Trans-replicating and self-replicating RNAS comprising a modified 5' UTR region

Abstract

This disclosure provides trans replicating RNAs (trRNA) and self- amplifying RNAs (saRNAs) with 5' Sindbis virus (SINV) untranslated region (UTR) extensions that modify trRNA / saRNA amplification and payload expression. This disclosure also describes a high throughput method for determining trRNA / saRNA amplification changes induced by 5' UTR extensions.

Description

[0001] TRANS-REPLICATING AND SELF-REPLICATING RNAS COMPRISING A MODIFIED 5’ UTR REGION

[0002] FIELD

[0003] This disclosure relates to self-amplifying (e.g., self-amplifying, trans amplifying) polynucleotides containing synthetic 5’ untranslated regions (UTRs).

[0004] RELATED APPLICATIONS

[0005] This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional Application No. 63 / 550,570, filed February 6, 2024, entitled “TRANS-REPLICATING AND SELF-REPLICATING RNAS COMPRISING A MODIFIED 5’ UTR REGION”, the content of which is hereby incorporated by reference herein in its entirety for all purposes.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The content of the electronic sequence listing (A1414700001WO00-SEQ-ROS.xml; Size: 1,277,038 bytes; and Date of Creation: February 5, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Amplifying ribonucleic acids (RNAs) are a class of RNA therapeutics where an RNA comprising or encoding a therapeutic is amplified in a target cell. Amplifying RNAs comprise two basic parts: (1) an mRNA encoding a replicase and (2) RNA comprising or encoding a pay load (e.g., a therapeutic) and a conserved sequence element (CSE) that is cognate the replicase. Upon entrance of amplifying RNAs into a target cell, a replicase is expressed by the cell. The expressed replicase, in turn, replicates (e.g., amplifies) a payload or a payload encoding sequence to be translated by the cell. Amplifying RNAs can be further broken down into two different categories: self-amplifying RNAs (saRNAs) and trans-amplifying RNAs (taRNAs). In saRNAs the mRNA encoding the replicase, the CSE, and the payload are all encoded by the same RNA polynucleotide. In 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. Typically, saRNAs and taRNAs comprise components derived from alphavirus genomes. For example, 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) may comprise a CSE. 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.

[0010] SUMMARY

[0011] A central challenge in developing saRNAs and taRNAs is tuning payload expression for a particular therapeutic. For example, in some applications 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. In different applications, 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. Thus, there is a general need to generate modifications that impact payload expression.

[0012] 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. For example, while 3N extensions typically amplified well with A or G at position 1 of the extension, 4N extensions typically amplified well with A or T / U in position 1 of the extension, and 6N extensions typically amplified well with only A at position 1 of the extension. Similar surprising results are seen at other positions and other extension lengths, as described herein.

[0013] In some aspects, 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)XIX2X3CGGC (REF ID NO: 1631; REF ID NO: 1638), wherein: Xi is A, T / U or G; X2is A, T / U or G; and X3is 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. In some embodiments, Xi is A or G; X2is A or G; and X3is A, T / U, or G. In some embodiments, the polynucleotide is an RNA polynucleotide and XiX2X3is any one of REF ID NOs: 272-276. In some embodiments, 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 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. In some embodiments, this disclosure provides, a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X1X2X3 is any one of REF ID NOs: 1-5.

[0014] In some aspects, 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)XIX2X3X4CGGC (SEQ ID NO: 1632; SEQ ID NO: 1639), wherein: Xi is A or T / U; X2is A, T / U or G; X3 is A, T / U, or G; and X4is A, T / U, or G, wherein the polynucleotide does not comprise a sequence of A(T / U)AAAA. In some embodiments, Xi is A; X2is A, T / U, or G; X3 is A or G; and X4is A or G. In some embodiments, the polynucleotide is an RNA polynucleotide and XiX2X3X4is REF ID NO: 277. In some embodiments, the 5’ SINV UTR comprises at least 90% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549. In some embodiments, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X 1 X2X3X4is REF ID NO: 6.

[0015] In some aspects, 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)XIX2X3X4XSCGGC (SEQ ID NO: 1633; SEQ ID NO: 1640), wherein: Xi is A, T / U or G; X2is A, T / U or G; X3is A, T / U, or G; X4is 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. In some embodiments, Xi is A; X2is A, T / U or G; X3 is A; X4is A or G; and X5 is A or G. In some embodiments, the polynucleotide is an RNA polynucleotide and XiX2X3X4Xs is any one of REF ID NOs: 278-281. In some embodiments, 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 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X1X2X3X4X5 is any one of REF ID NOs: 7-10.

[0016] In some aspects, 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. In some embodiments, 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. In some embodiments, the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692). In some embodiments, 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. In some embodiments, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6 is any one of REF ID NOs: 11-12 (SEQ ID NOs: 1693-1694).

[0017] In some aspects, 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; X2is A or G; X3is A, T / U, or G; X4is 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. In some embodiments, 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. In some embodiments, the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6X7 is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701). In some embodiments, 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. In some embodiments, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7 is any one of REF ID NOs: 13- 19 (SEQ ID NO: 1702-1708).

[0018] In some aspects, 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; X5is A, T / U, G, or C; X6is A, T / U, G, or C; X7is A, T / U, C, or G; and X8is A, T / U, C or G. In some embodiments, X2 is A or G; X3 is A; X4 is A, G, T / U or C; X5 is A; Xf> is T / U; X7 is G; and Xs is G. In some embodiments, the polynucleotide is an RNA polynucleotide and XiX2X3X4X5X6X7X8is REF ID NO: 291 (SEQ ID NO: 1709). In some embodiments, 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. In some embodiments, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7X8 is REF ID NO: 20 (SEQ ID NO: 1710).

[0019] In some aspects, 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; X2is A, T / U or G; X3is A, T / U, C or G; X4is A, T / U, C, or G; X5is A, T / U, C, or G; X6is A, T / U, C or G; X7is A, T / U, C or G; X8is A, T / U, C or G; and X9 is A, T / U, C or G. In some embodiments, Xi is A; X2 is A or G; X3 is A; X4 is A; X5 is A, T / U, G, or C; X<> is A; X7 is T / U; X8is G; and X9 is G. In some embodiments, the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6X7X8X9 is any one of REF ID NO: 292-306 (SEQ ID NOs: 1711-1725). In some embodiments, X1X2X3X4X5X6X7X8X9 is REF ID NO: 294. In some embodiments, the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 836-850 or to a transcript of any one of SEQ ID NOs: 564-578. In some embodiments, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7X8X9 is any one of REF ID NOs: 21-35 (SEQ ID NOs: 1726-1740). In some embodiments, XiX2X3X4X5X6X7X8X9 is REF ID NO: 23 (SEQ ID NO: 1728).

[0020] In some aspects, 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. In some embodiments, Xi is A; X2 is A or G; X3 is A or T / U; X4 is C; and X5 is T / U. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, this disclosure provides a selfamplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and AX1X2X3X4AX5GG is any one of REF ID NOs: 23, 25, or 27.

[0021] In some aspects, 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. In some embodiments, 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. In some embodiments, the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5AX6GG is any one of SEQ ID NO: 307-333. In some embodiments, 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, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5AX6GG is any one of SEQ ID NOs: 36-62.

[0022] In some aspects, 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. In some embodiments, 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. In some embodiments, the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5X6AX7GG is any one of SEQ ID NO: 334-384. In some embodiments, 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, 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. In some embodiments, this disclosure provides a selfamplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5X6AX7GG is any one of SEQ ID NOs: 63-113.

[0023] In some aspects, 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)AXIX2X3X4XSX6X7AX8GGCGGC (SEQ ID NO: 1657; SEQ ID NO: 1661), wherein: Xi is A or G; X2is A, T / U, or G; X3is A, T / U, C, or G; X4is A, T / U, C, or G; X5is A, T / U, or G; X6is A, T / U, C or G; X7is A, T / U, C or G; and X8is A, T / U, C, or G. In some embodiments, Xi is A; X2 is A or G; X3 is A or G; X4 is A or G; X5 is A; X<> is A; X7 is C and X8is T / U. In some embodiments, the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5X6AX7GG is any one of SEQ ID NO: 385-542. In some embodiments, the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 929-1086 or to a transcript of any one of SEQ ID NOs: 657-814. In some embodiments, 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. In some embodiments, this disclosure provides a self-amplifying RNA comprising the polynucleotide. In some embodiments, the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5X6AX7GG is any one of SEQ ID NOs: 114-271.

[0024] In some aspects, this disclosure provides a polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR) comprising a polynucleic acid described herein. In some embodiments, the polynucleic acid comprises a 5’ UTR extension provided herein. In some embodiments, the polynucleotide is an RNA polynucleotide and comprises any one of REF ID NO: 1638, SEQ ID NOs: 1639-1640, REF ID NOs: 1641-1644, or SEQ ID NOs: 1658-1661.

[0025] In some aspects, 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. In some embodiments, 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.

[0026] In some aspects, this disclosure provides a self-amplifying RNA comprising a 5’ SINV UTR comprising a 5’ UTR extension disclosed herein. In some embodiments, 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

[0027] In some aspects, 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. In some embodiments, 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.

[0028] In some aspects, 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.

[0029] In some embodiments, 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. In some embodiments, 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. In some embodiments, the 3’ alphavirus UTR is a VEEV 3’-UTR, SFV 3’-UTR, SINV 3’-UTR, or CHIKV 3’-UTR. In some embodiments, the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR. In some embodiments, the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR. In some embodiments, the payload comprises a reporter protein. In some embodiments, 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. In some embodiments, the payload is not a reporter protein. In some embodiments, cells of the plurality of cells comprise a polynucleotide encoding the replicase that is cognate to the CSE. In some embodiments, the method further comprises contacting cell of the plurality of cell with a polyribonucleotide encoding the replicase that is cognate to the CSE. In some embodiments, 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.

[0030] In some embodiments, obtaining the plurality of RNA polynucleotides comprises synthesizing the plurality of RNA polynucleotides using in vitro transcription.

[0031] In some embodiments, 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.

[0032] In some embodiments, 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.

[0033] In some embodiments, the contacting comprises transfecting trRNAs of the plurality of trRNAs into cells of the plurality of cells.

[0034] In some embodiments, the obtaining comprises obtaining a plurality of self-amplifying RNAs (saRNAs) that comprise the trRNA and a replicase construct. In some aspects, provided herein is 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.

[0035] In some aspects, provided herein is a DNA 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.

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] FIG. 6 shows logo plots for 5N extensions (AUNNNNNCGGC) based on enrichment of 5’ UTR extension in cells over time. The top panel shows the in vitro transcribed 5N trRNA library distribution before transfection. The middle panel shows the 5N trRNA library distribution after 1 transfection. The bottom panel shows the 5N trRNA library distribution after 4 serial transfections of the 5N trRNA library.

[0043] FIG. 7 shows logo plots for 6N extensions (AUNNNNNNCGGC) based on enrichment of 5’ UTR extension in cells over time. The top panel shows the in vitro transcribed 6N trRNA library distribution before transfection. The bottom panel shows the 6N trRNA library distribution after 4 serial transfections of the 6N trRNA library.

[0044] FIG. 8 shows logo plots for 7N extensions (AUNNNNNNNCGGC) based on enrichment of 5’ UTR extension in cells over time. The top panel shows the in vitro transcribed 7N trRNA library distribution before transfection. The middle panel shows the 7N trRNA library distribution after 1 transfection. The bottom panel shows the 7N trRNA library distribution after 4 serial transfections of the 7N trRNA library.

[0045] 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.

[0046] FIG. 10 shows logo plots for 9N extensions (AUNNNNNNNNNCGGC) 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.

[0047] 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.

[0048] 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.

[0049] FIGs. 13A-13B show oeSTR trRNA 5’-UTR extension library design and assay design. oeSTR extensions were designed based on findings from the 3N-9N screens. FIG. 13A shows an example of 5’ UTR extension library design for oeSTR extensions (e.g., oe5N, oe6N, oe7N, and oe8N). N represents A, T / U, G, or C. About 80,000 different 5’-UTR extensions were assayed. FIG. 13B shows a flow chart of the steps in determining amplification of the different trRNAs in the library.

[0050] 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.

[0051] 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. In this logo plot, positions with few or no nucleotide symbols shown are A, T / U, C, or G.

[0052] 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. In this logo plot, positions with few or no nucleotide symbols shown are A, T / U, C, or G.

[0053] 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. In this logo plot, positions with few or no nucleotide symbols shown are A, T / U, C, or G.

[0054] 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. In this logo plot, positions with few or no nucleotide symbols shown are A, T / U, C, or G.

[0055] DETAILED DESCRIPTION

[0056] In some embodiments, 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...XnCGGC wherein each X is a different nucleotide in the sequence, and 3 < n >12.

[0057] A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A), uridine (U) / deoxythymidine (T), guanosine / deoxyguanosine (G), cytidine / deoxycytidine (C)) and joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications. In some sequences described herein, T / U is used to denote a particular nucleotide may be a T or U depending on whether the polynucleotide is an RNA polynucleotide (U) or a DNA polynucleotide (T).

[0058] 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).

[0059] In some embodiments, 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. Typically, 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. As a skilled artisan will understand, 5 ’-UTRs may comprise sequence elements which play roles in regulation of expression (e.g., Kozak sequences) or structural elements which alter stability of the molecule (e.g., 5’ cap structures). A UTR located directly downstream of a stop codon operably linked to a gene or transgene is referred to herein as a 3 ’-UTR. 3 ’-UTRs may comprise structural elements which alter the stability of a construct and / or provide transcriptional control, including, but not limited to AU- rich elements and polyA tails. A variety of 5 ’-UTRs and a 3 ’-UTRs are known to those of ordinary skill in the art. UTRs may be naturally occurring or synthetic. In some embodiments, a polynucleotide comprises a 5 ’-UTR and / or a 3 ’-UTR.

