Alphavirus replicase variants

Abstract

Disclosed herein are Semliki Forest Virus (SFV) replicase variants, and trans-amplifying RNAs and self-amplifying RNA encoding SFV replicase variants.

Description

[0001] ALPHA VIRUS REPLICASE VARIANTS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional Application No. 63 / 631,322, filed April 8, 2024, entitled “ALPHA VIRUS REPLICASE VARIANTS”, the content of which is hereby incorporated by reference herein in its entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (A141470005WO00-SEQ-KGC.xml; Size: 157,109 bytes; and Date of Creation: April 2, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] A replicase is a ribonucleic acid (RNA)-dependent RNA polymerase capable of transcribing (i.e., reading) an RNA template to produce a copy of the RNA template (e.g., “amplify” the RNA template). Replicases can be encoded in self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA) (e.g., as part of saRNA and / or taRNA therapeutics). When transfected into a cell, the saRNA or taRNA expresses the replicase, which in turn amplifies an RNA payload, or RNA encoding a payload, that is encoded by the taRNA or saRNA. However, expression of a replicase in host cell (e.g., a human being treated with a taRNA) can trigger an undesirable host cell immune response. For example, the host cell immune response can trigger host cell death.

[0008] SUMMARY

[0009] In some aspects, this disclosure provides Semliki Forest Virus (SFV) replicase variants. In some embodiments, the SFV replicase variants have decreased immunogenicity as compared to wild-type SFV replicase. In some embodiments, this disclosure provides taRNAs and saRNAs that encode a SFV replicase variant described herein.

[0010] In some embodiments, this disclosure provides a Semliki Forest Virus (SFV) replicase variant wherein: (i) a position corresponding to 1211 of SEQ ID NO: 1 does not comprise an alanine; (ii) a position corresponding to 1212 of SEQ ID NO: 1 does not comprise an aspartic acid; (iii) a position corresponding to 1213 of SEQ ID NO: 1 does not comprise an alanine; (iv) an amino acid insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1; (v) a position corresponding to 1360 of SEQ ID NO: 1 does not comprise an asparagine; and / or (vi) a position corresponding to 1317 of SEQ ID NO: 1 does not comprise a glutamine.

[0011] In some embodiments, the SFV replicase variant comprises: (i) an arginine or asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine or glycine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a glutamic acid or lysine at a position corresponding to A1213 of SEQ ID NO: 1; (iv) a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1; (v) a threonine at a position corresponding to N1360 of SEQ ID NO: 1; or (vi) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1.

[0012] In some embodiments, the SFV replicase variant comprises: (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; and (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1.

[0013] In some embodiments, the SFV replicase variant comprises: (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; and (iii) a lysine acid at a position corresponding to A1213 of SEQ ID NO: 1.

[0014] In some embodiments, the SFV replicase variant comprises a threonine at a position corresponding to N1360 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises an arginine at a position corresponding to Q1317 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-10. In some embodiments, the SFV replicase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 2-10. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 3. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the SFV replicase variant further comprises an affinity tag. In some embodiments, this disclosure provides a polynucleotide comprising a nucleic acid encoding the SFV replicase variant. In some embodiments, the polynucleotide is a DNA polynucleotide. In some embodiments, the polynucleotide is an RNA polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 11-25.

