Recombinant adeno-associated virus vector and application thereof in gene delivery

Abstract

The invention relates to a recombinant adeno-associated virus vector and application thereof in gene delivery, and belongs to the technical field of biology. The invention provides a recombinant adeno-associated virus vector, the recombinant adeno-associated virus vector comprises an adeno-associated virus capsid protein VP1 mutant, and compared with an adeno-associated virus capsid protein VP1 of which the starting amino acid sequence is shown as SEQ ID NO.1, the adeno-associated virus capsid protein VP1 mutant has the advantages that the adeno-associated virus capsid protein VP1 mutant has the nucleotide sequence shown as SEQ ID NO.1; the arginine at the 447 site is mutated into methionine and the threonine at the 456 site is mutated into serine, or the arginine at the 459 site is deleted and the threonine at the 456 site is mutated into isoleucine; the mutation of the adeno-associated virus capsid protein VP1 significantly increases the transduction efficiency of the recombinant adeno-associated virus vector in the central nervous system (increased by 10-20 times), and the recombinant adeno-associated virus vector has great application prospects in establishment of central nervous system related disease models or preparation of central nervous system related gene therapy drugs.

Description

[0001] This application is a divisional application of the application with the application date of December 30, 2022, the application number of 202211741124.6, and the invention name of a recombinant adeno-associated virus vector and its application in gene delivery. TECHNICAL FIELD

[0002] The present application relates to a recombinant adeno-associated virus vector and its application in gene delivery, and belongs to the technical field of biotechnology. BACKGROUND

[0003] Central nervous system diseases include central nervous system degenerative diseases and brain tumors. Among them, central nervous system degenerative diseases refer to a group of diseases caused by chronic progressive degeneration of central nervous tissue, and pathological changes of brain and / or spinal cord are observed, mainly including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS) and the like. Brain tumors refer to tumors originating from intracranial tissues and brain metastases, and the clinical manifestations are intracranial hypertension and various nervous system symptoms, mainly including glioblastoma, meningioma and pituitary adenoma and the like. Central nervous system diseases are usually caused by genetic mutations, and these mutations cause abnormal development of the nervous system, neurodegeneration or impaired neuronal function, etc. Gene therapy can deliver therapeutic genes to specific tissues or cells using gene delivery vectors to correct abnormalities caused by defective genes. At present, gene therapy has been used for the treatment of various diseases including nervous system diseases.

[0004] Adeno-associated virus (AAV) is a single-stranded DNA replication-deficient parvovirus. The genome of adeno-associated virus is 4.7 kb in size, composed of terminal inverted repeats ITR and intermediate Rep, Cap genes. Among them, ITR plays an important role in the replication and packaging of adeno-associated virus; Rep gene encodes non-structural proteins involved in adeno-associated virus replication, packaging and genome integration, Cap gene encodes structural proteins VP1, VP2 and VP3, which are assembled into virus capsid in the ratio of 1:1:10, respectively, to serve as a virus gene delivery vector. In addition, another open reading frame embedded in Cap gene encodes assembly activating protein AAP, which is involved in the targeting and assembly of capsid proteins. Adeno-associated virus transduction of cells mainly undergoes cell surface receptor-mediated endocytosis, escape from endosome, nuclear entry, de-capsidation and double-stranded conversion processes. After entering the cell, it can enter its replication cycle in the presence of helper virus.

[0005] Recombination adeno-associated virus (rAAV) is a gene vector modified on the basis of non-pathogenic wild-type adeno-associated virus. Compared with other gene therapy vectors such as lentiviral vectors, adenoviral vectors and retroviral vectors, adeno-associated virus vectors have the advantages of high gene delivery efficiency, safety and stability, and low cytotoxicity. So far, adeno-associated virus vectors have been applied to many gene therapy drugs, such as Glybera, Luxturna, Zolgensma, Upstaza and Roctavian. There are many adeno-associated virus serotypes, among which adeno-associated virus type 2 (AAV2) is the most widely studied adeno-associated virus, and Luxturna and Upstaza use adeno-associated virus type 2 vectors. However, the infection range of adeno-associated virus type 2 is limited in the central nervous system (see literature: Tordo et.al., 2018; Han et.al., 2022), so it is necessary to develop adeno-associated virus type 2 with higher transduction efficiency in the central nervous system in order to better apply adeno-associated virus type 2 vectors to gene delivery in the central nervous system. SUMMARY