[0060] In some embodiments, a UTR is a 5’ Sindbis virus (SINV) UTR. A “5’ SINV UTR” refers to a wildtype or modified 5’ SINV UTR. In some embodiments, a wildtype 5’ SINV UTR comprises the sequence set forth in SEQ ID NO: 815. Unless otherwise stated, 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. In some embodiments, 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). In some embodiments, the mutation is in the 5’-UTR extension. In some embodiments, the mutation is not in the 5 ’-UTR extension. In some embodiments, the modified 5’ SINV UTR comprises the sequence set forth in any one of SEQ ID NOs: 816-1086. In some embodiments, the 5’ SINV UTR comprises a polynucleic acid having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 816-1086. In some embodiments, the 5’ SINV UTR consists of the sequence set forth in any one of SEQ ID NOs: 816-1086. In some embodiments, a polynucleotide provided herein (e.g., a trRNA, an saRNA) comprises a modified 5’ SINV UTR comprising a polynucleic acid having a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence set forth in any one of SEQ ID NOs: 816-1086. In some embodiments, a polynucleotide provided herein (e.g., a trRNA, an saRNA) comprises a modified 5’ SINV UTR consisting of a sequence set forth in any one of SEQ ID NOs: 816-1086.

[0061] 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...XnCGGC). In some embodiments, 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).

[0062] In some embodiments, a polynucleotide (e.g., a trRNA, an saRNA) further comprises a 3’ Semliki Forest Virus (SFV) UTR. In some embodiments, a 3’ SFV UTR comprises the sequence set forth in SEQ ID NO: 1651.

[0063] Amplifying RNAs

[0064] In some embodiments, a polynucleotide (e.g., a trRNA, an saRNA) comprising a modified 5’ SINV UTR (e.g., a 5’ SINV UTR comprising a 5’ UTR extension) is an amplifying RNA. An “amplifying RNA” is an RNA polynucleotide comprising a gene (i.e., a nucleic acid encoding a payload or a nucleic acid payload) operably linked to a conserved sequence element (CSE). A “conserved sequence element (CSE),” refers to a recognition site for an alphavirus replicase. Typically, a CSE functions as a core promoter or enhancer for initiation of replication of a downstream sequence, such that a 5’-CSE may initiate synthesis of a plus-strand and a 3’- CSE may initiate synthesis of a minus-strand. An RNA polynucleotide may comprise one or more 5’-CSEs and / or 3’-CSEs. CSEs may be comprised in a UTR, for example a 5’-UTR and / or a 3’ UTR. In some embodiments, a CSE forms one or more secondary structure(s), such as one or more stem-loops. Non-limiting examples of CSEs include CSE1, CSE2, CSE3, CSE4, and variants or derivatives thereof. In some embodiments, the CSE is a CSE derived from SFV, SINV, VEEV, or CHIKV alphavirus. CSEs are known in the art, e.g., as described in Hyde JL, Virus Res. 2015 Aug 3;206:99-107.

[0065] The term “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). Typically, an alphaviral replicase comprises a complex formed by the non-structural proteins nsPl, nsP2, nsP3, and nsP4. Once expressed, an alphaviral replicase may interact with a RNA polynucleotide comprising one or more CSEs which are cognate to the replicase and generate mirrored copies of the RNA polynucleotide, which can be subsequently translated (e.g., by a host cell).

[0066] 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). In some embodiments, a replicase construct encodes a replicase derived from an SFV (SFV replicase). In some embodiments, a replicase construct encodes a replicase derived from a SINV (SINV replicase). In some embodiments, a replicase and CSE from the same species are cognate; for example, an SFV replicase is assumed to be cognate to a CSE derived from an SFV. In some embodiments, a replicase and CSE from different species are cognate.

[0067] In some embodiments, the amplifying RNA is a trans-replicating RNA (trRNA). A “trans-replicating RNA (trRNA)” refers to an RNA polynucleotide comprising nucleic acids encoding a payload or nucleic acids comprising a payload, wherein the nucleic acids are operably linked to a CSE , and wherein the trRNA does not encode a replicase. In some embodiments, 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. In some embodiments, 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.

[0068] In some embodiments, 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). The term “trans-amplifying RNA” (taRNA) is used herein to refer to a trRNA and a separate RNA encoding a replicase (i.e., a replicase construct). A “replicase construct” is an RNA polynucleotide comprising nucleic acids encoding a replicase from an RNA virus, for example, an alphavirus. In some embodiments, 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.

[0069] In some embodiments, an amplifying RNA is a self-amplifying RNA (saRNA). “Selfamplifying RNAs (saRNAs),” unlike trRNAs, comprise certain trRNA elements (e.g., CSE and a nucleic acid encoding a payload or a nucleic acid payload) and nucleic acids encoding a replicase (e.g., a replicase construct) in the same polynucleotide. saRNAs are known, e.g., as described in Comes JDG et al., Trends Biotechnol. 2023 Nov;41(l l):1417-1429.

[0070] When using amplifying RNAs to express a payload in a cell (e.g., when administering a taRNA or saRNA to a subject), a cognate replicase must be provided by another source (e.g., by the same cell, or by a separate RNA polynucleotide encoding the replicase).

[0071] In some embodiments, a polynucleotide encoding a replicase (e.g., a replicase construct) comprises one or more UTRs. In some embodiments, a replicase construct comprises a 5 ’-UTR. In some embodiments, a replicase construct comprises a 3 ’-UTR. In some embodiments, a replicase construct comprises a 5 ’-UTR and a 3 ’-UTR. In some embodiments, a replicase construct comprises a 5 ’-UTR derived from human alpha-globin (5’-HBA-UTR). An exemplary 5’-HBA-UTR is provided in SEQ ID NO: 1646. In some embodiments, a replicase construct comprises a 3 ’-UTR derived from human alpha- globin (3’-HBA-UTR). An exemplary 3’-HBA- UTR is provided in SEQ ID NO: 1648. In some embodiments, a replicase construct comprises a 5’-HBA-UTR and a 3’-HBA-UTR. In some embodiments, a replicase construct comprises a 5’- HBA-UTR, a SFV replicase-encoding sequence, and a 3’-HBA-UTR (5’-HBA-UTR-SFV replicase-3’-HBA-UTR). An exemplary 5’-HBA-UTR-SFV replicase-3’-HBA-UTR construct is provided in SEQ ID NO: 1649.

[0072] In some embodiments, a trRNA comprises nucleic acids encoding a pay load. A “payload” refers to one or more gene products of for delivery to or expression by an organism. A payload may be a functional nucleic acid (e.g., RNA), a protein, a peptide or protein fragment, or a fusion protein. In some embodiments, a payload is an antigen. In some embodiments, a payload is an infectious disease antigen (e.g., a protein from a pathogen). In some embodiments, an antigen is viral antigen (e.g., a spike or fusion protein). In some embodiments, an antigen is a respiratory virus antigen.

[0073] In some embodiments, a payload is a selectable marker. As used herein, a “selectable marker” is a peptide or protein that can be used to screen cells by artificial selection. Nonlimiting examples of selectable markers include antibiotic resistance proteins (e.g., ampicillin, puromycin) and negative selection markers (e.g., thymidine kinase). In some embodiments, a payload is a reporter. A “reporter” is a peptide or protein which alters the appearance of a cell such that cells can be visually or optically screened for presence or absence of the peptide or protein. In some embodiments, a reporter is an enzyme which alters the appearance of a cell, such as beta-galactosidase. In some embodiments, a reporter is a peptide or peptide fragment (e.g., secreted embryonic alkaline phosphatase (SEAP)) which can be detected in combination with additional reagents (e.g., as say- specific media). In some embodiments, a reporter is a fluorophore, such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or any derivative thereof.

[0074] In some embodiments, a payload is not a reporter (e.g., the payload is an antigen or other peptide or protein).

[0075] In some embodiments, 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. In some embodiments, 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. In some embodiments, an RNA polynucleotide comprises a 5 ’-UTR and 3 ’-UTR from the same virus. In some embodiments, an RNA polynucleotide comprises a 5’-SINV UTR (e.g., a modified 5’ SINV UTR), and a 3’-SINV UTR. In some embodiments, an RNA polynucleotide comprises a 5’-UTR and 3’-UTR from different alphaviruses. In some embodiments, an RNA polynucleotide comprises a 5’-SINV UTR (e.g., a modified 5’ SINV UTR), and a 3 ’-SFV UTR. In some embodiments, 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.

[0076] 5’ SINV UTRs Comprising 3N Extensions

[0077] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “3N” extension. 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)XIX2X3CGGC).

[0078] In some embodiments, 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)XIX2X3CGGC (REF ID NO: 1631, REF ID NO: 1638), wherein: Xi is A, T / U or G; X2is A, T / U or G; and X3is A, T / U, or G. In some embodiments, the polynucleic acid does not comprise a sequence of A(T / U)(T / U)GA. In some embodiments, 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)XIX2X3CGGC (REF ID NO: 1631, REF ID NO: 1638), wherein: Xi is A or G; X2is A or G; and X3is A, T / U, or G.

[0079] 3N Extension taRNA

[0080] In some embodiments, 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 AUXIX2X3CGGC (REF ID NO: 1638), wherein: Xi is A, U or G; X2is A, U or G; and X3is A, U, or G; and wherein the polynucleic acid does not comprise a sequence of AUUGA. In some embodiments, 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 AUXIX2X3CGGC (REF ID NO: 1638), wherein: Xi is A or G; X2is A or G; and X3is A, U, or G; and wherein the polynucleic acid does not comprise a sequence of AUUGA.

[0081] In some embodiments, 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 AUXIX2X3CGGC (REF ID NO: 1638), wherein XiX2X3is any one of REF ID NOs: 272-276. In some embodiments, 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 AUXIX2X3CGGC (REF ID NO: 1638), wherein XiX2X3is any one of REF ID NOs: 272-276. In some embodiments, 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: 272.

[0082] In some embodiments, 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: 273.

[0083] In some embodiments, 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: 274.

[0084] In some embodiments, 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.

[0085] In some embodiments, 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.

[0086] In some embodiments, 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.

[0087] In some embodiments, 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. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% 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, 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 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. In some embodiments, 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.

[0088] 3N Extension saRNA

[0089] In some embodiments, 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. In some embodiments, 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.

[0090] In some embodiments, 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. In some embodiments, 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.

[0091] In some embodiments, 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.

[0092] In some embodiments, 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. In some embodiments, 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.

[0093] In some embodiments, 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: 275.

[0094] In some embodiments, 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.

[0095] In some embodiments, 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. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0096] 5’ SINV UTRs Comprising 4N Extensions

[0097] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “4N” extension. 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)XIX2X3X4CGGC).

[0098] In some embodiments, 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)XIX2X3X4CGGC (SEQ ID NO: 1632, SEQ ID NO: 1639), wherein: Xi is A or T / U; X2 is A, T / U or G; X3 is A, T / U, or G; and X4is A, T / U, or G. In some embodiments, the polynucleic acid does not comprise a sequence of A(T / U)AAAA. In some embodiments, 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)XIX2X3X4CGGC (SEQ ID NO: 1632, SEQ ID NO: 1639), wherein: Xi is A; X2is A, T / U, or G; X3is A or G; and X4is A or G. 4N Extension taRNA

[0099] In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein: Xi is A or U; X2 is A, U or G; X3 is A, U, or G; and X4 is A, U, or G, wherein the polynucleic acid does not comprise a sequence of AUAAAA. In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein: Xi is A; X2 is A, U, or G; X3 is A or G; and X4 is A or G, wherein the polynucleic acid does not comprise a sequence of AUAAAA.

[0100] In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein XiX2X3X4is REF ID NO: 277. In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein XiX2X3X4is REF ID NO: 6.

[0101] In some embodiments, the 5’ SINV UTR comprises at least 90% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549. In some embodiments, the 5’ SINV UTR comprises at least 95% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549.

[0102] In some embodiments, the trRNA comprises REF ID NO: 1365. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to REF ID NO: 1365 or to a transcript of REF ID NO: 1094. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to REF ID NO: 1365 or to a transcript of REF ID NO: 1094. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to REF ID NO: 1365 or to a transcript of REF ID NO: 1094. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to REF ID NO: 1365 or to a transcript of REF ID NO: 1094.

[0103] In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein X1X2X3X4 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 4N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR. In some embodiments, 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 REF ID NO: 1365.

[0104] 4N Extension saRNA

[0105] In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein: Xi is A or U; X2 is A, U or G; X3 is A, U, or G; and X4 is A, U, or G, wherein the polynucleic acid does not comprise a sequence of AUAAAA. In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein: Xi is A; X2 is A, U, or G; X3 is A or G; and X4 is A or G, wherein the polynucleic acid does not comprise a sequence of AUAAAA.

[0106] In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639) wherein XiX2X3X4is REF ID NO: 277. In some embodiments, 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 XiX2X3X4is REF ID NO: 6.

[0107] In some embodiments, 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 AUX1X2X3X4CGGC (SEQ ID NO: 1639), wherein X1X2X3X4 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 4N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a payload; and a 3’ SFV UTR. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase. 5’ SINV UTRs Comprising 5N Extensions

[0108] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “5N” extension. 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).

[0109] In some embodiments, 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)XIX2X3X4X5CGGC (SEQ ID NO: 1633; SEQ ID NO: 1640), wherein: Xi is A, T / U or G; X2is A, T / U or G; X3is A, T / U, or G; X4is A, T / U, or G; and X5is A, T / U, C, or G. In some embodiments, the polynucleic acid does not comprise a sequence of A(T / U)AAAAA. In some embodiments, 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)XIX2X3X4X5CGGC (SEQ ID NO: 1633; SEQ ID NO: 1640), wherein: Xi is A; X2 is A, T / 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 A(T / U)AAAAA.

