CN116063403A - Adeno-associated virus mutant and application thereof - Google Patents

Abstract

The present invention provides an adeno-associated virus mutant and an application thereof. The coat protein of the adeno-associated virus mutant includes the amino acid sequence shown in SEQ ID NO: 1-5, or has more than 98% of SEQ ID NO: 1-5 Amino acid sequences that are identical and have the same or similar biological functions. The invention constructs an adeno-associated virus library by splicing and recombining the coat protein coding gene of the wild-type adeno-associated virus, and obtains an adeno-associated virus mutant by screening with a neutralizing antibody against the AAV coat protein, which has a strong infective ability to human liver cells , and can avoid the neutralizing effect of neutralizing antibodies, and specifically solve the technical obstacles in the field of hemophilia treatment.

Description

Adeno-associated virus mutant and its application

This application is a divisional application of patent application No. 202010442092.4 (the filing date of the original application is May 22, 2020, and the name of the invention is adeno-associated virus mutants and their applications).

Technical Field

The invention belongs to the technical field of genetic engineering and bioengineering, and relates to adeno-associated virus mutants and applications thereof.

Background Art

In recent years, adeno-associated virus (AAV) has been used as a gene therapy vector in the clinical treatment of various gene defect diseases, such as hemophilia B, DMD progressive muscular atrophy, SMA movement disorder, etc., and has achieved exciting results. In particular, the successful application of AAV in the treatment of hemophilia is expected to save thousands of patients who are ineffective with conventional treatments. It has been reported in the literature that after the injection of AAV carrying the factor IX gene, hemophilia patients did not have a serious immune response, and the damage to the liver by AAV was also transient; after the injection of AAV, the level of factor IX in the patient's peripheral blood recovered to about 10% of the normal level, and was expressed stably for a long time, basically breaking away from the dependence on the recombinant protein of factor IX. Preliminary clinical trials are underway for hemophilia A caused by the loss of factor VIII. However, the gene encoding coagulation factor VIII is relatively long, about 4.5kbp, and with the promoter and termination sequences, the gene length of the entire sequence has exceeded the packaging range of AAV. This is the main obstacle encountered by the AAV gene therapy method in the treatment of hemophilia A, and it is also a current research hotspot.

However, AAV gene therapy also has some limitations in the treatment of hemophilia. One of them is that a certain proportion of anti-AAV capsid protein neutralizing antibodies exist in the peripheral blood of normal people and hemophilia patients. Therefore, before evaluating whether a patient can undergo AAV gene therapy, it is necessary to first detect whether there are neutralizing antibodies in the peripheral blood. If the patient has anti-AAV neutralizing antibodies in the body, he is not suitable for AAV gene therapy. Considering that AAV gene therapy is a promising hemophilia treatment method, it is particularly important to help AAV evade the clearance of neutralizing antibodies in the body.

In response to this problem, researchers have proposed a variety of methods, including empty shell protein neutralization, immunosuppressant pretreatment, small fragment DNA neutralization, AAV capsid protein mutation, etc. The empty shell protein neutralization method refers to the mixing of a large amount of AAV empty shell protein when injecting AAV to offset the pressure of AAV being cleared by neutralizing antibodies in the body, thereby increasing the infection efficiency. However, studies have found that AAV empty shell protein is more easily presented by antigen-presenting cells, stimulating a strong T cell immune response, enhancing the clearance effect of T cells on AAV, and ultimately leading to a lower transduction efficiency of AAV. The immunosuppressant pretreatment method refers to injecting an appropriate amount of immunosuppressants, such as dexamethasone, into patients before injecting AAV. Although this method can improve the transduction efficiency of AAV to a certain extent, the degree of improvement is limited because dexamethasone has a wide range of immune suppression and cannot specifically eliminate neutralizing antibodies against AAV and T/B cells that recognize AAV. The small fragment DNA neutralization method refers to the use of small fragments of DNA that can specifically block AAV neutralizing antibodies. It has good application prospects, but there have been no reports of related clinical applications. The AAV capsid protein mutation method refers to mutating certain sites in the amino acid sequence of the AAV capsid protein recognized by neutralizing antibodies, thereby reducing the ability of neutralizing antibodies to recognize AAV and thus evading the clearance effect of neutralizing antibodies. There are currently a large number of research reports, but the reasons and methods for evading neutralizing antibodies have not yet been clarified.

Therefore, modifying AAV to evade the clearance of neutralizing antibodies in the body and improving the applicability of AAV are of great significance and have broad application prospects in the field of gene defect disease treatment.