[0006] To solve the above problems, the present application provides an adeno-associated virus capsid protein VP1 mutant, wherein the adeno-associated virus capsid protein VP1 mutant has a deletion of arginine at position 459, a mutation of arginine at position 447 and / or a mutation of threonine at position 456 compared with the adeno-associated virus capsid protein VP1 having the amino acid sequence shown in SEQ ID NO. 1.

[0007] In an embodiment of the present application, the adeno-associated virus capsid protein VP1 mutant has a mutation of arginine at position 447 to methionine and a mutation of threonine at position 456 to serine compared with the adeno-associated virus capsid protein VP1 having the amino acid sequence shown in SEQ ID NO. 1; or the adeno-associated virus capsid protein VP1 mutant has a deletion of arginine at position 459 and a mutation of threonine at position 456 to isoleucine compared with the adeno-associated virus capsid protein VP1 having the amino acid sequence shown in SEQ ID NO. 1.

[0008] In an embodiment of the present application, the amino acid sequence of the adeno-associated virus capsid protein VP1 mutant is shown in SEQ ID NO. 2 or SEQ ID NO. 4.

[0009] The application also provides an adeno-associated virus capsid mutant, which comprises the adeno-associated virus capsid protein VP1 mutant described above.

[0010] The application also provides an adeno-associated virus mutant, which comprises the adeno-associated virus capsid mutant described above and a genome.

[0011] In an embodiment of the application, the nucleotide sequence of the Cap gene in the genome is as shown in SEQ ID NO. 3 or SEQ ID NO. 5.

[0012] The application also provides a recombinant adeno-associated virus vector, which is characterized in that the recombinant adeno-associated virus vector comprises the adeno-associated virus mutant described above, and the genome of the adeno-associated virus mutant is integrated with a target gene encoding a target protein and / or a functional RNA.

[0013] In an embodiment of the application, the target protein comprises at least one of a marker protein or an active protein.

[0014] In an embodiment of the application, the marker protein comprises at least one of a fluorescent protein or an enzyme reaction color developing protein.

[0015] In an embodiment of the application, the marker protein comprises at least one of BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP, tagBFP, HRP, Firefly luciferase or Renilla luciferase.

[0016] In an embodiment of the application, the active protein comprises at least one of an activating neuron protein, an inhibiting neuron protein, a calcium ion signal probe protein, a small molecule signal probe protein, an apoptosis mediating protein, a disease related mutant protein, a normal protein under physiological conditions, a cytokine, an anti-virus factor, a virus infection auxiliary receptor, a recombinase or a gene editing tool protein.

[0017] In an embodiment of the application, the functional RNA comprises at least one of a small RNA, a small interfering RNA, a small hairpin RNA, a small guide RNA, an organelle localization RNA, a Barcode RNA for RNA sequencing or a Barcode RNA for in situ hybridization analysis.

[0018] In an embodiment of the present application, the genome of the adeno-associated virus mutant further integrates a promoter.

[0019] In an embodiment of the present application, the promoter comprises at least one of CAG, CMV, hUbC, Ef1a, nEF, hSyn, CaMKIIa, Vgat, Thyl, TRE, UAS, GFAP, gfaABC1D, TH, RPE65, TRE, CBA, PGK, E-SARE, C-fos, RAM, SST, PV, mDlx, NPY, CR, TCAP, SFRP2, ChAT, TPH2, mTH, GAD67, GFAP104, CD68, Nestin, MBP, TRPV1, L7 / Pcp2, mOXT, RK, hGRK1, CAR, Grm6, ROH, Nrl, MCK, dMCK or tMCK.

