Adeno-associated virus mutant and use thereof

Abstract

Provided are an adeno-associated virus mutant and the use thereof. The amino acid sequence of the adeno-associated virus capsid protein mutant comprises a sequence shown as any one of SEQ ID Nos. 1-6. Further provided is the use of the adeno-associated virus capsid protein mutant and an expression vector, host cell and recombinant adeno-associated virus thereof in the preparation of a drug delivery tool for preventing and / or treating muscle or cardiac diseases. The provided adeno-associated virus capsid protein mutant has muscle or heart targeting ability, the muscle targeting ability is increased by a maximum of about 496.41 times, the liver and spleen tropism is nearly 100 times lower than that of a control group, and said mutant has good specificity, and exhibits good effect in NHPs.

Description

Adeno-associated virus mutants and their applications Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an adeno-associated virus mutant and its applications. Background Technology

[0002] Adeno-associated virus (AAV) is a class of non-enveloped small viruses containing a linear single-stranded DNA genome. Belonging to the genus *Dependovirus* in the family Parvoviridae, it requires a helper virus (usually adenovirus) for replication. The AAV genome is a single-stranded DNA fragment contained within a non-enveloped viral capsid and can be divided into three functional regions: two open reading frames (Rep gene and Cap gene) and terminal inverted repeats (ITRs). Recombinant adeno-associated virus vectors (rAAV) are derived from non-pathogenic wild-type adeno-associated viruses. Due to their advantages such as broad host range, non-pathogenicity, low immunogenicity, long-term stable expression of exogenous genes, good diffusion properties, and physical stability, they are widely used as gene transfer vectors in gene therapy and vaccine research. In medical research, rAAV is used for gene therapy research on various diseases (including in vivo and in vitro experiments), such as gene function studies, disease model construction, and gene knockout mouse creation.

[0003] In recent years, gene therapy has become a novel approach for treating muscle diseases, with AAV (autotrophic muscular atrophy) serving as an effective gene vector widely used. Duchenne muscular dystrophy (DMD) is a rare and fatal neuromuscular genetic disease affecting approximately one in 3,500-5,000 males worldwide. DMD is caused by alterations or mutations in the gene encoding dystrophin. Symptoms of DMD typically appear in infants and young children, with affected patients experiencing developmental delays such as difficulty walking, climbing stairs, or standing up from a sitting position. Elevidys (trade name), generically known as delandistrogene moxeparvovec, formerly known as SRP-9001, is a gene therapy delivered via the AAVrh74 vector, using the MHCK7 promoter to express a truncated DMD gene (micro-dystrophin) in DMD patients. It was launched in June 2023 for use in 4-5 year old DMD patients who can walk independently (contraindicated in individuals with deletion mutations in exons 8 and / or 9). On June 20, 2024, the FDA granted full approval to ELEVIDYS for patients aged 4 years and older with DMD who can walk independently, and accelerated approval (conditional) for patients aged 4 years and older with DMD who cannot walk independently. In addition, several other AAV treatment cases and clinical studies are underway. However, like any drug treatment, AAV treatment carries some potential risks. For example, excessively high doses may trigger an immune system response, leading to side effects. Furthermore, high doses also mean greater manufacturing complexity and higher costs. Therefore, the main purpose of AAV serotyping is to develop drugs with higher targeting to reduce dosage, or to make drugs more specific to avoid adverse reactions.

[0004] In summary, while AAV is one of the most widely used and safest gene therapy vectors currently available, further improvements are needed in areas such as lower-dose muscle targeting in vivo. Developing serotypes with better therapeutic effects, lower dosages, fewer side effects, and lower costs is crucial. Therefore, there is an urgent need to develop a novel AAV gene therapy product with lower dosage requirements and costs to meet the needs of more diverse patients and to promote the large-scale, socialized application of AAV-based gene therapy methods. Summary of the Invention

[0005] This invention provides an adeno-associated virus mutant with muscle or heart targeting capabilities and its applications.

[0006] The technical solution adopted in this invention is as follows:

[0007] In a first aspect, the present invention provides an adeno-associated virus capsid protein mutant whose amino acid sequence includes any of the sequences shown in SEQ ID No. 7-12.

[0008] The adeno-associated virus capsid protein mutant of the present invention has muscle or heart targeting. The mutant has good targeting of different muscle tissues (quadriceps, biceps, and abdominal muscles, etc.). Compared with the control group AAV9, the muscle targeting is increased by up to about 496.41 times, and the liver tropism is also nearly 100 times lower than that of the control group. It has good specificity and also shows good effects in NHP (Nonhuman primate).

[0009] In some embodiments, a targeting peptide is inserted into the amino acid sequence; the amino acid sequence of the targeting peptide is any of the sequences shown in SEQ ID No. 1 to 6.

[0010] Secondly, the present invention provides a nucleic acid encoding the adeno-associated virus capsid protein mutant.

[0011] In some embodiments, the nucleotide sequence comprises any of the nucleotide sequences shown in SEQ ID No. 13 to 18.

[0012] Thirdly, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0013] Fourthly, the present invention provides a host cell comprising the aforementioned expression vector.

[0014] Fifthly, the present invention provides a host cell that expresses the aforementioned adeno-associated virus capsid protein mutant.

[0015] In a sixth aspect, the present invention provides a recombinant adeno-associated virus, including the aforementioned adeno-associated virus capsid protein mutant.

[0016] The recombinant adeno-associated virus vector constructed using the AAV capsid protein mutant of this invention has higher specificity, better safety, and a wider range of applications.

[0017] In some implementations, a heterologous target gene is also included.

[0018] In some embodiments, the heterologous target gene encodes any one of the following gene products: interfering RNA, aptamer, endonuclease, or guide RNA.

[0019] In a seventh aspect, the present invention provides a method for preparing recombinant adeno-associated virus, comprising introducing at least the following components into a host cell: 1) the nucleic acid or the expression vector, 2) an adeno-associated virus helper plasmid, and 3) a plasmid containing two terminal inverted repeat sequences.

[0020] Eighthly, the present invention provides rAAV prepared by the method described above.

[0021] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the recombinant adeno-associated virus or the rAAV, and a pharmaceutically acceptable carrier.