Summary of the invention

In view of the deficiencies in the prior art and actual needs, the present invention provides an adeno-associated virus mutant and its application. The adeno-associated virus mutant has a strong ability to infect human liver cells and can evade the neutralizing effect of neutralizing antibodies, thereby specifically solving the obstacles encountered by hemophilia patients with anti-AAV neutralizing antibodies when receiving gene therapy.

To achieve this object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides an adeno-associated virus mutant, wherein the capsid protein of the adeno-associated virus mutant comprises an amino acid sequence shown in SEQ ID NOs: 1 to 5;

SEQ ID NO: 1:

MAADGYLPDWLEDTLSEGIRQWWKLKPGAPPKPKANQQKQDDGRGLLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGGTESVPDPQPLGEPPAAPSGVGPNTMAAGGGAP MADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDKERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGI VADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPHPIGTRYLTRPL;

SEQ ID NO:2:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMASGGGAPVADNNE GADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYNLNGRNSLANPGIAMASHKDDKERFFPSNGILIFGKQNAARDADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPHPIGTRYLTRPL;

SEQ ID NO:3:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDRQLKAGDNPYLRYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADN NEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMASHKDDKERFFPSNGILIFGKQNAARDADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL;

SEQ ID NO:4:

MAADGYLPDWLEDNLSEGIREWWDLKPGAPPKPKANQQKQDDGRGLLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMASHKDDKERFFPSNGILIFGKQNAARDADYSDVMLTSEEEIKTTNPVATEEY GIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPHPIGTRYLTRPL;

SEQ ID NO:5:

MAADGYLPDWLEDNLSEGIREWWALKPGAPPKPKANQQKQDDGRGLLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMA DNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDKERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGI VADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL.

In the present invention, an adeno-associated virus library is constructed by splicing and recombining the coat protein encoding gene of the wild-type adeno-associated virus, and five adeno-associated virus mutants are obtained by screening with neutralizing antibodies against AAV coat proteins. The coat proteins of the adeno-associated virus mutants include the amino acid sequences shown in SEQ ID NOs: 1 to 5. The obtained adeno-associated virus mutants have the ability to evade anti-AAV neutralizing antibodies and have efficient and specific infection ability for human liver cells, and have broad application prospects in the field of hemophilia gene therapy.

The five adeno-associated virus mutants screened by the present invention have high sequence homology. After sequence comparison with the wild-type AAV9, it was found that the 100th to 200th amino acids located at the N-terminus of the AAV capsid protein amino acid sequence may be a high-frequency recognition region for anti-AAV neutralizing antibodies.

Preferably, the capsid protein of the adeno-associated virus mutant also includes an amino acid sequence that has more than 98% identity with SEQ ID NO: 1 to 5 and has the same or similar biological function. For example, the capsid protein of the adeno-associated virus mutant may have an identity of 98% with SEQ ID NO: 1 to 5 and has the ability to evade anti-AAV neutralizing antibodies.

In a second aspect, the present invention provides a nucleic acid molecule encoding the adeno-associated virus mutant described in the first aspect, or encoding a protein having the same or similar biological function as the adeno-associated virus mutant described in the first aspect.

Preferably, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NOs: 6 to 10 and/or the complementary sequence of SEQ ID NOs: 6 to 10;

SEQ ID NO:6:

GCGATCTGGTCATGTGGATTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGC GGCGGATGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCA AGAAGATACGTCTTTTGGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCCAAAAAGAGACTCAATTTCGGTCAGACTGGCGGCACAGAGTCAGTTCCAGACCCTCAACCTCTCGGAGA ACCTCCAGCAGCGCCCTCTGGTGTGGGACCTAATACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGC GATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGTCAAGGAGGT CACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCG GCTCCGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAATGGCAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGcaACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACC AGTACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGG ACCCTGTTACCGCCAACAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCTATGGCAACACACAAAGACGACAAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACCACTAACC CTGTGGCTACAGAGGAATACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGCCAGGGGGCCTTACCCGGTATGGT CTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGC GCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACTAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCACCCATTGGCACCCGTTAC CTCACCCGTCCCCTGTAATTGTCTGTTAATCAATAAACCGGTTGATTCGTTTCAGTTGAACTTTGGTGTCGTCTAGAGGGCCGCTCGATAAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCACGAAAGAACATGTGAGCAAAACGCCAGCAAAAGCCAGGAACCGTAAAAAGCCGCGTTGCTGGCGTTTTTCCATAGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCC TGGAAGCTCCCTCGTGCGCTCTCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGATCTCA;

SEQ ID NO:7:

GGTCATGTGGTTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACG CCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAA GATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCCAAGAAGCGGGTTTCTTGAACCTCTTGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCCAAAAAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTTCCAGACCCTCAACCTCTCGGAGAACC TCCAGCAGCGCCCTCTGGTGTGGGACCTAATACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGC GATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACTTACAACAACCATCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAACTCTTCAACATTCAGGTCAAAG AGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCGGAGTACCAGTTGCCGTACGTC CTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCTCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATT GACCAGTACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGC TGCCAGGACCCTGTTACCGCCAACAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCAAATACAATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCTATGGCCTCACACAAAGACGACAAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACC ACTAACCCTGTGGCTACAGAGGAATACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGCCAGGGGGCCTTAC CCGGTATGGTCTGGCAGAACCGGGACGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAG GAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCACCCCA TTGGCACCCGTTACCTCACCCGTCCCCTGTAATTGTCTGTTAATCAATAAACCGGTTGATTCGTTTCAGTTGAACTTTGGTGTCGTCTAGAGGGCCGCTCGATAAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCA CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCCTGACGAGCATCACAAAATCGACGCTCAAGTCAGAGTGGCGAAACCCGACAGGACT ATAAAGATAACAGGCGTTTCCCCCTGGAGCTCCCTCGTGCGCTCCTCCTGTTCCGACCCTGCCGCTACCGGATACCTGTCCGCCGTTCCCCCTTCCGGGAGCGTGCGCCTT;

SEQ ID NO:8:

GGTCATGTGGATTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGGCGGCGGATGCA GCGGCCCTCGAGCACGACAAGGCCTACGACCGGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTT GGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTTTTGGTCTGGTTGAGGAAGGCGCTAAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCTC AGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGA GTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACTTACAACAACCATCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAA GACCATCGCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCGGAGTACCAGTTGCCGTACGTCCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTC CCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTCTCGGACTCAAACAACA GGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAACAACGCGTCTCAACGACAACCGGGC AAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCAAATACCATCTGAATGGAAGAAAATTCATTGGCTAATCCTGGCATCGCTATGGCCTCACACAAAGACGACAAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATACGGTATCGTGGCAGATA ACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAAGATTC CTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGT GGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGA ACTTTGGTGTCGTCTAGAGGGCCGCTCGATAAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAGGCGGTAATA CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGCCAGCAAAACGCCAGGAACCGTAAAAAGCCGCGTTGCTGGCGTTTTTCCATAGCTCCGCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGAAGCTCCCTCGTGCGCTCTCTGTCCGACCCTGCCGCTTACCGGATACTGTCCCGCTTTTCCCCTTCG GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGTATCTCAGTTCGTGTAGTCGTCGCTCAGCTGGGCTGGGTGCACGACCCCGTTCAGCCCGACGCTGCGCCTTATCGGTACTATCGTCT;

SEQ ID NO:9:

GGTCATGTGGATTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGGCGGCGGATG CAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTT TGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTTTTGGTCTGGTTGAGGAAGGTGCTAAGACGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCC TCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACA GAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACTTACAACAACCATCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAACTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGA GTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCGGAGTACCAGTTGCCGTACGTCCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCG TTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTTAACAATGGCAGCCAGGCAGTGGGACGGTCATCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTCTCGGACTCAA ACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAACAACGCGTCTCAACGACAAC CGGGCAAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCTATGGCCTCACACAAAGACGACAAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATACGGTATCGTGGCAG ATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCA AGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTA CAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCACCCATTGGCACCCGTTACCTCACCCGTCCCCTGTAATTGTCTGTTAATCAATAAACCGGTTGATTCGTT TCAGTTGAACTTTGGTGTCGTCTAGAGGGCCGCTCGATAAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAAGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTCGGTGTAGTCGTCGCTCCAAGCTGGGCTGGGTGCACGAACCCCGTCAGCCCGAC;

SEQ ID NO:10:

GGTCATGTGGATTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGCGCTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGA CGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAA GATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCGGAGAACCTC CAGCAACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCG ATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGCAGCCGACTCATCAACACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAG AGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCGGAGTACCAGTTGCCGTACGTCC TCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATT GACCAGTACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCT GCCAGGACCCTGTTACCGCCAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCTATGGCAACACACAAAGACGACAAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACCACT AACCCTGTGGCTACAGAGGAATACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGCCAGGGGGCCTTACC CGGTATGGTCTGGCAGAACCGGGACGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGA AAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATT GGCACCCGTTACCTCACCCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTGTCGTCTAGAGGGCCGCTCGATAAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCA CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCT CTTCCGCTTCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCTGGAAGCTCCCTCGTGCGCTCTCTGTTCCGACCTGCCGCTTACCGGATACTGTCCGCTTTCTCCCTTCGGGAGCGTGCGCTTTCTCATAGCT.