[0015] In some embodiments, this disclosure provides a trans amplifying ribonucleic acid (RNA) (taRNA) comprising: (i) a first RNA polynucleotide comprising a nucleic acid encoding the SFV replicase variant; and (ii) a second RNA polynucleotide comprising: a conserved sequence element (CSE) that is cognate to the replicase encoded by the first RNA polynucleotide; and a nucleic acid payload or a nucleic acid encoding a payload. In some embodiments, the second RNA polynucleotide comprises: (i) a 5’ alphavirus untranslated region (UTR); (ii) the CSE that is cognate to the replicase encoded by the first RNA polynucleotide; (iii) the nucleic acid payload or the nucleic acid encoding the pay load; and (iv) a 3’ alphavirus UTR. In some embodiments, the 5’ alphavirus UTR is a VEEV 5’-UTR, SFV 5’-UTR, SINV 5’- UTR, or CHIKV 5’-UTR. In some embodiments, the 3’ alphavirus UTR is a VEEV 3’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR. In some embodiments, the 5’ alphavirus UTR comprises a mutation relative to a wild- type 5’ alphavirus UTR. In some embodiments, the 3’ alphavirus UTR comprises a mutation relative to a wild-type 3’ alphavirus UTR. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide comprising the nucleic acid encoding the SFV replicase variant.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGs. 1A-1B show that “NGK” replicase and “RLE” replicase (both SFV replicase variants) result in less cell death than wild-type SFV replicase. FIG. 1A shows cell death and apoptotic phenotype of Vero cells transfected with trans-amplifying RNAs (taRNAs) encoding either wild-type replicase or RLE replicase. FIG. IB shows the percent of healthy, dying and dead Vero cells that have been transfected with different SFV replicase variants including NGK replicase and RLE replicase.

[0018] FIG. 2 shows that NGK replicase and RLE replicase can decrease cell death while maintaining expression of the taRNA payload, green fluorescent protein (GFP).

[0019] FIG. 3 shows that SFV replicase variants (RLE + nsp3 N24T, nspl K425insK, npsl K424insK RLE + nsp3 N24T, and nsp2 Q780R + RLE + nsp3 N24T) all decrease BHK-21 cell death compared to wild- type SFV 120 hours after transfection with a self-amplifying RNA comprising the SFV wild-type or one of the SFV replicase variants. The pay load is GFP. DETAILED DESCRIPTION

[0020] In some aspects, this disclosure provides Semliki Forest Virus (SFV) replicase variants. Semliki Forest Virus is an alphavirus originally found in Asia. SFV replicases typically comprise four non- structural proteins (nspl, nsp2, nsp3 and nsp4). In some embodiments, the non-structural proteins are expressed and form a replicase complex. In some embodiments, the non-structural proteins are part of the same polyprotein. In some embodiments, the polyprotein is cleaved into the four different non-structural proteins, which form complex that has replicase activity.

[0021] In some embodiments, this disclosure provides SFV replicase variants. In some embodiments, SFV replicase variants comprise an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase. In some embodiments, the reference SFV replicase is a wildtype SFV replicase. In some embodiments, a wild-type SFV replicase comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more mutations are independently selected from a substitution (e.g., a single nucleotide polymorphism), deletion, or insertion of one or more amino acids. A substitution or insertion may comprise any amino acid (e.g., any natural amino acid, any unnatural amino acid, a modified amino acid). In some embodiments, an insertion comprises an insertion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more). In some embodiments, an insertion is an insertion of a single amino acid. In some embodiments, a deletion comprises a deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more). In some embodiments, a deletion is a deletion of a single amino acid.

[0022] In some embodiments, a mutation (e.g., substitution, deletion, or insertion) is described by referencing a position numbered according to an alignment of an amino acid sequence of an SFV replicase variant to an amino acid of a reference SFV replicase. In some embodiments, aligning a variant sequence relative to a reference sequence comprises introducing gaps to the variant sequence and / or the reference sequence, e.g., to account for insertions and deletions. In some embodiments, an SFV replicase variant comprises an amino acid sequence having a certain identity to a reference amino acid sequence (e.g., of a reference SFV replicase). As used herein, the term “identity” refers to a relationship between at least one variant sequence (e.g., amino acid sequence, nucleotide sequence) and one reference sequence of the same type, characterized by a measure of identical matches between the variant sequence and the reference sequence, e.g., after aligning the sequences and introducing gaps as necessary. In some embodiments, determining the identity of a variant sequence relative to a reference sequence comprises aligning the variant sequence to the reference sequence. In some embodiments, identity is provided as a percent of identity; one of ordinary skill in the art will understand that percent identity typically represents a calculation based on a number of identical nucleotides and gap penalties. Methods for aligning sequences and / or calculating identity of sequences are well known in the art, for example, using publicly available computer software such as Basic Local Alignment Search Tool (BLAST; e.g., as available on the world wide web at blast.ncbi.nlm.nih.gov / Blast.cgi), ClustalW (e.g., as available on the world wide web at genome.jp / tools-bin / clustalw), and TCoffee (e.g., as available on the world wide web at tcoffee . erg . eu / app s / tcoffee / all .html) .

