Encrypted RNA and Methods of Using the Same

Abstract

Provided herein are encrypted RNAs that enable an increase in the translation of a polypeptide, including a therapeutic polypeptide, after contact with a translation activator, DNAs encoding the encrypted RNAs, and methods of using them.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit under 35 U.S.C. § 119 of U.S. Provisional Patent Application No. 63 / 388,110, filed Jul. 11, 2022, entitled "ENCRYPTED RNA AND METHODS OF ITS USE"; U.S. Provisional Patent Application No. 63 / 390,139, filed Jul. 18, 2022, entitled "ENCRYPTED RNA AND METHODS OF ITS USE"; U.S. Provisional Patent Application No. 63 / 390,245, filed Jul. 18, 2022, entitled "ENCRYPTED RNA AND METHODS OF ITS USE"; and U.S. Provisional Patent Application No. 63 / 493,906, filed Apr. 3, 2023, entitled "ENCRYPTED RNA AND METHODS OF ITS USE", the entire disclosure of each of which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (A137870001WO00 - SEQ - CEW.xml; size: 647,938 bytes; and created on Jul. 11, 2023) is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to encrypted RNA that enables an increase in the translation of a polypeptide comprising a therapeutic polypeptide after contact with a translation activator, DNA encoding the encrypted RNA, and methods of using them.

Background Art

[0004] Currently, the two major structural classes of FDA-approved drugs are small molecules and proteins. Small molecule drugs, which consist mainly of hydrophobic organic compounds, typically act by inactivating or inhibiting target proteins through competitive binding. However, the proteins that can have such binding pockets are estimated to account for only 2-5% of the protein-coding human genome (Hopkins AL.et al.Nat Rev Drug Discov.2002;1:727-30). In contrast, protein-based drugs (e.g., antibodies) can bind to various targets with high specificity or can be used to replace mutant or defective proteins (e.g., delivery of insulin for diabetes). However, the size, specificity, and stability of proteins limit their usefulness for many potential disease targets.

[0005] However, the mRNA precursors and DNA precursors of proteins can be specifically targeted via Watson-Crick base pairing and, in the case of gene editing aimed at permanently altering the host DNA, are therapeutically promising in that they can not only treat gene defects but also serve as a means to treat gene defects. Over the past few decades, RNA drugs have emerged as candidates for addressing diseases at the gene and RNA levels. Since 1990, it has been known that nucleic acids can be used to regulate protein production in vivo (Wolff JA,et al.Science.1990;247:1465-8), but therapeutic RNA delivery has been limited by many factors. Naked single-stranded RNA is susceptible to nuclease degradation, can activate the immune system, and is too large and too negatively charged to passively cross the cell membrane and may require additional means of cell entry and escape from endosomes that transport extracellular nanoparticles to the cytoplasm (Sahay G,et al.J Control Release.2010;145:182-95). Therefore, the nucleic acid delivery field has focused on the design of delivery methods and materials to transport RNA drugs to the site of interest.

[0006] Despite the recent successes of RNA therapeutics and vaccines, there remains a need in the art to identify safe and effective RNA pharmaceuticals for the treatment of diseases, including RNA pharmaceuticals with increased disease or target specificity.

SUMMARY OF THE INVENTION

[0007] Provided herein is an isolated RNA polynucleotide comprising a coding region having a coding sequence encoding one or more therapeutic polypeptides, wherein the template region comprises two different regions, the left flanking region of the virus (“L region”) and the right flanking region of the virus (“R region”). The present disclosure relates to “coded RNA” that encodes a polypeptide of interest that is translated at a low level until contacted with a “target-specific translational activator”. The target-specific translational activator directs an increase in the translation of the polypeptide of interest by transcribing the coded RNA into a separate mRNA species that is more translatable by the cellular ribosome machinery. In some embodiments, the coded RNA encodes a therapeutic polypeptide of interest. The present disclosure also relates to DNA encoding the coded RNA. In some embodiments, the target-specific translational activator comprises an RNA-dependent RNA polymerase or an RNA-dependent DNA polymerase.

[0008] Aspects of the present disclosure provide an isolated RNA polynucleotide comprising a coding region having a coding sequence encoding one or more therapeutic polypeptides, a template region, wherein the template region comprises two different regions, namely the left flanking region of the virus (“L region”) and the right flanking region of the virus (“R region”), the L region being adjacent to and contiguous with the 5' end of the coding region, the R region being adjacent to and contiguous with the 3' end of the coding region, the coding sequence being in an antisense orientation, the therapeutic polypeptide being heterologous to the virus, and the template region interacting with and initiating a polymerase in a cell containing an RNA-dependent polymerase.

[0009] In some embodiments, the present disclosure provides reverse complements of the isolated RNA polynucleotides described herein.

[0010] In some embodiments, the virus is selected from the group consisting of viruses of the orders Amarillovirales, Articulavirales, Blubervirales, Bunyavirales, Hepelivirales, Martellivirales, Mononegavirales, Nidovirales, and Picornavirales. In some embodiments, the virus is selected from the group consisting of viruses of the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, Rhabdoviridae, and Togaviridae.In some embodiments, the virus is selected from the group consisting of alpha coronavirus 229E, alpha coronavirus NL63, alpha coronavirus WA2028, turkey metapneumovirus (AMPV), beta coronavirus HKU1, beta coronavirus HKU15, beta coronavirus HKU33, beta coronavirus OC43, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, eastern equine encephalitis virus (EEEV), enterovirus D68 (EV-D68), foot-and-mouth disease virus, hantavirus, Hendra virus, hepatitis B virus, hepatitis C virus, HMPV, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), infectious salmon anemia virus, influenza A virus, influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, norovirus, rabies virus, respiratory syncytial virus, Reston Ebola virus, rhinovirus, Rift Valley fever virus, rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan equine encephalitis virus (VEEV), vesicular stomatitis virus, western equine encephalitis virus (WEEV), yellow fever virus, Zika virus, and Zika virus.

[0011] In some embodiments, the virus is not an alphavirus. In some embodiments, the template region is native to the virus. In some embodiments, the template region is a variant of a template region native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the template region native to the virus. In some embodiments, each of the L and R regions of the template region contains less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the template region native to the virus. In some embodiments, each of the L and R regions of the template region is mutated from the template region native to the virus by 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer substitutions that do not participate in 5' capping. In some embodiments, each of the L and R regions of the template region is mutated from the template region native to the virus by 1 or fewer substitutions that do not participate in 5' capping.

[0012] In some embodiments, the isolated RNA polynucleotide comprises at least one nucleoside modification. In some embodiments, the level of nucleoside modification can refer to the level of modification across the fully isolated polynucleotide or a portion thereof (e.g., the template region). In some embodiments, the template region is nucleoside-modified, and the percentage of modified nucleosides is 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less. In some embodiments, the template region is nucleoside-modified, and the percentage of modified nucleosides is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 100%.

[0013] In some embodiments, the nucleoside modification is a non-immunogenic uridine modification, and the proportion of the modified uridine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less. In some embodiments, the nucleoside modification is a non-immunogenic uridine modification, and the proportion of the modified uridine modification exceeds 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%, or is 100%.

[0014] In some embodiments, the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of the modified cytidine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less. In some embodiments, the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of the modified cytidine modification exceeds 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%, or is 100%.

[0015] In some embodiments, the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of the modified adenosine modification is 1% to 30%. In some embodiments, the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of the modified adenosine modification is about 1%, 5%, 10%, 15%, 20%, 25% or 30%.

[0016] In some embodiments, the isolated polynucleotide comprises a 5' cap structure. In some embodiments, the 5' end of the L region comprises a 5' cap structure. In some embodiments, the 5' end of the L region comprises one or more mutations associated with the 5' cap structure. In some embodiments, the 5' cap structure is selected from the group consisting of Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structures.

[0017] In some embodiments, the isolated polynucleotide does not comprise a 5' cap structure (uncapped). In some embodiments, the 5' end of the L region does not comprise a 5' cap structure (uncapped). In some embodiments, the 5' end of the isolated polynucleotide comprises a 5'-monophosphate, 5'-diphosphate or 5'-triphosphate. In some embodiments, the 5' end of the isolated polynucleotide does not comprise 5'-phosphate (dephosphorylated).

[0018] In some embodiments, the template region is the reverse complement of the template region that is native to the virus. In some embodiments, the template region is a variant of the reverse complement of the template region that is native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reverse complement of the template region that is native to the virus. In some embodiments, each reverse complement of the L region and the R region varies from the reverse complement of the template region that is native to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping. In some embodiments, each reverse complement of the L region and the R region varies from the reverse complement of the template region that is native to the virus by 1 or fewer substitutions that do not participate in 5' capping.

[0019] In some embodiments, the isolated RNA polynucleotide comprises at least one nucleoside modification. In some embodiments, the level of nucleoside modification can refer to the level of modification across the fully isolated polynucleotide or a portion thereof (e.g., the template region). In some embodiments, the template region is nucleoside-modified, and the percentage of modified nucleotides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less. In some embodiments, the 5' end of the reverse complement of the R region encodes a cap structure. In some embodiments, the 5' end of the R region is capped.

[0020] In some embodiments, the therapeutic polypeptide is a secreted polypeptide. In some embodiments, the therapeutic polypeptide is selected from the group consisting of interferon, interferon-stimulated gene, cytokine, chemokine, antibody, signaling molecule, cytotoxic protein, protein that causes cell death, anti-neoplastic protein, immunomodulatory protein, protein toll-like receptor agonist or dominant negative protein. In some embodiments, the cytokine is an inflammatory cytokine. In some embodiments, the inflammatory cytokine is TNF-α. In some embodiments, the cytokine is an anti-inflammatory cytokine. In some embodiments, the anti-inflammatory cytokine is interleukin-1 receptor antagonist (IL-1RN). In some embodiments, the therapeutic polypeptide is interleukin or caspase. In some embodiments, the interleukin is IL-12A, IL-12B or IL-2. In some embodiments, the secreted protein is an antibody.

[0021] In some embodiments, the therapeutic polypeptide is interferon. In some embodiments, the interferon is IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-γ or IFN-λ. In some embodiments, the interferon is IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β1, IFN-ε, IFN-κ, IFN-ω1, IFN-γ, IFN-λ1 (IL28A), IFN-λ2 (IL28B), IFN-λ3 (IL29) or IFN-λ4. In some embodiments, the interferon is IFN-α, IFN-β, IFN-κ, IFN-λ1 (IL28A), IFN-λ2 (IL28B) or IFN-λ3 (IL29).

[0022] In some embodiments, the coding sequence encodes two or more therapeutic polypeptides that can be separated by one or more ribosome skip sequences. In some embodiments, the coding region further comprises one or more regulatory elements selected from the group consisting of ribosome binding sites, Kozak sequences, Shine-Dalgarno sequences, ribozymes, riboswitches, promoters, microRNA binding sites, and internal ribosome entry sites (IRES). In some embodiments, one or more regulatory elements are operably linked to the coding sequence. In some embodiments, the RNA polynucleotide further comprises a polyadenylation signal and / or a 3' poly(A) tail.

[0023] In some embodiments, the RNA-dependent polymerase is an RNA-dependent RNA polymerase. In some embodiments, the RNA-dependent polymerase is an RNA-dependent DNA polymerase. In some embodiments, the RNA-dependent polymerase is a virus-derived polymerase.

[0024] In some embodiments, the isolated RNA polynucleotide is single-stranded RNA. In some embodiments, the isolated polynucleotide is in a linear form. In some embodiments, the isolated polynucleotide is in a covalently closed circular form.

[0025] In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 2 or a variant of SEQ ID NO: 2, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 20, 21, 22, or 23 or a variant of any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the variant of SEQ ID NO: 2 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 26 of SEQ ID NO: 2. In some embodiments, the variant comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 15 of any one of SEQ ID NO: 20, 21, 22, or 23. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 3 or a variant of SEQ ID NO: 3, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 24, 25, 26, or 27. In some embodiments, (i) the variant of SEQ ID NO: 3 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 35 of SEQ ID NO: 3. In some embodiments, (i) the variant of SEQ ID NO: 24 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 24, (ii) the variant of SEQ ID NO: 25 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 25, (iii) the variant of SEQ ID NO: 26 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 26, or (iv) the variant of SEQ ID NO: 27 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 27. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 4 or a variant of SEQ ID NO: 4, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 28, 29, 30, or 31 or a variant of any one of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31.In some embodiments, the variant of SEQ ID NO: 4 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 14 to 50 of SEQ ID NO: 4. In some embodiments, any one variant of SEQ ID NO: 28, 29, 30, or 31 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of any one of SEQ ID NO: 28, 29, 30, or 31.

[0026] In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 1 or 5 or a variant of SEQ ID NO: 1 or 5, and wherein the R region comprises the nucleotide sequence shown as SEQ ID NO: 18 or 19 or a variant of SEQ ID NO: 18 or 19. In some embodiments, the variant of SEQ ID NO: 1 or 5 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 37 of SEQ ID NO: 1 or 5. In some embodiments, the variant of SEQ ID NO: 18 or 19 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 20 of SEQ ID NO: 18 or 19. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 6 or a variant of SEQ ID NO: 6, and wherein the R region comprises the nucleotide sequence shown as SEQ ID NO: 32 or 33, or a variant of SEQ ID NO: 32 or 33. In some embodiments, the variant of SEQ ID NO: 6 comprises mutations at one or more nucleotide positions selected from positions 14 or 15 of SEQ ID NO: 6. In some embodiments, the variant of SEQ ID NO: 32 or 33 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 33 of SEQ ID NO: 32 or 33. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 7 or a variant of SEQ ID NO: 7, and wherein the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 34, 35, 36, or 37 or a variant of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, the variant of SEQ ID NO: 7 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 20 of SEQ ID NO: 7. In some embodiments, the variant is any one of SEQ ID NO: 34, 35, 36, or 37, and the variant comprises mutations at one or more nucleotide positions selected from the group consisting of positions 5 to 8 of SEQ ID NO: 34, 35, 36, or 37.In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 8 or a variant of SEQ ID NO: 8, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 38, 39, 40, or 41 or a variant of any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. In some embodiments, the variant of SEQ ID NO: 8 comprises mutations at one or more nucleotide positions selected from positions 14 or 15 of SEQ ID NO: 8. In some embodiments, the variant of any one of SEQ ID NO: 38, 39, 40, or 41 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 13 of SEQ ID NO: 38, 39, 40, or 41. In some embodiments, the virus is an influenza virus, the L region is the nucleotide sequence shown as SEQ ID NO: 9 or a variant of SEQ ID NO: 9, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 42 or 43 or a variant of SEQ ID NO: 42 or 43. In some embodiments, the variant of SEQ ID NO: 9 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 18 of SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 42 or 43 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 14 of SEQ ID NO: 42 or 43. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 11 or a variant of SEQ ID NO: 11, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 46 or 47 or a variant of SEQ ID NO: 46 or 47. In some embodiments, the variant of SEQ ID NO: 11 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 81 of SEQ ID NO: 11. In some embodiments, the variant of SEQ ID NO: 46 or 47t comprises mutations at one or more nucleotide positions selected from the group consisting of positions 5 to 9 of SEQ ID NO: 46 or 47.In some embodiments, the virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 12 or a variant of SEQ ID NO: 12, and here, the R region contains the nucleotide sequence shown as either SEQ ID NO: 48 or 49, or is a variant of either SEQ ID NO: 48 or 49. In some embodiments, the variant of SEQ ID NO: 12 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 52 of SEQ ID NO: 12. In some embodiments, the variant of either SEQ ID NO: 48 or 49 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 11 of SEQ ID NO: 48 or 49. In some embodiments, the virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 13 or a variant of SEQ ID NO: 13, and here, the R region contains the nucleotide sequence shown as SEQ ID NO: 50 or 51 or a variant of SEQ ID NO: 50 or 51. In some embodiments, the variant of SEQ ID NO: 13 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 87 of SEQ ID NO: 13. In some embodiments, the variant of SEQ ID NO: 50 or 51 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 17 of SEQ ID NO: 50 or 51. In some embodiments, the virus is an influenza virus, the L region contains the nucleotide sequence shown as any one of SEQ ID NO: 10 or a variant of SEQ ID NO: 10, and here, the R region contains the nucleotide sequence shown as SEQ ID NO: 44 or 45 or a variant of SEQ ID NO: 44 or 45. In some embodiments, the variant of SEQ ID NO: 10 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 86 of SEQ ID NO: 10. In some embodiments, the variant of SEQ ID NO: 44 or 45 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 21 of SEQ ID NO: 44 or 45.In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 14 or a variant of SEQ ID NO: 14, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 52 or 53, or is a variant of either SEQ ID NO: 52 or 53. In some embodiments, the variant of SEQ ID NO: 14 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 93 of SEQ ID NO: 14. In some embodiments, the variant of either SEQ ID NO: 52 or 53 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 48 of SEQ ID NO: 52 or 53. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 15 or a variant of SEQ ID NO: 15, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 54 or 55, or a variant of either SEQ ID NO: 54 or 55. In some embodiments, the variant of SEQ ID NO: 15 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 95 of SEQ ID NO: 15. In some embodiments, the variant of either SEQ ID NO: 54 or 55 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 34 of SEQ ID NO: 54 or 55.

[0027] In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 16 or a variant of SEQ ID NO: 16, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 56 or 57, or is a variant of either SEQ ID NO: 56 or 57. In some embodiments, the variant of SEQ ID NO: 16 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 81 of SEQ ID NO: 16. In some embodiments, the variant of either SEQ ID NO: 56 or 57 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 56 or 57. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 17 or a variant of SEQ ID NO: 17, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 58 or 59 or a variant of either SEQ ID NO: 58 or 59. In some embodiments, the variant of SEQ ID NO: 17 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 22 of SEQ ID NO: 17. In some embodiments, the variant of either SEQ ID NO: 58 or 59 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 32 of SEQ ID NO: 58 or 59.

[0028] In some embodiments, the virus is a simian becovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 137 or a variant of SEQ ID NO: 137, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 128 or any one variant of SEQ ID NO: 128. In some embodiments, the variant of SEQ ID NO: 137 contains mutations at one or more nucleotide positions selected from the group consisting of positions 40 to 1557 of SEQ ID NO: 137. In some embodiments, any one variant of SEQ ID NO: 128 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 30 of SEQ ID NO: 128. In some embodiments, the virus is a simian becovirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 138, 139, 140, 141, 142, 143 or 144 or a variant of any one of SEQ ID NO: 138, 139, 140, 141, 142, 143 or 144, and the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 130, 136, 145, 146 or 147 or a variant of any one of SEQ ID NO: 130, 136, 145, 146 or 147.In some embodiments, (i) the variant of SEQ ID NO: 138 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 312 of SEQ ID NO: 138, (ii) the variant of SEQ ID NO: 139 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1567 of SEQ ID NO: 139, (iii) the variant of SEQ ID NO: 140 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1488 of SEQ ID NO: 140, (iv) the variant of SEQ ID NO: 141 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1593 of SEQ ID NO: 141, (v) the variant of SEQ ID NO: 142 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1570 of SEQ ID NO: 142, (vi) the variant of SEQ ID NO: 143 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1488 of SEQ ID NO: 143, and (vii) the variant of SEQ ID NO: 144 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1593 of SEQ ID NO: 144. In some embodiments, (i) the variant of any one of SEQ ID NOs: 130, 136, 145, 146 or 147 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130, (ii) the variant of SEQ ID NO: 136 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 33 of SEQ ID NO: 136, (iii) the variant of SEQ ID NO: 145 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 1461 of SEQ ID NO: 145, (iv) the variant of SEQ ID NO: 146 contains mutations at one or more nucleotide positions selected from the group consisting of positions 60 to 1441 of SEQ ID NO: 146, or (v) the variant of SEQ ID NO: 147 contains mutations at one or more nucleotide positions selected from the group consisting of positions 60 to 897 of SEQ ID NO: 147.

[0029] In some embodiments, the virus is respiratory syncytial virus (RSV), the L region comprises a nucleotide sequence represented as any one of SEQ ID NO: 158, 163, 165, 166 or 419 or a variant of any one of SEQ ID NO: 158, 163, 165, 166 or 419, and the R region comprises a nucleotide sequence represented as any one of SEQ ID NO: 169, 170, 176, 177 or 420 or a variant of any one of SEQ ID NO: 169, 170, 176, 177 or 420. In some embodiments, (i) the variant of SEQ ID NO: 158 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 207 of SEQ ID NO: 158, (ii) the variant of SEQ ID NO: 163 contains mutations at one or more nucleotide positions selected from the group consisting of positions 18 to 210 of SEQ ID NO: 163, (iii) the variant of SEQ ID NO: 165 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 147 of SEQ ID NO: 165, (iv) the variant of SEQ ID NO: 166 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 32 of SEQ ID NO: 166, or (v) the variant of SEQ ID NO: 419 contains mutations at one or more nucleotide positions selected from the group consisting of positions 18 to 35 of SEQ ID NO: 419. In some embodiments, (i) the variant of SEQ ID NO: 169 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 78 of SEQ ID NO: 169, (ii) the variant of SEQ ID NO: 170 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 80 of SEQ ID NO: 170, (iii) the variant of SEQ ID NO: 176 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 36 of SEQ ID NO: 176, (iv) the variant of SEQ ID NO: 177 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 33 of SEQ ID NO: 177, or (v) the variant of SEQ ID NO: 420 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 35 of SEQ ID NO: 420.

[0030] In some embodiments, the virus is a parainfluenza virus, where the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 181, 182 or 183 or a variant of any one of SEQ ID NO: 181, 182 or 183, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 184 or a variant of SEQ ID NO: 184. In some embodiments, (i) the variant of SEQ ID NO: 181 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 181, (ii) the variant of SEQ ID NO: 182 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 142 of SEQ ID NO: 182, or (iii) the variant of SEQ ID NO: 183 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 183. In some embodiments, the variant of SEQ ID NO: 184 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 98 of SEQ ID NO: 184. In some embodiments, the virus is a parainfluenza virus, where the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 187, 188 or 189 or a variant of any one of SEQ ID NO: 187, 188 or 189, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 190 or a variant of SEQ ID NO: 190. In some embodiments, (i) the variant of SEQ ID NO: 187 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 187, (ii) the variant of SEQ ID NO: 188 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 101 of SEQ ID NO: 188, or (iii) the variant of SEQ ID NO: 189 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 189. In some embodiments, the variant of SEQ ID NO: 190 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 93 of SEQ ID NO: 190.

[0031] In some embodiments, the virus is a metapneumovirus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NO: 196, 197 or 199 or a variant of any one of SEQ ID NO: 196, 197 or 199, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 201 or a variant of SEQ ID NO: 201. In some embodiments, (i) the variant of SEQ ID NO: 196 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 224 of SEQ ID NO: 196, (ii) the variant of SEQ ID NO: 197 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 230 of SEQ ID NO: 197, or (iii) the variant of SEQ ID NO: 199 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 140 of SEQ ID NO: 199. In some embodiments, the variant of SEQ ID NO: 201 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 201. In some embodiments, the virus is a metapneumovirus, the L region comprises a nucleotide sequence represented as SEQ ID NO: 19, or a variant of SEQ ID NO: 195, wherein the R region comprises a nucleotide sequence represented as SEQ ID NO: 200 or a variant of SEQ ID NO: 200. In some embodiments, the variant of SEQ ID NO: 195 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 224 of SEQ ID NO: 195. In some embodiments, the variant of SEQ ID NO: 200 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 200.

[0032] In some embodiments, the virus is a henipavirus, where the L region is the nucleotide sequence shown as SEQ ID NO: 204 or a variant of SEQ ID NO: 204, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 206 or a variant of SEQ ID NO: 206. In some embodiments, the variant of SEQ ID NO: 204 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 204. In some embodiments, the variant of SEQ ID NO: 206 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 206. In some embodiments, the virus is a henipavirus, where the L region is the nucleotide sequence shown as SEQ ID NO: 209 or 210 or a variant of SEQ ID NO: 209 or 210, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 211 or a variant of SEQ ID NO: 211. In some embodiments, (i) the variant of SEQ ID NO: 209 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 209, or (ii) the variant of SEQ ID NO: 210 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 83 of SEQ ID NO: 210. In some embodiments, the variant of SEQ ID NO: 211 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 211.

[0033] In some embodiments, the virus is a hepadnavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 222 or 223 or a variant of SEQ ID NO: 222 or 223, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 225 or a variant of SEQ ID NO: 225. In some embodiments, (i) the variant of SEQ ID NO: 222 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 639 of SEQ ID NO: 222, or (ii) the variant of SEQ ID NO: 223 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 186 of SEQ ID NO: 223. In some embodiments, the variant of SEQ ID NO: 225 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 1023 of SEQ ID NO: 225.

[0034] In some embodiments, the virus is a filovirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 227, 228, 229 or 230 or a variant of any one of SEQ ID NO: 227, 228, 229 or 230, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 231 or a variant of SEQ ID NO: 231. In some embodiments, (i) the variant of SEQ ID NO: 227 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 710 of SEQ ID NO: 227, (ii) the variant of SEQ ID NO: 228 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 713 of SEQ ID NO: 228, (iii) the variant of SEQ ID NO: 229 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 707 of SEQ ID NO: 229, or (iv) the variant of SEQ ID NO: 230 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 707 of SEQ ID NO: 230. In some embodiments, the variant of SEQ ID NO: 231 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 449 of SEQ ID NO: 231. In some embodiments, the virus is a filovirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 232, 233, 234 or 235 or a variant of any one of SEQ ID NO: 232, 233, 234 or 235, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 236 or a variant of SEQ ID NO: 236. In some embodiments, (i) the variant of SEQ ID NO: 232 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 678 of SEQ ID NO: 232, (ii) the variant of SEQ ID NO: 233 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 681 of SEQ ID NO: 233, (iii) the variant of SEQ ID NO: 234 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 678 of SEQ ID NO: 234, or (iv) the variant of SEQ ID NO: 235 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 678 of SEQ ID NO: 235.In some embodiments, the variant of SEQ ID NO: 236 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 437 of SEQ ID NO: 236.

[0035] In some embodiments, the virus is a filovirus, the L region contains the nucleotide sequence shown as any one of SEQ ID NO: 237, 238 or 239 or a variant of any one of SEQ ID NO: 237, 238 or 239, and the R region contains the nucleotide sequence shown as SEQ ID NO: 240 or a variant of SEQ ID NO: 240. In some embodiments, (i) the variant of SEQ ID NO: 237 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 605 of SEQ ID NO: 237, (ii) the variant of SEQ ID NO: 238 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 606 of SEQ ID NO: 238, or (iii) the variant of SEQ ID NO: 239 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 605 of SEQ ID NO: 239. In some embodiments, the variant of SEQ ID NO: 240 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 83 of SEQ ID NO: 240. In some embodiments, the virus is a filovirus, the L region contains the nucleotide sequence shown as SEQ ID NO: 241 or a variant of SEQ ID NO: 241, and here, the R region contains the nucleotide sequence shown as SEQ ID NO: 242 or a variant of any one of SEQ ID NO: 242. In some embodiments, the variant of SEQ ID NO: 241 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 34 of SEQ ID NO: 241. In some embodiments, the variant of SEQ ID NO: 242 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 593 of SEQ ID NO: 242. In some embodiments, the virus is a filovirus, the L region contains the nucleotide sequence shown as SEQ ID NO: 243 or a variant of SEQ ID NO: 243, and here, the R region contains the nucleotide sequence shown as SEQ ID NO: 244 or a variant of SEQ ID NO: 244. In some embodiments, the variant of SEQ ID NO: 243 contains mutations at one or more nucleotide positions selected from the group consisting of positions 30 to 45 of SEQ ID NO: 243.In some embodiments, the variant of SEQ ID NO: 244 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 677 of SEQ ID NO: 244. In some embodiments, the virus is a filovirus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NO: 245, 246 or 247 or a variant of any one of SEQ ID NO: 245, 246 or 247, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 248 or a variant of SEQ ID NO: 248. In some embodiments, (i) the variant of SEQ ID NO: 245 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 29 to 171 of SEQ ID NO: 245, (ii) the variant of SEQ ID NO: 246 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 30 to 171 of SEQ ID NO: 246, (iii) the variant of SEQ ID NO: 247 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 29 to 171 of SEQ ID NO: 247. In some embodiments, the variant of SEQ ID NO: 248 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 91 of SEQ ID NO: 248.

[0036] In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 249 or a variant of SEQ ID NO: 249, and wherein the R region comprises the nucleotide sequence shown as SEQ ID NO: 250 or 251 or a variant of SEQ ID NO: 250 or 251. In some embodiments, the variant of SEQ ID NO: 249 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 274 of SEQ ID NO: 249. In some embodiments, (i) the variant of SEQ ID NO: 250 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 183 of SEQ ID NO: 250, or (ii) the variant of SEQ ID NO: 251 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 375 of SEQ ID NO: 251. In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 255 or a variant of SEQ ID NO: 255, and wherein the R region comprises the nucleotide sequence shown as SEQ ID NO: 256 or 257 or a variant of SEQ ID NO: 256 or 257. In some embodiments, the variant of SEQ ID NO: 255 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 35 of SEQ ID NO: 255. In some embodiments, (i) the variant of SEQ ID NO: 256 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 600 to 273 of SEQ ID NO: 256, or (ii) the variant of SEQ ID NO: 257 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 377 of SEQ ID NO: 257. In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 261 or a variant of SEQ ID NO: 261, and wherein the R region comprises the nucleotide sequence shown as SEQ ID NO: 262 or 263 or a variant of SEQ ID NO: 262 or 263. In some embodiments, the variant of SEQ ID NO: 261 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 215 of SEQ ID NO: 261.In some embodiments, (i) the variant of SEQ ID NO: 262 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 166 of SEQ ID NO: 262, or (ii) the variant of SEQ ID NO: 263 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 379 of SEQ ID NO: 263.

[0037] Aspects of the disclosure provide an isolated RNA polynucleotide comprising a coding region having a coding sequence encoding one or more polypeptides, and a template region comprising two different regions, namely a viral left flanking region ("L region") and a viral right flanking region ("R region"), wherein the L region is adjacent and contiguous to the 5' end of the coding region, and the R region is adjacent and contiguous to the 3' end of the coding region, and wherein the coding sequence is in a sense orientation, and wherein the template region interacts with and initiates the RNA-dependent polymerase activity of a polymerase in a cell containing the RNA-dependent polymerase, provided that the virus is not an alphavirus or the polypeptide is heterologous to the virus.

[0038] In some embodiments, the disclosure provides the reverse complement of the isolated RNA polynucleotide described herein.

[0039] In some embodiments, the virus is selected from the group consisting of viruses of the orders Amarillovirales, Articulavirales, Blubervirales, Bunyavirales, Hepelivirales, Mononegavirales, Nidovirales, and Picornavirales. In some embodiments, the virus is selected from the group consisting of viruses of the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, and Rhabdoviridae.In some embodiments, the virus is from the group consisting of alphacoronavirus 229E, alphacoronavirus NL63, alphacoronavirus WA2028, avian metapneumovirus (AMPV), betacoronavirus HKU1, betacoronavirus HKU15, betacoronavirus HKU33, betacoronavirus OC43, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, enterovirus D68 (EV-D68), foot-and-mouth disease virus, hantavirus, Hendra virus, hepatitis B virus, hepatitis C virus, HMPV, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), infectious salmon anemia virus, influenza A, influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, norovirus, rabies virus, respiratory syncytial virus, Reston Ebola virus, rhinovirus, Rift Valley fever virus, rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, vesicular stomatitis virus, yellow fever virus, Zaire Ebola virus, and Zika virus.