In the present invention, the nucleic acid sequence shown in SEQ ID NO:6 is the coding sequence of the amino acid sequence shown in SEQ ID NO:1, the nucleic acid sequence shown in SEQ ID NO:7 is the coding sequence of the amino acid sequence shown in SEQ ID NO:2, the nucleic acid sequence shown in SEQ ID NO:8 is the coding sequence of the amino acid sequence shown in SEQ ID NO:3, the nucleic acid sequence shown in SEQ ID NO:9 is the coding sequence of the amino acid sequence shown in SEQ ID NO:4, and the nucleic acid sequence shown in SEQ ID NO:10 is the coding sequence of the amino acid sequence shown in SEQ ID NO:5.

In a third aspect, the present invention provides an expression vector, comprising a wild-type adeno-associated virus vector into which the nucleic acid molecule described in the second aspect is inserted.

Preferably, the wild-type adeno-associated virus vector includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV2.

In a fourth aspect, the present invention provides an adeno-associated virus library, wherein the adeno-associated virus library includes the adeno-associated virus mutant described in the first aspect.

Preferably, the adeno-associated virus library also includes wild-type and/or mutant adeno-associated viruses that do not have the ability to evade anti-adeno-associated virus coat protein neutralizing antibodies.

In a fifth aspect, the present invention provides a method for constructing the adeno-associated virus library according to the fourth aspect, the method comprising:

(1) PCR amplification of the coat protein encoding gene of wild-type adeno-associated virus;

(2) After the amplified product is digested with DNase, DNA fragments with a length of 100 to 300 bp are selected for reassembly;

(3) inserting the spliced product into a wild-type adeno-associated virus vector to obtain an adeno-associated virus vector library;

(4) The adeno-associated virus vector library is co-transfected with a helper plasmid into mammalian cells to prepare the adeno-associated virus library.

Preferably, the PCR primers in step (1) include the nucleic acid sequences shown in SEQ ID NOs: 11 to 12;

SEQ ID NO: 11: 5’-ATAAAGCGAGTAGTC-3’;

SEQ ID NO: 12: 5'-GAGGGTATGCGACAT-3'.

Preferably, the splicing method in step (2) is to mix DNA fragments with a length of 100 to 300 bp with DNA polymerase, perform gradient cooling in the range of 96 to 41° C., and obtain the splicing product by Shuffling PCR.

In the present invention, DNA fragments with a length of 100 to 300 bp are selected as splicing fragments, which not only enriches the sequence diversity of the splicing products and increases the virus amount in the adeno-associated virus library, but also ensures the splicing efficiency of the shuffling PCR, improves the splicing success rate of the splicing products, and helps to efficiently screen for adeno-associated virus mutants that can evade the neutralization effect of neutralizing antibodies.

Preferably, the wild-type adeno-associated virus vector in step (3) includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV2.

In a sixth aspect, the present invention provides a method for screening adeno-associated virus mutants according to the first aspect, the method comprising:

The adeno-associated virus library described in the fourth aspect is mixed and incubated with human immunoglobulin for human intravenous injection, and then introduced into liver cell humanized mice;

Adenovirus was introduced, and after a period of feeding, human cells in the mouse liver were isolated and DNA was extracted;

Using the primer pair shown in SEQ ID NO: 13-14 for PCR amplification, inserting the amplified product into a wild-type adeno-associated virus vector, and co-transfecting mammalian cells with a helper plasmid; lysing the mammalian cells after culturing to obtain the adeno-associated virus mutant;

SEQ ID NO:13: CAACTCCATCACTAGGGGTTC;

SEQ ID NO: 14: CATGGGAAAGGTGCCAGA.

In the present invention, the upstream primer SEQ ID NO: 13 is designed according to the AAV2-rep gene, and the downstream primer SEQ ID NO: 14 is designed according to the downstream consensus sequence of the AAV2-ITR plasmid.

In the seventh aspect, the present invention provides a method for preparing an adeno-associated virus mutant, the method comprising co-transfecting mammalian cells with the expression vector described in the third aspect and an auxiliary plasmid; lysing the mammalian cells after culturing to obtain the adeno-associated virus mutant.

In an eighth aspect, the present invention provides a pharmaceutical composition, comprising any one or a combination of at least two of the adeno-associated virus mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the adeno-associated virus library described in the fourth aspect;

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In the ninth aspect, the present invention provides a use of the adeno-associated virus mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the adeno-associated virus library described in the fourth aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of drugs for treating gene defect diseases.