[0022] In a tenth aspect, the present invention uses the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, and the rAAV in the preparation of a drug or formulation for delivering a gene product to the cells or tissues of a subject.

[0023] In some embodiments, the cells are muscle cells or heart cells; the tissue is muscle tissue or heart tissue.

[0024] In the eleventh aspect, the present invention utilizes the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, and the rAAV in the preparation of drug delivery tools for the prevention and / or treatment of muscle or heart diseases.

[0025] In some embodiments, the muscle diseases include, but are not limited to, any one of Duchenne muscular dystrophy, Becker muscular dystrophy, X-linked myotubular myopathy, limb girdle muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy; the cardiac diseases include, but are not limited to, any one of arrhythmic cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, angina pectoris, coronary heart disease, myocardial infarction, and heart failure.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention employs a motif-based method to construct an AAV virus library for muscle-targeting screening. This method discovers effective AAV variants with fewer screening cycles, overcoming the drawbacks of conventionally used random libraries, such as large number of variants, poor screening accuracy, and the need for multiple rounds of repeated screening and validation. The adeno-associated virus (AAV) capsid protein mutants screened by this invention exhibit muscle or cardiac targeting. These mutants show good targeting specificity to different muscle tissues (quadriceps, biceps, and abdominal muscles, etc.), with muscle targeting increased by up to approximately 496.41 times compared to the control group AAV9. Furthermore, their liver tropism is nearly 100 times lower than the control group, demonstrating good specificity and showing good efficacy in NHP. The recombinant AAV vector constructed using the AAV capsid protein mutants of this invention has higher specificity, better safety, and a wider range of applications. This will be of great significance in improving the effectiveness of gene therapy and serving a large number of patients in the future. Attached Figure Description

[0028] Figure 1 shows the in vivo imaging results of Balb / c mice infected with different serotypes. In Figure 1, A: 14 days, B: 21 days.

[0029] Figure 2 shows the targeting analysis of different serotypes on the muscle (biceps brachii) of Balb / c mice (21 days). In Figure 2, A represents the relative mRNA expression level, and B represents the protein expression level.

[0030] Figure 3 shows the targeting analysis of different serotypes on the muscle (triceps brachii) of Balb / c mice (21 days). In Figure 3, A represents the relative mRNA expression level, and B represents the protein expression level.

[0031] Figure 4 shows the targeting analysis of different serotypes on the muscle (quadriceps femoris) of Balb / c mice (21 days). In Figure 4, A represents the relative mRNA expression level, and B represents the protein expression level.

[0032] Figure 5 shows the targeting analysis of different serotypes on the muscle (abdominal muscle) of Balb / c mice (21 days). In Figure 5, A represents the relative mRNA expression level, and B represents the protein expression level.

[0033] Figure 6 shows the targeting analysis of different serotypes on the muscle (gastrocnemius) of Balb / c mice (21 days). In Figure 6, A represents the relative mRNA expression level, and B represents the protein expression level.

[0034] Figure 7 shows the cardiac targeting analysis of different serotypes in Balb / c mice (21 days). In Figure 7, A represents the relative mRNA expression level, and B represents the protein expression level.

[0035] Figure 8 shows the liver targeting analysis of different serotypes in Balb / c mice (21 days). In Figure 8, A represents the relative mRNA expression level, and B represents the protein expression level.

[0036] Figure 9 shows the NGS analysis of the muscle targeting and liver tropism of different serotypes in cynomolgus monkeys. In Figure 9, A represents the puncture at 14 days and B represents the puncture at 28 days. Detailed Implementation

[0037] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0038] Unless the context explicitly indicates otherwise, the term "or" refers to a single element among the listed alternatives, and the term "and / or" refers to any one, any two, any three, any more, or all of the listed alternatives.

[0039] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the elements, integers, or steps mentioned are constituted. For example, when referring to a polypeptide that “comprising” a specific sequence, it is also intended to cover polypeptides composed of that specific sequence.

[0040] Adeno-associated virus (AAV) is a non-enveloped icosahedral capsid virus belonging to the Parvoviridae family, comprising a single-stranded DNA viral genome. The Parvoviridae family includes the Dependent Virus genus, which includes AAV, and relies on the presence of helper viruses such as adenoviruses for replication. Due to its relatively simple structure, ability to infect a variety of cells (including quiescent and dividing cells) without integrating into the host genome, and its relatively mild immunogenicity, AAV has been shown to be useful as a biological tool for expressing target genes in vitro or in vivo. This article also considers AAV-based expression vectors, including recombinant AAV (rAAV) carrying target genes for therapeutic purposes.

[0041] The wild-type AAV virus genome is a linear, single-stranded DNA (ssDNA) molecule, approximately 5000 nucleotides (nt) in length. The AAV genome typically includes two inverted terminal repeats (ITRs), which cap the viral genome at the 5' and 3' ends, respectively, providing the origin of replication. These ITRs have a characteristic T-shaped hairpin structure and perform multiple functions, including, but not limited to, acting as primers for the endogenous DNA polymerase complex of the host viral replication cell, thus serving as the origin of DNA replication.

[0042] The wild-type AAV virus genome also includes the Rep and Cap genes, which encode four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40) and three capsid or structural proteins (VP1, VP2, VP3), respectively. The Rep proteins are involved in viral replication and packaging, while the capsid proteins assemble to form the protein coat or AAV capsid. Alternating splicing and alternating start codons and promoters result in four distinct Rep proteins generated from a single open reading frame in the Rep gene and three capsid proteins generated from a single open reading frame in the Cap gene.

[0043] When referring to AAV, the term "viral capsid protein" or "capsid protein" refers to the protein in AAV that is capable of self-assembling to produce AAV particles, also known as the outer coat protein or VP protein. The VP protein comprises three subunits: VP1, VP2, and VP3. Therefore, changes in VP protein mutants relative to wild-type VP protein can be reflected in changes in the amino acid sequences of the VP1, VP2, and VP3 subunits. Accordingly, in this article, "capsid protein mutant" includes VP protein mutants, as well as VP1, VP2, and / or VP3 subunit mutants. Due to the amino acid sequence consistency among the VP1, VP2, and VP3 subunits expressed from the same Cap gene, modifications to the coding sequence in the Cap gene, such as modifications to the coding sequence of the VP1 subunit, simultaneously alter the amino acid sequences of the expressed VP2 and VP3 subunits.