[0040] In some embodiments, the template region is native to the virus. In some embodiments, the template region is a variant of a template region native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the template region native to the virus. In some embodiments, each of the L region and the R region of the template region contains fewer than 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the template region native to the virus. In some embodiments, each of the L region and the R region of the template region varies from the template region native to the virus by 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer substitutions that do not participate in 5' capping. In some embodiments, each of the L region and the R region of the template region varies from the template region native to the virus by 1 or fewer substitutions that do not participate in 5' capping.

[0041] In some embodiments, the isolated RNA polynucleotide comprises at least one nucleoside modification. In some embodiments, the level of nucleoside modification can refer to the level of modification across the fully isolated polynucleotide or a portion thereof (e.g., the template region). In some embodiments, the template region is nucleoside-modified and the percentage of modified nucleosides is 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less. In some embodiments, the template region is nucleoside-modified and the percentage of modified nucleosides is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 100%.

[0042] In some embodiments, the nucleoside modification is a non-immunogenic uridine modification, and the proportion of the modified uridine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less. In some embodiments, the nucleoside modification is a non-immunogenic uridine modification, and the proportion of the modified uridine modification exceeds 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%, or is 100%.

[0043] In some embodiments, the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of the modified cytidine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less. In some embodiments, the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of the modified cytidine modification exceeds 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%, or is 100%.

[0044] In some embodiments, the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of the modified adenosine modification is 1% to 30%. In some embodiments, the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of the modified adenosine modification is about 1%, 5%, 10%, 15%, 20%, 25% or 30%.

[0045] In some embodiments, the isolated polynucleotide comprises a 5' cap structure. In some embodiments, the 5' end of the L region comprises a 5' cap structure. In some embodiments, the 5' end of the L region comprises one or more modifications associated with the 5' cap structure. In some embodiments, the 5' cap structure is selected from the group consisting of Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structures.

[0046] In some embodiments, the isolated polynucleotide does not comprise a 5' cap structure (uncapped). In some embodiments, the 5' end of the L region does not comprise a 5' cap structure (uncapped). In some embodiments, the 5' end of the isolated polynucleotide comprises a 5'-monophosphate, 5'-diphosphate or 5'-triphosphate. In some embodiments, the 5' end of the isolated polynucleotide does not comprise a 5'-phosphate (dephosphorylated).

[0047] In some embodiments, the template region is the reverse complement of the template region that is native to the virus. In some embodiments, the template region is a variant of the reverse complement of the template region that is native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the reverse complement of the template region that is native to the virus. In some embodiments, each reverse complement of the L region and the R region varies from the reverse complement of the template region that is native to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping. In some embodiments, each reverse complement of the L region and the R region varies from the reverse complement of the template region that is native to the virus by 1 or fewer substitutions that do not participate in 5' capping.

[0048] In some embodiments, the isolated RNA polynucleotide comprises at least one nucleoside modification. In some embodiments, the level of nucleoside modification can refer to the level of modification across the fully isolated polynucleotide or a portion thereof (e.g., the template region). In some embodiments, the template region is nucleoside-modified, and the percentage of modified nucleotides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less. In some embodiments, the 5' end of the reverse complement of the R region encodes a cap structure. In some embodiments, the 5' end of the R region is capped.

[0049] In some embodiments, the therapeutic polypeptide is a secreted polypeptide. In some embodiments, the therapeutic polypeptide is selected from the group consisting of interferon, interferon-stimulated gene, cytokine, chemokine, antibody, signaling molecule, cytotoxic protein, protein that causes cell death, anti-neoplastic protein, immunomodulatory protein, protein toll-like receptor agonist or dominant negative protein. In some embodiments, the cytokine is an inflammatory cytokine. In some embodiments, the inflammatory cytokine is TNF-α. In some embodiments, the cytokine is an anti-inflammatory cytokine. In some embodiments, the anti-inflammatory cytokine is interleukin-1 receptor antagonist (IL-1RN). In some embodiments, the therapeutic polypeptide is an interleukin or a caspase. In some embodiments, the interleukin is IL-12A, IL-12B or IL-2. In some embodiments, the secreted protein is an antibody.

[0050] In some embodiments, the therapeutic polypeptide is interferon. In some embodiments, the interferon is IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-γ or IFN-λ. In some embodiments, the interferon is IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β1, IFN-ε, IFN-κ, IFN-ω1, IFN-γ, IFN-λ1 (IL28A), IFN-λ2 (IL28B), IFN-λ3 (IL29) or IFN-λ4. In some embodiments, the interferon is IFN-α, IFN-β, IFN-κ, IFN-λ1 (IL28A), IFN-λ2 (IL28B) or IFN-λ3 (IL29).

[0051] In some embodiments, the coding sequence encodes two or more therapeutic polypeptides that can be separated by one or more ribosome skip sequences. In some embodiments, the coding region further comprises one or more regulatory elements selected from the group consisting of a ribosome binding site, a Kozak sequence, a Shine-Dalgarno sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site, and an internal ribosome entry site (IRES). In some embodiments, one or more regulatory elements are operably linked to the coding sequence. In some embodiments, the RNA polynucleotide further comprises a polyadenylation signal and / or a 3' poly(A) tail.

[0052] In some embodiments, the RNA-dependent polymerase is an RNA-dependent RNA polymerase. In some embodiments, the RNA-dependent polymerase is an RNA-dependent DNA polymerase. In some embodiments, the RNA-dependent polymerase is a virus-derived polymerase.

[0053] In some embodiments, the isolated RNA polynucleotide is single-stranded RNA. In some embodiments, the isolated polynucleotide is in a linear form. In some embodiments, the isolated polynucleotide is in a covalently closed circular form.

[0054] In some embodiments, the virus is a simian retrovirus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67 or a variant of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 129 or a variant of SEQ ID NO: 129. In some embodiments, the variant comprises mutations at one or more nucleotide positions selected from the group consisting of positions 66-67 or 1426-1493 of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 39, or 789. In some embodiments, the variant of SEQ ID NO: 129 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 129.

[0055] In some embodiments, the virus is a simian virus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77 or a variant of any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 68 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 68, (ii) the variant of SEQ ID NO: 69 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 69, (iii) the variant of SEQ ID NO: 70 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 70, (iv) the variant of SEQ ID NO: 71 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 71, (v) the variant of SEQ ID NO: 72 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 72, (vi) the variant of SEQ ID NO: 73 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 73, (vii) the variant of SEQ ID NO: 74 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1485-1552 of SEQ ID NO: 74, (viii) the variant of SEQ ID NO: 75 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1686-1753 of SEQ ID NO: 75, (ix) the variant of SEQ ID NO: 76 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1704-1771 of SEQ ID NO: 76, or (x) the variant of SEQ ID NO: 77 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1720-1787 of SEQ ID NO: 77.In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0056] In some embodiments, the virus is a simian virus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88 or a variant of any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 78 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1734 to 1801 of SEQ ID NO: 78, (ii) the variant of SEQ ID NO: 79 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1687 to 1754 of SEQ ID NO: 79, (iii) the variant of SEQ ID NO: 80 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1695 to 1762 of SEQ ID NO: 80, (iv) the variant of SEQ ID NO: 81 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 81, (v) the variant of SEQ ID NO: 82 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1443 to 1510 of SEQ ID NO: 82, (vi) the variant of SEQ ID NO: 83 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1459 to 1526 of SEQ ID NO: 83, (vii) the variant of SEQ ID NO: 85 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 85, (viii) the variant of SEQ ID NO: 86 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 86, (ix) the variant of SEQ ID NO: 87 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1435 to 1502 of SEQ ID NO: 87, or (x) the variant of SEQ ID NO: 88 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1463 to 1530 of SEQ ID NO: 88.In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0057] In some embodiments, the virus is a simian vesicular virus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108 or a variant of any one of SEQ ID NOs: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 89 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1466-1533 of SEQ ID NO: 89, (ii) the variant of SEQ ID NO: 90 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 90, (iii) the variant of SEQ ID NO: 91 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 91, (iv) the variant of SEQ ID NO: 92 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 92, (v) the variant of SEQ ID NO: 96 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-769 or 1471-1471 of SEQ ID NO: 96, (vi) the variant of SEQ ID NO: 104 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 104, (vii) the variant of SEQ ID NO: 105 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 105, (viii) the variant of SEQ ID NO: 106 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 106, (ix) the variant of SEQ ID NO: 107 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 107, or (x) the variant of SEQ ID NO: 108 contains mutations at one or more nucleotide positions selected from the group consisting of positions 89-839 or 1485-1552 of SEQ ID NO: 108.In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0058] In some embodiments, the virus is a simian vesicular virus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117 or 118 or a variant of any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117 or 118, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 130 or a variant of SEQ ID NO: 130.In some embodiments, (i) the variant of SEQ ID NO: 109 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1686 to 1753 of SEQ ID NO: 109, (ii) the variant of SEQ ID NO: 110 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1704 to 1771 of SEQ ID NO: 110, (iii) the variant of SEQ ID NO: 111 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1720 to 1787 of SEQ ID NO: 111, (iv) the variant of SEQ ID NO: 112 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1734 to 1801 of SEQ ID NO: 112, (v) the variant of SEQ ID NO: 113 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1687 to 1754 of SEQ ID NO: 113, (vi) the variant of SEQ ID NO: 114 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1695 to 1762 of SEQ ID NO: 114, (vii) the variant of SEQ ID NO: 115 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 115, (viii) the variant of SEQ ID NO: 116 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 116, (ix) the variant of SEQ ID NO: 117 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 117, or (x) the variant of SEQ ID NO: 118 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 118. In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0059] In some embodiments, the virus is a simian virus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126 or 127 or a variant of any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126 or 127, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 119 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1443-1510 of SEQ ID NO: 119, (ii) the variant of SEQ ID NO: 120 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1459-1526 of SEQ ID NO: 120, (iii) the variant of SEQ ID NO: 122 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 122, (iv) the variant of SEQ ID NO: 123 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 123, (v) the variant of SEQ ID NO: 124 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 124, (vi) the variant of SEQ ID NO: 125 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1463-1530 of SEQ ID NO: 125, (vii) the variant of SEQ ID NO: 126 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1466-1533 of SEQ ID NO: 126, (viii) the variant of SEQ ID NO: 127 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1425-1492 of SEQ ID NO: 127. In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130.

[0060] In some embodiments, the virus is a respiratory syncytial virus (RSV), the L region comprises a nucleotide sequence shown as any one of SEQ ID NOs: 148, 149, 150, 151 or 152 or a variant of any one of SEQ ID NOs: 148, 149, 150, 151 or 152, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 154 or 155. In some embodiments, (i) the variant of SEQ ID NO: 148 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 78 of SEQ ID NO: 148, (ii) the variant of SEQ ID NO: 149 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 33 of SEQ ID NO: 149, (iii) the variant of SEQ ID NO: 150 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 35 of SEQ ID NO: 150, (iv) the variant of SEQ ID NO: 151 contains mutations at one or more nucleotide positions selected from the group consisting of positions 18 to 36 of SEQ ID NO: 151, or (v) the variant of SEQ ID NO: 152 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 38 of SEQ ID NO: 152. In some embodiments, (i) the variant of SEQ ID NO: 154 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 207 of SEQ ID NO: 154, or (ii) the variant of SEQ ID NO: 155 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 32 of SEQ ID NO: 155.

[0061] In some embodiments, the virus includes a parainfluenza virus, where the L region includes the nucleotide sequence shown as SEQ ID NO: 180 or a variant of SEQ ID NO: 180, and where the R region includes the nucleotide sequence shown as SEQ ID NO: 179 or a variant of SEQ ID NO: 179. In some embodiments, the variant of SEQ ID NO: 180 includes a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 180. In some embodiments, the variant of SEQ ID NO: 179 includes a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 98 of SEQ ID NO: 179. In some embodiments, the virus is a parainfluenza virus, where the L region includes the nucleotide sequence shown as SEQ ID NO: 186 or a variant of SEQ ID NO: 186, and where the R region includes the nucleotide sequence shown as SEQ ID NO: 185 or a variant of SEQ ID NO: 185. In some embodiments, the variant of SEQ ID NO: 186 includes a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 186. In some embodiments, the variant of SEQ ID NO: 185 includes a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 93 of SEQ ID NO: 185.

[0062] In some embodiments, the virus is a metapneumovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 194 or a variant of SEQ ID NO: 194, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 192 or a variant of any one of SEQ ID NO: 192. In some embodiments, the variant of SEQ ID NO: 194 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 220 of SEQ ID NO: 194. In some embodiments, the variant of SEQ ID NO: 192 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 192. In some embodiments, the virus is a metapneumovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 193 or a variant of SEQ ID NO: 193, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 191 or a variant of SEQ ID NO: 191. In some embodiments, the variant of SEQ ID NO: 193 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 220 of SEQ ID NO: 193. In some embodiments, the variant of SEQ ID NO: 191 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 191.

[0063] In some embodiments, the virus is a henipavirus, where the L region comprises the nucleotide sequence shown as SEQ ID NO: 203 or a variant of SEQ ID NO: 203, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 202 or a variant of SEQ ID NO: 202. In some embodiments, the variant of SEQ ID NO: 203 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 203. In some embodiments, the variant of SEQ ID NO: 202 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 202. In some embodiments, the virus is a henipavirus, where the L region comprises the nucleotide sequence shown as SEQ ID NO: 207 or a variant of SEQ ID NO: 207, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 208 or a variant of SEQ ID NO: 208. In some embodiments, the variant of SEQ ID NO: 207 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 207. In some embodiments, the variant of SEQ ID NO: 208 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 208.

[0064] In some embodiments, the virus is a hepadnavirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 212, 213, 214, 215 or 216 or a variant of any one of SEQ ID NO: 212, 213, 214, 215 or 216, and the R region comprises a nucleotide sequence shown as any one of SEQ ID NO: 217, 218, 219 or 220 or a variant of any one of SEQ ID NO: 217, 218, 219 or 220. In some embodiments, (i) the variant of SEQ ID NO: 212 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1326 of SEQ ID NO: 212, (ii) the variant of SEQ ID NO: 213 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1291 of SEQ ID NO: 213, (iii) the variant of SEQ ID NO: 214 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1325 of SEQ ID NO: 214, (iv) the variant of SEQ ID NO: 215 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 15 of SEQ ID NO: 215, or (v) the variant of SEQ ID NO: 216 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 211 of SEQ ID NO: 216. In some embodiments, (i) the variant of SEQ ID NO: 217 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 754 of SEQ ID NO: 217, (ii) the variant of SEQ ID NO: 218 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 790 of SEQ ID NO: 218, (iii) the variant of SEQ ID NO: 219 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 892 of SEQ ID NO: 219, or (iv) the variant of SEQ ID NO: 220 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 2309 of SEQ ID NO: 220.

[0065] In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as either SEQ ID NO: 252 or SEQ ID NO: 253 or a variant described in either SEQ ID NO: 252 or SEQ ID NO: 253, and wherein the R region comprises the nucleotide sequence described in any one of SEQ ID NO: 254 or a variant of any one of SEQ ID NO: 254. In some embodiments, (i) the variant of SEQ ID NO: 252 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 223 of SEQ ID NO: 252, or (ii) the variant of SEQ ID NO: 253 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 415 of SEQ ID NO: 253. In some embodiments, (i) the variant of SEQ ID NO: 254 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 321 of SEQ ID NO: 254. In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as either SEQ ID NO: 258 or SEQ ID NO: 259 or a variant described in either SEQ ID NO: 258 or SEQ ID NO: 259, and wherein the R region comprises the nucleotide sequence described in any one of SEQ ID NO: 260 or a variant of any one of SEQ ID NO: 260. In some embodiments, (i) the variant of SEQ ID NO: 258 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 323 of SEQ ID NO: 258, or (ii) the variant of SEQ ID NO: 259 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 427 of SEQ ID NO: 259. In some embodiments, the variant of SEQ ID NO: 260 contains mutations at one or more nucleotide positions selected from the group consisting of positions 60 to 84 of SEQ ID NO: 260.

[0066] In some embodiments, the virus is an alphavirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 264 or 265 or a variant described in any one of SEQ ID NO: 264 or 265, and wherein the R region comprises a nucleotide sequence described in any one of SEQ ID NO: 266 or a variant of any one of SEQ ID NO: 266. In some embodiments, (i) the variant of SEQ ID NO: 264 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 216 of SEQ ID NO: 264, or (ii) the variant of SEQ ID NO: 265 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 429 of SEQ ID NO: 265. In some embodiments, the variant of SEQ ID NO: 266 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 262 of SEQ ID NO: 266.

[0067] Aspects of the present disclosure provide an isolated RNA polynucleotide comprising a coding region having a coding sequence encoding one or more polypeptides, and a template region comprising two different regions, namely the left flanking region of the virus (“L region”) and the right flanking region of the virus (“R region”), wherein the L region is adjacent and continuous with the 5' end of the coding region, and the R region is adjacent and continuous with the 3' end of the coding region, and wherein at least 30% of the uridine nucleotides are modified, at least 30% of the cytidine nucleotides are modified, and / or 1 to 30% of the adenosine nucleotides are modified, and wherein the template region interacts with and initiates the RNA-dependent polymerase activity of a polymerase in a cell containing the RNA-dependent polymerase.

[0068] In some embodiments, the coding sequence is in an antisense orientation. In some embodiments, the coding sequence is in a sense orientation.

[0069] In some embodiments, the polypeptide is a secreted protein. In some embodiments, the polypeptide is selected from the group consisting of a pharmaceutical, a therapeutic polypeptide, an antigen, and a reporter.

[0070] Also provided herein is any reverse complement of the isolated RNA polynucleotides described herein.

[0071] In some embodiments, the virus is selected from the group consisting of viruses of the orders Amarillovirales, Articulavirales, Blubervirales, Bunyavirales, Hepelivirales, Martellivirales, Mononegavirales, Nidovirales, and Picornavirales. In some embodiments, the virus is selected from the group consisting of viruses of the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, Rhabdoviridae, and Togaviridae.In some embodiments, the virus is selected from the group consisting of alpha coronavirus 229E, alpha coronavirus NL63, alpha coronavirus WA2028, turkey metapneumovirus (AMPV), beta coronavirus HKU1, beta coronavirus HKU15, beta coronavirus HKU33, beta coronavirus OC43, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, eastern equine encephalitis virus (EEEV), enterovirus D68 (EV-D68), foot-and-mouth disease virus, hantavirus, Hendra virus, hepatitis B virus, hepatitis C virus, HMPV, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), infectious salmon anemia virus, influenza A virus, influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, norovirus, rabies virus, respiratory syncytial virus, Reston Ebola virus, rhinovirus, Rift Valley fever virus, rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan equine encephalitis virus (VEEV), vesicular stomatitis virus, western equine encephalitis virus (WEEV), yellow fever virus, Zaïre Ebola virus, and Zika virus.

[0072] In some embodiments, the template region is native to the virus. In some embodiments, the template region is a variant of a template region native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the template region native to the virus. In some embodiments, each of the L region and the R region of the template region contains less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the template region native to the virus. In some embodiments, each of the L region and the R region of the template region varies from the template region native to the virus by 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer substitutions that do not participate in 5' capping. In some embodiments, each of the L region and the R region of the template region varies from the template region native to the virus by 1 or fewer substitutions that do not participate in 5' capping.

[0073] In some embodiments, the template region is nucleoside-modified, and the proportion of modified nucleosides is 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less. In some embodiments, the template region is nucleoside-modified, and the proportion of modified nucleosides is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 100%.

[0074] In some embodiments, the nucleoside modification is a non-immunogenic uridine modification, and the proportion of modified uridine modification exceeds 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100%. In some embodiments, the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of modified cytidine modification exceeds 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100%.

[0075] In some embodiments, the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of modified adenosine modification is about 1%, 5%, 10%, 15%, 20%, 25% or 30%.

[0076] In some embodiments, the isolated polynucleotide comprises a 5' cap structure. In some embodiments, the 5' end of the L region comprises a 5' cap structure. In some embodiments, the 5' end of the L region comprises one or more mutations associated with the 5' cap structure. In some embodiments, the 5' cap structure is selected from the group consisting of Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structures.

[0077] In some embodiments, the isolated polynucleotide does not comprise a 5' cap structure (uncapped). In some embodiments, the 5' end of the L region does not comprise a 5' cap structure (uncapped). In some embodiments, the 5' end of the isolated polynucleotide comprises a 5'-monophosphate, 5'-diphosphate or 5'-triphosphate. In some embodiments, the 5' end of the isolated polynucleotide does not comprise a 5'-phosphate (dephosphorylated).

[0078] In some embodiments, the template region is the reverse complement of the template region that is natural to the virus. In some embodiments, the template region is a variant of the reverse complement of the template region that is natural to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reverse complement of the template region that is natural to the virus. In some embodiments, each reverse complement of the L region and the R region varies from the reverse complement of the template region that is natural to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping. In some embodiments, each reverse complement of the L region and the R region varies from the reverse complement of the template region that is natural to the virus by 1 or fewer substitutions that do not participate in 5' capping. In some embodiments, the 5' end of the reverse complement of the R region encodes a 5' cap structure. In some embodiments, the 5' end of the R region is capped.

[0079] In some embodiments, the coding sequence encodes two or more polypeptides that can be separated by one or more ribosome skip sequences. In some embodiments, the coding region further comprises one or more regulatory elements selected from the group consisting of a ribosome binding site, a Kozak sequence, a Shine-Dalgarno sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site, and an internal ribosome entry site (IRES). In some embodiments, one or more regulatory elements are operably linked to the coding sequence. In some embodiments, the RNA polynucleotide further comprises a polyadenylation signal and / or a 3' poly(A) tail.

[0080] In some embodiments, the RNA-dependent polymerase is an RNA-dependent RNA polymerase. In some embodiments, the RNA-dependent polymerase is an RNA-dependent DNA polymerase. In some embodiments, the RNA-dependent polymerase is a virus-derived polymerase.

[0081] In some embodiments, the isolated RNA polynucleotide is single-stranded RNA. In some embodiments, the isolated polynucleotide is in a linear form. In some embodiments, the isolated polynucleotide is in a covalently closed circular form.

[0082] In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 2 or a variant of SEQ ID NO: 2, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 20, 21, 22, or 23 or a variant of any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the variant of SEQ ID NO: 2 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 26 of SEQ ID NO: 2. In some embodiments, the variant comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 15 of any one of SEQ ID NO: 20, 21, 22, or 23. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 3 or a variant of SEQ ID NO: 3, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 24, 25, 26, or 27. In some embodiments, (i) the variant of SEQ ID NO: 3 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 35 of SEQ ID NO: 3. In some embodiments, (i) the variant of SEQ ID NO: 24 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 24, (ii) the variant of SEQ ID NO: 25 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 25, (iii) the variant of SEQ ID NO: 26 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 26, or (iv) the variant of SEQ ID NO: 27 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 27. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 4 or a variant of SEQ ID NO: 4, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 28, 29, 30, or 31 or a variant of any one of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31.In some embodiments, the variant of SEQ ID NO: 4 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 50 of SEQ ID NO: 4. In some embodiments, any one variant of SEQ ID NO: 28, 29, 30, or 31 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of any one of SEQ ID NO: 28, 29, 30, or 31.

[0083] In some embodiments, the virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 1 or 5, or a variant of SEQ ID NO: 1 or 5, and here, the R region contains the nucleotide sequence shown as SEQ ID NO: 18 or 19, or a variant of SEQ ID NO: 18 or 19. In some embodiments, the variant of SEQ ID NO: 1 or 5 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 37 of SEQ ID NO: 1 or 5. In some embodiments, the variant of SEQ ID NO: 18 or 19 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 20 of SEQ ID NO: 18 or 19. In some embodiments, the virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 6 or a variant of SEQ ID NO: 6, and here, the R region contains the nucleotide sequence shown as SEQ ID NO: 32 or 33, or a variant of SEQ ID NO: 32 or 33. In some embodiments, the variant of SEQ ID NO: 6 contains mutations at one or more nucleotide positions selected from positions 14 or 15 of SEQ ID NO: 6. In some embodiments, the variant of SEQ ID NO: 32 or 33 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 33 of SEQ ID NO: 32 or 33. In some embodiments, the virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 7 or a variant of SEQ ID NO: 7, and here, the R region contains the nucleotide sequence shown as any one of SEQ ID NO: 34, 35, 36, or 37, or a variant of any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, the variant of SEQ ID NO: 7 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 20 of SEQ ID NO: 7. In some embodiments, it is a variant of any one of SEQ ID NO: 34, 35, 36, or 37, wherein the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 5 to 8 of SEQ ID NO: 34, 35, 36, or 37.In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 8 or a variant of SEQ ID NO: 8, and here, the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 38, 39, 40, or 41 or a variant of any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 41. In some embodiments, the variant of SEQ ID NO: 8 contains mutations at one or more nucleotide positions selected from positions 14 or 15 of SEQ ID NO: 8. In some embodiments, the variant of any one of SEQ ID NO: 38, 39, 40, or 41 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 13 of SEQ ID NO: 38, 39, 40, or 41. In some embodiments, the virus is an influenza virus, the L region is the nucleotide sequence shown as SEQ ID NO: 9 or a variant of SEQ ID NO: 9, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 42 or 43 or a variant of SEQ ID NO: 42 or 43. In some embodiments, the variant of SEQ ID NO: 9 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 18 of SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 42 or 43 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 14 of SEQ ID NO: 42 or 43. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 11 or a variant of SEQ ID NO: 11, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 46 or 47 or a variant of SEQ ID NO: 46 or 47. In some embodiments, the variant of SEQ ID NO: 11 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 81 of SEQ ID NO: 11. In some embodiments, the variant of SEQ ID NO: 46 or 47t contains mutations at one or more nucleotide positions selected from the group consisting of positions 5 to 9 of SEQ ID NO: 46 or 47.In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 12 or a variant of SEQ ID NO: 12, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 48 or 49, or is a variant of either SEQ ID NO: 48 or 49. In some embodiments, the variant of SEQ ID NO: 12 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 52 of SEQ ID NO: 12. In some embodiments, the variant of either SEQ ID NO: 48 or 49 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 11 of SEQ ID NO: 48 or 49. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 13 or a variant of SEQ ID NO: 13, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 50 or 51 or a variant of SEQ ID NO: 50 or 51. In some embodiments, the variant of SEQ ID NO: 13 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 87 of SEQ ID NO: 13. In some embodiments, the variant of SEQ ID NO: 50 or 51 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 17 of SEQ ID NO: 50 or 51. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 10 or a variant of SEQ ID NO: 10, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 44 or 45 or a variant of SEQ ID NO: 44 or 45. In some embodiments, the variant of SEQ ID NO: 10 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 86 of SEQ ID NO: 10. In some embodiments, the variant of SEQ ID NO: 44 or 45 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 21 of SEQ ID NO: 44 or 45.In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 14 or a variant of SEQ ID NO: 14, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 52 or 53, or is a variant of either SEQ ID NO: 52 or 53. In some embodiments, the variant of SEQ ID NO: 14 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 93 of SEQ ID NO: 14. In some embodiments, the variant of either SEQ ID NO: 52 or 53 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 48 of SEQ ID NO: 52 or 53. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 15 or a variant of SEQ ID NO: 15, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 54 or 55, or a variant of either SEQ ID NO: 54 or 55. In some embodiments, the variant of SEQ ID NO: 15 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 95 of SEQ ID NO: 15. In some embodiments, the variant of either SEQ ID NO: 54 or 55 contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 34 of SEQ ID NO: 54 or 55.

[0084] In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 16 or a variant of SEQ ID NO: 16, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 56 or 57, or is a variant of either SEQ ID NO: 56 or 57. In some embodiments, the variant of SEQ ID NO: 16 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 81 of SEQ ID NO: 16. In some embodiments, the variant of either SEQ ID NO: 56 or 57 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 56 or 57. In some embodiments, the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 17 or a variant of SEQ ID NO: 17, and here, the R region comprises the nucleotide sequence shown as either SEQ ID NO: 58 or 59 or a variant of either SEQ ID NO: 58 or 59. In some embodiments, the variant of SEQ ID NO: 17 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 22 of SEQ ID NO: 17. In some embodiments, the variant of either SEQ ID NO: 58 or 59 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 32 of SEQ ID NO: 58 or 59.

[0085] In some embodiments, the virus is a simian becovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 137 or a variant of SEQ ID NO: 137, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 128 or any one variant of SEQ ID NO: 128. In some embodiments, the variant of SEQ ID NO: 137 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 40 to 1557 of SEQ ID NO: 137. In some embodiments, any one variant of SEQ ID NO: 128 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 30 of SEQ ID NO: 128. In some embodiments, the virus is a simian becovirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 138, 139, 140, 141, 142, 143 or 144 or a variant of any one of SEQ ID NO: 138, 139, 140, 141, 142, 143 or 144, and the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 130, 136, 145, 146 or 147 or a variant of any one of SEQ ID NO: 130, 136, 145, 146 or 147.In some embodiments, (i) the variant of SEQ ID NO: 138 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 312 of SEQ ID NO: 138, (ii) the variant of SEQ ID NO: 139 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1567 of SEQ ID NO: 139, (iii) the variant of SEQ ID NO: 140 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1488 of SEQ ID NO: 140, (iv) the variant of SEQ ID NO: 141 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1593 of SEQ ID NO: 141, (v) the variant of SEQ ID NO: 142 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1570 of SEQ ID NO: 142, (vi) the variant of SEQ ID NO: 143 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1488 of SEQ ID NO: 143, and (vii) the variant of SEQ ID NO: 144 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1593 of SEQ ID NO: 144. In some embodiments, (i) the variant of any one of SEQ ID NOs: 130, 136, 145, 146 or 147 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130, (ii) the variant of SEQ ID NO: 136 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 33 of SEQ ID NO: 136, (iii) the variant of SEQ ID NO: 145 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1461 of SEQ ID NO: 145, (iv) the variant of SEQ ID NO: 146 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 1441 of SEQ ID NO: 146, or (v) the variant of SEQ ID NO: 147 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 897 of SEQ ID NO: 147.

[0086] In some embodiments, the virus is respiratory syncytial virus (RSV), the L region comprises a nucleotide sequence represented as any one of SEQ ID NO: 158, 163, 165, 166 or 419 or a variant of any one of SEQ ID NO: 158, 163, 165, 166 or 419, and the R region comprises a nucleotide sequence represented as any one of SEQ ID NO: 169, 170, 176, 177 or 420 or a variant of any one of SEQ ID NO: 169, 170, 176, 177 or 420. In some embodiments, (i) the variant of SEQ ID NO: 158 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 207 of SEQ ID NO: 158, (ii) the variant of SEQ ID NO: 163 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 18 to 210 of SEQ ID NO: 163, (iii) the variant of SEQ ID NO: 165 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 147 of SEQ ID NO: 165, (iv) the variant of SEQ ID NO: 166 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 32 of SEQ ID NO: 166, or (v) the variant of SEQ ID NO: 419 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 18 to 35 of SEQ ID NO: 419. In some embodiments, (i) the variant of SEQ ID NO: 169 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 78 of SEQ ID NO: 169, (ii) the variant of SEQ ID NO: 170 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 80 of SEQ ID NO: 170, (iii) the variant of SEQ ID NO: 176 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 36 of SEQ ID NO: 176, (iv) the variant of SEQ ID NO: 177 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 33 of SEQ ID NO: 177, or (v) the variant of SEQ ID NO: 420 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 35 of SEQ ID NO: 420.

