Muscle-targeting adeno-associated virus capsid variants, recombinant adeno-associated viruses and uses thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to muscle-targeting AAV capsid variants, recombinant AAV, and their applications. To improve the muscle tissue transduction efficiency of AAV and reduce liver tropism, this invention provides a muscle-targeting AAV capsid variant protein, which is a polypeptide that enhances the muscle cell or tissue targeting of AAV capsid protein and reduces liver tropism by substituting, inserting, and / or deleting at least one polypeptide in the variable region VIII of the parental AAV capsid protein VP1. Experimental results demonstrate that the AAV capsid protein variant of this invention has high transduction efficiency, significantly increases gene expression in muscle tissue, and possesses cross-species characteristics, enabling efficient transduction of muscle tissue and reducing liver tropism in mice and non-human primates. Clinical data indicate that AAVM101.μDys prepared based on the AAV capsid protein variant of this invention has good safety and efficacy in the treatment of DMD, laying a clinical foundation for further research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to muscle-targeting adeno-associated virus capsid variants and their recombinant adeno-associated viruses and their applications. Background Technology

[0002] Gene therapy is a method of treating or preventing diseases by modifying or manipulating genes. Its core lies in correcting or compensating for the function of abnormal genes, and it has shown great potential in the treatment of major genetic diseases. Recombinant adeno-associated virus (AAV) is currently the most promising gene delivery vector due to its high safety, wide adaptability, and low immunogenicity; nine recombinant AAV gene therapy products have already been approved for marketing. AAV is a naturally replication-defective non-pathogenic virus and is the simplest, non-enveloped, single-stranded linear DNA virus discovered to date. The AAV genome consists of inverted repeat sequences (ITRs) at both ends and REP and CAP genes in the middle. Recombinant AAV consists of the capsid of wild-type AAV and a modified genome sequence. Its genome sequence retains only the ITR sequences necessary for viral replication and packaging, while replacing the REP and CAP gene sequences with expression cassettes containing therapeutic genes and their regulatory elements. This maximizes gene packaging capacity while reducing immunogenicity and cytotoxicity during in vivo delivery.

[0003] However, long-term research and clinical practice have found that commonly used natural AAV serotypes, such as AAV1, AAV2, AAV8, and AAV9, while having some affinity for muscle tissue, have less than ideal targeting and transduction efficiency. After systemic administration, a large number of viral particles accumulate in non-target organs such as the liver, leading to hepatotoxicity and reduced transduction efficiency to target muscle tissue. Simultaneously, neutralizing antibodies against natural AAV serotypes are prevalent in the population, which can cause gene therapies based on these serotypes to fail. Furthermore, due to low targeting efficiency, effective treatment for myopathy requires injections of extremely high doses of recombinant AAV drugs (≥1×10⁻⁶). 14 This not only poses a challenge to the large-scale production of the virus, but also places higher demands on the transduction efficiency and immunogenicity of recombinant AAV. Several adverse events caused by high-dose injections have been reported in clinical trials. For example, in the Phase I / II clinical trial ASPIRO (NCT03199469) conducted by Astellas, three children received a high dose (3 × 10⁻⁶ g / kg), which resulted in adverse events in three children. 14Following a single intravenous injection of AT132 (recombinant AAV8-MTM1) at a dose of (vg / kg), liver failure led to death. This occurred in a study conducted by Solid Biosciences using the AAV9 vector (SGT001, 2×10⁻⁶ vg / kg). 14 In a phase I / II clinical trial using high-dose AAV9 vectors for the treatment of Duchenne muscular dystrophy (DMD) (Fordadistrogenemovaparvovec, 3×10), adverse events such as complement activation and thrombocytopenia led to kidney and cardiopulmonary injury. Furthermore, in other clinical trials using high-dose AAV9 vectors for the treatment of DMD (Fordadistrogenemovaparvovec, 3×10), adverse events such as complement activation and thrombocytopenia led to kidney and cardiopulmonary injury. 14 Patient deaths have also been reported in doses of (vg / kg). These clinical reports highlight the significant challenges of muscle delivery and underscore the necessity of developing highly efficient muscle-targeting AAV variants. Recently, a genetically engineered capsid AAV-SLB101 (NCT06138639) was used in a clinical trial for DMD, demonstrating the practical value of genetically engineered AAV capsids in clinical research.

[0004] The targeting ability of AAVs largely depends on the specific interaction between the viral capsid and target cell surface receptors, as well as their intracellular transport after endocytosis. Current research has attempted to enhance muscle targeting by systematically optimizing key steps in the interaction between the AAV capsid and the host through strategies such as rational design, directed evolution, and computer-aided design. However, these variants still require further improvement in targeting specificity, transduction efficiency, and the ability to evade pre-existing immunity. For example, some variants of AAV9 have shown improved muscle targeting, but AAV9's blood-brain barrier penetration and the high pre-existing neutralizing antibody positivity rate in humans limit its application. While the AAVrh74 serotype derived from rhesus monkeys does not cross the blood-brain barrier and shows some immunological improvements, achieving high-level, uniformly distributed therapeutic protein expression in systemic muscle tissue requires further improvement. Therefore, there is an urgent need in this field to develop novel AAV capsid variants with higher muscle tissue transduction efficiency, lower liver tropism, and good cross-species consistency, as well as recombinant AAV vectors and particles with superior therapeutic effects. Summary of the Invention

[0005] To overcome the design and application deficiencies of existing adeno-associated virus (AAV) vectors, improve AAV muscle tissue transduction efficiency, and reduce liver tropism, this invention provides a novel muscle-targeting AAV capsid variant derived from the AAVrh74 parental serotype. The innovation lies in the insertion of a polypeptide sequence. Compared to the parent, the engineered AAV capsid variant protein provided by this invention can efficiently target muscle tissue (such as skeletal muscle and diaphragm) in various mammalian species (including rodents and non-human primates), while significantly reducing accumulation in non-target organs such as the liver. The recombinant adeno-associated virus (recombinant AAV) particles packaged with the target gene can be used for the treatment of neuromuscular diseases.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a muscle-targeting AAV capsid variant protein, wherein the variant protein is a polypeptide that has been substituted, inserted, and / or deleted in the variable region VIII of the parental AAV capsid protein VP1.

[0007] Furthermore, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1 and SEQ ID NO.2, or has more than 80% homology with SEQ ID NO.1 and SEQ ID NO.2 and has the same or similar biological activities.

[0008] Preferably, the polypeptide has the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, or is a polypeptide obtained by substituting and / or deleting and / or inserting 1 to 3 amino acids in the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] Further, the parental AAV capsid protein is selected from at least one of AAVrh74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0010] Preferably, the parental AAV capsid protein is AAVrh74 capsid protein, and its amino acid sequence is shown in SEQ ID NO.5.

[0011] Furthermore, the polypeptide substitution, insertion, and / or deletion sites are any two or more amino acid positions between positions 586 and 599 of the parental AAV capsid protein VP1 amino acid sequence.

[0012] Preferably, the polypeptide substitution, insertion, and / or deletion sites are between positions 586 and 587, 586 and 598, 586 and 599, 586 and 590, 586 and 591, 586 and 592, 587 and 588, 587 and 589, 587 and 590, 587 and 591, 587 and 592, 588 and 589, 588 and 591, 588 and 592, 589 and 590, 589 and 591, 589 and 592, 590 and 591, 590 and 592, and 591 and 592, respectively, in the amino acid sequence of the parental AAV capsid protein VP1.

[0013] Most preferably, the amino acid sequence of the AAV capsid variant protein is as shown in SEQ ID NO.3 and SEQ ID NO.4, or has more than 90% homology with SEQ ID NO.3 and SEQ ID NO.4 and has the same or similar biological activities.

[0014] In a second aspect, the present invention provides an isolated polynucleotide that encodes the aforementioned muscle-targeting AAV capsid variant protein.

[0015] Preferably, the sequence of the polynucleotide is as shown in SEQ ID NO.6 or SEQ ID NO.7.

[0016] Thirdly, the present invention provides a muscle-targeting recombinant AAV particle containing the AAV capsid variant protein described in the first aspect, and a target gene or target gene expression cassette packaged within the AAV capsid variant protein; the target gene is capable of treating or preventing diseases affecting muscle cells / tissues.

[0017] Furthermore, the target gene is selected from at least one of the following groups: therapeutic genes for muscle cell / tissue diseases, genes encoding therapeutic proteins or peptides, and genes encoding therapeutic RNA.

[0018] Preferably, the gene encoding the therapeutic protein or peptide is selected from genes encoding therapeutic antibodies or antibody fragments, or genes encoding genome editing enzymes.

[0019] Preferably, the gene encoding therapeutic RNA is an interfering RNA, a gRNA for genome editing, an exon-jumping antisense RNA, or a repressive tRNA gene.

[0020] More preferably, the target gene is a gene encoding "mini" dystrophin (μDys) or N-acetylgalactosamine-6-sulfatase (GALNS). Most preferably, the gene sequence encoding μDys is shown in SEQ ID NO.8.

