CN118725048B

CN118725048B - Adeno-associated virus mutant and application thereof

Info

Publication number: CN118725048B
Application number: CN202410952063.0A
Authority: CN
Inventors: 李华鹏; 钟育健; 卜晔; 潘越; 陈欢; 张有为; 代志勇; 陈君霖
Original assignee: Guangzhou Packgene Biotech Co ltd
Current assignee: Guangzhou Packgene Biotech Co ltd
Priority date: 2024-07-16
Filing date: 2024-07-16
Publication date: 2024-12-20
Anticipated expiration: 2044-07-16
Also published as: CN118725048A; CN119751595A; CN119751597A; CN119751596A; CN119751599A; CN119751598A

Abstract

The present invention belongs to the field of biomedicine technology, and discloses an adeno-associated virus mutant and its application. The amino acid sequence of the adeno-associated virus capsid protein mutant of the present invention includes a sequence as shown in any one of SEQ ID No.1‑6. The present invention uses the adeno-associated virus capsid protein mutant and its expression vector, host cell, and recombinant adeno-associated virus in the preparation of a drug delivery tool for preventing and/or treating muscle or heart diseases. The adeno-associated virus capsid protein mutant of the present invention has muscle or heart targeting, and 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, with good specificity, and also shows good effects in NHP. The present invention can promote the AAV-based gene therapy method towards large-scale and socialized application.

Description

Adeno-associated virus mutant and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to an adeno-associated virus mutant and application thereof.

Background

Adeno-associated virus (AAV) is a type of non-enveloped small virus that encapsulates a linear single-stranded DNA genome, belonging to the genus lentivirus (Parvoviridae) dependent virus (Dependovirus), which requires helper virus (usually adenovirus) to participate in replication. AAV genomes are single-stranded DNA fragments contained in non-enveloped viral capsids and can be divided into three functional regions, two open reading frames (Rep gene, cap gene) and an Inverted Terminal Repeat (ITR). The recombinant adeno-associated virus vector (rAAV) is derived from a non-pathogenic wild adeno-associated virus, and is widely applied to gene therapy and vaccine research as a gene transfer vector due to the advantages of wide host range, non-pathogenicity, low immunogenicity, long-term stable expression of exogenous genes, good diffusion performance, stable physical properties and the like. In medical research, rAAV is used in research (including in vivo and in vitro experiments) for gene therapy of various diseases, such as gene function research, construction of disease models, preparation of gene knockout mice, and the like.

In recent years, gene therapy has become a novel approach to the treatment of muscle and meat diseases, wherein AAV has been widely used as an effective gene vector. Taking Dunaliella Muscular Dystrophy (DMD) as an example, it is a rare fatal neuromuscular genetic disease that occurs every 3500-5000 men worldwide. DMD is caused by alterations or mutations in the gene encoding the dystrophin protein. Symptoms of DMD are often present in infants and the affected patient may experience developmental delays, such as walking, climbing stairs, or standing from a seated position. Elevidys (trade name), commonly known as delandistrogene moxeparvovec, earlier known as SRP-9001, is a gene therapy delivered by AAVrh74 vector, expressing a truncated DMD gene (mini-protein gene micro-dystrophin) in DMD patients using MHCK promoter. Market 6 at 2023 for DMD populations that were able to walk independently between 4 and 5 years of age (disabled populations with deletion mutations at exons 8 and/or 9). 2024, 6 and 20, FDA complete approval ELEVIDYS was for DMD patients over 4 years old who were not independently ambulatory, while accelerated approval (conditional marketing) of the drug was for DMD patients over 4 years old who were not independently ambulatory. In addition, there are other AAV therapeutic cases or clinical studies in progress. However, as with any drug therapy, AAV therapy presents some potential risks. For example, overdosing may cause a reaction to the immune system, leading to side effects. Furthermore, high doses also mean higher production difficulties and higher cost. Therefore, development of drugs with higher targeting to reduce drug dosage or better specificity to avoid adverse reactions is a main aim of AAV serotype engineering.

In view of the foregoing, although AAV is one of the most widely used and safe gene therapy vectors at present, there is still a need for further improvement in terms of lower dose muscle targeting in vivo, and the like. It is important to develop serotypes of better therapeutic efficacy, lower therapeutic doses, reduced side effects and cost of use. Therefore, there is a need to develop a new AAV gene therapy product with lower dosage requirements and costs to meet the needs of more diverse patients and to drive AAV-based gene therapy approaches toward large-scale, social applications.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an adeno-associated virus mutant with muscle or heart targeting and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the invention provides an adeno-associated virus capsid protein mutant, the amino acid sequence of which comprises a sequence as shown in any one of SEQ ID No. 7-12.

The adeno-associated virus capsid protein mutant has muscle or heart targeting, the mutant has good targeting to different muscle tissues (quadriceps femoris, biceps brachii, abdominal muscle and the like), compared with a control group AAV9, the muscle targeting is improved by about 496.41 times at most, the liver tropism is also lower than that of the control group by nearly hundred times, the specificity is good, and the mutant also has good effect in NHP (Nonhuman primate, non-human primate).

As a preferred embodiment of the adeno-associated virus capsid protein mutant, a targeting peptide is inserted into the amino acid sequence, and the amino acid sequence of the targeting peptide is shown as any one of SEQ ID No. 1-6.

In a second aspect, the invention provides a nucleic acid encoding a mutant of the adeno-associated viral capsid protein.

As a preferred embodiment of the nucleic acid of the present invention, the nucleotide sequence thereof comprises the nucleotide sequence shown in any one of SEQ ID Nos. 13 to 18.

In a third aspect, the invention provides an expression vector comprising said nucleic acid.

In a fourth aspect, the invention provides a host cell comprising said expression vector.

In a fifth aspect, the invention provides a host cell expressing said adeno-associated viral capsid protein mutant.

In a sixth aspect, the invention provides a recombinant adeno-associated virus comprising said adeno-associated virus capsid protein mutant.

