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WO2025171152A1 - Arn trans-réplicatifs et auto-réplicatifs comprenant une région 5'utr modifiée - Google Patents

Arn trans-réplicatifs et auto-réplicatifs comprenant une région 5'utr modifiée

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Publication number
WO2025171152A1
WO2025171152A1 PCT/US2025/014823 US2025014823W WO2025171152A1 WO 2025171152 A1 WO2025171152 A1 WO 2025171152A1 US 2025014823 W US2025014823 W US 2025014823W WO 2025171152 A1 WO2025171152 A1 WO 2025171152A1
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WIPO (PCT)
Prior art keywords
utr
seq
sinv
polynucleotide
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/014823
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English (en)
Inventor
Kevin CREAMER
Adam DINGENS
Cory Dane SAGO
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Amplitude Therapeutics Inc
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Amplitude Therapeutics Inc
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Filing date
Publication date
Application filed by Amplitude Therapeutics Inc filed Critical Amplitude Therapeutics Inc
Publication of WO2025171152A1 publication Critical patent/WO2025171152A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses

Definitions

  • This disclosure relates to self-amplifying (e.g., self-amplifying, trans amplifying) polynucleotides containing synthetic 5’ untranslated regions (UTRs).
  • self-amplifying e.g., self-amplifying, trans amplifying
  • RNAs can be further broken down into two different categories: self-amplifying RNAs (saRNAs) and trans-amplifying RNAs (taRNAs).
  • saRNAs self-amplifying RNAs
  • taRNAs trans-amplifying RNAs
  • the mRNA encoding the replicase, the CSE, and the payload are all encoded by the same RNA polynucleotide.
  • taRNAs the mRNA encoding the replicase (e.g., replicase construct) and the RNA encoding the CSE and the payload (trans replicating RNA (trRNA)) are different RNA molecules.
  • saRNAs and taRNAs comprise components derived from alphavirus genomes.
  • the nucleic acids encoding the pay load may be flanked by a 5’-UTR and/or a 3’- UTR derived from one or more alphaviruses.
  • Alphavirus UTRs e.g., 5’-UTR, 3’-UTR
  • Alphavirus 5’-UTR and 3’-UTR sequences can also influence amplification of a payload-encoding sequence by a replicase, thereby modulating expression of a payload.
  • a central challenge in developing saRNAs and taRNAs is tuning payload expression for a particular therapeutic.
  • increasing payload expression from a taRNA or saRNA is advantageous because it reduces the amount of saRNA or taRNA that needs to be administered to a subject to induce a therapeutic response.
  • decreasing payload expression from a taRNA or saRNA is advantageous, e.g., when a high level of payload expression is associated with adverse side effects.
  • the inventors of this disclosure measured the effects of over 500,000 synthetic sequence extensions inserted into 5’ Sindbis virus (SINV) UTRs on trRNA amplification using a novel high throughput screen. Amplification of a plurality of trRNAs comprising a plurality of different 5’-UTR extensions from 3 nucleotide extensions to 12 nucleotide extensions was measured using the screen and resulted in identification of many different 5’-UTR extensions that modify trRNA amplification. Surprisingly, different extension lengths were associated with unique properties.
  • this disclosure provides a polynucleotide comprising a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX 2 X 3 CGGC (REF ID NO: 1631; REF ID NO: 1638), wherein: Xi is A, T/U or G; X 2 is A, T/U or G; and X 3 is A, T/U, or G, wherein the 5’ terminal of the 5’ SINV UTR does not comprise a sequence of A(T/U)(T/U)GA.
  • the polynucleotide is an RNA polynucleotide and XiX 2 X 3 is any one of REF ID NOs: 272-276.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 816-820 or to a transcript of any one of SEQ ID NOs: 544-548.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ Semliki Forest Virus (SFV) UTR.
  • this disclosure provides, a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and X1X2X3 is any one of REF ID NOs: 1-5.
  • this disclosure provides a polynucleotide comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX 2 X 3 X 4 CGGC (SEQ ID NO: 1632; SEQ ID NO: 1639), wherein: Xi is A or T/U; X 2 is A, T/U or G; X3 is A, T/U, or G; and X 4 is A, T/U, or G, wherein the polynucleotide does not comprise a sequence of A(T/U)AAAA.
  • Xi is A; X 2 is A, T/U, or G; X3 is A or G; and X 4 is A or G.
  • the polynucleotide is an RNA polynucleotide and XiX 2 X 3 X 4 is REF ID NO: 277.
  • the 5’ SINV UTR comprises at least 90% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ SFV UTR.
  • this disclosure provides a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and X 1 X 2 X 3 X 4 is REF ID NO: 6.
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX 2 X3X 4 XSCGGC (SEQ ID NO: 1633; SEQ ID NO: 1640), wherein: Xi is A, T/U or G; X 2 is A, T/U or G; X 3 is A, T/U, or G; X 4 is A, T/U, or G; and X5 is A, T/U, C, or G, wherein the polynucleotide does not have a sequence of A(T/U)AAAAA.
  • SINV UTR Sindbis virus
  • Xi is A; X 2 is A, T/U or G; X3 is A; X 4 is A or G; and X5 is A or G.
  • the polynucleotide is an RNA polynucleotide and XiX 2 X3X 4 Xs is any one of REF ID NOs: 278-281.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 822-825 or to a transcript of any one of SEQ ID NOs: 550-553.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ SFV UTR.
  • this disclosure provides a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and X1X2X3X4X5 is any one of REF ID NOs: 7-10.
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX2X3X4XSX6CGGC (REF ID NO: 1634; REF ID NO: 1641), wherein: Xi is A or T/U; X2 is A or G; X3 is A, T/U, or G; X4 is A, T/U, G or C; X5 is A, T/U, G, or C; and Xf> is A, T/U, G, or C.
  • SINV UTR Sindbis virus
  • Xi is A; X2 is A or G; X3 is A, T/U, or G; X4 is A, T/U, or G; X5 is A, T/U, or G; and Xf> is A, T/U, or G.
  • the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 826-827 or to a transcript of any one of SEQ ID NOs: 554-555.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ SFV UTR.
  • this disclosure provides a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6 is any one of REF ID NOs: 11-12 (SEQ ID NOs: 1693-1694).
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX2X3X4 X5X6X7CGGC (REF ID NO: 1635; REF ID NO: 1642), wherein: Xi is A, T/U or G; X 2 is A or G; X 3 is A, T/U, or G; X 4 is A, T/U, C, or G; X5 is A, T/U, G, or C; X ⁇ > is A, T/U, G, or C; and X7 is A, T/U, G, or C.
  • SINV UTR Sindbis virus
  • Xi is A; X2 is A or G; X3 is A or G; X4 is A, T/U, or G; X5 is A, T/U, G, or C; X ⁇ > is A, T/U, G, or C; and X7 is A, T/U, G, or C.
  • the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6X7 is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701).
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 828-834 or to a transcript of any one of SEQ ID NOs: 556-562.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ SFV UTR.
  • this disclosure provides a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7 is any one of REF ID NOs: 13- 19 (SEQ ID NO: 1702-1708).
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX2X3X4X5X6X7XSCGGC (REF ID NO: 1636; REF ID NO: 1643), wherein: Xi is A or G; X2 is A or G; X3 is A, T/U, or G; X4 is A, T/U, G, or C; X 5 is A, T/U, G, or C; X 6 is A, T/U, G, or C; X 7 is A, T/U, C, or G; and X 8 is A, T/U, C or G.
