Methods of preventing or treating rna virus infection in subject

Abstract

The present invention relates generally to the field of molecular biology. In particular, the specification teaches a method of preventing or treating an RNA virus infection in a subject comprising administering to the subject a recombinant adeno-associated virus (AAV) at a dose of about 5 * 1011 to about 5 * 1013 vgs / kg wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs. In one embodiment, the RNA virus infection is an infection of enterovirus 71.

Description

Technical Field

[0001] The present invention relates generally to the field of molecular biology. In particular, the present specification teaches methods of preventing or treating RNA virus infection in a subject. Background Art

[0002] Enterovirus 71 (EV71), Coxsackievirus (CAV16 and CAV6) and echovirus are all non-enveloped positive single-stranded RNA viruses that are highly contagious and spread through body fluids. EV71 infection is most common in children less than 5 years old, with about 50-80% of children testing seropositive for EV71, and is also observed to a lesser extent in adults. Due to the symptoms associated with infection, EV71 is also a major contributor to a disease called hand, foot and mouth disease (HFMD). Infection may occasionally lead to severe neurological disease or death. To date, there is no commercially available vaccine or therapeutic agent for preventing or eliminating EV71 infection. Clinical trials are only conducted for vaccine modes rather than treatment modes. In recent years, EV71 infection has reached record incidences in several countries, and this highlights the urgent need to develop vaccines and therapeutic agents for currently incurable infections.

[0003] It would be desirable to overcome or ameliorate at least one of the problems discussed above, or at least provide a useful alternative. Summary of the invention

[0004] Disclosed herein is a method for preventing or treating an RNA virus infection in a subject, the method comprising administering to the subject a dose of about 5x10 11 About 5x10 13 vgs / kg dose of a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0005] Disclosed herein is a recombinant adeno-associated virus (AAV) for treating an RNA viral infection in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0006] Disclosed herein is the use of a recombinant adeno-associated virus (AAV) in the manufacture of a medicament for treating an RNA viral infection in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0007] Disclosed herein is a method for inhibiting RNA viral nucleic acid in a subject, the method comprising administering to the subject about 5x10 11 About 5x10 13 vgs / kg of a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0008] In one embodiment, the method comprises inhibiting RNA viral nucleic acid in skeletal or central nervous system (CNS) cells or tissues of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Embodiments of the invention are described hereinafter, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0010] Figure 1. Design of novel antiviral AAV-CRISPR-CasRx therapeutics for RNA virus targeting. A. Schematic diagram of the mechanism of the hypothetical CRISPR-CasRx antiviral activity against RNA viruses such as EV-A71. Positive-strand RNA viruses such as EV-A71 release genomes when entering host cells, which serve as templates to prepare negative-strand genomes and subgenomic templates, which are then used in turn to produce more copies of positive-strand viral genomes and viral mRNAs. CasRx and guide RNA delivered by AAV expressed in EV-A71 infected cells can specifically target and cut both viral genomes and viral mRNAs to disrupt viral replication and viral function. B. Design schematic diagram of novel AAV genome carrier plasmids (cargo plasmids) for antiviral applications. Plasmid of pZac2.1-CMV-CasRx-3xHA-PolyA-U6-gRNA. Between the ITR sequences, the CMV promoter drives the expression of CasRx, which has a 3xHA tag and does not contain an NLS sequence, followed by a rabbit polyadenylation sequence. This is followed by a U6 promoter that drives transcription of the inserted guide sequence and guide scaffold, followed by another ITR sequence. The guide sequence can be cloned into this backbone using BbsI restriction enzyme digestion. C. To verify that the packaged virus is able to transduce mammalian cells and express the CasRx vector, 10K human immortalized corneal endothelial cells B4G12 were transduced at an MOI of 100K in a 48-well plate and plated for 72 hours. Anti-HA staining was performed at 1:200 for 2 hours, and anti-GFP was performed at 1:1500 for 2 hours. Secondary antibody staining was performed at 1:1000 for 2 hours. Images were taken at an exposure of 15ms and a gain set to 6400 for the bright field and 488 channels, and the exposure was set to 800us and the gain was set to 6400 for the DAPI channel. D. AAV validation with new vector plasmids carrying CasRx and gRNA. The human muscle immortalized cell line RD was transduced with AAV2-GFP at MOI 10K for GFP expression. The GFP knockdown efficiency of guide1 only, guide2 only, and guide1+guide2 was tested by transducing RD cells expressing GFP with AAV2-CasRx carrying a GFP targeting guide at MOI 100K (D) or MOI 1000K (E) for 72 hours.

[0011] Figure 2. CRISPR-CasRx gRNA selection and antiviral targeting strategy. A. Schematic diagram of the bioinformatics pipeline for gRNA prediction and scoring as applied to the enterovirus genome. B. Scoring and visualization of gRNAs for 795 enterovirus target genome sequences. Top: Entropy score for each nucleotide of the aligned viral genome. Middle: Percentage of conservation for each nucleotide between the aligned viral genomes. Bottom: Frequency of gRNAs for targeting enteroviruses that are within the best fourth quartile. C. Selected gRNAs targeting enterovirus RNA genomes. Blue arrows: gRNAs with high scores. Red arrows: gRNAs with medium / low scores. D. Evaluation of low-scoring guides - inhibitory activity of EV-A71 replication under single-guide single-target conditions or multi-guide single-target conditions. In a 96-well plate, 10K RD cells were seeded in each well and transduced with AAVDJ-eGFP without a guide or AAVDJ-CasRx carrying an eGFP guide 2 or EV-A71 guide for 72 hours, then subjected to EV-A71 infection at MOI 1 for 12 hours, and then the supernatant was harvested for viral plaque assay. The control AAV-CasRx did not use a guide or used an eGFP guide 2. The comparison between the two groups (CasRx-eGFPg2 and CasRx-EV71_3D guide) was analyzed by Student's t-test (two-tailed) using the software Prism9. *p<0.05, **p<0.005. E. Evaluation of high-scoring guides-inhibitory activity of EV-A71 replication under single-guide single-target conditions or single / double guide / target accompanied by multiple target conditions. In a 96-well plate, 10K RD cells were seeded in each well and transduced with AAVDJ-eGFP without a guide or AAVDJ-CasRx carrying eGFP guide 2 or EV-A71 guide for 72 hours, then subjected to EV71 infection at MOI 1 for 12 hours, and then the supernatant was harvested for viral plaque assay. Control AAV-CasRx did not use a guide or used eGFP guide 2. Comparison between the two groups (CasRx-eGFPg2 and CasRx-EV71_3D guide) was analyzed by Student's t-test (two-tailed) using the software Prism9. *p<0.05, **p<0.005. F. Evaluation of the number of multiplexed CasRx guides on a single target for the effective inhibitory activity of EV-A71 replication. In a 96-well plate, 10K RD cells were seeded in each well and transduced with AAV2-eGFP without a guide or AAV2-CasRx carrying a GFP guide2 or EV-A71 guide at an MOI of 1000K for 72 hours, and then subjected to EV71 infection at an MOI of 1 for 12 hours, and the supernatant was harvested for viral plaque assay. Control AAV2-CasRx used no guide or eGFP guide2.Comparison between the two groups (CasRx-eGFPg2 and CasRx-EV71_3D guide) was analyzed by Student's t-test (two-tailed) using the software Prism 9. *p<0.05. G. Percentage of EV-A71 plaques in the presence of different numbers of multiplexed CasRx guides at MOI 1000K.

[0012] Figure 3 .Bio-panning of AAV serotypes to identify the most effective AAV serotype capsid for transduction. A. Human skeletal muscle has been proposed as a target organ that supports persistent infection and replication of EV71. Using the human RD rhabdomyosarcoma cell line (ATCC), an AAV panel bio-panning experiment was performed to determine the most suitable AAV serotype that can effectively transduce human muscle cells to deliver CRISPR-CasRx tools for viral RNA targeting. 10K cells were plated in each well of a 96-well plate and transduced with each serotype of AAV at an MOI of 100K, and the expression of GFP protein was quantified 72 hours after transduction. The experiment was performed in duplicate. B. Mouse skeletal muscle is one of the main target organs that supports persistent infection and replication of EV71. Using the mouse C2C12 skeletal muscle cell line (ATCC), an AAV panel bio-panning experiment was performed to determine the most suitable AAV serotype that can effectively transduce mouse muscle cells to deliver CRISPR-CasRx tools for viral RNA targeting in a mouse model. 10K cells were plated in each well of a 96-well plate and transduced with each serotype of AAV at an MOI of 10K, and the expression of GFP protein was quantified 72 hours after transduction. Experiments were performed in duplicate.

[0013] Figure 4 .Profiling of human innate and adaptive immune responses in AAV2-transduced human RD cells. One million human skeletal RD cells were inoculated in a 6-well dish and transduced with AAV2 serotypes at MOI 100K and 1000K. RNA from transduced cells was harvested 72 hours after transduction using the RNeasyuniversal plus mini kit (Qiagen). cDNA was synthesized and the immune response gene panel was quantified using the RT2 profiling kit (Qiagen). The results showed that in AAV2-transduced RD cells, there was a minimal antiviral cellular response, with only two genes (NLRP3 and SLC11A1) significantly upregulated in a dose-dependent manner. Both NLRP3 and SLC11A1 are involved in macrophage activation or recruitment, but have not been shown to exhibit intracellular antiviral activity.

[0014] Figure 5. In vivo evaluation of the antiviral efficacy of AAVDJ-CRISPR-CasRx in a mouse model of hand, foot and mouth disease. A. BALB / c mice were treated with 1x10 11 vgs or 1x10 12 vgs AAVDJ-EV71gRNAs / mouse were injected intraperitoneally and then treated with 2×10 7 EV-A71 was injected intraperitoneally at a dose of 1x10 PFU / mouse. 12 vgs AAVDJ-GFPgRNA was used as a treatment control. The survival of mice was recorded for 19 days post infection (dpi). B. The weight percentage of each mouse in each treatment group was recorded for 19dpi. C. The clinical score of each mouse was recorded for up to 19dpi using the mouse clinical assessment scoring system (M-CASS), which involves observation of five markers: activity, respiration, movement, body weight and dehydration. D. Virus titers in the hind limbs of mice from different treatment groups, as determined using a plaque formation assay. E. Virus titers in the brains of mice from different treatment groups, as determined using a plaque formation assay. F. H&E staining of mouse hind limbs and spinal cord. Polymorphonuclear meningitis in the spinal cord (black arrow). Necrotic and focal interstitial mononuclear cell infiltration (blue arrow) in the hind limbs and spinal cord. G. Immunohistochemical staining for EV-A71-specific antigens. The presence of viral antigens in the anterior horn cells of the hind limbs and cervical spinal cord (red arrow).

[0015] Figure 6. In vivo evaluation of the antiviral efficacy of AAVDJ-CRISPR-CasRx in a mouse model of hand, foot and mouth disease. A. BALB / c mice were treated with 2×10 7 PFU EV-A71 / mouse was injected intraperitoneally. After 2 hours, mice were treated with 1x10 11 vector genome (vgs) or 1x10 12 vgs AAVDJ-EV71gRNAs / mouse were injected intraperitoneally. 1x10 12 vgs AAVDJ-GFPgRNA was used as a treatment control. The survival of mice was recorded for 19 days post infection (dpi). B. The body weight percentage of each mouse in each treatment group was recorded for 19 dpi. C. For up to 19 dpi, the clinical score of each mouse was recorded using the Mouse Clinical Assessment Scoring System (M-CASS), which involves the observation of five markers: activity, respiration, movement, body weight, and dehydration.