[0023] In some embodiments, this disclosure provides a SFV replicase variant, wherein: (i) a position corresponding to 1211 of SEQ ID NO: 1 does not comprise an alanine; (ii) a position corresponding to 1212 of SEQ ID NO: 1 does not comprise an aspartic acid; (iii) a position corresponding to 1213 of SEQ ID NO: 1 does not comprise an alanine; (iv) a position corresponding to 1360 of SEQ ID NO: 1 does not comprise an asparagine; (v) a single amino acid insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1; or (vi) a position corresponding to 1317 of SEQ ID NO: 1 does not comprise a glutamine. “Immediately c-terminal to a position” refers to the amino acid that is covalently attached to the carboxy group of an amino acid at the position. For example, amino acid 426 is immediately c- terminal to amino acid 425.

[0024] In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations are independently selected from:

[0025] (i) a mutation at position 1211;

[0026] (ii) a mutation at position 1212;

[0027] (iii) a mutation at position 1213;

[0028] (iv) a mutation at position 1360;

[0029] (v) a mutation at a position immediately C-terminal of position 425; and / or

[0030] (vi) a mutation at position 1317.

[0031] In some embodiments, the positions are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1.

[0032] In some embodiments, the one or more mutations are independently selected from:

[0033] (i) a substitution at position 1211;

[0034] (ii) a substitution at position 1212;

[0035] (iii) a substitution at position 1213; (iv) a substitution at position 1360;

[0036] (v) an insertion at a position immediately C-terminal of position 425; and / or

[0037] (vi) a substitution at position 1317.

[0038] In some embodiments, the one or more mutations are independently selected from:

[0039] (i) a substitution at position A1211 ;

[0040] (ii) a substitution at position D1212;

[0041] (iii) a substitution at position A1213;

[0042] (iv) a substitution at position N1360;

[0043] (v) an insertion at a position immediately C-terminal of position K425; and / or

[0044] (vi) a substitution at position Q1317.

[0045] In some embodiments, the one or more mutations are independently selected from:

[0046] (i) A1211R or A1211N;

[0047] (ii) D1212L or D1212G;

[0048] (iii) A1213E or A1213K;

[0049] (iv) N1360T;

[0050] (v) K425insK; and / or

[0051] (vi) Q780R.

[0052] In some embodiments, the SFV replicase variant comprises: (i) a single nucleotide polymorphism (SNP) at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a single nucleotide polymorphism (SNP) at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a single nucleotide polymorphism (SNP) at a position corresponding to A1213 of SEQ ID NO: 1;

[0053] (iv) an amino acid insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1; (v) a single nucleotide polymorphism (SNP) corresponding to the amino acid at a position corresponding to N1360 of SEQ ID NO: 1; or (vi) a single nucleotide polymorphism (SNP) corresponding to the amino acid at a position corresponding to Q1317 of SEQ ID NO: 1.

[0054] In some embodiments, the SFV replicase variant comprises: (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1; (iv) a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1;

[0055] (v) a threonine at a position corresponding to N1360 of SEQ ID NO: 1; or (vi) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1.

[0056] In some embodiments, the SFV replicase variant comprises: (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises a leucine at a position corresponding to D1212 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises: a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1.

[0057] In some embodiments, the SFV replicase variant comprises: (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; and (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0058] (i) A1211R;

[0059] (ii) D1212L; and

[0060] (iii) A1213E; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 2. In some embodiments, the SFV replicase comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 2. In some embodiments, the SFV replicase comprises an amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 is also referred to as “RLE replicase”.