The recombinant adeno-associated virus vector constructed by the AAV capsid protein mutant has higher specificity, better safety and wide application range.

As a preferred embodiment of the recombinant adeno-associated virus of the invention, a heterologous gene of interest is also included.

As a preferred embodiment of the recombinant adeno-associated virus of the invention, the heterologous gene of interest encodes any one of the gene products interference RNA, aptamer, endonuclease, guide RNA.

In a seventh aspect, the invention provides a method of producing a recombinant adeno-associated virus comprising introducing into a host cell at least 1) said nucleic acid or said expression vector, 2) an adeno-associated virus helper plasmid, 3) a plasmid comprising two inverted terminal repeats.

In an eighth aspect, the invention provides a rAAV prepared by the method.

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

In a tenth aspect, the invention uses the adeno-associated viral capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, the rAAV in the preparation of a medicament or formulation for delivering a gene product into a cell or tissue of a subject.

As a preferred embodiment of the use according to the invention, the cells are muscle cells or heart cells and the tissue is muscle tissue or heart tissue.

In an eleventh aspect, the invention provides use of the adeno-associated virus capsid protein mutant, the expression vector, the host cell, the recombinant adeno-associated virus, the rAAV in the manufacture of a drug delivery vehicle for the prevention and/or treatment of muscle or heart-like diseases.

As a preferred embodiment of the application of the present invention, the muscular diseases include, but are not limited to, any one of Dunaliella muscular dystrophy, becker muscular dystrophy, X-linked myotubular muscular disease, limb-girdle muscular dystrophy, myotonic muscular dystrophy, facial shoulder brachial muscular dystrophy, and cardiac diseases include, but are not limited to, any one of arrhythmia cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, dilated diseases, angina pectoris, coronary heart disease, myocardial infarction, and heart failure.

Compared with the prior art, the invention has the beneficial effects that:

The invention adopts a method based on specific motif to construct AAV virus library for muscle targeting screening, and can discover effective AAV variants through less times of screening processes, thereby overcoming the defects of large variety number of commonly used random libraries, poor screening precision and need of repeated screening and verification for multiple rounds. The adeno-associated virus capsid protein mutant screened by the invention has muscle or heart targeting, the mutant has good targeting to different muscle tissues (quadriceps femoris, biceps brachii, abdominal muscle and the like), compared with a control group AAV9, the muscle targeting is improved by about 496.41 times at most, the liver tropism is also lower than that of the control group by nearly hundred times, the specificity is good, and the mutant also shows good effect in NHP. The recombinant adeno-associated virus vector constructed by the AAV capsid protein mutant has higher specificity, better safety and wide application range. This will be of great importance in the future for improving the benefits of gene therapy and serving a wide range of patients.

Drawings

FIG. 1 shows the results of in vivo imaging of Balb/c mice infected with different serotypes, in FIG. 1, day A14 and day B21;

FIG. 2 shows the muscle (biceps brachii) targeting analysis (21 days) of Balb/c mice by different serotypes, wherein A is the relative mRNA expression level and B is the protein expression level in FIG. 2;

FIG. 3 shows the muscle (triceps brachii) targeting analysis (21 days) of Balb/c mice by different serotypes, in FIG. 3, A is the relative mRNA expression level and B is the protein expression level;

FIG. 4 shows the muscle (quadriceps femoris) targeting analysis (21 days) of Balb/c mice for different serotypes, where A is the relative mRNA expression level and B is the protein expression level in FIG. 4;

FIG. 5 shows muscle (abdominal muscle) targeting analysis (21 days) of Balb/c mice for different serotypes, where A is the relative mRNA expression level and B is the protein expression level in FIG. 5;

FIG. 6 shows the analysis of muscle (gastrocnemius) targeting of Balb/c mice by different serotypes (21 days), in FIG. 6, A is the relative expression level of mRNA and B is the protein expression level;

FIG. 7 shows heart targeting analysis (21 days) of Balb/c mice with different serotypes, wherein A is mRNA relative expression level and B is protein expression level in FIG. 7;

FIG. 8 shows liver targeting analysis (21 days) of Balb/c mice with different serotypes, wherein A is mRNA relative expression level and B is protein expression level in FIG. 8;

Fig. 9 is a graph of NGS detection analysis of muscle targeting and liver tropism of different serotypes on cynomolgus monkeys, in fig. 9, a is a 14 day puncture and B is a 28 day puncture.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The term "or" refers to a single element of a list of selectable elements and the term "and/or" refers to any one, any two, any three, any more or all of the list of selectable elements unless the context clearly indicates otherwise.

The terms "comprises" or "comprising" are intended to include the recited element, integer or step, but not to exclude any other element, integer or step. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, are also intended to cover the circumstance that the recited elements, integers or steps consist of them. For example, when referring to a polypeptide "comprising" a particular sequence, it is also intended to encompass polypeptides consisting of that particular sequence.

An "adeno-associated virus (AAV)" is a non-enveloped icosahedral capsid virus of the parvoviridae family, including a single-stranded DNA virus genome. Parvoviridae include dependoviridae, which include AAV, which rely on the presence of a helper virus, such as an adenovirus, for its replication. Due to their relatively simple structure, AAV has proven useful as a biological tool for expressing genes of interest in vitro or in vivo, being able to infect a wide variety of cells (including resting and dividing cells) without integration into the host genome, and its relatively gentle immunogenic characteristics. AAV-based expression vectors are also contemplated herein, including recombinant AAV (rAAV) with a gene of interest for therapeutic purposes.

The wild-type AAV viral genome is a linear, single stranded DNA (ssDNA) molecule of about 5000 nucleotides (nt) in length. AAV viral genomes typically include two Inverted Terminal Repeats (ITRs) that terminate the viral genome at the 5 'and 3' ends, respectively, providing an origin of replication for the viral genome. These ITRs have a characteristic T-shaped hairpin structure and serve a variety of functions, including but not limited to serving as an origin of DNA replication by serving as a primer for the endogenous DNA polymerase complex of the host virus replicating cell.