[0016] Figure 7. AAVDJ-CRISPR-CasRx prevented mortality and reduced pathology when applied 6 hours after EV-A71 infection. A. BALB / c mice were treated with 2×10 7 PFU EV-A71 / mouse were injected intraperitoneally. After 6 hours, mice were treated with 1x10 11 Viral genomes (vgs) or 1x10 12 vgs AAVDJ-EV71gRNAs / mouse were injected intraperitoneally. 1x10 12 vgs AAVDJ-GFPgRNA was used as a treatment control. B. Survival of mice was recorded for 19 days post infection (dpi). Comparison between the two groups (CasRx-GFPg2 and CasRx-EV71_3D guide) was analyzed by the logarithmic (Mantel-Cox) test. **p<0.005. C. The body weight of each mouse in each treatment group was recorded for 19dpi. D. The clinical score of each mouse was recorded using the mouse clinical assessment scoring system (M-CASS) for up to 19dpi. E. Virus titers in mouse hind limbs as determined using a plaque formation assay. F. Virus titers in mouse brains as determined using a plaque formation assay; Kruskal-Wallis test with Dunn's multiple comparison post hoc test. **p<0.005. G. Immunohistochemical staining for EV-A71-specific antigens (red arrows). H. H&E staining of mouse hind limbs and spinal cord. Polymorphonuclear meningitis in the spinal cord (arrow). Necrosis and focal interstitial mononuclear infiltration in the hindlimb (arrows).

[0017] Figure 8. AAVDJ-CRISPR-CasRx prevented mortality and reduced pathology when applied 24 hours after EV-A71 infection. A. BALB / c mice were treated with 2×10 7 PFU EV-A71 / mouse was injected intraperitoneally. 24 hours later, mice were treated with 1x10 11 Viral genomes (vgs) or 1x10 12 vgs AAVDJEV71gRNA / mouse was injected intraperitoneally. 1x10 12vgs AAVDJ-GFPgRNA was used as a treatment control. B. The survival of mice was recorded for 19 days post infection (dpi). The comparison between the two groups (CasRx-GFPg2 and CasRx-EV71_3D guide) was analyzed by the time series (Mantel-Cox) test. *p<0.05. C. The body weight of each mouse in each treatment group was recorded for 19dpi. D. The clinical score of each mouse was recorded using the mouse clinical assessment scoring system (M-CASS). E. The viral titer in the hind limbs of mice from different treatment groups was determined using a plaque formation assay. F. The viral titer in the brain of mice from different treatment groups was determined using a plaque formation assay; the comparison between the two groups (CasRx-GFPg2 and CasRx-EV71_3D guide) was analyzed by the Kruskal-Wallis test with Dunn's multiple comparison post hoc test. **p<0.005. G. Immunohistochemical staining for EV-A71-specific antigens. The presence of viral antigens in the hind limbs (arrows). H. H&E staining of mouse hind limb and spinal cord. Polymorphonuclear meningitis in the spinal cord (arrow). Necrosis and focal interstitial mononuclear cell infiltration (arrow) in the hind limb and spinal cord.

[0018] Fig. 9 .AAVDJ-CRISPR-CasRx did not induce significant toxicity at therapeutic doses. A.BALB / c mice were treated with saline, 1x10 12 vgs AAVDJ-GFPgRNA, 1x10 11 vgs AAVDJ-EV71gRNAs, or 1x10 12 vgsAAVDJEV71gRNAs / 2-day-old mice were injected intraperitoneally. Survival was recorded for 19 days post infection (dpi). B. The body weight of each mouse in each treatment group was recorded for 19 dpi. C. For up to 19 dpi, the clinical scores of mice were recorded using the Mouse Clinical Assessment Scoring System (MCASS), which involves the observation of five markers: activity, respiration, movement, body weight, and dehydration.

[0019] Fig.10 .CasRx-HA expression in mice transduced with AAVDJCasRx-EV71 gRNA. Tissue sections were stained with anti-HA antibody and counterstained with DAPI. CasRx-HA staining was observed in all groups for both spinal cord and hindlimb tissues (upper row). 11 vgs AAVDJ-EV71gRNA transduced mouse tissue (middle column) compared to the 1x10 12 vgs AAVDJ-GFPgRNA (left column) and 1x10 12Stronger CasRx staining was observed in tissues from mice transduced with vgs AAVDJ-EV71gRNA (right column).

[0020] Figure 11. Whole-gut virus inhibition guided by AAV2-CasRx-EV71_3D. A. Evaluation of inhibitory activity of six gRNA pools targeting HFM41 strains against three other EV71 variants (H strain, B5 strain, and C4 strain). 10K RD cells were transduced with AAV2-CasRx carrying GFP guide 2 or EV-A71 guide at MOI10K, 100K, and 1000K for 72 hours, and then subjected to EV-A71 infection at MOI1 for 12 hours, after which the supernatant was harvested for viral plaque assay. B. Pools of six gRNAs designed for HFM41 strains showed antiviral inhibition against three other EV71 variants (H strain, B5 strain, and C4 strain) at MOI 1000K. Each pool of C.6 gRNAs was modified specifically for the sequence of H, B5, or C4 strains. D. Each reengineered pool of 6 gRNAs showed antiviral inhibition against its respective H, B5 or C4 strain. In all experimental panel groups, the comparison between the two groups (CasRx-GFPg2 and CasRx-EV71_3D guides) was analyzed by Student's t-test (two-tailed). *p<0.05, **p<0.005, ***p<0.001. DETAILED DESCRIPTION

[0021] This specification teaches a method for preventing or treating RNA virus infection in a subject. The method may include administering a recombinant adeno-associated virus (AAV) to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs. In one embodiment, the AAV comprises a construct encoding the following: at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0022] In one embodiment, a method for preventing or treating an RNA virus infection in a subject is provided, the method comprising administering to the subject a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs. The method may comprise administering to the subject about 5x10 11 About 5x10 13 vgs / kg of recombinant adeno-associated virus (AAV).

[0023] Without being bound by theory, the inventors have shown for the first time that CRISPR-Cas can eliminate RNA viruses in vivo. The present disclosure demonstrates that disease progression can be prevented in an infected mouse model, and discloses that disease progression can be treated in an infected mouse model. CRISPR-Cas can also inhibit RNA viral nucleic acids in bone or central nervous system (CNS) cells or tissues.

[0024] Disclosed herein is a method for preventing or treating an RNA virus infection in a subject, the method comprising administering to the subject a dose of about 5x10 11 About 5x10 13 Vector genomes (vgs) / kg dose of a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0025] RNA virus infection can be infection by a single-stranded RNA virus. The single-stranded RNA virus can be selected from enterovirus, coxsackievirus and epileptovirus. Enterovirus can be enterovirus 71. Coxsackievirus can be selected from CAV16 and CAV6. Epileptovirus can be selected from epileptovirus A, epileptovirus B, epileptovirus C, epileptovirus D, epileptovirus E and epileptovirus F.

[0026] Cas nuclease

[0027] The terms "Cas nuclease", "CRISPR-associated protein", "Cas protein" or "CRISPR-associated nuclease" refer to a wild-type Cas protein, a fragment thereof, or a mutant or variant thereof. The term "Cas mutant" or "Cas variant" refers to a protein or polypeptide derivative of a wild-type Cas protein, such as a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein or a combination thereof. In certain embodiments, the Cas mutant or Cas variant substantially retains the nuclease activity of the Cas protein. In certain embodiments, the Cas nuclease is mutated such that one or more nuclease domains are inactive. In some embodiments, the Cas nuclease is mutated such that it lacks some or all of the nuclease activity of its wild-type counterpart. The term "Cas nuclease" also contemplates the use of natural and modified Cas nucleases.

[0028] The term "cleavage" or "cleaving" refers to the break of covalent phosphodiester bonds in the ribosylphosphodiester backbone of a polynucleotide. The term "cleavage" or "cleaving" encompasses both single-strand breaks and double-strand breaks. A "nuclease cleavage site" or "genomic nuclease cleavage site" is a region of nucleotides comprising a nuclease cleavage sequence recognized by a specific nuclease, which acts on the nucleotide sequence of the cleavage polynucleotide.

[0029] In one embodiment, the Cas nuclease is a Cas13 nuclease. The Cas nuclease may be a Cas13a, Cas13b, Cas13c or Cas13d nuclease. The Cas13 nuclease may be a Cas13d or CasRx nuclease (which is a Cas 13d from Ruminococcus flavefaciens). The Cas13 nuclease may, for example, show a consistent and powerful target-specific cleavage effect. It also has a small size suitable for the AAV delivery system in the clinic. The Cas13 nuclease may be a modified Cas13 nuclease comprising, for example, one or more amino acid substitutions, truncations and / or cyclic substitutions. In one embodiment, the endonuclease function of the Cas13 nuclease is retained in the modified Cas13 nuclease.

[0030] In one embodiment, the Cas nuclease is codon optimized for expression in a cell, such as a human cell.

[0031] Guide RNA

[0032] In one embodiment, at least one guide RNA is provided, which comprises i) a first nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence encoded by one of the nucleic acid sequences shown in SEQ ID NOs: 1-10, or ii) a first nucleic acid sequence having at least 70% sequence identity to one of the nucleic acid sequences shown in SEQ ID NOs: 11-20.

[0033] The method as defined herein may comprise administering 1, 2, 3, 4, 5, 6 or more different guide RNAs to the subject. The method may comprise administering 6 different guide RNAs, wherein each of the guide RNAs comprises i) a first nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence encoded by one of the nucleic acid sequences shown in SEQ ID NOs: 1-6, or ii) a first nucleic acid sequence having at least 70% sequence identity to one of the nucleic acid sequences shown in SEQ ID NOs: 11-16.

[0034] As used herein, the term "guide RNA" or "guide RNA" refers to an RNA that is specific to a target nucleic acid and can form a complex with a Cas protein (e.g., Cas13) and bring the Cas protein to the target nucleic acid. The guide RNA may include or consist of a spacer sequence (i.e., a first nucleic acid sequence) specific to the target nucleic acid and a direct repeat sequence (i.e., a second nucleic acid sequence) that promotes binding to a Cas protein (e.g., a Cas13 protein).

[0035] The terms "nucleic acid" and "polynucleotide" used interchangeably herein refer to the polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Therefore, these terms include but are not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. These terms further include but are not limited to mRNA or cDNA comprising intron sequences. The backbone of a polynucleotide may include sugar and phosphate groups (as can be found in RNA or DNA generally), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may include a polymer of synthetic subunits such as phosphoramidites, and therefore may be oligodeoxynucleoside phosphoramidites or mixed phosphoramidite-phosphodiester oligomers. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars, and linking groups such as fluororibose and thioesters, and nucleotide branches. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more naturally occurring nucleotides with analogs, and introduction means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid supports. The term "polynucleotide" also encompasses peptide nucleic acids, PNAs and LNAs. The polynucleotide may further comprise genomic DNA, cDNA or DNA-RNA hybrids.

[0036] In the context of forming a CRISPR complex, a "target sequence" or "target nucleic acid" refers to a sequence to which a guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may comprise an RNA polynucleotide. In some embodiments, the target sequence is localized in the nucleus or cytoplasm of a cell. In one embodiment, the "target sequence" is a viral nucleic acid sequence. In one embodiment, the "target sequence" is an enteroviral nucleic acid sequence.