Preferably, the gene defect disease includes hemophilia.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention constructs an adeno-associated virus library by splicing and recombining the coat protein encoding gene of the wild-type adeno-associated virus, and screens five adeno-associated virus mutants using neutralizing antibodies against the AAV coat protein, which have the ability to escape anti-AAV neutralizing antibodies and have high-efficiency and specific infection ability for human liver cells;

(2) The five adeno-associated virus mutants screened by the present invention have high sequence homology. After sequence comparison with wild-type AAV9, it was found that the amino acids 100 to 200 located at the N-terminus of the amino acid sequence of the AAV capsid protein may be a high-frequency recognition region for anti-AAV neutralizing antibodies;

(3) The adeno-associated virus mutant of the present invention has broad application prospects and huge market value in the field of hemophilia gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the origin of the coat protein gene sequence in the AAV mutant and the point mutations present in the sequence;

Figure 2 shows the evolutionary relationship between AAV mutants and wild type;

FIG3 shows the packaging efficiency of wild-type and mutant AAV in HEK293 cell lines;

FIG4 shows the infection efficiency of wild-type and mutant AAV on Huh7 cells in vitro;

FIG5 shows the ability of wild-type and mutant AAV to evade IVIG neutralizing antibodies in vitro;

FIG6 (A) shows the ability of AAV wild type and mutants to escape neutralizing antibodies in normal human peripheral blood serum in vitro, and FIG6 (B) shows the ability of AAV wild type and mutants to escape neutralizing antibodies in hemophilia patient peripheral blood serum in vitro;

FIG7 shows the infection efficiency of mutant 2-10 on human liver cells in vivo;

FIG8(A) is a schematic diagram of the gene therapy process, and FIG8(B) is a diagram of the therapeutic effect.

DETAILED DESCRIPTION

To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

Example 1 Acquisition of adeno-associated virus mutants

In this example, PCR primers (SEQ ID NOs: 11-12) containing specific sequences and restriction sites were first used for ordinary PCR to amplify the coat protein coding genes of wild-type adeno-associated viruses (AAV) 1-10 purchased from BioAsk, USA, so that the mass of the PCR product reached more than 1 μg; then the obtained PCR products were mixed and treated with a Shuffling PCR kit to obtain an AAV coat protein mutant library, and the specific steps were as follows:

10 μg of wild-type AAV capsid protein gene amplification mixed product was incubated with 1U DNase at 37°C for 5 min, 1 μL 100mM EDTA was used to terminate the reaction, and the mixture was inactivated at 75°C for 10 min; the fragments with a length of 100 to 300 bp in the DNase digested product were purified using a DNA purification kit; the purified DNA fragments were reassembled using the high-fidelity enzyme PFU, and the specific splicing conditions are shown in Table 1; the obtained splicing products were recombinantly inserted into the AAV2-ITR plasmid to obtain the AAV vector library;

Table 1 Shuffling PCR conditions

The auxiliary plasmid pXX6-80 and the AAV vector library were transfected into the HEK293 cell line at a ratio of 1:2; after 48 hours, the cells were collected, and the cells were repeatedly frozen and thawed and ultrasonically lysed; ultracentrifuged overnight in a cesium chloride solution of a certain density, and the cesium chloride solutions at different levels in the centrifuge tube were collected. The cesium chloride solution containing AAV was placed in a dialysis bag and dialyzed in PBS, and finally a PBS solution containing AAV was obtained, which was the AAV library;

100 μL AAV library containing 1×10 ¹³ viruses was incubated with 100 μL 1 mg/mL human immunoglobulin (IVIG) for intravenous injection at 4°C for 2 h, and then the mixture of AAV and IVIG was injected into liver cell humanized mice by medial canthal vein injection; on the third day, 1×10 ⁷ μg adenovirus dl309 was injected by medial canthal vein; on the fifth day, human cells in the mouse liver were isolated and DNA was extracted, and the capsid protein gene was amplified using primer pair F1 (SEQ ID NO: 13): CAACTCCATCACTAGGGGTTC and R1 (SEQID NO: 14): CATGGGAAAGGTGCCAGA and high-fidelity PFU enzyme, and re-ligated and integrated into the AAVrep-cap packaging plasmid.

The above steps were repeated three times to obtain an adeno-associated virus mutant, which can escape the neutralization of neutralizing antibodies against AAV capsid protein in vivo. The capsid protein amino acid sequence of the mutant is shown in SEQ ID NOs: 1 to 5, and the nucleic acid sequence is shown in SEQ ID NOs: 6 to 10.

