim 1, wherein the second RNA polynucleotide of (b) comprises, from 5’ to 3’: i. the nucleic acid encoding the IMP; ii. a linker; and iii. the nucleic acid encoding a payload.

68. The taRNA of claim 67, wherein the IMP is E3L, PR8 NS1, or SOCS1.

69. The taRNA of claim 67, wherein the linker is a CVB3 IRES element or a nucleic acid encoding T2A.

70. The taRNA of any one of claims 67, wherein the nucleic acid(s) of the second RNA polynucleotide is (are) flanked by a 5’ UTR and a 3’ UTR.

71. The taRNA of claim 1, wherein the second RNA polynucleotide of (b) comprises, from 5’ to 3’: i. the nucleic acid encoding a payload; ii. a linker; and iii. the nucleic acid encoding the IMP.

72. The taRNA of claim 71 , wherein the IMP is E3L, SOCS 1 , or PR8 NS 1.

73. The taRNA of claim 71, wherein the linker is CVB3 IRES element or T2A.

74. The taRNA of claim 71, wherein the nucleic acid(s) of the second RNA polynucleotide of (b) is (are) flanked by a 5’ UTR and a 3’ UTR.

75. The taRNA of claim 2, wherein the first RNA polynucleotide of (a) comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii).

76. The taRNA of claim 75, wherein the IMP is E3L, dnPKR, or SOCS1.

77. The taRNA of claim 75, wherein the linker is a nucleic acid encoding a 2A peptide.

78. The taRNA of claim 1 or claim 2, wherein the payload comprises an antigen.

79. The taRNA of claim 1 or claim 2, wherein the payload comprises a reporter.

80. The taRNA of claim 79, wherein the reporter is SEAR81. The taRNA of claim 1 or 2, wherein the first RNA polynucleotide of (a) comprises a sequence of any one of SEQ ID NO: 148-176.

82. The taRNA of claim 81, wherein the second RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 177-220.

83. A composition comprising a deoxyribonucleic acid (DNA) polynucleotide that can be transcribed to produce the second RNA polynucleotide of (b) of claim 1 or claim 2.

84. The composition of claim 83, further comprising an additional DNA polynucleotide that can be transcribed to produce the first RNA polynucleotide of (a) of any one of claims 1 to 79.

85. A cell comprising the taRNA of claim 1 or claim 2.

86. A method of expressing a payload in a cell, the method comprising: transfecting a cell with the taRNA of claim 1 or claim 2.

87. A method of expressing a pay load in a subject, the method comprising: administering to a subject the taRNA of claim 1 or claim 2.

88. The method of claim 87, wherein the subject is a mammal.

89. The method of claim 88, wherein the subject is a human.

90. A fusion protein comprising, from N-terminal to C-Terminal, a replicase and a protein or peptide.

91. The fusion protein of claim 90, wherein the replicase and the protein or peptide are joined by a 2 A peptide.

92. The fusion protein of claim 90, wherein the protein or peptide comprises an immune modulating protein (IMP).

93. The fusion protein of claim 90, wherein the protein or peptide comprises a first IMP, a second IMP and / or a third IMP.

94. A fusion protein comprising, from N-terminal to C-terminal, a first protein or peptide, a replicase, and a second protein or peptide.

95. The fusion protein of claim 94, wherein the first protein or peptide comprises a first immune modulating protein (IMP).

96. The fusion protein of claim 94, wherein the second protein or peptide comprises a second IMP.

97. The fusion protein of claim 94, wherein the first protein or peptide comprises a first IMP and the second protein or peptide comprises a second IMP.

98. The fusion protein of claim 97, wherein the first IMP comprises an Influenza A / Puerto Rico / 8 / 34 virus non- structural protein 1 (PR8 NS1) protein and the second IMP comprises VACV RNA-binding protein E3 (E3L).

