Self-amplifying mRNA nucleic acid sequences with enhanced expression levels of foreign genes

Abstract

The present invention relates to an improved self-amplifying mRNA comprising a mutant replicase, wherein the mutant replicase comprises a mutant macrodomain. The present invention also relates to a preparation method and application of the self-amplifying nucleic acid molecule. The alphavirus RNA replicase mutation and self-amplifying mRNA of the present invention have good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a self-amplifying RNA nucleic acid sequence with enhanced exogenous gene expression level. Background Art

[0002] Self-amplifying mRNA (saRNA) is a type of mRNA that can self-amplify in vivo. It can be used in vaccines to produce more antigens, and due to the immunostimulatory effect brought by replication, it can stimulate a stronger immune response. SaRNA is regarded as the next generation of mRNA molecules. Due to its long expression cycle and self-adjuvant effect, it has shown its advantages in effectively inducing specific immunity in infectious diseases and tumor vaccines. In addition, saRNA also has unique potential in protein replacement therapies with expected long half-lives. In 2023, Japan approved the saRNA-based new crown vaccine, further proving the safety of saRNA as a vaccine. Compared with non-replicating mRNA, an important advantage of saRNA is that the expression level of exogenous genes is high and the duration is long, which can significantly reduce the administration dose and reduce side effects. Therefore, further improving the expression efficiency has always been one of the directions of saRNA sequence optimization.

[0003] For the optimization of non-replicating mRNA, the use of nucleotide modification to circumvent the recognition of mRNA by pattern recognition receptors has been proven to be an extremely successful strategy aimed at improving translation efficiency by reducing the innate immune response. However, since saRNA essentially mimics the replication and expression mechanism of viruses, it is more complex than non-replicating mRNA. In addition to the presence of unmodified nucleotides, saRNA replication products including double-stranded RNA, daughter RNA with Cap0 structure, and uncapped 5'-triphosphate daughter RNA can be recognized by pattern recognition receptors (PRR) and initiate antiviral immune responses including but not limited to interferon or NF-KB signaling pathways. The interferon-stimulated factors induced by this, such as protein kinase R (PKR), 2-5-oligoadenylate synthetase (OAS), and interferon induced proteins with tetratricopeptide repeats (IFIT), will perform functions such as host translation inhibition and induction of apoptosis to limit saRNA replication and expression. Therefore, reducing the immunogenicity of saRNA itself has a positive effect on improving the expression efficiency of saRNA, and is therefore the main idea for optimizing the expression efficiency of saRNA.

[0004] Generally, in vitro synthesis of mRNA is mainly achieved by in vitro transcription (IVT) using linearized plasmid DNA or PCR amplification products as templates using RNA polymerase. Due to the long length of self-amplified RNA, linearized plasmid DNA is usually selected as the transcription template to ensure sequence accuracy. Therefore, the plasmid needs to be linearized using a specific DNA endonuclease (such as BspQI or XbaI). However, due to or partially due to the non-specific digestion of the DNA endonuclease, unexpected linearization products are produced, resulting in non-target in vitro transcription products, which ultimately reduce the integrity and expression efficiency of the self-amplified RNA product.

[0005] There is still a need to develop new self-amplifying mRNAs with enhanced expression levels of exogenous genes. Summary of the invention

[0006] The present disclosure provides improved saRNA constructs, and methods for preparing and using the constructs. The present disclosure also provides the use of the saRNA to carry target genes for treatment, prevention and / or diagnosis. Specifically, after in-depth research and creative work, the present invention has obtained an optimized self-amplified mRNA backbone sequence, which can significantly reduce the intensity of the induced natural immune response, significantly reduce the translation inhibition, and significantly reduce cell apoptosis, thereby significantly improving the expression efficiency.

[0007] In one aspect, the present invention provides an mRNA molecule encoding an RNA replicase composed of nonstructural proteins 1, 2, 3 and 4 derived from an alphavirus, wherein the nonstructural protein 3 comprises a macrodomain at its N-terminus as shown in the amino acid sequence of SEQ ID NO: 3 or 5. In some embodiments, the nonstructural proteins 1, 2, 3 and 4 are derived from Venezuelan equine encephalitis virus.

[0008] In some embodiments, the mRNA molecule encodes a nonstructural protein 1 having an amino acid sequence as shown in SEQ ID NO: 17 or at least 90% identical to SEQ ID NO: 17, a nonstructural protein 2 having an amino acid sequence as shown in SEQ ID NO: 18 or at least 90% identical to SEQ ID NO: 18, and a nonstructural protein 4 having an amino acid sequence as shown in SEQ ID NO: 21 or at least 90% identical to SEQ ID NO: 21. Further, the mRNA molecule encodes a nonstructural protein 3 having an amino acid sequence at least 90% identical to SEQ ID NO: 19 or 20 and comprising a macrodomain as shown in the amino acid sequence of SEQ ID NO: 3 or 5 at the N-terminus.

[0009] In some embodiments, the mRNA molecule comprises a nucleotide sequence encoding the macrodomain as shown in SEQ ID NO: 14 or 16.

[0010] In some embodiments, the mRNA molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 13 or 15.

[0011] In some embodiments, the mRNA molecule further comprises a target gene coding sequence and, optionally, an RNA promoter upstream of the target gene coding sequence.

[0012] In some embodiments, the mRNA molecule further comprises:

[0013] 5' untranslated region, RNA promoter, target gene coding sequence, 3' untranslated region and polyadenylation sequence,

[0014] Optionally, the mRNA molecule further comprises a 5' cap sequence, a signal peptide coding sequence, a Kozak sequence and / or a restriction enzyme cleavage site. The Kozak sequence may be connected to the 3' end of the RNA promoter.

[0015] In some embodiments, the mRNA molecule comprises the following operably linked nucleotide sequences in order from 5' to 3': a 5' untranslated region, coding sequences of nonstructural proteins 1, 2, 3 and 4, an RNA promoter, a target gene coding sequence, a 3' untranslated region and a polyadenylation sequence,

[0016] Optionally, the nucleotide sequences may be connected via a linker sequence.

[0017] In some embodiments, the coding sequences of non-structural proteins 1, 2, 3 and 4 comprise a nucleotide sequence encoding non-structural protein 1, a nucleotide sequence encoding non-structural protein 2, a nucleotide sequence encoding non-structural protein 3 and a nucleotide sequence encoding non-structural protein 4 that are operably linked.

[0018] In some embodiments, the coding sequences of non-structural proteins 1, 2, 3 and 4 comprise, from 5' to 3', a nucleotide sequence encoding non-structural protein 1, a nucleotide sequence encoding non-structural protein 2, a nucleotide sequence encoding non-structural protein 3 and a nucleotide sequence encoding non-structural protein 4 that are operably linked.

[0019] In some embodiments, the 5' untranslated region is derived from an alphavirus; preferably, derived from Venezuelan equine encephalitis virus; for example, the 5' untranslated region comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:9.

[0020] In some embodiments, the 3' untranslated region is derived from an alphavirus; preferably, derived from Venezuelan equine encephalitis virus; for example, the 3' untranslated region comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:10.

[0021] In some embodiments, the RNA promoter is a subgenomic promoter, such as a subgenomic promoter derived from an alphavirus. Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus.

[0022] In some embodiments, the RNA promoter is a 26S promoter. For example, the sequence of the RNA promoter is shown in SEQ ID NO:11.

[0023] In some embodiments, the sequence of the poly(A) sequence is as shown in SEQ ID NO:12.

[0024] In some embodiments, the target gene coding sequence is a sequence encoding a therapeutic polypeptide, a preventive polypeptide, a diagnostic polypeptide, a reporter gene, an antigen, or a sequence encoding a regulatory structure (eg, a non-coding gene).

[0025] In some embodiments, the mRNA molecule comprises a nucleotide sequence as shown in any one of SEQ ID NO:4, SEQ ID NO:6 to SEQ ID NO:8.

[0026] In one aspect, the invention provides a DNA molecule encoding an mRNA molecule as disclosed herein.

[0027] In one aspect, the present invention provides a recombinant vector comprising a DNA molecule as disclosed herein; preferably, the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0028] In one aspect, the present invention provides a recombinant host cell comprising an mRNA molecule, a DNA molecule or a recombinant vector as disclosed herein.

[0029] In one aspect, the present invention provides a pharmaceutical composition comprising an mRNA molecule or a DNA molecule as disclosed herein, and one or more pharmaceutically acceptable carriers; preferably, the carrier is a liposome delivery system.

[0030] In one aspect, the present invention provides a vaccine formulation comprising an mRNA molecule or a DNA molecule as disclosed herein; preferably, the mRNA molecule or the DNA molecule is encapsulated by a liposome delivery system. The vaccine formulation may also contain one or more vaccine adjuvants. Accordingly, the target gene encoded by the mRNA may encode a vaccine antigen. For example, the vaccine formulation is a vaccine formulation for preventing viral infection such as novel coronavirus infection or preventing severe illness caused by novel coronavirus infection. Accordingly, the target gene encoded by the mRNA may encode an immunogenic peptide of the novel coronavirus.

[0031] In one aspect, the present invention provides the use of mRNA molecules or DNA molecules as disclosed herein in the preparation of drugs for treating or preventing viral infections, drugs for treating or preventing tumors, or drugs for protein replacement therapy. Accordingly, the target gene encoded by the mRNA can be a vaccine antigen gene, a tumor killing gene, a therapeutic protein gene, an antibody gene, etc.

[0032] In one aspect, the invention provides a kit comprising an mRNA molecule or a DNA molecule as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1:The mutated amino acid positions of the macrodomain in saRNA and the amino acid sequence comparison results of different virus macrodomains. Macro is the macrodomain, AUD is the alphavirus-unique domain, HVD is the hypervariable domain, different shapes represent different mutations, and amino acids with more than 50% homology in the amino acid comparison results are marked with shades. The full names of the viruses listed in the figure are as follows: SARS-CoV: Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2; MERS-CoV: Middle East Respiratory Syndrome Coronavirus; SFV: Semliki Forest Virus; CHIKV: Chikungunya Virus; SINV: Sindbis Virus; MAYV: Mayaro virus; EEEV: Eastern Equine Encephalitis Virus; VEEV: Venezuelan Equine Encephalitis Virus.

[0034] Figure 2 : Schematic diagram of the protein structure of the saRNA macrodomain. The arrows mark the positions of ADP ribose, glutamine at position 48 (abbreviated Q), and isoleucine at position 113 (abbreviated I) bound to the macrodomain.

[0035] Figure 3 :The results of immunoblotting for the effect of macrodomain amino acid mutation on total cellular protein ADP-ribosylation. Mock is a simulated transfection group, in which only transfection reagent was added without transfection of any RNA molecules. GAPDH protein was used as a cellular protein internal reference. The bottom value is the ratio of total protein ADP-ribosylation level of each group relative to the control group.

[0036] Figure 4: Comparison of the subgenomic RNA replication capacity of mutant saRNA in HeLa cells. The significant differences between the groups were statistically analyzed using two-way ANOVA. **** and ^^^^ represent P values ​​less than 0.0001 for WT-saRNA and 190-saRNA or 191-saRNA groups, respectively.

[0037] Figure 5 : Comparison of the double-stranded RNA formation ability of mutant saRNA during hela cell replication. Figure 5 a is a schematic diagram of flow cytometry results 24 hours after transfection. Figure 5 b is a statistical bar graph of the proportion of double-stranded RNA.

[0038] Figure 6 : Principal component analysis of transcriptome levels 24 hours after HeLa cells were transfected with different saRNAs.

[0039] Figure 7 : Venn diagram of the number of genes upregulated or downregulated in different saRNAs relative to the control group.

[0040] Figure 8 : Ridge plot of differential gene clustering analysis in wild-type saRNA relative to the control group. Figure 8 a is the biological function cluster analysis of differentially expressed genes; Figure 8 b is the molecular function clustering analysis of differentially expressed genes; the horizontal axis is the log2 value of the change fold, green represents the adjusted P value, blue dots represent each down-regulated gene, and red dots represent each up-regulated gene.

[0041] Fig. 9 : Heat map of genes at the transcriptome level of the interferon pathway after transfection with different saRNAs.

[0042] Fig.10 : Fluorescence quantitative PCR verification results of genes related to the innate immune pathway after HeLa cells were transfected with different saRNAs for 2, 6, and 24 hours.