[0110] 5N Extension taRNA

[0111] In some embodiments, 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: Xi is A, U or G; X2is 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. In some embodiments, 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: 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.

[0112] In some embodiments, 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. In some embodiments, 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 AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 is any one of REF ID NOs: 7-10.

[0113] In some embodiments, 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 REF ID NO:

[0114] 278.

[0115] In some embodiments, 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 REF ID NO:

[0116] 279.

[0117] In some embodiments, 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 REF ID NO:

[0118] 280.

[0119] In some embodiments, 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 REF ID NO:

[0120] 281.

[0121] In some embodiments, 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.

[0122] In some embodiments, 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. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% 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 99% identity to any one of REF ID NOs: 1366-1369 or to a transcript of any one of REF ID NOs: 1095-1097.

[0123] In some embodiments, 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 AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 5N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR. In some embodiments, 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: 1366-1369.

[0124] 5N Extension saRNA

[0125] In some embodiments, 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; X2is 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. In some embodiments, 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.

[0126] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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:

[0127] 278.

[0128] In some embodiments, 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:

[0129] 279.

[0130] In some embodiments, 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:

[0131] 280.

[0132] In some embodiments, 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:

[0133] 281.

[0134] In some embodiments, 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 AUX1X2X3X4X5CGGC (SEQ ID NO: 1640), wherein X1X2X3X4X5 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 5N Extensions”); a CSE that is cognate to the replicase; a nucleic acid pay load or nucleic acids encoding a pay load; and a 3’ SFV UTR. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0135] 5’ SINV UTRs Comprising 6N Extensions

[0136] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “6N” extension. 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)XIX2X3X4X5X6CGGC).

[0137] In some embodiments, 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)XIX2X3X4X5X6CGGC (REF ID NO: 1634; REF ID NO: 1641), wherein: Xi is A or T / U; X2is A or G; X3is A, T / U, or G; X4is A, T / U, G or C; X5is A, T / U, G, or C; and X<> is A, T / U, G, or C. In some embodiments, 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)XIX2X3X4X5X6CGGC (REF ID NO: 1634; REF ID NO: 1641), wherein: Xi is A; X2is A or G; X3is A, T / U, or G; X4is A, T / U, or G; Xs is A, T / U, or G; and X6is A, T / U, or G (SEQ ID NOs: 1741-1742).

[0138] 6N Extension taRNA

[0139] In some embodiments, 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 AUXIX2X3X4X5X6CGGC (REF ID NO: 1641), wherein: Xi is A or U; X2is A or G; X3is A, U, or G; X4is A, U, G or C; Xs is A, U, G, or C; and Xe is A, U, G, or C. In some embodiments, 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 AUXIX2X3X4XSX6CGGC (REF ID NO: 1641), wherein: Xi is A; X2is A or G; X3is A, U, or G; X4is A, U, or G; X5is A, U, or G; and X6is A, U, or G (SEQ ID NO: 1741).

[0140] In some embodiments, 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 AUXIX2X3X4X5X6CGGC (REF ID NO: 1641), wherein XiX2X3X4X5X6is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692). In some embodiments, 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 AUXIX2X3X4X5X6CGGC (REF ID NO: 1641), wherein XiX2X3X4X5X6is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).

[0141] In some embodiments, 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 AUXIX2X3X4X5X6CGGC (REF ID NO: 1641), wherein XiX2X3X4X5X6is REF ID NO: 282 (SEQ ID NO: 1691).

[0142] In some embodiments, 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 AUXIX2X3X4X5X6CGGC (REF ID NO: 1641), wherein XiX2X3X4X5X6is REF ID NO: 283 (SEQ ID NO: 1692). In some embodiments, 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. In some embodiments, 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.

[0143] In some embodiments, 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. In some embodiments, 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.

[0144] In some embodiments, 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. In some embodiments, 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.

[0145] 6N Extension saRNA

[0146] In some embodiments, 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; X2is 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. In some embodiments, 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; X2is A or G; X3is A, U, or G; X4is A, U, or G; X5is A, U, or G; and X6is A, U, or G (SEQ ID NO: 1741).

[0147] In some embodiments, 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 XiX2X3X4X5X6is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692). In some embodiments, 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 XiX2X3X4X5X6is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).

[0148] In some embodiments, 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 X1X2X3X4X5X6 is REF ID NO: 282 (SEQ ID NO: 1691).

[0149] In some embodiments, 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 X1X2X3X4X5X6 is REF ID NO: 283 (SEQ ID NO: 1692).

[0150] In some embodiments, 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 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 payload; and a 3’ SFV UTR. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0151] 5’ SINV UTRs Comprising 7N Extensions

[0152] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “7N” extension. 7N extensions are 7-nucleotide long extensions (e.g., X1X2X3X4X5X6X7) inserted between the A(T / U) and CGGC of an alphaviral (e.g., SINV) 5’- UTR (e.g., A(T / U)XIX2X3X4X5X6X7CGGC).

[0153] In some embodiments, 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)XIX2X3X4X5X6X7CGGC (REF ID NO: 1635; REF ID NO: 1642), wherein: Xi is A, T / U or G; X2is A or G; X3is A, T / U, or G; X4is A, T / U, C, or G; X5is A, T / U, G, or C; Xe is A, T / U, G, or C; and X7 is A, T / U, G, or C. In some embodiments, 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)XIX2X3X4XSX6X7CGGC (REF ID NO: 1635; REF ID NO: 1642), wherein: Xi is A; X2is A or G; X3is A or G; X4is A, T / U, or G; X5is A, T / U, G, or C; X6is A, T / U, G, or C; and X7is A, T / U, G, or C (SEQ ID NO: 1743; SEQ ID NO: 1744).

[0154] 7N Extension taRNA

[0155] In some embodiments, 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 AUXIX2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein: Xi is A, U or G; X2is A or G; X3 is A, U, or G; X4is A, U, C, or G; X5 is A, U, G, or C; Xe is A, U, G, or C; and X7 is A, U, G, or C. In some embodiments, the polynucleotide comprises 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 AUXIX2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein: Xi is A; X2is A or G; X3 is A or G; X4is A, U, or G; X5 is A, U, G, or C; Xe is A, U, G, or C; and X7 is A, U, G, or C.

[0156] In some embodiments, 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 AUXIX2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein XiX2X3X4X5X6X7 is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701). In some embodiments, 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 AUXIX2X3X4XSX6X73CGGC (REF ID NO: 1642), wherein X^XsX^XeX? is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701).

[0157] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 284 (SEQ ID NO: 1695).

[0158] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 285 (SEQ ID NO: 1696).

[0159] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 286 (SEQ ID NO: 1697).

[0160] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 287 (SEQ ID NO: 1698).

[0161] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 288 (SEQ ID NO: 1699).

[0162] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 289 (SEQ ID NO: 1700).

[0163] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 290 (SEQ ID NO: 1701).

[0164] In some embodiments, 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. In some embodiments, the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 828-834 or to a transcript of any one of SEQ ID NOs: 556-562. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 828-834 or to a transcript of any one of SEQ ID NOs: 556-562. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 828-834 or to a transcript of any one of SEQ ID NOs: 556-562.

[0165] In some embodiments, the trRNA comprises any one of REF ID NOs: 1372-1378. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1372-1378 or to a transcript of any one of REF ID NOs: 1100-1106. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1372-1378 or to a transcript of any one of REF ID NOs: 1100-1106. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1372-1378 or to a transcript of any one of REF ID NOs: 1100-1106. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1372-1378 or to a transcript of any one of REF ID NOs: 1100-1106.

[0166] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 7N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR. In some embodiments, 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: 1372-1378.

[0167] 7N Extension saRNA

[0168] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein: Xi is A, U or G; X2is A or G; X3 is A, U, or G; X4 is A, U, C, or G; X5 is A, U, G, or C; Xf> is A, U, G, or C; and X7 is A, U, G, or C. In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein: Xi is A; X2is A or G; X3 is A or G; X4 is A, U, or G; X5 is A, U, G, or C; Xf> is A, U, G, or C; and X7 is A, U, G, or C (SEQ ID NO: 1743; SEQ ID NO: 1744). In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642) wherein X1X2X3X4X5X6X7 is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701). In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642) wherein X1X2X3X4X5X6X7 is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701).

[0169] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 284 (SEQ ID NO: 1695).

[0170] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 285 (SEQ ID NO: 1696).

[0171] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 286 (SEQ ID NO: 1697).

[0172] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 287 (SEQ ID NO: 1698).

[0173] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 288 (SEQ ID NO: 1699).

[0174] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 289 (SEQ ID NO: 1700). In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 is REF ID NO: 290 (SEQ ID NO: 1701).

[0175] In some embodiments, 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 AUX1X2X3X4X5X6X7CGGC (REF ID NO: 1642), wherein X1X2X3X4X5X6X7 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 7N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a payload; and a 3’ SFV UTR. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0176] 5’ SINV UTRs Comprising 8N Extensions

[0177] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “8N” extension. 8N extensions are 8-nucleotide long extensions (e.g., X1X2X3X4X5X6X7X8) inserted between the A(T / U) and CGGC of an alphaviral (e.g., SINV) 5’- UTR (e.g., A(T / U)XIX2X3X4X5X6X7X8CGGC).

[0178] In some embodiments, 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)XIX2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein: Xi is A or G; X2is A or G; X3is A, T / U, or G; X4is A, T / U, G, or C;X5is A, T / U, G, or C; X6is A, T / U, G, or C; X7 is A, T / U, C, or G; and Xs is A, T / U, C or G. In some embodiments, 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)XIX2X3X4X5X6X7XSCGGC (REF ID NO: 1643), wherein: Xi is A; X2is A or G; X3is A; X4is A, G, T / U or C; X5is A; X6is T / U; X7is G; and X8is G (SEQ ID NO: 1745; SEQ ID NO: 1746).

[0179] 8N Extension taRNA

[0180] In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein: Xi is A or G; X2is A or G; X3is A, U, or G; X4is A, U, G, or C;X5is A, U, G, or C; X6is A, U, G, or C; X7is A, U, C, or G; and Xs is A, U, C or G. In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein: Xi is A; X2 is A or G; X3 is A; X4 is A, G, U or C; X5 is A; Xe is U; X7 is G; and Xs is G (SEQ ID NO: 1746).

[0181] In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein X1X2X3X4X5X6X7X8 is REF ID NO: 291 (SEQ ID NO: 1709). In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein X1X2X3X4X5X6X7X8 is REF ID NO: 291 (SEQ ID NO: 1709).

[0182] In some embodiments, the 5’ SINV UTR comprises at least 90% identity to SEQ ID NO: 835 or to a transcript of SEQ ID NO: 563. In some embodiments, the 5’ SINV UTR comprises at least 95% identity to SEQ ID NO: 835 or to a transcript of SEQ ID NO: 563. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to SEQ ID NO: 835 or to a transcript of SEQ ID NO: 563. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to SEQ ID NO: 835 or to a transcript of SEQ ID NO: 563.

[0183] In some embodiments, the trRNA comprises the nucleic acid sequence set forth in REF ID NO: 1379. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to REF ID NO: 1379 or to a transcript of REF ID NO: 1107. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to REF ID NO: 1379 or to a transcript of REF ID NO: 1107. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NO: 1379 or to a transcript of REF ID NO: 1107. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to REF ID NO: 1379 or to a transcript of REF ID NO: 1107.

[0184] In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC, wherein X1X2X3X4X5X6X7X8 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 8N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR. In some embodiments, 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 REF ID NO: 1379.

[0185] 8N Extension saRNA

[0186] In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein: Xi is A or G; X2is A or G; X3is A, U, or G; X4is A, U, G, or C;X5is A, U, G, or C; X6is A, U, G, or C; X7is A, U, C, or G; and Xs is A, U, C or G. In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein: Xi is A; X2 is A or G; X3 is A; X4 is A, G, U or C; X5 is A; Xe is U; X7 is G; and Xs is G (SEQ ID NO: 1746).

[0187] In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643) wherein X1X2X3X4X5X6X7X8 is REF ID NO: 291 (SEQ ID NO: 1709). In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643) wherein X1X2X3X4X5X6X7X8 is REF ID NO: 291 (SEQ ID NO: 1709).

[0188] In some embodiments, 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 AUX1X2X3X4X5X6X7X8CGGC (REF ID NO: 1643), wherein X1X2X3X4X5X6X7X8 are as described in an embodiment of this section (“5’ SINV UTRs Comprising 8N Extensions”); a CSE that is cognate to the replicase; nucleic acids encoding a pay load; and a 3’ SFV UTR. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0189] 5’ SINV UTRs Comprising 9N Extensions Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising a “9N” extension. 9N extensions are 9-nucleotide long extensions (e.g., X1X2X3X4X5X6X7X8X9) inserted between the A(T / U) and CGGC of an alphaviral (e.g., SINV) 5 ’-UTR (e.g., A(T / U)XIX2X3X4X5X6X7X8X9CGGC).

[0190] In some embodiments, 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)XIX2X3X4X5X6X7X8X9CGGC (REF ID NO: 1637; REF ID NO: 1644), wherein: Xi is A, T / U, C, or G; X2is A, T / U or G; X3is A, T / U, C or G; X4is A, T / U, C, or G; X5is A, T / U, C, or G; X6is A, T / U, C or G; X7is A, T / U, C or G; X8is A, T / U, C or G; and X9is A, T / U, C or G. In some embodiments, 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)XIX2X3X4XSX6X7X8X9CGGC, wherein: Xi is A, T / U or G; X2 is A or G; X3is A, T / U, or G; X4is A, T / U, C, or G; X5is A, T / U, C, or G; X6is A, T / U, C or G; X7is A, T / U, C or G; Xs is A, T / U, C or G; and X9 is A, T / U, C or G. In some embodiments, 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)XIX2X3X4X5X6X7X8X9CGGC (REF ID NO: 1637; REF ID NO: 1644), wherein: Xi is A; X2is A or G; X3is A; X4is A; X5is A, T / U, G, or C; X6is A; X7is T / U; X8is G; and X9is G (SEQ ID NO: 1747; SEQ ID NO: 1748).