[0087] In some embodiments, the virus is a parainfluenza virus, where the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 181, 182, or 183 or a variant of any one of SEQ ID NO: 181, 182, or 183, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 184 or a variant of SEQ ID NO: 184. In some embodiments, (i) the variant of SEQ ID NO: 181 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 181, (ii) the variant of SEQ ID NO: 182 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 142 of SEQ ID NO: 182, or (iii) the variant of SEQ ID NO: 183 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 183. In some embodiments, the variant of SEQ ID NO: 184 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 98 of SEQ ID NO: 184. In some embodiments, the virus is a parainfluenza virus, where the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 187, 188, or 189 or a variant of any one of SEQ ID NO: 187, 188, or 189, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 190 or a variant of SEQ ID NO: 190. In some embodiments, (i) the variant of SEQ ID NO: 187 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 187, (ii) the variant of SEQ ID NO: 188 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 101 of SEQ ID NO: 188, or (iii) the variant of SEQ ID NO: 189 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 189. In some embodiments, the variant of SEQ ID NO: 190 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 93 of SEQ ID NO: 190.

[0088] In some embodiments, the virus is a metapneumovirus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NO: 196, 197 or 199 or a variant of any one of SEQ ID NO: 196, 197 or 199, and the R region comprises a nucleotide sequence represented as SEQ ID NO: 201 or a variant of SEQ ID NO: 201. In some embodiments, (i) the variant of SEQ ID NO: 196 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 224 of SEQ ID NO: 196, (ii) the variant of SEQ ID NO: 197 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 230 of SEQ ID NO: 197, or (iii) the variant of SEQ ID NO: 199 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 140 of SEQ ID NO: 199. In some embodiments, the variant of SEQ ID NO: 201 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 201. In some embodiments, the virus is a metapneumovirus, the L region comprises the nucleotide sequence represented as SEQ ID NO: 195 or a variant of SEQ ID NO: 195, wherein the R region comprises the nucleotide sequence represented as SEQ ID NO: 200 or a variant of SEQ ID NO: 200. In some embodiments, the variant of SEQ ID NO: 195 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 224 of SEQ ID NO: 195. In some embodiments, the variant of SEQ ID NO: 200 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 200.

[0089] In some embodiments, the virus is a henipavirus, where the L region is the nucleotide sequence shown as SEQ ID NO: 204 or a variant of SEQ ID NO: 204, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 206 or a variant of SEQ ID NO: 206. In some embodiments, the variant of SEQ ID NO: 204 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 204. In some embodiments, the variant of SEQ ID NO: 206 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 206. In some embodiments, the virus is a henipavirus, where the L region is the nucleotide sequence shown as SEQ ID NO: 209 or 210 or a variant of SEQ ID NO: 209 or 210, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 211 or a variant of SEQ ID NO: 211. In some embodiments, (i) the variant of SEQ ID NO: 209 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 209, or (ii) the variant of SEQ ID NO: 210 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 83 of SEQ ID NO: 210. In some embodiments, the variant of SEQ ID NO: 211 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 211.

[0090] In some embodiments, the virus is a hepadnavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 222 or 223 or a variant of SEQ ID NO: 222 or 223, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 225 or a variant of SEQ ID NO: 225. In some embodiments, (i) the variant of SEQ ID NO: 222 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 639 of SEQ ID NO: 222, or (ii) the variant of SEQ ID NO: 223 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 186 of SEQ ID NO: 223. In some embodiments, the variant of SEQ ID NO: 225 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 1023 of SEQ ID NO: 225.

[0091] In some embodiments, the virus is a filovirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 227, 228, 229 or 230 or a variant of any one of SEQ ID NO: 227, 228, 229 or 230, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 231 or a variant of SEQ ID NO: 231. In some embodiments, (i) the variant of SEQ ID NO: 227 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 710 of SEQ ID NO: 227, (ii) the variant of SEQ ID NO: 228 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 713 of SEQ ID NO: 228, (iii) the variant of SEQ ID NO: 229 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 707 of SEQ ID NO: 229, or (iv) the variant of SEQ ID NO: 230 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 707 of SEQ ID NO: 230. In some embodiments, the variant of SEQ ID NO: 231 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 449 of SEQ ID NO: 231. In some embodiments, the virus is a filovirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 232, 233, 234 or 235 or a variant of any one of SEQ ID NO: 232, 233, 234 or 235, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 236 or a variant of SEQ ID NO: 236. In some embodiments, (i) the variant of SEQ ID NO: 232 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 678 of SEQ ID NO: 232, (ii) the variant of SEQ ID NO: 233 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 681 of SEQ ID NO: 233, (iii) the variant of SEQ ID NO: 234 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 678 of SEQ ID NO: 234, or (iv) the variant of SEQ ID NO: 235 contains mutations at one or more nucleotide positions selected from the group consisting of positions 23 to 678 of SEQ ID NO: 235.In some embodiments, the variant of SEQ ID NO: 236 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 437 of SEQ ID NO: 236.

[0092] In some embodiments, the virus is a filovirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 237, 238 or 239 or a variant of any one of SEQ ID NO: 237, 238 or 239, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 240 or a variant of SEQ ID NO: 240. In some embodiments, (i) the variant of SEQ ID NO: 237 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 605 of SEQ ID NO: 237, (ii) the variant of SEQ ID NO: 238 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 606 of SEQ ID NO: 238, or (iii) the variant of SEQ ID NO: 239 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 605 of SEQ ID NO: 239. In some embodiments, the variant of SEQ ID NO: 240 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 83 of SEQ ID NO: 240. In some embodiments, the virus is a filovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 241 or a variant of SEQ ID NO: 241, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 242 or a variant of any one of SEQ ID NO: 242. In some embodiments, the variant of SEQ ID NO: 241 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 34 of SEQ ID NO: 241. In some embodiments, the variant of SEQ ID NO: 242 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 593 of SEQ ID NO: 242. In some embodiments, the virus is a filovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 243 or a variant of SEQ ID NO: 243, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 244 or a variant of SEQ ID NO: 244. In some embodiments, the variant of SEQ ID NO: 243 contains mutations at one or more nucleotide positions selected from the group consisting of positions 30 to 45 of SEQ ID NO: 243.In some embodiments, the variant of SEQ ID NO: 244 contains mutations at one or more nucleotide positions selected from the group consisting of positions 100 to 677 of SEQ ID NO: 244. In some embodiments, the virus is a filovirus, and the L region contains the nucleotide sequence shown as any one of SEQ ID NO: 245, 246, or 247 or a variant of any one of SEQ ID NO: 245, 246, or 247, and the R region contains the nucleotide sequence shown as SEQ ID NO: 248 or a variant of SEQ ID NO: 248. In some embodiments, (i) the variant of SEQ ID NO: 245 contains mutations at one or more nucleotide positions selected from the group consisting of positions 29 to 171 of SEQ ID NO: 245, (ii) the variant of SEQ ID NO: 246 contains mutations at one or more nucleotide positions selected from the group consisting of positions 30 to 171 of SEQ ID NO: 246, (iii) the variant of SEQ ID NO: 247 contains mutations at one or more nucleotide positions selected from the group consisting of positions 29 to 171 of SEQ ID NO: 247. In some embodiments, the variant of SEQ ID NO: 248 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 91 of SEQ ID NO: 248.

[0093] In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 249 or a variant of SEQ ID NO: 249, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 250 or 251 or a variant of SEQ ID NO: 250 or 251. In some embodiments, the variant of SEQ ID NO: 249 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 274 of SEQ ID NO: 249. In some embodiments, (i) the variant of SEQ ID NO: 250 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 183 of SEQ ID NO: 250, or (ii) the variant of SEQ ID NO: 251 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 375 of SEQ ID NO: 251. In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 255 or a variant of SEQ ID NO: 255, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 256 or 257 or a variant of SEQ ID NO: 256 or 257. In some embodiments, the variant of SEQ ID NO: 255 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 35 of SEQ ID NO: 255. In some embodiments, (i) the variant of SEQ ID NO: 256 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 600 to 273 of SEQ ID NO: 256, or (ii) the variant of SEQ ID NO: 257 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 377 of SEQ ID NO: 257. In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 261 or a variant of SEQ ID NO: 261, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 262 or 263 or a variant of SEQ ID NO: 262 or 263. In some embodiments, the variant of SEQ ID NO: 261 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 215 of SEQ ID NO: 261.In some embodiments, (i) the variant of SEQ ID NO: 262 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 166 of SEQ ID NO: 262, or (ii) the variant of SEQ ID NO: 263 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 379 of SEQ ID NO: 263.

[0094] In some embodiments, the virus is a simian virus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77 or a variant of any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 68 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 68, (ii) the variant of SEQ ID NO: 69 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 69, (iii) the variant of SEQ ID NO: 70 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 70, (iv) the variant of SEQ ID NO: 71 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 71, (v) the variant of SEQ ID NO: 72 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 72, (vi) the variant of SEQ ID NO: 73 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 73, (vii) the variant of SEQ ID NO: 74 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1485-1552 of SEQ ID NO: 74, (viii) the variant of SEQ ID NO: 75 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1686-1753 of SEQ ID NO: 75, (ix) the variant of SEQ ID NO: 76 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1704-1771 of SEQ ID NO: 76, or (x) the variant of SEQ ID NO: 77 contains mutations at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1720-1787 of SEQ ID NO: 77.In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0095] In some embodiments, the virus is a simian retrovirus, the L region comprises a nucleotide sequence represented by any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88 or a variant of any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88, and the R region comprises the nucleotide sequence represented by SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 78 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1734-1801 of SEQ ID NO: 78, (ii) the variant of SEQ ID NO: 79 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1687-1754 of SEQ ID NO: 79, (iii) the variant of SEQ ID NO: 80 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1695-1762 of SEQ ID NO: 80, (iv) the variant of SEQ ID NO: 81 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 81, (v) the variant of SEQ ID NO: 82 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1443-1510 of SEQ ID NO: 82, (vi) the variant of SEQ ID NO: 83 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1459-1526 of SEQ ID NO: 83, (vii) the variant of SEQ ID NO: 85 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 85, (viii) the variant of SEQ ID NO: 86 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 86, (ix) the variant of SEQ ID NO: 87 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1435-1502 of SEQ ID NO: 87, or (x) the variant of SEQ ID NO: 88 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1463-1530 of SEQ ID NO: 88.In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0096] In some embodiments, the virus is a simian virus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108 or a variant of any one of SEQ ID NO: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 89 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1466-1533 of SEQ ID NO: 89, (ii) the variant of SEQ ID NO: 90 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 90, (iii) the variant of SEQ ID NO: 91 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 91, (iv) the variant of SEQ ID NO: 92 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 92, (v) the variant of SEQ ID NO: 96 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-769 or 1471-1471 of SEQ ID NO: 96, (vi) the variant of SEQ ID NO: 104 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 104, (vii) the variant of SEQ ID NO: 105 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 105, (viii) the variant of SEQ ID NO: 106 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 106, (ix) the variant of SEQ ID NO: 107 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 107, or (x) the variant of SEQ ID NO: 108 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 89-839 or 1485-1552 of SEQ ID NO: 108.In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0097] In some embodiments, the virus is a simian vesicular virus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117 or 118 or a variant of any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117 or 118, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130.In some embodiments, (i) the variant of SEQ ID NO: 109 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1686 to 1753 of SEQ ID NO: 109, (ii) the variant of SEQ ID NO: 110 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1704 to 1771 of SEQ ID NO: 110, (iii) the variant of SEQ ID NO: 111 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1720 to 1787 of SEQ ID NO: 111, (iv) the variant of SEQ ID NO: 112 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1734 to 1801 of SEQ ID NO: 112, (v) the variant of SEQ ID NO: 113 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1687 to 1754 of SEQ ID NO: 113, (vi) the variant of SEQ ID NO: 114 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1695 to 1762 of SEQ ID NO: 114, (vii) the variant of SEQ ID NO: 115 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 115, (viii) the variant of SEQ ID NO: 116 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 116, (ix) the variant of SEQ ID NO: 117 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 117, or (x) the variant of SEQ ID NO: 118 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 118. In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130.

[0098] In some embodiments, the virus is a simian virus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126 or 127 or a variant of any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126 or 127, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130. In some embodiments, (i) the variant of SEQ ID NO: 119 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1443-1510 of SEQ ID NO: 119, (ii) the variant of SEQ ID NO: 120 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1459-1526 of SEQ ID NO: 120, (iii) the variant of SEQ ID NO: 122 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 122, (iv) the variant of SEQ ID NO: 123 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 123, (v) the variant of SEQ ID NO: 124 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 124, (vi) the variant of SEQ ID NO: 125 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1463-1530 of SEQ ID NO: 125, (vii) the variant of SEQ ID NO: 126 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1466-1533 of SEQ ID NO: 126, (viii) the variant of SEQ ID NO: 127 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1425-1492 of SEQ ID NO: 127. In some embodiments, the variant of SEQ ID NO: 130 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130.

[0099] In some embodiments, the virus is respiratory syncytial virus (RSV), the L region comprises a nucleotide sequence shown as any one of SEQ ID NOs: 148, 149, 150, 151 or 152 or a variant of any one of SEQ ID NOs: 148, 149, 150, 151 or 152, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 154 or 155. In some embodiments, (i) the variant of SEQ ID NO: 148 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 78 of SEQ ID NO: 148, (ii) the variant of SEQ ID NO: 149 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 33 of SEQ ID NO: 149, (iii) the variant of SEQ ID NO: 150 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 35 of SEQ ID NO: 150, (iv) the variant of SEQ ID NO: 151 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 18 to 36 of SEQ ID NO: 151, or (v) the variant of SEQ ID NO: 152 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to ¾ of SEQ ID NO: 152. In some embodiments, (i) the variant of SEQ ID NO: 154 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 207 of SEQ ID NO: 154, or (ii) the variant of SEQ ID NO: 155 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 32 of SEQ ID NO: 155.

[0100] In some embodiments, the virus includes a parainfluenza virus, where the L region includes the nucleotide sequence shown as SEQ ID NO: 180 or a variant of SEQ ID NO: 180, and where the R region includes the nucleotide sequence shown as SEQ ID NO: 179 or a variant of SEQ ID NO: 179. In some embodiments, the variant of SEQ ID NO: 180 includes mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 180. In some embodiments, the variant of SEQ ID NO: 179 includes mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 98 of SEQ ID NO: 179. In some embodiments, the virus is a parainfluenza virus, where the L region includes the nucleotide sequence shown as SEQ ID NO: 186 or a variant of SEQ ID NO: 186, and where the R region includes the nucleotide sequence shown as SEQ ID NO: 185 or a variant of SEQ ID NO: 185. In some embodiments, the variant of SEQ ID NO: 186 includes mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 186. In some embodiments, the variant of SEQ ID NO: 185 includes mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 93 of SEQ ID NO: 185.

[0101] In some embodiments, the virus is a metapneumovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 194 or a variant of SEQ ID NO: 194, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 192 or any one variant of SEQ ID NO: 192. In some embodiments, the variant of SEQ ID NO: 194 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 220 of SEQ ID NO: 194. In some embodiments, the variant of SEQ ID NO: 192 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 192. In some embodiments, the virus is a metapneumovirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 193 or a variant of SEQ ID NO: 193, and here, the R region comprises the nucleotide sequence shown as SEQ ID NO: 191 or a variant of SEQ ID NO: 191. In some embodiments, the variant of SEQ ID NO: 193 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 220 of SEQ ID NO: 193. In some embodiments, the variant of SEQ ID NO: 191 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 191.

[0102] In some embodiments, the virus is a henipavirus, where the L region comprises the nucleotide sequence shown as SEQ ID NO: 203 or a variant of SEQ ID NO: 203, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 202 or a variant of SEQ ID NO: 202. In some embodiments, the variant of SEQ ID NO: 203 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 203. In some embodiments, the variant of SEQ ID NO: 202 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 202. In some embodiments, the virus is a henipavirus, where the L region comprises the nucleotide sequence shown as SEQ ID NO: 207 or a variant of SEQ ID NO: 207, and where the R region comprises the nucleotide sequence shown as SEQ ID NO: 208 or a variant of SEQ ID NO: 208. In some embodiments, the variant of SEQ ID NO: 207 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 207. In some embodiments, the variant of SEQ ID NO: 208 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 208.

[0103] In some embodiments, the virus is a hepadnavirus, the L region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 212, 213, 214, 215 or 216 or a variant of any one of SEQ ID NOs: 212, 213, 214, 215 or 216, and the R region comprises a nucleotide sequence represented as any one of SEQ ID NOs: 217, 218, 219 or 220 or a variant of any one of SEQ ID NOs: 217, 218, 219 or 220. In some embodiments, (i) the variant of SEQ ID NO: 212 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 1326 of SEQ ID NO: 212, (ii) the variant of SEQ ID NO: 213 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 1291 of SEQ ID NO: 213, (iii) the variant of SEQ ID NO: 214 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 1325 of SEQ ID NO: 214, (iv) the variant of SEQ ID NO: 215 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 15 of SEQ ID NO: 215, or (v) the variant of SEQ ID NO: 216 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 211 of SEQ ID NO: 216. In some embodiments, (i) the variant of SEQ ID NO: 217 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 754 of SEQ ID NO: 217, (ii) the variant of SEQ ID NO: 218 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 790 of SEQ ID NO: 218, (iii) the variant of SEQ ID NO: 219 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 892 of SEQ ID NO: 219, or (iv) the variant of SEQ ID NO: 220 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 2309 of SEQ ID NO: 220.

[0104] In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 252 or 253 or a variant described in any one of SEQ ID NO: 252 or 253, and wherein the R region comprises the nucleotide sequence described in any one of SEQ ID NO: 254 or a variant of any one of SEQ ID NO: 254. In some embodiments, (i) the variant of SEQ ID NO: 252 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 223 of SEQ ID NO: 252, or (ii) the variant of SEQ ID NO: 253 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 415 of SEQ ID NO: 253. In some embodiments, (i) the variant of SEQ ID NO: 254 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 321 of SEQ ID NO: 254. In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 258 or 259 or a variant described in any one of SEQ ID NO: 258 or 259, and wherein the R region comprises the nucleotide sequence described in any one of SEQ ID NO: 260 or a variant of any one of SEQ ID NO: 260. In some embodiments, (i) the variant of SEQ ID NO: 258 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 323 of SEQ ID NO: 258, or (ii) the variant of SEQ ID NO: 259 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 427 of SEQ ID NO: 259. In some embodiments, the variant of SEQ ID NO: 260 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 84 of SEQ ID NO: 260.

[0105] In some embodiments, the virus is an alphavirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NO: 264 or 265 or a variant described in any one of SEQ ID NO: 264 or 265, and wherein the R region comprises the nucleotide sequence described in any one of SEQ ID NO: 266 or a variant of any one of SEQ ID NO: 266. In some embodiments, (i) the variant of SEQ ID NO: 264 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 216 of SEQ ID NO: 264, or (ii) the variant of SEQ ID NO: 265 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 429 of SEQ ID NO: 265. In some embodiments, the variant of SEQ ID NO: 266 comprises a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 262 of SEQ ID NO: 266.

[0106] Aspects of the present disclosure provide an isolated DNA polynucleotide encoding any of the isolated RNA polynucleotides described herein.

[0107] Other aspects provide a cell or cell line comprising any of the isolated DNA polynucleotides described herein.

[0108] Other aspects provide a vector comprising any of the isolated RNA polynucleotides or isolated DNA polynucleotides described herein. In some embodiments, the vector is a viral vector or an expression vector. In some embodiments, the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentivirus vector, a herpesvirus vector, an alphavirus vector, and a baculovirus vector.

[0109] Another aspect of the disclosure provides an RNA-protein complex comprising any of the isolated RNA polynucleotides described herein and an RNA-binding protein, wherein the isolated RNA polynucleotide of the RNA-protein complex has increased stability compared to an isolated RNA polypeptide that does not contain the RNA-binding protein. In some embodiments, the RNA-binding protein is a viral capsid protein (N) or a viral capsid protein. In some embodiments, the RNA-binding protein is a viral capsid protein (N) or a viral capsid protein of a virus. In some embodiments, the viral nucleocapsid protein or a viral capsid protein derived from influenza virus, Sendai virus, pneumovirus, paramyxovirus, henipavirus or hepadnavirus.

[0110] Another aspect of the disclosure provides a composition comprising any of the isolated RNA polynucleotides, isolated DNA polynucleotides, cells or cell lines, vectors, or RNA-protein complexes described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0111] Yet another aspect provides nanoparticles comprising any of the isolated RNA polynucleotides, isolated DNA polynucleotides, or RNA-protein complexes described herein.

[0112] Aspects of the present disclosure provide a method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the isolated RNA polynucleotides, isolated DNA polynucleotides, cells or cell lines, vectors, RNA-protein complexes, compositions or nanoparticles described herein. In some embodiments, the method further comprises administering to a subject in need thereof a therapeutically effective amount of any one of a second isolated RNA polynucleotide, a second isolated DNA polynucleotide, a second cell or cell line, a second vector, a second RNA-protein complex, a second composition, or a second nanoparticle, wherein the second entity is different from the first entity (e.g., the second isolated RNA polynucleotide is different from the first isolated RNA polynucleotide administered to the subject). In some embodiments, the subject is a human, bovine, porcine, ovine, equine, deer, rodent, fish or poultry.

[0113] In some embodiments, the subject has a disease or disorder resulting from a viral infection. In some embodiments, the subject has an infection by a virus.

[0114] In some embodiments, the administration is by parenteral delivery including intratracheal or inhalation, intranasal, oral, rectal, vaginal, transmucosal, or enteral administration; intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous or intraperitoneal administration.

[0115] Various aspects of the present disclosure provide a method comprising contacting a cell with any one of the isolated RNA polynucleotides, isolated DNA polynucleotides, cells or cell lines, vectors, RNA-protein complexes, compositions or nanoparticles described herein. In some embodiments, the contacting step is in vitro or ex vivo.

[0116] Aspects of the present disclosure provide a method comprising administering to a subject in need thereof (i) a therapeutically effective amount of any one of the isolated RNA polynucleotides, isolated DNA polynucleotides, cells or cell lines, vectors, RNA-protein complexes, compositions or nanoparticles described herein and (ii) a therapeutic polypeptide or a second polynucleotide encoding a polymerase that interacts with the therapeutic polypeptide or polypeptide and is capable of initiating transcription or translation of the therapeutic polypeptide or polypeptide. In some embodiments, the method further comprises administering to the subject (iii) one or more accessory proteins associated with polymerase activity. In some embodiments, the accessory protein is a nucleocapsid protein. In some embodiments, the polymerase and / or accessory protein is administered in the form of one or more nucleic acids encoding the polymerase and / or accessory protein. In some embodiments, (i) and (ii) are administered sequentially or simultaneously. In some embodiments, (i) and (ii) are present on the same polynucleotide. In some embodiments, (i) and (ii) are present on separate polynucleotides.

[0117] In some embodiments, the method further comprises administering to a subject in need thereof a therapeutically effective amount of any one of the isolated RNA polynucleotides, isolated DNA polynucleotides, cells or cell lines, vectors, RNA-protein complexes, compositions or nanoparticles described herein. In some embodiments, the subject is a human, bovine, porcine, ovine, equine, deer, rodent, fish or poultry.

[0118] Other aspects of the disclosure provide a method comprising: (a) providing a DNA vector encoding any of the isolated RNA polynucleotides described herein; (b) linearizing the DNA vector to produce a linear DNA vector; and (c) contacting the linear DNA vector with an RNA polymerase to thereby produce an isolated RNA polynucleotide. In some embodiments, the method further comprises (d) subjecting the isolated RNA polynucleotide produced in (c) to one or more purification steps. In some embodiments, the one or more purification steps in (d) comprise contacting the isolated RNA polynucleotide with a DNAse under conditions suitable for digestion of the DNA vector, and are selected from tangential flow filtration. In some embodiments, the DNA vector comprises a promoter capable of directing the activity of an RNA polymerase and / or restriction endonuclease recognition site. In some embodiments, the RNA polymerase is T7 RNA polymerase and the promoter is a T7 promoter. In some embodiments, linearizing the DNA vector comprises contacting the DNA vector with a restriction endonuclease that recognizes a restriction endonuclease recognition site. In some embodiments, the contacting in (c) is performed at about 50 °C. In some embodiments, the contacting in (c) is performed in the presence of one or more additional factors selected from the group consisting of ribonucleotide triphosphates, modified nucleotide triphosphates, cap analogs, inorganic pyrophosphatase, and RNase inhibitors. In some embodiments, the method further comprises formulating the isolated RNA polynucleotide into nanoparticles.

[0119] Other aspects of the disclosure provide a method of making a transgenic animal or plant, comprising inserting any of an isolated RNA polynucleotide, an isolated DNA polynucleotide, a cell or cell line, a vector, an RNA-protein complex or composition, or a nanoparticle into an animal or plant, thereby producing a transgenic animal or plant. In some embodiments, the coding sequence encodes an antiviral polypeptide. In some embodiments, the transgenic animal or plant has increased resistance to viral infection. In some embodiments, the transgenic animal or plant is a bird, pig, fish, cow, horse, camel, dog, cat, mouse, rat, guinea pig, hamster, ferret, primate, or other commercially valuable animal or plant species.

[0120] In one aspect, the disclosure provides an isolated ribonucleic acid (RNA) polynucleotide comprising (a) a coding region encoding a therapeutic polypeptide of interest, and (b) a template region for binding to a target-specific translation activator. The isolated polynucleotide interacts with the translation activator to cause increased transcription and ultimately translation of the therapeutic polypeptide of interest in a cell containing the RNA polynucleotide. In some embodiments, the translation activator is a polymerase. In some embodiments, the polymerase is an RNA-dependent RNA polymerase or an RNA-dependent DNA polymerase. In some embodiments, the template region of the isolated RNA or translation activator is not derived from an alphavirus genome. In some embodiments, the RNA is a single-stranded RNA polynucleotide. In some embodiments, the coding region of the polypeptide of interest is on the sense strand or the antisense strand.

[0121] Isolated ribonucleic acid (RNA) polynucleotides can incorporate nucleosides that are not adenosine, cytidine, guanosine, or uridine in some embodiments. In some embodiments, the 5'-end of the isolated ribonucleic acid (RNA) polynucleotide is capped. In some embodiments, it is not capped. In some embodiments, the isolated ribonucleic acid (RNA) polynucleotide is 5'-monophosphorylated or 5'-unphosphorylated.

[0122] The isolated ribonucleic acid (RNA) polynucleotides of the present disclosure can be linear or in a covalently closed circular form.

[0123] In some embodiments, the isolated ribonucleic acid (RNA) polynucleotide has increased immunogenicity after contact with a translation activator.

[0124] In some embodiments, the isolated ribonucleic acid (RNA) polynucleotide has a coding region encoding an interferon, an interferon-stimulated gene, an antibody, a signaling molecule, a cytotoxic protein, a protein that causes cell death, an anti-neoplastic protein, an immunomodulatory protein, or a dominant negative protein. In some embodiments, the coding region encodes both a pro-inflammatory cytokine and an anti-inflammatory cytokine. In some embodiments, the coding region encodes an interleukin-1 receptor antagonist. In some embodiments, the coding region encodes an interleukin or a caspase. In some embodiments, the coding region encodes a protein having antiviral activity. In some embodiments, the coding region encodes a secreted protein that can be an antibody or an interferon including IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-γ, or IFN-λ, IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β1, IFN-ε, IFN-κ, IFN-ω1, IFN-γ, IFN-λ1 (IL28A), IFN-λ2 (IL28B), IFN-λ3 (IL29), or IFN-λ-4. In some embodiments, the interferon is IFN-α, IFN-β, IFN-λ1 (IL28A), IFN-λ2 (IL28B), or IFN-λ3 (IL29).

[0125] In some embodiments, the target-specific translation activator comprises a viral RNA-dependent RNA polymerase. The viral RNA-dependent RNA polymerase or RNA-dependent DNA polymerase in some embodiments is produced from the viral genome during viral infection. In some embodiments, the target-specific translation activator is influenza A polymerase, influenza B polymerase, respiratory syncytial virus (RSV) polymerase, coronavirus polymerase, simian betacoronavirus polymerase, metapneumovirus polymerase, parainfluenza virus polymerase, or henipavirus polymerase. In some embodiments, the polymerase is NL63, OC43, 229E, HKU-1, SARS-CoV-1, SARS-CoV-2 or MERS-CoV polymerase. In some embodiments, the target-specific translation activator comprises a hepadnavirus polymerase or hepatitis B virus polymerase. The target-specific translation activator may comprise additional polypeptides required for mRNA synthesis, such as matrix proteins or nucleoproteins.

[0126] In some embodiments, the isolated RNA comprises a left flanking region (「L」) consisting of cis-acting sequences; a central region (「C」) consisting of the coding region of the polypeptide of interest; and a right flanking region (「R」) consisting of cis-acting sequences, where the regions L and R together enable a target-specific translational activator to direct the transcription of an mRNA different from the isolated RNA encoding the therapeutic polypeptide of interest. In some embodiments, region L consists of the sequences of Table 1 where the flank is identified as 「L」 and the encoding is identified as 「antisense」. In some embodiments, region C consists of an antisense protein coding sequence. In some embodiments, region R consists of the sequences of Table 1 where the flank is identified as 「R」 and the encoding is identified as 「antisense」. In some embodiments, region L consists of the sequences of Table 1 where the flank is identified as 「L」 and the encoding is identified as 「sense」, region C consists of a sense protein coding sequence, and region R consists of the sequences of Table 1 where the flank is identified as 「R」 and the encoding is identified as 「sense」.

[0127] In some embodiments, region L consists of sequence L’, region R consists of sequence R’, L’ is the L sequence from Table 2, R’ is the R sequence from Table 2, and L’ and R’ share the same coding RNA scaffold. In some embodiments, the coding RNA scaffold is antisense and in some embodiments is sense.

[0128] According to the present disclosure, region C can encode one or two or more polypeptides of interest. In some embodiments, two or more polypeptides of interest are separated by a ribosome skip site. In some embodiments involving two or more polypeptides of interest, the isolated RNA has a first central region ("C1") consisting of the coding regions of the polypeptides of interest; internal flanking regions ("I"), each consisting of cis-acting sequences, that separate the preceding polypeptide of interest from the subsequent polypeptide of interest, one or more additional coding regions; and a structure of a subsequent region consisting of the coding region of the subsequent polypeptide of interest. In some embodiments, the encryption of the encrypted RNA is antisense, and the internal flanking region ("I") is selected from paramyxovirus or pneumovirus gene start sequences. In some embodiments, region L consists of sequence L', region R consists of sequence R', L' is the L sequence from Table 2, R' is the R sequence from Table 2, and L' and R' share the same encrypted RNA scaffold, and the target virus is RSV. In some embodiments, the internal adjacent region ("I") is selected from Table 1 where the target virus is specified as "RSV" and the flank is specified as "I1".

[0129] The present disclosure also encompasses an isolated DNA encoding the above isolated RNA. The present disclosure also encompasses a viral vector comprising the above isolated RNA or the isolated DNA encoding that RNA. In some embodiments, the viral vector is an adenovirus, adeno-associated virus, poxvirus, retrovirus, lentivirus, herpesvirus, alphavirus, or baculovirus.