  • SINV UTR Sindbis virus
  • the polynucleotide is an RNA polynucleotide and XiX2X3X 4 X 5 X6X7X8is REF ID NO: 291 (SEQ ID NO: 1709).
  • the 5’ SINV UTR comprises at least 90% identity to SEQ ID NO: 835 or to a transcript of any one of SEQ ID NO: 563.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a pay load, and a 3’ SFV UTR.
  • this disclosure provides a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7X8 is REF ID NO: 20 (SEQ ID NO: 1710).
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX2X3X4XSX6X7X8X9CGGC (REF ID NO: 1637; REF ID NO: 1644), wherein: Xi is A, T/U, C or G; X 2 is A, T/U or G; X 3 is A, T/U, C or G; X 4 is A, T/U, C, or G; X 5 is A, T/U, C, or G; X 6 is A, T/U, C or G; X 7 is A, T/U, C or G; X 8 is A, T/U, C or G; and X9 is A, T/U, C or G.
  • SINV UTR Sindbis virus
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X4AXSGGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is A or G; X2 is A or G; X3 is A or T/U; X4 is A, T/U, C, or G; X5 is T/U or C.
  • SINV Sindbis virus
  • Xi is A; X2 is A or G; X3 is A or T/U; X4 is C; and X5 is T/U.
  • the polynucleotide is an RNA polynucleotide and AX1X2X3X4AX5GG is any one of AGAAGAUGG (REF ID NO: 294), AGAAGACGG (REF ID NO: 296), or AGAACAUGG (REF ID NO: 298).
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 839, 841, or 843 or to a transcript of any one of SEQ ID NOs: 566, 568, or 570.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ SFV UTR.
  • this disclosure provides a selfamplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and AX1X2X3X4AX5GG is any one of REF ID NOs: 23, 25, or 27.
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X4XSAX6GGCGGC (SEQ ID NO: 1655; SEQ ID NO: 1659), wherein: Xi is A or G; X2 is A or G; X3 is A or G; X4 is A, T/U, C, or G; X5 is A, T/U, C, or G; Xf> is T/U.
  • SINV UTR Sindbis virus
  • Xi is A or G; X2 is A; X3 is A; X4 is A or G; X5 is T/U or C; and X ⁇ > is T/U.
  • the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5AX6GG is any one of SEQ ID NO: 307-333.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 851-877 or to a transcript of any one of SEQ ID NOs: 579-605.
  • this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X4XSX6AX7GGCGGC (SEQ ID 1656; SEQ ID NO: 1660), wherein: Xi is A or G; X2 is A, T/U, or G; X3 is A, T/U, C, or G; X4 is A or G; X5 is A, T/U, C or G; Xf> is A, T/U, C or G; and X7 is T/U or C.
  • SINV UTR Sindbis virus
  • Xi is A; X2 is A or G; X3 is A, C, or G; X4 is A or G; X5 is A or T/U; X ⁇ > is T/U or C; and X7 is T/U.
  • the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5X6AX7GG is any one of SEQ ID NO: 334-384.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 878-928 or to a transcript of any one of SEQ ID NOs: 606-656.
  • the polynucleotide is comprised in a taRNA, wherein the taRNA comprises: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, the polynucleotide comprising the 5’ SINV UTR, nucleic acids encoding a payload, and a 3’ SFV UTR.
  • this disclosure provides a self-amplifying RNA comprising the polynucleotide.
  • the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5X6AX7GG is any one of SEQ ID NOs: 114-271.
  • this disclosure provides a taRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising, from 5’ to 3’, a 5’ SINV UTR comprising a 5’ UTR extension disclosed herein, nucleic acids encoding a pay load, and a 3’ SFV UTR.
  • the taRNA comprises any one of REF ID NO: 1638, SEQ ID NOs: 1639-1640, REF ID NOs: 1641-1644, or SEQ ID NOs: 1658-1661.
  • this disclosure provides a self-amplifying RNA comprising a 5’ SINV UTR comprising a 5’ UTR extension disclosed herein.
  • the saRNA comprises any one of REF ID NO: 1638, SEQ ID NOs: 1639-1640, REF ID NOs: 1641-1644, or SEQ ID NOs: 1658-1661
  • this disclosure provides a DNA polynucleotide that, when transcribed, produces an RNA polynucleotide comprising a 5’ SINV UTR comprising a 5’ UTR extension disclosed herein.
  • the DNA polynucleotide comprises any one of REF ID NO: 1631, SEQ ID NOs: 1632-1633, REF ID NOs: 1634-1637, or SEQ ID NOs: 1654-1657.
  • this disclosure provides method of determining amplification of a plurality of trans-replicating ribonucleic acids (RNAs) (trRNAs) in cells, the method comprising: obtaining a plurality of trRNAs, wherein trRNAs of the plurality of trRNAs comprise a conserved sequence element (CSE) and a 5’ untranslated region (UTR) extension (5’-UTR extension), wherein at least two of the trRNAs comprise different 5’-UTR extensions; contacting trRNAs of the plurality of trRNAs with a plurality of cells, wherein cells of the plurality of cells comprise a replicase that is cognate to the CSE; determining amplification of the plurality of contacted trRNAs of the plurality of trRNAs, the determining comprising detecting the at least two different 5’-UTR extensions in the plurality of cells or cell lysate of the plurality of cells.
  • CSE conserved sequence element
  • each trRNA of the plurality of trRNAs (a) a 5’ alphavirus UTR comprising a 5 ’-UTR extension; (b) a CSE; (c) nucleic acids encoding a pay load; and (d) a 3’ alphavirus UTR.
  • the 5’ alphavirus UTR is a Venezuelan Equine Encephalitis Virus (VEEV) 5 ’-UTR, Semliki Forest Virus (SFV) 5 ’-UTR, Sindbis Virus (SINV) 5’-UTR, or Chikungunya virus (CHIKV) 5’-UTR.
  • VEEV Venezuelan Equine Encephalitis Virus
  • SFV Semliki Forest Virus
  • SINV Sindbis Virus
  • CHIKV Chikungunya virus
  • the reporter protein is enhanced green fluorescent protein (EGFP), mCherry, mKate, anchored heavy chain variable domain (aVHH), nano luciferase (nanoLuc), firefly luciferase (Flue), secreted embryonic alkaline phosphatase (SEAP), or Cre recombinase.
  • the payload is not a reporter protein.
  • cells of the plurality of cells comprise a polynucleotide encoding the replicase that is cognate to the CSE.
  • the method further comprises contacting cell of the plurality of cell with a polyribonucleotide encoding the replicase that is cognate to the CSE.
  • the plurality of trRNAs comprises at least 10 different trRNAs. In some embodiments, the at least 10 different trRNAs each comprise at different 5’ UTR extension.
  • obtaining the plurality of RNA polynucleotides comprises synthesizing the plurality of RNA polynucleotides using in vitro transcription.
  • determining amplification of the plurality of trRNAs comprises sequencing 5 ’-UTR extensions of the plurality of trRNAs before the contacting to produce control data; and sequencing 5’ UTR extensions of the plurality of trRNAs after the contacting to produce enrichment data.
  • determining amplification further comprises using the control data and the enrichment data to determine a relative increase in the amount of 5 ’-UTR extension before and after the contacting.