[0021] More preferably, the target gene expression cassette contains a gene encoding μDys or GALNS, a promoter, and / or a polyA signal. Most preferably, the μDys target gene expression cassette sequence is shown in SEQ ID NO. 9.

[0022] Furthermore, the muscle-targeting recombinant AAV particle is a virus formed by co-transfecting a recombinant AAV vector containing the target gene or the target gene expression cassette with the AAV capsid variant protein and helper plasmid into host cells, packaging, and purifying the virus.

[0023] Most preferably, the recombinant AAV vector containing the target gene or the target gene expression cassette is selected from at least one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0024] Fourthly, the present invention provides a pharmaceutical composition comprising the recombinant AAV particles described in the third aspect of the present invention and a pharmaceutically acceptable auxiliary carrier.

[0025] Fifthly, the present invention provides the use of the above-mentioned AAV capsid variant protein, recombinant AAV particles, or pharmaceutical composition in the preparation of medicaments for treating and / or preventing diseases affecting muscle cells / tissues.

[0026] Furthermore, the diseases affecting muscle cells / tissues include, but are not limited to, at least one of neuromuscular genetic diseases, cardiomyopathy, rhabdomyosarcoma, polymyositis, and dermatomyositis.

[0027] Preferably, the diseases affecting muscle cells / tissues include at least one of: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, spinal muscular atrophy, myotonic dystrophy type 1 and 2, central nucleus myopathy, Pompe disease, glycogen storage disease III, amyotrophic lateral sclerosis, or congenital myasthenia gravis.

[0028] Preferably, the therapeutic genes in the neuromuscular genetic diseases targeted by the drug include at least one of DMD, BMD, DYSF, FKPP, SMN1, ASAH1, DMPK, CNBP, DNM2, BIN1, GAA, AGL, SOD, ALS2, CHAT, or AGRN.

[0029] Furthermore, the drug is delivered via intravenous or intramuscular injection.

[0030] Beneficial effects: This invention develops a novel AAV capsid protein variant by substituting, inserting, and / or deleting at least one polypeptide in the variable region VIII of the parental AAV capsid protein VP1, resulting in higher muscle tissue transduction efficiency, lower liver tropism, and good cross-species consistency. The target gene is then packaged with the AAV capsid protein variant to obtain recombinant AAV virus particles.

[0031] Experimental results demonstrate that the recombinant AAV virus particles obtained from the AAV capsid protein variant and its packaging target gene of this invention have the following advantages: 1. The AAV capsid protein variants AAVM101 and AAVM102 developed based on the parental AAVrh74 capsid protein of this invention have shown significantly higher transduction efficiency in mouse myoblasts and human skeletal muscle cells than wild-type AAV9 and AAVrh74 in in vitro cell experiments. In in vivo experiments, after systemic administration, the gene expression level of recombinant AAV particles obtained based on the AAV capsid variants in muscle tissue is 2 to 10 times higher than that of wild-type AAVrh74, while the enrichment in non-target organs such as the liver is significantly reduced.

[0032] 2. Reduced dosage and toxicity: Due to its high targeting efficiency, only a lower dose of recombinant AAV virus is needed to achieve therapeutic effects in muscle tissue, thereby significantly reducing the risk of immune response and hepatotoxicity caused by high viral load and improving the safety window of treatment.

[0033] 3. Broad clinical application prospects: The capsid variant AAVM101 of this invention has cross-species characteristics, enabling efficient transduction of muscle tissue in mice and non-human primates, reducing liver tropism, and providing a novel, efficient and safe gene delivery tool for a variety of hereditary and acquired muscle-related diseases, with clinical translational value and market potential.

[0034] 4. Human data confirm that the recombinant AAV virus particles AAVM101-μDys prepared in this invention have good safety and functional improvement in the treatment of DMD: In the first human phase I clinical trial for children aged 5-6 years with DMD, AAVM101-μDys provided by this invention showed good safety and preliminary efficacy, laying a key clinical foundation for its further development. Attached Figure Description

[0035] Figure 1 The diagrams show the design and screening of AAV capsid variants in Example 1: a) Schematic diagram of library plasmid construction; b) Schematic diagram of directed evolution screening of AAV capsid variants.

[0036] Figure 2The sequence comparison and structure prediction of the AAV capsid variant in Example 1 are as follows: a) Schematic diagram of sequence comparison between the AAV capsid variant and the parental AAV capsid; b) Schematic diagram of the molecular structure of AAVM101, including monomers (peptide insertion is shown in red), trimers (peptide insertion is shown in yellow), and icosahedrons (peptide insertion is shown in yellow); c) Schematic diagram of the molecular structure of AAVM102, including monomers (peptide insertion is shown in red), trimers (peptide insertion is shown in green), and icosahedrons (peptide insertion is shown in green).

[0037] Figure 3 For the yield and identification of the AAV capsid variant in Example 2: a, AAV capsid packaging yield, expressed as viral genome copies produced per 50 mL Erlenmeyer flask (vg / flask); b, SYPRO™ Ruby protein gel staining image of AAV capsid protein.

[0038] Figure 4 The transduction efficiency of the AAV capsid variant in in vitro cells was evaluated as follows: a) Fluorescence imaging of recombinant AAV-EGFP 24 hours after infection of C2C12 and HSKMC, scale bar: 200 µm; b) Determination of the relative expression level of EGFP mRNA; c) Determination of AAV virus copy number.

[0039] Figure 5 This is an evaluation of the transduction efficiency of in vivo whole-body delivery of recombinant AAV-ffLuc in Example 4.

[0040] Figure 6 This is a tissue fluorescence imaging of recombinant AAV-EGFP delivered in vivo and systemically in Example 5.

[0041] Figure 7 This is a determination of the mRNA expression of recombinant AAV-EGFP delivered in vivo and systemically in Example 5.

[0042] Figure 8 This study investigates the biodistribution of recombinant AAV-EGFP delivered in vivo and systemically in Example 5.

[0043] Figure 9 Serum biochemical indicators were analyzed in the DMD disease model treated with systemic delivery of recombinant AAV-μDys in Example 6: a) serum CK level; b) serum ALT level; c) serum AST level.

[0044] Figure 10 Immunofluorescence analysis of muscle tissue from a DMD disease model treated with systemic delivery of recombinant AAV-μDys in Example 6.

[0045] Figure 11For the protein immunoblotting analysis of the whole-body delivery of recombinant AAV-μDys to treat DMD disease model in Example 6: a, immunoblotting map of µDys protein expression in muscle tissue; b, semi-quantitative analysis of µDys protein expression in muscle tissue.

[0046] Figure 12 H&E staining analysis of muscle tissue from the DMD disease model treated with systemic delivery of recombinant AAV-μDys in Example 6: Black arrows indicate pathological features such as inflammatory cell infiltration or myofibrillary necrosis. Scale bar: 100 µm.

[0047] Figure 13 Masson's trichrome staining analysis of muscle tissue from the DMD disease model treated with systemic delivery of recombinant AAV-μDys in Example 6: a, Masson staining map of muscle tissue; b, quantitative analysis of collagen fiber deposition. Blue staining represents collagen fibers. Scale bar: 100 µm.

[0048] Figure 14 This is an assessment of muscle function recovery in the DMD disease model treated with systemic delivery of recombinant AAV-μDys in Example 6.

[0049] Figure 15 Example 7 shows the evaluation of recombinant AAV transduction efficiency in non-human primates: a) Schematic diagram of gene delivery transduction efficiency evaluation in non-human primates; b) Deep sequencing analysis of mRNA expression carrying the delivered gene in non-human primates.

[0050] Figure 16 Evaluation of µDys protein expression in muscle biopsies of DMD patients treated with systemic delivery of recombinant AAV-μDys in Example 8: a) Immunofluorescence images of gastrocnemius muscle biopsy sections from each patient before and 6 months after treatment, scale bar: 100 µm; b) Western blot images of µDys protein expression in gastrocnemius muscle from each patient before and 6 months after treatment; c) Semi-quantitative analysis of µDys protein expression in gastrocnemius muscle.

[0051] All statistical analyses were performed using GraphPad Prism 10 software. Data are expressed as mean ± standard deviation (SD), and Dunnett's test or t-test was used for comparisons between groups. P values ​​are expressed as follows: * P < 0.05, ** P < 0.01. Detailed Implementation

[0052] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0053] The term "adeno-associated virus (AAV)" refers to a naturally replication-defective, non-pathogenic virus that is currently the simplest, non-enveloped, single-stranded linear DNA virus discovered.

[0054] Existing natural AAV serotypes have low targeting efficiency, and effective treatment for myopathy requires injection of extremely high doses of recombinant AAV drugs, leading to excessive liver burden. In order to improve the muscle tissue transduction efficiency of AAV and reduce liver tropism, in one embodiment of the present invention, a novel muscle-targeting AAV capsid variant is provided, wherein the variant protein is at least one polypeptide that has been substituted, inserted, and / or deleted in the variable region VIII of the parental AAV capsid protein VP1.

[0055] The polypeptide described in this invention can enhance the targeting of AAV capsid protein to muscle cells or tissues and reduce its hepatic orientation.