The wild-type AAV viral genome also includes a Rep gene and a Cap gene, encoding four non-structural Rep proteins (Rep 78, rep68, rep52, rep 40) and encoding three capsid or structural proteins (VP 1, VP2, VP 3), respectively. Rep proteins are associated with viral replication and packaging, while capsid proteins assemble to form the protein coat or AAV capsid of an AAV. Alternate splicing and alternate initiation codons and promoters result in four different Rep proteins being produced from a single open reading frame in the Rep gene and three capsid proteins being produced from a single open reading frame in the Cap gene.

In the context of AAV, the term "viral capsid protein" or "capsid protein" as used herein refers to a protein of AAV that is capable of self-assembly to produce AAV particles, also referred to as coat protein or VP protein. The VP protein comprises three subunits VP1, VP2 and VP3, and thus changes in VP protein mutants relative to the wild-type VP protein can be manifested in amino acid sequence changes of the VP1, VP2 and VP3 subunits. Accordingly, herein, "capsid protein mutants" include VP protein mutants, as well as VP1 VP2 and/or VP3 subunit mutants. Because of the identity of amino acid sequences between VP1, VP2, and VP3 subunits expressed from the same Cap gene, the amino acid sequences of the expressed VP2 and VP3 subunits are simultaneously altered when the coding sequence in the Cap gene is altered, e.g., when the coding sequence of the VP1 subunit is altered.

The term "serotype" as used in reference to AAV is used to refer to the difference in serology of the capsid protein of AAV from other AAV serotypes. The determination of serological uniqueness is based on the reactivity of one antibody with one AAV, while the lack of cross-reactivity with other or another AAV. This cross-reactivity difference is typically due to differences in capsid protein sequence (or subunit sequence thereof)/antigenic determinants (e.g., due to VP1, VP2 and/or VP3 sequence differences in serotype AAV 9). A variety of AAV serotypes have been discovered, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12, and mutants thereof.

Reference to the capsid protein of AAV or a subunit thereof, a "variable region" refers to a region whose amino acid sequence varies relatively widely between serotypes. Generally, the sequence between the relatively conserved regions can be the variable region sequence by aligning the amino acid sequences of numerous serotype AAV capsid proteins. The variable region may be involved in the binding of AAV to cell surface receptors.

"Recombinant AAV vector" refers to an AAV genome derived by removing portions of wild-type genes (e.g., rep genes and Cap genes) from the AAV genome using molecular biological methods, and replacing them with heterologous nucleic acid sequences (e.g., coding sequences for proteins or RNAs for therapeutic purposes). Typically, for recombinant AAV vectors, one or both Inverted Terminal Repeat (ITR) sequences of the AAV genome remain therein. In most cases, recombinant AAV vectors are replication defective, lacking sequences encoding functional Rep and Cap proteins in their viral genomes. These replication defective AAV particles may lack most of the parental coding sequences and carry substantially only one or two AAV ITR sequences and the target nucleic acid for delivery to a cell, tissue, organ or organism. AAV comprising a recombinant AAV vector is referred to herein as a recombinant AAV (rAAV).

"Amino acid changes" herein include amino acid substitutions, deletions or insertions. The number of amino acid changes that occur in the mutant sequence relative to the parent sequence may be calculated as the sum of the number of deleted amino acids and the number of inserted amino acids in the number of amino acid substitutions.

The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide. An "isolated nucleic acid molecule" refers to a nucleic acid molecule that is free of the natural environment in which it exists (e.g., the intracellular environment), substantially free of one or more substances typically associated with its nature, such as proteins, nucleic acids, lipids, carbohydrates, cell membranes, etc., or is a nucleic acid molecule that is prepared manually (e.g., synthetically produced).

The term "expression vector" refers to a nucleic acid molecule comprising various expression elements for expressing a protein of interest or an RNA of interest in a host cell. For expression vectors for expressing a protein of interest in eukaryotic cells, these expression elements typically include promoters, enhancers, polyadenylation signal sequences, and the like. To facilitate amplification in E.coli, the expression vector will typically also include E.coli replicon sequences. In addition, the expression vector may further include antibiotic resistance genes or selectable marker genes (e.g., ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), neomycin resistance gene (NeoR), etc.) for selection and Multiple Cloning Sites (MCS) for insertion of the gene of interest.

The term "host cell" refers to cells in which an expression vector can be maintained and/or replicated, including prokaryotic and eukaryotic cells, for example, bacteria (e.g., E.coli), fungi (yeast), insect cells (e.g., SF 9), and mammalian cells (e.g., HEK-293T).

The term "pharmaceutically acceptable carrier" as used in reference to pharmaceutical compositions refers to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers and the like which may be safely administered and which are suitable for administration to humans and/or animals without undue adverse side effects, while maintaining the viability of the drug or active agent located therein. Depending on the route of administration, a variety of different carriers well known in the art may be used, including, but not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifying agents, isotonic saline, and/or pyrogen-free water and the like.

"Targeting" of AAV or rAAV refers to the phenomenon in which AAV or rAAV, when introduced into the body, relatively aggregates in a specific tissue or organ. For example, targeting may be manifested as a higher concentration in a tissue than in B tissue. This targeting can be reflected by measuring the amount or concentration of its genome in different tissues or organs.

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

The test methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available. The nucleotide sequence of the low hepadnavicular serotype capsid is shown in SEQ ID No.32 of Chinese patent document CN 116041443B.

EXAMPLE 1 screening of novel mutants

(1) Construction of low hepadnavirus mutant pool backbone plasmid

The low hepadnavirus mutant library backbone vector comprises a CAG promoter, an Intron, a mutated low hepadnavirus capsid protein sequence [ VP1 sequence after T580 is deleted, the T580 nucleic acid sequence ACC is mutated to ACT, thereby constituting with the polyA pre-stretch sequence the cleavage site Bsrg I (TGTACA) for subsequent backbone enzyme tangentially ] and polyA. The sequences are synthesized by a gene synthesis mode and inserted between ITRs of AAV vector plasmids to form the low-hepadnavirus mutant library skeleton vector.