[0044] The term "serotype" as used in the context of AAV refers to the serological difference between the capsid protein of an AAV and other AAV serotypes. Serological uniqueness is determined based on the reactivity of an antibody with one AAV and the lack of cross-reactivity with other AAVs. This difference in cross-reactivity is typically due to differences in the capsid protein sequence (or its subunit sequences) / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of serotype AAV9). Several AAV serotypes have been identified, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12, as well as their mutants.

[0045] When referring to the capsid protein or subunits of AAV, the "variable region" refers to the area where its amino acid sequence varies relatively significantly across different serotypes. Typically, relatively conserved regions are identified by comparing the amino acid sequences of AAV capsid proteins from numerous serotypes; the sequences located between these regions are the variable regions. The variable region may be related to the binding of AAV to cell surface receptors.

[0046] "Recombinant AAV vector" refers to an AAV genome derived by removing parts of the wild-type genes (e.g., the Rep and Cap genes) from the AAV genome using molecular biology methods and replacing them with heterologous nucleic acid sequences (e.g., sequences encoding proteins or RNA for therapeutic purposes). Typically, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in recombinant AAV vectors. In most cases, recombinant AAV vectors are replication-deficient, lacking sequences encoding functional Rep and Cap proteins in their viral genome. These replication-deficient AAV particles may lack most of the parental coding sequences and essentially carry only one or two AAV ITR sequences and the target nucleic acid for delivery to cells, tissues, organs, or organisms. AAVs including recombinant AAV vectors are referred to herein as recombinant AAV (rAAV).

[0047] "Amino acid alterations" in this article include amino acid substitutions, deletions, or insertions. The number of amino acid alterations in a mutant sequence relative to the parent sequence can be expressed as the sum of the number of amino acid substitutions, deletions, and insertions.

[0048] In this document, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably to refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include (but are not limited to) DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide. “Isolated nucleic acid molecule” refers to a nucleic acid molecule that has been removed from its natural environment (such as the intracellular environment) and is substantially free of one or more substances typically associated with its natural state, such as proteins, nucleic acids, lipids, carbohydrates, cell membranes, etc., or is an artificially prepared (e.g., synthetically produced) nucleic acid molecule.

[0049] The term "expression vector" refers to a nucleic acid molecule containing various expression elements for expressing a target protein or RNA in a host cell. For expression vectors used to express a target protein in eukaryotic cells, these expression elements typically include promoters, enhancers, polyadenylation signal sequences, etc. To facilitate amplification in *E. coli*, the expression vector usually also includes an *E. coli* replicon sequence. In addition, the expression vector may also include antibiotic resistance genes or selection marker genes for screening (e.g., ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), neomycin resistance gene (NeoR), etc.) and multiple cloning sites (MCS) for inserting the target gene.

[0050] The term "host cell" refers to the cell in which the expression vector can be maintained and / or replicated, including prokaryotic and eukaryotic cells, such as bacteria (e.g., Escherichia coli), fungi (yeast), insect cells (e.g., SF9), and mammalian cells (e.g., HEK-293T).

[0051] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to solid or liquid diluents, fillers, antioxidants, stabilizers, and other substances that can be safely administered to humans and / or animals without excessive adverse side effects, while also being suitable for maintaining the activity of the drug or active agent contained therein. Depending on the route of administration, a variety of carriers well known in the art can be used, including, but not limited to, sugars, starches, cellulose and their derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and / or pyrogen-free water.

[0052] The "targeting" of AAV or rAAV refers to the phenomenon that, when introduced into the body, it accumulates relatively in specific tissues or organs. For example, targeting can manifest as a higher concentration in tissue A than in tissue B. This targeting can be reflected by detecting the amount or concentration of its genome in different tissues or organs.

[0053] "Amino acid alterations" in this article include amino acid substitutions, deletions, or insertions. The number of amino acid alterations in a mutant sequence relative to the parent sequence can be counted as the sum of the number of amino acid substitutions, the number of amino acid deletions, and the number of amino acid insertions.

[0054] This application also provides the following technical solution: an adeno-associated virus capsid protein mutant containing a targeting peptide sequence, wherein the amino acid sequence of the targeting peptide is AG(X1). a RGD(X1) b X2X3X4X5X6;

[0055] Among them, X1-X6 are each independently selected from any one of the 20 basic amino acids; and

[0056] a = 0 or 1, b = 0 or 1, and a + b = 1.

[0057] The 20 basic amino acids are: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0058] In some implementations, X6 is selected from A or R.

[0059] In some implementations, X1 is selected from any one of L, R, G, A, and S.

[0060] In some implementations, when a = 1 and b = 0, X1 is selected from any one of L, R, G, A, and S; when a = 0 and b = 1, X1 is selected from L.

[0061] In some implementations, X2X3X4X5 is selected from any one of RSVQ, LREV, LSTH, ISIL, SRNT, and YLYS.

[0062] In some implementations, X2X3X4X5X6 is selected from any one of RSVQA, LREVA, LSTHR, ISILR, SRNTA, and YLYSR.

[0063] In some embodiments, the adeno-associated virus capsid protein mutant contains sequence a; the targeting peptide is inserted between position 583Q and position 584Q of sequence a or into the mutant sequence of sequence a; the mutant sequence of sequence a has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity with sequence a, and the insertion position of the targeting peptide in the mutant sequence of sequence a corresponds to the position between position 583Q and position 584Q of sequence a; the amino acid sequence of sequence a is as follows:

[0064] In some embodiments, the amino acid sequence of the targeting peptide is as shown in any of SEQ ID Nos. 1 to 6, or has one or two amino acid changes compared to SEQ ID Nos. 1 to 6. In some embodiments, the one or two amino acid changes do not occur at positions 1-2 (AG) of the targeting peptide. In some embodiments, the one or two amino acid changes do not occur at positions RGD of the targeting peptide. In some embodiments, the one or two amino acid changes do not occur at positions X1 and / or X6 of the targeting peptide.

[0065] In some embodiments, the amino acid sequence of the adeno-associated virus capsid protein mutant includes any of the sequences shown in SEQ ID No. 7-12, or includes a homologous sequence to any of the sequences in SEQ ID No. 7-12; the homologous sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID No. 7-12. In some embodiments, the differences between the homologous sequence and SEQ ID No. 7-12 do not alter the sequence of the targeting peptide.