[0056] The term "targeting" refers to the specificity of the AAV capsid protein, present in AAV viral particles, for infecting specific types of cells or tissues. The term "hepatic tropism" refers to the tropism of the liver or liver tissues and cells (including hepatocytes).

[0057] Studies have shown that when the amino acid sequence of a polypeptide is as shown in SEQ ID NO.1 or SEQ ID NO.2, or has more than 80% homology with SEQ ID NO.1 or SEQ ID NO.2 and has the same or similar biological activities, it enhances the targeting of AAV capsid protein to muscle cells or tissues.

[0058] More preferably, the polypeptide is a polypeptide obtained by substituting and / or deleting and / or inserting 1 to 3 amino acids in the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0059] In some embodiments of the present invention, the parental AAV capsid protein is selected from at least one of AAVrh74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0060] The application of AAV9 is limited by its ability to cross the blood-brain barrier and the high pre-existing positive rate of neutralizing antibodies against AAV9 in the human population. Furthermore, the AAVrh74 serotype derived from rhesus monkeys does not cross the blood-brain barrier, and research on it is limited. Therefore, in some specific embodiments of this invention, the parental AAV capsid protein is the AAVrh74 capsid protein, whose amino acid sequence is shown in SEQ ID NO. 5.

[0061] In some embodiments of the present invention, the polypeptide substitution, insertion, and / or deletion sites are between positions 586 and 599 of the parental AAV capsid protein VP1 amino acid sequence. In some preferred embodiments of the present invention, the amino acid sequence of the AAV capsid variant protein is as shown in SEQ ID NO. 3 and SEQ ID NO. 4, or has more than 90% homology with SEQ ID NO. 3 and SEQ ID NO. 4 and has the same or similar biological activities.

[0062] The above 90% homology and the same or similar biological activity refer to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO.3 or SEQ ID NO.4.

[0063] In another embodiment of the present invention, a muscle-targeting recombinant AAV particle is also provided, which contains the AAV capsid variant protein and a target gene or target gene expression cassette packaged within the AAV capsid variant protein; the target gene is capable of treating or preventing diseases affecting muscle cells / tissues.

[0064] The “muscle tissue” mentioned in this invention includes the heart, skeletal muscle, diaphragm, etc.; “muscle cells” refer to myocytes, myotubes, myoblasts, or satellite cells.

[0065] The target genes for treating or preventing diseases affecting muscle cells / tissues as described in this invention include, but are not limited to: therapeutic genes for muscle cell / tissue diseases, genes encoding therapeutic proteins or peptides (such as genes encoding therapeutic antibodies or antibody fragments, genes encoding genome editing enzymes), and genes encoding therapeutic RNA (such as interfering RNA, gRNA for genome editing, exon-jumping antisense RNA, and repressive tRNA genes).

[0066] Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder caused by a mutation in the DMD gene that blocks the synthesis of its encoded product, dystrophin. Therefore, in some embodiments of this invention, the gene encoding "mini" dystrophin (μDys) is used as the target gene and packaged with an AAV capsid variant protein to treat diseases affecting muscle cells / tissues. The gene sequence encoding μDys is shown in SEQ ID NO. 8.

[0067] To more efficiently package and produce recombinant adenovirus particles for infecting target cells, in some embodiments of the present invention, a target gene expression cassette is designed containing an encoding μDys gene, a promoter, and / or a polyA signal. Most preferably, the μDys target gene expression cassette sequence is shown in SEQ ID NO. 9.

[0068] In this invention, diseases affecting muscle cells / tissues include, but are not limited to, at least one of neuromuscular genetic diseases, cardiomyopathy, rhabdomyosarcoma, polymyositis, and dermatomyositis.

[0069] More specifically, the diseases affecting muscle cells / tissues include at least one of: DMD, Becker muscular dystrophy, limb-girdle muscular dystrophy, spinal muscular atrophy, myotonic dystrophy type 1 and 2, central nucleus myopathy, Pompe disease, glycogen storage disease III, amyotrophic lateral sclerosis, or congenital myasthenia gravis.

[0070] The therapeutic genes in neuromuscular genetic diseases targeted by the drug include at least one of the following: DMD, BMD, DYSF, FKPP, SMN1, ASAH1, DMPK, CNBP, DNM2, BIN1, GAA, AGL, SOD, ALS2, CHAT, or AGRN.

[0071] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0072] ddPCR Implementation Procedure: Treat 5 µL of the sample to be tested with DNase I. Serially dilute the DNase I-treated sample to prepare ddPCR Mix. Add the ddPCR Mix, diluted sample, control AAV, and nuclease-free water to each PCR tube and mix well. Then place the tube in a droplet generator to begin droplet generation. Turn on the PX1 heat sealer: set the temperature to 180℃ to preheat the instrument, then seal the tube and run the PCR program as follows: Stage 1: Step 1, 95℃, 10 s, 1 cycle; Stage 2: Step 1, 95℃, 15 s; Step 2, 60℃, 1 min; Step 3, 72℃, 25 s; 42 cycles; Stage 3, Step 1, 98℃, 10 min, 1 cycle; Stage 4, Step 1, 4℃, time hold, 1 cycle. After the reaction is complete, read the droplets using a droplet reader (QuantaSoft). TM The software performs data analysis.

[0073] RT-PCR Implementation Procedure: RNA was extracted from cells or tissues using the Qiagen RNA Extraction Kit, following the manufacturer's instructions. Then, Takara's PrimeScript was used for the extraction. TM The RT reagent kit with gDNA Eraser was used to reverse transcribe 800 ng of RNA into cDNA. The library insert fragment enriched in the cDNA was amplified using the Q5 enzyme with primers 74-F (SEQ ID NO. 20): 5'-TGATGCTAACCAGCGAGGAA-3'; 74-R (SEQ ID NO. 21): 5'-GAAAGTTGCCGTCCGTATGA-3'.

[0074] RT-qPCR Implementation Procedure: RNA was extracted from cells or tissues using the Qiagen RNA Extraction Kit, following the manufacturer's instructions. Then, Takara's PrimeScript was used for RNA extraction. TM The RT reagent kit with gDNA Eraser was used to reverse transcribe RNA into cDNA. Real-time PCR was performed using Takara's TB Green Premix EX Taq reagent for detection. The relative expression level of the target gene was calculated using the 2^-ΔΔCT method. The primers used were... EGFP_F_Primer (SEQ ID NO.22): ATCATGGCCGACAAGCAGAA; EGFP_R_Primer (SEQ ID NO. 23): TCTCGTTGGGGTCTTTGCTC; Mus_GAPDH_F_Primer(SEQ ID NO.24):TGTGAACGGATTTGGCCGTA; Mus_GAPDH_R_Primer(SEQ ID NO.25):ACTGTGCCGTTGAATTTGCC; Human_GAPDH_F_Primer (SEQ ID NO.26): AGAAGGCTGGGGCTCATTTG; Human_GAPDH_R_Primer (SEQ ID NO. 27): AGGGGCCATCCACAGTCTTC.

[0075] qPCR: Genomic DNA was extracted from cells or tissues using a total DNA extraction kit from Qiagen. Absolute quantification of the viral genome was performed using Taqman qPCR on a Thermo Scientific QuantStudio3 Real-time PCR system, with parallel assays performed using standards of known DNA concentrations. The primers and probes used were: GFP_F_Primer (SEQ ID NO.28): GAGGCGCACCATCTTCTTCAA; GFP_R_Primer (SEQ ID NO.29): TCTTGAAGTCGATGCCCTT; GFP_Probe (FAM) (SEQ ID NO.30): ACAAGACCCGCGCCGAGGTG.

[0076] Example 1: Design and Screening of AAV Capsid Variant Proteins Using AAVrh74 serotype as the parental capsid, NNK primers 74-lib-F (SEQ ID NO.31): 5'-ctacccggtccctgctaccggcagcaacgcgtctcca-3'; 74-lib-R (SEQ ID NO.32): 5'-ggcccctacaataggagcggcMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNttgcaggttatcggccaccacg-3' were designed to insert random 10 peptides into positions 587 and 591 of the AAVrh74 capsid protein (SEQ ID NO:5), while simultaneously selecting the tissue-specific promoter CK8 (SEQ ID NO:10) to drive the expression of the capsid library. Figure 1 a). Subsequently, following the method of Viviana Gradinaru et al. (NatNeurosci, 2021), the constructed library plasmids were used for library virus packaging. Specifically, only 10 ng of plasmid library was added per 150 mm culture dish to prevent chimeric capsid formation, and PUC19, AAV2 / rh74-REP-AAP-Δcap (SEQ ID NO:11) and helper plasmid were combined in a 1:1:2 ratio (40 µg total DNA per 150 mm culture dish). PEI pro transfection was used, and the virus was collected after 60 h to limit secondary transduction of producing cells. Finally, purification was performed by iodixanol density gradient ultracentrifugation, and viral genome titer was determined using droplet digital PCR (ddPCR).