[0191] 9N Extension taRNA

[0192] In some embodiments, 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: Xi is A, U or G; X2is A or G; X3is A, U, or G; X4is A, U, C, or G; X5is A, U, C, or G; X6is A, U, C or G; X7is A, U, C or G; Xs is A, U, C or G; and X9 is A, U, C or G. In some embodiments, 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: Xi is A; X2is A or G; X3is A; X4is A; X5is A, U, G, or C; X6is A; X7is U; X8is G; and X9is G (SEQ ID NO: 1748).

[0193] In some embodiments, 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 any one of REF ID NOs: 292-297, 299, or 304 (SEQ ID NOs: 1711- 1716, 1718, or 1723). In some embodiments, 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 AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644), wherein X1X2X3X4X5X6X7X8X9 is any one of REF ID NOs: 292-297, 299, or 304 (SEQ ID NOs: 1711-1716, 1718, or 1723).

[0194] In some embodiments, 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: 292 (SEQ ID NO: 1711).

[0195] In some embodiments, 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: 293 (SEQ ID NO: 1712).

[0196] In some embodiments, 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: 294 (SEQ ID NO: 1713).

[0197] In some embodiments, 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).

[0198] In some embodiments, 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: 296 (SEQ ID NO: 1715).

[0199] In some embodiments, 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: 297 (SEQ ID NO: 1716).

[0200] In some embodiments, 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: 298 (SEQ ID NO: 1717).

[0201] In some embodiments, 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 SEQ ID NO: 299 (SEQ ID NO: 1718).

[0202] In some embodiments, 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: 300 (SEQ ID NO: 1719).

[0203] In some embodiments, 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: 301 (SEQ ID NO: 1720).

[0204] In some embodiments, 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: 302 (SEQ ID NO: 1721).

[0205] In some embodiments, 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: 303 (SEQ ID NO: 1722).

[0206] In some embodiments, 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: 304 (SEQ ID NO: 1723).

[0207] In some embodiments, 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: 305 (SEQ ID NO: 1724).

[0208] In some embodiments, 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: 306 (SEQ ID NO: 1725).

[0209] In some embodiments, the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 836-850 or to a transcript of any one of SEQ ID NOs: 564-578. In some embodiments, wherein the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 836-850 or to a transcript of any one of SEQ ID NOs: 564-578. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 836-850 or to a transcript of any one of SEQ ID NOs: 564-578. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 836-850 or to a transcript of any one of SEQ ID NOs: 564-578.

[0210] In some embodiments, the trRNA comprises any one of REF ID NOs: 1380-1394. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1380-1394 or to a transcript of any one of REF ID NOs: 1108-1122. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1380-1394 or to a transcript of any one of REF ID NOs: 1108-1122. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1380-1394 or to a transcript of any one of REF ID NOs: 1108-1122. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1380-1394 or to a transcript of any one of REF ID NOs: 1108-1122.

[0211] In some embodiments, 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 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. In some embodiments, 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.

[0212] 9N Extension saRNA In some embodiments, 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: Xi is A, U or G; X2is A or G; X3is A, U, or G; X4is A, U, C, or G; X5is A, U, C, or G; X6is A, U, C or G; X7is A, U, C or G; Xs is A, U, C or G; and X9 is A, U, C or G. In some embodiments, 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: Xi is A; X2is A or G; X3is A; X4is A; X5is A, U, G, or C; X6is A; X7is U; X8is G; and X9is G (SEQ ID NO: 1748).

[0213] In some embodiments, 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 XiX2X3X4X5X6X7X8X9is any one of REF ID NOs: 292-306 (SEQ ID NOs: 1711-1725). In some embodiments, 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 AUX1X2X3X4X5X6X7X8X9CGGC (REF ID NO: 1644) wherein X1X2X3X4X5X6X7X8X9 is any one of REF ID NOs: 292-306 (SEQ ID NOs: 1711-1725).

[0214] In some embodiments, 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).

[0215] In some embodiments, 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: 293 (SEQ ID NO: 1712).

[0216] In some embodiments, 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: 294 (SEQ ID NO: 1713).

[0217] In some embodiments, 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).

[0218] In some embodiments, 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).

[0219] In some embodiments, 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).

[0220] In some embodiments, 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).

[0221] In some embodiments, 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).

[0222] In some embodiments, 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).

[0223] In some embodiments, 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).

[0224] In some embodiments, 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).

[0225] In some embodiments, 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).

[0226] In some embodiments, 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).

[0227] In some embodiments, 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).

[0228] In some embodiments, 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).

[0229] In some embodiments, 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. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0230] 5’ SINV UTRs Comprising oe5N Extensions

[0231] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising an “oe5N” extension. 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.

[0232] In some embodiments, 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)AXIX2X3X4AX5GGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is A or G; X2is A or G; X3 is A or T / U; X4is A, T / U, C, or G; and X5 is T / U or C. In some embodiments, 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)AXIX2X3X4AX5GGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is A; X2is A or G; X3 is A or T / U; X4is C; and X5 is T / U. In some embodiments, 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)AXIX2X3X4AXSGGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein: Xi is G; X2is A or G; X3is A; X4is C; and X5is T / U or C. oe5N Extension taRNA

[0233] In some embodiments, 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 AUAXIX2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein: Xi is A or G; X2is A or G; X3 is A or U; X4is A, U, C, or G; and X5 is U or C. In some embodiments, 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 AUAXIX2X3X4AXSGGCGGC (SEQ ID NO: 1658), wherein: Xi is A; X2 is A or G; X3 is A or U; X4is C; and X5 is U. In some embodiments, 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 AUAXIX2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein: Xi is G; X2is A or G; X3is A; X4is C; and X5 is U or C.

[0234] In some embodiments, 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 AUXAXIX2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AXIX2X3X4AX5GG is any one of SEQ ID NOs: 293-294, 296, or 298-306. In some embodiments, 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 AUAXIX2X3X4AX5GGCGGC (SEQ ID NO: 1658), wherein AXIX2X3X4AX5GG is any one of SEQ ID NOs: 293-294, 296, or 298-306.

[0235] In some embodiments, 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.

[0236] In some embodiments, 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.

[0237] In some embodiments, 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.

[0238] In some embodiments, 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.

[0239] In some embodiments, 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.

[0240] In some embodiments, 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.

[0241] In some embodiments, 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.

[0242] In some embodiments, 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.

[0243] In some embodiments, 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.

[0244] In some embodiments, 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.

[0245] In some embodiments, 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.

[0246] In some embodiments, 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.

[0247] In some embodiments, 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. In some embodiments, 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.

[0248] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0249] In some embodiments, 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. In some embodiments, 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

[0250] In some embodiments, 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; X2is A or G; X3 is A or U; X4 is A, U, C, or G; and X5 is U or C. In some embodiments, 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; X2 is A or G; X3 is A or U; X4 is C; and X5 is U. In some embodiments, 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 G; X2is A or G; X3is A; X4is C; and X5 is U or C.

[0251] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0252] In some embodiments, 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.

[0253] In some embodiments, 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.

[0254] In some embodiments, 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.

[0255] In some embodiments, 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.

[0256] In some embodiments, 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: 300.

[0257] In some embodiments, 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.

[0258] In some embodiments, 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. In some embodiments, 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.

[0259] In some embodiments, 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: 304.

[0260] In some embodiments, 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.

[0261] In some embodiments, 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.

[0262] In some embodiments, 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. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0263] 5’ SINV UTRs Comprising oe6N Extensions

[0264] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising an “oe6N” extension. oe6N extensions are extensions of the following generic sequence: (AX1X2X3X4X5AX6GG) inserted between the A(T / U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR, in which Xi, X2, X3, X4, X5, and Xf> were randomly varied as A, T / U, G or C in the screen.

[0265] In some embodiments, 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)AXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1655; SEQ ID NO: 1659), wherein: Xi is A or G; X2 is A or G; X3 is A or G; X4is A, T / U, C, or G; X5 is A, T / U, C, or G, and X<> is T / U. In some embodiments, 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)AXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1655; SEQ ID NO: 1659), wherein: Xi is A or G; X2 is A; X3 is A; X4is A or G; X5 is T / U or C, and Xe is T / U. oe6N Extension taRNA

[0266] In some embodiments, 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 AUAXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein: Xi is A; X2is A or G; X3 is A or G; X4is A or G; X5 is A, U, C, or G; and Xe is A, U, C or G. In some embodiments, 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) AXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein: Xi is A; X2is A or G; X3is A or G; X4is A or G; X5 is A; and Xe is A, U, C, or G.

[0267] In some embodiments, 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 AUAXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X4XSAX6GG is any one of SEQ ID NOs: 307-333. In some embodiments, 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 AUAXIX2X3X4XSAX6GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X4X5AX6GG is any one of SEQ ID NOs: 307-333.

[0268] In some embodiments, 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 AXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X4X5AX6GG is SEQ ID NO: 307.

[0269] In some embodiments, 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 AXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1659), wherein AXIX2X3X4X5AX6GG is SEQ ID NO: 308. In some embodiments, 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.

[0270] In some embodiments, 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.

[0271] In some embodiments, 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.

[0272] In some embodiments, 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.

[0273] In some embodiments, 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.

[0274] In some embodiments, 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: 314.

[0275] In some embodiments, 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.

[0276] In some embodiments, 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. In some embodiments, 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.

[0277] In some embodiments, 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.

[0278] In some embodiments, 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: 319.

[0279] In some embodiments, 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.

[0280] In some embodiments, 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.

[0281] In some embodiments, 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.

[0282] In some embodiments, 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.

[0283] In some embodiments, 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: 324. In some embodiments, 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: 325.

[0284] In some embodiments, 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.

[0285] In some embodiments, 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.

[0286] In some embodiments, 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.

[0287] In some embodiments, 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.

[0288] In some embodiments, 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.

[0289] In some embodiments, 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.

[0290] In some embodiments, 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. In some embodiments, 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.

[0291] In some embodiments, 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.

[0292] In some embodiments, 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. In some embodiments, 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.

[0293] In some embodiments, 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. In some embodiments, 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

[0294] In some embodiments, 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; X2is 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. In some embodiments, 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; X2is A or G; X3is A or G; X4 is A or G; X5 is A; and Xf> is A, T / U, C, or G.

[0295] In some embodiments, 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. In some embodiments, 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.

[0296] In some embodiments, 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.

[0297] In some embodiments, 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.

[0298] In some embodiments, 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.

[0299] In some embodiments, 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. In some embodiments, 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.

[0300] In some embodiments, 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.

[0301] In some embodiments, 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.

[0302] In some embodiments, 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.

[0303] In some embodiments, 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.

[0304] In some embodiments, 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.

[0305] In some embodiments, 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.

[0306] In some embodiments, 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. In some embodiments, 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: 319.

[0307] In some embodiments, 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.

[0308] In some embodiments, 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: 321.

[0309] In some embodiments, 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: 322.

[0310] In some embodiments, 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: 323.

[0311] In some embodiments, 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: 324.

[0312] In some embodiments, 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.

[0313] In some embodiments, 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: 326. In some embodiments, 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: 327.

[0314] In some embodiments, 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: 328.

[0315] In some embodiments, 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: 329.

[0316] In some embodiments, 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.

[0317] In some embodiments, 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: 331.

[0318] In some embodiments, 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: 332.

[0319] In some embodiments, 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.

[0320] In some embodiments, 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. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0321] 5’ SINV UTRs Comprising oe7N Extensions

[0322] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising an “oe7N” extension. oe7N extensions are extensions of the following generic sequence: (AX1X2X3X4X5X6AX7GG) inserted between the A(T / U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR, in which Xi, X2, X3, X4, X5, Xe, and X7 were randomly varied as A, T / U, G or C in the screen.

[0323] In some embodiments, 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)AXIX2X3X4X5X6AX7GGCGGC (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; X4is 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. In some embodiments, 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)AXIX2X3X4XSX6AX7GGCGGC (SEQ ID NO: 1656; SEQ ID NO: 1660), wherein: Xi is A or G; X2is A or G; X3is A, C, or G; X4is A or G; X5is A or T / U; X6is T / U or C; and X7is T / U. oe7N Extension taRNA

[0324] In some embodiments, 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 AUAXIX2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein: Xi is A or G; X2is A, U, or G; X3 is A, U, C, or G; X4is A or G; X5 is A, U, C, or G; Xe is A, U, C, or G; and X7 is U or C. In some embodiments, 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 AUAXiX2X3X4X5XeAX7GGCGGC (SEQ ID NO: 1660), wherein: Xi is A or G; X2 is A or G; X3 is A, C, or G; X4is A or G; X5 is A or U; Xe is U or C; and X7 is U.

[0325] In some embodiments, 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 AUAXIX2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1660), wherein AXiX2X3X4XsXeAX7GG is any one of SEQ ID NOs: 334-384. In some embodiments, 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.

[0326] In some embodiments, 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.

[0327] In some embodiments, 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.

[0328] In some embodiments, 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.

[0329] In some embodiments, 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.

[0330] In some embodiments, 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.

[0331] In some embodiments, 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.

[0332] In some embodiments, 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.

[0333] In some embodiments, 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.

[0334] In some embodiments, 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.

[0335] In some embodiments, 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.