[0037] The term "at least 80% sequence identity" includes at least 81% to 99% and all integer percentages therebetween.

[0038] As used herein, the expression "sequence identity" or, for example, "a sequence having 50% identity to..." refers to the degree to which a sequence is identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis over a comparison window. Therefore, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I, U) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, G1n, Cys, and Met) occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, typically wherein the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0039] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12 monomer units in length, but frequently 15 to 18 monomer units, and often at least 25 monomer units, including nucleotides and amino acid residues. Because two polynucleotides may each contain (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) a sequence that is different between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6, typically about 50 to about 100, and more typically about 100 to about 150 contiguous positions, in which a sequence is compared to a reference sequence having the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may include about 20% or less additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The best sequence alignment for aligning the comparison window may be performed by computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by checking the best alignment generated by any of the various methods selected (i.e., resulting in the highest percentage homology over the comparison window). Reference may also be made to the BLAST program series disclosed, for example, by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis may be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0040] "Stringent conditions" refer to conditions under which nucleic acid can hybridize with its target polynucleotide sequence. Preferably, under stringent conditions, nucleic acid hybridizes with its target polynucleotide sequence, but not with other sequences. That is, under stringent conditions, hybridization is specific for the target sequence. Stringent conditions are sequence-dependent (e.g., longer sequences specifically hybridize at higher temperatures). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting temperature (Tm) of a specific sequence under a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and polynucleotide concentration) at which 50% of probes complementary to the target sequence hybridize with the target sequence under equilibrium. Typically, stringent conditions are conditions wherein for short probes (e.g., 10 to 50 nucleotides), the salt concentration is at least about 0.01 to about 1.0 M sodium ion concentration (or other salts) at about pH 7.0 to about pH 8.3, and the temperature is at least about 30°C.

[0041] The first nucleic acid sequence (or spacer sequence) can be modified to hybridize with any desired sequence in the target nucleic acid.The first nucleic acid sequence can have a length of about 10 nucleotides to about 100 nucleotides.For example, it can have a length of about 10 nucleotides (nt) to about 90nt, about 10nt to about 80nt, about 10nt to about 70nt, about 10nt to about 60nt, about 10nt to about 50nt, about 10nt to about 40nt, about 10nt to about 30nt.For example, it can have a length of about 14nt to about 30nt, about 15nt to about 30nt, about 16nt to about 30nt, about 17nt to about 30nt, about 18nt to about 30nt, about 19nt to about 30nt, about 20nt to about 30nt, about 21nt to about 30nt, about 22nt to about 30nt or about 23nt to about 30nt. For example, it can have a length of about 23nt to about 30nt, 23nt to about 29nt, about 23nt to about 28nt, about 23nt to about 27nt, about 23nt to about 26nt, about 23nt to about 25nt, or about 23nt to about 24nt. The length of the first nucleic acid sequence can be 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt or more.

[0042] In one embodiment, the guide RNA is complementary to a viral nucleic acid or a variant thereof. The length of the viral nucleic acid can be, for example, 10 to 30 nucleotides. In one embodiment, the viral nucleic acid is a single-stranded RNA. In one embodiment, the viral nucleic acid is a genomic RNA or a genomic RNA fragment. In another embodiment, the viral nucleic acid is an RNA transcript or an RNA transcript fragment from an enterovirus.

[0043] In one embodiment, the guide RNA is complementary to an enterovirus nucleic acid or a variant thereof. The length of the enterovirus nucleic acid can be, for example, 10 to 30 nucleotides. In one embodiment, the enterovirus nucleic acid is a single-stranded RNA. In one embodiment, the enterovirus nucleic acid is a genomic RNA or genomic RNA fragment from an enterovirus. In another embodiment, the enterovirus nucleic acid is an RNA transcript or an RNA transcript fragment from an enterovirus. The enterovirus nucleic acid may comprise or consist of a gene or corresponding mRNA from an enterovirus, such as an enterovirus 2A protease (2A), an enterovirus viral protein 3 (VP3), and a 3D polymerase (3Dpol).

[0044] In one embodiment, a guide RNA is provided, which comprises a first nucleic acid sequence complementary to a viral nucleic acid and a second nucleic acid sequence capable of promoting the binding of a Cas13 nuclease to the viral nucleic acid.

[0045] In one embodiment, a guide RNA is provided, comprising a first nucleic acid sequence complementary to a viral nucleic acid and a second nucleic acid sequence capable of promoting the binding of a Cas13 nuclease to a viral nucleic acid. The first nucleic acid sequence may have at least 70% sequence identity with a nucleic acid sequence encoded by one of the nucleic acid sequences shown in SEQ ID NOs: 1-10. The first nucleic acid may have at least 70% sequence identity with one of the nucleic acid sequences shown in SEQ ID NOs: 11-20.

[0046] The first nucleic acid sequence may be adjacent to (or placed near) the second nucleic acid sequence. Alternatively, the first nucleic acid sequence may be connected to the second nucleic acid sequence via a linker sequence.

[0047] In one embodiment, the guide RNA is chemically modified. Examples of guide RNA chemical modifications include, but are not limited to, incorporation of 2'-O-methyl (M), 2'-O-methyl 3' thiophosphate (MS), S-constrained ethyl (cEt) or 2'-O-methyl 3' thio PACE (MSP) at one or more terminal nucleotides. Compared to unmodified guide RNA, such chemically modified guide RNAs may include increased stability and increased activity. Chemically modified guide RNAs further include, but are not limited to, RNAs with thiophosphate bonds, and locked nucleic acids (LNA) nucleotides containing methylene bridges between 2' and 4' carbons of the ribose ring.

[0048] The first nucleic acid sequence can be placed upstream or downstream of the second nucleic acid sequence.

[0049] In one embodiment, the gRNA of the present invention comprises non-naturally occurring nucleic acids and / or non-naturally occurring nucleotides and / or nucleotide analogs, and / or chemical modifications. Non-naturally occurring nucleic acids may include, for example, a mixture of natural and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and / or nucleotide analogs may be modified at ribose, phosphate and / or base moieties. In one embodiment of the present invention, the guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one embodiment, the guide RNA comprises one or more ribonucleotides and one or more deoxyribonucleotides. In one embodiment, the guide RNA comprises one or more non-naturally occurring nucleotides or nucleotide analogs, such as nucleotides with thiophosphate bonding, nucleotides with borane phosphate bonding, locked nucleic acids (LNA) nucleotides or bridged nucleic acids (BNA) included in a methylene bridge between 2' and 4' carbons of a ribose ring. Other examples of modified nucleotides include 2'-O-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs or 2'-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromouridine, pseudouridine (Ψ), N1-methylpseudouridine (me1Ψ), S-methoxyuridine (SmoU), inosine, and 7-methylguanosine.

[0050] treat

[0051] The present disclosure relates to methods of preventing or treating a viral infection in a subject.

[0052] As used herein, the term "treating" may refer to (1) preventing or delaying the onset of one or more symptoms of a disease; (2) inhibiting the development of a disorder or one or more symptoms of a disorder; (3) relieving a disorder, i.e., causing the disorder or at least one or more symptoms of a disorder to subside; and / or (4) causing a reduction in the severity of one or more symptoms of a disorder.

[0053] The term "administering" refers to contacting, applying, injecting, infusing or providing the composition of the present invention to a subject.

[0054] As used throughout the specification, the term "subject" is understood to mean a human, or may be a domesticated animal or a companion animal. Although the methods of the present invention are particularly contemplated for use in treating humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domesticated animals such as horses, cattle and sheep, or zoo animals such as primates, felines, canines, bovines and ungulates. A "subject" may include an individual, patient or individual, and may be of any age or gender. A "subject" may be a pediatric subject.

[0055] The method as defined herein may comprise administering to the subject an effective amount of a recombinant adeno-associated virus (AAV). In one embodiment, the method comprises administering to the subject about 5x10 11 About 1x10 13 Dosage of vector genomes (vgs) / kg recombinant adeno-associated virus (AAV).

[0056] The term "effective amount" as defined herein means to administer an amount of a medicament to an individual in need thereof, either in a single dose or as part of a series of doses effective for the initiation, treatment or prevention. The effective amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the evaluation of the medical condition, and other relevant factors. It is expected that the amount will fall within a relatively wide range that can be determined by routine experiments.

[0057] Nucleic acid / construct

[0058] Provided herein are nucleic acids comprising or encoding a guide RNA as defined herein.

[0059] Provided herein are constructs comprising nucleic acids encoding guide RNAs. The constructs may further comprise nucleic acids encoding Cas nucleases.

[0060] Disclosed herein are vectors comprising nucleic acid sequences encoding one or more guide RNAs as defined herein. The vector may further comprise a nucleic acid encoding a Cas nuclease. The vector may be designed to target the viral genome at multiple locations, thereby increasing the chance of disabling the virus in the cell.

[0061] The term "construct" refers to a recombinant genetic molecule comprising one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule, and includes any construct containing: (1) nucleic acid sequences including regulatory sequences and coding sequences that are not found together in nature (i.e., at least one nucleotide sequence is heterologous relative to at least one other nucleotide sequence thereof), or (2) sequences encoding portions of non-naturally contiguous functional RNA molecules or proteins, or (3) portions of non-naturally contiguous promoters. Representative constructs include any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule derived from any source, capable of genomic integration or autonomous replication, including nucleic acid molecules to which one or more nucleic acid molecules have been operably linked. Constructs of the present invention generally include the necessary elements to direct the expression of a nucleic acid sequence of interest, such as a guide RNA sequence, also contained in the construct. Such elements can include control elements, such as promoters operably connected to the nucleotide sequence of interest (so as to instruct its transcription), and often also include polyadenylation sequences. In certain embodiments of the present invention, constructs can be contained in vectors. In addition to the components of constructs, vectors can also include, for example, one or more selectable markers, one or more replication origins such as prokaryotic and eukaryotic origins, at least one multiple cloning site and / or promote the stable integration of constructs into the genome of host cells. Two or more constructs can be included in a single nucleic acid molecule, such as a single vector, or can be included in two or more nucleic acid molecules separated, such as two or more vectors separated. "Expression construct" generally includes at least one control sequence operably connected to the nucleotide sequence of interest. In this way, for example, a promoter operably connected to the nucleotide sequence to be expressed is provided in the expression construct for including the expression in the host cell in biology or its part. For the practice of the present invention, conventional compositions and methods for making and using constructs and host cells are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Vols. 1, 2, and 3, JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0062] As used herein, "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been connected. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends, no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DMA segment can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by viruses or virus-like particles for transduction into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial replication origin). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are thereby replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Common expression vectors utilized in recombinant DNA technology are often in the form of plasmids.

[0063] In some embodiments, the vector is circular. In other embodiments, the vector is linear. In some embodiments, the vector is encapsulated in a lipid nanoparticle, a liposome, a non-lipid nanoparticle, or a viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0064] As used herein, the terms "encode", "encoding", etc. refer to the ability of a nucleic acid to provide another nucleic acid or polypeptide. For example, if a nucleic acid sequence can be transcribed and / or translated to produce a polypeptide, or if a nucleic acid sequence can be processed into a form that can be transcribed and / or translated to produce a polypeptide, it is said to "encode" a polypeptide. Such nucleic acid sequences can include coding sequences or both coding sequences and non-coding sequences. Therefore, the terms "encode", "encoding", etc. include RNA products resulting from transcription of DNA molecules, proteins resulting from translation of RNA molecules, proteins resulting from transcription of DNA molecules to form RNA products and subsequent translation of RNA products, or proteins resulting from transcription of DNA molecules to provide RNA products, processing of RNA products to provide processed RNA products (e.g., mRNA), and subsequent translation of processed RNA products.

