Adeno-associated virus mutant and use thereof

Abstract

The present invention belongs to the technical field of biopharmaceuticals, and disclosed are an adeno-associated virus mutant, and a use thereof. An amino acid sequence of the adeno-associated virus capsid protein mutant comprises any one of the sequences shown in SEQ ID No. 1 to SEQ ID No. 4. The adeno-associated virus capsid protein mutant, and an expression vector, host cell and recombinant adeno-associated virus thereof, are used in the preparation of a drug delivery tool for preventing and / or treating muscular or cardiac disease. The adeno-associated virus capsid protein mutant has muscle or cardiac targeting properties, with muscle targeting improved by up to about 258 times, and hepatic tropism nearly 100 times lower than that of the control group, exhibiting good specificity. It can promote the development of AAV-based gene therapy methods toward large-scale and societal application.

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 and gaining widespread application. 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 the US 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 U.S. Food and Drug Administration (FDA) granted full approval to ELEVIDYS for patients aged 4 years and older with DMD who can walk independently, and accelerated approval (conditional marketing authorization) 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, AAV treatment also 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.

[0004] In addition, the current AAV screening strategy, which is mainly based on directed evolution, can obtain some effective mutants in large-scale screening. However, the screening process is cumbersome and expensive. In particular, the targeting ability failure caused by species differences and the huge cost of screening in monkeys not only increase the threshold for researchers to participate, but also the high development cost is ultimately reflected in the cost of drug use.

[0005] Therefore, the main purpose of AAV serotyping is to develop serotypes that have better therapeutic effects, lower treatment doses, fewer side effects, and lower usage costs, so as to obtain drugs with higher targeting and thus lower drug doses, or to make drugs more specific and thus avoid adverse reactions. Summary of the Invention

[0006] This invention provides an adeno-associated virus mutant with muscle or heart targeting capabilities and its applications.

[0007] The technical solution adopted in this invention is as follows:

[0008] In a first aspect, the present invention provides an adeno-associated virus capsid protein mutant, the amino acid sequence of which includes any of the sequences shown in SEQ ID No. 1 to 4.

[0009] The adeno-associated virus capsid protein mutant of the present invention has muscle or heart targeting, especially good targeting to different muscle tissues (quadriceps, biceps and abdominal muscles, etc.). Compared with the control group AAV9, the muscle targeting is increased by up to about 258 times, and the liver tropism is also nearly 100 times lower than that of the control group, with good specificity.

[0010] 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. 5 to 8.

[0011] Secondly, the present invention provides a nucleic acid encoding the adeno-associated virus capsid protein mutant.

[0012] In some embodiments, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID No. 9-12.

[0013] Thirdly, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0014] Fourthly, the present invention provides a host cell comprising the aforementioned expression vector.

[0015] Fifthly, the present invention provides a host cell that expresses the aforementioned adeno-associated virus capsid protein mutant.

[0016] In a sixth aspect, the present invention provides a recombinant adeno-associated virus, including the aforementioned adeno-associated virus capsid protein mutant.

[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 adenovirus 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 inserts the RGD peptide into a low-hepatophilia backbone, resulting in an adeno-associated virus (AAV) capsid protein mutant with muscle or cardiac targeting, particularly showing good targeting of different muscle tissues (quadriceps, biceps, and abdominal muscles, etc.). Compared to the control group AAV9, muscle targeting is increased by up to approximately 258 times, and hepatophilia is nearly 100 times lower, demonstrating good specificity, safety, and broad applicability. This invention develops a novel AAV gene therapy product with lower dosage requirements and costs to meet the needs of more diverse patients and promote the large-scale, socialized application of AAV-based gene therapy methods. This will be of great significance in improving the efficacy of gene therapy and serving a wider range 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 represents mice infected for 14 days and B represents mice infected for 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 muscles (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 abdominal muscles 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 gastrocnemius muscle 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 targeting analysis of different serotypes on other organs of Balb / c mice (21 days, relative mRNA expression level); in Figure 9, A is the lung, B is the kidney, and C is the brain.

[0037] Figure 10 shows the NGS detection analysis of muscle targeting and liver tropism of different serotypes in cynomolgus monkeys; in Figure 10, A is the puncture at 14 days and B is the puncture at 28 days. Detailed Implementation

[0038] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] "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).

[0048] "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.

[0049] 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.

[0050] 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.

[0051] 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).

[0052] 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.

[0053] 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.

[0054] "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.