[0077] AAV libraries were used to infect mouse myoblasts (C2C12) and human skeletal muscle myoblasts (HSKMCs) with a specific MOI. RNA was extracted 24 h after infection and RT-PCR was performed to amplify the first round of enriched dominant sequences. High-throughput sequencing was then used to analyze the dominant capsid sequences. Subsequent rounds of screening collected mRNA from C2C12 or HSKMCs obtained in the previous round. Using the reverse-transcribed cDNA as a template, PCR amplification was performed to enrich the dominant capsid sequences, forming a new library for the next round of screening. A total of four rounds of screening were conducted. Figure 1 b). High-throughput sequencing analysis yielded two highly enriched dominant sequences, and the resulting AAV capsid variant proteins were named AAVM101 (SEQ ID NO:3) and AAVM102 (SEQ ID NO:4). Figure 2 a). Structural modeling revealed that the insertion of its 10-peptide forms the second tallest apex of the three protrusions on the variable region VIII, a change that may affect interactions with receptors, etc. Figure 2 bc).

[0078] Example 2: Production of recombinant adeno-associated virus (recombinant AAV) containing AAVM101 and AAVM102 capsid variant proteins Using homologous recombination, serotype capsid plasmids of AAV capsid variants AAVM101 and AAVM102 were constructed using the parental AAVrh74 capsid protein as a template. Virus packaging and production were performed according to the method of She et al. (Singal Transduct Target Ther, 2023). Specifically, HEK293 cells were expanded and seeded into a 10-layer cell factory. After 72 h, when cell confluence reached 89-90%, three-plasmid co-transfection was performed. Helper plasmid pAdΔF6, pAAV-REP-Cap trans plasmid (containing the capsid variant protein AAVM101 (SEQ ID NO.6) / AAVM102 gene (SEQ ID NO.7)), and cis expression plasmid were prepared in 50 ml DMEM at a ratio of 10:10:1 to form a DNA Mix. PEI pro was prepared in 48 ml DMEM to form a PEI mix. After vortexing, the PEI mix was added to the DNA mix at a constant speed and immediately vortexed for about 10 s. The mixture was then incubated at room temperature for 15 min. The transfection complex was added to 1000 ml DMEM and mixed. The culture medium in the cell factory was discarded, and the transfection complex was added along the cell factory wall. The cells were transferred to a 37°C, 5% CO2 incubator and incubated for 144 h. The cell culture supernatant was collected and concentrated by tangential flow filtration and purified by iodixanol density gradient ultracentrifugation. The purity of the final viral stock solution was assessed by SYPRO™ Ruby protein gel staining, and the viral genome titer was absolutely quantified by digital PCR (ddPCR). The obtained virus was used for subsequent in vitro cell and in vivo animal experiments.

[0079] SYPRO™ Ruby protein gel staining results showed that the serotypes were consistent with wild-type AAV9 and AAVrh74. The VP1, VP2, and VP3 bands of AAVM101 and AAVM102 were clear and free of impurities, indicating that the purified virus was of acceptable quality. Figure 3 a). ddPCR quantification showed no significant difference in viral yield among viral particles packaged from AAV capsid variants AAVM101 (SEQ ID NO.3), AAVM102 (SEQ ID NO.4), and AAVrh74 (SEQ ID NO.5), indicating that the inserted fragment did not affect packaging efficiency. Figure 3 b).

[0080] Example 3: Evaluation of transduction efficiency of AAVM101 and AAVM102 capsid variants in in vitro muscle cell lines 1. Preparation of self-complementary recombinant AAV plasmid vector and virus carrying EGFP expression The pAAVsc.CMV.EGFP.W3.bGH cis plasmid vector (SEQ ID NO:12) contains the CMV promoter (SEQ ID NO:13) and the EGFP gene (SEQ ID NO:14). Following the recombinant AAV virus production method of Example 2, recombinant self-complementary AAV viruses (scAAV) were produced using AAVrh74, AAV9, AAVM101 (the capsid variant shown in SEQ ID NO:3), and AAVM102 (the capsid variant shown in SEQ ID NO:4) capsid plasmids, along with the pAAVsc.CMV.EGFP.W3.bGH cis plasmid vector and the helper pAdΔF6 plasmid. The produced recombinant AAV viruses were named scAAVrh74.EGFP, scAAV9.EGFP, scAAVM101.EGFP, and scAAVM102.EGFP, respectively.

[0081] 2. Four types of viruses, scAAV9.EGFP, scAAVrh74.EGFP, scAAVM101.EGFP, and scAAVM102.EGFP, were mixed at a ratio of 5 × 10⁻⁶. 5 The infection multiples (MOIs) were used to infect C2C12 and HSKMC cells, respectively. After 24 hours, fluorescence imaging was performed, and DNA and RNA were collected for RT-qPCR assay.

[0082] Fluorescence image results ( Figure 4 a) showed that, compared with the AAV9 serotype and the parental serotype AAVrh74, the capsid variants AAVM101 and AAVM102 exhibited significantly improved transduction efficiency in C2C12 and HSKMCs. RT-qPCR results for mRNA expression assays ( Figure 4 (b) showed that AAVM101 and AAVM102 significantly improved transduction efficiency in muscle cells, increasing by 20.5-fold and 8.3-fold, respectively, in C2C12 cells, and by 17.7-fold and 13.6-fold, respectively, compared to AAVrh74. Further viral genome copy number analysis indicated that the improved transduction efficiency of the new capsid variants may be due to increased DNA levels of AAV capsid entering the cell. Figure 4 c).

[0083] Example 4: In vivo gene expression kinetics of AAVM101 and AAVM102 capsid variants after systemic injection In wild-type C57BL / 6J mice (4-6 weeks old), recombinant AAV viruses expressing luciferase (scAAV9.ffLuc, scAAVrh74.ffLuc, scAAVM101.ffLuc, and scAAVM102.ffLuc, respectively) were injected via tail vein at a dose of 3 × 10⁻⁶.13 vg / kg. After a period of time following administration, the biodistribution and tissue tropism of the virus in vivo were dynamically monitored using an IVIS instrument. The preparation method of the cis-plasmid vector expressing Luciferase driven by the CMV promoter and the corresponding recombinant virus was the same as in Example 3, except that the EGFP gene sequence was replaced with the Luciferase gene sequence (SEQ ID NO:15).

[0084] In vivo imaging results showed that AAVM101 exhibited significant systemic tissue transduction capacity over time, especially with more efficient transgene expression in limb muscle tissue; in contrast, AAVM102 showed weaker systemic transduction. Figure 5 ).

[0085] Example 5: Evaluation of in vivo tissue distribution and transduction efficiency of AAVM101 and AAVM102 capsid variants after systemic injection In wild-type C57BL / 6J mice (4-6 weeks old), the following recombinant AAV viruses, the same as those used in Example 3, were injected via the tail vein: scAAV9.EGFP, scAAVrh74.EGFP, scAAVM101.EGFP, and scAAVM102.EGFP, at a dose of 3 × 10⁻⁶. 13 vg / kg.

[0086] 1. Tissue fluorescence imaging analysis IVIS fluorescence imaging results showed that, compared with AAV9 and the parental AAVrh74, AAVM101-mediated EGFP transgene expression was significantly enhanced in various skeletal muscles (including tibialis anterior, triceps brachii, quadriceps femoris, gastrocnemius, and soleus) and the diaphragm; at the same time, its liver transduction capacity was significantly reduced compared with AAVrh74. Figure 6 ).

[0087] 2. Measurement of transgenic expression levels in vivo Quantitative analysis of viral vector-mediated transgenic mRNA expression levels by RT-qPCR showed that AAVM101 exhibited significantly higher transduction efficiency than AAV9 and AAVrh74 in all skeletal muscles tested. Specifically, in the tibialis anterior, triceps brachii, and diaphragm, AAVM101-mediated transgenic expression levels reached 4.9, 3.8, and 8.7 times that of the parental AAVrh74, respectively. Meanwhile, AAVM101 expression in the liver was 0.8 times that of the parental AAVrh74. In the spleen, lungs, kidneys, and non-target organs of the brain, its transduction efficiency was similar or decreased. Figure 7 ).

[0088] 3. In vivo biodistribution studies Tissue distribution analysis of viral genome copy number by RT-qPCR showed that AAVM101 delivered the EGFP gene to the muscle of C57 mice more efficiently than AAV9 and AAVrh74, and significantly reduced the vector genome in the liver. Figure 8 The results showed that the capsid variant AAVM101 of the present invention exhibited more specific muscle tissue targeting. Figure 8 ).

[0089] The above results indicate that after systemic administration, the AAVM101 variant allows viral particles to reach more muscle tissue and reduces accumulation in the liver; it can more efficiently target and transduce muscle tissue (including skeletal muscle, myocardium, etc.), and its gene expression level in muscle tissue is significantly higher than that of existing AAV serotypes (AAVrh74 and AAV9).