(2) Construction of mutant Rep-CAP vectors

By introducing a stop codon at the N-terminal of VP1, VP2 and VP3 proteins of CAP sequences in low hepadnavicular serotypes, a Rep-CAP vector can normally express Rep proteins and AAP proteins, but can not express VP1, VP2 and VP3 proteins of CAP, so that pollution of CAP sequences in parents is avoided. The above sequences were synthesized by means of gene synthesis and inserted into CAP sequences replacing the low hepadnavirus type Rep-CAP vector.

(3) Construction of random polypeptide vector libraries comprising RGD motifs

The design method comprises the steps of taking TNLQ583 and Q588AAT sequences of the low hepadnavicular serotype CAP as insertion and modification sequences, combining ：AGRGDXXXXXR,AGXRGDXXXXR,AGXXRGDXXXR,AGXXXRGDXXR,AGXXXXRGDXR,AGXXXXXRGDR,AGRGDXXXXXA,AGXRGDXXXXA,AGXXRGDXXXA,AGXXXRGDXXA,AGXXXXRGDXA,AGXXXXXRGDA, upstream primer sequences comprising homologous arm sequences, the combined sequences and primer matching sequences to form 12 sequence primers, taking the same sequence as a downstream primer, taking the primers as an upstream primer and a downstream primer to form a primer pair, amplifying a target fragment library under the condition that the low hepadnavicular serotype CAP carrier is taken as a template, and carrying out homologous recombination on the fragment library with the skeleton plasmid of the low hepadnavicular serotype mutant library after enzyme digestion to form a carrier library.

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

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCAGAGGAGACNNKNNKNNKNN KNNKAGACAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKAGAGGAGACNNKNNKNN KNNKAGACAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKAGAGGAGACNNKNN KNNKAGACAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKNNKAGAGGAGACNN KNNKAGACAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKNNKNNKAGAGGAGA CNNKAGACAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKNNKNNKNNKAGAGG AGACAGACAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCAGAGGAGACNNKNNKNNKNN KNNKGCTCAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKAGAGGAGACNNKNNKNN KNNKGCTCAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKAGAGGAGACNNKNN KNNKGCTCAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKNNKAGAGGAGACNN KNNKGCTCAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKNNKNNKAGAGGAGA CNNKGCTCAAGCAGCTACCGCAGAT;

CAGTATGGTTCTGTATCTACTAACCTCCAGGCTGGCNNKNNKNNKNNKNNKAGAGG AGACGCTCAAGCAGCTACCGCAGAT。

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

CGGTTTATTGATTAACAATCGATTACAGATTACGAGTCAGGTATCTGGTG。

The specific operation steps are that a carrier containing low hepadnavim CAP is used as a template, and the primer is used for PCR amplification to obtain fragments containing random sequences. And (3) connecting the nucleic acid fragments into the low hepatism serotype mutant library skeleton vector constructed in the step (1) through a Gibson homologous recombination connection mode (BsrG I enzyme digestion and glue recovery purification), purifying the connected vector through a PCR product purification kit, then digesting the connected vector through a Plasmid-SAFE DNASE enzyme to remove the fragments which are not connected, and finally purifying the connected vector through the PCR product purification kit to obtain the constructed low hepatism serotype mutant random polypeptide vector library containing RGD motif, namely the low hepatism serotype mutant Plasmid library.

(4) Production of low hepadnavirus pool

And (3) co-transferring the mutant Rep-Cap plasmid constructed in the step (2), the low hepatism serotype mutant plasmid library constructed in the step (3) and the pHelper plasmid into HEK-293T cells, purifying adeno-associated virus by iodixanol gradient ultra-high speed centrifugation, measuring the virus titer to be proper titer at 10 ¹²GC/mL～10¹³ GC/mL, obtaining the low hepatism serotype mutant virus library, and standing at-80 ℃ for later use.

(5) Screening mutants

(5.1) Animal injection and dissection

The cynomolgus monkey intravenous injection low hepadnavirus mutant virus library is used, animal dissection and organ material taking are carried out after 28 days of injection, liquid nitrogen quick freezing is carried out immediately after sample material taking, and the obtained sample is used for subsequent RNA extraction experiments.

(5.2) Total RNA extraction and RT-PCR

And (3) grinding the sample, namely pre-cooling the grinder 10min in advance and setting grinding parameters. The animal tissue samples stored in-80℃refrigerator were removed, about 50-100mg of tissue was cut into Huang Douli pieces in sterile petri dishes and transferred to 1.5mL RNase-free EP tubes. Proper TransZol Up is added according to the proportion of 1mL TransZol Up per 50-100mg of tissue, then two clean sterile 3mm grinding steel balls are added, and a sealing film is wound. The sample was placed in a 24-well grind adapter and trimmed, the screw was tightened, and the cap closing button was pressed. And starting a grinding program, taking out the sample after the operation of the instrument is finished, and observing the grinding granularity of the sample, wherein the subsequent extraction operation can be performed if no massive tissue residue exists. The milled samples were centrifuged at 4℃at 12,000Xg for 2min and the supernatant was pipetted into a new 1.5mL RNase-free EP tube with corresponding labeling.