  • the contacting comprises transfecting trRNAs of the plurality of trRNAs into cells of the plurality of cells.
  • the obtaining comprises obtaining a plurality of self-amplifying RNAs (saRNAs) that comprise the trRNA and a replicase construct.
  • saRNAs self-amplifying RNAs
  • a DNA polynucleotide that, when transcribed, produces a ribonucleic acid RNA polynucleotide comprising a 5’ SINV UTR, wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of any one of REF ID NO: 1638, SEQ ID NOs: 1639-1640, REF ID NOs: 1641-1644, or SEQ ID NOs: 1658-1661.
  • RNA polynucleotide that, when transcribed, produces a RNA polynucleotide comprising a 5’ UTR comprising the sequence set forth in any one of SEQ ID NOs: 816-1086.
  • FIG. 1 is a schematic of a screen for measuring amplification of trans replicating ribonucleic acids (trRNAs) comprising different 5’ untranslated region (5 ’-UTR) extensions.
  • trRNAs trans replicating ribonucleic acids
  • FIGs. 2A-2B show trRNA 5’-UTR extension library design and assay design.
  • FIG. 2A shows an example of 5’ UTR extension library design for even length extensions (e.g., 4N, 6N and 8N).
  • N represents A, T/U, G, or C.
  • Hundreds of thousands of different 5 ’-UTR extensions were assayed.
  • the same design strategy was used for odd number 5’ UTR extensions (e.g., 3N, 5N, 7N and 9N).
  • FIG. 2B is a flow chart of the steps in determining amplification of the different trRNAs in the library.
  • FIGs. 3A-3B show performance of four control trRNAs.
  • FIG. 3A shows EGFP expression by each control trRNA.
  • FIG. 3B shows enrichment of these same sequences in a pooled screen experiment. There is agreement between rank order of controls in individual expression assessment and in pooled screens.
  • FIG. 4 shows logo plots for 3N extensions (AUNNNCGGC) based on enrichment of 5’ UTR extension in cells over time.
  • the top panel shows the in vitro transcribed 3N trRNA library distribution before transfection.
  • the middle panel shows the 3N trRNA library distribution after 1 transfection.
  • the bottom panel shows the 3N trRNA library distribution after 4 serial transfections of the 3N trRNA library.
  • FIG. 5 shows logo plots for 4N extensions (AUNNNNCGGC) based on enrichment of 5’ UTR extension in cells over time.
  • the top panel shows the in vitro transcribed 4N trRNA library distribution before transfection.
  • the bottom panel shows the 4N trRNA library distribution after 4 serial transfections of the 4N trRNA library.
  • FIG. 9 shows logo plots for 8N extensions (AUNNNNNNNNCGGC) based on enrichment of 5’ UTR extension in cells over time.
  • the top panel shows the in vitro transcribed 8N trRNA library distribution before transfection.
  • the bottom panel shows the 8N trRNA library distribution after 4 serial transfections of the 8N trRNA library.
  • FIG. 10 shows logo plots for 9N extensions (AUNNNNNNNCGGC) based on enrichment of 5’ UTR extension in cells over time.
  • the top panel shows the in vitro transcribed 9N trRNA library distribution before transfection.
  • the bottom panel shows the 9N trRNA library distribution after 4 serial transfections of the 9N trRNA library.
  • FIGs. 11A-1 IB show validation of enrichment data from the screen shown in FIG. 1.
  • FIG. 11A shows that 5’ UTR extensions that increased enrichment in the screen also increase trRNA payload expression in vitro.
  • FIG. 1 IB shows tRNA payload expression of trRNA 5’ UTR extension hits from the screen in BHK-21 cells.
  • FIG. 12 shows in vivo validation of trRNA hits from the screen, as determined by SEAP expression. Particularly, results show that a top performing trRNA 5’ UTR extension from the screen A(T/U)AGAAGA(T/U)GG (SEQ ID NO: 1667; SEQ ID NO: 1668) (“optimized trRNA”) shows greatly increased payload expression in in vitro compared to a T3A 5’ UTR extension, which has been shown to express better than a trRNA without a 5’ UTR extension.
  • FIG. 14 shows fold enrichment of oeSTR trRNA 5’-UTR extensions compared to a T3A 5’-UTR, WT 5’-UTR, and oeSTR (which is one of the top hits from the 3N-9N screens). Many oeSTR extensions show greatly increased enrichment over other 5’-UTRs extensions including oeSTR.
  • FIG. 15 shows a logo plot for the oe5N extension AUANNNNANGGCGGC (SEQ ID NO: 1669) based on enrichment of 5’ UTR after 4 serial transfections of in vitro transcribed oe6N trRNA library.
  • positions with few or no nucleotide symbols shown are A, T/U, C, or G.
  • FIG. 16 shows a logo plot for the oe6N extension AUANNNNNANGGCGGC (SEQ ID NO: 1670) based on enrichment of 5’ UTR after 4 serial transfections of in vitro transcribed oe6N trRNA library.
  • positions with few or no nucleotide symbols shown are A, T/U, C, or G.
  • FIG. 17 shows a logo plot for the oe7N extension AUANNNNNNANGGCGGC (SEQ ID NO: 1671) based on enrichment of 5’ UTR after 4 serial transfections of in vitro transcribed oe7N trRNA library.
  • positions with few or no nucleotide symbols shown are A, T/U, C, or G.
  • FIG. 18 shows a logo plot for the oe8N extension AUANNNNNNNANGGCGGC (SEQ ID NO: 1672) based on enrichment of 5’ UTR after 4 serial transfections of in vitro transcribed oe8N trRNA library.
  • positions with few or no nucleotide symbols shown are A, T/U, C, or G.
  • this disclosure describes polynucleotides (e.g., a trRNA or saRNA) comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid (e.g., 5’ SINV UTR extension) having a sequence A(T/U)XIX2...X n CGGC wherein each X is a different nucleotide in the sequence, and 3 ⁇ n >12.
  • SINV Sindbis virus
  • 5’ SINV UTR extension having a sequence A(T/U)XIX2...X n CGGC wherein each X is a different nucleotide in the sequence, and 3 ⁇ n >12.
  • 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.
  • 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.
  • 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).
  • a sequence, including a polynucleotide or amino acid sequence, is found to have a specified percent identity to a reference sequence, such as a sequence disclosed in this application and/or recited in the claims when sequence identity is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993 (e.g., BLAST, NBLAST, XBLAST or Gapped BLAST programs, using default parameters of the respective programs).
  • polynucleotides of the instant disclosure comprise an untranslated region (UTR).
  • UTRs may act as stabilizing elements and/or provide regulation of transcription of a gene or transgene.
  • UTRs are found upstream and/or downstream of a gene (e.g., 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.
  • 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.
  • a polynucleotide comprises a 5 ’-UTR and/or a 3 ’-UTR.
  • a UTR is a 5’ Sindbis virus (SINV) UTR.
  • SINV UTR refers to a wildtype or modified 5’ SINV UTR.
  • a wildtype 5’ SINV UTR comprises the sequence set forth in SEQ ID NO: 815.
  • modified 5’ SINV UTRs described herein are described relative to wildtype 5’ SINV UTR (e.g., SEQ ID NO: 815); however, the ordinarily skilled artisan will appreciate that minor variations (e.g., naturally occurring variants) may occur within the 5’ SINV UTR, and the teachings provided herein may be applicable to these variants.