[0065] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers comprising or consisting of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.

[0066] As used herein, the term "recombinant" when applied to "nucleic acid molecules", "polynucleotides" and the like is understood to mean an artificial nucleic acid structure (i.e., a non-replicating cDNA or RNA; or a replicon, a self-replicating cDNA or RNA), which can be transcribed and / or translated in a host cell or a cell-free system as described herein. Recombinant nucleic acid molecules or polynucleotides can be inserted into a vector. Non-viral vectors, such as plasmid expression vectors or viral vectors can be used. The types of vectors and the insertion techniques of nucleic acid constructs are known to those skilled in the art. Nucleic acid molecules or polynucleotides according to the present disclosure do not occur in nature in the arrangement described by the present invention. In other words, the heterologous nucleotide sequence does not naturally combine with elements of the parental viral genome (e.g., promoters, ORFs, polyadenylation signals, DNA recognition moieties, endonucleases).

[0067] As used herein, the term "operably connected" or "operably linked" refers to a juxtaposition in which the components so described are in a relationship that permits them to function in their intended manner. For example, a regulatory element or regulatory sequence that is "operably linked" to a coding sequence refers to the positioning and / or orientation of the regulatory sequence relative to the coding sequence to allow expression of the coding sequence under conditions compatible with the regulatory sequence.

[0068] Control sequences need not be contiguous with the nucleotide sequence of interest, as long as they function to direct its expression. Thus, for example, untranslated but transcribed intervening sequences may be present between the promoter and coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[0069] "Regulatory elements", "regulatory sequences", "control elements" or "control sequences" mean nucleic acid sequences (e.g., DNA) necessary for the expression of an operably linked coding sequence in a particular host cell. Regulatory sequences suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailer sequences that regulate mRNA stability, and targeting sequences that target the product encoded by the transcribed polynucleotide to an intracellular compartment within the cell or to the extracellular environment.

[0070] "Promoter" refers to a nucleotide sequence usually upstream (5') of its coding sequence, which controls the expression of the coding sequence by providing recognition of RNA polymerase and other factors required for correct transcription."Promoter regulatory sequence" consists of proximal and more distal upstream elements. The promoter regulatory sequence affects the transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, untranslated leader sequences, introns and polyadenylation signal sequences. They include natural sequences and synthetic sequences and sequences that may be combinations of synthetic sequences and natural sequences. "Enhancer" is a DNA sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. It can operate in two orientations (normal or flipped), and can function even when moving from the promoter upstream or downstream. The meaning of the term "promoter" includes "promoter regulatory sequence".

[0071] In one embodiment, the regulatory element is a promoter.

[0072] The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between the translation start and stop codons of a coding sequence. The terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides ('codons') in a coding sequence that specify initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0073] deliver

[0074] In some embodiments, the invention relates to introducing or delivering a guide RNA or vector as defined herein, or any therapeutic agent described herein, into a cell of interest. It should be understood that the agent can be introduced into the cell in an in vitro model or an in vivo model.

[0075] In some embodiments, guide RNA or vector as defined herein can be transfected into cells by various methods, including viral vectors and non-viral vectors. Viral vectors can include retroviruses, slow viruses, adenoviruses and adeno-associated viruses (AAV). It should be understood that any viral vector can be incorporated into the present invention to achieve guide RNA or vector delivery into cells. Depending on the guide RNA or vector designed for digestion or incapacity, some viral vectors may be more effective than other viral vectors. In one embodiment, the vector contains essential components necessary for replication and maintenance of the vector in the host cell, such as a replication origin.

[0076] Retrovirus is a single-stranded RNA virus, which stores its nucleic acid in the form of mRNA genome (including 5' cap and 3' poly A tail), and targets host cells as obligate parasites. In some methods in the art, retrovirus has been used to introduce nucleic acid into cells. Once located in the host cell cytoplasm, the virus uses its own reverse transcriptase to produce DNA by its RNA genome, which is contrary to the usual pattern, and is therefore reverse (reverse). Then, this new DNA is incorporated into the host cell genome by integrase, and now retroviral DNA is referred to as provirus. For example, recombinant retroviruses such as Moloney murine leukemia virus have the ability to be integrated into the host genome in a stable manner. They contain reverse transcriptases that allow integration into the host genome. Retroviral vectors can be replicative or replication-defective. In some embodiments, retrovirus is incorporated to achieve transfection into cells, but Cas / gRNA complexes are designed to target viral genomes.

[0077] In some embodiments of the present invention, a lentivirus, which is a subclass of a retrovirus, is used as a viral vector. In view of its ability to integrate into the genome of non-dividing cells, lentiviruses may be suitable as delivery vehicles (vectors), which are unique features of lentiviruses because other retroviruses can only infect dividing cells. When the virus enters the cell, the viral genome in the form of RNA is reverse transcribed to produce DNA, which is then inserted into the genome at random positions by viral integrase. The vector currently referred to as the provirus remains in the genome and is passed to its offspring when the cell divides.

[0078] In contrast to lentivirus, adenoviral DNA is not integrated into the genome and is not replicated during cell division. Adenovirus and related AAV can be used as delivery vectors because they are not integrated into the host's genome. In some aspects of the invention, only the viral genome to be targeted, rather than the host's cells, is affected by the Cas / gRNA complex. Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. AAV can infect both dividing cells and non-dividing cells, and may incorporate its genome into the genome of the host cell. For example, due to its potential use as a gene therapy vector, researchers have created a modified AAV called self-complementary adeno-associated virus (scAAV). AAV packages single-stranded DNA and requires a second-strand synthesis process, while scAAV packages anneal together to form two chains of double-stranded DNA. By skipping the second-strand synthesis, scAAV allows rapid expression in cells. Otherwise, scAAV carries many characteristics of its AAV counterpart. The method of the present invention can incorporate herpes virus, poxvirus, alphavirus or vaccinia virus as a means of delivery vector.

[0079] Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vectors, lentiviral vectors, adenoviral vectors, helper-dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, and retroviral vectors. In some embodiments, the viral vector is an AAV vector. The AAV vector can be AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV9, AAV10, AAV11, AAV12, AAV13, rh10, AAVDJ, AAV-PHP.S, AAV-PHP.B, AAVPHP.eB, and Anc80. The AAV vector can be AAV2, AAVDJ, or AAV1.

[0080] In some embodiments, the vector comprises a nucleotide sequence encoding a Cas nuclease. In some embodiments, the nuclease encoded by the vector can be a Cas protein. In some embodiments, the vector system comprises a copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system comprises more than one copy of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease is operably connected to at least one transcription or translation control sequence. In some embodiments, the nucleotide sequence encoding the nuclease is operably connected to at least one promoter.

[0081] In some embodiments, the promoter is constitutive or inducible. In some embodiments, the promoter can be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-α (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, its functional fragment or any combination of the foregoing. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a truncated CMV promoter. In some embodiments, the promoter can be an EF1a promoter. In some embodiments, the promoter is an inducible promoter. Non-limiting exemplary inducible promoters include promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics or ethanol. In some embodiments, an inducible promoter is a promoter with a low basal (non-induced) expression level, e.g. Promoter (Clontech).

[0082] The vector may comprise a nucleotide sequence encoding a guide RNA as described herein. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be different so that they target different target sequences, or may be the same so that they target the same target sequence. In some embodiments in which the vector comprises more than one guide RNA, each guide RNA may have other different properties, such as activity or stability in a complex with a Cas nuclease. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence, such as a promoter, a 3′UTR, or a 5′UTR. In one embodiment, the promoter may be a tRNA promoter, such as a tRNA L ys 3 Or tRNA chimera. In some embodiments, the promoter can be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA can be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA can be operably linked to a mouse or human H1 promoter. In embodiments with more than one guide RNA, the promoters used to drive expression can be the same or different.

[0083] In some embodiments, the nucleotide sequence encoding the guide RNA can be located on the same vector containing the nucleotide sequence encoding the Cas nuclease. In some embodiments, the expression of the guide RNA and the Cas nuclease can be driven by the corresponding promoters thereof. In some embodiments, the expression of the guide RNA can be driven by the same promoter that drives the expression of the Cas nuclease, such as the Cas protein. In some embodiments, the guide RNA and the Cas nuclease, such as the Cas protein transcript, can be contained in a single transcript. For example, the guide RNA can be in the untranslated region (UTR) of the Cas nuclease, such as the Cas protein transcript. In some embodiments, the guide RNA can be in the 5′UTR of the transcript. In other embodiments, the guide RNA can be in the 3′UTR of the transcript. In some embodiments, the intracellular half-life of the transcript can be reduced by including the guide RNA in its 3′UTR and thereby shortening the length of its 3′UTR. In other embodiments, the guide RNA can be located in the intron of the transcript. In some embodiments, a suitable splicing site can be added at the intron where the guide RNA is located so that the guide RNA is correctly spliced ​​out of the transcript. In some embodiments, expression of a Cas nuclease, such as a Cas protein and a guide RNA from the same vector in close temporal proximity can promote more efficient formation of the CRISPRRNP complex.

[0084] In some embodiments, the composition comprises a vector system. In some embodiments, the vector system can comprise a single vector. In other embodiments, the vector system can comprise two vectors. In other embodiments, the vector system can comprise three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of guide RNAs are used, the vector system can comprise more than three vectors.

[0085] In certain embodiments of the present invention, non-viral vectors can be used to achieve transfection. The method of non-viral delivery of nucleic acid includes lipofection, nuclear transfection, microinjection, biolistics, virosomes, liposomes, micelles, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial virions and medicament enhanced DNA uptake. Lipofection is described in, for example, U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cations and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides include those lipids in U.S. Patent Nos. 7,166,298 or U.S. Patent No. 6,890,554.

[0086] CRISPR enzyme and / or any of the RNA herein, such as guide RNA, can be delivered using any suitable vector, such as a plasmid or viral vector, such as adeno-associated virus (AAV), lentivirus, adenovirus or other viral vector type or combination thereof. Effector protein and one or more guide RNAs can be packaged into one or more vectors, such as plasmid or viral vectors. In some embodiments, a vector such as a plasmid or viral vector is delivered by, for example, intramuscular injection, while other times delivery is via intravenous, transdermal, intranasal, oral, mucosal or other delivery methods. In some embodiments, the vector is delivered to the subject intravenously, intraperitoneally or orally. Such delivery can be via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be delivered herein may vary greatly depending on various factors, such as vector selection, target cells, organisms or tissues, the general condition of the subject to be treated, the degree of transformation / modification sought, route of administration, mode of administration, type of transformation / modification sought, etc.

[0087] Such dosages may further contain, for example, carriers (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharmaceutically acceptable carriers (e.g., phosphate buffered saline), pharmaceutically acceptable excipients, and / or other compounds known in the art. The dosage may further contain one or more pharmaceutically acceptable salts, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspending agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc., may also be present, especially when the dosage form is a reconstituted form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, p-chlorophenol, gelatin, albumin, and combinations thereof. A detailed discussion of pharmaceutically acceptable excipients can be found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), which is incorporated herein by reference.