[0043] Fig.11 : Luciferase expression levels in HeLa cells after 2, 6, 24 and 48 hours of transfection with different saRNAs.

[0044] Fig.12 :The results of immunoblotting detection of cell translation activity after 2, 6, and 24 hours of transfection with different saRNAs. GAPDH was used as an internal reference.

[0045] Fig.13 : Detection of ribosomal RNA integrity after HeLa cells were transfected with different saRNAs for 2, 6, 24 and 48 hours. Fig.13a is a schematic diagram of the detection of ribosomal RNA integrity 24 hours after saRNA transfection using capillary electrophoresis, and the arrow marks the degraded ribosomal RNA. Fig.13 b is the statistical results of the detection of ribosomal RNA integrity at different times.

[0046] Fig.14 :Detection of eIF2α phosphorylation level after 6 and 24 hours of transfection with different saRNAs. peIF2α is phosphorylated eIF2α, and total eIF2α protein and GAPDH are used as internal references.

[0047] Fig.15 : Fluorescence microscopy images of different saRNA transfections 24 hours later, red arrows mark cells with nuclear shrinkage and their EGFP expression. DAPI represents stained cell nuclei, and EGFP represents cells expressing EGFP.

[0048] Fig.16 : Statistical results of the proportion of live cells that did not undergo apoptosis 24 and 48 hours after transfection with different saRNAs.

[0049] Fig.17 : Detection results of activated caspase 8 and caspase 3 proteins that were cleaved after 24 hours of transfection with different saRNAs, and the uncleaved full-length caspase was used as a control. GAPDH was used as an internal reference.

[0050] Fig.18 : Detection of cell apoptosis and EGFP expression intensity in HeLa cells transfected with wild-type saRNA treated or not with Caspase inhibitors.

[0051] Fig.19 : Schematic diagram of detection of in vivo expression levels of different mRNA-LNPs. Fig.19 a is a schematic diagram of the detection process, and D0 is the immunization time. Fig.19 b is the fluorescence images of in vivo imaging of mice on days 1, 3, and 7 after immunization.

[0052] Fig. 20 : Time curve of luciferase expression level in mice after mRNA-LNP immunization.

[0053] Fig.21 : Statistical results of the area under the curve of the in vivo expression level in mice after mRNA-LNP immunization. DETAILED DESCRIPTION OF THE INVENTION

[0055] The present application constructs an improved saRNA, which contains specific mutations in the macrodomain of the encoded replicase, so that the saRNA has reduced ADP ribose hydrolysis activity. At the same time, the mutation moderately weakens the replication ability of the replicase encoded by the sgRNA, thereby causing a decrease in the proportion of double-stranded RNA formed in saRNA replication. Since double-stranded RNA is a byproduct in the saRNA replication process, and is often recognized by pattern recognition receptors in cells to stimulate the innate immune response of cells, the reduction of double-stranded RNA will reduce the activation of natural immune receptors, thereby appropriately reducing the intensity of the natural immune response induced by saRNA. As verified in the embodiment, compared to the original wild-type saRNA, the translation inhibition of the sgRNA containing mutations is significantly weakened, and cell apoptosis is significantly reduced, so the expression efficiency of saRNA is significantly improved after mutation.

[0056] Accordingly, this improved sgRNA may contain nucleotide sequences encoding various target proteins (e.g., antigenic proteins), and saRNA that effectively induces specific immunity in vivo can be obtained and prepared into infectious disease and tumor vaccines.

[0057] definition

[0058] As used herein, the terms "self-amplifying RNA" or "saRNA" or "sa-mRNA" are used interchangeably and refer to an mRNA that encodes an RNA replicase domain sequence and has the ability to self-replicate and amplify after entering a cell. saRNA is similar to mRNA, and generally comprises a 5'UTR, a 3'UTR, and a poly (A) tail, but also comprises a non-structural protein sequence derived from a virus (e.g., an alpha virus) and a subgenomic promoter (sgPr) located upstream of the target protein coding sequence. saRNA is a positive-strand RNA molecule that is first translated into several non-structural protein components by the ribosome after entering the cell and assembled into an RNA replicase. RNA replicase first uses the saRNA that enters the cell for the first time to synthesize the saRNA negative strand, and then synthesizes a new saRNA copy using the negative strand as a template, thereby achieving self-amplification of saRNA. At the same time, RNA replicase also recognizes sgPr, and then begins to synthesize subgenomic RNA from its downstream, which accumulates in large quantities in the host cell. When the target protein encoded by saRNA is an antigen protein (for example, used as a vaccine), the antigen gene encoded by the subgenomic RNA will translate a large number of antigen molecules and trigger cellular antigen presentation. The saRNA described herein includes both saRNA backbone sequences without a target protein coding sequence and saRNA that operably connects the saRNA backbone sequence to the target protein coding sequence.

[0059] As used herein, the term "macrodomain" refers to a conserved protein domain encoded by viruses such as alphavirus, coronavirus, and hepatitis E virus, which can recognize and remove ADP ribosylation, and is therefore also considered an ADP ribosyl hydrolase. Many studies have shown that the reduction of ADP ribose hydrolysis activity in the virus macrodomain will lead to a reduction in the ability of the virus to replicate, so the macrodomain is one of the potential hotspots for the development of antiviral drugs in the near future. In the alphavirus family represented by VEEV, the macrodomain is present at the N-terminus of nonstructural protein 3, such as Figure 1 The amino acid sequences of the macrodomain are relatively conservative among different viruses, with a homology of more than 50%.

[0060] As used herein, the term "RNA replicase" is also called RNA-dependent RNA replicase (RdRp), which is a type of RNA polymerase that can synthesize RNA using RNA as a template. It is present in most RNA viruses and plays a role in replicating viral RNA and synthesizing mRNA. It is an enzyme required for the replication of other RNA viruses and viroids except retroviruses. The RNA replicase used for saRNA is usually derived from alphaviruses and contains nonstructural proteins 1, 2, 3, and 4 (nsP1, nsP2, nsP3, and nsP4).

[0061] As used herein, the term "subgenomic promoter" (Subgenomic Promoter, abbreviated as SGP) refers to a nucleic acid sequence upstream (5' end) of a nucleic acid sequence (e.g., a coding sequence) that controls the transcription of the nucleic acid sequence by providing recognition and structural sites for RNA polymerase, typically RNA-dependent RNA polymerase, particularly functional alphavirus nonstructural proteins. SGP may contain additional recognition or binding sites for additional factors. Subgenomic promoters are typically genetic elements of positive-strand RNA viruses (e.g., alphaviruses). The subgenomic promoter of an alphavirus is a nucleic acid sequence contained in the viral genomic RNA. The general feature of a subgenomic promoter is that it allows transcription (RNA synthesis) to begin in the presence of an RNA-dependent RNA polymerase (e.g., a functional alphavirus nonstructural protein). The RNA (-) strand (i.e., the complementary strand of the alphavirus genomic RNA) serves as a template for the synthesis of (+) strand subgenomic transcripts, and the synthesis of (+) strand subgenomic transcripts typically begins at or near the subgenomic promoter.

[0062] As used herein, the term "subgenomic" refers to a nucleotide sequence (such as RNA or DNA) that is smaller in length or size than the genomic nucleotide sequence from which it is derived. For example, a subgenomic region may be a region encoding a VEEV structural protein, and a subgenomic RNA may be transcribed from a subgenomic region using an internal subgenomic promoter, the sequence of which is located in the genomic viral RNA or its complement. Transcription of the subgenomic region may be mediated by a virally encoded polymerase associated with a host cell-encoded protein (e.g., nsP1-4).

[0063] As used herein, the term "effective amount" refers to the amount of the saRNA molecule of any one of the present invention that can prevent or treat the indications or symptoms described in the present invention, or reduce and / or alleviate the indications or symptoms in a subject.

[0064] Composition of saRNA

[0065] The present application reduces the ADP ribose hydrolysis activity by amino acid mutation of the macro domain in the replicase protein domain encoded by saRNA, thereby reducing the replication efficiency of saRNA, thereby reducing the immunogenicity compared to conventional saRNA, and achieving the improvement of expression efficiency. Therefore, the present invention provides a mutation macro domain and a nucleic acid molecule (such as an mRNA molecule or a DNA molecule) encoding the mutation macro domain. The present invention also provides a replicase comprising the mutation macro domain and a nucleic acid molecule (such as an mRNA molecule or a DNA molecule) encoding the replicase. The present invention further provides a nucleic acid molecule (such as a saRNA molecule or a DNA molecule) constructed by combining the nucleic acid sequence encoding the replicase with the sequence encoding the target gene.

[0066] In some embodiments, the saRNA disclosed herein comprises a nucleotide sequence encoding an RNA replicase, wherein the RNA replicase comprises a mutant macrodomain, and the mutant macrodomain has a Q48P or I113F substitution (numbering according to SEQ ID NO: 1) compared to the wild-type macrodomain from VEEV (as shown in SEQ ID NO: 1). Specifically, the mutant macrodomain encoded by the saRNA may comprise an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 5. More specifically, the saRNA may comprise a nucleotide sequence encoding a mutant macrodomain as shown in SEQ ID NO: 14 or SEQ ID NO: 16. The replicase may be an alphavirus replicase, for example, comprising alphavirus nonstructural proteins 1, 2, 3 and 4.

[0067] In some embodiments, the saRNA disclosed herein comprises nucleotide sequences encoding replicase constituent proteins nsP1, nsP2, nsP3 and nsP4 derived from alphavirus, wherein the four replicase constituent proteins are capable of assembling into an RNA replicase complex, wherein the mutant macrodomain is located at the N-terminus of nsP3. The four non-structural protein components nsP1, nsP2, nsP3 and nsP4 are assembled into an RNA replicase complex in the form of a polyprotein. nsP1-4 all have their unique functions, wherein nsP4 plays the role of an RNA polymerase with RNA as a template. The sequence of the replicase constituent protein encoded by the saRNA may be derived from, for example, Venezuelan equine encephalitis virus (VEEV) and forest encephalitis virus (SFV), etc. In some embodiments, the domain sequence of at least one non-structural replicase comprises a sequence selected from Group IV RNA viruses, including Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEE), Sindbis virus, forest encephalitis virus (SFV), etc. In some embodiments, the multiple non-structural protein sequences that form the replicase are derived from an alphavirus such as VEEV. Specifically, the saRNA may comprise a nucleotide sequence encoding a replicase constituent protein as shown in SEQ ID NO: 13 or SEQ ID NO: 15.

[0068] Although the native alphavirus genome encodes structural proteins in addition to the non-structural replicase, in some embodiments, the alphavirus-based saRNA of the present invention does not encode alphavirus structural proteins.

[0069] In some embodiments, in addition to the nucleotide sequences encoding the four replicase proteins nsP1, nsP2, nsP3 and nsP4, the saRNA further comprises a 5' untranslated region, a 3' untranslated region and a poly (A) tail, wherein the nucleotide sequences encoding the four replicase proteins nsP1, nsP2, nsP3 and nsP4 are located between the 5' untranslated region and the 3' untranslated region. It will be readily understood by those skilled in the art that the 5'UTR, 3'UTR and poly (A) sequences are not limited to those exemplified herein, and sequences conventionally used for mRNA construction can be used for the saRNA of the present invention. The 5' untranslated region may be derived from a variety of viruses, such as alphaviruses and coronaviruses. In some embodiments, the 5' untranslated region is from Venezuelan equine encephalitis virus. Specifically, the 5' untranslated region may comprise a nucleotide sequence as shown in SEQ ID NO: 9. The 3' untranslated region may be derived from a variety of viruses, such as alphaviruses and coronaviruses. In some embodiments, the 3' untranslated region is from Venezuelan equine encephalitis virus. Specifically, the 3' untranslated region may comprise a nucleotide sequence as shown in SEQ ID NO: 10. In some embodiments, the poly(A) sequence comprises 20-200 adenylic acids. The poly(A) sequence may comprise a restriction enzyme site inside or at the 3' end.

[0070] In some embodiments, the saRNA further comprises a nucleotide sequence encoding a target protein or a target gene (GOI) coding sequence, wherein the target protein is or the target gene encodes any one selected from the following: a therapeutic polypeptide, a preventive polypeptide, a diagnostic polypeptide, a reporter gene, an antigen, or a gene encoding a regulatory structure. For example, the target protein can be an infectious disease antigen, an allergic antigen, or a tumor antigen. Alternatively, the target gene can encode a non-coding gene, such as siRNA, microRNA, gRNA, etc.