[0130] The present disclosure also encompasses a cell line comprising the above DNA. It further encompasses nanoparticles comprising the above isolated RNA or DNA.

[0131] According to another aspect of the present disclosure, a method of inducing cell death is provided. The method comprises administering a therapeutically effective amount of the above isolated RNA or isolated DNA, wherein the coding region encodes an anti-neoplastic agent.

[0132] According to another aspect of the present disclosure, both a viral vector and a target-specific translation activator are introduced into a cell.

[0133] According to another aspect of the present disclosure, a method of inducing an immunogenic response in a subject is provided. The method includes administering to the subject a therapeutically effective amount of any one of the isolated RNA, isolated DNA, viral vector, cell line, or nanoparticle described above, and a target-specific translation activator.

[0134] According to another aspect of the present disclosure, a method of treating a viral infection in a subject is provided. The method includes administering to the subject a therapeutically effective amount of any one of the isolated RNA, isolated DNA, viral vector, cell line, or nanoparticle described above.

[0135] In some embodiments, the isolated RNA is produced after in vivo administration of the isolated DNA encoding the isolated RNA. In some embodiments, the isolated RNA or the DNA encoding the isolated RNA is delivered as an inhaled nanoparticle or an inhaled viral vector.

[0136] In any of the embodiments described herein, the polypeptide of interest can be an anti-neoplastic protein.

[0137] In some embodiments, the isolated RNA or the DNA encoding the isolated RNA and the target-specific translation activator are co-administered. In some embodiments, the isolated RNA or the DNA encoding the isolated RNA or the target-specific translation activator is administered via viral infection. In some embodiments, the subject is human.

[0138] In some embodiments, any one of the isolated RNA, isolated DNA, viral vector, or nanoparticle described above is administered to a cell. In some embodiments, the cell is a human cell, an animal cell, or a plant cell. In some embodiments, the isolated RNA or the isolated DNA or the viral vector or the nanoparticle is administered ex vivo.

[0139] According to another aspect of the present disclosure, a method of producing a transgenic animal or a transgenic plant is provided. This method includes inserting any of the above isolated RNA, isolated DNA, viral vector, or nanoparticles into the animal or the plant. In some embodiments, the coding region of the RNA polynucleotide encodes an antiviral polypeptide of interest. In some embodiments, the transgenic animal or plant has increased resistance to viral infection. In some embodiments, the transgenic animal or plant is a bird, pig, fish, cow, horse, camel, dog, cat, mouse, rat, guinea pig, hamster, ferret, primate, or other commercially valuable animal or plant species.

[0140] In some embodiments, the polypeptide of interest includes an antiviral polypeptide. In some embodiments, the transgenic animal or transgenic cell has increased resistance to viral infection.

[0141] According to another aspect of the present disclosure, a method of increasing the activation of encrypted RNA is provided by complexing any of the above isolated RNA with an RNA-binding protein. In some embodiments, the RNA-binding protein is a viral nucleocapsid protein or a capsid protein. In some embodiments, the RNA-binding protein is the viral nucleocapsid protein or capsid protein of the target virus of the encrypted RNA. In some embodiments, the viral nucleocapsid protein or capsid protein is obtained from influenza virus, simian virus, pneumovirus, paramyxovirus, henipavirus, or hepadnavirus.

[0142] Each of the limitations of the compositions and methods described in this disclosure can embrace various described embodiments. Thus, it is contemplated that each of the limitations of the invention that include any one element or combination of elements can be included in each aspect of the invention. This disclosure is not limited in its application to the details of the structure and arrangement of components described in the following description or shown in the drawings.

Brief Description of the Drawings

[0143] The accompanying drawings are not drawn to scale. For clarity, the drawings are merely illustrative and not required to enable the practice of this disclosure. Not all components are labeled in all of the drawings.

FIG. 1A - 1B

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[0210] Definitions: To facilitate a better understanding of the present disclosure, certain terms are first defined. As used herein, each of the following terms shall have the meaning set forth below, unless expressly provided otherwise herein. Further definitions are set forth throughout the specification.

[0211] The use of alternative forms (e.g., "or") should be understood to mean any, both, or any combination of the alternative forms. As used herein, the term "and / or" has the same meaning as "or".

[0212] As used herein, the indefinite articles "a", "an", or "some" should be understood to refer to "one or more" of the recited or recited components. Thus, the terms "a", "an", "some", "one or more", and "at least one" can be used interchangeably.

[0213] The terms "comprise", "have", and "include" are open-ended conjunctive verbs. Any one or more forms or tenses of these verbs such as "comprises", "comprising", "has", "having", "includes", and "including" are also open-ended. For example, any method that "comprises", "has", or "includes" one or more steps is not limited to having only those one or more steps, but can also include other unrecited steps. Similarly, any composition that "comprises", "has", or "includes" one or more features is not limited to having only those one or more features, but can cover other unrecited features. When an aspect is described herein in terms of "comprising", "having", or "including", it is always understood that other similar aspects described in terms of "consisting of" or "consisting essentially of" are also provided.

[0214] The use of any examples or exemplary language (e.g., "etc.") provided herein with respect to particular embodiments is merely intended to better clarify the present disclosure and does not limit the scope of the claimed present disclosure. The present disclosure is not limited to the specific methodologies, protocols, reagents, etc. described herein and can thus vary. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure, which is defined only by the claims.

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2 nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3 rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide one of ordinary skill in the art with general dictionaries of many of the terms used in this disclosure.

[0216] Units, prefixes, and symbols are denoted in the accepted form of the Systeme International de Unites (SI). Numerical ranges include the numbers defining the range. The headings provided herein are not limitations of the various aspects of the present disclosure which can be had by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0217] The terms "about," "substantially," "approximately," or "comprising essentially of" refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, e.g., the limitations of the measurement system. For example, "about," "substantially," "approximately," or "comprising essentially of" can mean within one standard deviation or beyond one standard deviation for each practice in the art. Alternatively, "about," "substantially," "approximately," or "comprising essentially of" can mean a range of up to 20%. Further, particularly with respect to biological systems or processes, this term can mean up to five-fold or up to ten-fold of a value. When a particular value or composition is provided in this application and the claims, unless otherwise specified, the meaning of "about," "substantially," "approximately," or "comprising essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0218] As used herein, any concentration range, ratio range, proportion range, or integer range should be understood to include any integer value within the recited range, and, when appropriate, fractions thereof (such as 1 / 10 or 1 / 100 of an integer), unless otherwise specified. Further, all ranges are intended to expressly include the boundaries of the range individually. For clarity, the range 3 to 6 is intended to include individually 3, 4, 5, and 6, as well as any fraction within that range.

[0219] As used herein, a "target-specific translation activator" is one or more polypeptides that direct the synthesis of the coding region of an encrypted RNA, the coding region of which comprises a coding sequence encoding a polypeptide of interest that is translated at an increased level when the target-specific translation activator contacts the encrypted RNA. As used herein, a "translation activator" means a "target-specific translation activator". In some embodiments, the target-specific translation activator is a polymerase. In some embodiments, the polymerase is an RNA-dependent RNA polymerase (RdRp). In some embodiments, the polymerase is an RNA-dependent DNA polymerase (RdDp, also called reverse transcriptase (RT)).

[0220] As used herein, an "encrypted RNA" is an isolated ribonucleic acid (RNA) polynucleotide that (a) comprises a "coding region" that comprises a coding sequence encoding a polypeptide of interest, and (b) comprises a "template region" for binding a target-specific translation activator, the target-specific translation activator directing the transcription of an mRNA that is different from the isolated RNA, and the translation of the polypeptide of interest increasing in the cell comprising the RNA polynucleotide when the RNA polynucleotide contacts the target-specific translation activator in the cell. As used herein, a "polypeptide of interest" or "protein of interest" is a polypeptide encoded within the coding sequence of the coding region of an encrypted RNA according to the present disclosure.

[0221] The template region is composed of two different regions, namely the left flanking region of the virus ("L region") and the right flanking region of the virus ("R region"). The L region is contiguous to the 5' side of the coding region, and the R region is contiguous to the 3' side of the coding region. Examples of the L region and the R region of various viruses and variants are provided in the sequence listing and in the following tables and examples. In some embodiments, the L region and the R region of the virus do not each contain a polynucleotide sequence encoding a polypeptide. In some embodiments, the L region or the R region may include a polynucleotide sequence encoding a polypeptide homologous to the virus. When the L or R region includes a polynucleotide sequence, the polynucleotide sequence contributes, as necessary, to the interaction between the L or R region and the translation activator.

[0222] The coding region includes one or more coding sequences. In some embodiments, the coding region includes two or more (e.g., 2, 3, 4, or more) coding sequences. In some embodiments, the coding region includes one coding sequence. Further, the coding region may include one or more non-coding sequences. Typically, the coding region includes a 5' untranslated region (5'UTR), a coding sequence, and a 3' untranslated region (3'UTR).

[0223] A "coding sequence" is a sequence of nucleotides that encodes the complete amino acid sequence of at least one polypeptide of interest. In some embodiments, the coding sequence encodes two or more (e.g., 2, 3, 4, or more) polypeptides. In some embodiments, the coding sequence encodes one polypeptide. As used herein, a "polypeptide of interest" is a polypeptide encoded by the coding sequence of the coding region. In some embodiments, the coding sequence of the coding region encodes a polypeptide that is heterologous to the virus from which the L region and R region of the coding RNA are derived. As used herein, "heterologous to a virus" means a coding sequence that encodes a polypeptide that is not naturally found in the species of the virus (not a native polypeptide). As used herein, "homologous to a virus" means that the coding sequence encodes a polypeptide that is naturally found in the species of the virus. A homologous sequence of a virus may also be referred to as "native" to the virus. The classification of virus species follows internationally recognized criteria established by the International Committee on Taxonomy of Viruses ("ICTV"). A coding sequence consists of a series of three-nucleotide units known as codons. The first three nucleotides of the coding sequence, the "start codon", initiate the translation of the polypeptide of interest and typically encode methionine or N-formylmethionine. An example of a start codon is "atg". The last three nucleotides of the coding sequence, the "stop codon", encode a stop codon or stop codons that terminate the translation elongation of the polypeptide of interest. Some examples of stop codons are "tag" (amber stop codon), "taa" (ochre stop codon), and "tga" (opal stop codon).

[0224] A "non-coding sequence" is a continuous nucleotide sequence that does not contain a coding sequence and does not encode a polypeptide. The expression of a polypeptide of interest can be altered using non-coding sequences. Non-limiting examples of non-coding sequences include 5' untranslated regions (UTRs), 3'-UTRs, promoters, introns, ribozymes, riboswitches, ribosome binding sites, Kozak sequences, Shine-Dalgarno sequences, internal ribosome entry sites (IRESs), polyadenylation signals, polyA sequences, microRNA binding sites, and other regulatory elements. As described above, in some embodiments, either the L region or the R region, or both the L and R regions, consist of non-coding sequences. In some embodiments, the L region or the R region can include a polynucleotide sequence that encodes one or more polypeptides homologous to the virus.

[0225] The "5'-UTR of a coding sequence" or "5' untranslated region of a coding sequence" is a non-coding sequence that is contiguous and adjacent to the 5' start codon of the coding sequence. When the coding sequence is 3' of the first coding sequence of the L region, the 5'-UTR of the coding sequence begins with the first nucleotide of the first 5' non-coding sequence within the coding region and ends at the nucleotide immediately preceding the start codon of the coding sequence. If there are two (or more) coding sequences within the coding region, the coding sequences can be separated by untranslated regions. When the coding sequence is not 3' of the first coding sequence of the L region, a second 5'-UTR can exist for the second coding sequence (the third 5'-UTR for the third coding sequence, etc.), which separates the coding sequences from each other. The 5'-UTR of a coding sequence can contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements include, for example, ribosome binding sites, Kozak sequences, Shine-Dalgarno sequences, ribozymes, riboswitches, promoters, microRNA binding sites, or IRES elements.

[0226] The "3'-UTR of the coding sequence" or "3' untranslated region of the coding sequence" is a non-coding sequence that is located adjacent and continuous to the 3' stop codon of the coding sequence. When the coding sequence is the first coding sequence adjacent to the 5' end of the R region, the 3'-UTR of the coding sequence starts with the first nucleotide following the stop codon of the coding sequence and ends with the last 3' nucleotide of the coding region before the 5' end of the R region. When there are two (or more) coding sequences within the coding region, the first and second coding sequences may be separated by an untranslated region. The first 3'-UTR of the first coding sequence can separate the first coding sequence from the next adjacent coding sequence closer to the R region (the second 3'UTR of the second coding sequence can separate the second coding sequence from the next adjacent coding sequence closer to the R region, and so on). The 3'-UTR of the coding sequence may contain one or more elements for controlling gene expression, also called regulatory elements. Such regulatory elements include, for example, ribozymes, microRNA binding sites, poly(A) sequences, and polyadenylation signals.

[0227] Translation of the polypeptide or protein of interest in the encrypted RNA of the present disclosure is increased when the encrypted RNA contacts / interacts with a target-specific translation activator of the encrypted RNA. Such interaction initiates the activity of the target-specific translation activator.

[0228] In some embodiments, the polypeptide of interest is a "therapeutic polypeptide." A therapeutic polypeptide, exemplified in more detail below, is a polypeptide that treats or ameliorates one or more symptoms of a disease or condition in a subject. In some embodiments, the treatment is of an existing condition. In some embodiments, the treatment is prophylactic treatment. In some embodiments, the therapeutic polypeptide encoded by the encrypted RNA is heterologous to the virus from which the L and R regions of the encrypted RNA are derived. In some embodiments, the coding sequence of the therapeutic polypeptide does not naturally occur at the same nucleotide position in the viral genome. In some embodiments, the therapeutic polypeptide is an immunomodulatory protein, such as a human immunomodulatory protein known to be active against the human immune system. Examples of immunomodulatory proteins include proteins such as chemokines, cytokines, interleukins, factors, antibodies, immune checkpoint inhibitors, or aptamers. In some embodiments, the therapeutic polypeptide is a native human protein or an analog of a human protein (e.g., a protein having one or more amino acid substitutions or a truncated form of a protein variant). In some embodiments, the therapeutic polypeptide is an antigen that includes a cancer antigen or an autoantigen that includes an antigen present in a pathogen.

[0229] A "polypeptide of interest for treatment," "therapeutic polypeptide," or "therapeutic protein" has an advantageous effect on the condition or disease state of a subject when administered to the subject in a therapeutically effective amount. In some aspects, the therapeutic polypeptide has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. The therapeutic polypeptide can have prophylactic properties and can be used to delay or prevent the onset of a disease or to reduce the severity of such a disease or pathological condition. The term therapeutic polypeptide includes the entire protein or polypeptide and can also refer to its active fragments. It can also include active analogs of the peptide or protein. A pharmaceutically active peptide or protein can also be referred to as a therapeutic peptide or protein.

[0230] In some embodiments, the polypeptide of interest is a polypeptide that does not induce or elicit a medically significant antigen-specific response against the polypeptide of interest when administered to a particular subject. In some embodiments, the polypeptide of interest is an immunostimulatory polypeptide. In some embodiments, the polypeptide of interest is a polypeptide that, when administered to a particular subject, induces or elicits a medically significant antigen-specific response against the polypeptide of interest. In some embodiments, the polypeptide of interest is an immunosuppressive polypeptide. In some embodiments, a polypeptide of interest that, when administered to a particular subject, induces or elicits a medically significant immunosuppressive immune response or inhibits or prevents an immunostimulatory or inflammatory immune response.

[0231] In some embodiments, the polypeptide of interest is a reporter polypeptide. Examples of reporter polypeptides are provided in the examples and are well known to those skilled in the art.

[0232] As used herein, "activation" or "activating" describes a process or action or series of processes or series of actions in which translation of a polypeptide of interest is increased when the encrypted RNA encoding the polypeptide of interest is contacted with a translation activator of the encrypted RNA. As used herein, encrypted RNA is said to be "activated" upon contact with a translation activator.

[0233] As shown in FIGS. 1A-1B and FIGS. 2A-2B, in some embodiments, contact between the encrypted RNA and the translation activator increases translation of the polypeptide of interest.

[0234] As used herein, "encrypted protein" or "encrypted polypeptide" is the polypeptide of interest encoded by the encrypted RNA.

[0235] As used herein, "therapeutic encrypted RNA" is encrypted RNA whose coding region encodes a therapeutic polypeptide. As used herein, "SHIELD" or "SHIELDRNA" or "SHIELD encrypted RNA" has the same meaning as "therapeutic encrypted RNA".

[0236] As used herein, "DNA-encoded encrypted RNA" is a DNA sequence that encodes an encrypted RNA cassette.

[0237] As used herein, "encrypted nucleic acid" means encrypted RNA or DNA-encoded encrypted RNA.

[0238] As used herein, "antisense encrypted RNA" means that the coding sequence encoding the polypeptide of interest within the coding region is arranged in an antisense orientation with respect to the encrypted RNA sequence. As used herein, "negative-sense encrypted RNA" and "(-)-sense encrypted RNA" correspond to "antisense encrypted RNA".

[0239] As used herein, "sense encrypted RNA" means that the coding sequence encoding the polypeptide of interest within the coding region is arranged in a sense orientation with respect to the encrypted RNA sequence. As used herein, "positive-sense encrypted RNA" and "(+)-sense encrypted RNA" correspond to "sense encrypted RNA".

[0240] As used herein, "influenza encrypted RNA" is encrypted RNA having a target-specific translation activator containing an influenza virus polypeptide. For the sake of clarity, encrypted RNA having a target-specific translation activator containing an influenza virus polypeptide means that the encrypted RNA is activated by an influenza virus polypeptide (e.g., influenza virus polymerase).

[0241] As used herein, "influenza A-encoded RNA" is an encrypted RNA having a target-specific translation activator that includes an influenza A virus polypeptide. For clarity, this means that the influenza A-encoded RNA is activated by an influenza A virus polypeptide (e.g., influenza A virus polymerase).

[0242] As used herein, "influenza B-encoded RNA" is an encrypted RNA that is activated by an influenza B virus polypeptide (e.g., influenza B virus polymerase).

[0243] As used herein, "therapeutic influenza-encoded RNA" or "influenza SHIELD" is an influenza-encoded RNA that is a therapeutic encrypted RNA.

[0244] As used herein, "influenza antisense-encoded RNA" or "influenza negative-sense-encoded RNA" or "influenza (-)-sense-encoded RNA" is an influenza-encoded RNA that is an antisense encrypted RNA.

[0245] As used herein, "influenza sense-encoded RNA" or "influenza positive-sense-encoded RNA" or "influenza (+)-sense-encoded RNA" is an influenza-encoded RNA that is a sense encrypted RNA.

[0246] As used herein, "SARS-CoV-encoded RNA" is an encrypted RNA that is activated by a SARS-CoV polypeptide (e.g., SARS-CoV virus polymerase).

[0247] As used herein, "therapeutic SARS-CoV-encoded RNA" or "SARS-CoV SHIELD" is a SARS-CoV-encoded RNA that is a therapeutic encrypted RNA.

[0248] As used herein, "SARS-CoV antisense-encoded RNA" or "SARS-CoV negative-sense-encoded RNA" or "SARS-CoV (-) sense-encoded RNA" is SARS-CoV encoded RNA that is antisense-encoded RNA.

[0249] As used herein, "SARS-CoV sense-encoded RNA" or "SARS-CoV positive-sense-encoded RNA" or "SARS-CoV (+) sense-encoded RNA" is SARS-CoV encoded RNA that is sense-encoded RNA.

[0250] As used herein, "SARS-2" is the SARS-CoV-2 virus.

[0251] As used herein, "RSV-encoded RNA" is RNA that is encoded and activated by a respiratory syncytial virus (RSV) polypeptide (e.g., RSV polymerase).

[0252] As used herein, "therapeutic RSV-encoded RNA" or "RSV SHIELD" is RSV-encoded RNA that is therapeutic-encoded RNA.

[0253] As used herein, "RSV antisense-encoded RNA" or "RSV negative-sense-encoded RNA" or "RSV (-) sense-encoded RNA" is RSV-encoded RNA that is antisense-encoded RNA.

[0254] As used herein, "RSV sense-encoded RNA" or "RSV positive-sense-encoded RNA" or "RSV (+) sense-encoded RNA" is RSV-encoded RNA that is sense-encoded RNA.

[0255] As used herein, a carrier or polymeric carrier is typically a compound that facilitates the transport or complex formation of another compound (cargo). A polymeric carrier is typically a carrier formed from a polymer. A carrier can associate with its cargo by covalent or non-covalent interactions. A carrier can transport a nucleic acid, such as RNA or DNA, to a target cell and / or can facilitate the uptake of the nucleic acid into the target cell. In some embodiments, the carrier may be a cationic component.

[0256] The term "cationic component" typically refers to a molecule that is positively charged (cationic) at a pH value typically between 1 and 9. Thus, a cationic component can be any positively charged compound or polymer such as a cationic peptide or protein or lipid that is positively charged under physiological conditions such as those occurring in vivo. A "cationic peptide or protein" can contain at least one positively charged amino acid or more than one positively charged amino acid selected, for example, from Arg, His, Lys or Asn. Thus, a "poly-cationic" component is also within the range of exhibiting two or more positive charges under a given condition.

[0257] The term "subject" refers to an animal, such as a human, to whom treatment, including prophylactic treatment, is provided by a method, polynucleotide (including coding RNA and DNA encoding coding RNA), and compositions described herein. For the treatment of a particular condition or disease state specific to a particular animal, such as a human subject, the term "subject" refers to that particular animal. Also included are cells, tissues obtained in vivo or cultured ex vivo or in vitro, and progeny of such cells or tissues. In addition to humans, subjects include cows, pigs, sheep, horses, deer, other ruminant animals, rodents, fish, and poultry (e.g., chickens and ducks).

[0258] The term "tissue" refers to a group or layer of similarly specialized cells that together perform a particular specialized function.

[0259] "Gene therapy" can typically be understood to mean the treatment of a patient's body or an isolated element of the patient's body, such as isolated tissue / cells, with a nucleic acid encoding a peptide or protein. It typically includes at least one of the following steps: a) directly administering the nucleic acid to the patient by any applicable route of administration, or administering it in vitro to the patient's isolated cells / tissue, such that the patient's cells are transfected either in vivo / ex vivo or in vitro; b) transcribing and / or translating the introduced nucleic acid molecule; and c) when the nucleic acid has not been directly administered to the patient, re-administering the isolated and transfected cells to the patient. As used herein, the term "gene therapy" typically encompasses the treatment, prevention, or prophylaxis of diseases or disorders.

[0260] As used herein, it is understood that RNA polynucleotides are composed of ribonucleotide monomers and DNA polynucleotides are composed of deoxyribonucleotide monomers. Since ribonucleotides are nucleotides and deoxyribonucleotides are nucleotides, the leading "ribo-" or "deoxyribo-" can be omitted when the meaning is clear. As an example, "an RNA polynucleotide composed of nucleotides" has the same meaning as "an RNA polynucleotide composed of ribonucleotides". Similarly, "a DNA polynucleotide composed of nucleotides" has the same meaning as "a DNA polynucleotide composed of deoxyribonucleotides".

[0261] "RNA" is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule or polynucleotide, i.e., a polymer consisting of ribonucleotides (nucleotides). These nucleotides are usually adenosine monophosphate (AMP), cytidine monophosphate (CMP), guanosine monophosphate (GMP), and uridine monophosphate (UMP) monomers, which are linked to each other along a so-called backbone or phosphodiester backbone. When the meaning is clear, RNA polynucleotides can be said to be composed of their nucleotide triphosphates, e.g., adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), or uridine triphosphate (UTP), which indicates that the RNA polynucleotides are synthesized or transcribed using nucleotide triphosphate monomers to form normal RNA polynucleotides.

[0262] The backbone is formed by a phosphodiester bond between the sugar of the first monomer, i.e., ribose, and the phosphate moiety of the second adjacent monomer. The specific sequence of monomers is called an RNA sequence. Usually, RNA can be obtained, for example, by transcription of a DNA sequence within a cell. In eukaryotic cells, transcription typically occurs within the nucleus or mitochondria. In vivo, transcription of DNA usually results in so-called immature RNA, which must be processed into so-called messenger RNA, usually abbreviated as mRNA. For example, processing of immature RNA in eukaryotes includes various post-transcriptional modifications such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called the maturation of RNA. Mature messenger RNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a specific polypeptide or protein. Typically, mature mRNA includes a 5'-UTR, an open reading frame, and a 3'-UTR. Apart from messenger RNA, there are several types of RNA that can be involved in the regulation of transcription or translation.

[0263] As used herein, "nucleoside modification" means that the RNA polynucleotide is composed of at least one nucleotide other than AMP, CMP, GMP, or UMP.

[0264] As used herein, the terms "nucleoside-modified RNA" or "nucleoside-modified encrypted RNA" or "nucleoside-modified therapeutic encrypted RNA" or "nucleoside-modified SHIELD" or "nucleoside-modified mRNA" refer to an RNA molecule or a part thereof that contains one, two, or more nucleoside modifications as compared to adenosine (A) ((2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol), guanosine (G) (2-amino-9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3H-purin-6-one), cytidine (C) (4-amino-1-[3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-one), or uridine (U) (1-[(3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2,4-dione), or as compared to AMP, GMP, CMP, or UMP. Non-limiting examples of nucleoside modifications are provided elsewhere herein. When the nucleotide sequence of a particular claimed RNA is identical in other respects to the sequence of a naturally occurring RNA molecule, the nucleoside-modified RNA is understood to be an RNA molecule having at least one modification that is different from the modifications present in the corresponding naturally occurring counterpart. The difference can be any of the chemical changes to the nucleoside / nucleotide.

[0265] In some embodiments, about 30% to 100% of the UMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides. In some embodiments, about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or about 100% of the UMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides. In some embodiments, about 30% - 100% of the CMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides. In some embodiments, about 30% - 100% of the CMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides. In some embodiments, about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or about 100% of the CMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides.

[0266] In some embodiments, about 1% to 30% of the AMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides. In some embodiments, about 1%, 2%, 3, 4%, 5%, 10%, 15%, 20%, 25%, or about 30% of the AMP nucleotides in the nucleoside-modified RNA are replaced with modified nucleosides.

[0267] In some embodiments, the nucleoside-modified RNA comprises at least one UMP modified to form N1-methyl-pseudo-UMP (N1-methylpseudouridine, N1m-pU). In some embodiments, the nucleoside-modified RNA comprises at least one UMP modified to form pseudo-UMP (pseudouridine, pU). In the nucleoside-modified RNA, it is not necessary to modify all nucleosides. In some embodiments, about 10% to about 100% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudo-UMP or N1-methyl-pseudo-UMP. In some embodiments, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or about 70% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudouridine or N1-methyl-pseudo-UMP. In some embodiments, about 10% to 35% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudo-UMP or N1-methyl-pseudo-UMP. In some embodiments, about 10%, 15%, 20%, 25%, 30% or about 35% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudo-UMP or N1-methyl-pseudo-UMP. In some embodiments, about 100% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudo-UMP or N1-methyl-pseudo-UMP. In some embodiments, about 70% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudo-UMP nucleotides. In some embodiments, about 100% of the UMP nucleotides in the nucleoside-modified RNA are replaced with pseudo-UMP nucleotides. In some embodiments, the nucleoside-modified RNA comprises at least one AMP modified to form N6-methyl-AMP. In some embodiments, the nucleoside-modified RNA comprises at least one CMP modified to form 5-methyl-CMP. In some embodiments, the nucleoside-modified RNA comprises at least one UMP modified to form 5-methoxy-UMP (moU). In some embodiments, the RNA does not contain nucleoside modifications (unmodified RNA).

[0268] As used herein, "capped RNA" or "5'-capped RNA" refers to an RNA molecule that has a Cap structure incorporated at its 5' end. Cap structures are present at the 5' ends of many mRNAs in eukaryotes as well as on the viral RNAs of some viruses.

[0269] Naturally occurring Cap structures typically contain a riboguanosine residue that is methylated at position N7 of the guanine base. This N7-methylguanosine (m 7 G) is linked to the 5' end of the mRNA molecule via a 5'-to-5'-triphosphate chain. The 5' cap of RNA can promote resistance to degradation by exonucleases and facilitate the transport of mRNA from the nucleus to the cytoplasm. Examples of naturally occurring Cap structures include Cap0, Cap1, and Cap2. When the only capping modification is N7-methylguanosine linked via a 5'-5'-triphosphate bond to the terminal nucleotide of the RNA, the structure is designated Cap0. When the RNA further incorporates 2'-O-methylation only at the 5' of the first nucleoside of Cap0 (i.e., the second-to-last nucleoside of the RNA containing m 7 G), the structure is called Cap1. When the RNA further incorporates 2'-O-methylation at the 5' of the first two nucleosides of Cap0 (i.e., both the second-to-last and the third-to-last nucleosides containing m 7 G), the structure is called Cap2.

[0270] Cap0(3'-O-Me) is Cap0 in which the 3'-OH (i.e., the 3'-hydroxyl group) of the 5' N7-methylguanosine (m 7 G) cap of Cap0 is replaced by -OCH3 (i.e., the 3'-methoxy group). Similarly, Cap1(3'-O-Me) and Cap2(3'-O-Me) are Cap1 and Cap2 structures that include 3'-O-methylation of the 5' N7-methylguanosine (m 7 G) cap for each respective Cap1 or Cap2.

[0271] In some embodiments, the capped RNA comprises a 5'-Cap structure that is a Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), or 2-azido-guanosine structure. All of these are nucleoside-modified RNA molecules.

[0272] As used herein, "uncapped RNA" or "non-capped RNA" refers to an RNA molecule lacking a 5'-Cap structure.

[0273] As used herein, "5'-phosphorylated" refers to the number of consecutive phosphate molecules attached to the 5'-end of an uncapped RNA. A "triphosphorylated" or "5'-triphosphorylated" RNA molecule is uncapped and has a 5'-terminal triphosphate (three phosphates). A "5'-diphosphorylated" or "5'-biphosphorylated" RNA molecule is uncapped and has a 5'-terminal diphosphate (two phosphates). A "monophosphorylated" or "5'-monophosphorylated" RNA molecule is uncapped and has a 5'-terminal monophosphate or 5'-terminal phosphate (one phosphate). An RNA molecule that is "non-phosphorylated" or "5'-non-phosphorylated" has no 5'-terminal phosphate (zero phosphates).

[0274] "Polymerase" generally refers to a molecular entity that can catalyze the synthesis of polymer molecules from monomeric components. "RNA polymerase" is a molecular entity that can catalyze the synthesis of RNA molecules from ribonucleotide components. "DNA polymerase" is a molecular entity that can catalyze the synthesis of DNA molecules from deoxyribonucleotide components. In the case of DNA polymerase or RNA polymerase, the molecular entity is typically a protein or an aggregate or complex of multiple proteins. Typically, DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, which is typically a DNA molecule. Some DNA polymerases are RNA-dependent DNA polymerases and synthesize DNA molecules based on a template nucleic acid. Some RNA-dependent DNA polymerases are called "reverse transcriptases". Typically, RNA polymerase synthesizes an RNA molecule based on a template nucleic acid that is either a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0275] "RNA-dependent RNA polymerase" or "RdRP" is a multi-domain (α and β) protein that catalyzes the RNA template-dependent formation of phosphodiester bonds between ribonucleotides in the presence of a divalent metal ion. Initiation of synthesis occurs at the 3' end of the template in a primer-dependent or independent manner and proceeds in the 5'→3' direction on the synthesized strand. The average length of the core RdRP domain is less than 500 amino acids and is folded into three subdomains. The active sites of RdRPs from different RNA viruses are conserved and are similar to the active sites of other enzymes such as reverse transcriptase and DNA polymerase, showing similar roles in the nucleotidyl transfer reaction.