[0090] Example 6: Evaluation of the therapeutic effects of AAVM101 and AAVM102 capsid variants in DMD model mice To investigate the feasibility of in vivo delivery of therapeutic transgenes using AAV variants, a cis-plasmid vector pAAV.CK8.μDys.rBG (SEQ ID NO:16) was constructed to drive the expression of "mini" dystrophin (μDys) using the tissue-specific promoter CK8. This vector contains the μDys gene expression cassette (SEQ ID NO:9), with the CK8 promoter shown in SEQ ID NO:10 and the μDys gene sequence shown in SEQ ID NO:8. Following the recombinant AAV production method of Example 2, AAV9.μDys, AAVrh74.μDys, AAVM101.μDys, and AAVM102.μDys recombinant AAV viruses were packaged and produced using AAV9, AAVrh74, AAVM101, and AAVM102 capsid plasmids, the pAAV.CK8.μDys.rBG cis-plasmid vector, and the helper pAdΔF6 plasmid, respectively. Male DMD (Mdx) mice aged 4-6 weeks were selected and injected via tail vein with AAV9.μDys, AAVrh74.μDys, AAVM101.μDys, and AAVM102.μDys, respectively, at a dose of 5 × 10⁻⁶. 13 vg / kg. Wild-type mice (WT) and Mdx-untreated mice were also included as control groups in the experiment.

[0091] 1. Serum biochemical index analysis Blood samples were collected at the treatment endpoint (12 weeks post-treatment) to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) levels. Results showed that the ALT / AST levels in the AAVM101 group were not significantly different from those in the WT group, and were significantly lower than those in the AAV9 and AAVrh74 groups, indicating that the AAVM101 group induced less hepatotoxicity. Figure 9 bc). CK, as a marker of muscle injury, showed that CK levels in the AAVM101 group decreased to near those in the WT group and were significantly lower than those in the AAV9 and AAVrh74 groups, indicating that AAVM101 treatment effectively protected the integrity of the fascia and significantly reduced persistent muscle damage. Figure 9 a).

[0092] 2. Expression assessment of the therapeutic protein μDys Muscle tissue was collected from mice 12 weeks after treatment, frozen-embedded, and sectioned. Double immunofluorescence staining of μDys and neuronal nitric oxide synthase (nNOS) showed that positive signals of μDys were observed in all examined muscle fibers in the AAVM101 treatment group, with better uniformity and extent of distribution than the control group. Furthermore, the μDys expressed in the AAVM101 group successfully recruited and bound nNOS to the muscle membrane, reconstructing the complete dystrophin-glycoprotein complex. Figure 10 Muscle tissue was collected from mice 12 weeks after treatment, and proteins were extracted and detected using an anti-dystrophin antibody. Results showed that μDys protein expression was highest in the AAVM101 treatment group, significantly higher than in the control AAVrh74 and AAV9 groups, in the tibialis anterior, triceps brachii, quadriceps femoris, gastrocnemius, and diaphragm muscles; in the myocardium, it was slightly higher than in the AAVrh74 group. Figure 11 ab).

[0093] 3. Assessment of improvement in histopathology Liver and muscle tissues were collected 12 weeks after treatment for histopathological assessment using H&E staining and Masson's trichrome staining. H&E staining showed that the morphology of muscle tissue in all experimental groups recovered to varying degrees compared to the untreated group after treatment. In the AAVM101 group, the muscle fiber morphology was more intact, including more uniform fiber size distribution, fewer necrotic fibers, a decreased proportion of central nuclei, and reduced inflammatory infiltration. In the liver tissue, except for the AAVM101 group, significant inflammatory cell infiltration was observed in all other groups, while no inflammatory cell infiltration was observed in the AAVM101 group, consistent with WT (whole-whole). Figure 12Masson staining showed that the AAVM101 group had the smallest area of ​​collagen fiber deposition (blue) in the tibialis anterior, triceps brachii, quadriceps femoris, gastrocnemius, and diaphragm muscles, significantly lower than AAV9 and AAVrh74. This indicates that the treatment with AAVM101 effectively inhibited the pathological fibrosis process and restored healthy muscle tissue structure. Figure 13 ab).

[0094] 4. Muscle function recovery assessment After 12 weeks of treatment, grip strength was assessed using a grip strength meter. The procedure was briefly as follows: mice were placed horizontally and dragged backward, allowing their forelimbs or hindlimbs to grasp a mesh strip. The grip strength meter automatically recorded the maximum grip strength of the mice during this process. Each mouse was tested three times, and the maximum value was recorded and included in further calculations. Results showed that grip strength in all four limbs was significantly improved in all treated groups compared to untreated mice, with the AAVM101 group exhibiting the most significant muscle strength recovery. Figure 14 ).

[0095] Example 7: Evaluation of transduction efficiency of AAVM101 and AAVM102 capsid variants in rhesus monkeys To investigate the transduction efficiency across species in vivo using AAV variants, 3-5 year old rhesus monkeys were selected to construct three cis plasmid vectors expressing human N-acetylgalactosamine 6-sulfatase driven by the broad-spectrum promoter CBh: pAAV.CBh.hGALNS.BC1.bGH (SEQ ID NO:17), pAAV.CBh.hGALNS.BC2.bGH (SEQ ID NO:18), and pAAV.CBh.hGALNS.BC3.bGH (SEQ ID NO:19). To quantify the transduction efficiency of different capsids, a unique barcode (BC) sequence was introduced for each capsid before the polyA vector. Following the recombinant adeno-associated virus production method of Example 2, recombinant AAV viruses AAVrh74.hGALNS.BC1, AAVM101.hGALNS.BC2, and AAVM102.hGALNS.BC3 were produced by packaging AAVrh74, AAVM101, and AAVM102 capsid plasmids with the aforementioned three cis plasmid vectors (SEQ ID NO: 17-19) and helper plasmid pAdΔF6. The virus libraries containing different capsids were then packaged at 2.5 × 10⁻⁶ units per virus. 13 A dose of vg / kg was mixed to form a virus pool. Forty-three days post-injection, NGS sequencing was used to analyze the mRNA expression levels corresponding to each barcode sequence to assess the delivery efficiency of different capsids. Figure 15a). The results showed that AAVM101 exhibited high transduction efficiency in the tibialis anterior, gastrocnemius, and diaphragm muscles, but its transduction efficiency in the liver was lower than that of AAVrh74 (a). Figure 15 b).

[0096] Example 8: Clinical study of AAVM101 capsid variant in patients with Duchenne muscular dystrophy (DMD) The applicant has initiated a single-center, open-label, single-arm Phase I clinical trial to evaluate the safety and preliminary efficacy of AAVM101.μDys (a virus formed by packaging the recombinant AAV vector pAAV.CK8.μDys.rBG shown in SEQ ID NO:16 with the AAVM101 capsid and helper plasmid, i.e., the AAVM101.μDys recombinant AAV virus in Example 6) (NCT06114056). Patients must meet the following criteria for enrollment: age between 5 and 10 years, genetically diagnosed with DMD, and below-normal muscle strength and motor function. Patients who have previously used any investigational drugs (such as exon skipping therapy) or have an AAV neutralizing antibody titer exceeding 1:400 are ineligible for participation.

[0097] The study ultimately included three pediatric patients aged 5-6 years diagnosed with DMD. Patient 1 carried a deletion in exons 46-53; Patient 2 had a nonsense mutation in exon 23; and Patient 3 had a nonsense mutation in exon 21. Prior to treatment, all patients had significantly elevated serum creatine kinase (CK) levels, with a mean of 20408 U / L. Functional assessments showed a mean North Star Outpatient Assessment Scale (NSAA) score of 26.7 and a mean Motor Function Measurement Scale (MFM) score of 89.0. Mean results for performance-based timing tests were: a mean time of 3.7 seconds for the 4-step stair climbing test (4SC), a mean distance of 462.3 meters for the 6-minute walk test (6MWT), and a mean time of 6.1 seconds for the 10-meter walk / run test (10MR). Each patient received a single dose of 1.0 × 10⁻⁶ U / L. 14 vg / kg. The primary endpoint was the safety and tolerability of the drug; secondary endpoints aimed to assess changes in motor function and the expression of therapeutic proteins. Motor function assessment indicators included NSAA, MFM, 4SC, 6MWT, and 10MR.

[0098] 1. Treatment safety assessment Adverse events (AEs) occurring during the one-year follow-up period were assessed and graded according to the NCI-CTCAE v.5.0 criteria. Forty-five adverse events were observed during the one-year follow-up period, of which 30 (66.7%) were considered treatment-related. The most common treatment-related adverse events were nausea (n=3), decreased appetite (n=3), and elevated liver enzymes (n=3). No serious adverse events occurred.

[0099] 2. Patient motor function assessment and CK level analysis During the one-year follow-up period, participants generally showed improvement across multiple motor function assessments (Table 1). Patient 1's NSAA score improved by 24.0% (from 25 to 31), Patient 2's score improved by 17.2% (from 29 to 34), and Patient 3's score improved by 11.5% (from 26 to 29). Patient 1's MFM score improved from 88 to 92, and Patient 3's MFM score also improved (from 85 to 87), while Patient 2's score remained at a high level throughout the follow-up period (from 94 to 94). In timed motor function assessments, Patient 1's 4SC time decreased by 58.3% (from 6.0 seconds to 2.5 seconds), Patient 2's by 33.3% (from 2.4 seconds to 1.6 seconds), and Patient 3's by 18.5% (from 2.7 seconds to 2.2 seconds). Similarly, patient 1's 10MR time decreased by 20.0%, patient 3's by 30.7%, while patient 2's time increased slightly (4.7%). Performance in the 6MWT test showed a different pattern: patient 1 achieved the greatest improvement (+19.3%, from 405 m to 483 m), patient 2 also improved (+8.9%, from 507 m to 552 m), while participant 3 showed a decrease relative to baseline (-6.3%, from 475 m to 445 m). Consistent with these functional changes, serum creatine kinase (CK) levels decreased in all three patients during follow-up. In patient 1, whose baseline CK level was the highest, the CK level decreased by 65.6% (from 22,183 U / L to 7,632 U / L); in patient 2, the CK level decreased by 59.0% (from 20,168 U / L to 8,277 U / L); and in patient 3, the CK level decreased by 71.2% (from 18,918 U / L to 5,448 U / L).