[0336] In some embodiments, 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.

[0337] In some embodiments, 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.

[0338] In some embodiments, 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.

[0339] In some embodiments, 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: 347.

[0340] In some embodiments, 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.

[0341] In some embodiments, 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: 349.

[0342] In some embodiments, 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.

[0343] In some embodiments, 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.

[0344] In some embodiments, 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.

[0345] In some embodiments, 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.

[0346] In some embodiments, 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.

[0347] In some embodiments, 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: 355.

[0348] In some embodiments, 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: 356.

[0349] In some embodiments, 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.

[0350] In some embodiments, 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: 358.

[0351] In some embodiments, 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: 359.

[0352] In some embodiments, 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.

[0353] In some embodiments, 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: 361.

[0354] In some embodiments, 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.

[0355] In some embodiments, 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: 363.

[0356] In some embodiments, 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.

[0357] In some embodiments, 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.

[0358] In some embodiments, 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: 366.

[0359] In some embodiments, 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: 367.

[0360] In some embodiments, 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: 368.

[0361] In some embodiments, 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.

[0362] In some embodiments, 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.

[0363] In some embodiments, 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: 371.

[0364] In some embodiments, 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: 372.

[0365] In some embodiments, 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: 373.

[0366] In some embodiments, 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: 374.

[0367] In some embodiments, 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.

[0368] In some embodiments, 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.

[0369] In some embodiments, 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.

[0370] In some embodiments, 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.

[0371] In some embodiments, 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.

[0372] In some embodiments, 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: 380.

[0373] In some embodiments, 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.

[0374] In some embodiments, 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.

[0375] In some embodiments, 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.

[0376] In some embodiments, 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.

[0377] In some embodiments, 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.

[0378] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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

[0379] In some embodiments, 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; X2is 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. In some embodiments, 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.

[0380] In some embodiments, 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. In some embodiments, 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.

[0381] In some embodiments, 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. In some embodiments, 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.

[0382] In some embodiments, 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.

[0383] In some embodiments, 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.

[0384] In some embodiments, 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: 338.

[0385] In some embodiments, 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: 339.

[0386] In some embodiments, 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: 340.

[0387] In some embodiments, 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: 341.

[0388] In some embodiments, 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: 342. In some embodiments, 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.

[0389] In some embodiments, 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.

[0390] In some embodiments, 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.

[0391] In some embodiments, 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.

[0392] In some embodiments, 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: 347.

[0393] In some embodiments, 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.

[0394] In some embodiments, 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: 349.

[0395] In some embodiments, 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: 350. In some embodiments, 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: 351.

[0396] In some embodiments, 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.

[0397] In some embodiments, 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.

[0398] In some embodiments, 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.

[0399] In some embodiments, 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.

[0400] In some embodiments, 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.

[0401] In some embodiments, 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.

[0402] In some embodiments, 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. In some embodiments, 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.

[0403] In some embodiments, 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.

[0404] In some embodiments, 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.

[0405] In some embodiments, 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.

[0406] In some embodiments, 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.

[0407] In some embodiments, 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.

[0408] In some embodiments, 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.

[0409] In some embodiments, 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. In some embodiments, 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.

[0410] In some embodiments, 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.

[0411] In some embodiments, 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.

[0412] In some embodiments, 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: 370.

[0413] In some embodiments, 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: 371.

[0414] In some embodiments, 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: 372.

[0415] In some embodiments, 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: 373.

[0416] In some embodiments, 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: 374. In some embodiments, 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: 375.

[0417] In some embodiments, 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 AUAX1X2X3X4X5X6AX7GGCGG (SEQ ID NO: 1660), wherein AX1X2X3X4X5X6AX7GG is SEQ ID NO: 376.

[0418] In some embodiments, 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: 377.

[0419] In some embodiments, 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: 378.

[0420] In some embodiments, 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: 379.

[0421] In some embodiments, 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: 380.

[0422] In some embodiments, 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: 381.

[0423] In some embodiments, 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: 382. In some embodiments, 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: 383.

[0424] In some embodiments, 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: 384.

[0425] In some embodiments, 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 AUX1X2X3X4X5X6X7X8X9X10X11CGGC (SEQ ID NO: 1660), wherein X1X2X3X4X5X6X7X8X9X10X11 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 pay load; and a 3’ SFV UTR. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0426] 5’ SINV UTRs Comprising oe8N Extensions

[0427] Described herein, in certain aspects, are polynucleotides (e.g., trRNA or saRNA) comprising an “oe8N” extension. oe8N extensions are extensions of the following generic sequence: (AX1X2X3X4X5X6X7AX8GG) inserted between the A(T / U) and CGGC of an alphaviral (e.g., SINV) 5’-UTR, in which Xi, X2, X3, X4, X5, Xe, X7 and Xs were randomly varied as A, T / U, G or C in the screen.

[0428] In some embodiments, 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)AXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1657; SEQ ID NO: 1661), wherein: Xi is A or G; X2 is A, T / U, or G; X3 is A, T / U, C, or G; X4 is A, T / U, C, or G; X5 is A, T / U, or G; Xf> is A, T / U, C, or G; X7 is A, T / U, C, or G; and Xs is A, T / U, C, or G. In some embodiments, 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)AXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1657; SEQ ID NO: 1661), wherein: Xi is A; X2 is A or G; X3 is A or G; X4 is A or G; X5 is A; Xf> is A; X7 is C; and Xs is T / U. oe8N Extension taRNA

[0429] In some embodiments, 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)AXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1657; SEQ ID NO: 1661), wherein: Xi is A or G; X2is A, T / U, or G; X3 is A, T / U, C, or G; X4 is A, T / U, C, or G; X5 is A, T / U, or G; Xf> is A, T / U, C, or G; X7 is A, T / U, C, or G; and X8is A, T / U, C, or G. In some embodiments, 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)AXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1657; SEQ ID NO: 1661), wherein: Xi is A; X2is A or G; X3 is A or G; X4 is A or G; X5 is A; Xf> is A; X7 is C; and X8is T / U.

[0430] In some embodiments, 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 AUAXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AXIX2X3X4XSX6X7AX8GG any one of SEQ ID NOs: 385-542. In some embodiments, 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 AUAXIX2X3X4XSX6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AXIX2X3X4X5X6X7AX8GG is any one of SEQ ID NOs: 385-542.

[0431] In some embodiments, 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 AUAXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AXIX2X3X4X5X6X7AX8GG is SEQ ID NO: 385.

[0432] In some embodiments, 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 AUAXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AXIX2X3X4X5X6X7AX8GG is SEQ ID NO: 386.

[0433] In some embodiments, 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 AUAXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1661), wherein AXIX2X3X4X5X6X7AX8GG is SEQ ID NO: 387.

[0434] In some embodiments, 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: 388.

[0435] In some embodiments, 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: 389.

[0436] In some embodiments, 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, wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 390.

[0437] In some embodiments, 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: 391.

[0438] In some embodiments, 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: 392.

[0439] In some embodiments, 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: 393.

[0440] In some embodiments, 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: 394.

[0441] In some embodiments, 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: 395.

[0442] In some embodiments, 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: 396.

[0443] In some embodiments, 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.

[0444] In some embodiments, 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: 398.

[0445] In some embodiments, 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: 399.

[0446] In some embodiments, 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: 400.

[0447] In some embodiments, 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: 401.

[0448] In some embodiments, 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.

[0449] In some embodiments, 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: 403.

[0450] In some embodiments, 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: 404.

[0451] In some embodiments, 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: 405.

[0452] In some embodiments, 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: 406.

[0453] In some embodiments, 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: 407.

[0454] In some embodiments, 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: 408.

[0455] In some embodiments, 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.

[0456] In some embodiments, 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: 410.

[0457] In some embodiments, 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: 411.

[0458] In some embodiments, 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.

[0459] In some embodiments, 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: 413.

[0460] In some embodiments, 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.

[0461] In some embodiments, 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.

[0462] In some embodiments, 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.

[0463] In some embodiments, 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.

[0464] In some embodiments, 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.

[0465] In some embodiments, 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.

[0466] In some embodiments, 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.

[0467] In some embodiments, 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.

[0468] In some embodiments, 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.

[0469] In some embodiments, 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.

[0470] In some embodiments, 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.

[0471] In some embodiments, 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.

[0472] In some embodiments, 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.

[0473] In some embodiments, 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.

[0474] In some embodiments, 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.

[0475] In some embodiments, 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.

[0476] In some embodiments, 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.

[0477] In some embodiments, 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.

[0478] In some embodiments, 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.

[0479] In some embodiments, 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.

[0480] In some embodiments, 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.

[0481] In some embodiments, 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.

[0482] In some embodiments, 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.

[0483] In some embodiments, 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: 437.

[0484] In some embodiments, 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: 438.

[0485] In some embodiments, 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.

[0486] In some embodiments, 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.

[0487] In some embodiments, 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.

[0488] In some embodiments, 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.

[0489] In some embodiments, 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.

[0490] In some embodiments, 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.

[0491] In some embodiments, 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.

[0492] In some embodiments, 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: 446.

[0493] In some embodiments, 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.

[0494] In some embodiments, 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: 448.

[0495] In some embodiments, 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: 449.

[0496] In some embodiments, 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: 450.

[0497] In some embodiments, 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: 451.

[0498] In some embodiments, 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: 452.

[0499] In some embodiments, 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.

[0500] In some embodiments, 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: 454.

[0501] In some embodiments, 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.

[0502] In some embodiments, 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: 456.

[0503] In some embodiments, 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: 457.

[0504] In some embodiments, 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.

[0505] In some embodiments, 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: 459.

[0506] In some embodiments, 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: 460.

[0507] In some embodiments, 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: 461.

[0508] In some embodiments, 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: 462.

[0509] In some embodiments, 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: 463.

[0510] In some embodiments, 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: 464.

[0511] In some embodiments, 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: 465.

[0512] In some embodiments, 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: 466.

[0513] In some embodiments, 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: 467.

[0514] In some embodiments, 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: 468.

[0515] In some embodiments, 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.

[0516] In some embodiments, 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.

[0517] In some embodiments, 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.

[0518] In some embodiments, 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.

[0519] In some embodiments, 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.

[0520] In some embodiments, 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: 474.

[0521] In some embodiments, 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.

[0522] In some embodiments, 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.

[0523] In some embodiments, 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.

[0524] In some embodiments, 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: 478.

[0525] In some embodiments, 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: 479.

[0526] In some embodiments, 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.

[0527] In some embodiments, 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: 481.

[0528] In some embodiments, 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: 482.

[0529] In some embodiments, 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: 483.

[0530] In some embodiments, 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: 484.

[0531] In some embodiments, 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: 485.

[0532] In some embodiments, 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: 486.

[0533] In some embodiments, 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: 487.

[0534] In some embodiments, 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: 488.

[0535] In some embodiments, 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: 489.

[0536] In some embodiments, 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: 490.

[0537] In some embodiments, 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: 491.

[0538] In some embodiments, 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: 492.

[0539] In some embodiments, 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: 493.

[0540] In some embodiments, 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: 494.

[0541] In some embodiments, 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: 495.

[0542] In some embodiments, 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: 496.

[0543] In some embodiments, 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: 497.

[0544] In some embodiments, 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: 498.

[0545] In some embodiments, 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: 499.

[0546] In some embodiments, 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: 500.

[0547] In some embodiments, 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: 501.

[0548] In some embodiments, 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: 502.

[0549] In some embodiments, 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: 503.

[0550] In some embodiments, 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: 504.

[0551] In some embodiments, 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: 505.

[0552] In some embodiments, 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: 506.

[0553] In some embodiments, 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: 507.

[0554] In some embodiments, 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: 508.

[0555] In some embodiments, 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: 509.

[0556] In some embodiments, 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: 510.

[0557] In some embodiments, 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: 511.

[0558] In some embodiments, 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: 512.

[0559] In some embodiments, 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: 513.

[0560] In some embodiments, 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: 514.

[0561] In some embodiments, 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: 515.

[0562] In some embodiments, 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: 516.

[0563] In some embodiments, 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: 517.

[0564] In some embodiments, 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: 518.

[0565] In some embodiments, 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: 519.

[0566] In some embodiments, 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: 520.

[0567] In some embodiments, 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: 521.

[0568] In some embodiments, 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: 522.

[0569] In some embodiments, 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: 523.

[0570] In some embodiments, 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: 524.

[0571] In some embodiments, 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: 525.

[0572] In some embodiments, 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: 526.

[0573] In some embodiments, 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: 527.

[0574] In some embodiments, 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: 528.

[0575] In some embodiments, 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: 529.

[0576] In some embodiments, 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: 530.

[0577] In some embodiments, 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: 531.

[0578] In some embodiments, 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: 532.

[0579] In some embodiments, 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: 533.

[0580] In some embodiments, 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: 534.

[0581] In some embodiments, 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: 535.

[0582] In some embodiments, 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: 536.

[0583] In some embodiments, 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: 537.

[0584] In some embodiments, 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: 538.

[0585] In some embodiments, 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: 539.

[0586] In some embodiments, 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: 540.

[0587] In some embodiments, 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: 541.

[0588] In some embodiments, 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: 542.

[0589] In some embodiments, the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 929-1086 or to a transcript of any one of SEQ ID NOs: 657-814. In some embodiments, wherein the 5’ SINV UTR comprises at least 95% identity to any one of SEQ ID NOs: 929-1086 or to a transcript of any one of SEQ ID NOs: 657-814. In some embodiments, the 5’ SINV UTR comprises at least 98% identity to any one of SEQ ID NOs: 929-1086 or to a transcript of any one of SEQ ID NOs: 657-814. In some embodiments, the 5’ SINV UTR comprises at least 99% identity to any one of SEQ ID NOs: 929-1086 or to a transcript of any one of SEQ ID NOs: 657-814.