Extraction of total RNA from samples referring specifically to TransZol Up Plus RNA Kit (Beijing full gold, cat# ER 501) instructions. Each time 1mL TranZol up was used, 0.2: 0.2ml RNA Extraction Agent was added, and the mixture was vigorously shaken for 5min, centrifuged at 12,000Xg and 4℃for 10min. The sample is divided into three layers, colorless aqueous phase is transferred into a new 1.5mL RNase-free EP tube, equal volume of absolute ethanol (precipitation may occur at the moment) is added, the mixture is gently inverted and uniformly mixed, the obtained solution and the precipitate are added into a centrifugal column, the centrifugal column is centrifuged at the room temperature for 30s at 12,000 Xg, the filtrate is discarded, 500 mu L CB9 is added, the centrifugal column is centrifuged at the room temperature for 30s at 12,000 Xg, the filtrate is discarded, the previous step is repeated once, 500 mu L WB9 is added, the centrifugal column is centrifuged at the room temperature for 30s at 12,000 Xg, the filtrate is discarded, the previous step is repeated once, the centrifugal column is centrifuged at the room temperature for 2min at 12,000 Xg, residual ethanol is thoroughly removed, the centrifugal column is placed into the 1.5mL RNase-free EP tube, 30-50 mu L RNase-FREE WATER (depending on the tissue size) is added at the center of the centrifugal column, the room temperature is 1min at the room temperature, the room temperature is centrifuged at the room temperature for 1min, and RNA is eluted;

sample nucleic acid concentration determination by detecting RNA concentration using a micro-scale nucleic acid quantitative detector, recording the concentration, OD260/280, OD260/230, and preserving RNA at-80 ℃.

RT-PCR extraction of RNA samples first strand cDNA synthesis was performed using PRIMESCRIPTTM IV A st strand cDNASynthesis Mix (Takara, 6215A). Then using NEB Q5 to carry out 2 rounds of PCR amplification (the first round of amplification is carried out by using an outer primer, the second round of amplification is carried out by using the first round of products recovered by gel as a template and using NGS primers), and the products of the PCR corresponding to the size of the bands are recovered by gel and sent to a company for NGS sequencing;

And (3) performing NGS sequencing, data analysis and candidate vector selection, namely performing sequencing data analysis after sequencing, selecting sequences which are arranged in front of the occurrence frequency and are repeatedly appeared in a plurality of samples as candidates, and performing subsequent construction and verification work of AAV mutants.

Example 2 construction of AAV capsid protein mutants and production of Virus

(1) Construction of mutant serotype vector and plasmid extraction

AAV9Rep-CAP plasmid (available from Guangzhou Pi Biotechnology Co., ltd.) was digested with Smi I and BshT I, gel-electrophoresed and the fragment band of about 5000bp was excised for gel recovery to obtain digested backbone fragments.

According to the Cap sequence of the mutant 1, designing primers, specifically, amplifying and gel-recovering a target product YJ573-1 by using a Cap-f+YJ573-R primer with a Rep-CAP plasmid of serotype 109 as a template, amplifying and gel-recovering a target product YJ573-2 by using a YJ573-F+cap-R primer with a Rep-CAP plasmid of serotype 109 as a template, and recombining and constructing the Rep-CAP plasmid of the mutant 1 by mixing framework fragments, YJ573-1 and YJ573-2 according to the following steps and proportions;

According to the Cap sequence of the mutant 2, the specific steps are that a Rep-CAP plasmid of a serotype 109 is used as a template to amplify and glue and recycle by using a Cap-f+YJ578-R primer to obtain a target product YJ578-1, a Rep-CAP plasmid of the serotype 109 is used as a template to amplify and glue and recycle by using a YJ578-F+cap-R primer to obtain a target product YJ578-2, and the Rep-CAP plasmid of the mutant 2 can be recombined and constructed by mixing a framework fragment, YJ578-1 and YJ578-2 according to the following steps and proportions;

according to the Cap sequence of the mutant 3, the specific steps are that a Rep-CAP plasmid of a serotype 109 is used as a template to amplify and glue and recycle by using a Cap-f+YJ588-R primer to obtain a target product YJ588-1, a Rep-CAP plasmid of the serotype 109 is used as a template to amplify and glue and recycle by using a YJ588-F+cap-R primer to obtain a target product YJ588-2, and the Rep-CAP plasmid of the mutant 3 can be constructed by mixing the framework fragments, YJ588-1 and YJ588-2 in the following steps and proportion;

According to the Cap sequence of the mutant 4, the specific steps are that a Rep-CAP plasmid of a serotype 109 is used as a template, a Cap-f+YJ581-R primer is used for amplification and gel recovery to obtain a target product YJ581-1, a Rep-CAP plasmid of the serotype 109 is used as a template, a YJ581-F+cap-R primer is used for amplification and gel recovery to obtain a target product YJ581-2, and the Rep-CAP plasmid of the mutant 4 can be constructed by mixing a framework fragment, YJ581-1 and YJ581-2 in the following steps and proportions;

according to the Cap sequence of the mutant 5, designing primers, specifically, amplifying and gel-recovering a target product YJ574-1 by using a Rep-CAP plasmid of a serotype 109 as a template and using a Cap-f+YJ574-R primer, amplifying and gel-recovering a target product YJ574-2 by using a Rep-CAP plasmid of the serotype 109 as a template and using a YJ574-F+cap-R primer, and recombining and constructing the Rep-CAP plasmid of the mutant 5 by mixing a framework fragment, YJ574-1 and YJ574-2 according to the following steps and proportions;

According to the Cap sequence of the mutant 6, the specific steps are that a Rep-CAP plasmid of a serotype 109 is taken as a template, a Cap-f+YJ585-R primer is used for amplification and gel recovery to obtain a target product YJ585-1, a Rep-CAP plasmid of the serotype 109 is taken as a template, a YJ585-F+cap-R primer is used for amplification and gel recovery to obtain a target product YJ585-2, and the Rep-CAP plasmid of the mutant 6 can be constructed by mixing a framework fragment, YJ585-1 and YJ585-2 in the following steps and proportions;

The primers involved in the construction of the Rep-CAP vector for the AAV capsid mutants described above are shown in Table 1:

TABLE 1 primer sequence information

1 Clean 200 mu L PCR tube is taken as a mark and placed on an ice box, the enzyme-digested skeleton and each target fragment are prepared into a reaction solution according to the mole ratio of the skeleton to the fragments of 1:3, and the reaction is carried out for 30min at 50 ℃ in a PCR instrument for recombination connection. 50. Mu.L of competent cells were thawed on ice, 10. Mu.L of ligation product was mixed with DH 5. Alpha. Competent cells, placed on ice for 20-30 min, heat shock at 42℃for 45 sec, placed rapidly on ice for 2 min, added 400. Mu.L of resuscitating SOC medium (without antibiotics), incubated at 37℃at 200rpm for 1h, and spread evenly on Amp-resistant plates (50. Mu.g/mL), incubated at 37℃for 14 h. Monoclonal bacteria were selected and grown in 4mL of liquid LB medium (Amp+ resistant) for 14 hours at 37 ℃.