[0055] This application also provides the following technical solution: an adeno-associated virus capsid protein mutant, which contains a targeting peptide sequence, wherein the amino acid sequence of the targeting peptide is SX1X2X3RGDX4X5X6X7X8A; wherein X1-X8 are each independently selected from any one of 20 basic amino acids.

[0056] 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.

[0057] 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:

[0058] In some embodiments, X3 is selected from any one of L, R, and A; and / or X4 is selected from any one of L and M. In some embodiments, X1X2X3 is selected from any one of PNL, ASA, TGR, and ISR. In some embodiments, X4X5X6X7X8 is selected from any one of LQVLA, LAHLT, LATIH, and MSSNP.

[0059] In some embodiments, the amino acid sequence of the targeting peptide is as shown in any of SEQ ID Nos. 5-8, or has one or two amino acid changes compared to SEQ ID Nos. 5-8. In some embodiments, the one or two amino acid changes do not occur at the RGD position 5-7 of the targeting peptide. In some embodiments, the one or two amino acid changes do not occur at the RGD position 5-7 of the targeting peptide, nor at the S position 1 and the A position 13.

[0060] 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. 1-4, or includes a homologous sequence to any of the sequences in SEQ ID No. 1-4; 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. 1-4. In some embodiments, the differences between the homologous sequence and SEQ ID No. 1-4 do not alter the sequence of the targeting peptide.

[0061] This application also provides a nucleic acid encoding any of the adeno-associated virus capsid protein mutants described in this application.

[0062] This application also provides an expression vector comprising the nucleic acid described in this application.

[0063] This application also provides a host cell that includes the expression vector described in this application.

[0064] This application also provides a host cell that expresses any of the adeno-associated virus capsid protein mutants described in this application.

[0065] This application also provides a recombinant adeno-associated virus, which includes any of the adeno-associated virus capsid protein mutants described in this application.

[0066] In some embodiments, the recombinant adeno-associated virus further includes the heterologous target gene described in this application.

[0067] 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.

[0068] This application also provides rAAV prepared by the preparation method described in this application.

[0069] This application also provides a pharmaceutical composition comprising the recombinant adeno-associated virus or the rAAV described in this application, and a pharmaceutically acceptable carrier.

[0070] 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.

[0071] 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 and / or heart diseases described in this application.

[0072] 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.

[0073] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0074] Example 1: Construction of AAV capsid protein mutant and production of virus

[0075] (1) Discovery and determination of target peptide sequences

[0076] This invention explores the structural and functional analysis of naturally occurring viruses or ligand peptides, applying different strategies to construct candidate serotype mutants in a more direct and cost-effective manner. The RGD motif can bind to integrin receptor motifs, and some integrin receptors are relatively specifically expressed in muscle cells. This invention screens for relevant ligands of integrin receptors and viruses capable of infecting muscles, identifying potential muscle-targeting peptides, particularly those containing the RGD motif, and then conducting relevant experimental verification analyses.

[0077] The peptide sequences selected in this invention are shown in Table 1:

[0078] Table 1 shows the selected peptide sequences.

[0079] The “S” and “A” at both ends of the amino acid sequence in Table 1 are used as the linking sequence between the serotype VP1 backbone and the target peptide. For example, the amino acid sequence of the target peptide is replaced by R584 to R587 of the low hepatotropic serotype (SEQ ID NO.13) VP1 or S584 to R594 of serotype 109 (SEQ ID NO.14) VP1 to form a new serotype VP1 sequence.

[0080] (2) Construction of mutant serotype vector and plasmid extraction

[0081] 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.

[0082] 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 as a template, the target product YJ561-1 was amplified and recovered by gel amplification using the Cap-f+YJ561-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ561-2 was amplified and recovered by gel amplification using the YJ561-F+cap-r primer. By mixing the backbone fragment, YJ561-1, and YJ561-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 1 can be recombined and constructed.

[0083] 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 YJ563-1 was amplified and recovered by gel amplification using the Cap-f+YJ563-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ563-2 was amplified and recovered by gel amplification using the YJ563-F+cap-r primer. By mixing the backbone fragment, YJ563-1, and YJ563-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 2 can be recombinantly constructed.

[0084] 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 YJ565-1 was amplified and recovered by gel amplification using the Cap-f+YJ565-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ565-2 was amplified and recovered by gel amplification using the YJ565-F+cap-r primer. By mixing the backbone fragment, YJ565-1, and YJ565-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 3 can be recombinantly constructed.

[0085] 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 YJ567-1 was amplified and recovered by gel amplification using the Cap-f+YJ567-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ567-2 was amplified and recovered by gel amplification using the YJ567-F+cap-r primer. By mixing the backbone fragment, YJ567-1, and YJ567-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 4 can be recombined and constructed.