[0061] In some embodiments, the SFV replicase variant comprises a threonine at a position corresponding to N1360 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises a threonine at position N1360 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising at least one mutation relative to an amino acid sequence of a reference SFV replicase, wherein the at least one mutation comprises: N1360T, wherein the position of the at least one mutation is numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase comprises a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity with SEQ ID NO: 3. In some embodiments, the SFV replicase comprises a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity with SEQ ID NO: 3. In some embodiments, the SFV replicase comprises amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 3 is also referred to as “nsp3 N24T replicase”.

[0062] In some embodiments, the SFV replicase variant comprises an arginine at a position corresponding to Q1317 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises an arginine at position Q1317 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising at least one mutation relative to an amino acid sequence of a reference SFV replicase, wherein the at least one mutation comprises: Q780R, wherein the position of the at least one mutation is numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase comprises an arginine at position Q1317 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity with SEQ ID NO: 5. In some embodiments, the SFV replicase comprises an arginine at position Q1317 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity with SEQ ID NO: 5. In some embodiments, the SFV replicase comprises an amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 5 is also referred to as “nsp2 Q780R Replicase”.

[0063] In some embodiments, the SFV replicase variant comprises a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises a lysine insertion at position immediately c- terminal to K425 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising at least one mutation relative to an amino acid sequence of a reference SFV replicase, wherein the at least one mutation comprises: K425insK, wherein the position of the at least one mutation is numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase comprises a lysine insertion immediately c-terminal to K425 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity with SEQ ID NO: 4. In some embodiments, the SFV replicase comprises a lysine insertion immediately c-terminal to K425 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity with SEQ ID NO: 4. In some embodiments, the SFV replicase comprises an amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 4 is also referred to as “nspl K425insK Replicase”.

[0064] “RLE” Replicase Variants

[0065] In some embodiments, the SFV replicase variant comprises (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1; and (iv) a threonine at a position corresponding to N1360 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; and (iv) a threonine at position corresponding N1360 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; and (iv) a threonine at position corresponding N1360 of SEQ ID NO: 1, and an amino acid sequence having at least 95% identity to SEQ ID NO: 6. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0066] (i) A1211R;

[0067] (ii) D1212L;

[0068] (iii) A1213E; and

[0069] (iv) N1360T; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; and (iv) a threonine at position corresponding N1360 of SEQ ID NO: 1, and an amino acid sequence having at least 99% identity to SEQ ID NO: 6. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 6 is also referred to as “RLE + nsp3 N24T Replicase”.

[0070] In some embodiments, the SFV replicase variant comprises (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1; and (iv) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; and an arginine at position Q1317 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0071] (i) A1211R;

[0072] (ii) D1212L;

[0073] (iii) A1213E; and

[0074] (iv) Q780R; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; and (iv) an arginine at position Q1317 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 7. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; and (iv) an arginine at position Q1317 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 7. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 7. SEQ ID NO: 7 is also referred to as “nsp2 Q780R + RLE Replicase”.

[0075] In some embodiments, the SFV replicase variant comprises (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1; (iv) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1; and (v) a threonine at position corresponding N1360 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; (iv) an arginine at position Q1317 of SEQ ID NO: 1; and (v) a threonine at position N1360 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0076] (i) A1211R;

[0077] (ii) D1212L;

[0078] (iii) A1213E;

[0079] (iv) Q780R; and

[0080] (v) N1360T; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; (iv) an arginine at position Q1317 of SEQ ID NO: 1; (v) a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1;

[0081] (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; (iv) an arginine at position Q1317 of SEQ ID NO: 1; (v) a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 8. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 8. SEQ ID NO: 8 is also referred to as “nsp2 Q780R + RLE + nsp3 N24T Replicase”.