[0590] In some embodiments, the trRNA comprises any one of REF ID NOs: 1473-1630 or a transcript of any one of REF ID NOs: 1201-1358. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 90% identity to any one of REF ID NOs: 1473-1630 or to a transcript of any one of REF ID NOs: 1201-1358. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 95% identity to any one of REF ID NOs: 1473-1630 or to a transcript of any one of REF ID NOs: 1201-1358. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 98% identity to any one of REF ID NOs: 1473-1630 or to a transcript of any one of REF ID NOs: 1201-1358. In some embodiments, the trRNA comprises a nucleic acid sequence having at least 99% identity to any one of REF ID NOs: 1473-1630 or to a transcript of any one of REF ID NOs: 1201-1358.

[0591] In some embodiments, 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. In some embodiments, 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: 1473-1630. oe8N Extension saRNA

[0592] In some embodiments, 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: Xi is A or G; X2is A, U, or G; X3is A, U, C, or G; X4is A, U, C, or G; X5is A, U, or G; X6is A, U, C, or G; X7 is A, U, C, or G; and Xs is A, U, C, or G. In some embodiments, 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: Xi is A; X2 is A or G; X3 is A or G; X4 is A or G; X5 is A; Xf> is A; X7 is C; and Xs is U.

[0593] In some embodiments, 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. In some embodiments, 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.

[0594] In some embodiments, 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: 385.

[0595] In some embodiments, 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. In some embodiments, 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: 387.

[0596] In some embodiments, 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.

[0597] In some embodiments, 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: 389.

[0598] In some embodiments, 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.

[0599] In some embodiments, 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.

[0600] In some embodiments, 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.

[0601] In some embodiments, 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.

[0602] In some embodiments, 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: 394. In some embodiments, 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: 395.

[0603] In some embodiments, 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: 396.

[0604] In some embodiments, 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.

[0605] In some embodiments, 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: 398.

[0606] In some embodiments, 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.

[0607] In some embodiments, 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.

[0608] In some embodiments, 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.

[0609] In some embodiments, 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: 402. In some embodiments, 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.

[0610] In some embodiments, 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: 404.

[0611] In some embodiments, 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: 405.

[0612] In some embodiments, 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.

[0613] In some embodiments, 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: 407.

[0614] In some embodiments, 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: 408.

[0615] In some embodiments, 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.

[0616] In some embodiments, 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: 410. In some embodiments, 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: 411.

[0617] In some embodiments, 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.

[0618] In some embodiments, 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.

[0619] In some embodiments, 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.

[0620] In some embodiments, 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: 415.

[0621] In some embodiments, 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.

[0622] In some embodiments, 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.

[0623] In some embodiments, 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. In some embodiments, 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.

[0624] In some embodiments, 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.

[0625] In some embodiments, 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: 421.

[0626] In some embodiments, 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.

[0627] In some embodiments, 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.

[0628] In some embodiments, 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.

[0629] In some embodiments, 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.

[0630] In some embodiments, 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: 426. In some embodiments, 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.

[0631] In some embodiments, 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.

[0632] In some embodiments, 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: 429.

[0633] In some embodiments, 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.

[0634] In some embodiments, 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.

[0635] In some embodiments, 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: 432.

[0636] In some embodiments, 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: 433.

[0637] In some embodiments, 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. In some embodiments, 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: 435.

[0638] In some embodiments, 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: 436.

[0639] In some embodiments, 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.

[0640] In some embodiments, 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.

[0641] In some embodiments, 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.

[0642] In some embodiments, 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: 440.

[0643] In some embodiments, 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: 441.

[0644] In some embodiments, 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: 442. In some embodiments, 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: 443.

[0645] In some embodiments, 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: 444.

[0646] In some embodiments, 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: 445.

[0647] In some embodiments, 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: 446.

[0648] In some embodiments, 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: 447.

[0649] In some embodiments, 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: 448.

[0650] In some embodiments, 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: 449.

[0651] In some embodiments, 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. In some embodiments, 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.

[0652] In some embodiments, 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.

[0653] In some embodiments, 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.

[0654] In some embodiments, 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: 454.

[0655] In some embodiments, 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: 455.

[0656] In some embodiments, 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.

[0657] In some embodiments, 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: 457.

[0658] In some embodiments, 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: 458. In some embodiments, 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: 459.

[0659] In some embodiments, 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: 460.

[0660] In some embodiments, 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: 461.

[0661] In some embodiments, 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: 462.

[0662] In some embodiments, 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: 463.

[0663] In some embodiments, 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.

[0664] In some embodiments, 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: 465.

[0665] In some embodiments, 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: 466. In some embodiments, 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: 467.

[0666] In some embodiments, 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: 468.

[0667] In some embodiments, 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: 469.

[0668] In some embodiments, 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: 470.

[0669] In some embodiments, 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.

[0670] In some embodiments, 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: 472.

[0671] In some embodiments, 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: 473.

[0672] In some embodiments, 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: 474. In some embodiments, 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: 475.

[0673] In some embodiments, 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: 476.

[0674] In some embodiments, 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: 477.

[0675] In some embodiments, 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: 478.

[0676] In some embodiments, 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: 479.

[0677] In some embodiments, 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: 480.

[0678] In some embodiments, 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: 481.

[0679] In some embodiments, 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: 482. In some embodiments, 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: 483.

[0680] In some embodiments, 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: 484.

[0681] In some embodiments, 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: 485.

[0682] In some embodiments, 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.

[0683] In some embodiments, 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: 487.

[0684] In some embodiments, 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: 488.

[0685] In some embodiments, 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: 489.

[0686] In some embodiments, 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: 490. - I l l -

[0687] In some embodiments, 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: 491.

[0688] In some embodiments, 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: 492.

[0689] In some embodiments, 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: 493.

[0690] In some embodiments, 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: 494.

[0691] In some embodiments, 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: 495.

[0692] In some embodiments, 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: 496.

[0693] In some embodiments, 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: 497.

[0694] In some embodiments, 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: 498. In some embodiments, 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: 499.

[0695] In some embodiments, 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: 500.

[0696] In some embodiments, 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: 501.

[0697] In some embodiments, 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: 502.

[0698] In some embodiments, 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: 503.

[0699] In some embodiments, 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: 504.

[0700] In some embodiments, 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: 505.

[0701] In some embodiments, 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. In some embodiments, 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: 507.

[0702] In some embodiments, 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: 508.

[0703] In some embodiments, 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: 509.

[0704] In some embodiments, 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: 510.

[0705] In some embodiments, 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: 511.

[0706] In some embodiments, 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, wherein AX1X2X3X4X5X6X7AX8GG is SEQ ID NO: 512.

[0707] In some embodiments, 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.

[0708] In some embodiments, 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: 514. In some embodiments, 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: 515.

[0709] In some embodiments, 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: 516.

[0710] In some embodiments, 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: 517.

[0711] In some embodiments, 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.

[0712] In some embodiments, 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: 519.

[0713] In some embodiments, 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: 520.

[0714] In some embodiments, 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: 521.

[0715] In some embodiments, 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: 522. In some embodiments, 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: 523.

[0716] In some embodiments, 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: 524.

[0717] In some embodiments, 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: 525.

[0718] In some embodiments, 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.

[0719] In some embodiments, 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: 527.

[0720] In some embodiments, 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.

[0721] In some embodiments, 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.

[0722] In some embodiments, 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: 530. In some embodiments, 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.

[0723] In some embodiments, 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: 532.

[0724] In some embodiments, 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: 533.

[0725] In some embodiments, 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: 534.

[0726] In some embodiments, 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: 535.

[0727] In some embodiments, 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: 536.

[0728] In some embodiments, 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: 537.

[0729] In some embodiments, 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: 538. In some embodiments, 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: 539.

[0730] In some embodiments, 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: 540.

[0731] In some embodiments, 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.

[0732] In some embodiments, 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: 542.

[0733] In some embodiments, 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 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. In some embodiments, the replicase is an SFV replicase. In some embodiments, the replicase is a SINV replicase.

[0734] Methods of Measuring trRNA Amplification

[0735] In some embodiments, this disclosure provides a method of determining amplification of a plurality of trans-replicating RNAs (trRNAs).

[0736] “Amplification” refers to producing one or more copies of an RNA polynucleotide (e.g., trRNA). In some embodiments, amplification refers to producing enough copies of an RNA polynucleotide, so that the copies of the RNA polynucleotide are detectable (e.g., via quantitative PCR (qPCR) or next-generation sequencing). In some embodiments, amplification does not always result in copies comprising sequences that are 100% identical to the sequence of the RNA polynucleotide. For example, a replicase producing copies of the RNA polynucleotide by amplifying the RNA polynucleotide may introduce a mutation into a copy of the RNA polynucleotide.

[0737] In some embodiments, 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.

[0738] A “plurality” refers to at least 2. In some embodiments, a plurality comprises at least 5 (e.g., at least 10, at least 100, at least 1,000, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10).

[0739] A “plurality of trRNAs” refers to at least 2 trRNAs. In some embodiments, a plurality of trRNAs comprises at least 5 (e.g., at least 10, at least 100, at least 1,000, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10) trRNAs. In some embodiments, the plurality of trRNAs comprises at least 2 trRNAs with different 5’-UTR extension sequences (e.g., at least 5 trRNAs with different 5’-UTR extension sequences, at least 10 trRNAs with different 5’-UTR extension sequences, at least 25 trRNAs with different 5’- UTR extension sequences, at least 50 trRNAs with different 5’-UTR extension sequences, at least 75 trRNAs with different 5’-UTR extension sequences, at least 100 trRNAs with 5’-UTR extension different sequences, at least 500 trRNAs with different 5’-UTR extension sequences, at least 1000 trRNAs with different 5’-UTR extension sequences, at least 5000 trRNAs with different 5’-UTR extension sequences, at least 10000 trRNAs with different 5’-UTR extension sequences, at least 50000 trRNAs with different 5’-UTR extension sequences, at least 100000 trRNAs with different 5’-UTR extension sequences, at least 250000 trRNAs with different 5’- UTR extension sequences, at least 500000 trRNAs with different 5’-UTR extension sequences, or at least 1000000 trRNAs with different 5’-UTR extension sequences).

[0740] In some embodiments, 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.

[0741] In some embodiments, the plurality of trRNAs comprises 100-50000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 100-100000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 100-200000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 100-500000 trRNAs with different 5’- UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 100- 1000000 trRNAs with different 5’-UTR extension sequences.

[0742] In some embodiments, the plurality of trRNAs comprises 1000-50000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 1000-100000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 1000-200000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 1000-500000 trRNAs with different 5’-UTR extension sequences. In some embodiments, the plurality of trRNAs comprises 1000-1000000 trRNAs with different 5’-UTR extension sequences.

[0743] In some embodiments, 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.

[0744] In some embodiments, trRNAs of the plurality of trRNAs comprise: (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. In some embodiments, most trRNAs of the plurality of trRNAs: (a) a 5’ alphavirus UTR comprising a 5 ’-UTR extension; (b) a CSE; (c) nucleic acids encoding a payload or a nucleic acid payload; and (d) a 3’ alphavirus UTR. In some embodiments, 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 or a nucleic acid pay load; and (d) a 3’ alphavirus UTR.

[0745] In some embodiments, the payload comprises a reporter protein (e.g., as described herein).

[0746] The plurality of trRNAs may be obtained in any suitable way. In some embodiments, obtaining the plurality of trRNAs comprises synthesizing the plurality of trRNAs. In some embodiments, obtaining the plurality of trRNAs comprises synthesizing a plurality of DNA sequences that when transcribed produces the plurality of trRNAs. Methods of DNA and RNA synthesis are known in the art and include, but are not limited to, silicon-based synthesis or phosphonamidite synthesis (e.g., as described in Hoose, A. Nat Rev Chem 7, 144-161 (2023)). Depending on the length of the trRNA, the method may comprise synthesizing portions of a given trRNA then ligating those portions together (e.g., as described in Hoose, A. Nat Rev Chem 7, 144-161 (2023)). In some embodiments, obtaining the plurality of trRNAs comprises determining the sequences of the plurality of trRNAs and obtaining synthesized trRNAs comprising the sequences (e.g., instructing a third party to synthesize the sequences). In some embodiments, obtaining the plurality of trRNA comprises obtaining a plurality of plasmids that comprise DNA, that when transcribed, produces the plurality of trRNAs. In some embodiments, obtaining the plurality of trRNA comprises obtaining a plurality of vectors (e.g., viral vectors) that comprise DNA, that when transcribed, produces the plurality of trRNAs. In some embodiments, obtaining the plurality of trRNAs comprises obtaining a plurality of vectors (e.g., viral vectors) that comprises the trRNAs of the plurality of trRNAs. In some embodiments, obtaining the plurality of trRNAs comprising obtaining a plurality of DNA polynucleotides (e.g., plasmids) that when transcribed produce the plurality of trRNAs, and performing in vitro transcription on the plurality of plasmids.

[0747] In some embodiments, 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).

[0748] In some embodiments, trRNAs of the plurality of trRNAs comprise: (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. In some embodiments of the method, the 5’ alphavirus UTR is a VEEV 5’- UTR, SFV 5 ’-UTR, SINV 5 ’-UTR, or CHIKV 5 ’-UTR. In some embodiments of the method, the 3’ alphavirus UTR is a VEEV 3 ’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR. In some embodiments, the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR. In some embodiments, the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR.

[0749] In some embodiments, obtaining the plurality of trRNAs comprises obtaining a plurality of saRNAs that comprise the trRNA and a replicase construct.

[0750] In some embodiments, the method comprises contacting 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.