[0276] Some viral polymerases have additional domains, such as methyltransferase or endonuclease domains, to perform functions related to RNA synthesis. The polymerase domain can also interact with other host factors for efficient polymerization and to discriminate activities such as genome replication and mRNA transcription. Host factors include translation factors, protein chaperones, RNA modifying enzymes, or other cellular proteins. Together with the RdRP, they form the viral replication complex (VRC). The VRCs differ in their composition, intracellular location, and interaction with the viral RNA template.

[0277] As defined herein, a "ribozyme" is a catalytic macromolecular complex that includes an RNA having catalytic activity. Examples of ribozymes include, but are not limited to, RNA molecules having self-splicing intron sequences, RNA molecules composed of the antigenomic ribozyme of hepatitis delta virus, RNA molecules composed of "hammerhead" ribozymes, and two-component ribonucleoprotein systems including guide RNAs complexed with Cas proteins ("CRISPR-Cas"). RNA molecules containing ribozymes with nuclease activity may cleave within the molecule in which they are embedded or may cleave RNA outside the molecule in which they are embedded.

[0278] As used herein, "sequence identity" is used to mean the relationship between two or more protein (polypeptide) sequences or two or more nucleic acid (polynucleotide) sequences, which is determined by comparing the sequences. Two or more sequences are identical if they show the same length and order of nucleotides or amino acids. The calculation of the percent identity (or identity %) of two nucleic acid sequences can be done, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, 96%, 97%, 98%, 99% or 100% of the length of the reference sequence. Then, the nucleotides at the corresponding nucleotide positions are compared. If the position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be achieved using an algorithm.For example, the percent identity between two nucleotide sequences or two polypeptide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference.

[0279] A polynucleotide sequence or polypeptide sequence can be compared by performing a sequence alignment, which may be with or without gaps. In a non-gapped alignment, two or more sequences are compared as "adjacent" sequences, i.e., one sequence is aligned with another sequence, and each nucleotide or amino acid in one sequence is directly compared one residue at a time with the corresponding nucleotide or amino acid in the other sequence. In a non-gapped alignment, otherwise identical sequence pairs can result in a potentially suboptimal overall alignment due to an insertion or deletion in one sequence that causes the other nucleotide or amino acid residue to fall out of alignment. In a gapped alignment, the sequences can be compared "non-adjacent" and insertions and deletions (collectively "gaps") can be inserted to optimally align the sequences.

[0280] As used herein, "sequence similarity" is used in a manner similar to "sequence identity", but incorporates aspects of the relatedness between two sequences, such as functional or phenotypic relatedness, that may not be fully explained by the method of determining sequence identity.

[0281] Methods for determining identity and similarity are embodied in publicly available algorithms or software, including BLAST, FASTA, T-COFFEE, and M-COFFEE. In some methods, a scaled similarity score matrix or equivalent can be used to assign scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix - the default matrix for the Basic Local Alignment Search Tool (BLAST) suite of programs. There are also alternative computational methods (e.g., INFERNAL or R-COFFEE) used to determine identity or similarity, which take into account aspects of sequence relatedness (covariance models, secondary structure, tertiary structure, etc.) in addition to the primary sequence (Eddy & Durbin, Nucleic Acids Research (1994); DOI: 10.1093 / nar / 22.11.2079) (Rivas et al., Bioinformatics (2020); DOI: 10.1093 / bioinformatics / btaa080) (Nawrocki & Eddy; Bioinformatics (2013); DOI: 10.1093 / bioinformatics / btt509).

[0282] In some embodiments, encrypted RNAs having different template regions can be activated by the same translation activator. Thus, the template regions can share a common structure and function even though their primary nucleotide sequences are different: i.e., the template regions for the same translation activator can be sequences that are not identical but are similar.

[0283] In some embodiments, an encrypted RNA having a variant template region has the same or similar activation as an encrypted RNA having a reference template region, in the presence or absence of a translation activator. Alternatively, the activation of an encrypted RNA having a variant template region may be changed (e.g., increased or decreased) compared to an encrypted RNA having a reference template region. Generally, a variant template region has at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity with its specific reference polynucleotide, but less than 100% sequence identity or similarity with the reference template region, as determined by sequence alignment programs and parameters described herein and known to those of ordinary skill in the art. As used herein, a "variant" of a nucleotide sequence has less than 100% sequence identity with a reference nucleotide sequence due to substitution of one nucleotide for another, addition (insertion) of one or more nucleotides, and / or deletion of one or more nucleotides relative to the reference sequence. As used herein, a "variant" of a polypeptide sequence has less than 100% sequence identity with a reference polypeptide sequence due to substitution of at least one amino acid for another, addition (insertion) of one or more amino acids, and / or deletion of one or more amino acids relative to the reference sequence.

[0284] In some embodiments, two different translation activators can activate the same encrypted RNA. Thus, translation activators can share a common structure and function, although their primary polypeptide sequences are different.

[0285] In some embodiments, a translation activator comprising a variant polypeptide (e.g., a variant polymerase) activates the encrypted RNA in the same manner as a translation activator comprising a reference polypeptide. Alternatively, the translation activator comprising the variant polypeptide may have a change in the activation of the encrypted RNA (e.g., an increase or decrease) as compared to the translation activator comprising the reference polypeptide. Generally, a variant polypeptide has at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity or similarity to a reference template region of a particular reference polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0286] A "stabilized nucleic acid molecule" is a nucleic acid molecule that has been modified to be more stable than an unmodified nucleic acid molecule against, for example, degradation or decomposition by environmental factors, or enzymatic digestion by exo- or endonucleases, typically a DNA or RNA molecule. In some embodiments, the stabilized nucleic acid molecule is stabilized against degradation in a cell such as a prokaryotic or eukaryotic cell. In some further embodiments, the stabilized nucleic acid molecule is stabilized against degradation in a mammalian cell such as a human cell. The stabilizing effect can also be exerted extracellularly, for example, in a buffer, for example, in the manufacturing process of a pharmaceutical composition comprising the stabilized nucleic acid molecule.

[0287] The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells such as eukaryotic cells. In the context of the present disclosure, the term "transfection" encompasses any method known to those skilled in the art for introducing nucleic acid molecules into cells such as mammalian cells. Such methods include, for example, electroporation, lipofection based on cationic lipids or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers such as DEAE-dextran or polyethyleneimine.

[0288] The term "vector" refers to a nucleic acid molecule. A vector in the context of the present disclosure is suitable for incorporating or holding a desired nucleic acid sequence, such as a nucleic acid sequence containing an open reading frame. Such vectors can be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that enables convenient storage of nucleic acid molecules, such as mRNA molecules. Thus, a vector can include, for example, a sequence corresponding to a desired mRNA sequence or a part thereof, such as a coding sequence and a sequence corresponding to the 3'-UTR of mRNA. An expression vector can be used for the production of expression products such as RNA, encoded RNA, mRNA, peptides, polypeptides, or proteins. An expression vector can include a promoter sequence, such as a sequence stretch of the vector that is necessary for transcription, such as an RNA polymerase promoter sequence. A cloning vector is typically a vector that contains a cloning site that can be used to incorporate a nucleic acid sequence into the vector. A cloning vector can be, for example, a plasmid vector or a bacteriophage vector. A transfer vector can be a vector suitable for transferring a nucleic acid molecule into a cell or an organism, such as a viral vector. In some embodiments, the viral vector is a lentiviral vector. A vector in the context of the present disclosure can be, for example, an RNA vector or a DNA vector. In some embodiments, the vector is a DNA molecule. In some embodiments, the vector includes a cloning site, a selection marker (such as an antibiotic resistance factor, etc.), and / or a sequence suitable for multiplexing of the vector (such as an origin of replication, etc.). In some embodiments, the vector is a plasmid vector, also called a plasmid.

[0289] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors because they can deliver a significant amount of genetic information into the DNA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0290] As used herein, "lentiviral vector" is a viral vector derived from a lentivirus.

[0291] A "vehicle" is typically understood to be a material suitable for storing, transporting, or administering a compound, such as a pharmaceutically active compound. For example, it can be a physiologically acceptable liquid suitable for storing, transporting, or administering a pharmaceutically active compound.

[0292] Encrypted RNA Aspects of the present disclosure are encrypted RNAs comprising DNA encoding the encrypted RNA,

[0293] In some embodiments, the encrypted RNA is single-stranded RNA (ssRNA).

[0294] In some embodiments, the encrypted RNA is capped ssRNA.

[0295] In some embodiments, the encrypted RNA is uncapped ssRNA.

[0296] In some embodiments, the encrypted RNA is 5'-triphosphorylated uncapped ssRNA.

[0297] In some embodiments, the encrypted RNA is 5'-diphosphorylated uncapped ssRNA.

[0298] In some embodiments, the encrypted RNA is a 5'-monophosphorylated uncapped ssRNA.

[0299] In some embodiments, the encrypted RNA is a 5'-unphosphorylated uncapped ssRNA.

[0300] In some embodiments, the encrypted RNA is an uncapped ssRNA having four or more 5' terminal phosphates (tetraphosphate, pentaphosphate).

[0301] In some embodiments, the encrypted RNA is a stabilized nucleic acid molecule.

[0302] In some embodiments, the encrypted RNA is a circular RNA.

[0303] In some embodiments, the template region of the encrypted RNA or the translation activator is not derived from an alphavirus genome.

[0304] In some embodiments, the encrypted RNA is delivered to a cell and translated at a low level until it contacts a polymerase encoded by an infectious virus, which then results in an increase in the translation of the encrypted RNA.

[0305] In some embodiments, the encrypted RNA is delivered to a cell together with a target-specific translation activator.

[0306] In some embodiments, DNA is used to encode the encrypted RNA.

[0307] In some embodiments, the encrypted nucleic acid is a stabilized nucleic acid molecule.

[0308] In some embodiments, the DNA sequences adjacent to the encrypted RNA within the DNA-encoded encrypted RNA cassette can have a desired effect on the production of the encrypted RNA by inducing one or more outcomes, including altering the level (abundance) of the produced encrypted RNA, altering the average molecular structure of the encrypted RNA, or altering the rate at which the encrypted RNA is produced from the DNA template.

[0309] In some embodiments, the DNA-encoded encrypted RNA cassette can be reused to produce other RNA species by replacing the encrypted RNA sequence with an alternative non-encrypted RNA sequence that encodes an RNA. In some embodiments, the DNA-encoded encrypted RNA cassette can be reused to encode a non-encrypted RNA sequence that encodes a viral genetic element. In some embodiments, the DNA-encoded encrypted RNA cassette can be reused to encode a non-encrypted RNA sequence that is antisense to a target sequence. In some embodiments, the DNA-encoded encrypted RNA cassette can be reused to encode a non-encrypted RNA sequence that encodes a guide RNA of the CRISPR-Cas system. In some embodiments, the DNA-encoded encrypted RNA cassette can be reused to encode a non-encrypted RNA sequence that encodes an mRNA sequence.

[0310] In some embodiments, the DNA encoding the encrypted RNA is delivered to cells in a viral vector.

[0311] In some embodiments, the DNA encoding the encrypted RNA is delivered to cells in a plasmid.

[0312] In some embodiments, the encrypted RNA or the DNA encoding the encrypted RNA encodes a therapeutic protein, such as an immunomodulatory protein.

[0313] In some embodiments, the encrypted RNA or DNA encoding the encrypted RNA encodes two or more target polypeptides. Strategies for encoding multiple polypeptides are well known to those of skill in the art (see, e.g., Liu et al., Scientific Reports (2017) DOI:10.1038 / s41598-017-02460-2). Such strategies include the use of multiple promoters, fusion proteins, proteolytic cleavage sites within polypeptides, internal ribosome entry sites, and "ribosome skipping" 2A peptides. In some embodiments, two or more target polypeptides are encoded in a coding sequence that is translated as two or more polypeptides via the action of one or more "2A"-like sequences present in the coding sequence (e.g., 2A sequences from porcine teschovirus-1, foot-and-mouth disease virus, equine rhinitis A virus, or Theileria equi virus). See, e.g., Liu et al. Scientific Reports (2017) 7:2193.

[0314] In some embodiments, the target-specific translation activator comprises a polymerase. In some embodiments, the translation activator comprises an RNA-dependent RNA polymerase ("RdRP") or an RNA-dependent DNA polymerase ("RdDP"). In some further embodiments, the translation activator comprises a further polypeptide that promotes mRNA synthesis. In some further embodiments, the additional polypeptide comprises a matrix protein, a nucleoprotein, or a non-structural protein.

[0315] RNA viruses are highly diverse in terms of viral particles, genomic structures, and viral entry and assembly mechanisms. However, they share fundamental features in their genome replication and transcription, and often use virus-encoded RdRPs to carry out the biosynthesis of RNA products directed by an RNA template. Genome replication mechanisms typically require the involvement of other factors during the initiation phase of synthesis, but RdRPs govern the elongation phase of synthesis, which includes thousands of efficient nucleotide addition cycles (NACs). Viral RdRPs vary widely in size and structural organization, ranging from picornavirus 3Dpol of approximately 50 kDa to flavivirus NS5 of approximately 100 kDa, which contains a naturally fused methyltransferase domain, the RSVL protein of approximately 250 kDa with at least three enzymatic domains, and the approximately 260 kDa three-subunit PA-PB1-PB2 influenza virus replicase complex. On the other hand, all RdRPs share a polymerase core of 50 - 70 kDa that forms a distinct right-handed structure surrounded by domains resembling the palm, fingers, and thumb. Among the seven classical RdRP catalytic motifs, A - E are within the most conserved palm domain, and F and G are located within the fingers, and they are all similarly arranged around the active site. The structural conservation of the RdRP polymerase core and the seven motifs forms the basis for understanding common features in the viral RdRP catalytic mechanism and for finding intervention strategies targeting these enzymes with a potentially broad spectrum of possibilities.

[0316] In some embodiments, the encrypted RNA is similar to RNA of viral origin but, instead of encoding a polypeptide (homologous) that is native to the virus, the encrypted RNA encodes a therapeutic polypeptide (heterologous) that is not native to the virus. In this case, the construct of encrypted RNA encoding the therapeutic polypeptide of interest is arranged with an adjacent L region and an adjacent R region, and this arrangement is not "native" to the virus.

[0317] In some embodiments, the encrypted RNA is similar to viral RNA and has cis - acting sequences sufficient to be encapsidated into viral particles. In some further embodiments, the encrypted RNA is infectious and has cis - acting sequences sufficient to be encapsidated into viral particles that can transmit and deliver the encrypted RNA to additional cells via viral infection. In some embodiments, the RNA species produced after contacting the encrypted RNA with a target - specific translation activator is competent for encapsidation into viral particles. In some further embodiments, the RNA species produced after contacting the encrypted RNA with a target - specific translation activator is infectious and has cis - acting sequences sufficient to be encapsidated into viral particles that can transmit and deliver the RNA species produced via viral infection to additional cells.

[0318] Animal negative - strand RNA viruses are grouped into several families and include well - known disease agents such as the pathogens of rabies, mumps, measles, and influenza, as well as more emerging pathogens such as Ebola virus. In all of these, the single - stranded RNA in the viral particle is complementary to the mRNA and is thus the minus strand. These viruses, although different in shape and structure, are similar in that they have an outer envelope derived from the membrane of the host cell in which they are assembled. Thus, the RNA of negative - strand RNA viruses is the antisense strand.

[0319] After infiltrating the cell, an important mission of the negative - strand RNA virus is to make its RNA double - stranded by synthesizing the corresponding positive RNA strand. Once double - stranded, both RNA strands are used as templates. The plus strand (or " + strand" ) is used as a template to produce more negative strands for the next generation of viral particles. The minus strand (or " - strand" ) is used as a template to produce multiple positive strands that act as mRNA molecules. This strategy is not only an effective division of labor but also avoids the problem of translating multiple reading frames on a single incoming viral RNA molecule.

[0320] Positive-strand RNA viruses, also known as sense-strand RNA viruses, are viruses whose genetic information consists of a single strand of RNA that is the plus (or sense) strand encoding mRNA and proteins. Replication in positive-strand RNA viruses proceeds through a minus-strand intermediate. Non-limiting examples of positive-strand RNA viruses include coronaviruses, polioviruses, coxsackieviruses, and echoviruses.

[0321] Some RNA viruses, including all retroviruses and lentiviruses, produce a DNA copy of their RNA genome during one aspect of their natural viral life cycle. The virus-encoded RdDP or reverse transcriptase is a polymerase that reverse transcribes the viral genomic RNA into a DNA copy, which can then be integrated into the host cell chromosome or maintained episomally outside the chromosome. The term proviral DNA or provirus DNA can be used to describe the DNA copy of the retroviral genome.

[0322] Some DNA viruses, such as viruses of the family Hepadnaviridae, whose members are hepatitis B virus, "HBV", replicate their DNA viral genomes through an RNA intermediate and possess an RdDP or reverse transcriptase to convert the RNA intermediate into a DNA template molecule for further genome amplification.

[0323] In some embodiments, the sequences within the encrypted RNA are converted to DNA by a translation activator consisting of an RdDP or reverse transcriptase. The DNA sequence can then be further transcribed to mRNA by the translation activator.

[0324] Some RNA viruses, such as hepatitis delta virus (HDV), are thought to transcribe their viral RNAs to produce mRNA using the low levels of RdRP activity of specific host RNA polymerases, including human RNA polymerase I (human PolI), human RNA polymerase II (human PolII), or human RNA polymerase III (human PolIII). These host RNA polymerases are typically thought to be primarily DNA-dependent RNA polymerases, but may be able to synthesize RNA from a DNA template or an RNA template.

[0325] In some embodiments, the translation activator of the encrypted RNA is composed of a polymerase that can synthesize RNA from a DNA template or an RNA template.

[0326] In some embodiments, the translation activator of the encrypted RNA consists of a viral RdRP. Without wishing to be bound by any particular theory, it is thought that activation of the encrypted RNA to mRNA occurs because the encrypted RNA contains a virus-derived sequence that can bind to the viral RdRP complex. In some embodiments, activation of the encrypted RNA occurs because the encrypted RNA contains a virus-derived sequence that can bind to the viral RdDP complex. In some embodiments, the polypeptide of interest of the therapeutic encrypted RNA is translated at low levels by the host cell ribosomal machinery in the absence of viral infection, and production of the therapeutic protein during viral infection is increased.

[0327] In some embodiments, the encrypted RNA may resemble a viral replication intermediate that the virus synthesizes into mRNA to replicate its genome. In other words, both the encrypted RNA and the reverse complement of the encrypted RNA can be activated by a translation activator. In some embodiments, when viral infection ends, the half-lives of the produced mRNA and the polypeptide of interest are short, and in the absence of the translation activator, new mRNA cannot be substantially produced, so translation of the polypeptide of interest of the encrypted RNA substantially ends.

[0328] In some embodiments, the encrypted RNA does not contain an internal ribosome entry site (IRES) sequence. In some embodiments, the encrypted RNA is engineered to lack features important for efficient protein translation by the host cell ribosome, such as a 5'-Cap or a 3' poly(A) tail. In some embodiments, the translation of the polypeptide of interest encoded by the encrypted RNA is at least 10-fold, 100-fold, 1000-fold, 10 4 -fold, 10 5 -fold or more increased in the presence of a viral infection (see, e.g., FIG. 4).

[0329] In some embodiments, the virus is selected from the group consisting of viruses of the orders Amarillovirales, Articulavirales, Blubervirales, Bunyavirales, Hepelivirales, Martellivirales, Mononegavirales, Nidovirales, and Picornavirales. In some embodiments, the virus is selected from the group consisting of viruses of the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, Rhabdoviridae, and Togaviridae.In some embodiments, the virus is selected from the group consisting of Alpha coronavirus 229E, Alpha coronavirus NL63, Alpha coronavirus WA2028, Avian metapneumovirus (AMPV), Beta coronavirus HKU1, Beta coronavirus HKU15, Beta coronavirus HKU33, Beta coronavirus OC43, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Eastern equine encephalitis virus (EEEV), Enterovirus D68 (EV-D68), Foot-and-mouth disease virus, Hantavirus, Hendra virus, Hepatitis B virus, Hepatitis C virus, HMPV, Human parainfluenza virus 1 (HPIV1), Human parainfluenza virus 3 (HPIV3), Infectious salmon anemia virus, Influenza A virus, Influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, Norwalk virus, Rabies virus, Respiratory syncytial virus, Reston Ebola virus, Rhinovirus, Rift Valley fever virus, Rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan equine encephalitis virus (VEEV), Vesicular stomatitis virus, Western equine encephalitis virus (WEEV), Yellow fever virus, Zaïre Ebola virus, and Zika virus.

[0330] In some embodiments, the virus does not belong to any of the orders selected from Amarillovirales, Articulavirales, Blubervirales, Bunyavirales, Hepelivirales, Martellivirales, Mononegavirales, Nidovirales, and Picornavirales. In some embodiments, the virus does not belong to any of the viral families of Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, Rhabdoviridae, and Togaviridae.

[0331] In some embodiments, the virus is not an alpha coronavirus. In some embodiments, the virus is not a metapneumovirus. In some embodiments, the virus is not a beta coronavirus. In some embodiments, the virus is not a chikungunya virus. In some embodiments, the virus is not a Crimean-Congo hemorrhagic fever virus. In some embodiments, the virus is not a dengue virus. In some embodiments, the virus is not an eastern equine encephalitis virus (EEEV). In some embodiments, the virus is not an enterovirus D68 (EV-D68). In some embodiments, the virus is not a foot-and-mouth disease virus. In some embodiments, the virus is not a hantavirus. In some embodiments, the virus is not a Hendra virus. In some embodiments, the virus is not a hepatitis B virus. In some embodiments, the virus is not a hepatitis C virus. In some embodiments, the virus is not an HMPV. In some embodiments, the virus is not a human parainfluenza virus 1 (HPIV1). In some embodiments, the virus is not an infectious salmon anemia virus. In some embodiments, the virus is not an influenza A virus. In some embodiments, the virus is not an influenza B virus. In some embodiments, the virus is not a Lassa virus. In some embodiments, the virus is not a Marburg virus. In some embodiments, the virus is not a Middle East respiratory syndrome coronavirus (MERS-CoV). In some embodiments, the virus is not a Newcastle disease virus (NDV). In some embodiments, the virus is not a Nipah virus. In some embodiments, the virus is not a Norwalk virus. In some embodiments, the virus is not a rabies virus. In some embodiments, the virus is not a respiratory syncytial virus. In some embodiments, the virus is not an Ebola virus. In some embodiments, the virus is not a rhinovirus. In some embodiments, the virus is not a Rift Valley fever virus. In some embodiments, the virus is not a rubella virus.In some embodiments, the virus is not SARS-CoV-1. In some embodiments, the virus is not SARS-CoV-2. In some embodiments, the virus is not Sudan Ebola virus. In some embodiments, the virus is not Venezuelan equine encephalitis virus (VEEV). In some embodiments, the virus is not vesicular stomatitis virus. In some embodiments, the virus is not Western equine encephalitis virus (WEEV). In some embodiments, the virus is not yellow fever virus. In some embodiments, the virus is not Zaire Ebola virus. In some embodiments, the virus is not Zika virus.

[0332] Methods for making any of the encrypted RNAs described herein are also within the scope of the disclosure. In some embodiments, the encrypted RNA is produced extracellularly via in vitro transcription (IVT) using an RNA polymerase and a DNA template molecule encoding the encrypted RNA. In some embodiments, the encrypted RNA is then processed and prepared via IVT as a precursor molecule that results in the encrypted RNA. Methods of using in vitro transcription to produce RNA, such as methods of producing RNA with reduced levels or that do not contain immunostimulatory by-products, will be apparent to those of skill in the art. See, for example, Dousis et al. Nature Biotechnology (2023) 41:560-568.

[0333] In some embodiments, the encrypted RNA is made by a method of in vitro transcription that includes (a) providing a DNA vector encoding any of the encrypted RNAs described herein, (b) linearizing the DNA vector to produce a linear DNA vector, and (c) contacting the linear DNA vector with an RNA polymerase (e.g., at about 50°C) to thereby produce an isolated RNA polynucleotide. In some embodiments, the contacting in (c) is performed in the presence of one or more additional factors (e.g., ribonucleotide triphosphates, modified nucleotide triphosphates, cap analogs, inorganic pyrophosphatase, and RNase inhibitors).

[0334] In some embodiments, the method further comprises subjecting the isolated RNA polynucleotide of (d)(c) to one or more purification steps, for example, contacting the isolated RNA polynucleotide with a DNAse under conditions suitable for digestion of the DNA vector. And tangential flow filtration is included.

[0335] In some embodiments, the DNA vector includes a promoter capable of directing the activity of an RNA polymerase and / or a restriction endonuclease recognition site. In some embodiments, the RNA polymerase is T7 RNA polymerase and the promoter is a T7 promoter. In some embodiments, linearizing the DNA vector comprises contacting the DNA vector with a restriction endonuclease that recognizes the restriction endonuclease recognition site. In some embodiments, the method further comprises formulating the isolated RNA polynucleotide into nanoparticles.

[0336] In some embodiments, the precursor-encrypted RNA is composed of an encrypted RNA portion and a ribozyme portion, and the ribozyme portion cleaves the precursor-encrypted RNA to produce two shorter RNA products comprising the encrypted RNA and the ribozyme. In some embodiments, after cleavage of the precursor-encrypted RNA by the ribozyme portion, the encrypted RNA is 5'-monophosphorylated.

[0337] Exemplary sequence elements of encrypted RNA or DNA-encoded encrypted RNA are listed in Table 1. Exemplary pairings of sequence elements that can be used together as elements of encrypted RNA are listed in Table 2. Exemplary coding sequences and reverse complements of the coding sequences (i.e., antisense coding sequences) are listed in Table 3. Some useful sequences for making some encrypted RNAs or for encrypted RNAs encoding DNA are listed in Table 4. Some exemplary amino acid sequences of the polypeptide of interest are listed in Table 5.

[0338] The following table should be read in order to list the next table or continue for potentially multiple pages until the table ends. For example, Table 1 continues over multiple pages until Table 2 begins. Similarly, Table 5 continues until the next section titled "Target Protein" begins. As long as DNA sequences are listed, it is understood that the sequences also disclose and embody their RNA counterparts (T→U). Similarly, when RNA sequences are listed, it is understood that the sequences also disclose and embody their DNA counterparts (U→T).

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 1-62

Table 1-63

Table 1-64

Table 1-65

Table 1-66

Table 1-67

Table 1-68

Table 1-69

Table 1-70

Table 1-71

Table 1-72

Table 1-73

Table 1-74

Table 1-75

Table 1-76

Table 1-77

Table 1-78

Table 1-79

Table 1-80

Table 1-81

Table 1-82

Table 1-83

Table 1-84

Table 1-85

Table 1-86

Table 1-87

Table 1-88

Table 1-89

Table 1-90

Table 1-91

Table 1-92

Table 1-93

Table 1-94

Table 1-95

Table 1-96

Table 1-97

Table 1-98

Table 1-99

Table 1-100

Table 1-101

Table 1-102

Table 1-103

Table 1-104

Table 1-105

Table 1-106

Table 1-107

Table 1-108

Table 1-109

Table 1-110

Table 1-111

Table 1-112

Table 1-113

Table 1-114

Table 1-115

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

[0339] As will be apparent to those skilled in the art, the nucleotide positions shown as allowing mutations correspond to the nucleotide positions in the reference sequence provided in that row. For example, in the first row, any of the nucleotides at positions 14 to 37 of the 5pIAV (SEQ ID NO: 1) can be mutated. In some embodiments, the encoded RNA contains one or more mutations at any of the positions shown in Table 6. Further exemplary mutations are shown in Table 17.

[0340] Protein of interest The terms "polypeptide", "peptide", "amino acid sequence" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched.

[0341] In some embodiments, the therapeutic polypeptide of interest comprises a "protein that causes cell death". When a protein that causes cell death is produced at a sufficient concentration within a cell, it increases the rate of cell death and thus decreases the expected lifespan of the cell. Examples of proteins that cause cell death include, but are not limited to, granzyme A and granzyme K, including granzyme; perforin; BCL-2 homology domain 3-only protein, B-cell lymphoma-2 (Bcl-2) family protein BIM, p53 upregulated apoptosis regulator (PUMA), Bid, Bcl2 modifying factor (BMF), Noxa, Bcl-2 interacting killer (BIK), BCL2-related X, apoptosis regulator (BAX), Bcl-2 cell death agonist (BAD), and other pro-apoptotic members of the BCL-2 family; herpes simplex virus thymidine kinase; vaccinia virus E3L; receptor-interacting protein kinase 3 (RIPK3) / mixed lineage kinase domain-like protein (MLKL); caspases including caspase-3, caspase-6, and caspase-7; gasdermin D.

[0342] In some embodiments, a protein that causes cell death can further increase the rate of cell death if the surrounding environment contains molecules at a concentration sufficient for the protein that causes cell death to contact and produce new cytotoxic molecules. Such enhancing proteins can include the use of herpes simplex virus thymidine kinase in combination with ganciclovir.

[0343] As used herein, "immune response" can be a specific response of the adaptive immune system to a particular antigen (i.e., a specific or adaptive immune response), a non-specific response of the innate immune system (i.e., a non-specific or innate immune response), or a combination thereof.

[0344] As used herein, "immunogenicity" refers to the ability of the polynucleotides of the present disclosure to induce an immune response. Some embodiments described herein involve reducing the immunogenicity of polynucleotides using RNA constructs having modified nucleotides in the absence of activation by a translational activator. One aspect of the present disclosure is the discovery that polynucleotides having an L region and an R region containing modified nucleotides can still be recognized by viral polymerases and efficiently replicated.

[0345] Nucleotide modifications can change the secondary or tertiary structure of polynucleotides, and these secondary and tertiary structures are important for the polynucleotide to react with and induce polymerase activity, so this discovery is surprising. Furthermore, there are prior art reports that viral RdRps, particularly alphavirus RdRps, cannot replicate a template without unmodified uridine nucleotides for efficient protein translation (see, for example, Beissert T et al., A Trans-amplifying RNA Vaccine Strategy for Induction of Potent Protective Immunity. Mol Ther. (2020) 28(1):119-128).