[0100] 3. Expression assessment of the therapeutic protein μDys Six months after treatment, post-treatment gastrocnemius muscle biopsy samples from all three patients were subjected to immunofluorescence and Western blot analysis to analyze the expression of "mini" dystrophin. Figure 16Immunofluorescence results showed that μDys expression was not detected in any samples before treatment, but after treatment, μDys was expressed to varying degrees in all three patients and co-localized with laminin 2α. Western blot analysis also confirmed μDys expression; semi-quantitative analysis showed that the μDys expression levels in these three patients, compared to normal tissues, were 102.4%, 45.7%, and 108.8%, respectively, with an average expression level of 85.6%.

[0101] In summary, in wild-type mice, the AAVM101 capsid variant of this invention exhibits higher muscle tissue transduction efficiency and reduced liver targeting. In DMD model mice, the overall efficacy of the AAVM101 capsid therapeutic vector of this invention is systematically and significantly superior to the commonly used AAVrh74 and AAV9 vectors. In the transduction efficiency evaluation in non-human primates, the AAVM101 capsid of this invention, compared with the parental AAVrh74 capsid, can achieve more efficient muscle transduction and reduce liver off-target effects, showing good clinical translational potential. In the clinical trial of DMD patients, AAVM101-μDys has good safety, with no serious adverse events, and all three patients showed improved motor function and a significant reduction in creatine kinase levels, supporting its further clinical development.