[0751] A “plurality of cells” refers to at least 2 cells. In some embodiments, a plurality of cells comprises at least 5 cells (e.g., at least 10 cells, at least 100 cells, at least 1,000 cells, at least IxlO4cells, at least IxlO5cells, at least IxlO6cells, at least IxlO7cells, at least IxlO8cells, at least IxlO9cells, or at least IxlO10cells). In some embodiments, cells of the plurality of cells are eukaryotic cells. In some embodiments, cells of the plurality of cells are mammalian cells. In some embodiments, cells of the plurality of cells are BHK21 cells. In some embodiments, contacting is performed in in vitro cell culture.

[0752] In some embodiment, contacting comprises physical interaction between an RNA of a plurality of RNA polynucleotides (e.g., a trRNA of a plurality of trRNAs) and a cell of the specific cell type. In some embodiments, the physical interaction is transitory. In some embodiments, the physical interaction results in the RNA polynucleotide entering the cell. In some embodiments, when a plurality of RNA polynucleotides is contacted with cells of the specific cell type, some physical interactions between an RNA polynucleotide and a cell are transitory and other physical interactions result in the RNA polynucleotide entering a cell. In some embodiments, contacting comprises transfecting the plurality of cells with the plurality of trRNAs. In some embodiments, contacting further comprises contacting (e.g., transfecting) cells of the plurality of cells with a replicase construct comprising a replicase that is cognate to a CSE of the plurality of trRNAs. In some embodiments, contacting comprises contacting the plurality of cells with a plurality of saRNAs that comprise the trRNA and a replicase construct.

[0753] In some embodiments, the method comprises multiple contacting steps. In some embodiments, the method comprises contacting the plurality of cells with the trRNA, incubating plurality of cells, extracting the trRNA from the incubated cells, and contacting the extracted trRNA from the incubated cells with a new plurality of cells. These contacting, incubating and extracting, contacting again steps may be repeated 1, 2, 3, 4, 5, 6 or more times. In some embodiments, these contacting, incubating and extracting, contacting again steps are repeated 2 times.

[0754] Determining amplification of the plurality of trRNAs may be performed using any suitable means. In some embodiments, the determining comprises measuring the amount of most of the trRNA (e.g., by sequencing the 5’ UTR extension) in the plurality of cells. Measuring may be performed using any suitable technique for measuring the amount of each 5’ UTR extension of the plurality of trRNAs. An “amount” of each 5’ UTR extension refers to either the actual amount of the 5’ UTR extension or the amount of trRNAs comprising the 5 ’UTR extension (e.g., in mols or grams), or a relative amount of each 5’ UTR extension (e.g., as compared to a control, or total amount of RNA sequenced). A control may be a known amount of an RNA polynucleotide standard. In some embodiments, measuring comprises performing qPCR to determine the amount of a 5’ UTR extension of a trRNA. In some embodiments, measuring comprises performing qPCR to determine an amount of most of the 5’ UTR extension of a plurality of trRNAs. In some embodiments, measuring comprises performing qPCR to determine amounts of each 5’ UTR extension of a plurality of trRNAs.

[0755] In some embodiments, measuring comprises performing next-generation sequencing (e.g., ILLUMINA sequencing) to determine an amount of a 5’ UTR extension of a trRNA. In some embodiments, measuring comprises performing next-generation sequencing to determine an amount of most of the 5’ UTR extensions of the plurality of trRNAs. In some embodiments, measuring comprises performing next-generation sequencing to determine an amount of each of the 5’ UTR extensions of the plurality of trRNAs. In some embodiments, the method comprises preparing the trRNAs extracted from the cells of the specific cell type for next-generation sequencing.

[0756] Preparing trRNAs for next-generation sequencing may be done using any suitable method. Methods of preparing RNA polynucleotides (e.g., trRNAs) for next generation sequencing are known in the art. In some embodiments, the method comprises reverse transcribing a 5’ UTR extension into DNA. In some embodiments, the method comprises ligating a unique molecule identifier (UMI) to extracted 5’ UTR extensions of the plurality of trRNAs or to DNA reverse transcribed from extracted 5’ UTR extensions of the plurality of trRNAs. In some embodiments, the method comprises amplifying the DNA polynucleotide (reverse transcribed from the trRNA) comprising the UMI.

[0757] Performing next-generation sequencing to measure the amount 5’ UTR extensions of the plurality of trRNAs produces sequencing data. In some embodiments, the sequencing data comprises counts of the number of times each 5’ UTR extension is observed in the sample that was sequenced. In some embodiments, the sequencing data comprises counts of the number of times a control sequence or 5’ UTR extension is observed in the sequencing data. In some embodiments, the sequencing data comprises a count of the total number of polynucleotides sequenced. In some embodiments, analyzing the sequencing data comprises normalizing the number of times a given 5’ UTR extension is observed by the number of times a control is observed to determine a relative amount of the given RNA barcode. In some embodiments, analyzing the sequencing data comprises normalizing the number of time a given 5’ UTR extension is observed by the total number of RNA polynucleotides (reversed transcribed to DNA) sequenced to determine a relative amount of the given RNA barcode. In some embodiments, the method further comprises sequencing 5’ UTR extension of the plurality of trRNAs before the contacting step and after the extracting step (e.g., before and after administration of the plurality of RNA polynucleotides to the subject), and then determining a change in the relative amount of at least some of the 5’ UTR extensions (e.g., most of the 5’ UTR extensions or all the 5’ UTR extensions) after extracting compared to before contacting.

[0758] An increase in the relative amount of a given 5’ UTR extension indicates that the trRNA comprising the 5’ UTR extensions has been amplified. In some embodiments, no change in the relative amount of a given 5’ UTR extensions indicates that the trRNA comprising the 5’ UTR extension has not been amplified. In some embodiments, a decrease in the relative amount of a given 5’ UTR extension indicates that the trRNA comprising the 5’ UTR extensions has degraded or not been delivered to cells of the plurality of cells.

[0759] EXAMPLES

[0760] Example 1- Validation of 5’ SINV UTR Extensions

[0761] A library of more than 250,000 different trRNAs having different 5’ SINV UTR extensions (ranging from 3-9) nucleotides in length were produced and screened in cells to determine how different 5’ SINV UTR extensions influenced trRNA amplification and pay load expression (FIGs 1, 2A and 2B). This library included the wildtype 5’SINV UTR and control 5’ SINV UTR extensions and deletions with known effects on trRNA amplification and payload expression (FIGs. 3A-3B). Control experiments showed that control trRNA expressed their payload as expected and were enriched as expected in in vitro cell culture.

[0762] 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 first step right after in vitro library production (before amplification). A second optional step after 1 round of transfection (for odd 5’ SINV UTR extension). And a third step after three serial transfections. The size of the nucleotide representation (e.g., T) 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. For example, 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. One conclusion from these results is that trRNAs with 3N extensions having C are not amplified well in vitro. Notably, C is found in higher frequencies as the extension length increases (FIGs. 7-8). For example, the frequency of C increases in the 6N extension (FIG. 7) at positions 5-7 (fourth N through sixth N) and in the 7N extension (FIG. 8) positions 5-8 (fourth N through 7thN). Surprisingly, C is less well tolerated at these positions in the 8N and 9N extensions (FIGs. 9-10).

[0763] Additionally, 5’ UTR extension length can influence tolerable nucleotides at a given position of the 5’ UTR extension. For example, A and G are at relatively high frequencies in the 3N extension at position 2 (first N), but as extension length G frequency decreases and A frequency increases indicating the G is less well tolerated (e.g., a G at position 2 results in decreased trRNA amplification). At position 4 (third N), A, T, and G are generally present in the logo plots, but at much different frequencies depending on the extension length.

[0764] From these data, 33 5’ SINV UTR extensions were selected for validation (see table 1 and table 2). FIG. 11 A shows a time course of trRNA payload expression for some of the selected 5’-UTR extensions. All three of the selected extensions outperform the wildtype. Additionally, payload expression of trRNAs with the 33 5’ SINV UTR extensions was measured in BHK-21 cells (FIG. 12B). These results indicate that most of the 5’ SINV UTR extensions identified in the screen as increasing trRNA amplification also increase trRNA payload expression.

[0765] One of the trRNA 5’ SINV UTR extensions A(T / U)AGAAGA(T / U)GG (SEQ ID NO: 1667; SEQ ID NO: 1668) was further validated in mice. Results show that this 5’ SINV UTR extension greatly increases pay load expression in mice as compared to mRNA alone and T3A trRNA, which is known to have better payload expression than wildtype trRNA.

[0766] Library creation

[0767] 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). Following PCR, 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).

[0768] In vitro Transcription

[0769] PCR products (800ng each) were separately used as template for in vitro transcription (IVT) using T7 RNA polymerase (NEB) in 20pl reactions. Template DNA was removed by addition of DNase I (NEB) and incubation for 30 minutes. RNA was purified using an equal volume of SPRI beads according to manufacture recommendations (CleanNGS DNA & RNA Clean-Up Magnetic Beads, Bulldog Bio) with final elution in RNase-free water. Purified IVT products were quantified by nanodrop and combined in ratios such that the resulting RNA library theoretically contained equimolar concentration of variants to be tested. Control transreplicating RNAs were prepared similarly and supplemented to this library at 1% each by mass.

[0770] Transfection / Serial Transfection

[0771] BHK21 cells were co-transfected with 6pg of the 5’ extension variant RNA library and 40pg SFV replicase mRNA using 150pl MessengerMax (ThermoFisher) per 60 million cells in biological duplicate. After 14 hours, total RNA was isolated using an RNeasy Plus kit (Qiagen). For serial RNA transfers, cells were co-transfected with 50pg SFV replicase mRNA and lOOpg total RNA isolated from the prior transfection using 250pl MessengerMax for 60 million cells. This was repeated twice for a total of three serial RNA transfers.

[0772] NGS prep

[0773] Rapid Amplification of cDNA Ends (RACE) 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.

[0774] Analysis After quality control and deduplication, 5’ variants were quantified in each sample with abundance in total RNA from the final serial transfection or enrichment relative to input IVT RNA library being the primary measure of replication efficiency for each variant.

[0775] 5’ SINV UTR extension sequences for validation Table 1: 5’ UTR Extension Sequences (DNA)

[0776] Table 2: 5’ UTR Extensions (RNA)

[0777] Example 2- Validation of 5’ SINV oeN UTR Extensions

[0778] A library of more than 80,000 different trRNAs having different “oeSTR” 5’ SINV UTR extensions (ranging from 10-12) nucleotides in length were produced and screened in cells to determine how oeSTR extensions influenced trRNA amplification and payload expression (FIGs. 13 A, 13B, and 14). The oeSTR extension sequences were designed based on the results from the 3N-9N screen, specifically the 5’ UTR extension sequence A(T / U)AGAAGA(T / U)GGC (SEQ ID NO: 1664; SEQ ID NO: 1665) (FIG. 13A). Position 1-3, 8, and 10-12 of A(T / U)AGAAGA(T / U)GGC (SEQ ID NO: 1664; SEQ ID NO: 1665) were not changed. Different length random extensions were added between positions 3 and 8. Position 9 was also random.

[0779] 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. For each extension length, position 1 (relative to the 5’ terminal) was fixed as A. For each extension length, the 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. For example, 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.

[0780] As in Example 1, the size of nucleotide representation (e.g., A) 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. With the tested extensions of 10, 11, or 12 nucleotides in length, 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. As shown in Table 3, several oeSTR extensions were enriched over the extensions tested in Example 1.

[0781] Table 3- Enrichment of 5’ UTRs with oeSTR Extension Over Comparable 5’ UTRs with Extensions in Example 1

[0782] EXEMPLARY SEQUENCES

[0783] Table 4: 5’ UTR Extensions (DNA) Table...

Claims

CLAIMSWhat is claimed is:

1. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)AXIX2X3X4AX5GGCGGC (SEQ ID NO: 1654; SEQ ID NO: 1658), wherein:Xi is A or G;X2is A or G;X3is A or T / U;X4is A, T / U, C, or G;X5is T / U or C.

2. The polynucleotide of claim 1, wherein:Xi is G;X2is A or G;X3is A;X4is G or C;X5is T / U or C.

3. The polynucleotide of claim 1, wherein the polynucleotide is an RNA polynucleotide and AXIX2X3X4AX5GG of SEQ ID NO: 1658 is any one of AGAAGAUGG (REF ID NO: 294), AGAAGACGG (REF ID NO: 296), or AGAACAUGG (REF ID NO: 298).

4. The polynucleotide of claim 1, wherein 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.

5. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 1-4, nucleic acids encoding a pay load, and a 3’ Semliki Forest virus (SFV) UTR.

6. A self-amplifying RNA comprising the polynucleotide of any one of claims 1-5.

7. The polynucleotide of claim 1, wherein the polynucleotide is a DNA polynucleotide and AX1X2X3X4AX5GG of SEQ ID NO: 1654 is any one of AGAAGATGG (REF ID NO: 23), AGAAGACGG (REF ID NO: 25), or AGAACATGG (REF ID NO: 27).

8. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3CGGC (REF ID NO: 1631; REF ID NO: 1638), wherein:Xi is A, T / U or G;X2is A, T / U or G; andX3is A, T / U, or G, wherein the polynucleic acid does not comprise a sequence of A(T / U)TGA.

9. The polynucleotide of claim 8, wherein:Xi is A or G;X2is A or G; andX3is A, T / U, or G.

10. The polynucleotide of claim 8, wherein the polynucleotide is an RNA polynucleotide and X1X2X3 of REF ID NO: 1638 is any one of REF ID NOs: 272-276.

11. The polynucleotide of claim 8, wherein 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.

12. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 8-11, nucleic acids encoding a pay load, and a 3’ Semliki Forest Virus (SFV) UTR.