[0088] dose

[0089] In one embodiment herein, delivery is via AAV. The dosage can be adjusted to balance the therapeutic benefit against any side effects. In one embodiment, AAV can be delivered at about 5x10 11 About 5x10 13 vgs / kg were administered.

[0090] Without wishing to be bound by theory, the inventors have discovered that the doses of AAV used in the present invention are both safe and effective in preventing or treating RNA viral infection in a subject.

[0091] In one embodiment, AAV is present in an amount of about 5×10 12 vgs / kg to about 5x10 13 vgs / kg. For example, AAV can be administered at a dose of about 5x10 12 vgs / kg, about 6x10 12 vgs / kg, about 7x10 12 vgs / kg, about 8x10 12 vgs / kg, about 9x10 12 vgs / kg, about 1x10 13 vgs / kg, about 2x10 13 vgs / kg, about 3x10 13 vgs / kg, about 4x10 13 vgs / kg or about 5x10 13 vgs / kg. In one embodiment, AAV is administered at a dose of about 5x10 12 vgs / kg to about 6x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 6x10 12 vgs / kg to about 7x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 7x10 12 vgs / kg to about 8x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 8x10 12 vgs / kg to about 9x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 9x10 12 vgs / kg to about 1x10 13 vgs / kg. In one embodiment, AAV is administered at a dose of about 1 x 10 13 vgs / kg is about 2x10 13 vgs / kg. In one embodiment, AAV is administered at a dose of about 2x10 13vgs / kg to about 3x10 13 vgs / kg. In one embodiment, AAV is administered at a dose of about 3x10 13 vgs / kg to about 4x10 13 vgs / kg. In one embodiment, AAV is administered at a dose of about 4 x 10 13 vgs / kg to about 5x10 13 vgs / kg were administered.

[0092] In one embodiment, AAV is present in an amount of about 5×10 11 vgs / kg to about 5x10 12 vgs / kg. For example, AAV can be administered at a dose of about 5x10 11 vgs / kg, about 6x10 11 vgs / kg, about 7x10 11 vgs / kg, about 8x10 11 vgs / kg, about 9x10 11 vgs / kg, about 1x10 12 vgs / kg, about 2x10 12 vgs / kg, about 3x10 12 vgs / kg, about 4x10 12 vgs / kg or about 5x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 5x10 11 vgs / kg to about 6x10 11 vgs / kg. In one embodiment, AAV is administered at a dose of about 6x10 11 vgs / kg to about 7x10 11 vgs / kg. In one embodiment, AAV is administered at a dose of about 7x10 11 vgs / kg to about 8x10 11 vgs / kg. In one embodiment, AAV is administered at a dose of about 8x10 11 vgs / kg to about 9x10 11 vgs / kg. In one embodiment, AAV is administered at a dose of about 9x10 11 vgs / kg to about 1x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 1 x 10 12 vgs / kg is about 2x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 2x10 12vgs / kg to about 3x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 3x10 12 vgs / kg to about 4x10 12 vgs / kg. In one embodiment, AAV is administered at a dose of about 4 x 10 12 vgs / kg to about 5x10 12 vgs / kg were administered.

[0093] In an alternative embodiment, AAV is present in an amount of about 5×10 9 vgs / kg to about 5x10 10 vgs / kg. For example, AAV can be administered at a dose of about 5x10 9 vgs / kg, about 6x10 9 vgs / kg, about 7x10 9 vgs / kg, about 8x10 9 vgs / kg, about 9x10 9 vgs / kg, about 1x10 10 vgs / kg, about 2x10 10 vgs / kg, about 3x10 10 vgs / kg, about 4x10 10 vgs / kg or about 5x10 10 vgs / kg were administered.

[0094] In an alternative embodiment, AAV is present in an amount of about 5×10 10 vgs / kg to about 5x10 11 vgs / kg. For example, AAV can be administered at a dose of about 5x10 10 vgs / kg, about 6x10 10 vgs / kg, about 7x10 10 vgs / kg, about 8x10 10 vgs / kg, about 9x10 10 vgs / kg, about 1x10 11 vgs / kg, about 2x10 11 vgs / kg, about 3x10 11 vgs / kg, about 4x10 11 vgs / kg or about 5x10 11 vgs / kg were administered.

[0095] Such concentrations may be delivered in about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution.

[0096] The AAV can be administered within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after infection in the subject.

[0097] In one embodiment, the method as defined herein comprises inhibiting RNA viral nucleic acid in the bone or central nervous system (CNS) cells or tissues of the subject. Without being bound by theory, it is found that the recombinant adeno-associated virus (AAV) as defined herein is able to eliminate EV-A71 directly from skeletal muscle or the central nervous system (CNS), which is an important reservoir and key tissue for EV-A71 tropism and pathological state. The method may comprise administering to the subject about 5x10 11 About 5x10 13 vgs / kg of recombinant adeno-associated virus (AAV).

[0098] In one embodiment, a method of inhibiting RNA viral nucleic acid in a subject's bone or central nervous system (CNS) cells or tissues is provided, the method comprising administering to the subject a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs. The method may comprise administering to the subject about 5x10 11 About 5x10 13 vgs / kg of recombinant adeno-associated virus (AAV). The method may be able to prevent or treat RNA viral infection in a subject.

[0099] Disclosed herein is a method for inhibiting viral nucleic acid in a subject, the method comprising administering to the subject about 5x10 11 About 5x10 13 vgs / kg of a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs. In one embodiment, the method comprises inhibiting RNA viral nucleic acids in bone or central nervous system (CNS) cells or tissues of a subject. Without being bound by theory, it was found that about 5x10 11 About 5x10 13 vgs / kg The recombinant adeno-associated virus (AAV) as defined herein is able to eliminate EV-A71 directly from skeletal muscle or central nervous system (CNS), which are important reservoirs and key tissues for EV-A71 tropism and pathological states.

[0100] In one embodiment, a recombinant adeno-associated virus (AAV) for use in inhibiting viral nucleic acid in a subject is provided, wherein about 5×10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0101] In one embodiment, a recombinant adeno-associated virus (AAV) is used in the manufacture of a medicament for inhibiting viral nucleic acid in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0102] Disclosed herein is a recombinant adeno-associated virus (AAV) for treating a viral infection in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0103] Disclosed herein is the use of a recombinant adeno-associated virus (AAV) in the manufacture of a medicament for treating a viral infection in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

[0104] Disclosed herein is a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) as defined herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0105] In one embodiment, a pharmaceutical composition is provided, comprising a guide RNA, a vector or a construct as defined herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0106] Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other treatment modalities. It should also be understood that, when necessary, the composition may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is substantially no restriction on other components that may also be included in the composition, provided that the additional agent does not negatively affect the composition.

[0107] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0108] "Pharmaceutically acceptable carrier" means a solid or liquid filler, diluent or encapsulated material that can be safely used for topical or systemic administration to animals, preferably mammals, including humans. Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials and combinations thereof (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in a pharmaceutical composition is considered.

[0109] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration are determined by such factors as the patient's condition and the type and severity of the patient's disease, although the appropriate dosage can be determined by clinical trials.

[0110] “About” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, position, or length that differs from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, position, or length by at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0111] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative (or).

[0112] As used in this application, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0113] Throughout this specification and the subsequent statements, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0114] Reference in this specification to any prior publication (or information derived therefrom) or any known matter is not and should not be taken as an acknowledgement or recognition or any form of implication that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0115] Those skilled in the art will appreciate that the invention described herein is susceptible to changes and modifications other than those specifically described. It should be understood that the present invention includes all such changes and modifications that fall within the spirit and scope. The present invention also includes all steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0116] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0117] Certain embodiments of the invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the general scope of the above description.

[0118] Example

[0119] Materials and methods

[0120] Design and construction of CRISPR-Cas delivery plasmid based on AAV vector

[0121] The CasRx sequence was a gift from Patrick Hsu (pXR001: EF1a-CasRX-2A-EGFP, Addgene No. 109049). Using a plasmid backbone with AAV2 ITRs, EcoRI digestion was performed to create a nick between the two ITR sequences for insertion of a custom-designed expression cassette. First, mammalian CMV promoter and enhancer sequence fragments were derived from PCR amplification using pAAV-SMVP-Cas9C. Then, CasRx (CasRx-HA poly A) with an HA tag and rabbit poly A tail downstream of the mammalian CMV promoter and enhancer was assembled into an AAV vector plasmid containing the AAV2 ITR sequence by using the Gibson reaction. Immediately downstream of the expression cassette, a gRNA backbone driven by the human U6 promoter was assembled using the Gibson reaction, and this sequence was a gift from Patrick Hsu (pXR003: CasRx gRNA cloning backbone, Addgene No. 109053). The expression cassettes were then assembled into an EcoRI digested plasmid with the AAV2 ITRs using Gibson assembly. Individual guide sequences derived from bioinformatics analysis were cloned via Gibson assembly of target guide sequences using BbsI digestion of the vector plasmid.

[0122] Bioinformatics-guided selection process (Cas13gRNAtor)

[0123] From NCBI Virus ( https: / / www.ncbi.nlm.nih.gov / labs / virus / ) were aligned using MAFFT, and Cas13gRNAtor was run on these sequences. A bash script was created to align the input sequences using the MAFFT command line. The length was kept the same as the reference genome. The conservation score at each nucleotide position was calculated as the percentage of the sequence matching the consensus sequence at that nucleotide position. A score of 1.0 indicates that the nucleotide position is completely conserved across all sequences. The Shannon entropy (SE) score calculates the tendency of each nucleotide position toward order and disorder 34 The entropy is proportional to the disorder rate, i.e., a higher SE score indicates a higher disorder at that particular nucleotide position. Shannon entropy is defined as follows: Where Pi is the probability of a given nucleotide (A, C, G, T, N; wherein N is an unspecified or missing nucleotide), and n is the number of sequences used in the alignment. At each nucleotide position, the sum is run for the 5 nucleotides in all aligned sequences. The entropy range is between 0 and log2(5)=2.32. A consensus sequence is generated based on the most conserved nucleotide at each position for downstream processing. In the next module within Cas13gRNAtor, Cas13gRNAtor uses the model and package developed in Wessel et al., 2020 (https: / / gitlab.com / sanjanalab / cas13) to generate additional scores for all possible gRNAs of the consensus sequence. The random forest model here itself uses several independent software, including the program RNAfold for predicting RNA secondary structure and minimum free energy (MFE), RNAplfold for measuring RNA accessibility (probability of target RNA unpairing), and RNAhybrid for calculating RNA-RNA hybridization between the guide RNA and its target site and RNA-RNA hybridization MFE for each gRNA nucleotide. Through this random forest model, based on the highest percentage increase of mean square error (%incMSE), several key features are inferred to help predict the target efficiency in Cas13d, including crRNA folding energy, local target 'C'-background and upstream target 'U'-background as the most important features. Cas13gRNAtor scores gRNA according to these features. Then, Cas13gRNAtor filters the best candidate gRNA from the fourth scoring quartile. Candidate gRNAs with T homopolymer (>3Ts) sequences are also removed. For each predicted gRNA, Cas13gRNAtor obtains the entropy and conservation scores of its nucleotide positions in the consensus sequence (including in the 'seed region', also known as the 'intolerant region', which is more intolerant of mutations), and derives the mean and standard deviation. If gRNA and seed region have an average conservation score of>0.9, gRNA is considered conservative. Cas13gRNAtor scores gRNA according to these features.