[0020] In an embodiment of the present application, the integration is insertion or substitution.

[0021] The present application also provides a gene delivery method, which is not for the purpose of diagnosis and treatment of diseases, for delivering a target gene encoding a target protein and / or functional RNA to a target site using the above-mentioned recombinant adeno-associated virus vector.

[0022] In an embodiment of the present application, the target site comprises the central nervous system.

[0023] The present application also provides the use of the above-mentioned adeno-associated virus capsid protein VP1 mutant, the above-mentioned adeno-associated virus capsid mutant, the above-mentioned adeno-associated virus mutant or the above-mentioned recombinant adeno-associated virus vector in gene delivery, preparation of a gene delivery reagent, establishment of a disease model or preparation of a gene therapy drug, which is not for the purpose of diagnosis and treatment of diseases.

[0024] In an embodiment of the present application, the gene delivery comprises delivering a target gene encoding a target protein and / or functional RNA to a target site.

[0025] In an embodiment of the present application, the target site comprises the central nervous system.

[0026] In an embodiment of the present application, the disease model comprises a central nervous system-related disease model.

[0027] In an embodiment of the present application, the gene therapy drug comprises a central nervous system-related disease gene therapy drug.

[0028] The technical solution of the present application has the following advantages:

[0029] 1. The application provides an adeno-associated virus capsid protein VP1 mutant, wherein the adeno-associated virus capsid protein VP1 mutant has the following mutations compared with the adeno-associated virus capsid protein VP1 with the amino acid sequence shown in SEQ ID NO. 1: the arginine at position 447 is mutated to methionine, and the threonine at position 456 is mutated to serine, or the arginine at position 459 is deleted, and the threonine at position 456 is mutated to isoleucine; the adeno-associated virus capsid protein VP1 mutant significantly increases the transduction efficiency of a recombinant adeno-associated virus vector in the central nervous system (by 10-20 times), and therefore, the adeno-associated virus capsid protein VP1 mutant has a great application prospect in establishing a central nervous system related disease model or preparing a central nervous system related gene therapy drug.

[0030] 2. The application provides a recombinant adeno-associated virus vector, wherein the recombinant adeno-associated virus vector comprises an adeno-associated virus capsid protein VP1 mutant, wherein the adeno-associated virus capsid protein VP1 mutant has the following mutations compared with the adeno-associated virus capsid protein VP1 with the amino acid sequence shown in SEQ ID NO. 1: the arginine at position 447 is mutated to methionine, and the threonine at position 456 is mutated to serine, or the arginine at position 459 is deleted, and the threonine at position 456 is mutated to isoleucine; the mutation of the adeno-associated virus capsid protein VP1 significantly increases the transduction efficiency of the recombinant adeno-associated virus vector in the central nervous system (by 10-20 times), and therefore, the recombinant adeno-associated virus vector has a great application prospect in establishing a central nervous system related disease model or preparing a central nervous system related gene therapy drug. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Map of plasmid pAAV2 / 2.A2.

[0032] Figure 2 : Map of plasmid pAAV2 / 2.A1.

[0033] Figure 3 : Brain tissue section imaging results of C57BL / 6 mice after positioning injection of rAAV2-CMV-EGFP-WPRE-hGH polyA and rAAV2.A2-CMV-EGFP-WPRE-hGH polyA viral vectors in brain regions.

[0034] Figure 4Imaging results of brain tissue sections of C57BL / 6 mice after rAAV2-CMV-EGFP-WPRE-hGH polyA and rAAV2.A1-CMV-EGFP-WPRE-hGH polyA viral vector injection into brain region. DETAILED DESCRIPTION

[0035] The following examples are provided to better enable those skilled in the art to further understand and practice the application, and are not intended to limit the scope of the application. Any product derived from the application or any product derived from the combination of the application and other prior art features, which is the same as or similar to the application, falls within the scope of the application.

[0036] The following examples are provided to better enable those skilled in the art to further understand and practice the application, and are not intended to limit the scope of the application. Any product derived from the application or any product derived from the combination of the application and other prior art features, which is the same as or similar to the application, falls within the scope of the application.