  • a modified 5’ SINV UTR may comprise a 5 ’-UTR extension as described herein.
  • a modified 5’ SINV UTR comprises a mutation relative to the wildtype 5’ SINV UTR (e.g., a single nucleotide polymorphism, an insertion, or a deletion).
  • the mutation is in the 5’-UTR extension.
  • the mutation is not in the 5 ’-UTR extension.
  • the modified 5’ SINV UTR comprises the sequence set forth in any one of SEQ ID NOs: 816-1086.
  • a “5’ terminal of a 5’ SINV UTR” refers to nucleotides at the 5’ end of the 5’ SINV UTR.
  • the 5’ terminal of wildtype 5’ SINV UTR e.g., the 5’ terminal of SEQ ID NO: 815) typically comprises six conserved nucleotides: A(T/U)CGGC.
  • the 5’-UTR extensions described herein are inserted between the A(T/U) and CGGC (e.g., A(T/U)XIX2...X n CGGC).
  • a polynucleotide comprising a 5’ SINV UTR (e.g., a modified 5’ SINV UTR described herein) additionally comprises polynucleotides that are upstream of the 5’ terminal of the 5’ SINV UTR (e.g., an RNA polymerase promoter).
  • alphavirus refers to an RNA virus belonging to the Togaviridae family. Alphaviruses generally comprise a single- stranded 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), Venezuelan equine encephalitis virus, and Chikungunya virus (CHIKV).
  • an alphaviral replicase comprises a complex formed by the non-structural proteins nsPl, nsP2, nsP3, and nsP4.
  • 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 that is “cognate” to a CSE refers to a replicase that is the capable of transcribing a portion of a polynucleotide comprising the CSE (e.g., capable of transcribing a polynucleotide comprising the payload).
  • a replicase construct encodes a replicase derived from an SFV (SFV replicase).
  • a trRNA comprises (a) a 5’ alphavirus UTR (e.g., a modified 5’ UTR); (b) a CSE; (c) nucleic acids encoding a pay load or a nucleic acid pay load; and (d) a 3’ alphavirus UTR.
  • a trRNA comprises (a) a 5’ SINV UTR comprising a 5’ UTR extension; (b) a CSE; (c) nucleic acids encoding a pay load or a nucleic acid pay load; and (d) a 3’ SFV alphavirus UTR.
  • the replicase cognate to the CSE comprised in a trRNA is provided by a replicase construct, such as the replicase construct of a trans-amplifying RNA (taRNA).
  • taRNA trans-amplifying RNA
  • a “replicase construct” is an RNA polynucleotide comprising nucleic acids encoding a replicase from an RNA virus, for example, an alphavirus.
  • the replicase of the replicase construct is modified (e.g., to comprise one or more mutations).
  • taRNAs do not comprise a single polynucleotide that comprises both a trRNA and a replicase construct.
  • 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., as say- specific media).
  • 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.
  • an RNA polynucleotide (e.g., a trRNA or saRNA) comprises a modified 5’-UTR described herein, and a 3’-UTR derived from a SFV, herein referred to as an “3’ SFV UTR”.
  • An exemplary 3’ SFV UTR is provided in SEQ ID NO: 1651.
  • an RNA polynucleotide (e.g., a trRNA or saRNA) comprises a modified 5 ’-UTR described herein, and a 3’-UTR derived from a SINV, herein referred to as a “3’ SINV UTR”.
  • an exemplary 3’ SINV UTR is provided in SEQ ID NO: 1653.
  • an RNA polynucleotide comprises a 5 ’-UTR and 3 ’-UTR from the same virus.
  • an RNA polynucleotide comprises a 5’-SINV UTR (e.g., a modified 5’ SINV UTR), and a 3’-SINV UTR.
  • an RNA polynucleotide comprises a 5’-UTR and 3’-UTR from different alphaviruses.
  • an RNA polynucleotide comprises a 5’-SINV UTR (e.g., a modified 5’ SINV UTR), and a 3 ’-SFV UTR.
  • an RNA polynucleotide comprises (a) a 5’ SINV UTR comprising a 5’ UTR extension; (b) a CSE; (c) a nucleic acid encoding a pay load or a nucleic acid pay load; and (e) a 3’ SFV UTR.
  • polynucleotides e.g., trRNA or saRNA
  • 3N extensions are 3-nucleotide long extensions (e.g., X1X2X3) inserted between the A(T/U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR (e.g., A(T/U)XIX 2 X 3 CGGC).
  • SINV alphaviral
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX 2 X 3 CGGC (REF ID NO: 1631, REF ID NO: 1638), wherein: Xi is A, T/U or G; X 2 is A, T/U or G; and X 3 is A, T/U, or G.
  • the polynucleic acid does not comprise a sequence of A(T/U)(T/U)GA.
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX 2 X 3 CGGC (REF ID NO: 1631, REF ID NO: 1638), wherein: Xi is A or G; X 2 is A or G; and X 3 is A, T/U, or G.
  • A(T/U)XIX 2 X 3 CGGC REF ID NO: 1631, REF ID NO: 1638
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 CGGC (REF ID NO: 1638), wherein: Xi is A, U or G; X 2 is A, U or G; and X 3 is A, U, or G; and wherein the polynucleic acid does not comprise a sequence of AUUGA.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 CGGC (REF ID NO: 1638), wherein XiX 2 X 3 is any one of REF ID NOs: 272-276.
  • the polynucleotide comprises a DNA polynucleotide that, when transcribed, produces a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 CGGC (REF ID NO: 1638), wherein XiX 2 X 3 is any one of REF ID NOs: 272-276.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 is REF ID NO: 275.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 is REF ID NO: 276.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 816-820 or to a transcript of any one of SEQ ID NOs: 544-548. In some embodiments, the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 816-820 or to a transcript of any one of SEQ ID NOs: 544-548. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 816-820 or to a transcript of any one of SEQ ID NOs: 544-548. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 816-820 or to a transcript of any one of SEQ ID NOs: 544-548.
  • the trRNA comprises any one of REF ID NOs: 1359-1364. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1359-1364 or to a transcript of any one of REF ID NOs: 1088-1092. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1359-1364 or to a transcript of any one of REF ID NOs: 1088-1092. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of SEQ ID NOs: 1359-1364.
  • the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. In some embodiments, the taRNA comprises a replicase construct of SEQ ID NO: 1649 and trRNA of any one of REF ID NOs: 1359-1364.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein: Xi is A, U or G; X2 is A, U or G; and X3 is A, U, or G, wherein the 5’ terminal of the 5’ SINV UTR does not comprise a sequence of AUUGA.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a poly nucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein: XI is A or G; X2 is A or G; and X3 is A, U, or G, wherein the polynucleic acid does not comprise a sequence of AUUGA.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638) wherein X1X2X3 is any one of REF ID NOs: 272-276.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638) wherein X1X2X3 is any one of REF ID NOs: 1-5.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 is REF ID NO: 272.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 is REF ID NO: 273.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 is REF ID NO: 274.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 is REF ID NO: 276.
  • this disclosure provides a saRNA comprising an RNA polynucleotide comprising: (a) a first nucleic acid encoding a replicase; and (b) a second nucleic acid comprising: a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3CGGC (REF ID NO: 1638), wherein X1X2X3 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 3N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a payload; and a 3’ SFV UTR.