[0071] In some embodiments, the saRNA further comprises a nucleotide sequence encoding one or more antigenic proteins for use as a vaccine. The antigenic protein can be a viral antigenic protein.

[0072] In some embodiments, the saRNA further comprises a nucleotide sequence encoding one or more therapeutic proteins or immunomodulators for use as disease therapeutic agents. The immunomodulators can be cytokines, chemokines or other immunostimulants or inhibitors.

[0073] In some embodiments, the saRNA disclosed herein comprises two expression units, wherein the first expression unit encodes a plurality of nonstructural domain sequences of an RNA replicase, the second expression unit encodes a protein of interest operably linked to a subgenomic promoter, the first and second expression units are operably linked and the first expression unit is located at the 5' end of the second expression unit. In some embodiments, the subgenomic promoter is a 26S promoter, for example, as shown in SEQ ID NO: 11.

[0074] Therefore, the saRNA of the present disclosure has at least two coding regions, the first coding region encodes multiple non-structural replicase domain sequences; the second coding region encodes a gene of interest operably linked to a subgenomic promoter.

[0075] More specifically, the saRNA disclosed herein may comprise the following 5' to 3' operably linked nucleic acid sequences: 5'UTR-nsP-SGP-GOI-3'UTR-Poly A,

[0076] More specifically, the saRNA disclosed herein may comprise the following nucleic acid sequences operably linked from 5' to 3': 5'UTR-nsP1-nsP2-nsP3-nsP4-SGP-GOI-3'UTR-Poly A,

[0077] Wherein, 5'UTR is 5' untranslated region, nsP (including nsP1, nsP2, nsP3, nsP4) is a plurality of non-structural protein sequences capable of forming replicase, SGP is a subgenomic promoter, GOI is one or more target protein encoding genes, 3'UTR is 3' untranslated region, Poly-A is 3' polyadenylic acid tail, and wherein nsP comprises a mutated macro domain. When there are multiple GOIs, each GOI can be operably linked to its respective SGP. In some embodiments, the SGP is a 26S promoter, for example as shown in SEQ ID NO: 11.

[0078] In some embodiments, the saRNA disclosed herein comprises a 5' non-translated sequence that is at least 85% identical to SEQ ID NO: 9, a non-structural protein coding sequence that is at least 85% identical to SEQ ID NO: 14 or 16 and comprises a nucleotide sequence encoding a macrodomain as shown in SEQ ID NO: 3 or 5, and / or a 3' non-translated sequence that is at least 85% identical to SEQ ID NO: 10. The at least 85% identical may be at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, and encompasses functional variants that retain the corresponding functions of these sequences.

[0079] In some embodiments, the 5'UTR, nsP, SGP and 3'UTR sequences are all derived from alphaviruses. The alphavirus genome encodes 4 nonstructural proteins and 6 structural proteins (capsid, E3, E2, 6K, TF and E1). Alphavirus structural proteins (core nucleocapsid protein C, envelope protein E2 and envelope protein E1, all components of viral particles) are usually encoded by a single open reading frame under the control of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562). The subgenomic promoter is recognized by cis-acting alphavirus nonstructural proteins. In particular, the alphavirus replicase uses the (-) strand complementary strand of the genomic RNA as a template to synthesize the (+) strand subgenomic transcript. (+) strand subgenomic transcripts encode alphavirus structural proteins (Kim et al., 2004, Virology, vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. vol. 278, pp. 41636-41645). Subgenomic RNA transcripts serve as templates for translation of open reading frames encoding structural proteins as a polyprotein, and polyproteins are cleaved to produce structural proteins.

[0080] The saRNA disclosed herein can linearize the DNA plasmid template before being prepared by in vitro transcription. To this end, a restriction enzyme site can be added to the 3' end of the saRNA to facilitate cutting. In addition, the saRNA can further contain other coding sequences, such as a signal peptide coding sequence at the 5' end, other sequences compatible with the replicase coding sequence, a Kozak sequence downstream of the SGP promoter, and other downstream coding regions, for example, for encoding other desired gene products.

[0081] In some specific embodiments, the saRNA disclosed herein comprises a saRNA backbone sequence as shown in SEQ ID NO: 7 or 8 (i.e., no target protein coding sequence or target gene sequence is added). In some specific embodiments, the saRNA disclosed herein comprises a nucleic acid sequence as shown in SEQ ID NO: 4 or 6, wherein luciferase and green fluorescent fusion protein coding sequences are used as exemplary target proteins.

[0082] Effects of saRNA

[0083] The saRNA of the present invention has the advantages of known saRNA over traditional mRNA, that is, it can achieve the same protein expression level as traditional mRNA with a very low dose, and prolong the existence time of antigen protein in the body, thereby possibly enhancing the immune response. In terms of production, a lower effective dose may reduce the production cost of saRNA. As a therapy, the dose and number of injections used in mRNA therapy can be reduced, thereby prolonging the therapeutic effect while reducing the possible toxic side effects of mRNA and delivery vectors.

[0084] Furthermore, the saRNA of the present invention is also improved to have one or more of the following characteristics:

[0085] (1) The replication efficiency is reduced, and the dsRNA formed during saRNA replication is significantly reduced;

[0086] (2) Reduced the intensity of the natural immune response induced by saRNA;

[0087] (3) Reduced translational inhibition of saRNA;

[0088] (4) Reduced saRNA-induced cell apoptosis;

[0089] (5) Improved the expression efficiency of saRNA.

[0090] The saRNA of the present disclosure contains a mutated macrodomain, so that the ADP ribose hydrolysis activity is weakened, thereby causing the virus to replicate and stimulate the innate immune system. Therefore, the saRNA of the present disclosure may have a reduced cytotoxic effect on host cells or subjects, providing enhanced safety. For example, the saRNA of the present disclosure can produce a high expression level of the encoded gene product while reducing the risk of undesirable effects (such as injection site irritation and / or pain). In addition, since the ability of the saRNA of the present disclosure to stimulate the innate immune system is reduced, it is particularly suitable for vaccine preparation to provide appropriate immunity to the host.

[0091] In addition, the saRNA of the present invention does not contain nucleotide sequences encoding viral structural proteins and therefore cannot lead to the production of alphavirus particles containing RNA. The inability to produce these viral particles means that the saRNA cannot self-perpetuate in an infectious form. The alphavirus structural proteins required for perpetuation in wild-type viruses are not present in the saRNA, and their positions are replaced by GOI, so that the saRNA encodes the required gene products, rather than the structural proteins of the alphavirus viral particles.

[0092] More specifically, the saRNA containing a mutant macrodomain of the present invention leads to a significant reduction in the dsRNA formed during replication. There are sensors in human cells that recognize foreign viral invasions, which are called pattern recognition receptors. One of the signals they recognize is double-stranded RNA that appears in the cytoplasm, because this may represent the replication of viral RNA in the cell. During the replication process, saRNA forms double-stranded RNA, which is very similar to the viral RNA in replication, and therefore may stimulate the innate immune response of the cell. This may further enhance the effect of the vaccine. However, this stimulated immune response may promote the immune response as a vaccine, but on the other hand, as a therapy, the stimulated immune response may cause side effects. If the stimulated innate immune response is too strong, the expression of mRNA may be inhibited, which in turn affects the efficacy of therapy and vaccines. In addition, the double-stranded RNA produced during saRNA replication is the key to activating the interferon or NFKB signaling pathway, thereby inducing a large number of downstream antiviral genes to inhibit saRNA replication. Therefore, the immunogenicity of saRNA requires precise design and adjustment, so as to moderately reduce the replication ability of saRNA and the production of double-stranded RNA, and the sgRNA disclosed in the present invention achieves this purpose.

[0093] At the same time, the translation inhibition and induced apoptosis of the saRNA containing the mutant macro domain of the present invention disclosed in the present invention are significantly reduced. mRNA usually triggers intrinsic receptors in cells, such as toll-like receptors (TLR), which lead to the production of type I interferon and the inhibition of translation. However, the translation inhibition of the saRNA of the present invention is reduced due to reduced immunogenicity, so although its replication ability is reduced, the final expression efficiency in the cell is improved compared to the control saRNA encoding the wild-type macro domain.

[0094] Methods for preparing saRNA

[0095] saRNA is generally obtained by in vitro transcription (IVT) by RNA polymerase. In one aspect, the present disclosure relates to a method for obtaining self-amplified mRNA, comprising: using a DNA molecule (e.g., a plasmid) encoding the saRNA of the present disclosure as a template and RNA polymerase to produce saRNA by in vitro transcription. For example, saRNA can be prepared by in vitro transcription of DNA encoding saRNA using a suitable DNA-dependent RNA polymerase, such as T7 bacteriophage RNA polymerase, SP6 bacteriophage RNA polymerase, T3 bacteriophage RNA polymerase, T5 bacteriophage RNA polymerase, RNA polymerase III, RNA polymerase II, Taq polymerase, etc., or mutants of these polymerases.

[0096] The transcription reaction will contain nucleotides as well as other components that support the activity of the selected polymerase, such as a suitable buffer and suitable salts. In addition, nucleotide analogs can also be incorporated into saRNA to, for example, alter the stability of such RNA molecules, increase resistance to ribonucleases, establish replication after introduction into appropriate host cells, and / or induce or reduce innate and adaptive immune responses.

[0097] Applications of saRNA

[0098] mRNA-based therapy is the delivery of in vitro synthesized mRNA to specific cells in the human body, where it is translated into the desired protein in the cytoplasm. As a vaccine or drug, mRNA can be used to prevent infectious diseases, treat tumors, and for protein replacement therapy. mRNA drugs have rich targets and are not restricted by the drugability of the target protein or intracellular / extracellular restrictions. The mRNA sequence is easy to design, and the patient's own cells are used to produce molecules, bypassing the challenges of chemical synthesis, and the self-induced molecules have stronger efficacy. The mRNA production platform has strong scalability and replicability, and is easy to scale up.

[0099] In one aspect, the present disclosure relates to a pharmaceutical composition, such as a vaccine composition comprising a saRNA of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a saRNA delivery system, preferably a nanoparticle composition. In some aspects, the nanoparticle composition comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

[0100] In one aspect, the sa-mRNA disclosed herein can be used for vaccination. The vaccine can be a preventive vaccine in the field of infectious diseases. For example, the target protein encoded in the saRNA is an antigen of a pathogen related to an mRNA vaccine encoding an infectious disease. After injection, specific antigens can be expressed in the body, which can simultaneously induce cellular immunity and humoral immunity, stimulate the production of corresponding antibodies and immune cells to prevent the corresponding pathogens. The vaccine can be a tumor therapeutic vaccine. For example, mRNA encoding tumor-specific antigen targets is delivered to the body in a specific manner, so that these tumor-specific antigens are translated and presented on the cell surface, thereby activating the immune system so that it can specifically recognize and kill tumor cells.

[0101] In one aspect, the saRNA disclosed herein can be used to treat diseases, for example, by providing therapeutic proteins or immune regulatory proteins for diseases. Typically, protein replacement therapy is used to treat rare monogenic diseases, aiming to restore enzyme function. Protein synthesis is difficult, administration presents many problems and is expensive, and using mRNA to turn the human body into a processing plant for its own proteins is theoretically an economically feasible and efficient way. The saRNA disclosed herein can theoretically synthesize any target protein and can be used as a protein supplement or replacement therapy to treat a variety of diseases.

[0102] In one aspect, the present disclosure provides a method of delivering a protein of interest to a subject, comprising administering to the subject a pharmaceutical composition containing one or more self-amplifying mRNAs of the present disclosure.