Figure 1 shows the origin of the coat protein gene sequence in each mutant and the point mutations present in the sequence; the Clustral W evolutionary analysis method was further used to calculate the evolutionary relationship between each AAV mutant and the wild-type AAV, and the results are shown in Figure 2. The mutants 2-10 (SEQ ID NO: 1), 2-19 (SEQ ID NO: 2), 4-24 (SEQ ID NO: 3), 1-1 (SEQ ID NO: 4), and 1-18 (SEQ ID NO: 5) were analyzed using the Alignment of the DNAMAN software. The homology matrix of the five sequences is shown in Table 2, and it was found that the homology of SEQ ID NO: 1 to 5 was 98.02%, thereby concluding that sequences with more than 98% homology to SEQ ID NO: 1 to 5 have the ability to escape neutralizing antibodies.

Table 2

LP1-1.seq 100% LP1-11.seq 96.7% 100% LP2-10.seq 97.0% 96.5% 100% LP3-19.seq 95.2% 97.0% 94.8% 100% LP4-24.seq 97.8% 98.4% 95.7% 97.1% 100%

Example 2 Packaging of adeno-associated virus mutants

Plasmids were extracted to obtain the Cap-Rep plasmids of each mutant, the luciferase plasmid pTR-CBh-luc and the auxiliary plasmid pXX6-80. Among them, pTR-CBh-luc needed to be digested by Sma1 to detect whether the palindromic sequences ITR at both ends of the AAV genome were intact. All plasmids were pre-tested by DNA agarose gel electrophoresis to detect whether the bands were single and whether they had supercoiled structures, and to ensure that the plasmid solution was not contaminated by a large amount of soluble proteins.

16 hours in advance, cultured HEK293 cells free of bacteria, viruses and pathogens were subcultured to new culture dishes (15 cm diameter) at a density of about 70% of the saturation density, and the culture medium was 10% FBS + DMEM;

Prepare the transfection solution. Each cell in a 15 cm dish needs 9 μg of pTR-CBh-luc plasmid, 12 μg of mutant Cap-Rep plasmid, and 15 μg of pXX6-80 plasmid. Add the three plasmids to 500 μL DMEM medium and mix well. Then add 150 μL of 1 μg/μL PEI dropwise while shaking. Let stand at room temperature for 10 min, and then add the mixture dropwise to the cell culture supernatant.

After 48 hours, HEK293 cells were pipetted into suspension, centrifuged at 2000 rpm for 5 min to collect the cell pellet, resuspended in 8.7 mL of ultrapure water, repeatedly frozen and thawed between dry ice and warm water three times, sonicated for 2 min to lyse the cells, 5 g of cesium chloride was added, sonicated for 2 min, and placed on ice;

Centrifuge at 4°C, 12,000 rpm for 20 min. The insoluble cell debris is suspended on the surface of the cesium chloride solution. Carefully transfer the lower transparent cesium chloride solution to an ultracentrifuge tube and centrifuge at 65,000 rpm, 15°C for 18 h in a Sorvall WX 80+Ultracentrifuge ultracentrifuge with the brake reduced to 5.

The cesium chloride solution after centrifugation is taken out layer by layer, and the volume of each layer is about 1 mL. The cesium chloride solution with a refractive index of about 1.37 is the cesium chloride solution containing AAV.

The cesium chloride solution containing AAV was transferred to a dialysis bag and placed in pre-cooled PBS for dialysis. The dialysis was repeated three times, and the PBS solution containing AAV was finally collected.

The concentration of the obtained AAV was measured by taking 90 μL of PBS solution containing AAV, adding 10 μL of DNase, digesting at 37°C for 1 hour, and adding 6 μL of 0.5M EDTA to terminate the reaction; then adding 100 μL of protease digestion solution, digesting at 55°C for 2 hours, and inactivating at 95°C for 5 minutes; the AAV solution was diluted 1000 times with ultrapure water and used as a template for real-time quantitative PCR. Quantitative PCR analysis was performed to calculate the concentration of AAV.

The results are shown in FIG3 . AAV2 and AAV3 have higher packaging efficiency, while the packaging efficiency of other wild-type and mutant AAVs is acceptable.

Example 3 In vitro infection efficiency of adeno-associated virus mutants

The concentrations of the wild-type and mutant AAV obtained in Example 2 were adjusted to the same concentration, and the in vitro infection efficiency of AAV was detected using the human liver tumor cell line Huh7.

First, Huh7 was evenly distributed into a 96-well plate. After culturing for 16 hours, a total of 1×10 ⁸ μg of AAV was added to each well, and three replicate wells were set up.

After Huh7 was cultured for 48 hours, the expression level of luciferase in each well was detected using the Promega luciferase assay kit.