[0124] Cell lines and viruses

[0125] Human RD cells and mouse C2C12 cells were purchased from the American Type Culture Collection (ATCC). Human B4G12 cells were purchased from Creative Bioarray. Cells were grown in culture media recommended by ATCC and Creative Bioarray, respectively. AAV is generated internally, as previously reported. In brief, AAV is packaged via triple transfection of 293AAV cell lines (Cell Biolabs AAV-100), which are plated in growth media in HYPERFlask'M' (Corning), and the culture media consists of DMEM+glutaMax+pyruvate+10%FBS (Thermo Fisher) supplemented with 1X MEM non-essential amino acids (Gibco). Cell coverage during transfection was 70-90%. Culture media was replaced with fresh preheated growth media before transfection. For each HYPERFlask'M', 200 μg pHelper (Cell Biolabs), 100 μg pRepCap [encoding capsid protein of serotype DJ or 2] and 100 μg pZac-CASI-GFP or pZac-CMV-CasRx-gRNAs were mixed in 5 ml DMEM, and 2 mg PEI "MAX" (Polysciences) (40 kDa, 1 mg / ml of H2O solution, pH 7.1) was added to achieve a PEI: DNA mass ratio of 5: 1. The mixture was incubated for 15 minutes and transferred dropwise to the cell culture medium. The next day after transfection, the culture medium was changed to DMEM + glutamax + pyruvate + 2% FBS. 48-72 hours after transfection, cells were harvested by scraping or dissociating with 1 × PBS (pH7.2) + 5mM EDTA, and pelleted at 1500g for 12 minutes. Cell mass is resuspended in 1-5ml lysis buffer (Tris HCl pH 7.5+2mM MgCl+150mM NaCl), and freeze-thaw 3× between dry ice-ethanol bath and 37 ℃ of water baths.Cell fragments are clarified via 4000g for 5 minutes, and supernatant is collected.The supernatant collected is processed 30 minutes at 37 ℃ with 50U / ml Benzonase (Sigma-Aldrich) and 1U / ml RNA enzyme mixture (Invitrogen), to remove unpackaged nucleic acid.After incubation, lysate is loaded onto the discontinuous density gradient composed of 15%, 25%, 40%, 60% Optiprep (Sigma-Aldrich) of each 6ml in 29.9ml Optiseal polypropylene tube (Beckman-Coulter).The tubes were ultracentrifuged at 54000 rpm at 18°C ​​for 1.5 hours in a 70-type Ti rotor. The 40% fraction was extracted and dialyzed against 1×PBS (pH 7.2) supplemented with 35 mM NaCl using an Amicon Ultra-15 (100 kDa MWCO) (Millipore). The titer of the purified AAV vector stock was determined using real-time qPCR with ITR sequence-specific primers and probes, referenced to ATCC Reference Standard Material 8 (ATCC). The EV-A71 laboratory strain was from strain 5865 / sin / 000009, GenBank accession number AF316321.

[0126] Immunofluorescence assay

[0127] 10,000 human immortalized corneal endothelial cells B4G12 were plated on glass slides in 48-well plates and transduced with AAVDJ-CasRx or AAVDJ-GFP at MOI 100K. At 3 days after transduction, cells were fixed and permeabilized with methanol for 10 minutes and blocked with 1X PBS with 5% BSA for 1 hour. This was followed by incubation with primary antibody anti-HA (abcam) at 1:200 dilution for 2 hours at RT, or anti-GFP (abcam) at 1:1500 for 2 hours at room temperature. Secondary antibody staining was performed at 1:1000 (ThermoFisher) for 2 hours at room temperature. The slides were then washed 3X with 1X PBS and fixed to the slides using ProLong mounting medium (Thermo). Images were captured using an Olympus microscope with exposure set to 15 ms and gain set to 6400 for the bright field and 488 channels and exposure set to 800 us and gain set to 6400 for the DAPI channel.

[0128] Biopanning assay for selection of AAV serotypes

[0129] Immortalized human RD cells or mouse C2C12 cells were seeded in 48-well plates at a density of 10,000 cells / well in 200 μl DMEM containing 10% FBS. Cells were cultured overnight at 37°C and allowed to adhere to the wells. Panels of AAV (1, 2, 6, 7, 8, 9, rh10, DJ, and Anc80) were used to transduce cells in triplicate at MOI 100K or 10K, respectively. 72 hours after transduction, cells were harvested and total GFP protein was quantified using a GFP quantification kit (Biovision) on a multiwell plate reader (Tecan).

[0130] RNA gene knockdown activity in AAVDJ-CasRx-transduced cells

[0131] Immortalized human RD cells were seeded in 48-well plates at a density of 10,000 cells / well in 200 μl DMEM containing 10% FBS. Cells were transduced with AAV2-GFP at MOI 10K for GFP expression. The GFP knockdown efficiency of only guide 1, only guide 2, and guide 1+guide 2 was tested by transducing RD cells expressing GFP with AAV2-CasRx carrying each guide at MOI 100K and MOI 1000K. 72 hours after transduction, cells were harvested and total GFP protein was quantified using a GFP quantification kit (Biovision) on a multiwell plate reader (Tecan).

[0132] In vitro antiviral plaque assay with EV-A71

[0133] To screen for anti-EV-A71 activity, RD cells were cultured at 10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and incubated overnight in an incubator. Dilutions of AAV2-CasRx carrying different guides were performed for transduction at MOI 1K, 10K, 100K and 1000K individually or in combination in a 100ul volume. 72 hours after transduction, cells were infected with EV-A71 virus at MOI 1. The plates were washed twice with 1X PBS and incubated with DMEM with 2% FBS for 12 hours. Supernatants from each well were harvested 12 hours after infection and used for subsequent viral plaque assays. For viral plaque assays, RD cells were seeded in 24-well plates and incubated with 10 to 10 6 The plates were washed twice with PBS and the overlay medium was added to each well and incubated for one hour with 15-minute shaking intervals and then allowed to stand in an incubator for 4 days. After 4 days of incubation, the overlay medium was removed and crystal violet stain was added to visualize plaques for counting and calculating the infectious virus titer expressed as the average number of PFU per milliliter of sample (PFU / ml).

[0134] RNA-seq library preparation, sequencing, and analysis

[0135] 8 hours after inoculation, RD cells were transduced with AAV2-CasRx-EV71 pools at MOI 1000K. After 6 days, total RNA was extracted from RD cells using the RNeasy Plus Mini kit from QIAGEN. Chained mRNA libraries were prepared using the NEBNext II Ultra Directional RNA Library Prep Kit (catalog # E7760S) from New England Biolabs, and sequenced with 150nt paired-end reads on 111umina HiSeq 4000. A total of ~ 25M ​​reads were demultiplexed under each condition. Sequencing reads were quality tested using FASTQC and mapped to the hg19 human genome using the 2.7.10a STAR aligner. Read alignment was performed using default parameters. Genome indexes were constructed using gene annotations and sjdbOverhang values ​​of 100 provided by the hg19 Illumina iGenomes collection. Quantification was performed using DESeq2 v1.34.0, triplicates were used to calculate intragroup dispersion, and contrast was used to compare treated (EV-A71 gRNA) and untreated conditions. Significantly differentially expressed genes were defined as having a false discovery rate of FDR < 0.01 and a log2 fold difference of > 0.75. Volcano plots were generated by Enhanced Volcano v1.12.0 in R4.1.2.

[0136] Flow cytometry

[0137] To titrate AAV2-GFP transduction, 10,000 cells were seeded in 48-well plates and transduced with AAV2-GFP at MOI 1K, 10K, 100K and 1000K or not transduced as a control. After 3 days, cells were harvested and suspended in flow cytometry buffer (0.5% BSA, 2 mM EDTA in 1x PBS) and passed through a 70 μm cell strainer before flow cytometric analysis by MACS Flow Cytometer (Miltenyi Biotec). To evaluate GFP knockdown by CasRx, 10,000 cells were seeded on 48-well plates and transduced with AAV2-GFP at MOI 10K, followed by transduction with AAV2-CasRx-GFP_gRNA1, AAV2-CasRx-GFP_gRNA2 or AAV2-CasRx-GFP_gRNA1+2 at MOI 100K or 1000K, or without AAV2-CasRx. After 3 days, cells were harvested and suspended in flow cytometry buffer (0.5% BSA, 2mM EDTA in 1x PBS) and passed through a 70 μm cell strainer before flow cytometric analysis by MACS Flow Cytometer (Miltenyi Biotec). To detect CasRx-HA expression, 100,000 RD cells / well were seeded on 12-well plates, and then they were transduced with AAV2-CasRx-HA at MOI 1000K. After 3 days, cells were harvested and rinsed once with 1x PBS before being fixed with Fixation Buffer (BioLegend #420801) for 20 minutes at room temperature. Cells were then rinsed three times with 1x Intracellular Staining Permeabilization Wash Buffer (BioLegend #421002) before staining for 30 minutes on ice with anti-HA primary antibody (abcam ab9110, 1:50). The cells were rinsed three times and then stained with Alexa FluorTM 488-conjugated secondary antibody (Invitrogen #A-21206, 1:200) on ice in the dark for 30 minutes. After rinsing 3 more times, cells were suspended in flow cytometry buffer (0.5% BSA, 2mM EDTA in 1x analysis by MACS Flow Cytometer (Miltenyi Biotec)). All flow cytometry results were analyzed using FlowJo software.

[0138] qPCR for profiling human innate and adaptive immune response genes

[0139] One million immortalized human bone RD cells were seeded in a 6-well dish and transduced with AAV2-CRISPR-CasRx at MOI 100K and 1000K. RNA of transduced cells was harvested 72 hours after transduction using the RNeasy universal plus mini kit (Qiagen). cDNA was synthesized using Superscript III (Thermo Fisher), and the immune response gene panel was quantified using the RT2 profiling kit (Qiagen).

[0140] Mouse infection and treatment

[0141] For toxicity analysis, BALB / c mice were treated with saline or 1x10 12 Viral genome (vgs) AAVDJGFPgRNA2 or 1x10 11 vgs or 1x10 12 vgs AAVDJ-EV71gRNAs / mouse were injected intraperitoneally. For prevention, BALB / c mice were injected with 1x10 11 Viral genomes (vgs) or 1x10 12 vgs AAVDJ-EV71gRNAs / mice were injected intraperitoneally, and then 2×10 7 PFU / mouse were intraperitoneally injected with EV-A71. For treatment analysis, BALB / c mice were treated with 2 × 10 7 PFU EV-A71 / mouse were injected intraperitoneally. After 6 hours or 24 hours, mice were treated with 1x10 11 Viral genomes (vgs) or 1x10 12 vgs AAVDJ EV71gRNA / mouse was injected intraperitoneally. 1x10 12vgs AAVDJ-GFPgRNA2 was used as a control. For up to 19 days post-infection (dpi), the survival of mice and the clinical score of each mouse in each treatment group were recorded daily. During the 19-day survival study, mice were scored in 5 categories for observed symptoms. The scoring system includes activity: 0 normal, 1 lethargy / abnormal posture, 2 curled / inactive, 3 no response to stimulation, severe tremor and inability to self-correct; breathing: 0 normal, 1 fast / shallow, 2 fast abdominal, 3 laborious, bluish; movement: 0 normal, 1 weak, uncoordinated, 2 single limb dragging / paralysis, 3 multi-limb dragging / paralysis; weight: 0 normal, 1 5% reduction within 24 hours, 2 more than 15% or up to 10% reduction within 24 hours, 3 more than 10% reduction within 24 hours or a total of 20% reduction, body condition 2 or less; dehydration: 0 normal skin tent, 1 skin tent present on the back, 2 moderate skin tent, 3 severe skin tent. A total of 6 or more points accumulated across all categories was defined as a humane endpoint, and mice with such scores were euthanized. Histological analysis was performed on mice sacrificed at 6 days post infection.