[0086] 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 YJ562-1 was amplified and recovered by gel amplification using the Cap-f+YJ562-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ562-2 was amplified and recovered by gel amplification using the YJ562-F+cap-r primer. By mixing the backbone fragment, YJ562-1, and YJ562-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 5 can be recombinantly constructed.

[0087] 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 YJ564-1 was amplified and recovered by gel amplification using the Cap-f+YJ564-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ564-2 was amplified and recovered by gel amplification using the YJ564-F+cap-r primer. By mixing the backbone fragment, YJ564-1, and YJ564-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 6 can be recombinantly constructed.

[0088] Based on the Cap sequence of mutant 7, 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 YJ566-1 was amplified and recovered by gel amplification using the Cap-f+YJ566-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ566-2 was amplified and recovered by gel amplification using the YJ566-F+cap-r primer. By mixing the backbone fragment, YJ566-1, and YJ566-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 7 can be recombined and constructed.

[0089] Based on the Cap sequence of mutant 8, 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 YJ568-1 was amplified and recovered by gel amplification using the Cap-f+YJ568-R primer. Using the Rep-CAP plasmid of serotype 109 as a template, the target product YJ568-2 was amplified and recovered by gel amplification using the YJ568-F+cap-r primer. By mixing the backbone fragment, YJ568-1, and YJ568-2 in the following steps and proportions, the Rep-CAP plasmid of mutant 8 can be recombined and constructed.

[0090] The primers used in the construction of the Rep-CAP vector for the above AAV capsid protein mutants are shown in Table 2:

[0091] Table 2 Primer sequences

[0092] 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 antibiotic-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.

[0093] 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.

[0094] 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 1-8), and GOI plasmid (ssAAV.CAG.Fluc-2a-eGFP.WPRE.SV40pA) were extracted.

[0095] (3) Packaging and purification of mutant serotype viruses

[0096] The Rep-Cap plasmids obtained from each group (control group serotype and AAV mutants 1-8), the GOI plasmids expressing firefly luciferase (Fluc) and green fluorescent protein (EGFP), and the pHelper plasmid were co-transfected into HEK-293T cells at appropriate amounts. The AAV virus was purified by gradient ultracentrifugation with iodixanol. The appropriate viral titer was measured to be between 1E+12GC / mL and 1E+13GC / mL. The cells were then stored at -80℃ for later use.

[0097] Example 2: Comparative testing of various indicators of mutant serotypes

[0098] (1) Mouse injection and dissection

[0099] 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 (mutant 5 was only used in 1 mouse due to its 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.

[0100] (2) In vivo imaging

[0101] 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 uL / 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.

[0102] (3) Detection of target gene mRNA expression level

[0103] (3.1) Total RNA extraction and reverse transcription:

[0104] 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 50-100mg tissue to 1ml TransZol Up, 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.

[0105] 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.

[0106] 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℃.

[0107] 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.

[0108] (3.2) Quantitative PCR (qPCR) experiment:

[0109] 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):

[0110] Table 3 qPCR system

[0111] Table 4 qPCR program settings

[0112] Table 5 qPCR primer sequences

[0113] (3.3) Data Analysis

[0114] Based on the Ct value of each group, according to Formula 2 -ΔΔct Calculate the relative expression level.

[0115] (4) Western blot analysis to detect the expression level of the target protein

[0116] Sample pretreatment: Cut the tissue into small fragments, weigh and record the weight, place them in 1.5 mL or 2 mL centrifuge tubes, label the tubes, freeze at -80℃ for later use, pre-cool the cryogenic grinder; dissolve RIPA (Beyotime, P0013B) lysis buffer (add PMSF a few minutes before use to make the final PMSF concentration 1 mM).

[0117] 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℃.

[0118] 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.

[0119] WB (Western Blot) detection:

[0120] 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.

[0121] 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.

[0122] 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;

[0123] 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.

[0124] (5) Injection, puncture and NGS analysis of cynomolgus monkeys

[0125] 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.