As shown in FIG4 , the wild-type AAV1, AAV2, AAV3, AAV6 and mutants 2-10 and 2-20 had high infection efficiency for Huh7 cells (AAV expression efficiency in huh7_48hour).

Example 4 Efficiency of AAV mutants in evading IVIG neutralizing antibodies

Previous experiments have found that there are neutralizing antibodies against all wild-type AAVs in human IVIG. In this example, the original concentration of 50 μg/μL IVIG was first diluted in a gradient ratio of 1:2, with the maximum dilution ratio being 1:8192, and the final volume of IVIG dilution was 20 μL; and the wild-type and mutant AAVs were adjusted to 20 μL containing 1×10 ⁸ μg of virus;

The IVIG gradient dilutions were incubated with different wild-type and mutant AAVs at 4°C for 30 min. After the incubation, the mixture was added to Huh7 cells (serum-free culture) pre-cultured in a 96-well plate and cultured for another 48 hours. After 48 hours, the expression level of luciferase was detected using the Promega luciferase assay kit.

As shown in Figure 5, wild-type AAV1, 2, 3, 6, and 8 are more easily neutralized by natural neutralizing antibodies in IVIG, and wild-type AAV9 has a strong resistance to neutralizing antibodies; mutants 1-1, 1-18, 3-19, and 4-24 have a strong resistance to neutralizing antibodies, and mutant 2-10 almost completely escapes the neutralization of IVIG. This shows that mutant AAVs that can escape neutralizing antibodies in IVIG have been successfully screened out from the mutant library.

Example 5 Efficiency of adeno-associated virus mutants in evading neutralizing antibodies in peripheral blood

In this example, the approximate range of AAV antibody concentrations in peripheral blood sera collected from 19 healthy subjects and 26 hemophilia patients was first determined, and then 8 gradient dilutions were performed at a ratio of 1:2, and a PBS control group without neutralizing antibodies was set up;

20 μL of serum dilution was incubated with 20 μL (1×10 ⁸ μg) of different wild-type and mutant AAVs at 4°C for 30 min. After incubation, the mixture was added to Huh7 cells (serum-free culture) pre-cultured in a 96-well plate and cultured for 48 hours. After 48 hours, the expression level of luciferase was detected using the Promega luciferase assay kit, and the titer of AAV neutralizing antibodies in the serum samples was calculated.

The titers of anti-AAV neutralizing antibodies in healthy serum samples and patient serum samples are shown in Figure 6 (A) and Figure 6 (B), respectively. The data are visualized and analyzed. The ruler shows the dilution multiple of the serum. The higher the dilution multiple, the darker the color, indicating that the serum contains more neutralizing antibodies. The results were analyzed and found that compared with the positive rate of anti-AAV neutralizing antibodies in the peripheral blood serum of healthy people, the positive rate of anti-AAV neutralizing antibodies in the peripheral blood serum of hemophilia patients was lower, but nearly one-third of hemophilia patients still had anti-AAV neutralizing antibodies in their peripheral blood serum; mutant 2-10 can significantly evade the neutralizing effect of neutralizing antibodies, reducing the positive rate of wild-type AAV neutralizing antibodies in serum from one-third to one-eighth. The use of mutant 2-10 is expected to enable more hemophilia patients to receive AAV gene therapy.

Since mutant 2-10 can effectively evade AAV neutralizing antibodies, comparison of the antibody expression profiles of wild-type AAV and mutant AAV revealed that the antibody expression profiles of AAV9 and 2-10 are relatively similar. According to the source analysis of each mutant in Figure 1, the 100th to 200th amino acids located at the N-terminus of the amino acid sequence of the capsid protein may be the high-frequency recognition region of anti-AAV neutralizing antibodies.

Example 6 In vivo infection efficiency of adeno-associated virus mutants 2-10

This example uses liver cell humanized mice for in vivo experiment verification. Liver cell humanized mice can represent the complex environment in the body and can directly detect the infection and expression efficiency of AAV on human liver cells. AAV8 has been successfully used in many clinical experiments as an AAV gene therapy vector. This example uses AAV8-GFP as a control to detect the infection and expression efficiency of mutant 2-10GFP in liver humanized mice.

1×10 ¹¹ μg AAV8-GFP and 2-10 GFP were intravenously injected into liver cell humanized mice. Seven days later, the livers of the mice were isolated and stained with immunofluorescence using TRITC-labeled anti-human albumin antibody. The livers were observed and photographed using a confocal fluorescence microscope.

As shown in Figure 7, there is no obvious difference in the ability of AAV8 and 2-10 to infect human liver cells in liver cell humanized mice, indicating that while 2-10 has evolved the ability to evade AAV neutralizing antibodies, its specific ability to infect human liver cells in vivo has not been weakened.