[0142] Histology of mouse tissues

[0143] Histological samples were fixed in 4% paraformaldehyde at 4°C, decalcified at room temperature for 2 hours, then embedded in paraffin and processed into 4 μm sections. Histopathology was assessed by hematoxylin and eosin (H&E) staining, and the EVA71 antigen was detected by immunohistochemical staining (1HC) using a commercially available anti-EV-A71 antibody (MAB979, 1:500 dilution), and images were captured using a Leica Bond-Max system. For staining of CasRx, histological slides were baked at 50°C for 10 minutes and dewaxed in xylene for 3x5 minutes. The slides were then rehydrated in a gradient of decreasing ethanol; 100% ethanol 2x5 minutes, 95% ethanol 5 minutes, 80% ethanol 3 minutes, 70% ethanol 3 minutes, 50% ethanol 3 minutes, rinsed with water, and rinsed with 1xPBS for 3x5 minutes. Antigen retrieval was performed by a 2100-Retriever steam cooker (Prestige Medical), and the slides were heated in 0.01M sodium citrate buffer pH 6.0 for 12-15 minutes, cooled for 3-4 hours, washed once in 1xPBS for 5 minutes, and then washed 3x for 5 minutes each using 1xPBS with 0.1% Triton. The slides were blocked in 2% BSA+5% goat serum-PBS at RT for 60 minutes, and then incubated overnight with a primary antibody 1:100 (anti-HA mouse, ab18181) in blocking buffer at 4°C. After washing 3x5 minutes with 1xPBS with 0.1% Triton, the slides were incubated with a secondary antibody 1:500 (Alexa Fluor 488, A21202) at RT in washing buffer for 2 hours, followed by washing 3x5 minutes with 6030.1% Triton-1xPBS, and counterstained with DAPI. Images were captured using a confocal microscope (Leica).

[0144] Quantification of EV-A71 titers in mouse tissues

[0145] EV-A71 infected mice were killed at 6 days post infection (dpi), and mouse tissues including hind limbs and brain were collected into CK14 homogenization tubes (Bertin Corp), homogenized and titrated by plaque formation assay. The tissue was weighed, and 1mL and 0.5mL DMEM for limb tissue and brain tissue were added to the tubes respectively, and then homogenized at 6000 × g for 10 seconds using an orbital oscillator. The process was repeated 3 times for brain tissue and 9 times for limb tissue. Homogenized tissue was centrifuged at 8000 × g, 4 ° C for 10 minutes to form a mass of tissue fragments. The supernatant was collected, and the viral load was titrated by viral plaque assay.

[0146] Ethics statement

[0147] Animal care and husbandry were provided in accordance with the National Advisory Committee for laboratory Animal Research (NACLAR) guidelines (Guidelines on the Care and Use of Animals for Scientific Purposes). Experiments with mice were designed and approved by the National University of Singapore Institutional Animal Care & Use Committee (IACUC) according to protocol 088 / 10 based on the NACLAR guidelines.

[0148] Example 1

[0149] Development of AAV-CRISPR-Cas antiviral paradigms for RNA virus targeting

[0150] The EV-A71 life cycle is typical in positive-stranded single-stranded RNA viruses, in which the virus enters the cell, releases its RNA genome into the cytoplasm, and synthesizes negative-sense genomes and subgenomic viral RNAs, from which new copies of positive-sense viral genomes and transcripts are synthesized (Figure 1A). In this study, it is proposed to use an AAV vector-based CRISPR-Cas13d system for direct targeting and cutting of viral genomes and resulting viral mRNAs in viral cells to reduce viral replication (Figure 1A). A new AAV carrier construct (Figure 1B) was constructed, which carries guide RNA (gRNA) and CasRx excluding NLS sequences, so that the expressed CasRx protein can approach the viral replication complex located outside the nuclear region. In order to verify the expression of CasRx protein, immortalized human corneal endothelial cells were transduced with AAV-CRISPR-CasRx-gRNA, and the expression and localization of HA-tagged CasRx protein in the cytoplasmic region were confirmed by immunofluorescence (Figure 1C). To test the efficacy of the new construct, AAV-CRISPR-CasRx-gRNAs targeting the GFP mRNA sequence were generated, and the virus was tested on cells expressing GFP (see Table 3 for gRNA sequences). The results confirmed that the CasRx-gRNAs without NLS were functional and reduced GFP expression to at least 1 / 2.2 (MOI = 100K) and at least 1 / 7 (MOI = 1000K) (Figures 1D and 1E). GFP gRNA2 was more effective for knocking down GFP gene expression and was therefore selected for use as a control gRNA in subsequent experiments.

[0151] In silico guide RNA design and testing strategies for CRISPR-Cas targeting of RNA viruses

[0152] To help the design and selection of antiviral CRISPR-gRNAs, Cas13gRNAtor was developed.Cas13gRNAtor is a bioinformatics tool that generates all possible gRNA candidates for a user-provided genetic or genomic sequence, and then scores each gRNA via multiple parameters: (i) multiple sequence alignments of target genes / genomes with defined conservative regions with entropy and conservation scores, (ii) structural information (RNA folding, hybridization energy), (iii) on-target scores for predicting efficacy, and (iv) off-target scores for specificity (Figures 2A and 2B).Cas13gRNAtor is applied to a list of candidate gRNAs on the computer (in silico), which target enteroviruses in highly conserved viral regions (in order to anticipate and avoid mutational escape), and are highly different from the human transcriptome and are therefore not expected to show off-targets of the human transcriptome. The gRNAs in the top 4 quartiles were filtered out based on the guide score, gRNAs predicted to have potential off-target sites in the human transcriptome (HG38; including non-coding RNAs) were eliminated, and gRNAs with T homopolymers (>3Ts) were eliminated to obtain a subset of 1265 candidate gRNAs. The conservation score at each nucleotide position is calculated as the percentage of the sequence matching the consensus sequence at the nucleotide position after performing a multiple sequence alignment. A score of 1.0 indicates that the nucleotide position is completely conserved across all sequences. Cas13gRNAtor was further narrowed down to 56 candidate gRNAs, which target highly conserved viral sequences, with an average conservation score of at least 0.9 for both gRNA and pre-spacer sequence intolerant regions.

[0153] To effectively deliver the CRISPR-Cas system into human skeletal RD cells, which serve as an in vitro model of EV-A71 infection, AAV serotype biopanning experiments were performed on these cells to identify the AAV serotype that is most suitable for transducing the cells. Among the 9 serotypes, AAV2 was identified as the most effective serotype for human skeletal RD cells ( Figure 3 A).

[0154] It was then investigated whether there were any differences in the activity of the individual guides based on their target genes or guide scores. AAV2-CRISPR-CasRx carrying each of the individual guides was used to transduce RD cells at MOI 1K, 10K, 100K, and 1000K, followed by infection of the cells with EV-A71 at MOI 1 72 hours after transduction. The supernatant was then harvested 12 hours after infection for plaque assays. The results suggest that for various high-scoring guides, significant viral inhibition was observed at low MOIs of 1K, 10K, and 100K for AAV-CRISPR-CasRx-gRNA. For gRNAs targeting VP3 and 3D genes, inhibition of up to 1 log reduction (up to 90.8% viral reduction) of viral titers was observed (Figure 2D). For low-scoring gRNAs, they did not show significant inhibition of viral replication at AAV-CRISPR-CasRx-gRNA MOIs of 1K, 10K, or 100K, but at AAV-CRISPR-CasRx-gRNA MOIs of 1000K, all gRNAs showed significant inhibition of viral replication with up to 1log reduction in viral titers (Figure 2E). This confirms that Cas13gRNAtor can identify a single gRNA for effective inhibition of viral replication. This result is also consistent with the following recent Cas13 antiviral studies: viral inhibition observed by a single guide that performs better can inhibit up to 90% of viral replication titers or reporter activity in vitro. In order to confirm that viral inhibition is not due to any intracellular antiviral activity stimulated by the AAV delivery vector, RD cells transduced with AAV2-CasRx-GFPgRNA2 were used to perform host human innate and adaptive immune response profile analysis at MOI 100K and 1000K. The results showed a minimal intracellular antiviral response in AAV2-CasRx-GFPgRNA2-transduced RD cells, with only two genes (NLRP3 and SLC11A1) significantly upregulated in a dose-dependent manner ( Figure 4 ). Both NLRP3 and SLC11A1 have been implicated in macrophage activation or recruitment but have not been shown to exhibit intracellular antiviral activity. Due to the minimal innate immunity induced intracellularly and the absence of viral plaque inhibitory activity from the AAV2-CasRx-GFPgRNA2 negative control, we concluded that the viral inhibitory function of the antiviral AAV-CasRx-gRNAs is specific and a consequence of the CRISPR-CasRx endonuclease activity targeted by the gRNA.

[0155] In order to develop more powerful AAV-CRISPR-Cas antiviral agents, different strategies for merging gRNA targeting, i.e., multi-guide multi-gene targeting or multi-guide single gene targeting methods, were explored. Four candidate gRNAs targeting different genes (2A, VP3 and 3Dpol) and four candidate gRNAs targeting only one gene (3Dpol) were shortlisted for comparison of antiviral efficacy. EV-A713Dpol serves as RNA-dependent RNA polymerase (RdRP) and is an essential gene for viral RNA synthesis in the replication cycle. The RdRP gene is a common target for developing viral inhibitors and antiviral therapeutics via viral replication blocking. The entropy and conservative scores of these individual guides are shown in Figure 5. The first pool of four candidate gRNAs targets the EV-A71 genome and subgenomic regions in 3D, VP3 and 2A, with a higher score predicted in silico, while the second pool of four candidate gRNAs targets the EV-A71 genome and subgenomic regions in 3Dpol, with a lower score predicted in silico (see Figure 2C and Table 1). In the multi-guide multi-gene approach, one or two gRNAs targeting each gene whose target site is located in the 2A, VP3 and 3D gene sequences were selected. This initial gRNA merger method showed significant inhibition of viral replication titers up to 1lOg virus titers and up to 91.1% virus reduction at MOI 100K and 1000K, but was not significantly better than the single-guide method (Figure 2D). In contrast to the multi-guide single-gene method in which 4 gRNAs target the same gene 3Dpol, significant inhibition of viral titer reduction up to 2logs or up to 98.8% was observed (Figure 2F). This result suggests that targeting all 4 gRNAs to the same 3D gene is more effective than 4 gRNAs with different genes on the targeting viral sequence. Next, by sequentially increasing the number of guides targeting the 3D gene, the differences in the inhibitory activity of different numbers of merged guides for the multi-guide single-gene approach were investigated. The results show that increasing the number of combined gRNAs for the same gene target until 3 gRNAs did not significantly improve inhibition, which remained at a viral titer reduction of about 110g, while increasing the number of combined gRNAs targeting the same gene target until 4 gRNAs significantly increased the inhibition of viral titer reduction up to 2logs or up to 98.8% (Figures 2F and 2G). Further increasing the number of gRNAs targeting the same gene to 5 increased inhibition up to 3logs or up to 99.7% (Figures 2F and 2G). For the number of gRNAs tested in this study, the most powerful mixture was 6, where up to 5logs or up to 99.99% of viral reduction was observed at MOI 100K or 1000K (Figures 2E, 2F and 2G). Even at a lower MOI of 10K, significant inhibition of viral titer up to 1log or up to 90.1% of viral reduction was observed (Figure 2E).Together, these data suggest that a multi-guide single-gene targeting strategy with up to six gRNAs could provide an effective approach to eliminate EV-A71 RNA viruses in vitro.