[0037] Example 1: Recombinant adeno-associated viral vector and method for preparing the same

[0038] The present example provides a recombinant adeno-associated viral vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA, which comprises an adeno-associated viral mutant AAV2.A2, the adeno-associated viral mutant AAV2.A2 comprises an adeno-associated viral capsid mutant, the adeno-associated viral capsid mutant comprises an adeno-associated viral capsid protein VP1 mutant, the adeno-associated viral capsid protein VP1 mutant has a methionine at position 447 and a serine at position 456, compared to the adeno-associated viral capsid protein VP1 having the starting amino acid sequence as shown in SEQ ID NO. 1, and the amino acid sequence is as shown in SEQ ID NO. 2.

[0039] The preparation process of the recombinant adeno-associated viral vector AAV2.A2 is as follows:

[0040] 1. Construction of plasmid pAAV2 / 2.A2

[0041] The plasmid pAAV2 / 2 (Addgene plasmid #104963) was used as a template, and Mu-A2-F (SEQ ID NO. 6) and Mu-A2-R (SEQ ID NO. 7) were used as primers. PCR was performed using a Fast Mutagenesis System kit (TransGen Biotech). The PCR product was digested with 1 μL of template digestion enzyme DMT (Beijing Quanshijin Biotechnology Co., Ltd.) at 37°C for 1 h to obtain the digestion product. 5 μL of the digestion product was transformed into Stbl3 competent cells (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the transformation product. The transformation product was plated on LB solid medium (Shenzhen Kangti Life Science and Technology Co., Ltd.) and incubated at 37°C for 16 h. Single colonies were picked and inoculated in 15 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L) and incubated at 37°C for 16 h to obtain the bacterial solution. The plasmid was extracted from the bacterial solution and sequenced. After sequencing, the plasmid pAAV2 / 2.A2 was obtained (see the map in FIG. 6). Figure 1 )。

[0042] The PCR reaction system was as follows: 2x TransStart FastPfu Fly PCR SuperMix, 25 μL; 10 μM forward primer, 1 μL; 10 M reverse primer, 1 μL; template pAAV2 / 2, 2 μL; ddH2O, 31 μL. The PCR reaction program was as follows: 98°C, 5 min; {98°C, 30 s; 60°C, 30 s; 72°C, 5 min; 72°C, 10 min} 36 cycles; 16°C, 30 min.

[0043] 2. Construction of recombinant adeno-associated virus vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA

[0044] Reference "AAV11 permits efficient retrograde targeting of projection neurons, Han et. al.; bioRxiv 2022.01.13.476170; doi: https: / / doi.org / 10.1101 / 2022.01.13.476170", using a three-plasmid packaging system to package adeno-associated virus, the core element loading plasmid pAAV-CMV-EGFP-WPRE-hGH polyA, adeno-associated virus element helper plasmid pAd-Helper and plasmid pAAV2 / 2.A2 were co-transfected into HEK-293T cells (American Type Culture Collection) at a plasmid molecular ratio of 1:1:1. After 72 hours of transfection, the supernatant and cell pellet were collected respectively, and concentrated and purified by iodixanol gradient centrifugation method to obtain recombinant adeno-associated virus vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA (in the genome of the recombinant adeno-associated virus vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA, the nucleotide sequence of the Cap gene is shown in SEQ ID NO. 3). The adeno-associated virus titer of the obtained recombinant adeno-associated virus vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA was detected by SYBR Green qPCR method, and the detection result was: 6.0 x 10 12 VG / mL.

[0045] Example 2: Recombinant adeno-associated virus vector and its preparation method

[0046] This example provides a recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA, which comprises an adeno-associated virus mutant AAV2.A1, the adeno-associated virus mutant AAV2.A1 comprises an adeno-associated virus capsid mutant, the adeno-associated virus capsid mutant comprises an adeno-associated virus capsid protein VP1 mutant, the adeno-associated virus capsid protein VP1 mutant has a deletion of arginine at position 459 and a mutation of threonine at position 456 to isoleucine compared to the adeno-associated virus capsid protein VP1 having the starting amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 4.