[0082] In some embodiments, the SFV replicase variant comprises (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1; (iv) a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1; and (v) a threonine at a position corresponding N1360 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0083] (i) A1211R;

[0084] (ii) D1212L;

[0085] (iii) A1213E;

[0086] (iv) K425insK; and

[0087] (v) N1360T; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1; and (v) a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 9. In some embodiments, the SFV replicase variant comprises (i) an arginine at position A1211 of SEQ ID NO: 1; (ii) a leucine at position D1212 of SEQ ID NO: 1; (iii) a glutamic acid at position A1213 of SEQ ID NO: 1; (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1; and (v) a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 9. SEQ ID NO: 9 is also referred to as “nspl K425insK + RLE + N24T”. “NGK” Variants

[0088] In some embodiments, the SFV replicase variant comprises (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; and (iii) a lysine at a position corresponding to A1213 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0089] (i) A1211N;

[0090] (ii) D1212G; and

[0091] (iii) A1213K; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; and (iii) a lysine at position A1213 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 10. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; and (iii) a lysine at position A1213 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 10. In some embodiments, the SFV replicase variant comprises an amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 10 is also referred to as “NGK replicase”.

[0092] In some embodiments, the SFV replicase variant comprises (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a lysine at a position corresponding to A1213 of SEQ ID NO: 1; and (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0093] (i) A1211N;

[0094] (ii) D1212G;

[0095] (iii) A1213K; and (iv) K425insK; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 1.

[0096] In some embodiments, the SFV replicase variant comprises (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a lysine at a position corresponding to A1213 of SEQ ID NO: 1; and (iv) a threonine at a position corresponding to N1360 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) a threonine at position N1360 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0097] (i) A1211N;

[0098] (ii) D1212G;

[0099] (iii) A1213K; and

[0100] (iv) N1360T; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) a threonine at position N1360 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 1.

[0101] In some embodiments, the SFV replicase variant comprises (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a lysine at a position corresponding to A1213 of SEQ ID NO: 1; and (iv) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) an arginine at position Q1317 of SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0102] (i) A1211N;

[0103] (ii) D1212G;

[0104] (iii) A1213K; and

[0105] (iv) Q780R; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) an arginine at position Q1317 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) an arginine at position Q1317 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 1.

[0106] In some embodiments, the SFV replicase variant comprises (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; (iii) a lysine at a position corresponding to A1213 of SEQ ID NO: 1; (iv) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1; (v) a threonine at a position corresponding to N1360 of SEQ ID NO: 1; and / or (vi) a lysine insertion immediately c- terminal to position K425 of SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and iv) an arginine at position Q1317 of SEQ ID NO: 1; (v) a threonine at position N1360 of SEQ ID NO: 1; and / or (vi) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1.

[0107] In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0108] (i) A1211N;

[0109] (ii) D1212G;

[0110] (iii) A1213K;

[0111] (iv) N1360T;

[0112] (v) K425insK; and / or

[0113] (vi) Q780R; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; (iv) an arginine at position Q1317 of SEQ ID NO: 1; (v) a threonine at position N1360 of SEQ ID NO: 1; and / or (vi) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1 and an amino acid sequence having at least 95% identity to SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; (iii) a lysine at position A1213 of SEQ ID NO: 1; and (iv) an arginine at position Q1317 of SEQ ID NO: 1; (v) a threonine at position N1360 of SEQ ID NO: 1; and / or (vi) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1 and an amino acid sequence having at least 99% identity to SEQ ID NO: 1. In some embodiments, an SFV replicase variant comprises an amino acid sequence comprising one or more mutations relative to an amino acid sequence of a reference SFV replicase, wherein the one or more mutations comprise:

[0114] (i) A1211N;

[0115] (ii) D1212G;

[0116] (iii) A1213K;

[0117] (iv) K425insK; and / or

[0118] (v) N1360T; wherein the positions of the mutations are numbered by alignment of the amino acid sequence of the reference SFV replicase to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SFV replicase variant comprises (i) an asparagine at position A1211 of SEQ ID NO: 1; (ii) a glycine at position D1212 of SEQ ID NO: 1; and (iii) a lysine at position A1213 of SEQ ID NO: 1 (iv) a lysine insertion immediately c-terminal to position K425 of SEQ ID NO: 1; and (v) a threonine at position N1360 of SEQ ID NO: 1; and an amino acid sequence having at least 99% identity to SEQ ID NO: 10.