[0156] Efficient in vivo viral elimination in a mouse model of EV-A71 infection

[0157] Next, we investigated the effectiveness of the multi-guided single gene approach for the AAV-CRISPR-CasRx antiviral model in the established EV-A71 mouse model of hand, foot and mouth disease. To ensure that the therapeutic agent is effectively delivered into mouse muscle tissue, which is the main replication target of the EV-A71 virus, an AAV serotype bio-panning experiment was performed using immortalized mouse C2C12 skeletal muscle cells, which identified the AAVDJ serotype as the most effective of the 11 serotypes evaluated ( Figure 3 B) To test the efficacy of the combined AAVDJ-CRISPR-CasRx-gRNAs in the mouse model, 2-day-old mice were first treated with 1x10 12 Total vector genomes (vgs) of AAVDJ-CRISPR-CasRx-GFPgRNA2 or 1x10 11 or 1x10 12 AAVDJ-CRISPR-CasRx-3D_gRNAs of total vgs were injected. After 3 days, mice were then treated with 2x10 7 The lethal dose of PFUEV-A71 / mouse was injected intraperitoneally, and the survival rate, clinical symptoms and body weight were monitored daily. As shown in Figure 5A, 20% survival was observed in the control group mice (n=5) at 11 days after infection. In contrast, at 13 days after infection and up to 19 days after infection, the survival rate of 1x10 11 80% survival was observed in the vgs AAVDJ-CRISPR-CasRx treatment group (n=5). 1x10 12 The higher treatment dose of vgs conferred a 100% protective effect in infected mice (n=7, log-rank Mantel-Cox test, *p=0.0003) until 19 days after lethal infection. Based on the physical symptoms of body weight, activity, respiration, movement and dehydration, higher clinical scores and lighter body weights of EV-A71-infected mice were observed in the control treatment group when compared with the two treatment groups showing minimal clinical symptoms (Figures 6B and 6C). EV-A71-infected mice in the control treatment group developed severe clinical symptoms, including inactivity, weight loss and hind limb paralysis.

[0158] To examine the effects of the AAV-CRISPR-CasRx antiviral mode on viral infection and pathological conditions within the established EV-A71 mouse model, muscle tissue and brain of EV-A71 infected mice were harvested at 6 dpi, and viral titers were quantified by viral plaque assay. The results showed that although viral titers were still detectable in the hind limbs and brain tissues after treatment in a few mice, a substantial reduction in viral titers was observed in most treated mice. In the hind limbs, viral titers showed a reduction of approximately 2 to 3 log in 4 mice compared to the control group, and viral titers were 1x10 12 Total vgs-treated mice (p = 0.0030, Kruskal-Wallis test) were not detected in 2 mice (Figure 5D). In brain tissue, the virus titer showed a 3-log reduction in 3 mice compared with the control group, and the virus titer was 1x10 12 Total vgs-treated mice were not detected in 3 mice (Figure 5E). Results from H&E staining of limb muscle and spinal cord tissue revealed that compared with the severe necrosis observed in the control group, the 1x10 12 There was no obvious muscle necrosis in AAVDJ-CRISPR-CasRx-3D_gRNAs of total vgs, while for 1x10 11 Mild necrosis was noted in mice treated with total VGS (Figure 5F). 12 No viral antigen distribution was observed in mice treated with total vgs, whereas in mice treated with 1x10 11 Minimal viral antigens were present in total vgs-treated mice, and severe viral antigen distribution was observed in control mice to the hindlimbs and mainly to the anterior horn neurons of the cervical spinal cord (Fig. 5G).

[0159] Importantly, the AAV-CRISPR-CasRx antiviral model was effective as a therapeutic intervention in mice already infected with EV-A71. To test the efficacy of the combined AAVDJ-CRISPR-CasRx-gRNAs in the mouse model, 5-day-old mice were first treated with 2x10 7 A lethal dose of 10 PFU EV-A71 / mouse was injected intraperitoneally. Then, 2 hours later, the mice were treated with 1x10 12 vgs AAVDJ-CRISPR-Cas-GFPgRNA2 or 1x10 11 or 1x10 12 Total vgs of AAVDJ-CRISPR-CasRx-3D_gRNAs were injected intraperitoneally (Figure 6). The survival rate, clinical symptoms and body weight of mice were monitored every day. These results confirmed that the use of 1x1011 vgs AAVDJ-CRISPR-CasRx-3D_gRNAs successfully conferred 50% of mice (n=8) survival, while 1x10 12 Total vgs conferred 62.5% of mice (n=8) survival until 19 days after lethal infection. Based on physical symptoms, 1x10 11 and most 1x10 12 Higher clinical scores and lighter body weights of EV-A71-infected mice were observed in the control group compared with the total VGS-treated group.

[0160] In another experiment, 5-day-old mice were treated with 2x10 7 A lethal dose of 10 PFU EV-A71 / mouse was injected intraperitoneally. After 6 hours, the mice were treated with 1x10 12 vgs AAVDJ-CRISPR-Cas-GFPgRNA2 or 1x10 11 or 1x10 12 Total vgs of AAVDJ-CRISPR-CasRx-3D_gRNAs were injected intraperitoneally. The survival rate, clinical symptoms and body weight of mice were observed every day (Figure 7A). The results indicate that the 1x10 11 vgs AAVDJ-CRISPR-CasRx-3D_gRNAs successfully conferred 80% mouse survival (n=5, log-rank Mantel-Cox test; *p=0.0039), while 1x10 12 Total vgs conferred 100% mouse survival (n=4, log-rank Mantel-291 Cox test; *p=0.0084) until 19 days after lethal infection (Fig. 7B). Milder clinical symptoms, better body weight, significantly reduced virus titers in hind limbs and brain, significantly reduced virus antigens in tissues, and reduced muscle necrosis were observed in the treatment group (Fig. 7C to 7H).

[0161] Importantly, even with treatment extended to 24 h post-infection (Fig. 8A), the 11 vgs AAVDJCRISPR-CasRx-3D_gRNA also successfully conferred 80% mouse survival (n=5, log-rank Mantel-Cox test; *p=0.053), and at 1x10 12 Total vgs conferred 100% mouse survival (n=5, log-rank Mantel-Cox test; *p=0.015) until 19 days after lethal infection (Figure 8B). The treated group again showed improved clinical scores and body weights (Figures 8C and 8D). Compared with the control group, the virus titer was 1x10 12Total vgs in the treated group of mice was significantly reduced by 3 logs in the hind limbs (p = 0.0018, Kruskal-Wallis test) (Figure 8E), and significantly reduced by 4 logs in the brain tissue (p = 0.0029, Kruskal-Wallis test) (Figure 8F). IHC in neurons of the hind limbs and anterior horn of the cervical spinal cord showed that the expression of Vgs in the treated group was significantly reduced by 3 logs (p = 0.0018, Kruskal-Wallis test) (Figure 8E), and significantly reduced by 4 logs (p = 0.0029, Kruskal-Wallis test) in the brain tissue (Figure 8F). 12 The minimal viral antigen distribution in mice treated with total VGS was observed in mice treated with 1x10 11 Moderate viral antigen distribution in mice treated with total vgs, and severe viral antigen distribution in control mice (Figure 8G). H&E staining performed on limb muscle and spinal cord tissue revealed that AAVDJ-CRISPR-CasRx-3D_gRNAs alleviated the severe muscle necrosis observed in the control group (Figure 8H). In short, the results indicate that the AAVDJ-CRISPR-CasRx-3D_gRNAs pattern is effective as a treatment for EV-A71 from beginning to end in the treatment window of 2 hours, 6 hours and 24 hours before infection and after infection. Importantly, AAVDJ-CRISPR-CasRx-3D_gRNAs treatment prevents death and eliminates up to 99.9% of EV-A71 RNA viruses in the body.

[0162] Table 1: EV-A71 gRNA sequences and scores

[0163]

[0164] Table 2 EV-A71 gRNA sequences

[0165]

[0166] Table 3: GFP gRNA sequences

[0167]

Claims

1. A method for preventing or treating an RNA virus infection in a subject, the method comprising administering to the subject a concentration of about 5 x 10 11 About 5x10 13 vgs / kg dose of a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

2. The method of claim 1, wherein the dose is about 5 x 10 12 About 5x10 13 vgs / kg.

3. The method according to claim 1 or 2, wherein the RNA viral infection is infection by a single-stranded RNA virus.

4. The method according to any one of claims 1 to 3, wherein the single-stranded RNA virus is selected from the group consisting of enterovirus, coxsackievirus and parechovirus.

5. The method of claim 4, wherein i) the enterovirus is enterovirus 71; ii) the coxsackievirus is selected from CAV16 and CAV6; and iii) the parechovirus is selected from parechovirus A, parechovirus B, parechovirus C, parechovirus D, parechovirus E and parechovirus F.

6. method according to claim 5, wherein the Cas13 nuclease is Cas13a, Cas13b, Cas13c or Cas13d nuclease.

7. The method according to any one of claims 1 to 6, wherein the AAV vector is an AAV2, AAVDJ or AAV1 vector.

8. The method according to any one of claims 1 to 7, wherein the at least one guide RNA comprises i) a first nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence encoded by one of the nucleic acid sequences shown in SEQ ID NOs: 1-10, or ii) a first nucleic acid having at least 70% sequence identity to one of the nucleic acid sequences shown in SEQ ID NOs: 11-20.

9. according to the method described in any one of claims 1 to 8, wherein the Cas13 nuclease is operably linked to a CMV promoter.

10. The method of any one of claims 1 to 9, wherein the guide RNA is operably linked to a U6 promoter.

11. The method of any one of claims 1 to 10, wherein preventing or treating RNA viral infection comprises inhibiting RNA viral nucleic acids in the subject's skeletal or central nervous system (CNS) cells or tissues.

12. A recombinant adeno-associated virus (AAV) for treating an RNA viral infection in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

13. Use of a recombinant adeno-associated virus (AAV) in the manufacture of a medicament for treating an RNA viral infection in a subject, wherein about 5x10 11 About 5x10 13 vgs / kg of a recombinant AAV is administered to a subject, wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

14. A method of inhibiting RNA viral nucleic acid in a subject, the method comprising administering to the subject about 5x10 11 About 5x10 13 vgs / kg of a recombinant adeno-associated virus (AAV), wherein the AAV comprises at least one heterologous nucleic acid sequence encoding a Cas13 nuclease and one or more guide RNAs.

15. The method of claim 14, wherein the method comprises inhibiting RNA viral nucleic acids in bone or central nervous system (CNS) cells or tissues of the subject.

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