The bacterial liquid is centrifuged at 12000rpm for 1 minute, the supernatant culture medium is poured out, 250 mu L of buffer P1/RNaseA mixed solution is added, bacteria are resuspended by high-speed vortex, 250 mu L of buffer P2 is added, the solution is thoroughly neutralized by reversing 8 to 10 times, 350 mu L of buffer P3 is added, the solution is immediately and evenly mixed for 8 to 10 times, the solution is centrifuged at 13000rpm for 10 minutes, the supernatant is taken and passes through a column, 12000 is centrifuged for 1 minute, waste liquid is poured out, 500 mu L of PW1,12000 is added for 1 minute, waste liquid is poured out, 600 mu L of PW2,12000 is added for 1 minute, 600 mu L of PW2,12000 is added for 1 minute, the supernatant is poured out, 12000 is idled for 2 minutes, 55 ℃ for 30 to 50 mu L of preheating eluent is added, the solution is left stand for 2 minutes, and 12000 is centrifuged at 1 minute. Concentration detection was performed using a micro nucleic acid quantitative instrument.

The obtained plasmid is subjected to concentration detection, 10 mu L of positive plasmid identified by enzyme digestion is taken and sequenced, and the positive plasmid is stored at-20 ℃. Sequencing results showed that the obtained plasmid was able to encode the variant capsid protein VP1. Finally, relevant Helper plasmids were extracted according to the amount of virus required for the post-test, and each group of Rep-Cap plasmids (control serotypes AAV2, AAV9, myoAAV A,109 and mutants 1-6) plasmids and GOI plasmids (ssaV.CAG.Fluc-2 a-eGFP.WPRE.SV40 pA).

(2) Packaging and purification of mutant serotype viruses

Rep-Cap plasmids of each group (control group serotypes and AAV mutants 1-6) are obtained, GOI plasmids expressing firefly luciferase (Fluc) and green fluorescent protein (EGFP) are co-transferred into HEK-293T cells in proper quantity, AAV viruses are purified by iodixanol gradient ultra-high speed centrifugation, and the virus titer is measured to be proper titer at about 1E+13GC/mL and is placed at-80 ℃ for standby.

Example 3 comparative testing of various indicators of mutant serotypes

(1) Mouse injection and dissection

Animal experiments were performed using Balb/c male mice of 6-8 weeks old, and related viruses were prepared according to designed experimental and control groups (mutant 5, 6 were only 2 mice due to low virus production), each group was injected with 2E11GC virus, living imaging was performed 14 days and 21 days after injection, animal dissection and organ sampling were performed 21 days after injection, liquid nitrogen quick freezing was performed immediately after sample sampling, and the samples were used for subsequent experiments such as RNA extraction and WB detection, respectively.

(2) Living body imaging

Mice were subjected to in vivo imaging at 14 days and 21 days, respectively, after injection. The mice were weighed prior to imaging, and an animal living imaging system (AniView, of the biological technology limited of egluTen, guangzhou) was started in advance and the small animal anesthesia system was commissioned. Setting an image storage path, shooting parameters and other information. Each mouse was injected intraperitoneally luciferin (15 mg/mL, promega, E1605) at a dose of 150mg/kg, i.e., 10. Mu.L/g, and imaging was started 10min after each group injection. Shooting of each batch of mice is completed sequentially according to the sequence of lying on the back, lying on the left side, lying on the prone position and lying on the right side. After shooting is completed, the mice are put back into the mouse cage to wait for anesthesia, and whether the states of the mice are abnormal or not is observed.

(3) Detection of mRNA expression level of target Gene

(3.1) Total RNA extraction and reverse transcription

Extraction of total RNA from samples referring specifically to TransZol Up Plus RNAKit (Beijing full gold, cat# ER 501) instructions. Each time 1mL TranZol up was used, 0.2: 0.2mL RNAExtraction Agent was added, and the mixture was vigorously shaken for 5min, centrifuged at 12,000Xg and 4℃for 10min. The sample is divided into three layers, colorless aqueous phase is transferred into a new 1.5mL RNase-free EP tube, equal volume of absolute ethanol (precipitation may occur at the moment) is added, the mixture is gently inverted and uniformly mixed, the obtained solution and the precipitate are added into a centrifugal column, the centrifugal column is centrifuged at the room temperature for 30s at 12,000 Xg, the filtrate is discarded, 500 mu L CB9 is added, the centrifugal column is centrifuged at the room temperature for 30s at 12,000 Xg, the filtrate is discarded, the previous step is repeated once, 500 mu L WB9 is added, the centrifugal column is centrifuged at the room temperature for 30s at 12,000 Xg, the filtrate is discarded, the previous step is repeated once, the centrifugal column is centrifuged at the room temperature for 2min at 12,000 Xg, residual ethanol is thoroughly removed, the centrifugal column is placed into the 1.5mL RNase-free EP tube, 30-50 mu L RNase-FREE WATER (depending on the tissue size) is added at the center of the centrifugal column, the room temperature is 1min at the room temperature, the room temperature is centrifuged at the room temperature for 1min, and RNA is eluted;

Reverse transcription Using per set of RNA samplesAll-in-One First-STRAND CDNA SYNTHESIS SuperMix for qPCR (One-Step gDNA Removal) (Beijing full gold, cat# AE 341-03), specific steps of which are referred to the specification.