[0119] Methods of Use

[0120] In some embodiments, a SFV replicase variant disclosed herein decreases the toxicity of the replicase inside of a cell (e.g., a mammalian cell) compared to the wild-type SFV replicase. Measurements of cell toxicity are known in the art. For example, cell toxicity can be measured by measuring ISG54 induction by cells. In another example, cell toxicity can be measured by measuring cell death or by measuring cell death markers (e.g., pro-apoptotic genes). In some embodiments, a SFV replicase variant comprising (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; and (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1 has decreased immunogenicity compared to a wild-type SFV replicase. In some embodiments, a SFV replicase comprising (i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1; (ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; and (iii) a lysine acid at a position corresponding to A1213 of SEQ ID NO: 1 has decreased immunogenicity compared to a wild-type SFV replicase. In some embodiments, RLE replicase, NGK replicase, nspl 425insK replicase, npsl K425insK + RLE + nsp3 N24T replicase and nsp2 Q780R + RLE + nsp3 N24T replicase all have decreased cell toxicity.

[0121] In some embodiments, the SFV replicase variant further comprises an affinity tag. Affinity tags are known in the art, and include but are not limited to, Flag tag, His tag, Tap tag, HALOTAG, and Myc tag. The SFV replicase variant may comprise the affinity tag on the n- terminal or c-terminal of the replicase.

[0122] Polynucleotides

[0123] In some aspects, this disclosure provides a polynucleotide encoding a SFV replicase variant described herein. In some embodiments, the polynucleotide is an RNA polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide that when transcribed (e.g., during in vitro transcription) produces an RNA polynucleotide encoding the SFV replicase. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID

[0124] NO: 11. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 12. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 13. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 14. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 15. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 16. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 17. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 18. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 19. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 20. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 21. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 22. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 23. In some embodiments, the SFV replicase variant encoded by a sequence of SEQ ID NO: 24. In some embodiments, the SFV replicase variant is encoded by a sequence of SEQ ID NO: 25.

[0125] Amplifying RNA

[0126] In some aspects, this disclosure provides a trans-amplifying RNA (taRNA) comprising a trans amplifying ribonucleic acid (RNA) (taRNA) comprising: (i) a first RNA polynucleotide comprising a nucleic acid encoding the SFV replicase variant described herein; and (ii) a second RNA polynucleotide comprising: a conserved sequence element (CSE) that is cognate to the replicase encoded by the first RNA polynucleotide; and a nucleic acid pay load or a nucleic acid encoding a payload.

[0127] A “trans-amplifying RNA”, hereinafter referred to as “taRNA,” comprises a first and second RNA polynucleotide, wherein the first RNA polynucleotide encodes a replicase (e.g., a SFV replicase variant), wherein the second RNA polynucleotide comprises a conserved sequence element (CSE) cognate to the replicase, and 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).

[0128] 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).

[0129] 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.

[0130] Replicase Constructs

[0131] 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 SFV replicase variant) 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 “nonreplicating 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, the 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). saRNAs, 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 the same polynucleotide. saRNAs are known, e.g., as described in Comes JDG et al., Trends Biotechnol. 2023 Nov;41(l l):1417-1429.

[0132] Once expressed, a replicase (e.g., a SFV replicase variant) 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 it, which can be subsequently translated (e.g., by a host cell). 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.

[0133] 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 polyA 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.

[0134] 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 DNA sequence is provided in SEQ ID NO: 31. In some embodiments, a replicase construct comprises a 3’-UTR derived from human alpha-globin (3’-HAG-UTR). An exemplary 3’-HAG-UTR DNA sequence is provided in SEQ ID NO: 32. In some embodiments, a replicase construct comprises a 5’-HAG-UTR and a 3’-HAG-UTR. trRNA The term “trans replicon construct” (z.e., second RNA polynucleotide), also known as a “trans replicating RNA”, hereinafter referred to as “trRNA” refers to an RNA construct capable of being replicated by a replicase of a taRNA. A trRNA comprises at least a nucleic acid encoding a pay load or a nucleic acid pay load (i.e., a pay load-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).

