Aav9 capsid protein variants and uses thereof

Abstract

The application provides an AAV9 capsid protein variant and use thereof. The inventors have found, through research, that by modifying the AAV9 capsid protein, changing the amino acid sequence of the capsid protein to a sequence as shown in SEQ ID NO: 3, the infection capacity can be significantly improved, especially the transduction efficiency in the liver, thereby providing a new prospect for gene therapy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an AAV9 capsid protein variant and its uses. Background Technology

[0002] AAV, or adeno-associated virus, is a small virus containing DNA particles. It is a single-stranded DNA virus that can infect humans and some other primates. AAV differs from adenovirus in its antigens and can only replicate in cell cultures when co-inoculated with adenovirus, making it a defective virus. AAV is a non-enveloped, icosahedral protein-coated, single-stranded DNA virus with a genome length of approximately 4.7 kb and a diameter of approximately 22 nm, making it a very small virus.

[0003] Gene therapy, a cutting-edge biomedical field, aims to treat a range of genetic and chronic diseases by adjusting, repairing, or replacing defective genes in patients. Since the 1990s, gene therapy has made significant progress and achieved success in several clinical trials. Adeno-associated virus (AAV) has become one of the most promising viral vectors in gene therapy due to its low immunogenicity, high safety, and non-integrating characteristics. AAV9 can infect a variety of tissues, including neurons, heart, liver, muscle, and lung cells, making it applicable to a wide range of conditions. Especially in neurological and muscular diseases, AAV9 is highly favored due to its efficient cross-tissue infection capabilities.

[0004] Wild-type AAV can achieve different tissue distributions after systemic administration depending on different serotypes, but its infection efficiency in specific tissues and cells is still not ideal, resulting in insufficient expression levels of the target gene and limiting its clinical efficacy. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an AAV9 capsid protein variant and its uses to improve AAV infection efficiency. As a key structure for AAV binding to host cells, the sequence and structure of the capsid protein directly determine the cell adhesion, internalization, and uncoating efficiency of AAV. Therefore, to improve AAV infection efficiency, the wild-type AAV9 capsid Cap sequence was mutated, and preferred AAVLYR2001 and AAVLYR2003 capsids were obtained through computer simulation and cell surface receptor binding. Among them, AAVLYR2003 has significantly higher infectivity than the wild-type.

[0006] The present invention provides an adeno-associated virus capsid protein variant, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0007] The capsid protein variant of the present invention was obtained by modifying the Cap sequence of AAV9. The Cap sequence of AAV9 mainly encodes VP1, VP2, and VP3, and the amino acid sequence of the Cap protein of AAV9 is shown in SEQ ID NO: 5.

[0008] Another aspect of the present invention provides a gene fragment encoding a capsid protein variant as described above.

[0009] In some embodiments, the nucleotide sequence encoding the gene fragment of the capsid protein variant shown in SEQ ID NO: 3 is as shown in SEQ ID NO: 4.

[0010] Another aspect of the present invention provides a vector containing the above-mentioned gene fragment.

[0011] Another aspect of the present invention provides a host cell comprising the above-described vector.

[0012] Another aspect of the present invention provides a recombinant adeno-associated virus (AAV9) variant containing a gene fragment encoding a capsid protein with the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the recombinant AAV9 variant further includes a gene fragment encoding a rep protein.

[0013] Furthermore, the recombinant adeno-associated virus (AAV9) variant also contains the target gene to be delivered. The target gene is used for gene therapy.

[0014] Another aspect of the present invention provides the use of the above-mentioned recombinant adeno-associated virus rAAV9 variant in the preparation of gene therapy drugs.

[0015] Another aspect of the present invention provides a gene therapy drug that uses the aforementioned recombinant adeno-associated virus (adeno-associated virus) rAAV9 variant as a vector. The target gene is carried by the recombinant adeno-associated virus rAAV9 variant and administered to patients via intravenous injection, intramuscular injection, subcutaneous injection, intrapleural injection, or inhalation to achieve the purpose of gene therapy and significantly improve AAV infection capacity.

[0016] Furthermore, the gene therapy drugs are mainly used to treat liver diseases, such as genetic disorders, non-alcoholic fatty liver disease (NAFLD), chronic hepatitis, and liver cancer.

[0017] A pharmaceutical composition comprising the gene therapy drug described above.

[0018] In addition to the gene therapy drugs mentioned above, the drug composition may also contain related adjuvant therapy drugs or other drugs for combination therapy, such as immunosuppressants, IgG degrading agents and controlled expression inducers.