[0103] Sequence Summary

[0104] The sequence involved in the present invention is as follows:

[0105] Sequence 1: Wild-type macrodomain amino acid sequence from VEEV

[0106] APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARRE(SEQ ID NO:1)

[0107] Sequence 3: Amino acid sequence of the macrodomain corresponding to saRNA-190 (introducing Q48P mutation, the corresponding RNA coding sequence changes from CAG to CCG)

[0108] APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLPPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARRE(SEQ ID NO:3)

[0109] Sequence 5: Amino acid sequence of the macrodomain corresponding to saRNA-191 (I113F mutation introduced, the corresponding RNA coding sequence changed from ATC to TTC)

[0110] APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGFFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARRE(SEQ ID NO:5)

[0111] Sequence 2: Wild-type saRNA sequence

[0112]

[0113]

[0114]

[0115]

[0116] Single underline: 5' and 3' UTR; Double underline: replicase domain sequence (shaded part is macro domain coding sequence); Box: 26S promoter and Kozak sequence; Italic: luciferase and green fluorescent fusion protein coding sequence, but can be replaced by any exogenous gene sequence; Dotted underline: BsQI restriction enzyme sequence

[0117] Sequence 4: saRNA-190 sequence (different from sequence 3 only by the introduction of the Q48P mutation in the macrodomain)

[0118] AUGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAUGGAGAAAGUUCACGUUGACA

[0119] UCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAG

[0120] CAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACG

[0121] GAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCAC

[0122] AAGUAUAUUGUAUCUCUCCGAUGAGAGUGCGGAAGAUCCGGACAGAUGUAUAAGUAUUGCAACUAAG

[0123] CUGAAGAAAAACUGUAAGGAAAUAACUGUAAGGAAUUGGACAAGAAAUGAAGGAGCUCGCCGCCGUC

[0124] AUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUGGUGUCGCUACGAAGG

[0125] GCAAGUCGCUGUUACCAGGAGAUGUAUACGCGGUUGACGGACCGCAAGUCUCUAUCAACCAAGCCAAUAA

[0126] GGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCUUUAUGUUAAGAACUUGGCUGGAG

[0127] CAUAUCCAUCAUACUCUACCAACUGGGCCGACGAAACCGUGUUAACGGCUCGUAAACUAGGCCUAUGCA

[0128] GCUCUGACGUUAUGGAGCGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCA

[0129] AAUGUUUUUUCUUGGCUCGACCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACC

[0130] UGCCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUAGUUGCG

[0131] ACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCUUCAGGCUAUGCUGCU

[0132] ACGAUGCACCGCGAGGAUUCUUGGCUGCAAAGUGACAGACACAUUGAACGGGGAGAGGGUCUCUUU

[0133] UCCCGUGUGCACGUAUUGGCCAGCCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAUGUCAG

[0134] UGCGGACGACGCGCAAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGA

[0135] GAAAACCAAUACCAUGAAAAAUAUACCUUUGCCCGUAGUGGCCCAGGCAAUUUGCUAGGUGGGGCAAAGG

[0136] AAUAUAAGGAAGAUCAAGAAGAUGAAAGGCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGU

[0137] UGUUGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAA

[0138] GUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAAUUGGAGAUCGGGCUGAG

[0139] AACAAGAAUCAGGAAAUGUUAGAGGAGCACAAGGAGCCGUCACCUCUCAUUACCGCCGAGGACGUACA

[0140] AGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAGUUGGCGCGCAGCUCUAC

[0141] CACCUUUGGCAGCUGAUGUGAGGAGCCCACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCU

[0142] GGGGCCGGCUCAGUGGAGACCACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAGAU

[0143] CGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUAUCUUGCAUCCACCCUCU

[0144] CGCUGAACAAGUCAUAGUGUAAACACACUCUGGCCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGG

[0145] UAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAGGACAUUUCAAGCUCUGAGUGAAAGUGCCA

[0146] CCAUUGUGUACAACGAACGUGAGUUCGUAAACAGGUACCUGCACCAAUAUUGCCACACAUGGAGGAGCGC

[0147] UGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGCGAGCACGAGCACGGCGAAUACCUGUACGACA

[0148] UCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUGGAU

[0149] CCUCCCUUCCAUGAAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCAACC

[0150] AUAGGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUCACCAAAAAAGA

[0151] UCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAAGGGACGUCAAGAAAAUGAAAGGGC

[0152] UGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGU

[0153] AUAUUGACGAAGCUUUUGCUUGUCAUGCAGGUACUCUCAGAGCGCCUCAUAGCCAUUAUAAAGACCUAAAA

[0154] AGGCAGUGCUCUGCGGGGAUCCCAAACAGUGCGGUUUUUUAACAUGAUGUGCCUGAAGUGCAUUUU

[0155] AACCACGAGAUUUGCACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCG

[0156] GUCGUCUCAACCUUGUUUACGAACAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUGAUU

[0157] GACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCUUUUCAUCAGAGGGUGGGUGAAG

[0158] CAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAUAGACGGCAGCUGCCCUCUCAAGGGCUGACCCGUAA

[0159] GGUGUGAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCUCUGUACGCACCACCUCAGAACAUGUGAAC

[0160] GUCCUACUGACCCGCACGGAGGACCGCAUCGUGUGGAAACACUAGCCGGCGACCCAUGGAUAAAAAACA

[0161] CUGACUGCCAAGUACCCUGGGAAUUUCACUGCCACGAUAGAGAGUGGCAAGCAGACAUGAUGCCAU

[0162] CAUGAGGCACAUCUUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGG

[0163] CCAAGGCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACACUGAACAAUGGAACACUGUG

[0164] GAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGCGUGAGGUUCUUU

[0165] GGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUUAGGAAUAUCACUGG

[0166] GAUAACUCCCCGUCGCCUAAACAUGACGGGCUGAAUAAAGAGUGGUCCGUCAGCUCUCUCCGCAGGUAC

[0167] CCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAU

[0168] GAUCCGCGCAUAAACCUAGUACCUGUAACAGAAGACUGCCUCAUUGCUUUAGUCCUCCCACCAUAAUGAA

[0169] CACCCACAGUGACUUUUCUUCAUUCGUCAGCAAAUUGAAGGCAGAACUGUCCUGGUGGUCGGGGA

[0170] AAAGUUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGACCU

[0171] GGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAUUGUGAGGACCCCAU

[0172] AUAAAUACCAUCACUAUCAGCAGUGUGAGACCAUGCCAUUAAGCUUAGCAUGUUGACCAAGAAAGCUU

[0173] GUCUGCAUCUGAAUCCCGGCGGGAACCUGUGUCAGCAUAGGUUAUGGUUACGCUGACAGGGCCAGCGAA

[0174] AGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGGUAUUGCAAACCGAAUCCUCACUUGAA

[0175] GAGACGGAAGUUCUGUUUGUAUUCAUGGGUACGAUCGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUU

[0176] UCAUCAACCUUGACCAAUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGUGCACCCUCUAUAUCAU

[0177] GUGGUGCGAGGGAUAUUGCCACGGCCACCGAAGGAGUGUGAUUAUAAAUGCUGCUAACAGCAAAGGACA

[0178] ACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAAAUUCCCGGAAAGCUUCGAUUUACCGCCGAUCG

[0179] AAGUAGGAAAAGCGCGACUGGUCAAAGGUGCAGCUAAACAUAUCAUGCCGUAGGACCAAACUUCA

[0180] ACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUC

[0181] AACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAU

[0182] CGACUAACCCAAUCAUUGACAUUUGCUGACAGCUUAGACACCACUGAUGCAGAUGUAGCCAUAUAC

[0183] UGCAGGGACAAGAAAUGGGAAUGAUCUCUCCAAGGAAGCAGUGGCUAGGAGAGAACAGUGGAGGAUA

[0184] UGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUC

[0185] UUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGCAAACUUUCUCAUAUUUGGAAGGGACCAAGU

[0186] UUCACCAGGCGGCCAAGGAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAG

[0187] CAGGUAUGCAUGUAUAUCCUCCGGAGAAAGCAUGAGCAGUAUAGGUCGAAAUGCCCCGUCGAAGAGUC

[0188] GGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUGGCAUCCAUGCCAUGACUCCAGAAAAGAGUACA

[0189] GCGCCUAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAUAGAAU

[0190] CACUGGUGUGCAGAAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUGCCUGCGUAUAUUCA

[0191] UCCAAGGAGAUAUCUCGGGAAACCACCGGUAGACGAGACUCCGGAGCCAUCGGGCAGAGAACCAAUC

[0192] CACAGAGGGGACACCUGAACAACCACCACUUAUAACCGAGGAUGAGACCAGGACUAGACGCCUGAGCC

[0193] GAUCAUCAUCGAAGGAAGGAAGGAAUAGCAUAAGUGUCUGUCAGAUGGCCCGACCCACCAGGUGC

[0194] UGCAAGUCGAGGCAGACAUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCCAUCCUCAUGCAU

[0195] CCGACUUUGAUGUGGACAGUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCA

[0196] ACGUCAGCCGAGACUAACUCUUAUCUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCC

[0197] UCGAACAGUAUUCAGGAACCCUCCAUCCCGCCUCGCGCACAAGAACACGUCACUUGCACCCAGCAG

[0198] GGCCUGCUCGAGAACCAGCCUAGUUUCCCACCCGCCAGGCGUGAGAUAGGGUGAUCACUAGAGAGGAGC

[0199] UCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAACCAGCCUGGUCUCCAACCCGC

[0200] CAGGCGUAAAUAGGGUGAUUACAAGAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUU

[0201] GAUGCGGGUGCAUACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAA

[0202] ACGGUGCUAUCCGAAGUGGUGUUGGAGAGGACGAAUGGAGAUUCGUAUGCCCCGCGCCUCGACCA

[0203] AGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCA

[0204] GUCCAGGAAGGUGGAGAACAUGAAGCCAUAACAGCUAGACGUAUUCUGCAAGGCUAGGGCAUUAUU

[0205] UGAAGGCAGAGGAAAAGUGGAGUGCUACCGAACCCUGCAUCCUGUUCCUUUGUAUUCAUCUAGUGUGA

[0206] ACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGUUGAAGAGACUUUCCGA

[0207] CUGUGGCUUCUACUGUAUAUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCU

[0208] GCUUAGACACUGCCAGUUUUUGCCUGCAAAGCUGCGCGAGCUUUCCAAAGAAACACUCCUAUUUGGAAC

[0209] CCACAAUACGAUCGGCAGUGCCUUCAGGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAA

[0210] AAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUGUGGAAU

[0211] GCCUUCAAGAAAUAUGCGUGAUAUAAUGAAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUG

[0212] AAGAAAACGUGGUAAAUUACAUUACCCAAAUUAAAAGGACCAAAAAGCUGCUGCUCUUUUUGCGAAGACAC

[0213] AUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGACAGGGUUUGUAAUGGACCUUAAAAGAGAGAGACGUGAAAG

[0214] UGACUCCAGGAACAAAACAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAG

[0215] CAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAUAGCGGUCCUGCUUCCGAAC

[0216] AUUCAUACACUGUUGAUAGUCGGGCUGAGAGACCUUGACCGCUAUAUAUAGCCGAGCACUUCCAGGCCUGGG

[0217] GAUGUGUUCUGGAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGACGCCAUGGCUCUGACCGC

[0218] GUUAAUGAUUCUGGAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCG

[0219] AAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGGAAUGU

[0220] UCCUCACACUGUUUGGAACACAGUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGACGGCUAA

[0221] CCGGAUCACCAUGUGCAGCAUUCAUUGGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAA

[0222] UGGCAGACAGGUGCCCACCUGGGUUGAAUAUGGAAGUCAAGAUAUAUAGCUGUGGUGGGCGAGAAA

[0223] GCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGUGGGCAGA

[0224] CCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAACAUGAUGAUGAGAGAGG

[0225] AAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGAGUGGGUAUUCUUUCAGAGCUGUGCAAGGCAG

[0226] UAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUCAUAUGUAUGGCCAUGACUACUCUAGCUAGCAGUG

[0227] UUAAUCAUUCAGCUACCUGAGAGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAU

[0228] AGUCUAGUCCGCCAAGGCCACCAUGACUCCUUCUCCACAAGCGCCUUCGGUCCAGUUGCCUUCUCCCU

[0229] GGGCCUGCUCCUGGUGUUGCCUGCUGCCUUCCCUGCCCCAGUGUUCACCCUGGAGGACUUCGUGGGGCG

[0230] ACUGGAGACAGACCGCCCGGCUACAACCUGGACCAAGUGCUGGAGCAAGGCGGCGUGAGCAGCCUGUUU

[0231] CAGAACCUGGCGUGAGCGUGACCCCCCAUCCAAAGAAUCGUGCUGAGCGGCGAGAACGGCCUGAAGAU

[0232] CGACAUCCACGUGAUCAUCCCCUACGAAGGCCUGAGCGGCGAUCAGAUGGGGCAGAUCGAGAAGAUCU

[0233] UCAAGGUGGUGUACCCCGUGGACGACCACCACUUCAAGGUGAUCCUGCACUACGGCACCCUGGUGAUC

[0234] GACGGCGUCACCCCCAACAUGAUCGACUACUUCGGCAGACCCUAUGAGGGCAUCGCUGUGUUCGACGG

[0235] CAAGAAGAUCACCGUGACCGGCACCCUGUGGAACGGCAACAAGAUCAUCGACGAGAGACUGAUCAACCC

[0236] CGACGGCAGCCUGCUGUUCAGAGUGACCAUCAACGGCGUGACCGGCUGGAGACUGUGCGAGAGAAUCC

[0237] UGGCCGCACCGCCACCAACUUCAGCCUGCUGAAGCAAGCCGGCGACGUGGAGGAGAACCCCGGCCCCg

[0238] UgAGCAAGGGCGAGGAGCUGUUCACCGGCGUGGUGCCCAUCCUGGUGGAGCUGGACGGCGACGUGAAC

[0239] GGCCACAAGUUCAGCGUGAGCGGCGAGGGCGAGGGCGACGCCACCUACGGCAAGCUGACCCUGAAGUU

[0240] CAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUGGUGACCACCCUGACCUACGGCGUGCA

[0241] GUGCUUCAGCAGAUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGAGCGCCAUGCCCGAGGGCUA

[0242] CGUGCAAGAGAGAACCAUCUUCUUCAAGGACGACGGCAACUACAAGACAAGAGCCGAGGUGAAGUUCG

[0243] AGGGCGACACCCUGGUGAACAGAAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUG

[0244] GGCCACAAGCUGGAGUACAACUACAACAGCCACAACGUGUACAUCAUGGCCGACAAGCAGAAGAAUGGC

[0245] AUCAAGGUGAACUUCAAGAUCAGACACAACAUCGAGGACGGCAGCGUGCAGCUGGCCGACCACUAUCAG

[0246] CAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACACAGAGCGCC

[0247] CUGAGCAAGGACCCCAACGAGAAGAGAGACCACAUGGUGCUGAAGGAGUUCGUGACCGCCGCCGGCAU

[0248] CACCCUGGGCAUGGACGAGCUGUACAAGUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCG

[0249] CGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUU

[0250] UUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA

[0251] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGC (SEQ ID NO:4) Sequence 6: saRNA-191 sequence (differs from sequence 3 only in the introduction of the I113F mutation in the macrodomain)

[0252] AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAUGGAGAAGUUCACGUUGACA

[0253] UCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCGCAGUUUGAGGUAGGAAGCCAAG

[0254] CAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUCAAAACUGAUCGAAACG

[0255] GAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCAC

[0256] AAGUAUAUUGUAUCUCUCCGAUGAGAGUGCGGAAGAUCCGGACAGAUGUAUAAGUAUUGCAACUAAG

[0257] CUGAAGAAAAACUGUAAGGAAAUAACUGUAAGGAAUUGGACAAGAAAUGAAGGAGCUCGCCGCCGUC

[0258] AUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUGGUGUCGCUACGAAGG

[0259] GCAAGUCGCUGUUACCAGGAGAUGUAUACGCGGUUGACGGACCGCAAGUCUCUAUCAACCAAGCCAAUAA

[0260] GGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCUUUAUGUUAAGAACUUGGCUGGAG

[0261] CAUAUCCAUCAUACUCUACCAACUGGGCCGACGAAACCGUGUUAACGGCUCGUAAACUAGGCCUAUGCA

[0262] GCUCUGACGUUAUGGAGCGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCA

[0263] AAUGUUUUUUCUUGGCUCGACCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACC

[0264] UGCCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUAGUUGCG

[0265] ACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCUUCAGGCUAUGCUGCU

[0266] ACGAUGCACCGCGAGGAUUCUUGGCUGCAAAGUGACAGACACAUUGAACGGGGAGAGGGUCUCUUU

[0267] UCCCGUGUGCACGUAUUGGCCAGCCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAUGUCAG

[0268] UGCGGACGACGCGCAAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGA

[0269] GAAAACCAAUACCAUGAAAAAUAUACCUUUGCCCGUAGUGGCCCAGGCAAUUUGCUAGGUGGGGCAAAGG

[0270] AAUAUAAGGAAGAUCAAGAAGAUGAAAGGCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGU

[0271] UGUUGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAA

[0272] GUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAAUUGGAGAUCGGGCUGAG

[0273] AACAAGAAUCAGGAAAUGUUAGAGGAGCACAAGGAGCCGUCACCUCUCAUUACCGCCGAGGACGUACA

[0274] AGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAGUUGGCGCGCAGCUCUAC

[0275] CACCUUUGGCAGCUGAUGUGAGGAGCCCACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCU

[0276] GGGGCCGGCUCAGUGGAGACCACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAGAU

[0277] CGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUAUCUUGCAUCCACCCUCU

[0278] CGCUGAACAAGUCAUAGUGUAAACACACUCUGGCCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGG

[0279] UAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAGGACAUUUCAAGCUCUGAGUGAAAGUGCCA

[0280] CCAUUGUGUACAACGAACGUGAGUUCGUAAACAGGUACCUGCACCAAUAUUGCCACACAUGGAGGAGCGC

[0281] UGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGCGAGCACGAGCACGGCGAAUACCUGUACGACA

[0282] UCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUGGAU

[0283] CCUCCCUUCCAUGAAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCAACC

[0284] AUAGGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUCACCAAAAAAGA

[0285] UCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAAGGGACGUCAAGAAAAUGAAAGGGC

[0286] UGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGU

[0287] AUAUUGACGAAGCUUUUGCUUGUCAUGCAGGUACUCUCAGAGCGCCUCAUAGCCAUUAUAAAGACCUAAAA

[0288] AGGCAGUGCUCUGCGGGGAUCCCAAACAGUGCGGUUUUUUAACAUGAUGUGCCUGAAGUGCAUUUU

[0289] AACCACGAGAUUUGCACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCG

[0290] GUCGUCUCAACCUUGUUUACGAACAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUGAUU

[0291] GACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCUAUUCUCUUUUCAUCAGAGGGUGGGUGAAG

[0292] CAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAUAGACGGCAGCUGCCCUCUCAAGGGCUGACCCGUAA

[0293] GGUGUGAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCUCUGUACGCACCACCUCAGAACAUGUGAAC

[0294] GUCCUACUGACCCGCACGGAGGACCGCAUCGUGUGGAAACACUAGCCGGCGACCCAUGGAUAAAAAACA

[0295] CUGACUGCCAAGUACCCUGGGAAUUUCACUGCCACGAUAGAGAGUGGCAAGCAGACAUGAUGCCAU

[0296] CAUGAGGCACAUCUUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGG

[0297] CCAAGGCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACACUGAACAAUGGAACACUGUG

[0298] GAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGCGUGAGGUUCUUU

[0299] GGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUUAGGAAUAUCACUGG

[0300] GAUAACUCCCCGUCGCCUAAACAUGACGGGCUGAAUAAAGAGUGGUCCGUCAGCUCUCUCCGCAGGUAC

[0301] CCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAU

[0302] GAUCCGCGCAUAAACCUAGUACCUGUAACAGAAGACUGCCUCAUUGCUUUAGUCCUCCCACCAUAAUGAA

[0303] CACCCACAGUGACUUUUCUUCAUUCGUCAGCAAAUUGAAGGCAGAACUGUCCUGGUGGUCGGGGA

[0304] AAAGUUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGACCU

[0305] GGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAUUGUGAGGACCCCAU

[0306] AUAAAUACCAUCACUAUCAGCAGUGUGAGACCAUGCCAUUAAGCUUAGCAUGUUGACCAAGAAAGCUU

[0307] GUCUGCAUCUGAAUCCCGGCGGGAACCUGUGUCAGCAUAGGUUAUGGUUACGCUGACAGGGCCAGCGAA

[0308] AGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGGUAUUGCAAACCGAAUCCUCACUUGAA

[0309] GAGACGGAAGUUCUGUUUGUAUUCAUGGGUACGAUCGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUU

[0310] UCAUCAACCUUGACCAAUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGUGCACCCUCUAUAUCAU

[0311] GUGGUGCGAGGGAUAUUGCCACGGCCACCGAAGGAGUGUGAUUAUAAAUGCUGCUAACAGCAAAGGACA

[0312] ACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAAUUCCCGGAAAGCUUCGAUUUACAGCCGAUCG

[0313] AAGUAGGAAAAGCGCGACUGGUCAAAGGUGCAGCUAAACAUAUCAUGCCGUAGGACCAAACUUCA

[0314] ACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUC

[0315] AACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCUUCUUUUCCGGGAACAAAGAU

[0316] CGACUAACCCAAUCAUUGACAUUUGCUGACAGCUUAGACACCACUGAUGCAGAUGUAGCCAUAUAC

[0317] UGCAGGGACAAGAAAUGGGAAUGAUCUCUCCAAGGAAGCAGUGGCUAGGAGAGAACAGUGGAGGAUA

[0318] UGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUC

[0319] UUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGCAAACUUUCUCAUAUUUGGAAGGGACCAAGU

[0320] UUCACCAGGCGGCCAAGGAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAG

[0321] CAGGUAUGCAUGUAUAUCCUCCGGAGAAAGCAUGAGCAGUAUAGGUCGAAAUGCCCCGUCGAAGAGUC

[0322] GGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACA

[0323] GCGCCUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAUAGAAU

[0324] CACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUGCCUGCGUAUAUUCA

[0325] UCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCGGCAGAGAACCAAUC

[0326] CACAGAGGGGACACCUGAACAACCACCACUUAUAACCGAGGAUGAGACCAGGACUAGAACGCCUGAGCC

[0327] GAUCAUCAUCGAAGAGGAAGAAGAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGC

[0328] UGCAAGUCGAGGCAGACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAU

[0329] CCGACUUUGAUGUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCA

[0330] ACGUCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCC

[0331] UCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCACUUGCACCCAGCAG

[0332] GGCCUGCUCGAGAACCAGCCUAGUUUCCCACCCGCCAGGCGUGAGAUAGGGUGAUCACUAGAGAGGAGC

[0333] UCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAACCAGCCUGGUCUCCAACCCGC

[0334] CAGGCGUAAAUAGGGUGAUUACAAGAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUU

[0335] GAUGCGGGUGCAUACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAA

[0336] ACGGUGCUAUCCGAAGUGGUGUUGGAGAGGACGAAUGGAGAUUCGUAUGCCCCGCGCCUCGACCA

[0337] AGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCA

[0338] GUCCAGGAAGGUGGAGAACAUGAAGCCAUAACAGCUAGACGUAUUCUGCAAGGCUAGGGCAUUAUU

[0339] UGAAGGCAGAGGAAAAGUGGAGUGCUACCGAACCCUGCAUCCUGUUCCUUUGUAUUCAUCUAGUGUGA

[0340] ACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGUUGAAGAGACUUUCCGA

[0341] CUGUGGCUUCUACUGUAUAUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCU

[0342] GCUUAGACACUGCCAGUUUUUGCCUGCAAAGCUGCGCGAGCUUUCCAAAGAAACACUCCUAUUUGGAAC

[0343] CCACAAUACGAUCGGCAGUGCCUUCAGGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAA

[0344] AAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUUGGGAAU

[0345] GCCUUCAAGAAAUAUGCGUGAUAUAAUGAAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUG

[0346] AAGAAAACGUGGUAAAUUACAUUACCCAAAUUAAAAGGACCAAAAAGCUGCUGCUCUUUUUGCGAAGACAC

[0347] AUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGACAGGGUUUGUAAUGGACCUUAAAAGAGAGAGACGUGAAAG

[0348] UGACUCCAGGAACAAAACAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCGAUCCGCUAG

[0349] CAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAUAGCGGUCCUGCUUCCGAAC

[0350] AUUCAUACACUGUUGAUAGUCGGGCUGAGAGACCUUGACCGCUAUAUAUAGCCGAGCACUUCCAGGCCUGGG

[0351] GAUGUGUUCUGGAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGACGCCAUGGCUCUGACCGC

[0352] GUUAAUGAUUCUGGAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCG

[0353] AAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGGAAUGU

[0354] UCCUCACACUGUUUGGAACACAGUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGACGGCUAA

[0355] CCGGAUCACCAUGUGCAGCAUUCAUUGGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAA

[0356] UGGCAGACAGGUGCCCACCUGGGUUGAAUAUGGAAGUCAAGAUAUAUAGCUGUGGUGGGCGAGAAA

[0357] GCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGUGGGCAGA

[0358] CCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAACAUGAUGAUGAGAGAGG

[0359] AAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGAGUGGGUAUUCUUUCAGAGCUGUGCAAGGCAG

[0360] UAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUCAUAUGUAUGGCCAUGACUACUCUAGCUAGCAGUG

[0361] UUAAUCAUUCAGCUACCUGAGAGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAU

[0362] AGUCUAGUCCGCCAAGGCCACCAUGAACUCCUUCUCCACAAGCGCCUUCGGUCCAGUUGCCUUCUCCCU

[0363] GGGCCUGCUCCUGGUGUUGCCUGCUGCCUUCCCUGCCCCAGUGUUCACCCUGGAGGACUUCGUGGGCG

[0364] ACUGGAGACAGACCGCCGGCUACAACCUGGACCAAGUGCUGGAGCAAGGCGGCGUGAGCAGCCUGUUU

[0365] CAGAACCUGGGCGUGAGCGUGACCCCCAUCCAAAGAAUCGUGCUGAGCGGCGAGAACGGCCUGAAGAU

[0366] CGACAUCCACGUGAUCAUCCCCUACGAAGGCCUGAGCGGCGAUCAGAUGGGGCAGAUCGAGAAGAUCU

[0367] UCAAGGUGGUGUACCCCGUGGACGACCACCACUUCAAGGUGAUCCUGCACUACGGCACCCUGGUGAUC

[0368] GACGGCGUCACCCCCAACAUGAUCGACUACUUCGGCAGACCCUAUGAGGGCAUCGCUGUGUUCGACGG

[0369] CAAGAAGAUCACCGUGACCGGCACCCUGUGGAACGGCAACAAGAUCAUCGACGAGAGACUGAUCAACCC

[0370] CGACGGCAGCCUGCUGUUCAGAGUGACCAUCAACGGCGUGACCGGCUGGAGACUGUGCGAGAGAAUCC

[0371] UGGCCGCACCGCCACCAACUUCAGCCUGCUGAAGCAAGCCGGCGACGUGGAGGAGAACCCCGGCCCCg

[0372] UgAGCAAGGGCGAGGAGCUGUUCACCCGGCGUGGUGCCCAUCCUGGUGGAGCUGGACGGCGACGUGAAC

[0373] GGCCACAAGUUCAGCGUGAGCGGCGAGGGCGAGGGCGACGCCACCUACGGCAAGCUGACCCUGAAGUU

[0374] CAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCCUGGUGACCACCCUGACCUACGGCGUGCA

[0375] GUGCUUCAGCAGAUACCCCGACCACAUGAAGCAGCACGACUUCUCAAGAGCGCCAUGCCCGAGGGCUA

[0376] CGUGCAAGAGAGAACCAUCUUCUUCAAGGACGACGCGCAACUACAAGACAAGACCGAGGUGAGUAUCG

[0377] AGGGCGACACCCUGGUGAACAGAAUCGAGCUGAAGGGCAUCGACCUUCAAGGAGGAGCGGCAACAUCCUG

[0378] GGCCACAAGCUGGAGUACAACUACAACAGCCACAACGUGUACAUCAUGGCCGACAAGCAGAAGAAUGGC

[0379] AUCAAGGUGACUUCAAGAUCAGAGACCAACAUCGAGGACGGCAGCGUGCAGCUGGCCGACCACAUUCAG

[0380] CAGAACACCCCAUCGGCGACGGCCCCGUGCUGCCCGACAACCACUACCUGAGCACACAGAGCGCC

[0381] CUGAGCAAGGACCCCAACGAGAAGAGAGACCACAUGGUGCUGAGGAGUUCGUGACCGCCGCGCGCAU

[0382] CACCCUGGGCAUGGACGAGCUGUACAAGUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCG

[0383] CGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUU

[0384] UUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGC (SEQ ID NO:6) Sequence 7: saRNA-190 backbone sequence

[0385] ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACAT

[0386] CGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCA

[0387] GGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGG

[0388] TGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTAT

[0389] CATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAA

[0390] AAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGAC

[0391] CCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTG

[0392] TTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC

[0393] GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCT

[0394] ACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGC

[0395] GGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTG

[0396] GCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACG

[0397] TGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAG

[0398] CTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGC

[0399] TGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACAT

[0400] TGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGG

[0401] GCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGC

[0402] CCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACT

[0403] AGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTT

[0404] ATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATA

[0405] GGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGT

[0406] CACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGA

[0407] AGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGAT

[0408] GTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCA

[0409] GCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAA

[0410] ATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATG

[0411] CCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGC

[0412] TCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCA

[0413] CACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGA

[0414] ATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGC

[0415] GAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACC

[0416] AAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCAC

[0417] CAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATG

[0418] AAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGA

[0419] CCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTA

[0420] AAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTT

[0421] AACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGT

[0422] CGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACA

[0423] CTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTT

[0424] GCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTG

[0425] TATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACT

[0426] GACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCC

[0427] AAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACA

[0428] TCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGT

[0429] GCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACG

[0430] GACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTC

[0431] CGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAA

[0432] CATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTT

[0433] GCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACC

[0434] TGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCAT

[0435] TCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGT

[0436] TGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTG

[0437] CCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGAC

[0438] CATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAG

[0439] CATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTT

[0440] CCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGC

[0441] AAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCA

[0442] CGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATT

[0443] ATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGG

[0444] AAAGCTTCGATTTACCGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATT

[0445] CATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATG

[0446] AGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATC

[0447] TTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGC

[0448] AGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAA

[0449] CAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCA

[0450] TCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGGAA

[0451] GGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGG

[0452] CCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAA

[0453] GAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAG

[0454] TACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGA

[0455] ATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCAT

[0456] CCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCA

[0457] CAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGAT

[0458] CATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAA

[0459] GTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTT

[0460] TGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCC

[0461] GAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTAT

[0462] TCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAG

[0463] AACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACC

[0464] CCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGG

[0465] TGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCATACAT

[0466] CTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTG

[0467] GTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTAC

[0468] GCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACAT

[0469] GAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAG

[0470] TGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGT

[0471] CGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAG

[0472] AGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCA

[0473] AAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGAT

[0474] CCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAA

[0475] TTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGG

[0476] GAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGG

[0477] ACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGT

[0478] TTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGT

[0479] ACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGG

[0480] AGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATT

[0481] ATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGG

[0482] ACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCT

[0483] GATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCAT

[0484] GATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGT

[0485] TGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAA

[0486] TCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGT

[0487] GGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGT

[0488] GTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATG

[0489] ACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAA

[0490] GGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCA

[0491] GTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACA

[0492] TAGTCTAGTCCGCCAAGGCCACCNNNNNNNNNNNNNNNNATACAGCAGCAATTGGCAAGCTGCTTACATAG

[0493] AACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGT

[0494] TTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGC (SEQ ID NO:7) Sequence 8: saRNA-191 backbone sequence

[0495] ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACAT

[0496] CGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCA

[0497] GGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGG

[0498] TGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTAT

[0499] CATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAA

[0500] AAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGAC

[0501] CCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTG

[0502] TTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC

[0503] GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCT

[0504] ACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGC

[0505] GGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTG

[0506] GCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACG

[0507] TGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAG

[0508] CTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGC

[0509] TGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACAT

[0510] TGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGG

[0511] GCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGC

[0512] CCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACT

[0513] AGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTT

[0514] ATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATA

[0515] GGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGT

[0516] CACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGA

[0517] AGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGAT

[0518] GTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCA

[0519] GCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAA

[0520] ATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATG

[0521] CCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGC

[0522] TCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCA

[0523] CACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGA

[0524] ATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGC

[0525] GAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACC

[0526] AAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCAC

[0527] CAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATG

[0528] AAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGA

[0529] CCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTA

[0530] AAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTT

[0531] AACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGT

[0532] CGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACA

[0533] CTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTT

[0534] GCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTG

[0535] TATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACT

[0536] GACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCC

[0537] AAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACA

[0538] TCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGT

[0539] GCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACG

[0540] GACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTC

[0541] CGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAA

[0542] CATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTT

[0543] GCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACC

[0544] TGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCAT

[0545] TCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGT

[0546] TGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTG

[0547] CCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGAC

[0548] CATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAG

[0549] CATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTT

[0550] CCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGC

[0551] AAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCA

[0552] CGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATT

[0553] ATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGG

[0554] AAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATT

[0555] CATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATG

[0556] AGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCTTC

[0557] TTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGC

[0558] AGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAA

[0559] CAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCA

[0560] TCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGGAA

[0561] GGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGG

[0562] CCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAA

[0563] GAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAG

[0564] TACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGA

[0565] ATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCAT

[0566] CCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCA

[0567] CAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGAT

[0568] CATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAA

[0569] GTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTT

[0570] TGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCC

[0571] GAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTAT

[0572] TCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAG

[0573] AACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACC

[0574] CCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGG

[0575] TGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCATACAT

[0576] CTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTG

[0577] GTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTAC

[0578] GCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACAT

[0579] GAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAG

[0580] TGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGT

[0581] CGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAG

[0582] AGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCA

[0583] AAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGAT

[0584] CCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAA

[0585] TTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGG

[0586] GAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGG

[0587] ACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGT

[0588] TTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGT

[0589] ACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGG

[0590] AGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATT

[0591] ATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGG

[0592] ACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCT

[0593] GATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCAT

[0594] GATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGT

[0595] TGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAA

[0596] TCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGT

[0597] GGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGT

[0598] GTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATG

[0599] ACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAA

[0600] GGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCA

[0601] GTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTTCTACGGCTAACCTGAATGGACTACGACA

[0602] TAGTCTAGTCCGCCAAGGCCACCNNNNNNNNNNNNNNNNATACAGCAGCAATTGGCAAGCTGCTTACATAG

[0603] AACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGT

[0604] TTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGC (SEQ ID NO: 8) in the above sequences 7-8:

[0605] “NNNNNNNNNNNNNNNN” indicates exogenous genes, including but not limited to: vaccine antigen genes, tumor killing genes, therapeutic protein genes, antibody genes; exogenous genes can be adjusted according to different purposes;

[0606] Here, "N" can be any base among A, T, C, and G, and the number of bases is not particularly limited; the 16 Ns here are only for illustration and are not limited to 16 bases.