[0102] Table 1: Results of patient motor function assessment and creatine kinase level analysis The following are the nucleotide and amino acid sequences involved in the embodiments of the present invention. SEQ ID NO.1: AAVM101 insert peptide amino acid sequence 10 aa RASRGDLPGA SEQ ID NO.2: AAVM102 insert peptide amino acid sequence 10 aa RASNNILPGA SEQ ID NO.3: AAVM101 capsid variant protein amino acid sequence 745 aa MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQRASRGDLPGAAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL SEQ ID NO.4: AAVM102 capsid variant protein amino acid sequence 745 aa MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQRASNNILPGAAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL SEQ ID NO.5: AAVrh74 capsid protein amino acid sequence, 738 aa MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL SEQ ID NO.6: Nucleotide sequence of AAVM101 capsid variant protein, 2235 bp atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtg ggacctgaaacctggagccccgaaacccaaagccaaccagcaaaagcaggacaacggccggggtctggtgcttcct ggctacaagtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcg agcacgacaaggcctacgaccagcagctccaagcgggtgacaatccgtacctgcggtataatcacgccgacgccga gtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgcgcagtcttccaggccaaaaagcgg gttctcgaacctctgggcctggttgaatcgccggttaagacggctcctggaaagaagagaccggtagagccatcac cccagcgctctccagactcctctacgggcatcggcaagaaaggccagcagcccgcaaaaaagagactcaattttgg gcagactggcgactcagagtcagtccccgaccctcaaccaatcggagaaccaccagcaggcccctctggtctggga tctggtacaatggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgacggagtgggtagttcct caggaaattggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgcacctgggccctgcc cacctacaacaaccacctctacaagcaaatctccaacgggacctcgggaggaagcaccaacgacaacacctacttc ggctacagcacccctgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcagcgac tcatcaacaacaactggggattccggcccaagaggctcaacttcaagctcttcaacatccaagtcaaggaggtcac gcagaatgaaggcaccaagaccatcgccaataaccttaccagcacgattcaggtctttacggactcggaataccag ctcccgtacgtgctcggctcggcgcaccagggctgcctgcctccgttcccggcggacgtcttcatgattcctcagt acgggtacctgactctgaacaatggcagtcaggctgtgggccggtcgtccttctactgcctggagtactttccttc tcaaatgctgagaacgggcaacaactttgaattcagctacaacttcgaggacgtgcccttccacagcagctacgcg cacagccagagcctggaccggctgatgaaccctctcatcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtactgcaggaactcagcagttgctattttctcaggccgggcctaacaacatgtcggctcaggccaagaa ctggctacccggtccctgctaccggcagcaacgcgtctccacgacactgtcgcagaacaacaacagcaactttgcc tggacgggtgccaccaagtatcatctgaatggcagagactctctggtgaatcctggcgttgccatggctacccaca aggacgacgaagagcgattttttccatccagcggagtcttaatgtttgggaaacagggagctggaaaagacaacgt ggactatagcagcgtgatgctaaccagcgaggaagaaataaagaccaccaacccagtggccacagaacagtacggc gtggtggccgataacctgcaacgtgcttcgagaggagacctgcctggggctgccgctcctattgtaggggccgtca atagtcaaggagccttacctggcatggtgtggcagaaccgggacgtgtacctgcagggtcccatctgggccaagat tcctcatacggacggcaactttcatccctcgccgctgatgggaggctttggactgaagcatccgcctcctcagatc ctgattaaaaacacacctgttcccgcggatcctccgaccaccttcaatcaggccaagctggcttctttcatcacgc agtacagtaccggccaggtcagcgtggagatcgagtgggagctgcagaaggagaacagcaaacgctggaacccaga gattcagtacacttccaactactacaaatctacaaatgtggactttgctgtcaatactgagggtacttattccgag cctcgccccattggcacccgttacctcacccgtaatctg SEQ ID NO.7: AAVM102 capsid variant protein nucleotide sequence 2235 bp atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtg ggacctgaaacctggagccccgaaacccaaagccaaccagcaaaagcaggacaacggccggggtctggtgcttcct ggctacaagtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcg agcacgacaaggcctacgaccagcagctccaagcgggtgacaatccgtacctgcggtataatcacgccgacgccga gtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgcgcagtcttccaggccaaaaagcgg gttctcgaacctctgggcctggttgaatcgccggttaagacggctcctggaaagaagagaccggtagagccatcac cccagcgctctccagactcctctacgggcatcggcaagaaaggccagcagcccgcaaaaaagagactcaattttgg gcagactggcgactcagagtcagtccccgaccctcaaccaatcggagaaccaccagcaggcccctctggtctggga tctggtacaatggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgacggagtgggtagttcct caggaaattggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgcacctgggccctgcc cacctacaacaaccacctctacaagcaaatctccaacgggacctcgggaggaagcaccaacgacaacacctacttc ggctacagcaccccctgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcagcgac tcatcaacaacaactggggattccggcccaagaggctcaacttcaagctcttcaacatccaagtcaaggaggtcac gcagaatgaaggcaccaagaccatcgccaataaccttaccagcacgattcaggtctttacggactcggaataccag ctcccgtacgtgctcggctcggcgcaccagggctgcctgcctccgttcccggcggacgtcttcatgattcctcagt acgggtacctgactctgaacaatggcagtcaggctgtgggccggtcgtccttctactgcctggagtactttccttc tcaaatgctgagaacgggcaacaactttgaattcagctacaacttcgaggacgtgcccttccacagcagctacgcg cacagccagagcctggaccggctgatgaaccctctcatcgaccagtacttgtactacctgtcccggactcaaagca cgggcggtactgcaggaactcagcagttgctattttctcaggccgggcctaacaacatgtcggctcaggccaagaa ctggctacccggtccctgctaccggcagcaacgcgtctccacgacactgtcgcagaacaacaacagcaactttgcc tggacgggtgccaccaagtatcatctgaatggcagagactctctggtgaatcctggcgttgccatggctacccaca aggacgacgaagagcgattttttccatccagcggagtcttaatgtttgggaaacagggagctggaaaagacaacgt ggactatagcagcgtgatgctaaccagcgaggaagaaataaagaccaccaacccagtggccacagaacagtacggc gtggtggccgataacctgcaacgtgcttcgaataatattctgcctggggctgccgctcctattgtaggggccgtca atagtcaaggagccttacctggcatggtgtggcagaaccgggacgtgtacctgcagggtcccatctgggccaagat tcctcatacggacggcaactttcatccctcgccgctgatgggaggctttggactgaagcatccgcctcctcagatc ctgattaaaaacacacctgttcccgcggatcctccgaccaccttcaatcaggccaagctggcttctttcatcacgc agtacagtaccggccaggtcagcgtggagatcgagtgggagctgcagaaggagaacagcaaacgctggaacccaga gattcagtacacttccaactactacaaatctacaaatgtggactttgctgtcaatactgagggtacttattccgag cctcgccccattggcacccgttacctcacccgtaatctg SEQ ID NO.8: μDys gene coding sequence 3561 bp atgctttggtgggaagaagtcgaggactgctacgagcgcgaggacgtgcagaagaaaaccttcaccaa atgggtcaacgcccagttcagcaagttcggcaagcagcacatcgagaacctgttcagcgacctccaggatggcaga aggctgctggatctgctggaaggactgaccggacagaagctgcccaaagagaagggcagcacaagagtgcacgccc tgaacaacgtgaacaaggccctgagagtgctccagaacaacaacgtggacctggtcaacatcggcagcaccgacat cgtggacggcaaccaaaactgaccctgggcctgatctggaacatcatcctgcactggcaaggaagaacgtgatg aagaaacatcatggcaggcctgcaacagacaaactctgaaaagattctgcttagctgggtcaggcagtccaccagga actacccccaagtgaatgtgatcaacttcaccaccagctggtctgatggcctggccctcaatgcccttatccacag ccacaggccagacctgtttgactggaacagtgtggtgtgtcagcagtcagccactcagagggctggagcatgctttt aacattgccagataccagcttggcatagagagagctgctggaccctgaggatgtggacaccacatacccgataaga agagcatcctgatgtatatcacaagcctgtttcaggtgctgcctcagcaggtgagcattgaggctatccaagaagt ggagatgctgcctagacctccaaaggtgaccaaggaggagcactttcagctgcaccaccagatgcactacagccag cagattacagtgtctctgggcccaaggctatgagaggaccagctctcctaaacctagattcaaatcctatgcctaca cccaggctgcctatgtgacaacctctgaccccaccagaagccccttcccttctcagcacctggaggctccagagga caagagctttggcagctcattgatggaaagtgaagtgaacctggatagataccagacagccctggagaagaagtactg agctggctggctgagtgcagaggacaccttgcaagctcagggggagatcagcaatgatgtggaggtggtcaaggacc agttccacacccatgagggctatatgatggacctgacagcccaccagggtaggtgggcaacattctgcagctggg cagcaaggctgataggcacaggcaaactgtctgaagatgaggagacagaggtgcaggagcagatgaacctgctgaac tcaagatgggaatgtctgagagtggccagcatggagaagcagtctaacctgcacagagaaatcagttatgtgccta gcacctacctgacagaaatcacccatgtgtcccaggctctcctggaggtggaacagctgcttaatgcccctgatct gtgtgccaaagattttgaggacctgtttaagcaggaggagagcctgaaaaacatcaaggactccctgcaacagagc tctggcagaattgacatcatccacagcaagaagacagctgccctgcagtctgccacaccagtggagagggtgaagc tgcaagaagctctgtcacagctggacttccagtgggaaaaggtgaacaaaatgtacaaggacagacagggcaggtt tgataggagtgtggagaagtggaggagattccactatgatatcaaaatcttcaatcagtggctgacagaagcagag cagttcctgagaaagacccaaatccctgaaaactgggagcatgccaagtacaagtggtacctgaaggaactgcagg atggcattggccagaggcagacagtggtgagaacattgaatgccacaggagaggagatcatacaacaaagcagcaa gacagatgcctctatcctgcaagaaaagctgggctccctgaatctgagatggcaggaggtatgcaagcagctgagt gatagaaagaaaagactggaagaaaccttggagagactgcaggagctgcaggaggctacagatgagctggacctga aactgaggcaggcagaagtgatcaagggctcctggcagcctgtgggagacctgctgattgactcactgcaggacca cctggaaaaggtgaaagccctgaggggagagattgcccctctgaaagagaatgtgagccatgtgaatgacctggct agacagctgaccaccctgggaatccagttgtcaccctacaacctgagcacactggaggatctgaacaccaggtgga agctgcttcaagtggctgtggaggacagggtgaggcagctgcatgaggcacacagggattttggcccagccagcca gcacttcctgagcacaagtgtgcaaggaccttgggagagagccatctcaccaaacaaggttccttactacataaac catgagacccagaccacctgctgggaccaccccaagatgacagagctgtaccagtccctggcagacctcaacaatg tcagattctctgcctacagaacagccatgaagctgagaagactgcaaaaagccctgtgtctggacctgctgagcct gtctgctgcctgtgatgctctggatcagcacaacctgaagcagaatgatcagccaatggatattctgcagatcatc aattgcctgactacaatctatgacagactggagcaggaacacaacaacctggtgaatgtccccctgtgtgtggaca tgtgcctgaactggctcctgaacgtgtacgacacaggaagaactggaagaattagagtgctttcttttaaaacagg aatcatctctctgtgcaaggcacacctggaagacaagtacaggtacctgttcaagcaggtggccagctcaactggc ttttgtgaccagagaaggcttggcctccttctgcatgactctatccagatccccaggcagcttggagaggtggcca gttttggaggctctaatattgagcccagtgtgaggagctgcttccagtttgccaacaacaaaccagagatagaggc agccctgttcctggactggatgagactggaacctcagagcatggtctggctgcctgtgctgcacagggtggcagct gcagagacagcaaaacaccaggccaagtgcaacatctgcaaggagtgccccatcattggcttcagatacagaagcc tgaaacactttaactatgacatctgccagagctgtttcttcagtggcagagtggccaaggggcacaagatgcacta tcctatggtggagtactgcacccctacaacctctggagaagatgttagggactttgccaaggttctgaagaacaag ttcagaaccaagagatactttgccaagcacccaaggatgggctacttgccagtgcagacagtgctggaaggagaca acatggagactggagaggaagacctgctctctcctcctcaggacacctctacaggcctggaggaagtgatggaaca gctgaacaatagcttccccagcagcaggggcagaaacacccctggcaagcctatgagggaagataccatgtga SEQ ID NO.9: μDys gene expression cassette 4584 bp ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgccc ggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgatt aacccgccatgctacttatctacttaagagcatgctgcccatgtaaggaggcaaggcctggggacacccgagatgc ctggttataattaacccagacatgtggctgcccccccccccccaacacctgctgcctctaaaaataaccctgcatg ccatgttcccggcgaagggccagctgtcccccgccagctagactcagcacttagtttaggaaccagtgagcaagtc agcccttggggcagcccatacaaggccatggggctgggcaagctgcacgcctgggtccggggtgggcacggtgccc gggcaacgagctgaaagctcatctgctctcaggggcccctccctggggacagcccctcctggctagtcacaccctg taggctcctctatataacccaggggcacaggggctgccctcattctaccaccacctccacagcacagacagacact caggagccagccagcagttaactgtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgc aaatcaaagaactgctcctcagtggatgttgcctttacttctaggccaccatgctttggtgggaagaagtcgagga ctgctacgagcgcgaggacgtgcagaagaaaaccttcaccaaatgggtcaacgcccagttcagcaagttcggcaag cagcacatcgagaacctgttcagcgacctccaggatggcagaaggctgctggatctgctggaaggactgaccggac agaagctgcccaaagagaagggcagcacaagagtgcacgccctgaacaacgtgaacaaggccctgagagtgctcca gaacaacaacgtggacctggtcaacatcggcagcaccgacatcgtggacggcaaccacaaactgaccctgggcctg atctggaacatcatcctgcactggcaagtgaagaacgtgatgaagaacatcatggcaggcctgcaacagacaaact ctgaaaagattctgcttagctgggtcaggcagtccaccaggaactacccccaagtgaatgtgatcaacttcaccac cagctggtctgatggcctggccctcaatgcccttatccacagccacaggccagacctgtttgactggaacagtgtg gtgtgtcagcagtcagccactcagaggctggagcatgcttttaacattgccagataccagcttggcatagagaagc tgctggaccctgaggatgtggacaccacataccctgataagaagagcatcctgatgtatatcacaagcctgtttca ggtgctgcctcagcaggtgagcattgaggctatccaagaagtggagatgctgcctagacctccaaaggtgaccaag gaggagcactttcagctgcaccaccagatgcactacagccagcagattacagtgtctctggcccaaggctatgaga ggaccagctctcctaaacctagattcaaatcctatgcctacacccaggctgcctatgtgacaacctctgaccccac cagaagccccttcccttctcagcacctggaggctccagaggacaagagctttggcagctcattgatggaaagtgaa gtgaacctggatagataccagacagccctggaagaagtactgagctggctgctgagtgcagaggacaccttgcaag ctcagggggagatcagcaatgatgtggaggtggtcaaggaccagttccacacccatgagggctatatgatggacct gacagcccaccagggtagagtgggcaacattctgcagctgggcagcaagctgataggcacaggcaaactgtctgaa gatgaggagacagaggtgcaggagcagatgaacctgctgaactcaagatgggaatgtctgagagtggccagcatgg agaagcagtctaacctgcacagagaaatcagttatgtgcctagcacctacctgacagaaatcacccatgtgtccca ggctctcctggaggtggaacagctgcttaatgcccctgatctgtgtgccaaagattttgaggacctgtttaagcag gaggagagcctgaaaaacatcaaggactccctgcaacagagctctggcagaattgacatcatccacagcaagaaga cagctgccctgcagtctgccacaccagtggagagggtgaagctgcaagaagctctgtcacagctggacttccagtg ggaaaaggtgaacaaaatgtacaaggacagacagggcaggtttgataggagtgtggagaagtggaggagattccac tatgatatcaaaatcttcaatcagtggctgacagaagcagagcagttcctgagaaagacccaaatccctgaaaact gggagcatgccaagtacaagtggtacctgaaggaactgcaggatggcattggccagaggcagacagtggtgagaac attgaatgccacaggagaggagatcatacaacaaagcagcaagacagatgcctctatcctgcaagaaaagctgggc tccctgaatctgagatggcaggaggtatgcaagcagctgagtgatagaaagaaaagactggaagaaaccttggaga gactgcaggagctgcaggaggctacagatgagctggacctgaaactgaggcaggcagaagtgatcaagggctcctg gcagcctgtgggagacctgctgattgactcactgcaggaccacctggaaaaggtgaaagccctgaggggagagatt gcccctctgaaagagaatgtgagccatgtgaatgacctggctagacagctgaccaccctgggaatccagttgtcac cctacaacctgagcacactggaggatctgaacaccaggtggaagctgcttcaagtggctgtggaggacagggtgag gcagctgcatgaggcacacagggattttggcccagccagccagcacttcctgagcacaagtgtgcaaggaccttgg gagagagccatctcaccaaacaaggttccttactacataaaccatgagacccagaccacctgctgggaccacccca agatgacagagctgtaccagtccctggcagacctcaacaatgtcagattctctgcctacagaacagccatgaagct gagaagactgcaaaaagccctgtgtctggacctgctgagcctgtctgctgcctgtgatgctctggatcagcacaac ctgaagcagaatgatcagccaatggatattctgcagatcatcaattgcctgactacaatctatgacagactggagc aggaacacaacaacctggtgaatgtccccctgtgtgtggacatgtgcctgaactggctcctgaacgtgtacgacac aggaagaactggaagaattagagtgctttcttttaaaacaggaatcatctctctgtgcaaggcacacctggaagac aagtacaggtacctgttcaagcaggtggccagctcaactggcttttgtgaccagagaaggcttggcctccttctgcatgactctatccagatccccaggcagcttggagaggtggccagttttggaggctctaatattgagcccagtgtgag gagctgcttccagtttgccaacaacaaaccagagatagaggcagccctgttcctggactggatgagactggaacct cagagcatggtctggctgcctgtgctgcacagggtggcagctgcagagacagcaaaacaccaggccaagtgcaaca tctgcaaggagtgccccatcattggcttcagatacagaagcctgaaacactttaactatgacatctgccagagctg tttcttcagtggcagagtggccaaggggcacaagatgcactatcctatggtggagtactgcacccctacaacctct ggagaagatgttagggactttgccaaggttctgaagaacaagttcagaaccaagagatactttgccaagcacccaa ggatgggctacttgccagtgcagacagtgctggaaggagacaacatggagactggagaggaagacctgctctctcc tcctcaggacacctctacaggcctggaggaagtgatggaacagctgaacaatagcttccccagcagcaggggcaga aacacccctggcaagcctatgagggaagataccatgtgatagatctttttccctctgccaaaaattatggggacat catgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaatttt ttgtgtctctcactcgagtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagtt ggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcc cgggcggcctcagtgagcgagcgagcgcgcag SEQ ID NO.10: CK8 promoter sequence 443bp agcatgctgcccatgtaaggaggcaaggcctggggacacccgagatgcctggttataattaacccagacatgtggctgcccccccccccccaacacctgctgcctctaaaaataaccctgcatgccatgttcccggcgaagggccagctgtcccccgccagctagactcagcacttagtttaggaaccagtgagcaagtcagcccttggggcagcccatacaaggccatggggctgggcaagctgcacgcctgggtccggggtgggcacggtgcccgggcaacgagctgaaagctcatctgctctcaggggcccctccctggggacagcccctcctggctagtcacaccctgtaggctcctctatataacccaggggcacaggggctgccctcattctaccaccacctccacagcacagacagacactcaggagccagccagc SEQ ID NO.11: AAV2 / rh74-REP-AAP-Δcap sequence 6887bp SEQ ID NO.12: pAAVsc.CMV.EGFP.W3.bGH vector sequence 4919bp SEQ ID NO.13: CMV promoter sequence 508 bp cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct SEQ ID NO.14: EGFP gene sequence 720 bp atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa SEQ ID NO.15: Luciferase gene sequence, 1659 bp SEQ ID NO.16: pAAV.CK8.μDys.rBG vector sequence 7453bp SEQ ID NO.17: pAAV.CBh.hGALNS.BC1.bGH vector sequence 5680 bp SEQ ID NO.18: pAAV.CBh.hGALNS.BC2.bGH vector sequence 5680 bp SEQ ID NO.19: pAAV.CBh.hGALNS.BC3.bGH vector sequence 5680 bp