[0066] This application also provides a nucleic acid encoding any of the adeno-associated virus capsid protein mutants described in this application.

[0067] This application also provides an expression vector comprising the nucleic acid described in this application.

[0068] This application also provides a host cell that includes the expression vector described in this application.

[0069] This application also provides a host cell that expresses any of the adeno-associated virus capsid protein mutants described in this application.

[0070] This application also provides a recombinant adeno-associated virus, which includes any of the adeno-associated virus capsid protein mutants described in this application.

[0071] In some embodiments, the recombinant adeno-associated virus further includes the heterologous target gene described in this application.

[0072] This application also provides a method for preparing recombinant adeno-associated virus, comprising introducing at least the following components into a host cell: 1) the nucleic acid or the expression vector described in this application, 2) an adeno-associated virus helper plasmid, and 3) a plasmid containing two terminal inverted repeat sequences.

[0073] This application also provides rAAV prepared by the preparation method described in this application.

[0074] This application also provides a pharmaceutical composition comprising the recombinant adeno-associated virus or rAAV described in this application, and a pharmaceutically acceptable carrier.

[0075] This application also provides the use of the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, and the rAAV described in this application in the preparation of a drug or formulation for delivering the gene product into the subject cells or tissues described in this application.

[0076] This application also provides the use of the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, and the rAAV described in this application in the preparation of drug delivery tools for the prevention and / or treatment of the muscle diseases or heart diseases described in this application.

[0077] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0078] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The hepatophilic capsid nucleotide sequence is given in Chinese Invention Patent Document CN116041443B, SEQ ID No. 32. The Rep-CAP vector was purchased from Guangzhou Paizhen Biotechnology Co., Ltd.

[0079] Example 1: Screening for novel mutants

[0080] (1) Constructing a backbone plasmid for a low hepatophilic serotype mutant library

[0081] The hepatophilic serotype mutant library backbone vector contains a CAG promoter, an intron, a mutated hepatophilic serotype capsid protein sequence [the VP1 sequence after T580 is removed, and the T580 nucleic acid sequence ACC is mutated to ACT, thus forming a BsrGI (TGTACA) restriction site with the polyA proximal sequence for subsequent backbone linearization], and polyA. These sequences are synthesized via gene synthesis and inserted between the ITRs of the AAV vector plasmid to form the hepatophilic serotype mutant library backbone vector.

[0082] (2) Construction of the mutant Rep-CAP vector

[0083] By introducing stop codons at the N-terminus of the VP1, VP2, and VP3 proteins in the CAP sequence from the hypohepatophilic serotype, the Rep-CAP vector can express Rep and AAP proteins normally, but not the VP1, VP2, and VP3 proteins of the CAP sequence, thus avoiding contamination of the parental CAP sequence. The above sequence was synthesized via gene synthesis and inserted to replace the CAP sequence in the hypohepatophilic Rep-CAP vector.

[0084] (3) Construction of random peptide vector libraries containing RGD motifs

[0085] Design Method: The TNLQ583 and Q588AAT sequences of the low hepatophilic CAP were used as insertion and modification sequences, combined as follows: AGRGDXXXXXR, AGXRGDXXXXR, AGXXRGDXXXR, AGXXXRGDXXR, AGXXXXRGDXR, AGXXXXXRGDR, AGRGDXXXXXA, AGXRGDXXXXA, AGXXRGDXXXA, AGXXXRGDXXA, AGXXXXRGDXA, AGXXXXXRGDA. The upstream primer sequence consisted of: homologous arm sequence + the above combined sequence + primer matching sequence, forming a total of 12 primer sequences. The downstream primer used the same sequence. These primers, as upstream and downstream primers, formed primer pairs. Using the low hepatophilic CAP vector as a template, the target fragment library was amplified. The fragment library has homologous arms at both ends, which can be homologously recombinated with the backbone plasmid of the low hepatophilic mutant library after enzyme digestion to form a vector library.

[0086] The base sequence of the upstream primer (5'->3') is as follows:

[0087] The base sequence of the downstream primer (5'->3') is as follows:

[0088] The specific steps are as follows: Using a vector containing the low hepatophilic serotype CAP as a template, PCR amplification is performed using the primers described above to obtain fragments containing random sequences. The fragments are subjected to gel electrophoresis and gel recovery to obtain purified nucleic acid fragments. The nucleic acid fragments are ligated into the low hepatophilic serotype mutant library backbone vector constructed in step (1) via Gibson homologous recombination (after BsrGI digestion and gel recovery purification). The ligated vector is purified using a PCR product purification kit and then digested with Plasmid-Safe DNase to remove unligated fragments. Finally, it is purified again using a PCR product purification kit to obtain the constructed low hepatophilic serotype mutant random polypeptide vector library containing the RGD motif, i.e., the low hepatophilic serotype mutant plasmid library.

[0089] (4) Production of low-hepatophilic mutant virus library

[0090] The mutated Rep-Cap plasmid constructed in step (2), the low hepatophilic serotype mutant plasmid library constructed in step (3), and the pHelper plasmid were co-transfected into HEK-293T cells. Adeno-associated virus was purified by gradient ultracentrifugation with iodixanol, and the viral titer was measured at 10. 12 GC / mL ~10 13 GC / mL is the appropriate titer to obtain a low hepatophilic serotype mutant virus library, which is then stored at -80℃ for later use.

[0091] (5) Screening for mutants

[0092] (5.1) Animal injection and dissection

[0093] The cynomolgus monkeys were intravenously injected with a low hepatophilic serotype mutant virus library. Animals were dissected and organs were collected 28 days after injection. The samples were immediately flash-frozen in liquid nitrogen and used for subsequent RNA extraction experiments.