99. A deoxyribonucleic acid (DNA) comprising nucleic acids encoding the fusion protein of claim 90 or 94.

100. A ribonucleic acid (RNA) comprising nucleic acids encoding the fusion protein of claim 90 or claim 94.

101. A self-amplifying ribonucleic acid (RNA) (saRNA) comprising:(i) a nucleic acid encoding, from 5’ to 3’, a replicase, a 2A peptide, and a first immune modulating protein (IMP); and(ii) a nucleic acid payload or a nucleic acid encoding a payload.

102. A self-amplifying ribonucleic acid (RNA) (saRNA) comprising:(i) a nucleic acid encoding, from 5’ to 3’, a first immune modulating protein (IMP), a 2A peptide, and a replicase; and(ii) a nucleic acid payload or a nucleic acid encoding a payload.

103. The saRNA of claim 101 or claim 102, further comprising:(iii) a nucleic acid encoding a second IMP.

104. The saRNA of claim 103, wherein the nucleic acid of (ii) and the nucleic acid of (iii) are joined by a nucleic acid sequence encoding a 2A peptide.

105. A trans-amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(b) a second RNA polynucleotide comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a nucleic acid encoding a first immune modulating protein (IMP), optionally wherein the second RNA polynucleotide comprises: a second IMP, a third IMP, a fourth IMP, a fifth IMP and / or a sixth IMP, optionally wherein adjacent IMPs in the second RNA polynucleotide are joined by a linker.

106. The taRNA of claim 105, wherein the second IMP, the third IMP, the fourth IMP, the fifth IMP and / or the sixth IMP are independently chosen from VACV B18R, VACV E3L, VACV F1L, VACV NIL, HSV ICP34.5, HSV US1, HSV US11, ICPO, PR8 NS1, TOSV NS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and EMCV Lpro.

107. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(b) a second RNA polynucleotide comprising, from 5’ to 3’:5’-CSE1-IMP1-L1-IMP2-L2-IMP3-L3-PCS-L4-IMP4-L5-IMP5-L6-IMP6-CSE2-3’; wherein:CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;IMP1, IMP2, IMP3, IMP4, IMP5, and IMP6are each independently an immune modulating protein (IMP);L1, L2, L3, L4, L5, and L6are each independently a linker;PCS is a nucleic acid payload or a nucleic acid encoding a payload; and IMP2-L2, IMP3-L3, L4-IMP4, L5-IMP5, and L6-IMP6are each independently optionally absent.

108. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(b) a second RNA polynucleotide comprising, from 5’ to 3’:5’-CSE1-IMP1-L1-IMP2-L2-IMP3-L3-PCS-L4-IMP4-L5-IMP5-L6-IMP6-CSE2-3’; wherein:CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;IMP1, IMP2, IMP3, IMP4, IMP5, and IMP6are each independently an immune modulating protein (IMP);L1, L2, L3, L4, L5, and L6are each independently a linker;PCS is a nucleic acid payload or a nucleic acid encoding a payload; andIMP1-!?, IMP2-L2, IMP3-L3, L5-IMP5, and L6-IMP6are each independently optionally absent.

109. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(b) a second RNA polynucleotide comprising, from 5’ to 3’:5’-CSE1-IMP1-L1-IMP2-L2-IMP3-L3-PCS-L4-IMP4-CSE2-3’; wherein:CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;IMP1, IMP2, IMP3, and IMP4are each independently an immune modulating protein (IMP);L1, L2, L3, and L4are each independently a linker;PCS is a nucleic acid payload or a nucleic acid encoding a payload; and IMP2-L2, IMP3-L3, and L4-IMP4are each independently optionally absent.

110. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase; and(b) a second RNA polynucleotide comprising, from 5’ to 3’:5’-CSE1-IMP1-L1-PCS-IMP2-L2-IMP3-L3-L4-IMP4-CSE2-3’; wherein:CSE1and CSE2are each independently a conserved sequence element (CSE) which is cognate to the replicase;IMP1, IMP2, IMP3, and IMP4are each independently an immune modulating protein (IMP);L1, L2, L3, and L4are each independently a linker;PCS is a nucleic acid payload or a nucleic acid encoding a payload; and IMP^L1, IMP3-L3, and L4-IMP4are each independently optionally absent.

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