[0346] Note that attempts to use nucleoside-modified alphavirus replicons (``self-amplifying RNAs'') encoding SARS-CoV-2 vaccine antigens have resulted in loss of antigen (protein) production in vivo (Voigt, E.A., et al. A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability. Npj Vaccines (2022). DOI: 10.1038 / s41541-022-00549-y). In contrast, in some embodiments, the encrypted RNAs described herein can be developed by modifying 100% of the uridine nucleotides and retain at least equivalent or greater protein production in the presence of viral polymerase in addition to a substantial reduction in immunogenicity in the absence of viral polymerase. It has been recognized that some viral RNAs are modified by cellular enzymes at selected positions during their natural life cycle. As an example, the adenosine of hepatitis C virus, Zika virus and feline leukemia virus is post-transcriptionally modified to N6-methyladenosine by cellular methyltransferases (Gokhale N. & Horner S; PLoS Pathog. 2017 Mar;13(3):e1006188). The modifications are thought to be rare; for example, the 5 kb RNA genome of Rous sarcoma virus is modified at only 15 positions (about 1% of the positions in the transcript). As used herein, an ``immunomodulatory polypeptide'' refers to a polypeptide that can alter the immune response, including inducing or suppressing the maturation of immune cells, inducing or suppressing cytokine biosynthesis, or altering humoral immunity by stimulating antibody production by B cells. Immunomodulatory polypeptides can have antiviral and antitumor activities and can also downregulate other aspects of the immune response, for example, diverting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases.

[0347] In some embodiments, the polypeptide of interest is an immunomodulatory polypeptide. In some further embodiments, the polypeptide of interest is an immunomodulatory polypeptide that is immunogenic in a subject, i.e., an immunomodulatory polypeptide that acts as an antigen in a subject to elicit an immune response. As used herein, the term "antigen" means an immunogenic compound that elicits an adaptive immune response in a subject being treated with the antigen. In particular, "antigen" relates to any substance that induces an antigen-specific antibody or T lymphocyte (T cell) response in the subject being treated. The term "antigen" includes any molecule that contains at least one epitope. In one aspect, an antigen is a molecule that, optionally after processing, induces an immune reaction specific for the antigen of the subject being treated. Antigens can include polypeptides derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or polypeptides derived from cancers including tumor antigens. In one aspect, an antigen corresponds to a naturally occurring product, e.g., a polypeptide that is naturally presented on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor. An antigen can elicit an immune response against a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor.

[0348] The term "pathogen" refers to a pathogenic biological agent that can cause disease in an organism. Pathogens include microorganisms such as bacteria, single-celled eukaryotes (protozoa), fungi, and viruses.

[0349] In some embodiments, the polypeptide of interest contains an antigen suitable for vaccination of a target organism. In some embodiments, the antigen is selected from the group consisting of self-antigens and non-self-antigens. A non-self antigen can be a viral antigen, a bacterial antigen, a fungal antigen, an allergen, or a parasite antigen.

[0350] In some embodiments, the antigen is a self - antigen, particularly a tumor antigen. The term "tumor antigen" or "tumor - associated antigen" refers to a protein that is specifically expressed under normal conditions in a limited number of tissues or organs or at specific developmental stages. For example, a tumor antigen can be specifically expressed under normal conditions in gastric tissue, such as gastric mucosa, reproductive organs, such as testis, trophoblast tissue, such as placenta, or germ - line cells, and is expressed or abnormally expressed in one or more tumors or cancer tissues. In this context, the "limited number" can be 3 or less, or 2 or less. Tumor antigens in the context of the present disclosure include, for example, differentiation antigens, such as cell - type - specific differentiation antigens, i.e., proteins that are specifically expressed in a specific cell type at a specific differentiation stage under normal conditions, cancer / testis antigens, i.e., proteins that are specifically expressed in the testis and sometimes in the placenta under normal conditions, and germ - line - specific antigens. In some embodiments, the tumor antigen is associated with the cell surface of cancer cells and is not expressed or rarely expressed in normal tissues. In some embodiments, the tumor antigen or the abnormal expression of the tumor antigen identifies cancer cells. In some embodiments, the tumor antigen expressed by cancer cells of a subject, such as a patient suffering from a cancer disease, is the subject's own protein. In some embodiments, the tumor antigen in the context of the present disclosure is expressed under normal conditions, particularly in tissues or organs that are not essential, i.e., tissues or organs that do not cause the death of the subject when damaged by the immune system, or in body organs or structures that are inaccessible or hardly accessible by the immune system.

[0351] As used herein, the "background translation" of a polypeptide of interest means the translation of the polypeptide of interest in the absence of a translation activator.

[0352] In some embodiments, the background translation of the polypeptide of interest is substantially non - immunogenic.

[0353] In some embodiments, the background translation of the polypeptide of interest is not substantially immunogenic. However, the translation of the polypeptide of interest in the presence of a translation activator (e.g., in viral infection or the expression of a polynucleotide encoding a translation activator) is substantially immunogenic.

[0354] Aspects of the present disclosure relate, at least in part, to an encrypted RNA comprising a coding sequence encoding a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an immunotherapy polypeptide. In some embodiments, the immunotherapy polypeptide of interest is a chemokine. In some embodiments, the immunotherapy polypeptide of interest is a cytokine. In some embodiments, the immunotherapy polypeptide of interest is a Toll-like receptor (TLR) agonist. In some embodiments, the therapeutic polypeptide of interest is a structural protein. In some embodiments, the therapeutic polypeptide of interest is a blood protein. In some embodiments, the immunotherapy polypeptide of interest is a programmed cell death polypeptide. In some embodiments, the immunotherapy polypeptide of interest is an antigen. In some embodiments, the immunotherapy polypeptide of interest is an antibody. In some embodiments, the therapeutic polypeptide of interest is an endocrine polypeptide. In some embodiments, the therapeutic polypeptide of interest is a human polypeptide. In some embodiments, the therapeutic polypeptide of interest is a receptor. In some embodiments, the therapeutic polypeptide of interest is a binding protein. In some embodiments, the therapeutic polypeptide of interest is a transcription factor. In some embodiments, the therapeutic polypeptide of interest is a tumor suppressor protein. In some embodiments, the therapeutic polypeptide of interest is a T cell receptor protein. In some embodiments, the therapeutic polypeptide of interest is a homing receptor. In some embodiments, the therapeutic polypeptide of interest is a translation factor. In some embodiments, the therapeutic polypeptide of interest is a membrane transporter. In some embodiments, the therapeutic peptide of interest is neither a viral peptide nor of viral origin.

[0355] In some embodiments, the immunotherapy polypeptide is a chemokine. As used herein, the term "chemokine" refers to a peptide that acts as a chemoattractant to guide cell movement. In some embodiments, the chemokine is chemokine ligand 1 (CCL1), CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, XCL1, XCL2, CX3CL1, IL-8, MCP-1, MIP-1 alpha.

[0356] In some embodiments, the immunotherapeutic polypeptide of interest is a cytokine. As used herein, the term "cytokine" refers to a peptide that is a signaling protein that aids in the control of immune system responses. In some embodiments, the cytokine is human tumor necrosis factor precursor (Homo sapiens), TNFα, TNFβ, prostaglandin, RANKL, VEGF, selectin, addressin. In some embodiments, the cytokine is an interleukin. As used herein, the term "interleukin" refers to a group of cytokines expressed and secreted by somatic cells. In some embodiments, the interleukin is interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, human interleukin-1 receptor antagonist precursor (Homo sapiens), human interleukin-12A and interleukin-12B precursor (bicistronic via P2A site) (Homo sapiens), human interleukin-2 precursor (Homo sapiens), mouse interleukin-12A and interleukin-12B precursor (bicistronic via P2A sequence) (Mus musculus), mouse interleukin-2 precursor (Mus musculus). In some embodiments, the cytokine is an interferon. As used herein, the term "interferon" refers to a group of cytokines expressed and secreted by somatic cells in response to infection.In some embodiments, the interleukin is human interferon-beta precursor (Homo sapiens), human interferon-lambda 3 precursor (Homo sapiens), human interferon-lambda 3 precursor (Homo sapiens), human interferon-lambda precursor (Homo sapiens), mouse interferon-kappa precursor (Mus musculus), mouse interferon-lambda 2 precursor (Mus musculus), mouse interferon-lambda 3 precursor (Mus musculus), Syrian hamster interferon-beta 1 precursor (Mesocricetus auratus), Syrian hamster interferon-lambda 3 precursor (Mesocricetus auratus), cotton rat interferon-alpha precursor (Sigmodon hispidus), cotton rat interferon-beta 1 precursor (Sigmodon hispidus), cotton rat interferon-lambda 2 precursor (Sigmodon hispidus), domestic ferret interferon-beta 1 precursor (Mustela putorius furo), IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα7, IFNα8, IFNα10, IFNα13, IFNα14, IFNα16, IFNα17, IFNα21, IFNβ1, IFN-ω, IFN-γ, IFN-κ, IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β1, IFN-ε, IFN-κ, IFN-ω1, IFN-γ, IFN-λ1 (IL28A), IFN-λ2 (IL28B), IFN-λ3 (IL29) or IFN-λ4. In some embodiments, the cytokine is a colony stimulating factor (CSF).As used herein, the term "colony stimulating factor" refers to a secreted glycoprotein that binds to a receptor protein on the surface of hematopoietic stem cells. In some embodiments, the colony stimulating factor is CSF-1, CSF-2, CSF-3, CSF-4, CSF-5, CSF-6, G-CSF, GM-CSF or M-CSF. In some embodiments, the immunotherapeutic polypeptide of interest is a Toll-like receptor (TLR) agonist. In some embodiments, the TLR agonist is of bacterial origin. In some embodiments, the TLR agonist of bacterial origin is BCSP31, FHA, MOMP, FomA, MymA (Rv3083), ESAT6, PorB, PVL, Porin, OmpA, PepO, OmpU, or flagellin. In some embodiments, the TLR agonist is of viral origin. In some embodiments, the TLR agonist of viral origin is glycoprotein F, envelope glycoprotein, glycoprotein GP, NS3, hemagglutinin H, gB, gH, gL, gp120, gp41, p24, or p17. In some embodiments, the immunotherapeutic polypeptide of interest is an interferon-stimulated gene. In some embodiments, the interferon-stimulated gene is BST2 (tettrin), C6orf150 (MB21D1), DDX58, EIFAK2, HPSE, IFIH1 (MDA5), IFIT1, IFIT2, IFIT3, IFIT5, IFITM1, IFITM2, IFITM3, IRF1, IRF7, ISG15, ISGS20, MX1, MX2, NAMPT (PBEF1), OAS1, OAS2, OAS3, RSAD2 (viperin), or ZC3HAV1 (ZAP).

[0357] In some embodiments, the therapeutic polypeptide of interest is a structural protein. In some embodiments, the structural protein is collagen, fibrin, fibrinogen, elastin, tubulin, actin, or myosin.

[0358] In some embodiments, the therapeutic polypeptide of interest is a blood protein. As used herein, the term "blood protein" refers to a protein present in plasma. In some embodiments, the blood protein is thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF) or modified factor VIII, or an anticoagulant.

[0359] In some embodiments, the immunotherapy polypeptide of interest is a programmed cell death polypeptide. As used herein, the term "programmed cell death polypeptide" refers to a polypeptide that initiates a series of intracellular molecular processes that lead to its death. In some embodiments, the programmed cell death polypeptide is a granzyme. In some embodiments, the granzyme is granzyme A. In some embodiments, the granzyme is granzyme K. In some embodiments, the programmed cell death polypeptide is perforin. In some embodiments, the programmed cell death polypeptide is an apoptosis protein. As used herein, the term "apoptosis protein" refers to a protein involved in cell death. In some embodiments, the apoptosis protein is a protein containing only the BCL-2 homology domain 3, BIM, PUMA, BID, BMF, NOXA, BIK, BAD, herpes thymidine kinase, vaccinia virus E3L, RIPK3 / MLKL, caspases including caspase-3, caspase-6, and caspase-7, or gasdermin D.

[0360] In some embodiments, the immunotherapeutic polypeptide of interest is an antigen. As used herein, the term "antigen" refers to a molecule or moiety or substance that can bind to a specific antibody or T cell receptor and elicit an immune response. In some embodiments, the antigen is a tumor antigen. In some embodiments, the tumor antigen is alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), MAGE, BAGE, GAGE, NY-ESO-1, HER2, HPV16E7, WT1, MART-1, gp100, tyrosinase, URLC10, VEGFR1, VEGFR2, MUC1, MUC2, survivin, TRP1 / gp75, TRP2, ganglioside, PSMA, or EphA3.

[0361] In some embodiments, the immunotherapeutic polypeptide of interest is an antibody. As used herein, the term "antibody" refers to a peptide used by the immune system to identify and counter foreign substances. In some embodiments, the antibody is a therapy for cancer. In some embodiments, the cancer antibody is trastuzumab, pembrolizumab, bevacizumab, cetuximab, ibritumomab, ofatumumab, or obinutuzumab. In some embodiments, the antibody is a therapy for viral infections. In some embodiments, the viral antibody is ansbuvimab, atorolimumab, mafosfamide, odesivimab, ibalizumab, obiltoxaximab, raxibacumab, sotrovimab, tixagevimab, cilgavimab, palivizumab, or immunoglobulin. In some embodiments, the antibody is a therapy for autoimmune disorders. In some embodiments, the autoimmune antibody is clazakizumab, clenoliximab, fezakinumab, fretkumab, gimsilumab, guselkumab, rituximab, or denosumab.

[0362] In some embodiments, the therapeutic polypeptide of interest is an endocrine polypeptide. In some embodiments, the endocrine polypeptide is a hormone. In some embodiments, the hormone is insulin, erythropoietin, thyroid hormone, catecholamine, gonadotropin, trophic hormone, human growth hormone, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, adrenocorticotropic hormone (ACTH), atropine, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, follicle-stimulating hormone (FSH), luteinizing hormone (LH), melanocyte-stimulating hormone (MSH), parathyroid hormone (PTH), renin, somatostatin, thyrotropin-releasing hormone (TRH), or vasopressin. In some embodiments, the therapeutic polypeptide of interest is an endocrine polypeptide. In some embodiments, the endocrine polypeptide is a growth factor. In some embodiments, the growth factor is epidermal growth factor (EGF), nerve growth factor (NGF), insulin-like growth factor, fibroblast growth factor (FGF), or platelet-derived growth factor (PDGF). In some embodiments, the therapeutic polypeptide of interest is an endocrine polypeptide. In some embodiments, the endocrine polypeptide is a growth factor receptor. In some embodiments, the growth factor receptor is a WNT receptor, Tie, neurotrophin receptor, Ephrin receptor, insulin-like growth factor receptor (IGF receptor), epidermal growth factor receptor (EGF receptor), fibroblast growth factor receptor (FGF receptor), platelet-derived growth factor receptor (PDGF receptor), or vascular endothelial growth factor receptor (VEGF receptor).

[0363] In some embodiments, the endocrine polypeptide is an enzyme. In some embodiments, the enzyme is tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative, steroidogenic enzyme, kinase, phosphodiesterase, methylase, demethylase, dehydrogenase, cellulase, protease, lipase, phospholipase, aromatase, cytochrome, adenylate or guanylate cyclase, or neuraminidase.

[0364] In some embodiments, the therapeutic polypeptide of interest is a human peptide. In some embodiments, the human peptide is for gene therapy. In some embodiments, the human peptide for gene therapy is PCCA, PCCB, MMUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, holocarboxylase synthetase, ACAT1, glutaryl-CoA dehydrogenase, OCTN2, SLC22A5, MCAD, VLCAD, LCHAD, HADHA, HADHB, arginosuccinate lyase, ASS1, SLC25A13, BCKDHA, BCKDHB, DBT, CBS, MTHFR, MTR, MTRR, MMADHC, phenylalanine hydroxylase, FAH, TAT, HPD, PAX8, TSHR, DUOX2, SLC5A5, TG, TPO, TSHB, HBB, TRDN.

[0365] In some embodiments, the therapeutic polypeptide of interest is a receptor. In some embodiments, the receptor is a steroid hormone receptor, a peptide receptor or an integrin.

[0366] In some embodiments, the therapeutic polypeptide of interest is a binding protein. In some embodiments, the binding protein is a growth hormone or growth factor binding protein, a single-stranded binding protein, calmodulin, gelsolin, polypyrimidine tract binding protein, maltose binding protein, metallothionein, FABP6, syntaxin binding protein 2, syntaxin binding protein 3, androgen binding protein, TATA binding protein, LTBP2, E3 binding protein, CREB, retinol binding protein 2, retinol binding protein 4, RNA binding protein FUS, or tropomodulin.

[0367] In some embodiments, the therapeutic polypeptide of interest is a transcription factor. In some embodiments, the transcription factor is OCT4, SOX2, KLF4, MYC (OSKM), TFIIA, TFIIB, TFIID, TFIIE, TFIIF, or TFIIH.

[0368] In some embodiments, the therapeutic polypeptide of interest is a tumor suppressor protein. As used herein, the term "tumor suppressor protein" refers to a protein that regulates cells during cell division and replication. In some embodiments, the tumor suppressor protein is angiopoietin-2 (Ang2), APC, MADR2, p53, TGF-β, BRCA1, p16, p14, CADM1, or FAS.

[0369] In some embodiments, the therapeutic polypeptide of interest is a T cell receptor protein. In some embodiments, the T cell receptor protein is TCR-α, TCR-β, CD2, CD3, CD4, CD5, CD7, CD8, ζ chain (CD27), or CD28.

[0370] In some embodiments, the therapeutic polypeptide of interest is a homing receptor. In some embodiments, the homing receptor is integrin α4β1, vascular cell adhesion molecule-1 (VCAM-1), BCR, CD34, or GLYCAM-1.

[0371] In some embodiments, the therapeutic polypeptide of interest is a translation factor. In some embodiments, the translation factor is eIF1A, eIF5B, eIF1, eIF5A, eIF2, or eIF6.

[0372] In some embodiments, the therapeutic polypeptide of interest is a membrane transporter. As used herein, the term "membrane transporter" refers to a membrane protein involved in the movement of ions, small molecules, and macromolecules, such as another protein, across a biological membrane. In some embodiments, the membrane transporter is a sugar transporter. In some embodiments, the sugar transporter is GLUT-1, GLUT-2, GLUT-3, GLUT-4, GLUT-5, GLUT-7, GLUT-9, GLUT-10, GLUT-11, GLUT-12, GLUT-14, SGLT-1, SGLT-2, SGLT-3, SGLT-5, SGLT-6, or HMIT. In some embodiments, the membrane transporter is an amino acid transporter. In some embodiments, the amino acid transporter is CAT-1, CAT-2, CAT-3, SNAT-1, SNAT-2, SNAT-3, SNAT-4, SNAT-5, LAT-2, LAT-4f2hc, EAAT-1, EAAT-2, EAAT-3, EAAT-4, EAAT-XCT, EAAT-4f2hc, or TATA-1. In some embodiments, the membrane transporter is a lipid transporter. In some embodiments, the lipid transporter is FABPpm, FATP-1, FATP-2, FATP-3, FATP-4, FATP-5, FATP-6, ABC, or NPC1L1. In some embodiments, the membrane transporter is a nucleoside transporter. In some embodiments, the nucleoside transporter is CNT1, CNT2, ENT-1, ENT-2, ENT-3, or ENT-14.

[0373] In some embodiments, the therapeutic polypeptide is a peptide that inhibits viral replication. In some embodiments, the peptide that inhibits viral replication is APOBEC3G, ISG15, OASL.OAS1, OAS2, OAS3, PML (TRIM19), SP100, tetherin (BST2), viperin (RSAD2), IFITM1, IFITM2, or IFITM3.

[0374] In some embodiments, the coding sequence further comprises a signal peptide. As used herein, the term "signal peptide" refers to a short peptide that binds to another peptide (e.g., a therapeutic peptide or an industrially applicable peptide) to translocate the other peptide. The terms "signal sequence", "targeting signal", "localization signal", "localization sequence", "transport peptide", "leader sequence", and "leader peptide" are used interchangeably herein. In some embodiments, the signal peptide is Sec / SPI, Sec / SPII, Sec / SPIII, Tat / SPI, or Tat / SPII.

[0375] In some embodiments, the nucleotide sequences encoding the protein of interest are listed in Table 3. In some embodiments, the amino acid sequences of the protein of interest are listed in Table 5.

[0376] In some embodiments, the therapeutic polypeptide of interest consists of cytokines involved in the regulation of lymphoid homeostasis, such as cytokines involved in the development, priming, proliferation, differentiation, or survival of T cells and that induce or enhance them. In some embodiments, the cytokine is an interleukin, such as IL-1, IL-2, IL-6, IL-6RA, IL-7, IL-12, IL-15, IL-21, or IL-23. In some embodiments, the interleukin is an anti-inflammatory cytokine (e.g., IL-1 receptor antagonist (IL-1RN or IL-1RA), IL-23RA, IL-36RA, or IL-37, etc.).

[0377] In some embodiments, the therapeutic polypeptide of interest consists of "antineoplastic proteins". Antineoplastic proteins are polypeptides effective in the treatment of cancer. Specific classes of antineoplastic proteins include, but are not limited to, monoclonal antibodies, nanobodies, hormones, proteins that cause cell death, immune checkpoint inhibitors, interleukins, and immunogens.

[0378] In some embodiments, the polypeptide of interest is an antagonist of programmed cell death ligand 1 (PD-L1) or programmed cell death 1 (PD-1).

[0379] As used herein, a PD-1 antagonist is, for example, an agent that inhibits or prevents PD-1 activity by binding to PD-1.

[0380] PD-1 activity can be interfered with by an antibody that selectively binds to PD-1 and blocks the activity of PD-1. PD-1 activity can also be inhibited or blocked by molecules other than an antibody that binds to PD-1. Such molecules include proteins (such as fusion proteins) and peptides that bind to PD-1 but do not activate PD-1, for example, peptidomimetics of PD-L1 and PD-L2.

[0381] Exemplary PD-1 antagonists include those described in U.S. Patent Application Publication Nos. 20130280265, 20130237580, 20130230514, 20130109843, 20130108651, 20130017199, 20120251537, and 20110271358, and European Patent EP2170959B1, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-1 antagonists are described in Curran et al., PNAS, 107, 4275 (2010); Topalian et al., New Engl. J. Med. 366, 2443 (2012); Brahmer et al., New Engl. J. Med. 366, 2455 (2012); Dolan et al., Cancer Control 21, 3 (2014); and Sunshine et al., Curr. Opin. in Pharmacol. 23 (2015).

[0382] Exemplary PD-1 antagonists include nivolumab, a fully human IgG4 monoclonal antibody that binds to PD-1 (e.g., OPDIVO® manufactured by Bristol-Myers Squibb); pidilizumab (e.g., CT-011 of CureTech), a humanized IgG1 monoclonal antibody that binds to PD-1; pembrolizumab, a humanized IgG4-kappa monoclonal antibody that binds to PD-1 (e.g., KEYTRUDA® of Merck); MEDI-0680 (AstraZeneca / MedImmune), a monoclonal antibody that binds to PD-1; and REGN2810 (Regeneron / Sanofi), a monoclonal antibody that binds to PD-1. Another exemplary PD-1 antagonist is AMP-224 (Glaxo Smith Kline and Amplimmune), a recombinant fusion protein composed of the extracellular domain of programmed cell death ligand 2 (PD-L2) and the Fc region of human IgG1 that binds to PD-1.

[0383] A PD-L1 antagonist, as used herein, is, for example, an agent that inhibits or prevents PD-L1 activity by binding to PD-L1.

[0384] PD-L1 activity can be blocked by a molecule that selectively binds to PD-L1 and blocks the activity of PD-L1, for example, by blocking the interaction with PD-1 and / or B7-1 and the activation of PD-1 and / or B7-1. PD-L1 activity can also be inhibited or blocked by a molecule other t...

Claims

1. An isolated RNA polynucleotide comprising: a coding region having a coding sequence encoding one or more therapeutic polypeptides; a template region comprising two different regions, a left flanking region of the virus ("L region") and a right flanking region of the virus ("R region"), wherein the L region is adjacent and contiguous to the 5' end of the coding region, and the R region is adjacent and contiguous to the 3' end of the coding region; and wherein the coding sequence is in an antisense orientation; wherein the therapeutic polypeptide is heterologous to the virus; and wherein the template region interacts with and initiates the RNA-dependent polymerase activity of a polymerase in a cell containing an RNA-dependent polymerase. An isolated RNA polynucleotide.

2. The reverse complement of the isolated RNA polynucleotide of claim 1.

3. The isolated RNA polynucleotide of claim 1 or 2, wherein the virus is selected from the group consisting of viruses in the order Amarylvirales, Articulavirales, Blubervirales, Bunyavirales, Hepelivirales, Martellivirales, Mononegavirales, Nidovirales, and Picornavirales in that order.

4. The isolated RNA polynucleotide according to any one of claims 1 to 3, wherein the virus is selected from the group consisting of viruses of the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, Rhabdoviridae and Togaviridae.

5. The isolated RNA polynucleotide according to any one of claims 1 to 4, wherein the virus is selected from the group consisting of alpha coronavirus 229E, alpha coronavirus NL63, alpha coronavirus WA2028, turkey metapneumovirus (AMPV), beta coronavirus HKU1, beta coronavirus HKU15, beta coronavirus HKU33, beta coronavirus OC43, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, eastern equine encephalitis virus (EEEV), enterovirus D68 (EV-D68), foot-and-mouth disease virus, hantavirus, Hendra virus, hepatitis B virus, hepatitis C virus, HMPV, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), infectious salmon anemia virus, influenza A virus, influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, Norwalk virus, rabies virus, respiratory syncytial virus, Reston Ebola virus, rhinovirus, Rift Valley fever virus, rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan equine encephalitis virus (VEEV), vesicular stomatitis virus, western equine encephalitis virus (WEEV), yellow fever virus, Zaire Ebola virus, and Zika virus.

6. The isolated RNA polynucleotide according to any one of claims 1 to 5, wherein the virus is not an alphavirus.

7. The isolated RNA polynucleotide according to any one of claims 1 to 6, wherein the template region is native to the virus.

8. The isolated RNA polynucleotide according to any one of claims 1 to 7, wherein the template region is a variant of the native template region for the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native template region for the virus.

9. The isolated RNA polynucleotide according to any one of claims 1 to 8, wherein each of the L region and the R region of the template region contains 10, 9, 8, 7, 6, 5, 4, 3, or less than 2 mutations compared to the template region natural to the virus.

10. The isolated RNA polynucleotide according to any one of claims 1 to 9, wherein each of the L region and the R region of the template region is mutated from the template region natural to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping.

11. The isolated RNA polynucleotide according to any one of claims 1 to 10, wherein each of the L region and the R region of the template region is mutated from the template region natural to the virus by 1 or fewer substitutions that do not participate in 5' capping.

12. The isolated RNA polynucleotide according to any one of claims 1 to 11, wherein the isolated RNA polynucleotide comprises at least one nucleoside modification.

13. The isolated RNA polynucleotide according to any one of claims 1 to 12, wherein the template region is nucleoside-modified and the proportion of modified nucleosides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less.

14. The isolated RNA polynucleotide according to any one of claims 1 to 12, wherein the template region is nucleoside-modified and the proportion of modified nucleosides is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or 100%.

15. The isolated RNA polynucleotide according to claim 12 or 13, wherein the nucleoside modification is a non-immunogenic uridine modification and the proportion of modified uridine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less.

16. The isolated RNA polynucleotide according to any one of claims 12 or 14, wherein the nucleoside modification is a non-immunogenic uridine modification and the proportion of modified uridine modification is more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%, or 100%.

17. The isolated RNA polynucleotide according to claim 12 or 13, wherein the nucleoside modification is a non-immunogenic cytidine modification and the proportion of the modified cytidine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less.

18. The isolated RNA polynucleotide according to any one of claims 12 or 14, wherein the nucleoside modification is a non-immunogenic cytidine modification and the proportion of the modified cytidine modification is more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%, or 100%.

19. The isolated RNA polynucleotide according to claim 12 or 13, wherein the nucleoside modification is a non-immunogenic adenosine modification and the proportion of the modified adenosine modification is 1% to 30%.

20. The isolated RNA polynucleotide according to claim 19, wherein the nucleoside modification is a non-immunogenic adenosine modification and the proportion of the modified cytidine modification is about 1%, 5%, 10%, 15%, 20%, 25% or 30%.

21. The isolated RNA polynucleotide according to any one of claims 1 to 20, wherein the isolated polynucleotide comprises a 5' cap structure.

22. The isolated RNA polynucleotide according to any one of claims 1 to 21, wherein the 5' end of the L region comprises a 5' cap structure.

23. The isolated RNA polynucleotide according to any one of claims 1 to 22, wherein the 5' end of the L region comprises one or more mutations related to the 5' cap structure.

24. The isolated RNA polynucleotide according to claim 22 or 23, wherein the 5' cap structure is selected from the group consisting of Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structures.

25. The isolated RNA polynucleotide according to any one of claims 1 to 20, wherein the isolated polynucleotide does not comprise a 5' cap structure (is uncapped).

26. The isolated RNA polynucleotide according to any one of claims 1 to 20 or 25, wherein the 5' end of the L region does not contain a 5' cap structure (is uncapped). **Claim 27** The isolated RNA polynucleotide according to claim 25 or 26, wherein the 5' end of the isolated polynucleotide contains a 5'-monophosphate, 5'-diphosphate or 5'-triphosphate. **Claim 28** The isolated RNA polynucleotide according to claim 25 or 26, wherein the 5' end of the isolated polynucleotide does not contain a 5'-phosphate (is dephosphorylated). **Claim 29** The isolated RNA polynucleotide according to claim 2, wherein the template region is the reverse complement of the template region natural to the virus. **Claim 30** The isolated RNA polynucleotide according to claim 29, wherein the template region is a variant of the reverse complement of the template region natural to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reverse complement of the template region natural to the virus. **Claim 31** The isolated RNA polynucleotide according to claim 29 or 30, wherein each reverse complement of the L region and the R region varies from the reverse complement of the template region natural to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping. **Claim 32** The isolated RNA polynucleotide according to any one of claims 29 to 31, wherein each reverse complement of the L region and the R region varies from the reverse complement of the template region natural to the virus by 1 or fewer substitutions that do not participate in 5' capping. **Claim 33** The isolated RNA polynucleotide according to any one of claims 29 to 32, wherein the isolated RNA polynucleotide contains at least one nucleoside modification. **Claim 34** The isolated RNA polynucleotide according to claim 33, wherein the template region is nucleoside-modified and the proportion of modified nucleotides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less. ​ ​ ​ The isolated RNA polynucleotide according to any one of claims 2 or 29 to 35, wherein the 5' end of the R region is capped.

37. The isolated RNA polynucleotide according to any one of claims 1 to 36, wherein the therapeutic polypeptide is a secreted polypeptide.

38. The isolated RNA polynucleotide according to any one of claims 1 to 37, wherein the therapeutic polypeptide is selected from the group consisting of interferon, interferon-stimulated gene, cytokine, chemokine, antibody, signaling molecule, cytotoxic protein, protein that causes cell death, anti-neoplastic protein, immunomodulatory protein, protein toll-like receptor agonist or dominant negative protein.

39. The isolated RNA polynucleotide according to claim 38, wherein the cytokine is an inflammatory cytokine.

40. The isolated RNA polynucleotide according to claim 38, wherein the inflammatory cytokine is TNF-α.

41. The isolated RNA polynucleotide according to claim 38, wherein the cytokine is an anti-inflammatory cytokine.

42. The isolated RNA polynucleotide according to claim 41, wherein the anti-inflammatory cytokine is interleukin-1 receptor antagonist (IL-1RN).

43. The isolated RNA polynucleotide according to any one of claims 1 to 42, wherein the therapeutic polypeptide is interleukin or caspase.