(3.2) Quantitative PCR (qPCR) experiments:

The qPCR system configuration was performed using each set of cDNAs as templates according to the instructions of 2x SYBR Green qPCR Master Mix (Bimake, cat# B21203):

TABLE 2qPCR System

Reagent(s)	Usage amount
		2x SYBR Green qPCR Master Mix	10μL
CDNA template	2μL
		Upstream primer (10. Mu.M)	1μL
Downstream primer (10. Mu.M)	1μL
		ROX Reference Dye	0.4μL
Deionized water	Up to 20μL

TABLE 3qPCR primer information

Primer name	Primer sequence (5 '- > 3')
		Fluc2-qPCR-F1	AACCAGCGCCATTCTGATCA
Fluc2-qPCR-R1	TCGGGGTTGTTAACGTAGCC
		GAPDH-F2	CAGGAGAGTGTTTCCTCGTCC
GAPDH-R2	TTCCCATTCTCGGCCTTGAC

Table 4qPCR program settings

3.3 Data analysis)

Based on the Ct value of each group, the relative expression amount is calculated according to equation 2 ^-ΔΔct.

(4) Detection of expression level of target protein by WB

Sample pretreatment, namely shearing the tissues into tiny fragments, weighing and recording the weight, placing the fragments into a 1.5ml or 2ml centrifuge tube, marking the tubes, freezing the fragments at-80 ℃ for later use, precooling a cryogrinder, dissolving RIPA (Biyun Tian, P0013B) lysate (PMSF is added in a few minutes before use to make the final concentration of PMSF be 1 mM);

The complete lysate is added according to the proportion of 150-250 mu L of lysate added per 20mg of tissue, then two sterilized zirconia grinding beads are added, and the samples (brain, spinal cord and other tissue samples: temperature-20 ℃ C., frequency 70Hz, time for shaking 50s and stopping 10s, circulation 3-4 times, muscle, liver and other samples: temperature-20 ℃ C., frequency 70Hz, time for shaking 50s and stopping 10s,5-7 times) are directly ground in the lysate. After grinding the sample, centrifuging the sample in a refrigerated centrifuge at 4 ℃ and 12,000Xg for 5-10min, and transferring the supernatant to a new sterilized EP tube for preservation at-20 ℃ or-80 ℃;

Protein concentration determination, namely, after protein concentration is determined according to the method in the modified BCA method protein concentration determination kit (manufacturer, cat# C503051), a proper amount of protein homogenate sample is taken according to the required dosage, and is mixed with a corresponding amount of 5X SDS-PAGE protein loading buffer, and the mixture is cooled in boiling water for 10min, centrifuged at a low speed for a moment, and then loaded.

WB (Western Blot) detection:

A SDS-PAGE electrophoresis, which comprises determining proper sample amount according to protein concentration and expression level, less than 20 mu L/hole, and tissue homogenate protein sample amount about 20-50 mu g, wherein the electrophoresis specific operation process comprises the steps of extracting a comb on a prefabricated gel, mounting the gel on an electrophoresis tank, adding electrophoresis buffer solution into an inner tank and an outer tank, adding newly prepared buffer solution into the inner tank, detecting leakage, adding electrophoresis buffer solution into the outer tank if no leakage exists, sampling a proper amount of treated protein sample, performing 100V constant pressure electrophoresis on a natural energy electrophoresis device by using a pre-dyed standard protein as a reference, and the electrophoresis time is 100min until bromophenol blue reaches the bottom of the gel. Closing the power supply, carefully removing the prefabricated rubber plate, taking down the gel, and placing the gel in a film transfer buffer solution to wait for subsequent operation;

B. transferring membrane, namely cutting 6 pieces of filter paper and 1 piece of PVDF membrane according to the glue area. The PVDF membrane is soaked in methanol for 5-10sec, then transferred to a transfer buffer solution for 5min, the filter paper is also pre-wetted in the transfer buffer solution, and a transfer device, namely a negative electrode (blackboard) -sponge-3 layers of wetted filter paper-gel-PVDF membrane-3 layers of wetted filter paper-sponge-positive electrode (transparent plate), is installed. Removing each layer of bubbles to avoid influencing transfer effect, clamping the bracket, placing into an electric transfer tank, transferring the film for 100min by using a 100V constant-pressure ice bath, judging whether the film transfer is successful or not according to whether the pre-dyed protein molecular weight standard strip is completely transferred to the PVDF film, soaking the transferred PVDF film in PBST solution, washing for 5min at room temperature, cutting the PVDF film according to the requirement, and taking care not to dry the PVDF film in the film cutting process;

C. Incubating PVDF membrane with blocking solution (5% skimmed milk powder) at room temperature for 2H or 4 ℃ overnight, transferring the blocked PVDF membrane into primary antibody hybridization solution (Luciferase Rabbit Polyclonal antibody (Proteintech, 27986-1-AP) according to 1:2000;GADPH Rabbit Polyclonal antibody (Proteintech, 10494-1-AP) according to 1:2000;Rabbit GFP tag Polyclonal antibody (Proteintech, 50430-2-AP) according to 1:2000, respectively adding into 4ml QuickBlock ^TM Western primary antibody dilution (Biyun, P0256) according to 1:2000, incubating at room temperature for 1H or 4 ℃ overnight, washing membrane with PBST for 3×5min, transferring the washed PVDF membrane into secondary antibody hybridization solution (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, SA 00001-2) according to 1:5000, adding into 4ml QuickBlock ^TM Western secondary antibody dilution (Biyun, P0258) according to 1:2000, incubating at room temperature for 1H, washing membrane with PBST for 3×5min;

D. And (3) color development, namely mixing the liquid A and the liquid B of the ECL chemiluminescence kit in equal volumes, vibrating and uniformly mixing, and then dripping the luminous liquid on the PVDF film to ensure that the PVDF film is covered with the luminous liquid, adjusting different exposure time to ensure that protein strips are clear, and photographing by an instrument.