[0047] The preparation process of the recombinant adeno-associated virus vector AAV2.A1 is as follows:

[0048] 1. Construction of plasmid pAAV2 / 2.A1

[0049] PCR was performed using the plasmid pAAV2 / 2 (Addgene plasmid #104963) as a template, Mu-A1-F (SEQ ID NO. 8) and Mu-A1-R (SEQ ID NO. 9) as primers, and a Fast Mutagenesis System kit (TransGen Biotech) to obtain a PCR product. The PCR product was digested with 1 μL of template digestion enzyme DMT (Beijing Quanshijin Biotechnology Co., Ltd.) at 37 °C for 1 h to obtain a digestion product. 5 μL of the digestion product was transformed into Stbl3 competent cells (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain a transformed product. The transformed product was plated on LB solid medium (Shenzhen Kangti Life Science and Technology Co., Ltd.) and incubated at 37 °C for 16 h. Single colonies were picked and inoculated in 15 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L) and incubated at 37 °C for 16 h to obtain a bacterial solution. The plasmid was extracted from the bacterial solution and sequenced. After sequencing, the plasmid pAAV2 / 2.A1 was obtained (see the map in FIG. 1). Figure 2

[0050] The PCR reaction system was as follows: 2 x TransStart FastPfu Fly PCR SuperMix, 25 μL; 10 μM forward primer, 1 μL; 10 M reverse primer, 1 μL; template pAAV2 / 2, 2 μL; ddH2O, 31 μL. The PCR reaction program was as follows: 98 °C, 5 min; {98 °C, 30 s; 60 °C, 30 s; 72 °C, 5 min; 72 °C, 10 min} 36 cycles; 16 °C, 30 min.

[0051] 2. Construction of recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA

[0052] ​Reference "AAV11 permits efficient retrograde targeting of projection neurons, Han et. al.; bioRxiv 2022.01.13.476170; doi: https: / / doi.org / 10.1101 / 2022.01.13.476170", using a three-plasmid packaging system to package adeno-associated virus, the core element loading plasmid pAAV-CMV-EGFP-WPRE-hGH polyA, adeno-associated virus element auxiliary plasmid pAd-Helper and plasmid pAAV2 / 2.A1 were co-transfected into HEK-293T cells (American Type Culture Collection) at a plasmid molecular ratio of 1:1:1. After 72 hours of transfection, the supernatant and cell precipitate were collected, respectively, and concentrated and purified by iodixanol gradient centrifugation to obtain recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA (in the genome of the recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA, the nucleotide sequence of the Cap gene is shown in SEQ ID NO. 5). The adeno-associated virus titer of the obtained recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA was detected by SYBR Green qPCR method, and the detection result was: 8.0 x 10 12 VG / mL.

[0053] Comparative Example 1: Recombinant adeno-associated virus vector and method for preparing the same

[0054] This comparative example provides a recombinant adeno-associated virus vector rAAV2-CMV-EGFP-WPRE-hGH polyA and a method for preparing the same, which is based on Example 1, and the plasmid pAAV2 / 2.A2 is replaced by the plasmid pAAV2 / 2. The adeno-associated virus titer of the obtained recombinant adeno-associated virus vector rAAV2-CMV-EGFP-WPRE-hGH polyA was detected by SYBR Green qPCR method, and the detection result was: 6.0 x 10 12 VG / mL.