[0094] (5.2) Total RNA extraction and RT-PCR

[0095] Sample Grinding: Pre-cool the grinder 10 minutes in advance and set the grinding parameters. Take out the animal tissue sample stored at -80℃, take about 50-100mg of tissue, cut it into soybean-sized pieces in a sterile culture dish, and transfer it to a 1.5mL RNase-free EP tube. Add an appropriate amount of TransZol Up at a ratio of 1mL TransZol Up per 50-100mg of tissue, then add two clean, sterile 3mm grinding steel balls, and wrap with sealing film. Place the sample in the 24-well grinder adapter, level it, tighten the screw, and press the cap button. Start the grinding program. After the instrument finishes running, remove the sample and observe the particle size. If there are no large tissue fragments remaining, the subsequent extraction operation can be performed. Centrifuge the ground sample at 4℃ and 12,000×g for 2 minutes, and transfer the supernatant to a new, appropriately labeled 1.5mL RNase-free EP tube.

[0096] Total RNA extraction from the sample: Refer to the TransZol Up Plus RNA Kit (Beijing TransGen, catalog number: ER501) instructions. For every 1 mL of TransZol Up used, add 0.2 mL of RNA Extraction Agent, vortex vigorously for 5 min; centrifuge at 12,000 × g, 4 °C for 10 min. At this point, the sample separates into three layers. Transfer the colorless aqueous phase to a new 1.5 mL RNase-free EP tube, add an equal volume of anhydrous ethanol (precipitation may occur at this point), and gently invert to mix. Add the resulting solution and precipitate together to a centrifuge column, centrifuge at 12,000 × g at room temperature for 30 s, and discard the filtrate. Add 500 μL of CB9, centrifuge at 12,000 × g at room temperature for 30 s, and discard the filtrate. Repeat the above step once. Add 500 μL of WB9, centrifuge at 12,000 × g at room temperature for 30 s, and discard the filtrate. Repeat the above step once. Centrifuge at 12,000 × g at room temperature for 2 min to completely remove residual ethanol. Place the centrifuge column in a 1.5 mL RNase-free EP tube, add 30-50 μL (depending on tissue size) of RNase-free water to the center of the centrifuge column, and let it stand at room temperature for 1 min. Centrifuge at 12,000 × g at room temperature for 1 min to elute RNA.

[0097] Nucleic acid concentration determination of samples: RNA concentration was detected using a micro-volume nucleic acid quantification instrument detector, and the concentration, OD260 / 280, and OD260 / 230 were recorded. The RNA was stored at -80℃.

[0098] RT-PCR: First-strand cDNA was synthesized from extracted RNA samples using PrimeScript™ IV 1st strand cDNA Synthesis Mix (Takara, 6215A). Two rounds of PCR amplification were then performed using NEB Q5 (the first round used outer primers; the second round used the gel-recovered product from the first round as a template for NGS primer amplification). PCR products corresponding to the band sizes were collected from the gel and sent to the company for NGS sequencing.

[0099] NGS sequencing, data analysis and candidate vector selection: Sequencing data analysis was performed after sequencing to select sequences that appeared frequently in multiple samples as candidates for subsequent construction and validation of AAV mutants.

[0100] Example 2: Construction of AAV capsid protein mutant and production of virus

[0101] (1) Construction of mutant serotype vector and plasmid extraction

[0102] The AAV9 Rep-CAP plasmid (purchased from Guangzhou Paizhen Biotechnology Co., Ltd.) was double-digested with Smi I and BshTI, and the fragment band of about 5000 bp was extracted by gel electrophoresis and recovered by gel electrophoresis to obtain the digested backbone fragment.

[0103] Based on the Cap sequence of mutant 1, the following primers were designed. The specific steps are as follows: using the Rep-CAP plasmid of serotype 109 (its sequence can be found in CN118725047A) as a template, the target product YJ573-1 was amplified and recovered by gel amplification using Cap-f+YJ573-R primers. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ573-2 was amplified and recovered by gel amplification using YJ573-F+cap-r primers. By mixing the backbone fragment, YJ573-1, and YJ573-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 1 can be recombined and constructed.

[0104] Based on the Cap sequence of mutant 2, the following primers were designed. The specific steps are as follows: using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ578-1 was amplified and recovered by gel amplification using the Cap-f+YJ578-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ578-2 was amplified and recovered by gel amplification using the YJ578-F+cap-r primer. By mixing the backbone fragment, YJ578-1, and YJ578-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 2 can be recombinantly constructed.

[0105] Based on the Cap sequence of mutant 3, the following primers were designed. The specific steps are as follows: using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ588-1 was amplified and recovered by gel amplification using the Cap-f+YJ588-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ588-2 was amplified and recovered by gel amplification using the YJ588-F+cap-r primer. By mixing the backbone fragment, YJ588-1, and YJ588-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 3 can be recombinantly constructed.

[0106] Based on the Cap sequence of mutant 4, the following primers were designed. The specific steps are as follows: using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ581-1 was amplified and recovered by gel amplification using the Cap-f+YJ581-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ581-2 was amplified and recovered by gel amplification using the YJ581-F+cap-r primer. By mixing the backbone fragment, YJ581-1, and YJ581-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 4 can be recombinantly constructed.

[0107] Based on the Cap sequence of mutant 5, the following primers were designed. The specific steps are as follows: using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ574-1 was amplified and recovered by gel amplification using the Cap-f+YJ574-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ574-2 was amplified and recovered by gel amplification using the YJ574-F+cap-r primer. By mixing the backbone fragment, YJ574-1, and YJ574-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 5 can be recombined and constructed.

[0108] Based on the Cap sequence of mutant 6, the following primers were designed. The specific steps are as follows: using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ585-1 was amplified and recovered by gel amplification using the Cap-f+YJ585-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ585-2 was amplified and recovered by gel amplification using the YJ585-F+cap-r primer. By mixing the backbone fragment, YJ585-1, and YJ585-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 6 can be recombined and constructed.

[0109] The primers used in the construction of the Rep-CAP vector for the above AAV capsid protein mutants are shown in Table 1:

[0110] Table 1 Primer sequence information

[0111] Label a clean 200 μL PCR tube and place it on an ice box. Prepare the reaction solution by mixing the enzyme digestion backbone and each target fragment at a backbone:fragment molar ratio of 1:3. Incubate the PCR tube at 50°C for 30 min to perform recombination ligation. Thaw 50 μL of competent cells on ice. Mix 10 μL of the ligation product with DH5α competent cells and incubate on ice for 20-30 minutes. Heat shock at 42°C for 45 seconds. Quickly place the tube on ice for 2 minutes. Add 400 μL of resuscitation SOC medium (antibiotic-free) and incubate at 37°C, 200 rpm for 1 h. Spread the mixture evenly on Amp-resistant plates (50 μg / mL) and incubate at 37°C for 14 h. Select single clones and expand the culture in 4 mL of liquid LB medium (Amp + antibiotic) at 37°C for 14 h.