[0135] 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.

[0136] 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).

[0137] 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.

[0138] 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).

[0139] 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. 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, Casl 1 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 payload 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).

[0140] 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 SEQ ID NOs: 26-28. In some embodiments, a 3’-SINV-UTR comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, a 3’-SFV-UTR comprises the sequence set forth in SEQ ID NO: 30.

[0141] 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., 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., 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 a trRNA comprising a CSE from a SINV. In some embodiments, a replicase derived from a SINV is capable of binding to a trRNA comprising a CSE from an SFV.

[0142] In some embodiments, the disclosure provides a self-amplifying RNA (saRNA) comprising a polynucleotide encoding a SFV replicase variant described herein.

[0143] Exemplary Sequences

[0144] Table 1: SFV Replicase Variant Protein Sequences -Z1 -

[0145] Table 2; SFV Replicase Variant Nucleic Acid Sequences

[0146] Table 3: SFV Replicase Variant in saRNA sequences

[0147] EXAMPLES

[0148] Example 1: SFV replicase variants “RLE replicase” and “NGK replicase” decrease cell death while maintaining payload expression when encoded in trans-amplifying RNAs.

[0149] One challenge faced when using taRNA or saRNA therapeutics is that the replicase expressed by the taRNA or saRNA (typically an alphavirus replicase like SFV replicase), is somewhat toxic to the cell and can result in cell death. This example describes SFV replicase variants that show decreased cell toxicity: RLE replicase (A1211R, D1212L, A1213E) and NGK replicase (A1211N, D1212G, A1213K).

[0150] Trans-amplifying RNAs (taRNAs) encoding wild-type SFV replicase, RLE replicase (or NGK replicase, and a GFP payload were tested for cell toxicity and payload expression in Vero cells. FIG. 1A show the percentage of cells that are dead or apoptotic after transfection with the taRNA. Results show that wild-type taRNA results in more apoptotic cells than RLE replicase. saRNA, mRNA and untreated were controls. FIG. IB shows that transfection of taRNAs encoding NGK replicase or RLE replicase resulted in more healthy cells and less dying or dead cells than transfection with a wild-type SFV replicase.

[0151] It was determined whether the mutations in RLE replicase and NGK replicase decreased expression of the payload (GFP) encoded by the taRNAs. Results show that RLE replicase and NGK replicase did not decrease expression of the GFP pay load (FIG. 2).

[0152] Overall, these results show that RLE replicase and NGK replicase can decrease taRNA replicase cytotoxicity while maintaining taRNA payload expression compared to wild-type SFV replicase.

[0153] Example 2: SFV replicase variants that decrease cell death in self-amplifying RNAs (saRNAs)

[0154] In contrast to taRNAs, the replicase amplifies both the RNA encoding the pay load and the RNA encoding the replicase, which results in increased replicase production, which in turn results increased cell death. This is particularly challenging for SFV replicase, which is more toxic than many other replicases (e.g., VEEV replicase) used in taRNAs or saRNAs. Described in this example are SFV replicase variants that have decreased cytotoxicity even when delivered to cells using saRNA (FIG. 3). 24 hours post transfection, most SFV replicase variants tested had similar effects as wild-type SFV replicase variant except nspl 425insK replicase, which appears to have an increased number of GFP positive cells. However, at 120 hours post transfection, npsl K425insK + RLE + nsp3 N24T replicase and nsp2 Q780R + RLE + nsp3 N24T replicase show much greater GFP expression and healthy cells. Overall, these results show that npsl K425insK + RLE + nsp3 N24T replicase and nsp2 Q780R + RLE + nsp3 N24T replicase have increased payload expression and decreased cytotoxicity at 120 hours post saRNA transfection compared to the other replicases tested including wild-type VEEV replicase, which is known to be less toxic than SFV replicase.