13. A self-amplifying RNA comprising the polynucleotide of any one of claims 8-12.

14. The polynucleotide of claim 8, wherein the polynucleotide is a DNA polynucleotide and X1X2X3 is any one of REF ID NOs: 1-5.

15. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3X4CGGC (SEQ ID NO: 1632; SEQ ID NO: 1639), wherein:Xi is A or T / U;X2is A, T / U or G;X3 is A, T / U, or G; andX4is A, T / U, or G wherein the polynucleic acid does not comprise a sequence of A(T / U)AAAA.

16. The polynucleotide of claim 15, wherein:Xi is A;X2is A, T / U, or G;X3 is A or G; andX4is A or G.

17. The polynucleotide of claim 15, wherein the polynucleotide is an RNA polynucleotide and XiX2X3X4of SEQ ID NO: 1639 is REF ID NO: 277.

18. The polynucleotide of claim 15, wherein the 5’ SINV UTR comprises at least 90% identity to SEQ ID NO: 821 or to a transcript of SEQ ID NO: 549.

19. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 15-18, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

20. A self-amplifying RNA comprising the polynucleotide of any one of claims 15-19.

21. The polynucleotide of claim 15, wherein the polynucleotide is a DNA polynucleotide and XiX2X3X4of SEQ ID NO: 1632 is REF ID NO: 6.

22. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3X4X5CGGC (SEQ ID NO: 1633; SEQ ID NO: 1640), wherein:Xi is A, T / U or G;X2is A, T / U or G;X3is A, T / U, or G;X4is A, T / U, or G; andX5is A, T / U, C, or G wherein the polynucleic acid does not have a sequence of A(T / U)AAAAA.

23. The polynucleotide of claim 22, wherein:Xi is A;X2is A, T / U or G;X3is A;X4is A or G; andXs is A or G.

24. The polynucleotide of claim 22, wherein the polynucleotide is an RNA polynucleotide and XiX2X3X4X5of SEQ ID NO: 1640 is any one of REF ID NOs: 278-281.

25. The polynucleotide of claim 22, wherein the 5’ SINV UTR comprises at least 90% identity to any one of REF ID NOs: 822-825 or to a transcript of any one of REF ID NOs: 550- 553.

26. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 22-25, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

27. A self-amplifying RNA comprising the polynucleotide of any one of claims 22-26.

28. The polynucleotide of claim 15, wherein the polynucleotide is a DNA polynucleotide and X1X2X3X4X5 of SEQ ID NO: 1633 is any one of REF ID NOs: 7-10.

29. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3X4X5X6CGGC (REF ID NO: 1634; REF ID NO: 1641), wherein:Xi is A or T / U;X2 is A or G;X3is A, T / U, or G;X4is A, T / U, G or C;X5 is A, T / U, G, or C; andX6is A, T / U, G, or C.

30. The polynucleotide of claim 29, wherein:Xi is A;X2 is A or G;X3is A, T / U, or G;X4is A, T / U, or G;X5 is A, T / U, or G; andX6is A, T / U, or G (SEQ ID NO: 1741; SEQ ID NO: 1742).

31. The polynucleotide of claim 29, wherein the polynucleotide is an RNA polynucleotide and XiX2X3X4X5X6of REF ID NO: 1641 is any one of REF ID NOs: 282-283 (SEQ ID NOs: 1691-1692).

32. The polynucleotide of claim 29, wherein 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.

33. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 29-32, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

34. A self-amplifying RNA comprising the polynucleotide of any one of claims 29-33.

35. The polynucleotide of claim 29, wherein the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6 of REF ID NO: 1634 is any one of REF ID NOs: 11-12 (SEQ ID NOs: 1693-1694).

36. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3X4X5X6X7CGGC (REF ID NO: 1635; REF ID NO: 1642), wherein:Xi is A, T / U or G;X2is A or G;X3is A, T / U, or G;X4is A, T / U, C, or G;X5is A, T / U, G, or C;Xe is A, T / U, G, or C; andX7is A, T / U, G, or C.

37. The polynucleotide of claim 36, wherein:Xi is A;X2is A or G;X3 is A or G;X4is A, T / U, or G;X5is A, T / U, G, or C;Xe is A, T / U, G, or C; andX7is A, T / U, G, or C (SEQ ID NO: 1743; SEQ ID NO: 1744).

38. The polynucleotide of claim 36, wherein the polynucleotide is an RNA polynucleotide and XiX2X3X4X5X6X7 of REF ID NO: 1642 is any one of REF ID NOs: 284-290 (SEQ ID NOs: 1695-1701).

39. The polynucleotide of claim 36, wherein 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.

40. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 36-39, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

41. A self-amplifying RNA comprising the polynucleotide of any one of claims 36-40.

42. The polynucleotide of claim 36, wherein the polynucleotide is a DNA polynucleotide and XiX2X3X4X5X6X70f REF ID NO: 1635 is any one of REF ID NOs: 13-19 (SEQ ID NOs: 1702-1708).

43. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3X4X5X6X7X8CGGC (REF ID NO: 1636; REF ID NO: 1643), wherein:Xi is A or G;X2is A or G;X3is A, T / U, or G;X4is A, T / U, G, or C;X5is A, T / U, G, or C;X6is A, T / U, G, or C;X7 is A, T / U, C, or G; andX8is A, T / U, C or G.

44. The polynucleotide of claim 43, wherein:Xi is A;X2is A or G;X3 is A;X4is A, G, T / U or C;X5 is A;X6is T / U;X7 is G; andX8is G (SEQ ID NO: 1745; SEQ ID NO: 1746).

45. The polynucleotide of claim 43, wherein the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6X7X8 of REF ID NO: 1643 is REF ID NO: 291 (SEQ ID NO: 1709).

46. The polynucleotide of claim 43, wherein 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.

47. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 43-46, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

48. A self-amplifying RNA comprising the polynucleotide of any one of claims 43-47.

49. The polynucleotide of claim 43, wherein the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7X8 of REF ID NO: 1636 is REF ID NO: 20 (SEQ ID NO: 1710).

50. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)XIX2X3X4X5X6X7X8X9CGGC (REF ID NO: 1637; REF ID NO: 1644), wherein:Xi is A, T / U, C or G;X2is A, T / U or G;X3is A, T / U, C or G;X4is A, T / U, C, or G;X5is A, T / U, C, or G;X6is A, T / U, C or G;X7is A, T / U, C or G;Xs is A, T / U, C or G; andX9is A, T / U, C or G.

51. The polynucleotide of claim 50, wherein:Xi is A;X2 is A or G;X3 is A;X4 is A;X5is A, T / U, G, or C;Xe is A;X7is T / U;Xs is G; andX9is G (SEQ ID NO: 1747; SEQ ID NO: 1748).

52. The polynucleotide of claim 50, wherein the polynucleotide is an RNA polynucleotide and X1X2X3X4X5X6X7X8X9 of REF ID NO: 1644 is any one of REF ID NO: 292-306 (SEQ ID NOs: 1711-1725).

53. The polynucleotide of claim 50, wherein X1X2X3X4X5X6X7X8X9 of REF ID NO: 1644 is REF ID NO: 294 (SEQ ID NO: 1713).

54. The polynucleotide of claim 50, wherein the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 836-850 or to a transcript of any one of SEQ ID NOs: 564- 578.

55. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 50-54, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

56. A self-amplifying RNA comprising the polynucleotide of any one of claims 50-55.

57. The polynucleotide of claim 50, wherein the polynucleotide is a DNA polynucleotide and X1X2X3X4X5X6X7X8X9 of REF ID NO: 1637 is any one of REF ID NOs: 21-35 (SEQ ID NOs: 1726-1740).

58. The polynucleotide of claim 57, wherein X1X2X3X4X5X6X7X8X9 of REF ID NO: 1637 is REF ID NO: 23 (SEQ ID NO: 1728).

59. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)AXIX2X3X4X5AX6GGCGGC (SEQ ID NO: 1655; SEQ ID NO: 1659), wherein:Xi is A or G;X2 is A or G;X3 is A or G;X4is A, T / U, C, or G;X5is A, T / U, C, or G;X6is T / U.

60. The polynucleotide of claim 59, wherein:Xi is A or G;X2 is A;X3 is A;X4 is A or G;X5is T / U or C;X6is T / U.

61. The polynucleotide of claim 59, wherein the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5AX6GG of SEQ ID NO: 1659 is any one of SEQ ID NO: 307-33362. The polynucleotide of claim 59, wherein 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.

63. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 59-62, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

64. A self-amplifying RNA comprising the polynucleotide of any one of claims 59-63.

65. The polynucleotide of claim 59, wherein the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5AX6GG of SEQ ID NO: 1655 is any one of SEQ ID NOs: 36-62.

66. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)AXIX2X3X4X5X6AX7GGCGGC (SEQ ID NO: 1656; SEQ ID NO: 1660), wherein:Xi is A or G;X2is A, T / U, or G;X3is A, T / U, C, or G;X4is A or G;X5is A, T / U, C or G;Xe is A, T / U, C or G; andX7is T / U or C.

67. The polynucleotide of claim 66, wherein:Xi is A;X2 is A or G;X3 is A, C, or G;X4is A or G;X5is A or T / U;Xe is T / U or C; andX7is T / U.

68. The polynucleotide of claim 66, wherein the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5X6AX7GG of SEQ ID NO: 1660 is any one of SEQ ID NO: 334-384.

69. The polynucleotide of claim 66, wherein 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.

70. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 66-69, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

71. A self-amplifying RNA comprising the polynucleotide of any one of claims 66-70.

72. The polynucleotide of claim 66, wherein the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5X6AX7GG of SEQ ID NO: 1656 is any one of SEQ ID NOs: 63-113.

73. A polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (UTR), wherein the 5’ SINV UTR comprises a 5’ terminal comprising a polynucleic acid having a sequence of A(T / U)AXIX2X3X4X5X6X7AX8GGCGGC (SEQ ID NO: 1657; SEQ ID NO: 1661), wherein:Xi is A or G;X2is A, T / U, or G;X3is A, T / U, C, or G;X4is A, T / U, C, or G;X5is A, T / U, or G;X6is A, T / U, C or G;X7 is A, T / U, C or G; andX8is A, T / U, C, or G.

74. The polynucleotide of claim 73, wherein:Xi is A;X2 is A or G;X3 is A or G;X4 is A or G;X5 is A;Xe is A;X7 is C andX8is T / U.

75. The polynucleotide of claim 73, wherein the polynucleotide is an RNA polynucleotide and AX1X2X3X4X5X6X7AX8GG of SEQ ID NO: 1661 is any one of SEQ ID NO: 385-542.

76. The polynucleotide of claim 73, wherein the 5’ SINV UTR comprises at least 90% identity to any one of SEQ ID NOs: 929-1086 or to a transcript of any one of SEQ ID NOs: 657- 814.

77. A trans amplifying ribonucleic acid (RNA) (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’, the polynucleotide of any one of claims 73-76, nucleic acids encoding a payload, and a 3’ Semliki Forest virus (SFV) UTR.

78. A self-amplifying RNA comprising the polynucleotide of any one of claims 73-77.

79. The polynucleotide of claim 73, wherein the polynucleotide is a DNA polynucleotide and AX1X2X3X4X5X6X7AX8GG of SEQ ID NO: 1657 is any one of SEQ ID NOs: 114-271.

80. A 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.

81. The method of claim 80, wherein each trRNA of the plurality of trRNAs:(a) a 5’ alphavirus UTR comprising a 5 ’-UTR extension;(b) a CSE;(c) a nucleic acid payload or a nucleic acid encoding a payload; and(d) a 3’ alphavirus UTR.

82. The method of claim 81, wherein 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.

83. The method of claim 81 or claim 82, wherein the 3’ alphavirus UTR is a VEEV 3 ’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR.

84. The method of any one of claims 81-83, wherein the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR.

85. The method of any one of claims 81-84, wherein the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR.

86. The method of any one of claims 81-85, wherein the payload comprises a reporter protein.

87. The method of claim 86, wherein the reporter protein is enhanced green fluorescent protein (EGFP), mCherry, mKate, anchored heavy chain variable domain (aVHH), nanoluciferase (nanoLuc), firefly luciferase (Flue), secreted embryonic alkaline phosphatase (SEAP), or Cre recombinase.

88. The method of any one of claims 81-85, wherein the payload is not a reporter protein.

89. The method of any one of claims 80-88, wherein cells of the plurality of cells comprise a polynucleotide encoding the replicase that is cognate to the CSE.

90. The method of any one of claims 80-88, further comprising contacting cell of the plurality of cell with a polyribonucleotide encoding the replicase that is cognate to the CSE.

91. The method of any one of claims 80-90, wherein the plurality of trRNAs comprises at least 10 different trRNAs.

92. The method of claim 91, wherein the at least 10 different trRNAs each comprise at different 5’ UTR extension.

93. The method of any one of claims 80-92, wherein obtaining the plurality of trRNAs comprises synthesizing the plurality of trRNAs using in vitro transcription.

94. The method of any one of claims 80-93, wherein 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.

95. The method of claim 92, wherein 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.

96. The method of any one of claims 80-95, wherein the contacting comprises transfecting trRNAs of the plurality of trRNAs into cells of the plurality of cells.

97. The method of any one of claims 80-96, wherein the obtaining comprises obtaining a plurality of self-amplifying RNAs (saRNAs) that comprise the trRNAs and a replicase construct.

98. A deoxyribonucleic acid (DNA) polynucleotide that, when transcribed, produces a ribonucleic acid (RNA) polynucleotide comprising a 5’ Sindbis virus (SINV) untranslated region (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.

99. A deoxyribonucleic acid (DNA) polynucleotide that, when transcribed, produces a ribonucleic acid (RNA) polynucleotide comprising a 5’ untranslated region (UTR) comprising the sequence set forth in any one of SEQ ID NOs: 816-1086.

Images