[0112] Centrifuge the bacterial culture at 12,000 rpm for 1 minute and discard the supernatant. Add 250 μL of buffer P1 / RNase A mixture and vortex to resuspend the bacteria. Add 250 μL of buffer P2 and invert 8-10 times. Add 350 μL of buffer P3 and immediately invert 8-10 times to thoroughly neutralize the solution. Centrifuge at 13,000 rpm for 10 minutes and pass the supernatant through a column. Centrifuge at 12,000 rpm for 1 minute and discard the waste liquid. Add 500 μL of PW1 and centrifuge at 12,000 rpm for 1 minute and discard the waste liquid. Add 600 μL of PW2 and centrifuge at 12,000 rpm for 1 minute and discard the supernatant. Centrifuge at 12,000 rpm for 2 minutes without loading. Add 30-50 μL of preheated elution buffer (55°C), let stand for 2 minutes, and centrifuge at 12,000 rpm for 1 minute. Concentration was detected using a micro-volume nucleic acid quantification instrument.

[0113] After concentration testing and enzyme digestion identification, 10 μL of the positive plasmid was sent for sequencing and stored at -20℃. Sequencing results showed that the obtained plasmid encoded the variant capsid protein VP1. Finally, based on the viral load required for subsequent testing, relevant Helper plasmids, Rep-Cap plasmids (control serotypes AAV2, AAV9, MyoAAV 4A, 109 and mutants 1-6), and GOI plasmid (ssAAV.CAG.Fluc-2a-eGFP.WPRE.SV40pA) were extracted.

[0114] (2) Packaging and purification of mutant serotype viruses

[0115] The Rep-Cap plasmids obtained from each group (control group serotype and AAV mutants 1-6) were used to express GOI plasmids of firefly luciferase (Fluc) and green fluorescent protein (EGFP). The pHelper plasmid was co-transfected into HEK-293T cells in an appropriate amount. The AAV virus was purified by gradient ultracentrifugation with iodixanol. The appropriate viral titer was measured to be around 1E+13GC / mL. The cells were then stored at -80℃ for later use.

[0116] Example 3: Comparative Test of Various Indicators of Mutant Serotypes

[0117] (1) Mouse injection and dissection

[0118] Animal experiments were conducted using 6-8 week old male Balb / c mice. The relevant viruses were prepared according to the designed experimental and control groups (mutants 5 and 6 were only tested in 2 mice due to their low viral yield). Each mouse in each group was injected with 2E11GC virus. In vivo imaging was performed 14 days and 21 days after injection. Animal dissection and organ sampling were performed 21 days after injection. The samples were immediately flash-frozen in liquid nitrogen and used for subsequent experiments such as RNA extraction and Western blot detection.

[0119] (2) In vivo imaging

[0120] In vivo imaging of mice was performed on days 14 and 21 post-injection. Mice were weighed before imaging, and the animal in vivo imaging system (AniView100, Guangzhou Boluteng Biotechnology Co., Ltd.) was turned on and the small animal anesthesia system was tested. Image save paths and imaging parameters were set. Each mouse was intraperitoneally injected with luciferin (15 mg / mL, Promega, E1605) at a dose of 150 mg / kg (10 μL / g). Imaging began 10 minutes after each injection. Each batch of mice was photographed sequentially in the supine, left lateral, prone, and right lateral positions. After imaging, the mice were returned to their cages to recover from anesthesia, and their condition was observed for any abnormalities.

[0121] (3) Detection of target gene mRNA expression level

[0122] (3.1) Total RNA extraction and reverse transcription

[0123] Sample Grinding: Pre-cool the grinder 10 minutes in advance and set the grinding parameters. Take out the animal tissue sample stored at -80℃, take about 50-100mg of tissue, cut it into soybean-sized pieces in a sterile culture dish, and transfer it to a 1.5mL RNase-free EP tube. Add an appropriate amount of TransZol Up at a ratio of 1mL TransZol Up per 50-100mg of tissue, then add two clean, sterile 3mm grinding steel balls, and wrap with sealing film. Place the sample in the 24-well grinder adapter, level it, tighten the screw, and press the cap button. Start the grinding program. After the instrument finishes running, remove the sample and observe the particle size. If there are no large tissue fragments remaining, the subsequent extraction operation can be performed. Centrifuge the ground sample at 4℃ and 12,000×g for 2 minutes, and transfer the supernatant to a new, appropriately labeled 1.5mL RNase-free EP tube.

[0124] Total RNA extraction from the sample: Refer to the TransZol Up Plus RNA Kit (Beijing TransGen, catalog number: ER501) instructions. For every 1 mL of TransZol Up used, add 0.2 mL of RNA Extraction Agent, vortex vigorously for 5 min; centrifuge at 12,000×g, 4℃ for 10 min. At this point, the sample separates into three layers. Transfer the colorless aqueous phase to a new 1.5 mL RNase-free EP tube, add an equal volume of anhydrous ethanol (precipitation may occur at this point), and gently invert to mix. Add the resulting solution and precipitate together to a centrifuge column, centrifuge at 12,000 × g at room temperature for 30 s, and discard the filtrate. Add 500 μL of CB9, centrifuge at 12,000 × g at room temperature for 30 s, and discard the filtrate. Repeat the above step once. Add 500 μL of WB9, centrifuge at 12,000 × g at room temperature for 30 s, and discard the filtrate. Repeat the above step once. Centrifuge at 12,000 × g at room temperature for 2 min to completely remove residual ethanol. Place the centrifuge column in a 1.5 mL RNase-free EP tube, add 30-50 μL (depending on tissue size) of RNase-free water to the center of the centrifuge column, and let it stand at room temperature for 1 min. Centrifuge at 12,000 × g at room temperature for 1 min to elute RNA.

[0125] Nucleic acid concentration determination of samples: RNA concentration was detected using a micro-volume nucleic acid quantification instrument detector, and the concentration, OD260 / 280, and OD260 / 230 were recorded. The RNA was stored at -80℃.