[0019] As described above, the AAV9 capsid protein variant of the present invention has the following beneficial effects:

[0020] The AAV9 capsid protein variant provided in this application exhibits significantly enhanced infection efficiency in the liver compared to wild-type AAV9. Attached Figure Description

[0021] Figure 1 The infection efficiency of novel AAV capsids AAVLYR2001 and AAVLYR2003 in mouse livers was compared with that of wild-type AAV9. Figure 1 The data presented in A are relative luciferase activities (reflecting infection efficiency), expressed as mean ± standard error (SEM). The sample size for each group is 3. The difference markers between groups are ns for p>0.05 and *** for p<0.001. Figure 1 The data presented in B are bioluminescent images of mouse liver tissue. Detailed Implementation

[0022] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the viral class associated with this name and belonging to the genus *Parvovirus-dependent* within the family Parvoviridae. AAV is a single-stranded DNA virus that grows exclusively in cells, where certain functions are provided by co-infecting helper viruses. All AAV serotypes exhibit remarkably similar replication characteristics mediated by homologous rep genes; and all possess three associated capsid proteins. At least 13 sequentially numbered naturally occurring AAV serotypes are known in the art.

[0023] The AAV genome consists of open reading frames Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). These ITR base pairs allow for the synthesis of complementary DNA strands. Rep and Cap are translated into a variety of different proteins (Rep78, Rep68, Rep52, Rep40 – proteins required for the AAV life cycle; VP1, VP2, VP3 – capsid proteins).

[0024] As used herein, the terms "nucleotide" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases, substantially composed of said bases, or composed of said bases. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 4.

[0025] As used herein, the terms "protein" or "peptide" refer to a polymeric form of amino acids of any length. In some embodiments, the protein comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 3.

[0026] As used herein, the term "vector" refers to a nucleic acid containing, substantially composed of, or composed of an intact replicon or terminal inverted repeat (ITR), such that when the vector is placed inside a cell by, for example, transfection, infection, or transformation, it can be preserved and / or replicated. It should be understood in the art that once inside a cell, a vector can be preserved and / or replicated as an extrachromosomal (free) element, or it can be integrated into the host cell chromosome.

[0027] In this document, for the purposes of this disclosure, recombinant AAV refers to AAV genomic DNA that has been genetically modified relative to wild-type AAV genomic DNA. It should be understood that, for the purposes of rAAV, the functional ITR sequence may be, but is not required to be, a wild-type nucleotide sequence, which may be altered, for example, by nucleotide insertion, deletion, or substitution, as long as it still provides the functions required for rescue, replication, and packaging. Therefore, in this document, an rAAV vector is a viral vector that contains at least a functional ITR that provides the functions required for viral replication and packaging in cis. Furthermore, it should be understood that the recombinant AAV viral genome may contain two or more (e.g., three) ITR sequences, and these ITR sequences may be identical or different.

[0028] As used herein, the term "capsid protein" refers to the proteins that constitute the outer shell of AAV viral particles, including VP1, VP2, and VP3, and determine the infectivity characteristics of the virus. In some embodiments, the capsid protein is a protein with an amino acid sequence such as SEQ ID NO: 3 or containing the amino acid sequence SEQ ID NO: 3.

[0029] As used herein, the term "treatment" refers to administering to a subject an effective amount of a polynucleotide, nucleic acid construct, vector, recombinant AAV, or pharmaceutical composition as described herein, such that the subject experiences a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Treatment may refer to extended survival compared to expected survival without treatment. Therefore, those skilled in the art will recognize that treatment may improve the disease condition but may not be a complete cure. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" in cases where disease progression is reduced or stopped. "Treatment" may also mean extended survival compared to expected survival without treatment. In some embodiments, patients requiring treatment include those already diagnosed with a hereditary or acquired disease.