(5) Cynomolgus monkey injection, puncture and NGS analysis

The animal experiment uses male cynomolgus monkey of about 4 years old, and the male cynomolgus monkey is used after the neutralizing antibody of AAV2 and AAV9 is detected to be qualified before the experiment. Different serotypes of mutants and control serotypes were packaged with different GOIs (ssav.cag.fluc.wpre.polya vectors carrying different Barcode) and given intravenous injection (total dose of mixed virus controlled to 3E13 GC/Kg) at equal viral amounts, muscle and liver punctures at different sites at 2 and 4 weeks respectively, and finally tissue RNA extraction, RT-PCR and NGS sequencing were performed and the fold ratio of each serotype mutant relative to control AAV9 was determined by analysis of NGS data.

Through the verification of different methods of mouse experiments, the obtained mutants 1-6 are found to have better muscle targeting than AAV9 and retain the low liver tropism characteristic of the skeleton (the result can be intuitively observed from the in-vivo imaging results of 14 days and 28 days in FIG. 1), wherein the mutants 1 and 3 even show stronger muscle targeting than the serotypes 109 and MyoAAV A. Mutant 1 had mRNA levels in gastrocnemius, quadriceps femoris, triceps brachii, biceps brachii, abdominal muscle and heart that were 75.97-fold, 37.27-fold, 186.02-fold, 13.79-fold, 496.41-fold and 10.06-fold, respectively, and mutant 3 had mRNA levels in gastrocnemius, quadriceps femoris, triceps brachii, biceps brachii, abdominal muscle and heart that were 71.95-fold, 43.25-fold, 112.55-fold, 4.99-fold, 247.96-fold and 9.46-fold, respectively, of AAV9 (fig. 2. A-7. A). The protein level results for mutant 1 and mutant 3 were substantially consistent with the trend of mRNA levels (fig. 2. B-7. B). Liver results (fig. 8) further confirm that serotype mutants based on low liver eosinophil backbones all showed low liver targeting, mRNA levels were 50-100 fold lower than AAV9, and still 9.5-19 fold reduced for MyoAAV a (obtained based on AAV9 backbone screening), showing very good targeting specificity.

To further illustrate the potential clinical utility of serotypes of the invention, mutant and control serotypes were intravenously injected into cynomolgus monkeys in an equal mix, and finally the relationship of expression levels of the different serotypes in muscle and liver tissue was determined by NGS analysis. Mutant 1 and mutant 3 showed good results in various muscles in cynomolgus monkeys, consistent with the results in mice, wherein the mRNA levels of mutant 1 in gastrocnemius, biceps brachii, triceps brachii, quadriceps femoris (4 weeks) were 5.75-fold, 13.31-fold, 35.05-fold, 15.76-fold, respectively, of AAV9 (fig. 9), except that the expression effect in gastrocnemius was slightly lower than MyoAAV a, the expression in other muscles was better than MyoAAV a, and the expression trend in 2 weeks was substantially consistent with 4 weeks. Slightly different from the effect of the mice, mutant 2, which is slightly inferior in the muscle of the mice, has a good effect on the muscle of the cynomolgus monkey, and the effect of part of the muscles (such as gastrocnemius and quadriceps femoris) is close to that of mutant 1. Furthermore, all muscle mutants obtained by screening, whether in mice or cynomolgus monkeys, exhibited liver tropism well below AAV9 and MyoAAV a, further demonstrating the identity and superiority of the scaffold in cross-species use.

In summary, the invention utilizes the strategy of constructing a small AAV mutant library to obtain a plurality of serotype mutants with better muscle targeting than AAV9, and respectively verifies that the serotype mutants have good effects in muscle tissues such as gastrocnemius, quadriceps femoris, triceps brachii, biceps brachii and abdominal muscles, and the like from mRNA and protein expression levels, and have lower hepatic tropism and better specificity. The mutants can further evaluate the clinical application value and safety, provide more useful and selectable carrier tools for gene therapy of muscle diseases, and benefit patients.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An adeno-associated virus capsid protein mutant, characterized in that its amino acid sequence is shown in SEQ ID No.7.

2. A nucleic acid encoding an adeno-associated virus capsid protein mutant, characterized in that its nucleotide sequence is shown in SEQ ID No.13.

3. An expression vector, characterized in that it comprises the nucleic acid according to claim 2.

A host cell, characterized in that it comprises the expression vector according to claim 3.

5. A host cell, characterized in that it expresses the adeno-associated virus capsid protein mutant according to claim 1.

6. A recombinant adeno-associated virus, characterized in that it comprises the adeno-associated virus capsid protein mutant according to claim 1.

7. The recombinant adeno-associated virus according to claim 6, further comprising a heterologous target gene.

8. The recombinant adeno-associated virus according to claim 7, characterized in that the heterologous target gene encodes any gene product of interfering RNA, aptamer, endonuclease, and guide RNA.

9. A method for preparing a recombinant adeno-associated virus, characterized in that it comprises introducing at least the following components into a host cell: 1) the nucleic acid described in claim 2 or the expression vector described in claim 3, 2) an adeno-associated virus helper plasmid, and 3) a plasmid containing two terminal inverted repeat sequences.

10. The rAAV prepared by the method according to claim 9.

11. A pharmaceutical composition comprising the recombinant adeno-associated virus according to any one of claims 6 to 8 or the rAAV according to claim 10, and a pharmaceutically acceptable carrier.

12. Use of the adeno-associated virus capsid protein mutant of claim 1, the expression vector of claim 3, the host cell of claim 4 or 5, the recombinant adeno-associated virus of any one of claims 6-8, and the rAAV of claim 10 in the preparation of a preparation for delivering gene products to cells or tissues of a subject, characterized in that the cells are muscle cells or heart cells; the tissues are muscle tissues or heart tissues.

13. Use of the adeno-associated virus capsid protein mutant of claim 1, the expression vector of claim 3, the host cell of claim 4 or 5, the recombinant adeno-associated virus of any one of claims 6-8, and the rAAV of claim 10 in the preparation of a drug for delivering gene products to cells or tissues of a subject, characterized in that the cells are muscle cells or heart cells; the tissues are muscle tissues or heart tissues.