[0126] The results of the above-mentioned validation experiments show that serotypes with different ligand peptide insertions and different insertion strategies, although all containing the RGD motif, exhibit significantly different effects. For example, serotype mutants 1-4, with longer inserted peptides, are significantly more effective than mutants 5-8, which have shorter insertion lengths. Furthermore, mutant 4 shows the best targeting effect on mouse muscle, followed by mutants 2 and 3. In the gastrocnemius, quadriceps, triceps, biceps, and abdominal muscles, the infection efficacy of mutant 4 is 53.84 times, 55.76 times, 257.58 times, 12.63 times, and 54.47 times that of AAV9, respectively. Its efficacy in the biceps and abdominal muscles is better than the control MyoAAV4A, which may be related to the previously developed low hepatophilicity scaffold (similar trends are observed with 109 using the same scaffold). In cardiac tissue, mutant 4 was less effective than mutants 2 and 3 (10.16 and 10.36 times that of AAV9, respectively) and the control MyoAAV4A, indicating stronger muscle specificity. In mouse liver, the low hepatophilicity of the scaffold was particularly pronounced. Different targeting peptides using this scaffold were significantly lower than those of AAV9 and MyoAAV4A based on the AAV9 scaffold. The liver expression of mutants 1-4 was 0.02, 0.01, 0.01, and 0.01 times that of AAV9, respectively. The mRNA effects of these serotype mutants on muscle and liver were largely consistent with in vivo imaging and Western blotting results, providing mutual corroboration. Furthermore, regarding targeting effects on different tissues such as the brain, lungs, and kidneys, except for a few serotypes that were slightly higher than AAV9 (e.g., mutant 2 in the brain was 1.85 times higher than AAV9, and mutant 5 in the kidney was 1.82 times higher than AAV9), the effects were generally lower than AAV9, especially in the brain and kidneys, where the differences between serotypes were not significant.

[0127] To further illustrate the potential clinical value of the serotypes in this invention, mutant and control serotypes 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. Significantly different from the mouse results, mutant serotype 4, which performed best in mouse muscle, was less effective in cynomolgus monkeys. Conversely, mutant serotype 3 showed the best effect among many mutant individuals; this difference may stem from species differences. Furthermore, consistent with the mouse results, mutants 1-4, with longer insert peptide lengths, were significantly more effective than mutants 5-8, which is one of the innovative aspects of this invention. Another point consistent with the mouse results is the liver tropism of different serotypes. Using the low-hepatic-tropy backbone 109 and other mutants, compared to AAV9 and MyoAAV4A using the AAV9 backbone, all exhibited low hepatic tropism, and the trends at 2 and 4 weeks were consistent.

[0128] In summary, by utilizing naturally occurring peptides containing the RGD motif, such as peptides from certain viruses or integrin ligands, and employing different construction strategies, we screened for peptides that better match and are compatible with the previously developed low-hepatophilic AAV backbone. This resulted in several serotype mutants with superior muscle targeting compared to 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 hepatophilicity and good 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, ultimately benefiting a wider range of patients.

[0129] 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, Its amino acid sequence includes any of the sequences shown in SEQ ID No. 1 to 4.

2. The adeno-associated virus capsid protein mutant according to claim 1, characterized in that, The amino acid sequence contains an inserted targeting peptide; the amino acid sequence of the targeting peptide is any one of the sequences shown in SEQ ID No. 5 to 8.

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

4. The nucleic acid according to claim 3, characterized in that, Its nucleotide sequence contains the nucleotide sequences shown in SEQ ID No. 9 to 12.

5. An expression carrier, characterized in that, It includes the nucleic acid as described in claim 3 or 4.

6. A host cell, characterized in that, It includes the expression vector as described in claim 5.

7. A host cell, characterized in that, It expresses the adeno-associated virus capsid protein mutant as described in claim 1 or 2.

8. A recombinant adeno-associated virus, characterized in that, Including the adeno-associated virus capsid protein mutant as described in claim 1 or 2.

9. The recombinant adeno-associated virus according to claim 8, characterized in that, It also includes heterologous target genes.

10. The recombinant adeno-associated virus according to claim 9, characterized in that, The heterologous target gene encodes any one of the following gene products: interfering RNA, aptamer, endonuclease, or guide RNA.

11. A method for preparing recombinant adeno-associated virus, characterized in that, Including the introduction of at least the following components into the host cell: 1) the nucleic acid of claim 3 or 4 or the expression vector of claim 5, 2) the adeno-associated virus helper plasmid, and 3) the 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 vector.

14. The 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, and the rAAV of claim 12 in the preparation of a drug or formulation for delivering a gene product to the cells or tissues of a subject.

15. The application according to claim 14, characterized in that, The cells are muscle cells or heart cells; the tissue is muscle tissue or heart tissue.

16. The 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, and the rAAV of claim 12 in the preparation of drug delivery tools for the prevention and / or treatment of muscle or heart diseases.

17. The application according to claim 16, characterized in that, The muscle diseases mentioned 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 heart diseases mentioned 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.

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