Claims

CLAIMSWhat is claimed is:

1. A Semliki Forest Virus (SFV) replicase variant wherein:(i) a position corresponding to 1211 of SEQ ID NO: 1 does not comprise an alanine;(ii) a position corresponding to 1212 of SEQ ID NO: 1 does not comprise an aspartic acid;(iii) a position corresponding to 1213 of SEQ ID NO: 1 does not comprise an alanine;(iv) an amino acid insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1;(v) a position corresponding to 1360 of SEQ ID NO: 1 does not comprise an asparagine; and / or(vi) a position corresponding to 1317 of SEQ ID NO: 1 does not comprise a glutamine.

2. The SFV replicase variant of claim 1, comprising:(i) an arginine or asparagine at a position corresponding to A1211 of SEQ ID NO: 1;(ii) a leucine or glycine at a position corresponding to D1212 of SEQ ID NO: 1;(iii) a glutamic acid or lysine at a position corresponding to A1213 of SEQ ID NO: 1;(iv) a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1;(v) a threonine at a position corresponding to N1360 of SEQ ID NO: 1; or(vi) an arginine at a position corresponding to Q1317 of SEQ ID NO: 1.

3. The SFV replicase variant of claim 2, comprising:(i) an arginine at a position corresponding to A1211 of SEQ ID NO: 1;(ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 1; and(iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 1.

4. The SFV replicase variant of claim 2, comprising:(i) an asparagine at a position corresponding to A1211 of SEQ ID NO: 1;(ii) a glycine at a position corresponding to D1212 of SEQ ID NO: 1; and(iii) a lysine acid at a position corresponding to A1213 of SEQ ID NO: 1.

5. The SFV replicase variant of any one of claims 1-4, comprising a threonine at a position corresponding to N1360 of SEQ ID NO: 1.

6. The SFV replicase variant of any one of claims 1-5, comprising an arginine at a position corresponding to Q1317 of SEQ ID NO: 1.

7. The SFV replicase variant of any one of claims 1-6, comprising a lysine insertion immediately c-terminal to a position corresponding to K425 of SEQ ID NO: 1.

8. The SFV replicase variant of claim 1, comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-10.

9. The SFV replicase variant of claim 1, comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 2-10.

10. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 2.

11. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 3.

12. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 4.

13. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 5.

14. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 6.

15. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 7.

16. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ IDNO: 8.

17. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 9.

18. The SFV replicase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 10.

19. The SFV replicase variant of any one of claims 1-18, further comprising an affinity tag.

20. A polynucleotide comprising a nucleic acid encoding the SFV replicase variant of any one of claims 1-19.

21. The polynucleotide of claim 20, wherein the polynucleotide is a DNA polynucleotide.

22. The polynucleotide of claim 20, wherein the polynucleotide is an RNA polynucleotide.

23. The polynucleotide of claim 22, comprising a nucleic acid sequence of SEQ ID NO: 11- 25.

24. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(i) a first RNA polynucleotide comprising a nucleic acid encoding the SFV replicase variant of any one of claims 1-5; and(ii) a second RNA polynucleotide comprising: a conserved sequence element (CSE) that is cognate to the replicase encoded by the first RNA polynucleotide; and a nucleic acid payload or a nucleic acid encoding a payload.

25. The taRNA of claim 24, wherein the second RNA polynucleotide comprises:(i) a 5’ alphavirus untranslated region (UTR);(ii) the CSE that is cognate to the replicase encoded by the first RNA polynucleotide;(iii) the nucleic acid payload or the nucleic acid encoding the payload; and(iv) a 3’ alphavirus UTR.

26. The taRNA of claim 25, wherein the 5’ alphavirus UTR is a SFV 5’-UTR or SINV 5’- UTR.

27. The taRNA of claim 25 or claim 26, wherein the 3’ alphavirus UTR is a 3 ’-UTR or SINV 3 ’-UTR.

28. The taRNA of any one of claims 25-27, wherein the 5’ alphavirus UTR comprises a mutation relative to a wild-type 5’ alphavirus UTR.

29. The taRNA of any one of claims 25-28, wherein the 3’ alphavirus UTR comprises a mutation relative to a wild-type 3’ alphavirus UTR.

30. A self-amplifying RNA comprising the polynucleotide of claim 22 or claim 23.

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