  • the replicase is an SFV replicase.
  • the replicase is a SINV replicase.
  • polynucleotides e.g., trRNA or saRNA
  • 4N extensions are 4-nucleotide long extensions (e.g., X1X2X3X4) inserted between the A(T/U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR (e.g., A(T/U)XIX2X 3 X 4 CGGC).
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX2X3X 4 CGGC (SEQ ID NO: 1632, SEQ ID NO: 1639), wherein: Xi is A; X 2 is A, T/U, or G; X 3 is A or G; and X 4 is A or G. 4N Extension taRNA
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4CGGC (SEQ ID NO: 1639) wherein XiX 2 X 3 X 4 is REF ID NO: 6.
  • polynucleotides e.g., trRNA or saRNA
  • 5N extensions are 5-nucleotide long extensions (e.g., X2X3X4X5) inserted between the A(T/U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR (e.g., A(T/U) X2X3X4X5CGGC).
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 is any one of REF ID NOs: 278-281.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 822-825 or to a transcript of any one of SEQ ID NOs: 550-553. In some embodiments, the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 822-825 or to a transcript of any one of SEQ ID NOs: 550-553. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 822-825 or to a transcript of any one of SEQ ID NOs: 550-553. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 822-825 or to a transcript of any one of SEQ ID NOs: 550-553.
  • the trRNA comprises any one of REF ID NOs: 1366-1369. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1366-1369 or to a transcript of any one of REF ID NOs: 1095-1097. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1366-1369 or to a transcript of any one of REF ID NOs: 1095-1097.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein: Xi is A, U or G; X 2 is A, U or G; X3 is A, U, or G; X4 is A, U, or G; and X5 is A, U, C, or G, wherein the polynucleic acid does not comprise a sequence of AUAAAAA.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein: Xi is A; X2 is A, U or G; X3 is A; X4 is A or G; and X5 is A or G, wherein the polynucleic acid does not comprise a sequence of AUAAAAA.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640) wherein X1X2X3X4X5 is any one of REF ID NOs: 278-281.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640) wherein X1X2X3X4X5 is any one of REF ID NOs: 278-281.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 is REF ID NO:
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 is REF ID NO:
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 is REF ID NO:
  • polynucleotides e.g., trRNA or saRNA
  • 6N extensions are 6-nucleotide long extensions (e.g., X1X2X3X4X5X6) inserted between the A(T/U) and CGGC of an alphaviral (e.g., SINV) 5 ’-UTR (e.g., A(T/U)XIX2X3X 4 X 5 X 6 CGGC).
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX2X3X 4 X 5 X 6 CGGC (REF ID NO: 1634; REF ID NO: 1641), wherein: Xi is A or T/U; X 2 is A or G; X 3 is A, T/U, or G; X 4 is A, T/U, G or C; X 5 is A, T/U, G, or C; and X ⁇ > is A, T/U, G, or C.
  • Xi is A or T/U
  • X 2 is A or G
  • X 3 is A, T/U, or G
  • X 4 is A, T/U, G or C
  • X 5 is A, T/U, G, or C
  • X ⁇ > is
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)XIX 2 X 3 X 4 X5X6CGGC (REF ID NO: 1634; REF ID NO: 1641), wherein: Xi is A; X 2 is A or G; X 3 is A, T/U, or G; X 4 is A, T/U, or G; Xs is A, T/U, or G; and X 6 is A, T/U, or G (SEQ ID NOs: 1741-1742).
  • Xi is A
  • X 2 is A or G
  • X 3 is A, T/U, or G
  • X 4 is A, T/U, or G
  • Xs is A, T/U, or G
  • X 6 is A, T/U, or
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 X 4 X 5 X 6 CGGC (REF ID NO: 1641), wherein: Xi is A or U; X 2 is A or G; X 3 is A, U, or G; X 4 is A, U, G or C; Xs is A, U, G, or C; and Xe is A, U, G, or C.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 X 4 XSX 6 CGGC (REF ID NO: 1641), wherein: Xi is A; X 2 is A or G; X 3 is A, U, or G; X 4 is A, U, or G; X 5 is A, U, or G; and X 6 is A, U, or G (SEQ ID NO: 1741).
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 X 4 X 5 X 6 CGGC (REF ID NO: 1641), wherein XiX 2 X 3 X 4 X 5 X 6 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces an trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a poly nucleic acid having a sequence of AUXIX 2 X 3 X 4 X 5 X 6 CGGC (REF ID NO: 1641), wherein XiX 2 X 3 X 4 X 5 X 6 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 X 4 X 5 X 6 CGGC (REF ID NO: 1641), wherein XiX 2 X 3 X 4 X 5 X 6 is REF ID NO: 282 (SEQ ID NO: 1691).
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXIX 2 X 3 X 4 X 5 X 6 CGGC (REF ID NO: 1641), wherein XiX 2 X 3 X 4 X 5 X 6 is REF ID NO: 283 (SEQ ID NO: 1692).
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 826-827 or to a transcript of any one of SEQ ID NOs: 554-555.
  • the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 826-827 or to a transcript of any one of SEQ ID NOs: 554-555. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 826-827 or to a transcript of any one of SEQ ID NOs: 554-555. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 826-827 or to a transcript of any one of SEQ ID NOs: 554-555.
  • the trRNA comprises any one of REF ID NOs: 1370-1371. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1370-1371 or to a transcript of any one of REF ID NOs: 1098-1099. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1370-1371 or to a transcript of any one of REF ID NOs: 1098-1099.
  • the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1370-1371 or to a transcript of any one of REF ID NOs: 1098-1099. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1370-1371 or to a transcript of any one of REF ID NOs: 1098-1099.
  • this disclosure provides a taRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising: a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6CGGC (REF ID NO: 1641), wherein X1X2X3X4X5X6 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 6N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR.
  • a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicas
  • the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. In some embodiments, the taRNA comprises replicase construct of any one of SEQ ID NOs: 1634-1636 and trRNA of any one of REF ID NOs: 1370-1371.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6CGGC (REF ID NO: 1641), wherein: Xi is A or U; X 2 is A or G; X3 is A, U, or G; X4 is A, U, G or C; X5 is A, U, G, or C; and Xf> is A, U, G, or C.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6CGGC (REF ID NO: 1641), wherein: Xi is A; X 2 is A or G; X 3 is A, U, or G; X 4 is A, U, or G; X 5 is A, U, or G; and X 6 is A, U, or G (SEQ ID NO: 1741).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6CGGC (REF ID NO: 1641) wherein XiX2X3X 4 X 5 X 6 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a poly nucleic acid having a sequence of AUX1X2X3X4X5X6CGGC (REF ID NO: 1641) wherein XiX2X3X 4 X 5 X 6 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 295 (SEQ ID NO: 1714).
  • the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. In some embodiments, the taRNA comprises replicase construct of any one of SEQ ID NOs: 1634-1636 and trRNA of any one of REF ID NOs: 1380-1394.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 292 (SEQ ID NO: 1711).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 295 (SEQ ID NO: 1714).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 296 (SEQ ID NO: 1715).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 297 (SEQ ID NO: 1716).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 298 (SEQ ID NO: 1717).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 299 (SEQ ID NO: 1718).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 300 (SEQ ID NO: 1719).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 301 (SEQ ID NO: 1720).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 302 (SEQ ID NO: 1721).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 303 (SEQ ID NO: 1722).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 304 (SEQ ID NO: 1723).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 305 (SEQ ID NO: 1724).