[0126] Reverse transcription: Used for each group of RNA samples All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (Beijing TransGen Biotech, Catalog No.: AE341-03). Please refer to the instruction manual for specific steps.

[0127] (3.2) Quantitative PCR (qPCR) experiment:

[0128] Using each cDNA group as a template, the qPCR system was prepared according to the instructions of the 2x SYBR Green qPCR Master Mix (Bimake, catalog number: B21203):

[0129] Table 2 qPCR system

[0130] Table 3 qPCR primer information

[0131] Table 4 qPCR program settings

[0132] 3.3) Data Analysis

[0133] Based on the Ct value of each group, according to Formula 2 -ΔΔct Calculate the relative expression level.

[0134] (4) Western blot analysis to detect the expression level of the target protein

[0135] Sample pretreatment: Cut the tissue into small fragments, weigh and record the weight, place them in 1.5ml or 2ml centrifuge tubes, label the tubes, freeze at -80℃ for later use, pre-cool the cryogenic homogenizer; dissolve RIPA (Beyotime, P0013B) lysis buffer (add PMSF a few minutes before use to make the final PMSF concentration 1mM).

[0136] Add the above-mentioned complete lysis buffer at a ratio of 150-250 μL of lysis buffer per 20 mg of tissue. Then add two sterilized zirconia grinding beads and grind the sample directly in the lysis buffer (for brain, spinal cord, and other tissue samples: temperature -20℃, frequency 70Hz, shake for 50 seconds, pause for 10 seconds, repeat 3-4 times; for muscle, liver, and other samples: temperature -20℃, frequency 70Hz, shake for 50 seconds, pause for 10 seconds, repeat 5-7 times). After grinding, centrifuge the sample in a refrigerated centrifuge at 4℃, 12,000×g for 5-10 min. Then transfer the supernatant to a new sterile EP tube and store at -20℃ or -80℃.

[0137] Protein concentration determination: After determining the protein concentration according to the method in the modified BCA protein concentration determination kit (Sangon Biotech, catalog number C503051), take an appropriate amount of protein homogenate sample according to the required amount, mix it with the corresponding amount of 5X SDS-PAGE protein loading buffer, boil in a water bath for 10 min, cool and centrifuge at low speed for a while, and wait for loading.

[0138] WB (Western Blot) detection:

[0139] A. SDS-PAGE electrophoresis: Determine the appropriate loading volume based on protein concentration and expression level, less than 20 μL / well. For tissue homogenate protein loading, the volume is approximately 20-50 μg. The specific electrophoresis procedure is as follows: Remove the comb from the precast gel, install the gel into the electrophoresis tank, and add electrophoresis buffer to both the inner and outer tanks. Add freshly prepared buffer to the inner tank and check for leaks. If there are no leaks, add electrophoresis buffer to the outer tank. Load an appropriate amount of the processed protein sample, using a pre-stained standard protein as a reference. Perform electrophoresis at a constant voltage of 100V on the Tianneng electrophoresis apparatus for 100 minutes, until bromophenol blue reaches the bottom of the gel. Turn off the power, carefully remove the precast gel plate, remove the gel, and place it in transfer buffer for subsequent operations.

[0140] B. Transfer: Cut 6 sheets of filter paper and 1 PVDF membrane according to the gel area. Soak the PVDF membrane in methanol for 5-10 seconds, then transfer it to transfer buffer and soak for 5 minutes. Pre-wet the filter paper in transfer buffer as well. Assemble the transfer apparatus: negative electrode (black plate) - sponge - 3 layers of wetted filter paper - gel - PVDF membrane - 3 layers of wetted filter paper - sponge - positive electrode (transparent plate). Remove air bubbles from each layer to avoid affecting the transfer effect, clamp the support, and place it in the electroporation tank. Use a 100V constant voltage ice bath for 100 minutes to transfer the membrane. Determine the success of the transfer by whether the pre-stained protein molecular weight standard band has been completely transferred to the PVDF membrane. Wash the transferred PVDF membrane in PBST solution at room temperature for 5 minutes. Cut the PVDF membrane as needed, taking care not to let it dry out during the cutting process.

[0141] C. Blocking and Antibody Incubation: Incubate the PVDF membrane with blocking buffer (5% skim milk powder) at room temperature for 2 hours or overnight at 4°C; transfer the blocked PVDF membrane to primary antibody hybridization buffer (Luciferase Rabbit Polyclonal antibody (Proteintech, 27986-1-AP) at a ratio of 1:2000; GADPH Rabbit Polyclonal antibody (Proteintech, 10494-1-AP) at a ratio of 1:2000; Rabbit GFP tag Polyclonal antibody (Proteintech, 50430-2-AP) at a ratio of 1:2000, adding each to 4 ml of QuickBlock buffer. TM Western blot primary antibody dilution buffer (Beyotime, P0256) is used to prepare the primary antibody hybridization solution. Incubate at room temperature for 1 hour or at 4°C overnight. Then wash the membrane with PBST 3×5 min. Transfer the washed PVDF membrane to the secondary antibody hybridization solution (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, SA00001-2) at a ratio of 1:5000 to 4 ml QuickBlock. TM In Western blot secondary antibody dilution buffer (Beyotime, P0258), prepare the secondary antibody hybridization solution, incubate at room temperature for 1 h, wash the membrane with PBST 3 × 5 min;

[0142] D. Color development: Mix equal volumes of solution A and solution B from the ECL chemiluminescence kit, shake to mix, and then drop the luminescent solution onto the PVDF membrane to ensure that the entire PVDF membrane is covered with the luminescent solution. Adjust different exposure times to make the protein bands clear, and then take pictures with the instrument.

[0143] (5) Injection, puncture and NGS analysis of cynomolgus monkeys

[0144] Animal experiments were conducted using male cynomolgus monkeys approximately 4 years old, which passed AAV2 and AAV9 neutralizing antibody tests before use. Different serotype mutants and control serotypes were packaged with different GOIs (ssAAV.CAG.Fluc.WPRE.polyA vectors carrying different barcodes) and intravenously injected with equal viral doses (total mixed viral dose controlled at 3E13GC / Kg). Muscle and liver biopsies were performed at different sites at 2 and 4 weeks, respectively. Finally, tissue RNA was extracted, RT-PCR and NGS sequencing were performed. By analyzing the NGS data, the fold increase of each serotype mutant relative to the control AAV9 was determined.