44. The isolated RNA polynucleotide according to claim 43, wherein the interleukin is IL-12A, IL-12B or IL-2.

45. The isolated RNA polynucleotide according to claim 38, wherein the secreted protein is an antibody.

46. The isolated RNA polynucleotide according to claim 38, wherein the therapeutic polypeptide is interferon.

47. The isolated RNA polynucleotide according to claim 46, wherein the interferon is IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-γ or IFN-λ.

48. The isolated RNA polynucleotide according to claim 47, wherein the interferon is IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β1, IFN-ε, IFN-κ, IFN-ω1, IFN-γ, IFN-λ1 (IL28A), IFN-λ2 (IL28B), IFN-λ3 (IL29) or IFN-λ4.

49. The isolated RNA polynucleotide according to claim 46, wherein the interferon is IFN-α, IFN-β, IFN-κ, IFN-λ1 (IL28A), IFN-λ2 (IL28B) or IFN-λ3 (IL29).

50. The isolated RNA polynucleotide according to any one of claims 1 to 49, wherein the coding sequence encodes two or more therapeutic polypeptides separated by one or more ribosome skip sequences.

51. The isolated RNA polynucleotide according to any one of claims 1 to 50, wherein the coding region further comprises one or more regulatory elements selected from the group consisting of a ribosome binding site, a Kozak sequence, a Shine-Dalgarno sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site and an internal ribosome entry site (IRES).

52. The isolated RNA polynucleotide according to any one of claims 1 to 51, wherein the one or more regulatory elements are operably linked to the coding sequence.

53. The isolated RNA polynucleotide according to any one of claims 1 to 52, further comprising a polyadenylation signal and / or a 3' poly(A) tail.

54. The isolated RNA polynucleotide according to any one of claims 1 to 53, wherein the RNA-dependent polymerase is an RNA-dependent RNA polymerase.

55. The isolated RNA polynucleotide according to any one of claims 1 to 53, wherein the RNA-dependent polymerase is an RNA-dependent DNA polymerase.

56. The isolated RNA polynucleotide according to any one of claims 1 to 55, wherein the RNA-dependent polymerase is a polymerase derived from the virus.

57. The isolated RNA polynucleotide according to any one of claims 1 to 56, wherein the isolated RNA polynucleotide is single-stranded RNA. [[ID=,1]]

58. The isolated RNA polynucleotide according to any one of claims 1 to 57, wherein the isolated polynucleotide is in a linear form.

59. The isolated RNA polynucleotide according to any one of claims 1 to 57, wherein the isolated polynucleotide is in a covalently closed circular form.

60. wherein the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 2 or a variant of SEQ ID NO: 2, the variant of SEQ ID NO: 2 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 26 of SEQ ID NO: 2, and the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 20, 21, 22 or 23 or a variant of any one of SEQ ID NO: 20, 21, 22 or 23, the variant comprising mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 15 of any one of SEQ ID NO: 20, 21, 22 or 23, The isolated RNA polynucleotide according to any one of claims 1 to 59.

61. wherein the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 3 or a variant of SEQ ID NO: 3, (i) the variant of SEQ ID NO: 3 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 35 of SEQ ID NO: 3, and the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 24, 25, 26 or 27 or a variant of any one of SEQ ID NO: 24, 25, 26 or 27, (i) the variant of SEQ ID NO: 24 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 24, (ii) the variant of SEQ ID NO: 25 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 25, (iii) the variant of SEQ ID NO: 26 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 26, or (iv) the variant of SEQ ID NO: 27 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 27, The isolated RNA polynucleotide according to any one of claims 1 to 59.

62. wherein the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 4 or a variant of SEQ ID NO: 4, the variant of SEQ ID NO: 4 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 50 of SEQ ID NO: 4, and the R region comprises the nucleotide sequence shown as any one of SEQ ID NO: 28, 29, 30 or 31 or a variant of any one of SEQ ID NO: 28, 29, 30 or 31, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of any one of SEQ ID NO: 28, 29, 30 or 31, The isolated RNA polynucleotide according to any one of claims 1 to 59.

63. wherein the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 1 or 5 or a variant of SEQ ID NO: 1 or 5, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 37 of SEQ ID NO: 1 or 5, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 18 or 19 or a variant of SEQ ID NO: 18 or 19, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 20 of SEQ ID NO: 18 or 19, The isolated RNA polynucleotide according to any one of claims 1 to 59.

64. wherein the virus is an influenza virus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 6 or a variant of SEQ ID NO: 6, the variant of SEQ ID NO: 6 contains mutations at one or more nucleotide positions selected from positions 14 or 15 of SEQ ID NO: 6, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 32 or 33 or a variant of SEQ ID NO: 32 or 33, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 33 of SEQ ID NO: 32 or 33, The isolated RNA polynucleotide according to any one of claims 1 to 59.

65. wherein the virus is an influenza virus, The L region contains the nucleotide sequence shown as SEQ ID NO: 7 or a variant of SEQ ID NO: 7, the variant of SEQ ID NO: 7 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 20 of SEQ ID NO: 7, and The R region contains the nucleotide sequence shown as any one of SEQ ID NO: 34, 35, 36 or 37 or a variant of any one of SEQ ID NO: 34, 35, 36 or 37, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 5 to 8 of SEQ ID NO: 34, 35, 36 or 37, The isolated RNA polynucleotide according to any one of claims 1 to 59.

66. The virus is an influenza virus, The L region contains the nucleotide sequence shown as SEQ ID NO: 8 or a variant of SEQ ID NO: 8, the variant of SEQ ID NO: 8 contains mutations at one or more nucleotide positions selected from positions 14 or 15 of SEQ ID NO: 8, and The R region contains the nucleotide sequence shown as any one of SEQ ID NO: 38, 39, 40 or 41 or a variant of any one of SEQ ID NO: 38, 39, 40 or 41, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 13 of SEQ ID NO: 38, 39, 40 or 41, The isolated RNA polynucleotide according to any one of claims 1 to 59.

67. The virus is an influenza virus, The L region contains the nucleotide sequence shown as SEQ ID NO: 9 or a variant of SEQ ID NO: 9, the variant of SEQ ID NO: 9 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 18 of SEQ ID NO: 9, and The R region contains the nucleotide sequence shown as SEQ ID NO: 42 or 43 or a variant of SEQ ID NO: 42 or 43, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 14 of SEQ ID NO: 42 or 43, The isolated RNA polynucleotide according to any one of claims 1 to 59.

68. The virus is an influenza virus, The L region contains the nucleotide sequence shown as SEQ ID NO: 11 or a variant of SEQ ID NO: 11, the variant of SEQ ID NO: 11 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 81 of SEQ ID NO: 11, and the R region contains the nucleotide sequence shown as SEQ ID NO: 46 or 47 or a variant of SEQ ID NO: 46 or 47, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 5 to 9 of SEQ ID NO: 46 or 47, The isolated RNA polynucleotide according to any one of claims 1 to 59.

69. The virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 12 or a variant of SEQ ID NO: 12, the variant of SEQ ID NO: 12 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 52 of SEQ ID NO: 12, and the R region contains the nucleotide sequence shown as any one of SEQ ID NO: 48 or 49 or a variant of any one of SEQ ID NO: 48 or 49, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 11 of SEQ ID NO: 48 or 49, The isolated RNA polynucleotide according to any one of claims 1 to 59.

70. The virus is an influenza virus, the L region contains the nucleotide sequence shown as SEQ ID NO: 13 or a variant of SEQ ID NO: 13, the variant of SEQ ID NO: 13 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 87 of SEQ ID NO: 13, and the R region contains the nucleotide sequence shown as SEQ ID NO: 50 or 51 or a variant of SEQ ID NO: 50 or 51, the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 17 of SEQ ID NO: 50 or 51, The isolated RNA polynucleotide according to any one of claims 1 to 59.

71. The virus is an influenza virus, the L region contains the nucleotide sequence shown as any one of SEQ ID NO: 10 or a variant of any one of SEQ ID NO: 10, the variant of SEQ ID NO: 10 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 86 of SEQ ID NO: 10, and The R region contains the nucleotide sequence shown as SEQ ID NO: 44 or 45, or a variant of SEQ ID NO: 44 or 45, and the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 21 of SEQ ID NO: 44 or 45. The isolated RNA polynucleotide according to any one of claims 1 to 59. **Claim 72** The virus is an influenza virus. The L region contains the nucleotide sequence shown as SEQ ID NO: 14 or a variant of SEQ ID NO: 14, the variant of SEQ ID NO: 14 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 93 of SEQ ID NO: 14, and The R region contains the nucleotide sequence shown as any one of SEQ ID NO: 52 or 53, or a variant of any one of SEQ ID NO: 52 or 53, and the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 48 of SEQ ID NO: 52 or 53. The isolated RNA polynucleotide according to any one of claims 1 to 59. **Claim 73** The virus is an influenza virus. The L region contains the nucleotide sequence shown as SEQ ID NO: 15 or a variant of SEQ ID NO: 15, the variant of SEQ ID NO: 15 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 95 of SEQ ID NO: 15, and The R region contains the nucleotide sequence shown as SEQ ID NO: 54 or 55, or a variant of any one of SEQ ID NO: 54 or 55, and the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 34 of SEQ ID NO: 54 or 55. The isolated RNA polynucleotide according to any one of claims 1 to 59. **Claim 74** The virus is an influenza virus. The L region contains the nucleotide sequence shown as SEQ ID NO: 16 or a variant of SEQ ID NO: 16, the variant of SEQ ID NO: 16 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 81 of SEQ ID NO: 16, and The R region contains a nucleotide sequence represented as any one of SEQ ID NO: 56 or 57, or a variant of any one of SEQ ID NO: 56 or 57, and the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 12 of SEQ ID NO: 56 or 57. The isolated RNA polynucleotide according to any one of claims 1 to 59. **Claim 75** The virus is an influenza virus. The L region contains a nucleotide sequence represented as SEQ ID NO: 17 or a variant of SEQ ID NO: 17, the variant of SEQ ID NO: 17 contains mutations at one or more nucleotide positions selected from the group consisting of positions 14 to 22 of SEQ ID NO: 17, and The R region contains a nucleotide sequence represented as any one of SEQ ID NO: 58 or 59, or a variant of any one of SEQ ID NO: 58 or 59, and the variant contains mutations at one or more nucleotide positions selected from the group consisting of positions 8 to 32 of SEQ ID NO: 58 or 59. The isolated RNA polynucleotide according to any one of claims 1 to 59. **Claim 76** The virus is a simian virus. The L region contains a nucleotide sequence represented as SEQ ID NO: 137 or a variant of SEQ ID NO: 137, the variant of SEQ ID NO: 137 contains mutations at one or more nucleotide positions selected from the group consisting of positions 40 to 1557 of SEQ ID NO: 137, and The R region contains a nucleotide sequence represented as SEQ ID NO: 128 or a variant of any one of SEQ ID NO: 128, and the variant of SEQ ID NO: 128 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 30 of SEQ ID NO:

128. The isolated RNA polynucleotide according to any one of claims 1 to 59. **Claim 77** The virus is a simian virus. The L region contains a nucleotide sequence represented as any one of SEQ ID NO: 138, 139, 140, 141, 142, 143 or 144, or a variant of any one of SEQ ID NO: 138, 139, 140, 141, 142, 143 or 144. (i) The variant of SEQ ID NO: 138 contains mutations at one or more nucleotide positions selected from the group consisting of positions 50 to 312 of SEQ ID NO:

138. (ii) the variant of SEQ ID NO: 139 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1567 of SEQ ID NO: 139, (iii) the variant of SEQ ID NO: 140 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1488 of SEQ ID NO: 140, (iv) the variant of SEQ ID NO: 141 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1593 of SEQ ID NO: 141, (v) the variant of SEQ ID NO: 142 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1570 of SEQ ID NO: 142, (vi) the variant of SEQ ID NO: 143 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1488 of SEQ ID NO: 143, or (vii) the variant of SEQ ID NO: 144 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1593 of SEQ ID NO: 144, and the R region contains the nucleotide sequence shown as any one of SEQ ID NOs: 130, 136, 145, 146 or 147 or a variant of any one of SEQ ID NOs: 130, 136, 145, 146 or 147, (i) the variant of SEQ ID NO: 130 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130, (ii) the variant of SEQ ID NO: 136 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 33 of SEQ ID NO: 136, (iii) the variant of SEQ ID NO: 145 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 50 to 1461 of SEQ ID NO: 145, (iv) the variant of SEQ ID NO: 146 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 1441 of SEQ ID NO: 146, or (v) the variant of SEQ ID NO: 147 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 897 of SEQ ID NO: 147, An isolated RNA polynucleotide according to any one of claims 1 to 59.

78. the virus is respiratory syncytial virus (RSV), The L region contains a nucleotide sequence represented as any one of SEQ ID NO: 158, 163, 165, 166 or 419, or a variant of any one of SEQ ID NO: 158, 163, 165, 166 or 419, wherein (i) the variant of SEQ ID NO: 158 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 207 of SEQ ID NO: 158, (ii) the variant of SEQ ID NO: 163 contains mutations at one or more nucleotide positions selected from the group consisting of positions 18 to 210 of SEQ ID NO: 163, (iii) the variant of SEQ ID NO: 165 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 147 of SEQ ID NO: 165, (iv) the variant of SEQ ID NO: 166 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 32 of SEQ ID NO: 166, or (v) the variant of SEQ ID NO: 419 contains mutations at one or more nucleotide positions selected from the group consisting of positions 18 to 35 of SEQ ID NO: 419, and the R region contains a nucleotide sequence represented as any one of SEQ ID NO: 169, 170, 176, 177 or 420, or a variant of any one of SEQ ID NO: 169, 170, 176, 177 or 420, wherein (i) the variant of SEQ ID NO: 169 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 78 of SEQ ID NO: 169, (ii) the variant of SEQ ID NO: 170 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 80 of SEQ ID NO: 170, (iii) the variant of SEQ ID NO: 176 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 36 of SEQ ID NO: 176, (iv) the variant of SEQ ID NO: 177 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 33 of SEQ ID NO: 177, or (v) the variant of SEQ ID NO: 420 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 35 of SEQ ID NO: 420, The isolated RNA polynucleotide according to any one of claims 1 to 59.

79. The virus is a parainfluenza virus, The L region contains a nucleotide sequence represented as any one of SEQ ID NO: 181, 182, or 183, or a variant of any one of SEQ ID NO: 181, 182, or 183, wherein (i) the variant of SEQ ID NO: 181 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 181, (ii) the variant of SEQ ID NO: 182 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 142 of SEQ ID NO: 182, or (iii) the variant of SEQ ID NO: 183 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 183, and the R region contains a nucleotide sequence represented as SEQ ID NO: 184 or a variant of SEQ ID NO: 184, and the variant of SEQ ID NO: 184 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 98 of SEQ ID NO: 184, An isolated RNA polynucleotide according to any one of claims 1 to 59.

80. The virus is a parainfluenza virus, the L region contains a nucleotide sequence represented as any one of SEQ ID NO: 187, 188, or 189, or a variant of any one of SEQ ID NO: 187, 188, or 189, wherein (i) the variant of SEQ ID NO: 187 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 187, (ii) the variant of SEQ ID NO: 188 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 101 of SEQ ID NO: 188, or (iii) the variant of SEQ ID NO: 189 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 189, and the R region contains a nucleotide sequence represented as SEQ ID NO: 190 or a variant of SEQ ID NO: 190, and the variant of SEQ ID NO: 190 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 93 of SEQ ID NO: 190, An isolated RNA polynucleotide according to any one of claims 1 to 59.

81. The virus is a metapneumovirus, The L region contains a nucleotide sequence represented as any one of SEQ ID NO: 196, 197, or 199, or a variant of any one of SEQ ID NO: 196, 197, or 199, wherein (i) the variant of SEQ ID NO: 196 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 224 of SEQ ID NO: 196, (ii) the variant of SEQ ID NO: 197 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 230 of SEQ ID NO: 197, or (iii) the variant of SEQ ID NO: 199 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 140 of SEQ ID NO: 199, and the R region contains a nucleotide sequence represented as SEQ ID NO: 201 or a variant of SEQ ID NO: 201, and the variant of SEQ ID NO: 201 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 201, The isolated RNA polynucleotide according to any one of claims 1 to 59.

82. The virus is a metapneumovirus, the L region contains a nucleotide sequence represented as SEQ ID NO: 195 or a variant of SEQ ID NO: 195, and the variant of SEQ ID NO: 195 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 224 of SEQ ID NO: 195, and the R region contains a nucleotide sequence represented as SEQ ID NO: 200 or a variant of SEQ ID NO: 200, and the variant of SEQ ID NO: 200 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO: 200, The isolated RNA polynucleotide according to any one of claims 1 to 59.

83. The virus is a henipavirus, the L region contains a nucleotide sequence represented as SEQ ID NO: 204 or a variant of SEQ ID NO: 204, and the variant of SEQ ID NO: 204 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 204, and the R region contains a nucleotide sequence represented as SEQ ID NO: 206 or a variant of SEQ ID NO: 206, and the variant of SEQ ID NO: 206 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 206, An isolated RNA polynucleotide according to any one of claims 1 to 59.

84. wherein the virus is a henipavirus, the L region comprises a nucleotide sequence shown as SEQ ID NO: 209 or 210 or a variant of SEQ ID NO: 209 or 210, (i) the variant of SEQ ID NO: 209 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 209, or (ii) the variant of SEQ ID NO: 210 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 83 of SEQ ID NO: 210, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 211 or a variant of SEQ ID NO: 211, and the variant of SEQ ID NO: 211 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 211, An isolated RNA polynucleotide according to any one of claims 1 to 59.

85. wherein the virus is a hepadnavirus, the L region comprises a nucleotide sequence shown as SEQ ID NO: 222 or 223 or a variant of SEQ ID NO: 222 or 223, (i) the variant of SEQ ID NO: 222 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 639 of SEQ ID NO: 222, or (ii) the variant of SEQ ID NO: 223 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 186 of SEQ ID NO: 223, and the R region comprises a nucleotide sequence shown as SEQ ID NO: 225 or a variant of SEQ ID NO: 225, and the variant of SEQ ID NO: 225 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1023 of SEQ ID NO: 225, An isolated RNA polynucleotide according to any one of claims 1 to 59.

86. wherein the virus is a filovirus, the L region comprises a nucleotide sequence shown as any one of SEQ ID NO: 227, 228, 229 or 230 or a variant of any one of SEQ ID NO: 227, 228, 229 or 230, (i) the variant of SEQ ID NO: 227 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 710 of SEQ ID NO: 227, (ii) the variant of SEQ ID NO: 228 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 23 to 713 of SEQ ID NO: 228, (iii) the variant of SEQ ID NO: 229 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 707 of SEQ ID NO: 229, or (iv) the variant of SEQ ID NO: 230 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 17 to 707 of SEQ ID NO: 230, and the R region contains the nucleotide sequence shown as SEQ ID NO: 231 or a variant of SEQ ID NO: 231, and the variant of SEQ ID NO: 231 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 449 of SEQ ID NO: 231, The isolated RNA polynucleotide according to any one of claims 1 to 59.

87. the virus is a filovirus, the L region contains the nucleotide sequence shown as any one of SEQ ID NOs: 232, 233, 234 or 235 or a variant of any one of SEQ ID NOs: 232, 233, 234 or 235, (i) the variant of SEQ ID NO: 232 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 678 of SEQ ID NO: 232, (ii) the variant of SEQ ID NO: 233 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 23 to 681 of SEQ ID NO: 233, (iii) the variant of SEQ ID NO: 234 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 23 to 678 of SEQ ID NO: 234, or (iv) the variant of SEQ ID NO: 235 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 23 to 678 of SEQ ID NO: 235, and the R region contains the nucleotide sequence shown as SEQ ID NO: 236 or a variant of SEQ ID NO: 236, and the variant of SEQ ID NO: 236 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 437 of SEQ ID NO: 236, The isolated RNA polynucleotide according to any one of claims 1 to 59.

88. the virus is a filovirus, the L region contains the nucleotide sequence shown as any one of SEQ ID NOs: 237, 238 or 239 or a variant of any one of SEQ ID NOs: 237, 238 or 239, (i) The variant of SEQ ID NO: 237 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 605 of SEQ ID NO: 237, (ii) The variant of SEQ ID NO: 238 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 21 to 606 of SEQ ID NO: 238, or (iii) The variant of SEQ ID NO: 239 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 605 of SEQ ID NO: 239, and The R region contains the nucleotide sequence shown as SEQ ID NO: 240 or a variant of SEQ ID NO: 240, and the variant of SEQ ID NO: 240 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 15 to 83 of SEQ ID NO: 240, The isolated RNA polynucleotide according to any one of claims 1 to 59.

89. The virus is a filovirus, The L region contains the nucleotide sequence shown as SEQ ID NO: 241 or a variant of SEQ ID NO: 241, and the variant of SEQ ID NO: 241 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 34 of SEQ ID NO: 241, and The R region contains the nucleotide sequence shown as SEQ ID NO: 242 or any one variant of SEQ ID NO: 242, and the variant of SEQ ID NO: 242 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 593 of SEQ ID NO: 242, The isolated RNA polynucleotide according to any one of claims 1 to 59.

90. The virus is a filovirus, The L region contains the nucleotide sequence shown as SEQ ID NO: 243 or a variant of SEQ ID NO: 243, and the variant of SEQ ID NO: 243 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 30 to 45 of SEQ ID NO: 243, and The R region contains the nucleotide sequence shown as SEQ ID NO: 244 or a variant of SEQ ID NO: 244, and the variant of SEQ ID NO: 244 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 100 to 677 of SEQ ID NO: 244, The isolated RNA polynucleotide according to any one of claims 1 to 59.

91. The virus is a filovirus, The L region contains the nucleotide sequence shown in any one of SEQ ID NOs: 245, 246, or 247, or a variant of any one of SEQ ID NOs: 245, 246, or 247, wherein (i) the variant of SEQ ID NO: 245 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 29 to 171 of SEQ ID NO: 245, (ii) the variant of SEQ ID NO: 246 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 30 to 171 of SEQ ID NO: 246, or (iii) the variant of SEQ ID NO: 247 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 29 to 171 of SEQ ID NO: 247, and the R region contains the nucleotide sequence shown as SEQ ID NO: 248 or a variant of SEQ ID NO: 248, and the variant of SEQ ID NO: 248 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 91 of SEQ ID NO: 248, An isolated RNA polynucleotide according to any one of claims 1 to 59.

92. The virus is an alphavirus, the L region contains the nucleotide sequence shown as SEQ ID NO: 249 or a variant of SEQ ID NO: 249, and the variant of SEQ ID NO: 249 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 274 of SEQ ID NO: 249, and the R region contains the nucleotide sequence shown as SEQ ID NO: 250 or 251 or a variant of SEQ ID NO: 250 or 251, (i) the variant of SEQ ID NO: 250 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 183 of SEQ ID NO: 250, or (ii) the variant of SEQ ID NO: 251 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 375 of SEQ ID NO: 251, An isolated RNA polynucleotide according to any one of claims 1 to 59.

93. The virus is an alphavirus, the L region contains the nucleotide sequence shown as SEQ ID NO: 255 or a variant of SEQ ID NO: 255, and the variant of SEQ ID NO: 255 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 35 of SEQ ID NO: 255, and The R region comprises the nucleotide sequence shown as SEQ ID NO: 256 or 257, or a variant of SEQ ID NO: 256 or 257, wherein (i) the variant of SEQ ID NO: 256 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 600 to 273 of SEQ ID NO: 256, or (ii) the variant of SEQ ID NO: 257 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 377 of SEQ ID NO: 257, an isolated RNA polynucleotide according to any one of claims 1 to 59.

94. wherein the virus is an alphavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 261 or a variant of SEQ ID NO: 261, and the variant of SEQ ID NO: 261 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 215 of SEQ ID NO: 261, the R region comprises the nucleotide sequence shown as SEQ ID NO: 262 or 263, or a variant of SEQ ID NO: 262 or 263, wherein (i) the variant of SEQ ID NO: 262 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 166 of SEQ ID NO: 262, or (ii) the variant of SEQ ID NO: 263 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 60 to 379 of SEQ ID NO: 263, an isolated RNA polynucleotide according to any one of claims 1 to 59.

95. An isolated RNA polynucleotide, comprising a coding region having a coding sequence encoding one or more polypeptides, a template region, the template region comprising two different regions, the left flanking region of the virus ("L region") and the right flanking region of the virus ("R region"), the L region being adjacent and continuous to the 5' end of the coding region, and the R region being adjacent and continuous to the 3' end of the coding region, wherein the coding sequence is in a sense orientation, the polypeptide is heterologous to the virus, the template region interacts with and initiates the RNA-dependent polymerase activity of the polymerase in a cell containing the RNA-dependent polymerase, and the virus is not an alphavirus, an isolated RNA polynucleotide.

96. The reverse complement of the isolated RNA polynucleotide according to claim 95.

97. The isolated RNA polynucleotide according to claim 95 or 96, wherein the virus is selected from the group consisting of viruses in the order Amarylivirales, Articulavirales, Blubervirales, Bunyavirales, Hepelevirales, Mononegavirales, Nidovirales, and Picornavirales in that order.

98. The isolated RNA polynucleotide according to any one of claims 95 to 97, wherein the virus is selected from the group consisting of viruses in the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, and Rhabdoviridae.

99. The isolated RNA polynucleotide according to any one of claims 95 to 98, wherein the virus is from the group consisting of alpha coronavirus 229E, alpha coronavirus NL63, alpha coronavirus WA2028, avian metapneumovirus (AMPV), beta coronavirus HKU1, beta coronavirus HKU15, beta coronavirus HKU33, beta coronavirus OC43, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, enterovirus D68 (EV-D68), foot-and-mouth disease virus, hantavirus, Hendra virus, hepatitis B virus, hepatitis C virus, HMPV, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), infectious salmon anemia virus, influenza A, influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, Norwalk virus, rabies virus, respiratory syncytial virus, Reston Ebola virus, rhinovirus, Rift Valley fever virus, rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, vesicular stomatitis virus, yellow fever virus, Zaire Ebola virus, and Zika virus.

100. The isolated RNA polynucleotide according to any one of claims 95 to 99, wherein the template region is native to the virus.

101. The isolated RNA polynucleotide according to any one of claims 95 to 99, wherein the template region is a variant of the template region native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the template region native to the virus.

102. The isolated RNA polynucleotide according to any one of claims 95 to 101, wherein each of the L region and the R region of the template region contains 10, 9, 8, 7, 6, 5, 4, 3, or less than 2 mutations compared to the template region native to the virus.

103. The isolated RNA polynucleotide according to any one of claims 95 to 101, wherein each of the L region and the R region of the template region is mutated from the template region natural to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping.

104. The isolated RNA polynucleotide according to any one of claims 95 to 103, wherein each of the L region and the R region of the template region is different from the template region natural to the virus by 1 or fewer substitutions that do not participate in 5' capping.

105. The isolated RNA polynucleotide according to any one of claims 95 to 104, wherein the isolated RNA polynucleotide comprises at least one nucleoside modification.

106. The isolated RNA polynucleotide according to any one of claims 95 to 105, wherein the template region is nucleoside-modified, and the proportion of modified nucleosides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less.

107. The isolated RNA polynucleotide according to any one of claims 95 to 105, wherein the template region is nucleoside-modified, and the proportion of modified nucleosides is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or 100%.

108. The isolated RNA polynucleotide according to claim 105 or 106, wherein the nucleoside modification is a non-immunogenic uridine modification, and the proportion of modified uridine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less.

109. The isolated RNA polynucleotide according to claim 105 or 107, wherein the nucleoside modification is a non-immunogenic uridine modification, and the proportion of modified uridine modification is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or more than 95%, or 100%.

110. The isolated RNA polynucleotide according to any one of claims 105 or 106, wherein the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of modified cytidine modification is 40%, 35%, 30%, 25%, 20%, 15% or 10% or less.

111. The isolated RNA polynucleotide according to any one of claims 105 or 107, wherein the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of the modified cytidine modification is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more than 95%, or 100%.

112. The isolated RNA polynucleotide according to claim 105 or 106, wherein the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of the modified adenosine modification is 1% to 30%.

113. The isolated RNA polynucleotide according to claim 112, wherein the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of the modified adenosine modification is about 1%, 5%, 10%, 15%, 20%, 25%, or 30%.

114. The isolated RNA polynucleotide according to any one of claims 95 to 113, wherein the isolated polynucleotide comprises a 5' cap structure.

115. The isolated RNA polynucleotide according to any one of claims 95 to 114, wherein the 5' end of the L region comprises a 5' cap structure.

116. The isolated RNA polynucleotide according to any one of claims 95 to 115, wherein the 5' end of the L region comprises one or more mutations related to the 5' cap structure.

117. The isolated RNA polynucleotide according to any one of claims 114 to 116, wherein the 5' cap structure is selected from the group consisting of Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analogue (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structures.

118. The isolated RNA polynucleotide according to any one of claims 95 to 113, wherein the isolated polynucleotide does not comprise a 5' cap structure (is uncapped).

119. The isolated RNA polynucleotide according to any one of claims 95 to 113 or 118, wherein the 5' end of the L region does not comprise a 5' cap structure (is uncapped).

120. The isolated RNA polynucleotide according to claim 118 or 119, wherein the 5' end of the isolated polynucleotide comprises a 5'-monophosphate, 5'-diphosphate or 5'-triphosphate.

121. The isolated RNA polynucleotide according to claim 118 or 119, wherein the 5' end of the isolated polynucleotide is free of a 5'-phosphate (dephosphorylated).

122. The isolated RNA polynucleotide according to claim 96, wherein the template region is the reverse complement of the template region natural to the virus.

123. The isolated RNA polynucleotide according to claim 96 or 122, wherein the template region is a variant of the reverse complement of the template region natural to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reverse complement of the template region natural to the virus.

124. The isolated RNA polynucleotide according to claim 122 or 123, wherein each reverse complement of the L region and the R region varies from the reverse complement of the template region natural to the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping.

125. The isolated RNA polynucleotide according to claim 123 or 124, wherein each reverse complement of the L region and the R region varies from the reverse complement of the template region natural to the virus by 1 or fewer substitutions that do not participate in 5' capping.

126. The isolated RNA polynucleotide according to any one of claims 122 to 125, wherein the isolated RNA polynucleotide comprises at least one nucleoside modification.

127. The isolated RNA polynucleotide according to claim 126, wherein the template region is nucleoside-modified and the proportion of modified nucleotides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less.

128. The isolated RNA polynucleotide according to any one of claims 96 or 122 to 127, wherein the 5' end of the reverse complement of the R region encodes a cap structure.

129. The isolated RNA polynucleotide according to any one of claims 96 or 122 to 128, wherein the 5' end of the R region is capped.

130. The isolated RNA polynucleotide according to any one of claims 95 to 129, wherein the therapeutic polypeptide is a secreted polypeptide.

131. The isolated RNA polynucleotide according to any one of claims 95 to 130, wherein the therapeutic polypeptide is selected from the group consisting of interferon, interferon-stimulated gene, cytokine, chemokine, antibody, signaling molecule, cytotoxic protein, protein that causes cell death, anti-neoplastic protein, immunomodulatory protein, protein toll-like receptor agonist or dominant negative protein.

132. The isolated RNA polynucleotide according to claim 131, wherein the cytokine is an inflammatory cytokine.

133. The isolated RNA polynucleotide according to claim 131, wherein the inflammatory cytokine is TNF-α.

134. The isolated RNA polynucleotide according to claim 131, wherein the cytokine is an anti-inflammatory cytokine.

135. The isolated RNA polynucleotide according to claim 134, wherein the anti-inflammatory cytokine is interleukin-1 receptor antagonist (IL-1RN).