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is REF ID NO: 306 (SEQ ID NO: 1725).
  • this disclosure provides a saRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising, a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 9N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR.
  • the replicase is an SFV replicase.
  • the replicase is an SFV replicas
  • polynucleotides e.g., trRNA or saRNA
  • oe5N extensions are extensions of the following generic sequence:(AXiX2X3X4AXsGG) inserted between the A(T/U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR, in which Xi, X2, X3, X4, and X5 were randomly varied as A, T/U, G or C in the screen.
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X 4 AX 5 GGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is A or G; X 2 is A or G; X3 is A or T/U; X 4 is A, T/U, C, or G; and X5 is T/U or C.
  • trRNA or saRNA comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X 4 AX 5 GGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is A or G
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX 2 X3X 4 AX 5 GGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is A; X 2 is A or G; X3 is A or T/U; X 4 is C; and X5 is T/U.
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X 4 AXSGGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is G; X 2 is A or G; X 3 is A; X 4 is C; and X 5 is T/U or C.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X 3 X 4 AX 5 GGCGGC (SEQ ID NO: 1658), wherein: Xi is A or G; X 2 is A or G; X3 is A or U; X 4 is A, U, C, or G; and X5 is U or C.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X3X 4 AXSGGCGGC (SEQ ID NO: 1658), wherein: Xi is A; X2 is A or G; X3 is A or U; X 4 is C; and X5 is U.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X 3 X 4 AX 5 GGCGGC (SEQ ID NO: 1658), wherein: Xi is G; X 2 is A or G; X 3 is A; X 4 is C; and X5 is U or C.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXAXIX2X 3 X 4 AX 5 GGCGGC (SEQ ID NO: 1658), wherein AXIX2X 3 X 4 AX 5 GG is any one of SEQ ID NOs: 293-294, 296, or 298-306.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a poly nucleic acid having a sequence of AUAXIX2X 3 X 4 AX 5 GGCGGC (SEQ ID NO: 1658), wherein AXIX2X 3 X 4 AX 5 GG is any one of SEQ ID NOs: 293-294, 296, or 298-306.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 293.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 294.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 296.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 298.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 299.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 300.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 301.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 302.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 303.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 304.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 305.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 306.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 837-838, 840, 842-850 or to a transcript of any one of SEQ ID NOs: 565-566, 568, 570-578. In some embodiments, the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 837-838, 840, 842-850 or to a transcript of any one of SEQ ID NOs: 565- 566, 568, 570-578.
  • the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 837-838, 840, 842-850 or to a transcript of any one of SEQ ID NOs: 565-566, 568, 570-578. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 837-838, 840, 842-850 or to a transcript of any one of SEQ ID NOs: 565-566, 568, 570-578.
  • the trRNA comprises any one of REF ID NOs: 1386, 1388-1391, or 1393-1394 or to a transcript of any one of REF ID NOs: 1114, 1116-1119, or 1121-1122. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1381-1382, 1384-1394 or to a transcript of any one of REF ID NOs: 1109-1110, 1112, 1114-1124.
  • the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1381-1382, 1384-1394 or to a transcript of any one of REF ID NOs: 1109-1110, 1112, 1114-1124. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1381-1382, 1384-1394 or to a transcript of any one of REF ID NOs: 1109-1110, 1112, 1114-1124.
  • the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1381-1382, 1384-1394 or to a transcript of any one of REEF ID NOs: 1109-1110, 1112, 1114-1124.
  • this disclosure provides a taRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising: a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC, wherein AX1X2X3X4AX5GG are as described in an embodiment of this section (“5’ SINV UTRs Comprising oe5N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR.
  • the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. In some embodiments, the taRNA comprises a replicase construct of SEQ ID NO: 1649 and a trRNA of any one of REF ID NOs: 1386, 1388-1391, or 1393-1394. oe5N Extension saRNA
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein: Xi is A or G; X 2 is A or G; X3 is A or U; X4 is A, U, C, or G; and X5 is U or C.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUXAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is any one of SEQ ID NOs: 293-294, 296, or 298-306.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a poly nucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is any one of SEQ ID NOs: 293-294, 296, or 298-306.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 293.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 294.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 296.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 298.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 299.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 301.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 302.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 303.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 305.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AX1X2X3X4AX5GG is SEQ ID NO: 306.
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1655; SEQ ID NO: 1659), wherein: Xi is A or G; X2 is A or G; X3 is A or G; X 4 is A, T/U, C, or G; X5 is A, T/U, C, or G, and X ⁇ > is T/U.
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a poly nucleic acid having a sequence of A(T/U)AXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1655; SEQ ID NO: 1659), wherein: Xi is A or G; X2 is A; X3 is A; X 4 is A or G; X5 is T/U or C, and Xe is T/U.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1659), wherein: Xi is A; X 2 is A or G; X3 is A or G; X 4 is A or G; X5 is A, U, C, or G; and Xe is A, U, C or G.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U) AXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1659), wherein: Xi is A; X 2 is A or G; X 3 is A or G; X 4 is A or G; X5 is A; and Xe is A, U, C, or G.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X 4 XSAX6GG is any one of SEQ ID NOs: 307-333.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces an trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X3X 4 XSAX6GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X 4 X 5 AX 6 GG is any one of SEQ ID NOs: 307-333.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X 4 X 5 AX 6 GG is SEQ ID NO: 307.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AXIX2X3X 4 X 5 AX 6 GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X 4 X 5 AX 6 GG is SEQ ID NO: 308.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 309.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 310.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 311.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 312.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 313.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 315.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 316.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 317.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 318.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 320.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 321.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 322.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 323.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 326.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 327.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 328.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 329.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 330.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 331.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 332.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 333.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 851-877 or to a transcript of any one of SEQ ID NOs: 579-605. In some embodiments, wherein the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 851-877 or to a transcript of any one of SEQ ID NOs: 579-605. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 851-877 or to a transcript of any one of SEQ ID NOs: 579-605. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 851-877 or to a transcript of any one of SEQ ID NOs: 579-605.
  • the trRNA comprises any one of REF ID NOs: 1395-1421. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1395-1421 or to a transcript of any one of REF ID NOs: 1123-1149. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1395-1421 or to a transcript of any one of REF ID NOs: 1123-1149.
  • the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1395-1421 or to a transcript of any one of REF ID NOs: 1123-1149. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1395-1421 or to a transcript of any one of REF ID NOs: 1123-1149.
  • this disclosure provides a taRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising: a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC, wherein AX1X2X3X4X5AX6GG are as described in an embodiment of this section (“5’ SINV UTRs Comprising oe6N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR.
  • a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct
  • the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. In some embodiments, the taRNA comprises a replicase construct of SEQ ID NO: 1649 and trRNA of any one of REF ID NOs: 1395-1421. oe6N Extension saRNA
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein: Xi is A; X 2 is A or G; X3 is A or G; X4 is A or G; X5 is A, T/U, C, or G; and Xf> is A, T/U, C or G.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U) AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein: Xi is A; X 2 is A or G; X 3 is A or G; X4 is A or G; X5 is A; and Xf> is A, T/U, C, or G.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659) wherein AX1X2X3X4X5AX6GG is any one of REF ID NOs: 292-306.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659) wherein AX1X2X3X4X5AX6GG is any one of SEQ ID NOs: 307-333.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 307.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 308.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 309.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 310.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 311.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 312.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 313.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 314.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 315.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 316.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 317.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 318.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 320.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 325.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 330.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AX1X2X3X4X5AX6GG is SEQ ID NO: 333.