[0030] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0032] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

[0033] Example 1 Preparation of AAV particles

[0034] The AAV9 capsid AAV-Cap9 sequence (nucleotide sequence as shown in SEQ ID NO: 6, amino acid sequence as shown in SEQ ID NO: 5) was mutated, and novel preferred AAV9 capsid protein variants, SEQ ID NO: 2 and SEQ ID NO: 4 (encoding polypeptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively), were obtained through computer simulation and binding to cell surface receptors. These were named AAV-CapR2001 and AAV-CapR2003, respectively. The novel capsid variant sequences were cloned into the capsid protein coding region of the pHAAV-RC9 packaging plasmid to construct novel AAV packaging plasmids LYR2P001 and LYR2P003. LYR2P001 or LYR2P003, the pHelper helper plasmid, and an AAV vector plasmid containing the firefly luciferase reporter gene were co-transfected into HEK293T cells. The wild-type AAV9 packaging plasmid pHAAV-RC9 was used as a control. Cells were collected 72 hours after transfection, and crude extracts of AAVR2001-Luci virus, AAVR2003-Luci virus, and AAV9-Luci virus were obtained by lysis. AAV particles were purified by iodixanol density gradient centrifugation and ultrafiltration, and the viral titer (vg / mL) was determined by ddPCR. The results showed that the plasmid yields per unit volume of the novel AAV-CapR2001 and AAV-CapR2003 capsid variants were slightly higher than those of wild-type AAV9, at 1.3 × 10⁻⁶. 11 vg / mL cells, 1.5 × 10 11 vg / mL cells and 1.2 ×10 11 vg / mL cells.