136. The isolated RNA polynucleotide according to any one of claims 95 to 135, wherein the therapeutic polypeptide is interleukin or caspase.

137. The isolated RNA polynucleotide according to claim 136, wherein the interleukin is IL-12A, IL-12B or IL-2.

138. The isolated RNA polynucleotide according to claim 131, wherein the therapeutic polypeptide is an antibody.

139. The isolated RNA polynucleotide according to claim 131, wherein the therapeutic polypeptide is interferon.

140. The isolated RNA polynucleotide according to claim 139, wherein the interferon is IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-γ or IFN-λ.

141. The isolated RNA polynucleotide according to claim 140, wherein the interferon is IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β1, IFN-ε, IFN-κ, IFN-ω1, IFN-γ, IFN-λ1 (IL28A), IFN-λ2 (IL28B), IFN-λ3 (IL29) or IFN-λ4.

142. The isolated RNA polynucleotide according to claim 139, wherein the interferon is IFN-α, IFN-β, IFN-κ, IFN-λ1 (IL28A), IFN-λ2 (IL28B) or IFN-λ3 (IL29).

143. The isolated RNA polynucleotide according to any one of claims 95 to 142, wherein the coding sequence encodes two or more therapeutic polypeptides separated by one or more ribosome skip sequences.

144. The isolated RNA polynucleotide according to any one of claims 95 to 143, wherein the coding region further comprises one or more regulatory elements selected from the group consisting of a ribosome binding site, a Kozak sequence, a Shine-Dalgarno sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site and an internal ribosome entry site (IRES).

145. The isolated RNA polynucleotide according to claim 144, wherein the one or more regulatory elements are operably linked to the coding sequence.

146. The isolated RNA polynucleotide according to any one of claims 95 to 145, further comprising a polyadenylation signal and / or a 3' poly(A) tail.

147. The isolated RNA polynucleotide according to any one of claims 95 to 146, wherein the RNA-dependent polymerase is an RNA-dependent RNA polymerase.

148. The isolated RNA polynucleotide according to any one of claims 95 to 146, wherein the RNA-dependent polymerase is an RNA-dependent DNA polymerase.

149. The isolated RNA polynucleotide according to any one of claims 95 to 148, wherein the RNA-dependent polymerase is a virus-derived polymerase.

150. The isolated RNA polynucleotide according to any one of claims 95 to 149, wherein the isolated RNA polynucleotide is single-stranded RNA. **Claim 151** The isolated RNA polynucleotide according to any one of claims 95 to 150, wherein the isolated polynucleotide is in a linear form. **Claim 152** The isolated RNA polynucleotide according to any one of claims 95 to 150, wherein the isolated polynucleotide is in a covalently closed circular form. **Claim 153** wherein the virus is a simian retrovirus, the L region comprises a nucleotide sequence represented by any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66 or 67 or a variant of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66 or 67, the variant comprising mutations at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1426 to 1493 of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66 or 67, and the R region comprises a nucleotide sequence represented by SEQ ID NO: 129 or a variant of SEQ ID NO: 129, the variant of SEQ ID NO: 129 comprising mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 129, The isolated RNA polynucleotide according to any one of claims 95 to 152. **Claim 154** wherein the virus is a simian retrovirus, the L region comprises a nucleotide sequence represented by any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76 or 77 or a variant of any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76 or 77, (i) the variant of SEQ ID NO: 68 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 68, (ii) the variant of SEQ ID NO: 69 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 69, (iii) the variant of SEQ ID NO: 70 comprises mutations at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1446 to 1513 of SEQ ID NO: 70, (iv) the variant of SEQ ID NO: 71 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1455 to 1522 of SEQ ID NO: 71, (v) the variant of SEQ ID NO: 72 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1462 to 1529 of SEQ ID NO: 72, (vi) the variant of SEQ ID NO: 73 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1469 to 1536 of SEQ ID NO: 73, (vii) the variant of SEQ ID NO: 74 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1485 to 1552 of SEQ ID NO: 74, (viii) the variant of SEQ ID NO: 75 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1686 to 1753 of SEQ ID NO: 75, (ix) the variant of SEQ ID NO: 76 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1704 to 1771 of SEQ ID NO: 76, or (x) the variant of SEQ ID NO: 77 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1720 to 1787 of SEQ ID NO: 77, and the R region contains the nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130, and the variant of SEQ ID NO: 130 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130, The isolated RNA polynucleotide according to any one of claims 95 to 152.

155. the virus is a simian becovirus, the L region contains the nucleotide sequence shown as any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88 or a variant of any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88, (i) the variant of SEQ ID NO: 78 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1734 to 1801 of SEQ ID NO: 78, (ii) the variant of SEQ ID NO: 79 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1687 to 1754 of SEQ ID NO: 79, (iii) the variant of SEQ ID NO: 80 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1695 to 1762 of SEQ ID NO: 80, (iv) the variant of SEQ ID NO: 81 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 81, (v) the variant of SEQ ID NO: 82 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1443 to 1510 of SEQ ID NO: 82, (vi) the variant of SEQ ID NO: 83 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1459 to 1526 of SEQ ID NO: 83, (vii) the variant of SEQ ID NO: 85 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 85, (viii) the variant of SEQ ID NO: 86 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1434 to 1501 of SEQ ID NO: 86, (ix) the variant of SEQ ID NO: 87 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1435 to 1502 of SEQ ID NO: 87, or (x) the variant of SEQ ID NO: 88 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1463 to 1530 of SEQ ID NO: 88, and the R region contains the nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130, and the variant of SEQ ID NO: 130 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130, The isolated RNA polynucleotide according to any one of claims 95 to 152.

156. the virus is a simian becovirus, the L region contains the nucleotide sequence shown as any one of SEQ ID NO: 89, 90, 91, 92, 96, 104, 105, 106, 107 or SEQ ID NO: 108 or a variant of any one of SEQ ID NO: 89, 90, 91, 92, 96, 104, 105, 106, 107 or SEQ ID NO: 108, (i) the variant of SEQ ID NO: 89 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1466 to 1533 of SEQ ID NO: 89, (ii) the variant of SEQ ID NO: 90 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1425 to 1492 of SEQ ID NO: 90, (iii) the variant of SEQ ID NO: 91 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1425 to 1492 of SEQ ID NO: 91, (iv) the variant of SEQ ID NO: 92 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1425 to 1492 of SEQ ID NO: 92, (v) the variant of SEQ ID NO: 96 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 769 or 1471 to 1471 of SEQ ID NO: 96, (vi) the variant of SEQ ID NO: 104 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1446 to 1513 of SEQ ID NO: 104, (vii) the variant of SEQ ID NO: 105 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1455 to 1522 of SEQ ID NO: 105, (viii) the variant of SEQ ID NO: 106 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1462 to 1529 of SEQ ID NO: 106, (ix) the variant of SEQ ID NO: 107 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 39 to 789 or 1469 to 1536 of SEQ ID NO: 107, or (x) the variant of SEQ ID NO: 108 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 89 to 839 or 1485 to 1552 of SEQ ID NO: 108, and the R region contains the nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130, and the variant of SEQ ID NO: 130 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO: 130, The isolated RNA polynucleotide according to any one of claims 95 to 152.

157. the virus is simian becovirus, The L region contains a nucleotide sequence represented as any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118, or a variant of any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118, wherein (i) the variant of SEQ ID NO: 109 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1686 to 1753 of SEQ ID NO: 109, wherein (ii) the variant of SEQ ID NO: 110 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1704 to 1771 of SEQ ID NO: 110, wherein (iii) the variant of SEQ ID NO: 111 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1720 to 1787 of SEQ ID NO: 111, wherein (iv) the variant of SEQ ID NO: 112 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1734 to 1801 of SEQ ID NO: 112, wherein (v) the variant of SEQ ID NO: 113 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1687 to 1754 of SEQ ID NO: 113, wherein (vi) the variant of SEQ ID NO: 114 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1695 to 1762 of SEQ ID NO: 114, wherein (vii) the variant of SEQ ID NO: 115 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 115, wherein (viii) the variant of SEQ ID NO: 116 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 116, wherein (ix) the variant of SEQ ID NO: 117 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 117, or wherein (xl) the variant of SEQ ID NO: 118 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO: 118, and The R region contains the nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130, and the variant of SEQ ID NO: 130 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO:

130. An isolated RNA polynucleotide according to any one of claims 95 to 152. **Claim 158** The virus is a simian becovirus. The L region contains the nucleotide sequence shown as any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126 or 127 or a variant of any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126 or 127. (i) The variant of SEQ ID NO: 119 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1443 to 1510 of SEQ ID NO:

119. (ii) The variant of SEQ ID NO: 120 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1459 to 1526 of SEQ ID NO:

120. (iii) The variant of SEQ ID NO: 122 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO:

122. (iv) The variant of SEQ ID NO: 123 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO:

123. (v) The variant of SEQ ID NO: 124 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1434 to 1501 of SEQ ID NO:

124. (vi) The variant of SEQ ID NO: 125 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1463 to 1530 of SEQ ID NO:

125. (vii) The variant of SEQ ID NO: 126 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1466 to 1533 of SEQ ID NO:

126. (viii) The variant of SEQ ID NO: 127 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 40 to 789 or 1425 to 1492 of SEQ ID NO:

127. And The R region contains the nucleotide sequence shown as SEQ ID NO: 130 or a variant of SEQ ID NO: 130, and the variant of SEQ ID NO: 130 contains mutations at one or more nucleotide positions selected from the group consisting of positions 20 to 320 of SEQ ID NO:

130. The isolated RNA polynucleotide according to any one of claims 95 to 152. **Claim 159** The virus is a respiratory syncytial virus (RSV), The L region contains the nucleotide sequence shown as any one of SEQ ID NO: 148, 149, 150, 151 or 152 or a variant of any one of SEQ ID NO: 148, 149, 150, 151 or 152, (i) The variant of SEQ ID NO: 148 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 78 of SEQ ID NO: 148, (ii) The variant of SEQ ID NO: 149 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 33 of SEQ ID NO: 149, (iii) The variant of SEQ ID NO: 150 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 35 of SEQ ID NO: 150, (iv) The variant of SEQ ID NO: 151 contains mutations at one or more nucleotide positions selected from the group consisting of positions 18 to 36 of SEQ ID NO: 151, or (v) The variant of SEQ ID NO: 152 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 38 of SEQ ID NO: 152, and The R region contains the nucleotide sequence shown as SEQ ID NO: 154 or 155 or a variant of SEQ ID NO: 154 or 155, (i) The variant of SEQ ID NO: 154 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 207 of SEQ ID NO: 154, or (ii) The variant of SEQ ID NO: 155 contains mutations at one or more nucleotide positions selected from the group consisting of positions 15 to 32 of SEQ ID NO: 155, The isolated RNA polynucleotide according to any one of claims 95 to 152. **Claim 160** The virus is a parainfluenza virus, The L region contains the nucleotide sequence shown as SEQ ID NO: 180 or a variant of SEQ ID NO: 180, and the variant of SEQ ID NO: 180 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 136 of SEQ ID NO: 180, and The R region contains the nucleotide sequence shown as SEQ ID NO: 179 or a variant of SEQ ID NO: 179, and the variant of SEQ ID NO: 179 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 98 of SEQ ID NO:

179. The isolated RNA polynucleotide according to any one of claims 95 to 152.

161. The virus is a parainfluenza virus, The L region contains the nucleotide sequence shown as SEQ ID NO: 186 or a variant of SEQ ID NO: 186, the variant of SEQ ID NO: 186 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 95 of SEQ ID NO: 186, and The R region contains the nucleotide sequence shown as SEQ ID NO: 185 or a variant of SEQ ID NO: 185, the variant of SEQ ID NO: 185 contains mutations at one or more nucleotide positions selected from the group consisting of positions 21 to 93 of SEQ ID NO:

185. The isolated RNA polynucleotide according to any one of claims 95 to 152.

162. The virus is a metapneumovirus, The L region contains the nucleotide sequence shown as SEQ ID NO: 194 or a variant of SEQ ID NO: 194, the variant of SEQ ID NO: 194 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 220 of SEQ ID NO: 194, and The R region contains the nucleotide sequence shown as SEQ ID NO: 192 or any one variant of SEQ ID NO: 192, the variant of SEQ ID NO: 192 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO:

192. The isolated RNA polynucleotide according to any one of claims 95 to 152.

163. The virus is a metapneumovirus, The L region contains the nucleotide sequence shown as SEQ ID NO: 193 or a variant of SEQ ID NO: 193, the variant of SEQ ID NO: 193 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 220 of SEQ ID NO: 193, and The R region contains the nucleotide sequence shown as SEQ ID NO: 191 or a variant of SEQ ID NO: 191, the variant of SEQ ID NO: 191 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 32 of SEQ ID NO:

191. The isolated RNA polynucleotide according to any one of claims 95 to 152.

164. wherein the virus is a henipavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 203 or a variant of SEQ ID NO: 203, the variant of SEQ ID NO: 203 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 203, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 202 or a variant of SEQ ID NO: 202, the variant of SEQ ID NO: 202 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 202, The isolated RNA polynucleotide according to any one of claims 95 to 152.

165. wherein the virus is a henipavirus, the L region comprises the nucleotide sequence shown as SEQ ID NO: 207 or a variant of SEQ ID NO: 207, the variant of SEQ ID NO: 207 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 77 of SEQ ID NO: 207, and the R region comprises the nucleotide sequence shown as SEQ ID NO: 208 or a variant of SEQ ID NO: 208, the variant of SEQ ID NO: 208 contains mutations at one or more nucleotide positions selected from the group consisting of positions 17 to 91 of SEQ ID NO: 208, The isolated RNA polynucleotide according to any one of claims 95 to 152.

166. wherein the virus is a hepadnavirus, the L region comprises the nucleotide sequence shown as any one of SEQ ID NOs: 212, 213, 214, 215 or 216 or a variant of any one of SEQ ID NOs: 212, 213, 214, 215 or 216, (i) the variant of SEQ ID NO: 212 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1326 of SEQ ID NO: 212, (ii) the variant of SEQ ID NO: 213 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1291 of SEQ ID NO: 213, (iii) the variant of SEQ ID NO: 214 contains mutations at one or more nucleotide positions selected from the group consisting of positions 101 to 1325 of SEQ ID NO: 214, (iv) The variant of SEQ ID NO: 215 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 15 of SEQ ID NO: 215, or (v) The variant of SEQ ID NO: 216 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 211 of SEQ ID NO: 216, and the R region contains a nucleotide sequence represented as any one of SEQ ID NO: 217, 218, 219 or 220 or a variant of any one of SEQ ID NO: 217, 218, 219 or 220, (i) The variant of SEQ ID NO: 217 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 754 of SEQ ID NO: 217, (ii) The variant of SEQ ID NO: 218 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 790 of SEQ ID NO: 218, (iii) The variant of SEQ ID NO: 219 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 892 of SEQ ID NO: 219, or (iv) The variant of SEQ ID NO: 220 contains a mutation at one or more nucleotide positions selected from the group consisting of positions 101 to 2309 of SEQ ID NO: 220, The isolated RNA polynucleotide according to any one of claims 95 to 152.

167. An isolated RNA polynucleotide comprising: A coding region having a coding sequence encoding one or more polypeptides, A template region, wherein the template region includes two different regions, the left flanking region of the virus ("L region") and the right flanking region of the virus ("R region"), the L region is adjacent to and continuous with the 5' end of the coding region, and the R region is adjacent to and continuous with the 3' end of the coding region, a template region, comprising At least 30% of the uridine nucleotides are modified, At least 30% of the cytidine nucleotides are modified, and / or 1 to 30% of the adenosine nucleotides are modified, and the template region interacts with and initiates the RNA-dependent polymerase activity of the polymerase in cells containing RNA-dependent polymerase, An isolated RNA polynucleotide.

168. The isolated RNA polynucleotide according to claim 167, wherein the coding sequence is in an antisense orientation.

169. The isolated RNA polynucleotide according to claim 167, wherein the coding sequence is in the sense orientation.

170. The isolated RNA polynucleotide according to any one of claims 167 to 169, wherein the polypeptide is a secreted protein.

171. The isolated RNA polynucleotide according to any one of claims 167 to 170, wherein the polypeptide is selected from the group consisting of a pharmaceutical, a therapeutic polypeptide, an antigen, and a reporter.

172. The reverse complement of the isolated RNA polynucleotide according to any one of claims 167 to 171.

173. The isolated RNA polynucleotide according to any one of claims 167 to 172, wherein the virus is selected from the group consisting of viruses in the order Amarylvirales, Articulavirales, Blubervirales, Bunyavirales, Hepelevirales, Martellivirales, Mononegavirales, Nidovirales, and Picornavirales in this order.

174. The isolated RNA polynucleotide according to any one of claims 167 to 173, wherein the virus is selected from the group consisting of viruses in the families Arenaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Matonaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Pneumoviridae, Rhabdoviridae, and Togaviridae.

175. The isolated RNA polynucleotide according to any one of claims 167 to 174, wherein the virus is selected from the group consisting of alpha coronavirus 229E, alpha coronavirus NL63, alpha coronavirus WA2028, avian metapneumovirus (AMPV), beta coronavirus HKU1, beta coronavirus HKU15, beta coronavirus HKU33, beta coronavirus OC43, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus, eastern equine encephalitis virus (EEEV), enterovirus D68 (EV-D68), foot-and-mouth disease virus, hantavirus, Hendra virus, hepatitis B virus, hepatitis C virus, HMPV, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 3 (HPIV3), infectious salmon anemia virus, influenza A virus, influenza B virus, Lassa virus, Marburg virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Newcastle disease virus (NDV), Nipah virus, Norwalk virus, rabies virus, respiratory syncytial virus, Reston Ebola virus, rhinovirus, Rift Valley fever virus, rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan equine encephalitis virus (VEEV), vesicular stomatitis virus, western equine encephalitis virus (WEEV), yellow fever virus, Zaire Ebola virus, and Zika virus.

176. The isolated RNA polynucleotide according to any one of claims 167 to 175, wherein the template region is native to the virus.

177. The isolated RNA polynucleotide according to any one of claims 167 to 175, wherein the template region is a variant of the template region native to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the template region native to the virus.

178. The isolated RNA polynucleotide according to any one of claims 167 to 177, wherein each of the L region and the R region of the template region contains less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the template region native to the virus.

179. The isolated RNA polynucleotide according to any one of claims 167 to 178, wherein each of the L region and the R region of the template region is mutated from the native template region for the virus by 10, 9, 8, 7, 6, 5, 4, 3 or 2 or fewer substitutions that do not participate in 5' capping.

180. The isolated RNA polynucleotide according to any one of claims 167 to 179, wherein each of the L region and the R region of the template region is mutated from the native template region for the virus by 1 or fewer substitutions that do not participate in 5' capping.

181. The isolated RNA polynucleotide according to any one of claims 167 to 180, wherein the template region is nucleoside-modified, and the proportion of modified nucleosides is 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less.

182. The isolated RNA polynucleotide according to any one of claims 167 to 181, wherein the template region is nucleoside-modified, and the proportion of modified nucleosides is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or 100%.

183. The isolated RNA polynucleotide according to any one of claims 167 to 182, wherein the nucleoside modification is a non-immunogenic uridine modification, and the proportion of modified uridine modification is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more than 95%, or 100%. 【Claim·184】 The isolated RNA polynucleotide according to any one of claims 167 to 183, wherein the nucleoside modification is a non-immunogenic cytidine modification, and the proportion of modified cytidine modification is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more than 95%, or 100%.

185. The isolated RNA polynucleotide according to any one of claims 167 to 184, wherein the nucleoside modification is a non-immunogenic adenosine modification, and the proportion of modified adenosine modification is about 1%, 5%, 10%, 15%, 20%, 25% or 30%.

186. The isolated RNA polynucleotide according to any one of claims 167 to 185, wherein the isolated polynucleotide contains a 5' cap structure.

187. The isolated RNA polynucleotide according to any one of claims 167 to 186, wherein the 5' end of the L region comprises a 5' cap structure.

188. The isolated RNA polynucleotide according to any one of claims 167 to 187, wherein the 5' end of the L region comprises one or more mutations related to the 5' cap structure.

189. The isolated RNA polynucleotide according to any one of claims 186 to 188, wherein the 5' cap structure is selected from the group consisting of Cap0, Cap0(3'-O-Me), Cap1, Cap1(3'-O-Me), Cap2, Cap2(3'-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structures.

190. The isolated RNA polynucleotide according to any one of claims 167 to 185, wherein the isolated polynucleotide does not contain (is not capped with) a 5' cap structure.

191. The isolated RNA polynucleotide according to any one of claims 167 to 190, wherein the 5' end of the L region does not contain (is not capped with) a 5' cap structure.

192. The isolated RNA polynucleotide according to claim 190 or 191, wherein the 5' end of the isolated polynucleotide comprises a 5'-monophosphate, 5'-diphosphate or 5'-triphosphate.

193. The isolated RNA polynucleotide according to claim 190 or 191, wherein the 5' end of the isolated polynucleotide does not contain (is dephosphorylated) a 5'-phosphate.

194. The isolated RNA polynucleotide according to claim 172, wherein the template region is the reverse complement of the template region natural to the virus.

195. The isolated RNA polynucleotide according to claim 172 or 194, wherein the template region is a variant of the reverse complement of the template region natural to the virus, and the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reverse complement of the template region natural to the virus.

196. The reverse complement of each of the L region and the R region is mutated from the reverse complement of the template region natural to the virus by 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer substitutions that do not participate in 5' capping. The isolated RNA polynucleotide according to claim 172, 194, or 195.

197. The reverse complement of each of the L region and the R region is mutated from the reverse complement of the template region natural to the virus by 1 or fewer substitutions that do not participate in 5' capping. The isolated RNA polynucleotide according to any one of claims 172 or 194 to 196.

198. The 5' end of the reverse complement of the R region encodes a 5' cap structure. The isolated RNA polynucleotide according to any one of claims 172 or 194 to 196.

199. The 5' end of the R region is capped. The isolated RNA polynucleotide according to any one of claims 172 or 194 to 198.

200. The coding sequence encodes two or more polypeptides separated by one or more ribosome skip sequences. The isolated RNA polynucleotide according to any one of claims 167 to 199.

201. The coding region further includes one or more regulatory elements selected from the group consisting of a ribosome binding site, a Kozak sequence, a Shine-Dalgarno sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site, and an internal ribosome entry site (IRES). The isolated RNA polynucleotide according to any one of claims 167 to 200.

202. The one or more regulatory elements are operably linked to the coding sequence. The isolated RNA polynucleotide according to claim 201.

203. Further including a polyadenylation signal and / or a 3' poly(A) tail. The isolated RNA polynucleotide according to any one of claims 167 to 202.

204. The RNA-dependent polymerase is an RNA-dependent RNA polymerase. The isolated RNA polynucleotide according to any one of claims 167 to 203.

205. The RNA-dependent polymerase is an RNA-dependent DNA polymerase. The isolated RNA polynucleotide according to any one of claims 167 to 203.

206. The isolated RNA polynucleotide according to any one of claims 167 to 205, wherein the RNA-dependent polymerase is the polymerase derived from the virus.

207. The isolated RNA polynucleotide according to any one of claims 167 to 206, wherein the isolated RNA polynucleotide is single-stranded RNA.

208. The isolated RNA polynucleotide according to any one of claims 167 to 207, wherein the isolated polynucleotide is in a linear form.

209. The isolated RNA polynucleotide according to any one of claims 167 to 208, wherein the isolated polynucleotide is in a covalently closed circular form.

210. An isolated DNA polynucleotide encoding the isolated RNA polynucleotide according to any one of claims 1 to 209.

211. A cell or cell line comprising the isolated DNA polynucleotide according to claim 210.

212. A vector comprising the isolated RNA polynucleotide according to any one of claims 1 to 209 or the isolated DNA polynucleotide according to claim 210.

213. The vector according to claim 212, wherein the vector is a viral vector or an expression vector.

214. The vector according to claim 213, wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentivirus vector, a herpesvirus vector, an alphavirus vector, and a baculovirus vector.

215. The isolated RNA polynucleotide according to any one of claims 1 to 209, an RNA-binding protein, and an RNA-protein complex, wherein the isolated RNA polynucleotide of the RNA-protein complex has increased stability compared to the isolated RNA polypeptide that does not contain the RNA-binding protein. RNA-protein complex.

216. The RNA-protein complex according to claim 215, wherein the RNA-binding protein is a viral nucleocapsid protein (N) or a viral capsid protein.

217. The RNA-protein complex according to claim 215 or 216, wherein the RNA-binding protein is the viral nucleocapsid protein (N) or the viral capsid protein of the virus.

218. The RNA-protein complex according to claim 216 or 217, wherein the viral nucleocapsid protein or the viral capsid protein is derived from an influenza virus, a simian becovirus, a pneumovirus, a paramyxovirus, a henipavirus or a hepadnavirus.

219. A composition comprising the isolated RNA polynucleotide according to any one of claims 1 to 209, the isolated DNA polynucleotide according to claim 210, the cell or cell line according to claim 211, the vector according to any one of claims 212 to 214, or the RNA-protein complex according to any one of claims 215 to 218.

220. The composition according to claim 219, further comprising a pharmaceutically acceptable carrier.

221. Nanoparticles comprising the isolated RNA polynucleotide according to any one of claims 1 to 209, the isolated DNA polynucleotide according to claim 210, or the RNA-protein complex according to any one of claims 215 to 218.

222. A method comprising administering to a subject in need thereof a therapeutically effective amount of the isolated RNA polynucleotide according to any one of claims 1 to 209, the isolated DNA polynucleotide according to claim 210, the cell or cell line according to claim 211, the vector according to any one of claims 212 to 214, the RNA-protein complex according to any one of claims 215 to 218, the composition according to claim 219 or 220, or the nanoparticles according to claim 221.

223. The method according to claim 222, further comprising administering to a subject in need thereof a therapeutically effective amount of a second isolated RNA polynucleotide according to any one of claims 1 to 209, a second isolated DNA polynucleotide according to claim 210, a second cell or cell line according to claim 211, a second vector according to any one of claims 212 to 214, a second RNA-protein complex according to any one of claims 215 to 218, a second composition according to claim 219 or 220, or a second nanoparticle according to claim 221.

224. The method according to claim 222 or 223, wherein the subject is a human, a cow, a pig, a sheep, a horse, a deer, a ruminant, a rodent, a fish or a poultry.

225. The method according to any one of claims 222 to 224, wherein the subject has a disease or disorder caused by a viral infection.

226. The method according to any one of claims 222 to 225, wherein the subject has an infection by a virus.

227. The method according to any one of claims 222 to 226, wherein the administration is by an intratracheal or inhaled, intranasal, oral, rectal, vaginal, transmucosal, or enteral administration; an intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and an intrathecal, direct intraventricular, intravenous or intraperitoneal administration by parenteral delivery.

228. A method comprising contacting a cell with an isolated RNA polynucleotide according to any one of claims Ⅰ to 209, an isolated DNA polynucleotide according to claim 210, a vector according to any one of claims 212 to 214, an RNA-protein complex according to any one of claims 215 to 218, or a composition according to claim 219 or 220, or a nanoparticle according to claim 221.

229. The method according to claim 228, wherein the contacting is in vitro or ex vivo.

230. To a subject in need (i) a therapeutically effective amount of an isolated RNA polynucleotide according to any one of claims Ⅰ to 209, an isolated DNA polynucleotide according to claim 210, a cell or cell line according to claim 211, a vector according to any one of claims 212 to 214, an RNA-protein complex according to any one of claims 215 to 218, or a composition according to claim 219 or 220, or a nanoparticle according to claim 221, and (ii) a second polynucleotide encoding a therapeutic polypeptide or a polymerase that interacts with the polypeptide and is capable of initiating its transcription or translation, A method comprising administering.

231. The method according to claim 230, further comprising administering to the subject (iii) one or more accessory proteins related to polymerase activity.

232. The method according to claim 231, wherein the accessory protein is a nucleocapsid protein.

233. The method according to claim 231 or 232, wherein the polymerase and / or accessory protein is administered in the form of one or more nucleic acids encoding the polymerase and / or accessory protein.

234. The method according to any one of claims 230 to 233, wherein (i) and (ii) are administered continuously or simultaneously.

235. The method according to any one of claims 230 to 234, wherein (i) and (ii) are present on the same polynucleotide.

236. The method according to any one of claims 230 to 234, wherein (i) and (ii) are present on separate polynucleotides.

237. The method according to any one of claims 230 to 236, further comprising administering to a subject in need thereof a therapeutically effective amount of a second isolated RNA polynucleotide according to any one of claims 1 to 209, a second isolated DNA polynucleotide according to claim 210, a second cell or cell line according to claim 211, a second vector according to any one of claims 212 to 214, a second RNA-protein complex according to any one of claims 215 to 218, a second composition according to claim 219 or 220, or a second nanoparticle according to claim 221.

238. The method according to any one of claims 230 to 237, wherein the subject is a human, bovine, porcine, ovine, equine, deer, ruminant, rodent, fish, or poultry.

239. (a) providing a DNA vector encoding an isolated RNA polynucleotide according to any one of claims 1 to 209; (b) linearizing the DNA vector to produce a linear DNA vector; (c) contacting the linear DNA vector with an RNA polymerase to thereby produce the isolated RNA polynucleotide; A method comprising:

240. The method according to claim 239, further comprising (d) subjecting the isolated RNA polynucleotide of (c) to one or more purification steps. 【Claim ​ ​ The method according to any one of claims 239 to 241, wherein the DNA vector comprises a promoter capable of directing the activity of the RNA polymerase and / or restriction endonuclease recognition site.

243. The method according to claim 242, wherein the RNA polymerase is T7 RNA polymerase and the promoter is a T7 promoter.

244. The method according to any one of claims 239 to 243, wherein linearizing the DNA vector comprises contacting the DNA vector with a restriction endonuclease that recognizes the restriction endonuclease recognition site.

245. The method according to any one of claims 239 to 244, wherein the contact in (c) is carried out at about 50°C.

246. The method according to any one of claims 239 to 245, wherein the contact in (c) is carried out in the presence of one or more additional factors selected from the group consisting of ribonucleotide triphosphates, modified nucleotide triphosphates, cap analogs, inorganic pyrophosphatase, and RNase inhibitors.

247. The method according to any one of claims 239 to 246, further comprising formulating the isolated RNA polynucleotide into nanoparticles.

248. A method for producing a transgenic animal or plant, comprising inserting the isolated RNA polynucleotide according to any one of claims 1 to 209, the isolated DNA polynucleotide according to claim 210, the cell or cell line according to claim 211, the vector according to any one of claims 212 to 214, the RNA-protein complex according to any one of claims 215 to 218, or the composition according to claim 219 or 220, or the nanoparticle according to claim 221 into an animal or plant, thereby producing a transgenic animal or plant.

249. The method according to claim 248, wherein the coding sequence encodes an antiviral polypeptide.

250. The transgenic animal or plant according to claim 248 or 249, wherein the transgenic animal or plant has increased resistance to virus infection.

251. The transgenic animal or plant according to any one of claims 248 to 205, which is a bird, pig, fish, cow, horse, camel, dog, cat, mouse, rat, cotton rat, hamster, ferret, primate, or other commercially valuable animal or plant species.

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