  • this disclosure provides a saRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising, a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AX1X2X3X4X5AX6GGCGGC, wherein AX1X2X3X4X5AX6GG are as described in an embodiment of this section (“5’ SINV UTRs Comprising oe6N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR.
  • the replicase is an SFV replicase.
  • the replicase is a SINV replicase.
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X 4 X 5 X 6 AX7GGCGGC (SEQ ID NO: 1656; SEQ ID NO: 1660), wherein: Xi is A or G; X2 is A, T/U, or G; X3 is A, T/U, C, or G; X 4 is A or G; X5 is A, T/U, C, or G; Xe is A, T/U, C, or G; and X7 is T/U or C.
  • Xi is A or G
  • X2 is A, T/U, or G
  • X3 is A, T/U, C, or G
  • X 4 is A or G
  • X5 is A, T/U,
  • the polynucleotide (e.g., trRNA or saRNA) comprises a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of A(T/U)AXIX2X3X 4 XSX6AX7GGCGGC (SEQ ID NO: 1656; SEQ ID NO: 1660), wherein: Xi is A or G; X 2 is A or G; X 3 is A, C, or G; X 4 is A or G; X 5 is A or T/U; X 6 is T/U or C; and X 7 is T/U.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of sequence of AUAXiX2X3X 4 X5XeAX7GGCGGC (SEQ ID NO: 1660), wherein: Xi is A or G; X2 is A or G; X3 is A, C, or G; X 4 is A or G; X5 is A or U; Xe is U or C; and X7 is U.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAXIX2X3X 4 X 5 X 6 AX7GGCGGC (SEQ ID NO: 1660), wherein AXiX2X3X 4 XsXeAX7GG is any one of SEQ ID NOs: 334-384.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces an trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is any one of SEQ ID NOs: 334-384.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 334.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 335.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 336.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 337.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 338.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 339.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 340.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 341.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 342.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 343.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 344.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 345.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 346.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 348.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 350.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 351.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 352.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 353.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 354.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 357.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 360.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 362.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 364.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 365.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 369.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 370.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 375.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 376.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 377.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 378.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 379.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 381.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 382.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 383.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 384.
  • the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 878-928 or to a transcript of any one of SEQ ID NOs: 606-656. In some embodiments, wherein the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 878-928 or to a transcript of any one of SEQ ID NOs: 606-656. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 878-928 or to a transcript of any one of SEQ ID NOs: 606-656. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 878-928 or to a transcript of any one of SEQ ID NOs: 606-656.
  • the trRNA comprises any one of REF ID NOs: 1422-1472 or a transcript of any one of REF ID NOs: 1150-1200. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1422-1472 or to a transcript of any one of REF ID NOs: 1150-1200. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1422-1472 or to a transcript of any one of REF ID NOs: 1150-1200.
  • the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1422-1472 or to a transcript of any one of REF ID NOs: 1150-1200. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1422-1472 or to a transcript of any one of REF ID NOs: 1150-1200.
  • this disclosure provides a taRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising: a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG are as described in an embodiment of this section (“5’ SINV UTRs Comprising oe7N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a payload; and a 3’ SFV UTR.
  • a first RNA polynucleotide comprising a nucleic acid encoding a replicas
  • the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. In some embodiments, the taRNA comprises a replicase construct of SEQ ID NO: 1649 and trRNA of any one of REF ID NOs: 1422-1472. oe7N Extension saRNA
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein: Xi is A or G; X 2 is A, U, or G; X3 is A, U, C, or G; X4 is A or G; X5 is A, U, C, or G; Xf> is A, U, C, or G; and X7 is U or C.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein: Xi is A or G; X2 is A or G; X3 is A, C, or G; X4 is A or G; X5 is A or U; Xf> is U or C; and X7 is U.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is any one of SEQ ID NOs: 334-384.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces an saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is any one of SEQ ID NOs: 334-384.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 334.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 335.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 336.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 337.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 343.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 344.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 345.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 346.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 348.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 352.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 353.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 354.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 355.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 356.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 357.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 358.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 359.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 360.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 361.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 362.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 363.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 364.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 365.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 366.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 367.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 368.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 369.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 397.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 402.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 409.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 412.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 414.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 415.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 416.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 417.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 418.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 419.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 420.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 421.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 422.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 423.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 424.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 425.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 426.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 427.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 428.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 429.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 430.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 431.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 432.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 433.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 434.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 435.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 436.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 439.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 440.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 441.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 442.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 443.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 444.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 445.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 447.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 453.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 455.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 458.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 469.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 470.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 471.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 472.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 473.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 475.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 476.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 477.
  • the polynucleotide comprises a trRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 480.
  • this disclosure provides a taRNA comprising: (a) a first RNA polynucleotide comprising a nucleic acid encoding a replicase (i.e., a replicase construct); and (b) a second RNA polynucleotide comprising: a 5’ SINV UTR wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG are as described in an embodiment of this section (“5’ SINV UTRs Comprising oe8N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a payload; and a 3’ SFV UTR.
  • a first RNA polynucleotide comprising a nucleic acid encoding
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG any one of SEQ ID NOs: 385-542.
  • the polynucleotide comprises a DNA polynucleotide that when transcribed produces a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is any one of SEQ ID NOs: 385-542.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 386.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 388.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 390.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 391.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 392.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 393.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 397.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 399.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 400.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 401.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 403.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 406.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 409.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 412.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 413.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 414.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 416.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 417.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 418.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 419.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 420.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 422.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 423.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 424.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 425.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 427.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 428.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 430.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 431.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 434.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 437.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 438.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 439.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 450.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 451.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 452.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 453.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 456.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 464.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 471.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 486.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 506.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 513.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 518.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 526.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 528.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 529.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 531.
  • the polynucleotide comprises a saRNA comprising a 5’ SINV UTR, wherein the 5’ terminal of the 5’ SINV UTR comprises a polynucleic acid having a sequence of AUAX1X2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 541.
  • the method comprises obtaining a plurality of trRNAs, wherein trRNAs of the plurality of trRNAs comprise a CSE and a 5’-UTR extension, wherein at least two of the trRNAs comprise different 5’-UTR extensions.
  • the plurality of trRNAs comprises 2-50000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 2- 100000 trRNAs with different sequences. In some embodiments, the plurality of trRNAs comprises 2-200000 trRNAs with different sequences.
  • the plurality of trRNAs comprises 10000-50000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 10000-100000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 10000-500000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 10000-200000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 10000-1000000 trRNAs with different 5’-UTR extension sequences.
  • obtaining the plurality of trRNAs comprises obtaining a plurality of trRNAs and obtaining additional polynucleotides that are not part of the plurality of trRNAs (e.g., polynucleotides that do not comprise an RNA barcode and/or a CSE that is cognate to the replicase).
  • the 3’ alphavirus UTR is a VEEV 3 ’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR.
  • the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR. In some embodiments, the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR.