[0035] Table 1 AAV capsid sequence

[0036] Sequence Name Amino Acid Sequence SEQ IDNO. Nucleotide Sequence SEQ IDNO. AAV-CapR2001 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQSSSHFQGAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL 1 atggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggtgggctttgaaacctggagcccctcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtcttgtgcttccgggttacaaataccttggacccggcaacggactcgacaagggggagccggtcaacgcagcagacgcggcggccctcgagcacgacaaggcctacgaccagcagctcaaggccggagacaacccgtacctcaagtacaaccacgccgacgccgagttccaggagcggctcaaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaaaaagaggcttcttgaacctcttggtctggttgaggaagcggctaagacggctcctggaaagaagaggcctgtagagcagtctcctcaggaaccggactcctccgcgggtattggcaaatcgggtgcacagcccgctaaaaagagactcaatttcggtcagactggcgacacagagtcagtcccagaccctcaaccaatcggagaacctcccgcagccccctcaggtgtgggatctcttacaatggcttcaggtggtggcgcaccagtggcagacaataacgaaggtgccgatggagtgggtagttcctcgggaaattggcattgcgattcccaatggctgggggacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaacaatcacctctacaagcaaatctccaacagcacatctggaggatcttcaaatgacaacgcctacttcggctacagcaccccctgggggtattttgacttcaacagattccactgccacttctcaccacgtgactggcagcgactcatcaacaacaactggggattccggcctaagcgactcaacttcaagctcttcaacattcaggtcaaagaggttacggacaacaatggagtcaagaccatcgccaataaccttaccagcacggtccaggtcttcacggactcagactatcagctcccgtacgtgctcgggtcggctcacgagggctgcctcccgccgttcccagcggacgttttcatgattcctcagtacgggtatctgacgcttaatgatggaagccaggccgtgggtcgttcgtccttttactgcctggaatatttcccgtcgcaaatgctaagaacgggtaacaacttccagttcagctacgagtttgagaacgtacctttccatagcagctacgctcacagccaaagcctggaccgactaatgaatccactcatcgaccaatacttgtactatctctcaaagactattaacggttctggacagaatcaacaaacgctaaaattcagtgtggccggacccagcaacatggctgtccagggaagaaactacatacctggacccagctaccgacaacaacgtgtctcaaccactgtgactcaaaacaacaacagcgaatttgcttggcctggagcttcttcttgggctctcaatggacgtaatagcttgatgaatcctggacctgctatggccagccacaaagaaggagaggaccgtttctttcctttgtctggatctttaatttttggcaaacaaggaactggaagagacaacgtggatgcggacaaagtcatgataaccaacgaagaagaaattaaaactactaacccggtagcaacggagtcctatggacaagtggccacaaaccaccagagtgcccaaTCTTCTTCTCATTTCCAGGGTgcacaggcgcagaccggctgggttcaaaaccaaggaatacttccgggtatggtttggcaggacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacggacggcaactttcacccttctccgctgatgggagggtttggaatgaagcacccgcctcctcagatcctcatcaaaaacacacctgtacctgcggatcctccaacggccttcaacaaggacaagctgaactctttcatcacccagtattctactggccaagtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaagcgctggaacccggagatccagtacacttccaactattacaagtctaataatgttgaatttgctgttaatactgaaggtgtatatagtgaaccccgccccattggcaccagatacctgactcgtaatctgtaa 2 AAV-CapR2003 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQHSKDPRSAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL 3 atggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggtgggctttgaaacctggagcccctcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtcttgtgcttccgggttacaaataccttggacccggcaacggactcgacaagggggagccggtcaacgcagcagacgcggcggccctcgagcacgacaaggcctacgaccagcagctcaaggccggagacaacccgtacctcaagtacaaccacgccgacgccgagttccaggagcggctcaaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaaaaagaggcttcttgaacctcttggtctggttgaggaagcggctaagacggctcctggaaagaagaggcctgtagagcagtctcctcaggaaccggactcctccgcgggtattggcaaatcgggtgcacagcccgctaaaaagagactcaatttcggtcagactggcgacacagagtcagtcccagaccctcaaccaatcggagaacctcccgcagccccctcaggtgtgggatctcttacaatggcttcaggtggtggcgcaccagtggcagacaataacgaaggtgccgatggagtgggtagttcctcgggaaattggcattgcgattcccaatggctgggggacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaacaatcacctctacaagcaaatctccaacagcacatctggaggatcttcaaatgacaacgcctacttcggctacagcaccccctgggggtattttgacttcaacagattccactgccacttctcaccacgtgactggcagcgactcatcaacaacaactggggattccggcctaagcgactcaacttcaagctcttcaacattcaggtcaaagaggttacggacaacaatggagtcaagaccatcgccaataaccttaccagcacggtccaggtcttcacggactcagactatcagctcccgtacgtgctcgggtcggctcacgagggctgcctcccgccgttcccagcggacgttttcatgattcctcagtacgggtatctgacgcttaatgatggaagccaggccgtgggtcgttcgtccttttactgcctggaatatttcccgtcgcaaatgctaagaacgggtaacaacttccagttcagctacgagtttgagaacgtacctttccatagcagctacgctcacagccaaagcctggaccgactaatgaatccactcatcgaccaatacttgtactatctctcaaagactattaacggttctggacagaatcaacaaacgctaaaattcagtgtggccggacccagcaacatggctgtccagggaagaaactacatacctggacccagctaccgacaacaacgtgtctcaaccactgtgactcaaaacaacaacagcgaatttgcttggcctggagcttcttcttgggctctcaatggacgtaatagcttgatgaatcctggacctgctatggccagccacaaagaaggagaggaccgtttctttcctttgtctggatctttaatttttggcaaacaaggaactggaagagacaacgtggatgcggacaaagtcatgataaccaacgaagaagaaattaaaactactaacccggtagcaacggagtcctatggacaagtggccacaaaccaccagagtgcccaaCATTCTAAAGACCCGCGTTCTgcacaggcgcagaccggctgggttcaaaaccaaggaatacttccgggtatggtttggcaggacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacggacggcaactttcacccttctccgctgatgggagggtttggaatgaagcacccgcctcctcagatcctcatcaaaaacacacctgtacctgcggatcctccaacggccttcaacaaggacaagctgaactctttcatcacccagtattctactggccaagtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaagcgctggaacccggagatccagtacacttccaactattacaagtctaataatgttgaatttgctgttaatactgaaggtgtatatagtgaaccccgccccattggcaccagatacctgactcgtaatctgtaa 4 AAV-Cap9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPCANQQHQDNARGLVLPGYKYLGPGNLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSNLFNMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRN 5 atggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggtgggctttgaaacctggagcccctcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtcttgtgcttccgggttacaaataccttggacccggcaacggactcgacaagggggagccggtcaacgcagcagacgcggcggccctcgagcacgacaaggcctacgaccagcagctcaaggccggagacaacccgtacctcaagtacaaccacgccgacgccgagttccaggagcggctcaaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaaaaagaggcttcttgaacctcttggtctggttgaggaagcggctaagacggctcctggaaagaagaggcctgtagagcagtctcctcaggaaccggactcctccgcgggtattggcaaatcgggtgcacagcccgctaaaaagagactcaatttcggtcagactggcgacacagagtcagtcccagaccctcaaccaatcggagaacctcccgcagccccctcaggtgtgggatctcttacaatggcttcaggtggtggcgcaccagtggcagacaataacgaaggtgccgatggagtgggtagttcctcgggaaattggcattgcgattcccaatggctgggggacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaacaatcacctctacaagcaaatctccaacagcacatctggaggatcttcaaatgacaacgcctacttcggctacagcaccccctgggggtattttgacttcaacagattccactgccacttctcaccacgtgactggcagcgactcatcaacaacaactggggattccggcctaagcgactcaacttcaagctcttcaacattcaggtcaaagaggttacggacaacaatggagtcaagaccatcgccaataaccttaccagcacggtccaggtcttcacggactcagactatcagctcccgtacgtgctcgggtcggctcacgagggctgcctcccgccgttcccagcggacgttttcatgattcctcagtacgggtatctgacgcttaatgatggaagccaggccgtgggtcgttcgtccttttactgcctggaatatttcccgtcgcaaatgctaagaacgggtaacaacttccagttcagctacgagtttgagaacgtacctttccatagcagctacgctcacagccaaagcctggaccgactaatgaatccactcatcgaccaatacttgtactatctctcaaagactattaacggttctggacagaatcaacaaacgctaaaattcagtgtggccggacccagcaacatggctgtccagggaagaaactacatacctggacccagctaccgacaacaacgtgtctcaaccactgtgactcaaaacaacaacagcgaatttgcttggcctggagcttcttcttgggctctcaatggacgtaatagcttgatgaatcctggacctgctatggccagccacaaagaaggagaggaccgtttctttcctttgtctggatctttaatttttggcaaacaaggaactggaagagacaacgtggatgcggacaaagtcatgataaccaacgaagaagaaattaaaactactaacccggtagcaacggagtcctatggacaagtggccacaaaccaccagagtgcccaagcacaggcgcagaccggctgggttcaaaaccaaggaatacttccgggtatggtttggcaggacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacggacggcaactttcacccttctccgctgatgggagggtttggaatgaagcacccgcctcctcagatcctcatcaaaaacacacctgtacctgcggatcctccaacggccttcaacaaggacaagctgaactctttcatcacccagtattctactggccaagtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaagcgctggaacccggagatccagtacacttccaactattacaagtctaataatgttgaatttgctgttaatactgaaggtgtatatagtgaaccccgccccattggcaccagatacctgactcgtaatctgtaa 6