Claims

1. A muscle-targeting AAV capsid variant protein, characterized by: The variant protein is at least one polypeptide that has been substituted, inserted, and / or deleted in the variable region VIII of the parental AAV capsid protein VP1.

2. The muscle-targeting AAV capsid variant protein according to claim 1, characterized in that: The amino acid sequence of the polypeptide is as shown in SEQ ID NO.1 and SEQ ID NO.2, or has more than 80% homology with SEQ ID NO.1 and SEQ ID NO.2 and has the same or similar biological activities; Preferably, the polypeptide has the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, or is a polypeptide obtained by substituting and / or deleting and / or inserting 1 to 3 amino acids in the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. The muscle-targeting AAV capsid variant protein according to claim 1, characterized in that: The parental AAV capsid protein is selected from at least one of AAVrh74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

4. The muscle-targeting AAV capsid variant protein according to claim 3, characterized in that: The parental AAV capsid protein is AAVrh74 capsid protein, and its amino acid sequence is shown in SEQ ID NO.

5.

5. The muscle-targeting AAV capsid variant protein according to claim 1, characterized in that: The polypeptide substitution, insertion, and / or deletion sites are any one or two or more amino acid positions between positions 586 and 599 of the parental AAV capsid protein VP1 amino acid sequence.

6. The muscle-targeting AAV capsid variant protein according to claim 1, characterized in that: The amino acid sequence of the AAV capsid variant protein is shown in SEQ ID NO.3 and SEQ ID NO.4, or has more than 90% homology with SEQ ID NO.3 and SEQ ID NO.4 and has the same or similar biological activities.

7. An isolated polynucleotide, characterized in that: Encodes the muscle-targeting AAV capsid variant protein as described in any one of claims 1 to 6.

8. The polynucleotide according to claim 7, characterized in that: The sequence of the polynucleotide is shown in SEQ ID NO.6 or SEQ ID NO.

7.

9. Muscle-targeted recombinant AAV particles, characterized in that: The invention comprises the AAV capsid variant protein according to any one of claims 1 to 6, and a target gene or target gene expression cassette packaged within the AAV capsid variant protein; the target gene is capable of treating or preventing diseases affecting muscle cells / tissues.

10. The recombinant AAV particles according to claim 9, characterized in that: The target gene is selected from at least one of the following groups: therapeutic genes for muscle cell / tissue diseases, genes encoding therapeutic proteins or peptides, and genes encoding therapeutic RNA; preferably, the gene encoding therapeutic proteins or peptides is selected from genes encoding therapeutic antibodies or antibody fragments and genes encoding genome editing enzymes; the gene encoding therapeutic RNA is a gene for interfering RNA, gRNA for genome editing, exon-jumping antisense RNA, or repressive tRNA.

11. The recombinant AAV particles according to claim 10, characterized in that: The target gene is a gene encoding μDys or GALNS; preferably, the gene sequence encoding μDys is shown in SEQ ID NO.

8.

12. The recombinant AAV particles according to claim 9, characterized in that: The target gene expression cassette contains a gene encoding μDys or GALNS, a promoter, and / or a polyA signal; preferably, the μDys target gene expression cassette sequence is shown in SEQ ID NO.

9.

13. A pharmaceutical composition, characterized in that: It contains the recombinant AAV particles as described in any one of claims 9 to 12 and a pharmaceutically acceptable auxiliary carrier.

14. The use of the AAV capsid variant protein according to any one of claims 1 to 6, the recombinant AAV particles according to any one of claims 9 to 12, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating and / or preventing diseases affecting muscle cells / tissues.

15. The application according to claim 14, characterized in that: The diseases affecting muscle cells / tissues include at least one of neuromuscular genetic diseases, cardiomyopathy, rhabdomyosarcoma, polymyositis, and dermatomyositis; preferably, the diseases affecting muscle cells / tissues include at least one of Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, spinal muscular atrophy, myotonic dystrophy type 1 and 2, central nucleus myopathy, Pompe disease, glycogen storage disease III, amyotrophic lateral sclerosis, or congenital myasthenia gravis; more preferably, the therapeutic genes in the neuromuscular genetic diseases targeted by the drug include at least one of DMD, BMD, DYSF, FKPP, SMN1, ASAH1, DMPK, CNBP, DNM2, BIN1, GAA, AGL, SOD, ALS2, CHAT, or AGRN.

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