Example 7 Gene therapy effect of adenovirus mutant LP2-10 on AAV neutralizing antibody-positive coagulation factor 9 gene-deficient mice

In this embodiment, we simulated the gene therapy process of hemophilia patients with AAV neutralizing antibodies in vivo, as shown in Figure 8 (A), first, AAVs (AAV1/2/3/6/8/9 mixture) were subcutaneously injected to immunize F9 KO mice, and peripheral blood was collected on the 13th day. The AAV neutralizing antibody detection system was used to detect whether anti-AAV neutralizing antibodies were successfully induced. The results showed that AAVs as antigens can induce anti-AAV1/2/3/6/8 neutralizing antibodies; AAV8/F9 and LP2-10-F9 were intravenously injected on the 14th day, and peripheral blood was collected on the 28th and 42nd days to detect the level of hF9. As shown in Figure 8 (B), in mice with neutralizing antibodies, the hF9 level of mice in the gene therapy group with LP2-10 as a vector was significantly higher than that in the AAV8 group. In mice without immunization, the gene therapy effect of LP2-10 was slightly lower than that of AAV8 but there was no significant difference.

In summary, the present invention constructs an adeno-associated virus library by splicing and recombining the capsid protein encoding gene of the wild-type adeno-associated virus, and obtains five adeno-associated virus mutants by screening with neutralizing antibodies against AAV capsid protein. The five mutants have the ability to evade anti-AAV neutralizing antibodies and have high and specific infection ability for human liver cells. Therefore, the mutants have broad application prospects and huge market value in the field of hemophilia gene therapy.

The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (10)

1. An adeno-associated virus mutant, characterized in that the amino acid sequence of the capsid protein of the adeno-associated virus mutant includes the sequence shown in SEQ ID NO:5. 2. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the adeno-associated virus mutant according to claim 1; Preferably, the nucleic acid sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO:10. 3. An expression vector, characterized in that the expression vector comprises a wild-type adeno-associated virus vector inserted with the nucleic acid molecule according to claim 2; Preferably, the wild-type adeno-associated virus vector includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV2. 4. An adeno-associated virus library, characterized in that the adeno-associated virus library comprises the adeno-associated virus mutant described in claim 1. 5. A method for constructing an adeno-associated virus library according to claim 4, characterized in that the method comprises: (1) PCR amplification of the coat protein encoding gene of wild-type adeno-associated virus; (2) After the amplified product is digested with DNase, DNA fragments with a length of 100 to 300 bp are selected for reassembly; (3) inserting the spliced product into a wild-type adeno-associated virus vector to obtain an adeno-associated virus vector library; (4) The adeno-associated virus vector library is co-transfected with a helper plasmid into mammalian cells to prepare the adeno-associated virus library. 6. The method according to claim 5, characterized in that the PCR primers in step (1) include the nucleic acid sequences shown in SEQ ID NOs: 11 to 12; Preferably, the splicing method in step (2) is to mix DNA fragments with a length of 100 to 300 bp with DNA polymerase, perform gradient cooling in the range of 96 to 41° C., and obtain the splicing product by Shuffling PCR; Preferably, the wild-type adeno-associated virus vector in step (3) includes any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV2. 7. A method for screening adeno-associated virus mutants according to claim 1, characterized in that the method comprises: The adeno-associated virus library of claim 4 is mixed and incubated with human immunoglobulin for human intravenous injection, and then introduced into liver cell humanized mice; Adenovirus was introduced, and after a period of feeding, human cells in the mouse liver were isolated and DNA was extracted; The primer pairs shown in SEQ ID NOs: 13-14 are used for PCR amplification, and the amplified product is inserted into a wild-type adeno-associated virus vector, and co-transfected with a helper plasmid into mammalian cells; after culturing, the mammalian cells are lysed to obtain the adeno-associated virus mutant. 8. A method for preparing an adeno-associated virus mutant, characterized in that the method comprises co-transfecting mammalian cells with the expression vector described in claim 3 and an auxiliary plasmid; and lysing the mammalian cells after culturing to obtain the adeno-associated virus mutant. 9. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises any one or a combination of at least two of the adeno-associated virus mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, or the adeno-associated virus library according to claim 4; Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent. 10. Use of the adeno-associated virus mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the adeno-associated virus library according to claim 4 or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating a gene defect disease; Preferably, the gene defect disease includes any one of hemophilia, congenital amaurosis, Duchenne muscular dystrophy or spinal muscular atrophy, or a combination of at least two thereof.

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