  • the screen in FIG. 1 was performed for extension ranging from 3N to 9N in length, where N is a nucleotide that could be A, T/U, C, or G.
  • the libraries were generated such that the vast majority of possible combinations 5’ UTR extensions ranging from 3N to 9N were present.
  • Results from the screen were displayed for extension lengths of 3N to 9N in logo plots (FIGs. 4- 10) at two or three different steps in the screen.
  • a third step after three serial transfections.
  • the size of the nucleotide representation indicates the frequency with that nucleotide was found at that position in that extension length at a given step.
  • the first two positions (0 and 1) in each logo plot were not altered.
  • FIG. 4 bottom which show results for the 3N extension after serial transfection, the Oth position is solely A, the first position is solely (T/U), the second position (first N) is primarily A with some G and a little T, the third position (second N) is primarily A and G with a small amount of T and C, and the fourth position (third N) is primarily A, T and G with a small amount of C.
  • DNA libraries of trans-replicating RNA constructs with highly diverse 5’ extensions were generated by PCR using forward oligos containing 1) T7 promoter sequence 2) AT, the highly conserved first two nucleotides of SINV and other alphaviruses 3) a variable number of mixed bases and 4) sequence complementary to the SINV 5’ viral UTR starting at nucleotide 4 as well as a reverse oligo downstream of the poly A sequence of a GFP-expressing trRNA reporter plasmid backbone (pUC57).
  • products were digested with Dpnl (NEB) for one hour to remove template plasmid and BspQI (NEB) overnight to trim PCR products to a constant polyA length. DNA was then purified using Clean and Concentrate kits (Zymo Research).
  • Rapid Amplification of cDNA Ends was then performed on input IVT RNA libraries and isolated total RNA using an oligo complimentary to GFP for first strand synthesis, a template- switching oligo (TSO), and M-MuLV reverse transcriptase (NEB).
  • cDNA corresponding to the 5’ end of target transcripts was then diluted and amplified using a TSO- specific oligo and a target- specific oligo containing unique molecule identifiers (UMIs) to facilitate sequencing fidelity and deduplication in downstream analysis as well as partial ILLUMINA adapters. Indexed, complete ILLUMINA adapter sequences were added and multiplexed next-generation sequencing was then performed on the MiSeq platform (Azenta) generating 250bp paired-end reads.
  • UMIs unique molecule identifiers
  • the screen in FIGs. 13A and 13B was performed for oe5N, oe6N, oe7N, and oe8N extensions of 9, 10, 11, or 12 nucleotides in length respectively, and in which 5, 6, 7, or 8 nucleotides (N) were varied, respectively.
  • position 1 (relative to the 5’ terminal) was fixed as A.
  • terminal position and (terminal - 1) position were fixed as GG, and (terminal - 3) position was fixed as A.
  • Varied positions (N) could be A, T/U, C, or G.
  • tested oe5N extensions consisted of 9 nucleotides having a pattern of ANNNNANGG, in which N could be any of A, T/U, C, or G.
  • the libraries were generated such that the vast majority of possible combinations 5’ UTR extensions ranging from oe6N to oe8N were present. Results from the screen were displayed for oe5N, oe6N, oe7N, and oe8N extensions in logo plots (FIGs. 15-17) after 4 serial transfections.
  • nucleotide representation indicates the frequency with that nucleotide was found at that position in that extension length after 4 serial transfections.
  • the first two positions (0 and 1) in each logo plot were not altered.
  • the final four nucleotides were found to converge on a pattern of AXGG, where X is T/U or C, while the first 6, 7, or 8 nucleotides were variable.
  • Table 3 several oeSTR extensions were enriched over the extensions tested in Example 1.
  • trRNA Sequences (DNA) (XXX in sequence of Table 6 refers to a payload or nucleic acids encoding a payload. XXX may represent more than 3 nucleotides). -2Z1 -
  • RNA sequences (XXX in sequence of Table 7 refers to a payload or nucleic acids encoding a payload. XXX may represent more than 3 nucleotides).
  • RNA sequences (XXX in sequence of Table 7 refers to a payload or nucleic acids encoding a payload. XXX may represent more than 3 nucleotides).

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Abstract

La présente invention concerne des ARN trans-réplicatifs (ARNt) et des ARN auto-amplifiés (ARNsA) avec des extensions de la région non traduite (UTR) 5' du virus Sindbis (SINV) qui modifient l'amplification des ARNt/ ARNsA et l'expression de la charge utile. La présente invention concerne également un procédé à haut débit pour déterminer des changements d'amplification de ARNtr/ARNsa induits par des extensions 5'UTR.
PCT/US2025/014823 2024-02-06 2025-02-06 Arn trans-réplicatifs et auto-réplicatifs comprenant une région 5'utr modifiée Pending WO2025171152A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055161A2 (fr) * 2002-12-12 2004-07-01 The Board Of Regents Of The University Of Texas System Production a grande echelle de replicons d'alphavirus encapsules
JP4729615B2 (ja) * 2005-03-29 2011-07-20 ニューヨーク・ユニバーシティ 欠陥シンドビスウイルスベクター
US8961995B2 (en) * 2012-09-20 2015-02-24 Uab Research Foundation Methods and compositions for alphavirus replicons

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055161A2 (fr) * 2002-12-12 2004-07-01 The Board Of Regents Of The University Of Texas System Production a grande echelle de replicons d'alphavirus encapsules
JP4729615B2 (ja) * 2005-03-29 2011-07-20 ニューヨーク・ユニバーシティ 欠陥シンドビスウイルスベクター
US8961995B2 (en) * 2012-09-20 2015-02-24 Uab Research Foundation Methods and compositions for alphavirus replicons

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
COMES JDG ET AL., TRENDS BIOTECHNOL., vol. 41, no. 11, November 2023 (2023-11-01), pages 1417 - 1429
HOOSE, A., NAT REV CHEM, vol. 7, 2023, pages 144 - 161
HYDE JL, VIRUS RES., vol. 206, 3 August 2015 (2015-08-03), pages 99 - 107
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 68
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 77
LUNDSTROM KENNETH: "Trans-amplifying RNA: Translational application in gene therapy", MOLECULAR THERAPY, vol. 31, no. 6, 1 June 2023 (2023-06-01), United States, pages 1507 - 1508, XP093097368, ISSN: 1525-0016, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10076252/pdf/main.pdf> DOI: 10.1016/j.ymthe.2023.03.015 *
NIESTERST HUBERT G ET AL: "Defined Mutations in the 5' Nontranslated Sequence of Sindbis Virus RNA", 1 September 1990 (1990-09-01), pages 4162 - 4168, XP093277461, Retrieved from the Internet <URL:https://pmc.ncbi.nlm.nih.gov/articles/PMC247880/pdf/jvirol00064-0134.pdf> *
PERKOVIC MARIO ET AL: "A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in mice", MOLECULAR THERAPY, vol. 31, no. 6, 1 June 2023 (2023-06-01), United States, pages 1636 - 1646, XP093242772, ISSN: 1525-0016, DOI: 10.1016/j.ymthe.2023.01.019 *
STRAUSS E G ET AL: "Complete nucleotide sequence of the genomic RNA of Sindbis virus", VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 133, no. 1, 1 February 1984 (1984-02-01), pages 92 - 110, XP023058388, ISSN: 0042-6822, [retrieved on 19840201], DOI: 10.1016/0042-6822(84)90428-8 *

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