[0037] Example 2: Detection of AAV intracapsular infection efficiency

[0038] In 8- to 10-week-old male C57BL / 6J mice, 5.0 × 10⁻⁶ mg / L was administered via tail vein. 12 The AAV vector, packaged and purified as described above, was administered at a dose of vg / kg, with 3 mice per group. Two weeks after administration, mice were anesthetized and injected intraperitoneally with the luciferase substrate. Twenty minutes later, the mice were euthanized, and liver tissue was collected. The peak emission signal was obtained using an IVIS system, and the emission intensity was quantified using imaging software. The data were then normalized.

[0039] Statistical analysis was performed using Prism 8 (Graph Pad) software. One-way ANOVA was used to analyze differences between groups. Significant differences and sample sizes are clearly indicated in the graphs and legends. Results are as follows: Figure 1 As shown, compared with wild-type AAV9, AAVLYR2003 showed significantly enhanced infection efficiency in mouse liver, while there was no significant difference in infection efficiency between AAVLYR2001 and wild-type AAV9 in mouse liver.

[0040] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. An adeno-associated virus capsid protein variant, characterized in that, The amino acid sequence of the capsid protein variant is shown in SEQ ID NO:

3.

2. The adeno-associated virus capsid protein variant according to claim 1, characterized in that: The parent of the adeno-associated virus capsid protein variant is the Cap protein of AAV9, the amino acid sequence of which is shown in SEQ ID NO:

5.

3. A polynucleotide, characterized in that: The polynucleotide encodes a capsid protein variant as described in claim 1 or 2.

4. The polynucleotide according to claim 3, characterized in that: The nucleotide sequence of the polynucleotide encoding the capsid protein variant shown in SEQ ID NO:3 is shown in SEQ ID NO:

4.

5. A carrier, characterized in that: The carrier contains the polynucleotide as described in claim 3 or 4.

6. A host cell, characterized by: The host cell contains the vector as described in claim 5.

7. A recombinant adeno-associated virus rAAV9 variant, characterized by: The recombinant adeno-associated virus rAAV9 variant contains a polynucleotide encoding a capsid protein with an amino acid sequence as shown in SEQ ID NO:

3.

8. The use of the recombinant adeno-associated virus rAAV9 variant as described in claim 7 in the preparation of a liver-targeted gene therapy drug.

9. A gene therapy drug, characterized in that: The gene therapy drug uses the recombinant adeno-associated virus rAAV9 variant as described in claim 7 as a vector.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains the gene therapy drug as described in claim 9.

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