[0145] Through different methods of mouse experiments, it was found that the obtained mutants 1-6 had better muscle targeting than AAV9 and retained the low liver tropism of the skeleton (this result can be observed more intuitively from the in vivo imaging results at 14 days and 28 days in Figure 1). Among them, mutants 1 and 3 even showed stronger muscle targeting than 109 and MyoAAV4A serotypes. The mRNA levels of mutant 1 in the gastrocnemius, quadriceps, triceps, biceps, abdominal muscles, and heart were 75.97, 37.27, 186.02, 13.79, 496.41, and 10.06 times that of AAV9, respectively. The mRNA levels of mutant 3 in the same muscle groups were 71.95, 43.25, 112.55, 4.99, 247.96, and 9.46 times that of AAV9, respectively (Figures 2.A-7.A). The protein level results for mutants 1 and 3 showed a generally consistent trend with the mRNA level results (Figures 2.B-7.B). The liver results (Figure 8) further confirmed that all serotype mutants based on the low liver tropism backbone showed low liver targeting, with mRNA levels 50-100 times lower than AAV9, and still 9.5-19 times lower for MyoAAV4A (obtained based on AAV9 backbone screening), showing very good targeting specificity.

[0146] To further illustrate the potential clinical value of the serotypes of this invention, mutant and control serotype viruses were intravenously injected into cynomolgus monkeys in equal volumes. NGS analysis was then used to determine the expression intensity of different serotypes in muscle and liver tissues. Mutants 1 and 3 showed good efficacy in various muscles of cynomolgus monkeys, consistent with results in mice. Specifically, the mRNA levels of mutant 1 in the gastrocnemius, biceps, triceps, and quadriceps muscles (at 4 weeks) were 5.75, 13.31, 35.05, and 15.76 times that of AAV9, respectively (Figure 9). Except for a slightly lower expression level in the gastrocnemius muscle compared to MyoAAV4A, its performance in other muscles was better than MyoAAV4A, and the expression trend at 2 weeks was basically consistent with that at 4 weeks. Slightly different from the mouse results, mutant 2, which performed slightly worse in mouse muscles, showed good efficacy in cynomolgus monkey muscles, with effects in some muscles (such as the gastrocnemius and quadriceps) approaching those of mutant 1. Furthermore, all the muscle mutants obtained through screening, whether in mice or cynomolgus monkeys, showed significantly lower liver tropism than AAV9 and MyoAAV 4A, further demonstrating the consistency and superiority of this scaffold in cross-species use.

[0147] In summary, this invention utilizes a strategy of constructing a small AAV mutant library to obtain multiple serotype mutants with better muscle targeting than AAV9. Verification at both mRNA and protein expression levels demonstrated their effectiveness in muscle tissues such as the gastrocnemius, quadriceps, triceps, biceps, and abdominal muscles, exhibiting lower liver tropism and better specificity. These mutants can be further evaluated for their clinical application value and safety, providing more useful and optional vector tools for gene therapy of muscle diseases, benefiting a wide range of patients.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An adeno-associated virus capsid protein mutant, characterized in that, The amino acid sequence thereof comprises a sequence as shown in any one of SEQ ID No. 7-12.

2. The adeno-associated virus capsid protein mutant of claim 1, wherein, The amino acid sequence is inserted with a targeting peptide; the amino acid sequence of the targeting peptide is a sequence as shown in any one of SEQ ID No. 1-6.

3. A nucleic acid encoding the adeno-associated virus capsid protein mutant of claim 1 or 2.

4. The nucleic acid of claim 3, wherein, The nucleotide sequence thereof comprises a nucleotide sequence as shown in any one of SEQ ID No. 13-18.

5. An expression vector, characterized by, The nucleic acid of claim 3 or 4.

6. A host cell, characterized in that, The expression vector of claim 5.

7. A host cell, characterized in that, The adeno-associated virus capsid protein mutant of claim 1 or 2.

8. A recombinant adeno-associated virus, characterized in that, The adeno-associated virus capsid protein mutant of claim 1 or 2.

9. The recombinant adeno-associated virus of claim 8, wherein, Further comprising a heterologous gene of interest.

10. The recombinant adeno-associated virus of claim 9, wherein, The heterologous gene of interest encodes any one of a gene product of interfering RNA, aptamer, endonuclease, guide RNA.

11. A method of producing a recombinant adeno-associated virus, characterized in that, Comprising introducing into a host cell at least the following components: 1) the nucleic acid of claim 3 or 4 or the expression vector of claim 5, 2) an adeno-associated virus helper plasmid, 3) a plasmid containing two terminal inverted repeat sequences.

12. The rAAV prepared by the method of claim 11.

13. A pharmaceutical composition comprising the recombinant adeno-associated virus of any one of claims 8-10 or the rAAV of claim 12, and a pharmaceutically acceptable carrier.

14. Use of the adeno-associated virus capsid protein mutant of claim 1 or 2, the expression vector of claim 5, the host cell of claim 6 or 7, the recombinant adeno-associated virus of any one of claims 8-10, or the rAAV of claim 12 in the preparation of a medicament or preparation for delivering a gene product into cells or tissues of a subject.

15. Use according to claim 14, characterized in that, The cells are muscle cells or heart cells; the tissues are muscle tissues or heart tissues.

16. Use of the adeno-associated virus capsid protein mutant of claim 1 or 2, the expression vector of claim 5, the host cell of claim 6 or 7, the recombinant adeno-associated virus of any one of claims 8-10, or the rAAV of claim 12 in the preparation of a drug delivery tool for preventing and / or treating muscle or heart diseases.

17. Use according to claim 16, characterized in that, The muscle diseases include but are not limited to any one of Duchenne muscular dystrophy, Becker muscular dystrophy, X-linked myotubular myopathy, limb girdle muscular dystrophy, myotonic muscular dystrophy, and facioscapulohumeral muscular dystrophy; the heart diseases include but are not limited to any one of arrhythmogenic cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, angina pectoris, coronary heart disease, myocardial infarction, and heart failure.

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