[0607] Sequence 9: 5' untranslated sequence

[0608] ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA(SEQ ID NO:9)

[0609] Sequence 10: 3' untranslated sequence

[0610] ATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTC (SEQ ID NO: 10)

[0611] Sequence 11: Subgenomic promoter sequence

[0612] CCTGAATGGACTACGACATAGTCTAGTCCGCCAAG(SEQ ID NO:11)

[0613] Sequence 12: Polyadenylation sequence

[0614] AAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 12)

[0615] Sequence 13: saRNA-190 replicase coding sequence

[0616]

[0617] GTTGAGGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAG

[0618] ACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTC

[0619] TCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAA

[0620] CACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACA

[0621] TGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTC

[0622] GTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAAC

[0623] TGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGA

[0624] ACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGT

[0625] CTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCA

[0626] AGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGC

[0627] AGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTC

[0628] TTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCT

[0629] CAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTT

[0630] TTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCT

[0631] CGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAA

[0632] TCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCA

[0633] CTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGC

[0634] CTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCA

[0635] CCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCG

[0636] GCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCA

[0637] AGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAG

[0638] GCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAAC

[0639] AATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGC

[0640] GTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAAT

[0641] AATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCG

[0642] CAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGC

[0643] AATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAAT

[0644] GAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGG

[0645] AAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCG

[0646] GCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAA

[0647] ATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGC

[0648] ATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATT

[0649] GGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAG

[0650] TTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA

[0651] CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGAT

[0652] ATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGT

[0653] GCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACCGCCGATCGAAGTAGGAAAAGCGCGACT

[0654] GGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAG

[0655] GTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTA

[0656] GCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTT

[0657] GCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTC

[0658] TCAAGGAAGCAGTGGCTAGGAGAGAACAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAA

[0659] CCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGC

[0660] GATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAA

[0661] TGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGC

[0662] AGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGT

[0663] GCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGC

[0664] TCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTC

[0665] TCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCC

[0666] GGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAG

[0667] ACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAG

[0668] ATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATC

[0669] CTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTA

[0670] GCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCG

[0671] ACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCA

[0672] CTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCA

[0673] CTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGT

[0674] CTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAA

[0675] TGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGT

[0676] AAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTC

[0677] GACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATA

[0678] CCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATT

[0679] TGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAAC

[0680] CGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTG

[0681] TGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAG

[0682] ACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATA

[0683] CGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATT

[0684] GCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAA

[0685] TATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGT

[0686] AAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTT

[0687] GCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAA

[0688] CATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCG

[0689] GAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATG

[0690] TCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACAT

[0691] CGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGT

[0692] GTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAA

[0693] AACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAA

[0694] CATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATG

[0695] ACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAA

[0696] GTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTC

[0697] CGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCA

[0698] GCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTG

[0699] GGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTAT

[0700] GGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAA(SEQ ID NO:13)

[0701] Sequence 14: saRNA-190 macro domain coding sequence

[0702] GCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGC

[0703] TAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGAT

[0704] TTACCGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGG

[0705] ACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCT

[0706] AAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAA

[0707] CAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAA (SEQ ID NO:14) Sequence 15: saRNA-191 replicase coding sequence

[0708] ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCC

[0709] CGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTG

[0710] GCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCC

[0711] GCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTG

[0712] TATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAA

[0713] GGAGCTCGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTC

[0714] GTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTAT

[0715] CACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAA

[0716] CTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAG

[0717] GCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAA

[0718] CCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTG

[0719] GCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTT

[0720] GCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCT

[0721] ACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTC

[0722] CCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCG

[0723] GACGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACA

[0724] CCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAG

[0725] GAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTT

[0726] TTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATT

[0727] TCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAA

[0728] AATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCA

[0729] GCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGAT

[0730] GTTGAGGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAG

[0731] ACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTC

[0732] TCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAA

[0733] CACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACA

[0734] TGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTC

[0735] GTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAAC

[0736] TGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGA

[0737] ACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGT

[0738] CTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCA

[0739] AGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGC

[0740] AGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTC

[0741] TTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCT

[0742] CAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTT

[0743] TTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCT

[0744] CGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAA

[0745] TCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCA

[0746] CTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGC

[0747] CTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCA

[0748] CCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCG

[0749] GCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCA

[0750] AGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAG

[0751] GCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAAC

[0752] AATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGC

[0753] GTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAAT

[0754] AATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCG

[0755] CAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGC

[0756] AATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAAT

[0757] GAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGG

[0758] AAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCG

[0759] GCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAA

[0760] ATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGC

[0761] ATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATT

[0762] GGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAG

[0763] TTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA

[0764] CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGAT

[0765] ATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGT

[0766] GCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACT

[0767] GGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAG

[0768] GTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTA

[0769] GCGATTCCACTGTTGTCCACCGGCTTCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTT

[0770] GCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTC

[0771] TCAAGGAAGCAGTGGCTAGGAGAGAACAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAA

[0772] CCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGC

[0773] GATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAA

[0774] TGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGC

[0775] AGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGT

[0776] GCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGC

[0777] TCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTC

[0778] TCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCC

[0779] GGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAG

[0780] ACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAG

[0781] ATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATC

[0782] CTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTA

[0783] GCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCG

[0784] ACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCA

[0785] CTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCA

[0786] CTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGT

[0787] CTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAA

[0788] TGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGT

[0789] AAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTC

[0790] GACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATA

[0791] CCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATT

[0792] TGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAAC

[0793] CGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTG

[0794] TGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAG

[0795] ACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATA

[0796] CGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATT

[0797] GCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAA

[0798] TATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGT

[0799] AAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTT

[0800] GCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAA

[0801] CATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCG

[0802] GAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATG

[0803] TCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACAT

[0804] CGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGT

[0805] GTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAA

[0806] AACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAA

[0807] CATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATG

[0808] ACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAA

[0809] GTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTC

[0810] CGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCA

[0811] GCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTG

[0812] GGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTAT

[0813] GGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAA(SEQ ID NO:15)

[0814] Sequence 16: Coding sequence of saRNA-191 macro domain

[0815] GCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGC

[0816] TAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGAT

[0817] TTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGG

[0818] ACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCT

[0819] AAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCTTCTTTTCCGGGAA

[0820] CAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAA(SEQ ID NO:16) Sequence 17: Amino acid sequence of non-structural protein 1

[0821] MEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEVDPSDTILDIGSAPARR

[0822] MYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCR

[0823] YEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSD

[0824] VMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKR

[0825] IAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLN

[0826] QRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRP

[0827] DTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDVQEAKCAADEAKEVREAEELRAAL

[0828] PPLAADVEEPTLEADVDLMLQEAGA

[0829] SEQ ID NO:18: Nonstructural protein 2 amino acid sequence

[0830] GSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC

[0831] Sequence 19: Amino acid sequence of non-structural protein 3 (corresponding to saRNA-190, with Q48P mutation introduced)

[0832] APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLPPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQRRFDAGA

[0833] Sequence 20: Amino acid sequence of non-structural protein 3 (corresponding to saRNA-191, with I133F mutation introduced)

[0834] APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGFFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQRRFDAGA

[0835] Sequence 21: Amino acid sequence of non-structural protein 4

[0836] YIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRKVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMV DGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTE ERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFFDKSEDDAMALTALMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLT GSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAADDEHDDDRRRALHEESTRWNRVGILSELCKAVESRYETVGTSIIVMAMTTLASSVKSFSYLRGAPITLYG Example

[0837] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0838] Example 1: Design of macrodomain mutations encoded by saRNA and detection of ADP ribose hydrolase activity

[0839] The present invention selects saRNA derived from Venezuelan equine encephalitis virus (VEEV) for subsequent design. In the alphavirus family represented by VEEV, the macrodomain exists at the N-terminus of nonstructural protein 3, such as Figure 1The amino acid sequences of the macrodomain are relatively conservative among different viruses, with a homology of more than 50%. Therefore, we selected amino acids 113 near the ADP-ribose hydrolysis active center and 48 far from the ADP-ribose hydrolysis active center for amino acid mutation. The specific positions are shown in Figure 2 . In coronaviruses and alphaviruses, position 113 encodes isoleucine (I) or valine (V), which is highly conserved. We infer that it is involved in the ADP ribose hydrolysis function. However, position 48 is quite different and is only encoded as glutamine (Q) in VEEV, so it may not be involved in the ADP ribose hydrolysis function. Since the side chains of phenylalanine and prolineamide are both cyclic structures, they have obvious structural differences from glutamine with only an amino group on the side chain or isoleucine with a methyl group and an ethyl group. Therefore, we constructed the following three saRNAs through homologous recombination and in vitro transcription:

[0840]

[0841]

[0842] Next, 200 ng of saRNA-190, saRNA-191 and wild-type saRNA were transfected into Hela cells. After 24 hours, the level of ADP-ribosylation of total protein in the cells was detected using an anti-mono-ADP-ribosylation antibody. Figure 3 The results showed that 24 hours after transfection, the ADP-ribosylation of 190 / 191-saRNA cell proteins was significantly stronger than that of the wild-type transfection group, indicating that the ADP ribosyl hydrolase activity of saRNA-190 and saRNA-191 was significantly reduced compared with that of wild-type saRNA. The data show that the design of amino acid mutations at positions 113 and 48 of the macrodomain successfully reduced the ADP ribosyl hydrolase activity.

[0843] Example 2: Detection of replication levels of mutant saRNAs with impaired ADP ribose hydrolase activity

[0844] After verifying that the ADP ribose hydrolase activity of the two mutant saRNAs was reduced, we tested whether the replication ability of the mutant saRNAs would change. After transfecting saRNA-190, saRNA-191 and wild-type saRNA into Hela cells for 2, 6, 24 and 48 hours, RNA was extracted and the EGFP gene encoded by saRNA was subjected to fluorescence quantitative PCR.

[0845] like Figure 4The results showed that 190 / 191-saRNA significantly reduced the level of RNA encoding EGFP compared to wild-type saRNA. At the same time, we used anti-dsRNA antibodies to perform flow cytometry on double-stranded RNA in Hela cells 6 hours and 24 hours after transfection with wild-type saRNA, saRNA-190, and saRNA-191. Figure 5 The results showed that compared with wild-type saRNA, saRNA-190 and saRNA-191 had a lower proportion of double-stranded RNA formed during replication 6 hours after transfection, and this difference was more significant 24 hours after transfection. More than half of the cells transfected with wild-type saRNA were detected with dsRNA 24 hours after transfection, which was much higher than the other two groups. There was no significant difference between 190-saRNA and 191-saRNA. In summary, the above results show that 190 / 191-saRNA with macrodomain amino acid mutations has a weakened ability to replicate in vitro cells.

[0846] Example 3: Detection of differences in natural immunity induced by mutant saRNA

[0847] Considering that double-stranded RNA can significantly trigger the innate immune response, it is necessary to examine the changes in the innate immune level induced by mutant saRNA and wild-type saRNA. After transfecting Hela cells with 190 / 191-saRNA and wild-type saRNA for 24 hours, we extracted total cell RNA for RNA sequencing and analyzed the differences in transcriptome levels.

[0848] According to the results of principal component analysis, compared with the control group, the transcriptome difference of the 190-saRNA transfected group was the smallest, while the wild-type saRNA group induced very significant transcriptome changes (see Figure 6 ). Further, we counted the number of genes that were differentially expressed in different saRNA groups relative to the control group. We found that in all saRNA groups, the number of upregulated genes was much greater than the number of downregulated genes, and most of the upregulated genes in groups 190 and 191 were almost the same as those in the wild-type saRNA group. However, we found that the number of genes that were commonly downregulated between different groups was relatively small (see Figure 7 ), suggesting that the biological functions of the genes upregulated by 190 / 191-saRNA and wild-type saRNA may be similar. Therefore, a cluster analysis of the differentially expressed genes in the wild-type saRNA group compared with the control group was performed. The results showed that the 10 groups with the most significant differences in biological process (GO:BP) were almost all related to immune response, and almost all of the genes were highly upregulated (see Figure 8a), indicating that wild-type saRNA induced a strong antiviral innate immune response in cells. Further analysis of the molecular function (GO: MF) of the differentially expressed genes revealed that the functions of the upregulated genes were related to cytokine / chemokine / growth factor / receptor binding and dsRNA / ssRNA binding (see Figure 8 b). It is known that the proportion of cells that form double-stranded RNA after 190 / 191-saRNA transfection is significantly lower than that of wild-type RNA. Therefore, it is not difficult to imagine that the difference in the proportion of double-stranded RNA formed during the replication of different saRNAs may affect the induction of natural immune responses.

[0849] Next, we examined the upregulated genes in the double-stranded RNA / single-stranded RNA binding proteins, and we found that the intracellular RNA sensor RIG-I, MDA-5 and the endosomal RNA sensor TLR3 as well as the downstream interferon-stimulated genes of the IFN pathway such as OAS family and EIF2AK2 were highly upregulated (see Fig. 9 ), indicating that the double-stranded RNA produced during saRNA replication is the key to activating the interferon or NFKB signaling pathway, thereby inducing a large number of downstream antiviral genes to inhibit saRNA replication. From the gene heat map of the interferon pathway between different groups, it can be seen that compared with the control group, the transcription levels of interferon pathway genes in all cells transfected with saRNA were significantly upregulated. In addition to the RNA-binding proteins mentioned, there are also interferon beta, transcription factors IRF7 / 9, STAT1 and many downstream interferon-stimulated genes. In addition, the upregulation of cytokines was also seen, suggesting that the NF-kb pathway was also effectively activated. As expected, the transcription levels of interferon signaling pathway genes induced by saRNA in different groups were significantly different. Compared with the control group, 190 induced the weakest IFN immune response, 191 was slightly stronger, and the wild type was the strongest. As Fig.10 , the mRNA levels of interferon pathway and cytokines were verified by fluorescence quantitative PCR 2, 6 and 24 hours after transfection. It was found that within 2 hours, the RNA transcription levels of each group did not change compared with the control group, and at 6 hours, compared with the control group, the IFN-beta, CXCL10, CCL5 and IFIT2 RNA levels of the wild-type saRNA group were significantly upregulated, and CXCL10, IFIT2 and PKR were also upregulated in the 190 and 191 groups, but there was no difference between 190 and 191. 24 hours after transfection, all RNAs including NF-KB and interferon pathways were most significantly upregulated in the wild-type saRNA group, followed by 191-saRNA, and the lowest in 190-saRNA.

[0850] In summary, it was detected that the transcription levels of genes in the innate immune pathway induced by different saRNAs were positively correlated with their replication ability. Since the ability of 190 / 191-saRNA to replicate and form double-stranded RNA was weakened, it induced a weaker innate immune response.

[0851] Example 4: Detection of protein translation activity of mutant saRNA in in vitro cell models

[0852] In order to detect the activity of different saRNAs expressing exogenous genes, different saRNAs encoding luciferase were transfected into HeLa cells. The results showed that the expression of wild-type saRNA was higher than that of mutant saRNA at 2 hours and 6 hours, but at 24 hours, the expression of 190 / 191-saRNA was slightly higher, and at 48 hours, the expression of 190-saRNA was 6 times higher than that of wild-type saRNA (see Fig.11 ). It is speculated that the natural immune response induced by saRNA will limit the virus from hijacking the cell translation mechanism for replication through host protein translation inhibition, so the cell translation activity after different saRNA transfection was detected. Before collecting the cells, puromycin was added to the culture medium to a final concentration of 5ug / mL and treated for 5 minutes to allow it to be incorporated into the polypeptide chain, and the intracellular labeled protein content was detected with an anti-puromycin antibody. The results showed that there was no significant difference in intracellular translation activity 2 hours after transfection, while at 6 hours and 24 hours, the translation activity of the wild-type saRNA group was weaker than that of the mutant saRNA group, and the 190-saRNA protein translation activity was slightly weaker than the control at 6 hours, but similar to the control group at 24 hours. In contrast, the translation activity of cells in the 191-saRNA transfection group was slightly weaker at 24 hours (see Fig.12 ). This result proves that after saRNA transfection, protein translation in cells is inhibited. Combined with the previous observation that PKR and OAS genes in dsRNA binding proteins are significantly upregulated after saRNA transfection, it is speculated that the activation of OAS / RNase L and PKR / eIF2α pathways may be involved in the inhibition of host protein translation, thereby limiting the expression of saRNA. Therefore, the integrity of ribosomal RNA and eIF2α phosphorylation at different time points after saRNA transfection were detected (see Fig.13 , 14 ). The results showed that the ribosomal RNA of cells transfected with wild-type saRNA was still relatively intact within 2 and 6 hours, but was severely degraded at 24 and 48 hours, while the integrity of the ribosomal RNA transfected with 190 / 191 saRNA was not significantly reduced compared with the control group. This result suggests that the ribosomal RNA was degraded after wild-type saRNA transfection, which is the result of the activation of the OAS / RNase L pathway (see Fig.13). At the same time, the results of eIF2α phosphorylation detection showed that after 6 hours of transfection, all groups of saRNA induced eIF2α phosphorylation, and it was positively correlated with the subgenomic level at 6h, with wild-type saRNA being the strongest and 191-saRNA being the weakest. Unexpectedly, 24 hours after transfection, the eIF2α phosphorylation level was significantly reduced, and the eIF2α phosphorylation induced by wild-type saRNA was even weaker than that of the control group (i.e., the mock group: only transfection reagent was added, and no RNA molecules were transfected), while the eIF2α phosphorylation degree of mutant saRNA was similar to that of the control group (see Fig.14 ). Based on the above results, host translation was not inhibited at 2 hours after transfection, but host translation mediated by PKR / eIF2α appeared at 6 hours. However, since the subgenomic RNA amplification level of wild-type saRNA was much higher than that of mutant saRNA, the expression level was still higher than that of the other two groups. At 24 hours, the phosphorylation level of wild-type eIF2α decreased, so host translation inhibition was largely promoted by rRNA degradation caused by OAS / RNase L activation, so the expression level of wild-type saRNA, which was most significantly inhibited, was the lowest.

[0853] The above results prove that saRNA transfection of cells activated PKR / eIF2α and OAS / RNase L-mediated host translation inhibition, but compared with wild-type saRNA, mutant saRNA had a lower degree of translation inhibition and therefore showed a higher expression level.

[0854] Example 5: Apoptosis induction detection of mutant saRNA in in vitro cell models

[0855] It was observed that 24 hours after transfection with wild-type saRNA, the cell nucleus underwent significant disintegration, suggesting that HeLa cells may have undergone apoptosis (see Fig.15 ). The cells transfected for 24 hours and 48 hours were stained with annexin-V and PI. Fig.16 As shown in the figure, all cells transfected with saRNA underwent apoptosis, but the percentage of dead cells in the group transfected with 190-saRNA was significantly lower than that in other groups. Western blot was performed to detect the protein expression levels and activation status of the apoptosis initiator molecule caspase 8 and the effector molecule caspase 3. The results showed that all caspases were significantly activated in cells transfected with saRNA, but the caspase activation in the wild-type saRNA group was significantly stronger than that in other groups (see Fig.17 ). In addition, if Fig.18, cells transfected with wild-type saRNA were treated with caspase inhibitors, and it was found that cell apoptosis was strongly inhibited after treatment, and the fluorescence intensity of EGFP expressed by saRNA increased by 3 times. These results show that saRNA transfection of Hela cells leads to apoptosis, and inhibiting apoptosis can improve the expression efficiency of saRNA.

[0856] Example 6: Detection of in vivo expression level of 190-saRNA

[0857] In order to verify whether the mutant saRNA also has a high expression level in vivo, 190-saRNA expressing the firefly luciferase gene, wild-type saRNA and non-replicating mRNA (nrmRNA) were encapsulated with lipid nanoparticles (LNPs). Fig.19 As shown in a, 4-6 week old balb / c mice were intramuscularly immunized with 5 μg of different mRNA-LNPs expressing the firefly luciferase gene, and live imaging was performed on days 1, 3, 7, 14, and 21. The results showed that the expression level of non-replicating mRNA was slightly higher than that of saRNA on the first day after immunization, but it began to decay afterwards and was almost undetectable after day 7 (see Fig.19 b, 20). The expression level of 190-saRNA continued to rise for at least 7 days and was higher than that of wild-type saRNA. Although it remained consistent with wild-type saRNA after 14 days, the total expression level was higher than that of wild-type saRNA and non-replicating mRNA (see Fig.21 ). Consistent with what was observed in vitro, 190-saRNA also showed higher expression levels in vivo.

[0858] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An mRNA molecule encoding nonstructural proteins 1, 2, 3 and 4 from Venezuelan equine encephalomyelitis virus, wherein the macro domain of the nonstructural protein 3 at its N-terminus is as shown in the amino acid sequence of SEQ ID NO: 3 or 5.

2. The mRNA molecule according to claim 1, comprising a nucleotide sequence encoding the macro domain as shown in SEQ ID NO: 14 or 16.

3. The mRNA molecule according to claim 1, wherein the mRNA molecule encodes a nonstructural protein 1 as shown in SEQ ID NO: 17, a nonstructural protein 2 as shown in SEQ ID NO: 18, a nonstructural protein 3 as shown in SEQ ID NO: 19 or 20, and a nonstructural protein 4 as shown in SEQ ID NO:

21.

4. The mRNA molecule according to claim 1, which comprises, in the 5' to 3' direction, an operably linked nucleotide sequence encoding nonstructural protein 1, a nucleotide sequence encoding nonstructural protein 2, a nucleotide sequence encoding nonstructural protein 3, and a nucleotide sequence encoding nonstructural protein 4.

5. The mRNA molecule according to claim 4, wherein the mRNA molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 13 or 15.

6. The mRNA molecule according to any one of claims 1 to 5, further comprising a target gene coding sequence.

7. The mRNA molecule according to claim 6, further comprising an RNA promoter upstream of the target gene coding sequence.

8. The mRNA molecule according to any one of claims 1 to 5, further comprising a 5' untranslated region, a 3' untranslated region and a polyadenylation sequence.

9. The mRNA molecule according to claim 8, further comprising a signal peptide coding sequence, a 5' cap sequence, a Kozak sequence and / or a restriction enzyme cleavage site.

10. The mRNA molecule according to claim 8, wherein the mRNA molecule comprises the following operably linked nucleotide sequences in the 5' to 3' direction: a 5' untranslated region, a nucleotide sequence encoding nonstructural proteins 1, 2, 3 and 4, an RNA promoter, a target gene coding sequence, a 3' untranslated region and a polyadenylation sequence. The mRNA molecule according to claim 10 , wherein the nucleotide sequences are connected via a linker sequence. The mRNA molecule according to claim 8 , wherein the 5′ untranslated region is derived from an alphavirus.

13. The mRNA molecule according to claim 12, wherein the 5' untranslated region is derived from Venezuelan equine encephalomyelitis virus. The mRNA molecule according to claim 12 , wherein the 5′ untranslated region comprises the sequence shown in SEQ ID NO:

9. The mRNA molecule according to claim 8 , wherein the 3′ untranslated region is derived from an alphavirus.

16. The mRNA molecule according to claim 15, wherein the 3' untranslated region is derived from Venezuelan equine encephalomyelitis virus.

17. The mRNA molecule according to claim 15, wherein the 3' untranslated region comprises the sequence shown in SEQ ID NO:

10.

18. The mRNA molecule of claim 7, wherein the RNA promoter is a subgenomic promoter.

19. The mRNA molecule of claim 18, wherein the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus.

20. The mRNA molecule of claim 18, wherein the RNA promoter is a 26S promoter.

21. The mRNA molecule of claim 18, wherein the RNA promoter comprises the sequence shown in SEQ ID NO:

11.

22. The mRNA molecule according to claim 8, wherein The polyadenylation sequence contains restriction enzyme sites.

23. The mRNA molecule of claim 22, wherein the poly(A) sequence comprises the sequence shown in SEQ ID NO:

12.

24. The mRNA molecule of claim 22, wherein the 3' end of the poly(A) sequence comprises a restriction enzyme site.

25. The mRNA molecule according to claim 6, wherein The target gene coding sequence is a sequence encoding a therapeutic polypeptide, a preventive polypeptide, a diagnostic polypeptide, a reporter gene, an antigen or a sequence encoding a regulatory structure.

26. The mRNA molecule according to claim 6, wherein the sequence thereof is shown in any one of SEQ ID NOs: 4 and 6-8.

27. A DNA molecule encoding the mRNA molecule of any one of claims 1 to 26. A recombinant vector comprising the DNA molecule according to claim 27. The recombinant vector according to claim 28 , wherein the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector.

30. A recombinant host cell comprising the mRNA molecule according to any one of claims 1 to 26, the DNA molecule according to claim 27, or the recombinant vector according to claim 28.

31. A pharmaceutical composition comprising the mRNA molecule of any one of claims 1 to 26 or the DNA molecule of claim 27, and one or more pharmaceutically acceptable carriers.

32. The pharmaceutical composition of claim 31, wherein the carrier is a liposome-based delivery system or a nanoparticle composition.

33. A vaccine preparation comprising the mRNA molecule according to any one of claims 1 to 26 or the DNA molecule according to claim 27.

34. The vaccine formulation of claim 33, wherein the mRNA molecule or DNA molecule is encapsulated by a liposome-based delivery system.

35. The vaccine formulation of claim 33, wherein the vaccine formulation further comprises one or more vaccine adjuvants.

36. The vaccine formulation according to claim 33, wherein the vaccine formulation is a vaccine formulation for preventing viral infection.

37. Use of the mRNA molecule according to any one of claims 1 to 26 or the DNA molecule according to claim 27 in the preparation of a drug for treating or preventing viral infection, a drug for treating or preventing tumors, or a drug for protein replacement therapy.

38. A kit comprising the mRNA molecule according to any one of claims 1 to 26 or the DNA molecule according to claim 27.

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