[0055] Experimental Example 1: Transduction efficiency of recombinant adeno-associated virus vector in central nervous system

[0056] This experimental example provides the transduction efficiency experiment of the recombinant adeno-associated virus vectors of Examples 1-2 and Comparative Example 1 in the central nervous system, and the experimental process is as follows:

[0057] The recombinant adeno-associated virus vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA of Example 1, the recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA of Example 2 and the recombinant adeno-associated virus vector rAAV2-CMV-EGFP-WPRE-hGH polyA of Comparative Example 1 were stereotaxically injected into two brain regions of C57BL / 6 mice (8-10 weeks old, purchased from Hunan SLEEK JINGDA Experimental Animal Co., Ltd.) at the caudoputamen nucleus (CPu) and the ventral hippocampus (vHPC) respectively, and the injection dose was 300 nL. After 3 weeks of injection, the mouse brain tissue was perfused. The mouse brain tissue was first fixed with a DEPC-treated PFA (paraformaldehyde) solution with a concentration of 4% (m / v, g / 100 mL) for 4 hours, and then dehydrated with a DEPC-treated PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH = 7.4) containing 30% (m / v, g / 100 mL) sucrose for 48 hours. The dehydrated mouse brain tissue was fully embedded with an embedding agent (SAKURA) and cut into 40 μm thick sections using a freezing microtome. The mouse brain tissue sections were laminated and microimaged using a slide scanner. The scanning results are shown in FIGS. 1-3. Figures 3-4 .

[0058] From Figure 3 It can be seen that, whether in the CPu brain region or the vHPC brain region, the recombinant adeno-associated virus vector rAAV2.A2-CMV-EGFP-WPRE-hGH polyA of Example 1 exhibited a larger transduction range (about 10 times larger) than the recombinant adeno-associated virus vector rAAV2-CMV-EGFP-WPRE-hGH polyA of Comparative Example 1.

[0059] From Figure 4 It can be seen that, in the CPu brain region, the recombinant adeno-associated virus vector rAAV2.A1-CMV-EGFP-WPRE-hGH polyA of Example 2 exhibited a larger transduction range (about 20 times larger) than the recombinant adeno-associated virus vector rAAV2-CMV-EGFP-WPRE-hGH polyA of Comparative Example 1.

[0060] Obviously, the above examples are merely illustrative examples for the sake of clarity, and are not intended to limit the embodiments. Based on the above description, those of ordinary skill in the art can also make other different forms of changes or modifications. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.

Claims

1. A mutant of adeno-associated virus capsid protein VP1, characterized in that, Compared with the adeno-associated virus capsid protein VP1 whose starting amino acid sequence is shown in SEQ ID NO.1, the mutant of adeno-associated virus capsid protein VP1 has arginine at position 447 mutated to methionine, and threonine at position 456 mutated to serine.

2. The adeno-associated virus capsid protein VP1 mutant as described in claim 1, characterized in that, The amino acid sequence of the adeno-associated virus capsid protein VP1 mutant is shown in SEQ ID NO.

2.

3. An adeno-associated virus capsid mutant, characterized in that, The adeno-associated virus capsid mutant comprises the adeno-associated virus capsid protein VP1 mutant as described in claim 1 or 2.

4. An adeno-associated virus mutant, characterized in that, The adeno-associated virus mutant comprises the adeno-associated virus capsid mutant and genome as described in claim 3.

5. The adeno-associated virus mutant as described in claim 4, characterized in that, The nucleotide sequence of the Cap gene in the genome is shown in SEQ ID NO.

3.

6. A recombinant adeno-associated virus vector, characterized in that, The recombinant adeno-associated virus vector comprises the adeno-associated virus mutant of claim 4 or 5, and the genome of the adeno-associated virus mutant is integrated with a target gene encoding the target protein and / or functional RNA.

7. The recombinant adeno-associated virus vector as described in claim 6, characterized in that, The genome of the adeno-associated virus mutant also integrates a promoter.

8. A gene delivery method, wherein the method is not for the purpose of disease diagnosis and treatment, characterized in that, The method uses the recombinant adeno-associated virus vector of claim 6 or 7 to deliver the target gene and / or functional RNA encoding the target protein to the target location.

9. The application of the adeno-associated virus capsid protein VP1 mutant of claim 1 or 2, or the adeno-associated virus capsid mutant of claim 3, or the adeno-associated virus mutant of claim 4 or 5, or the recombinant adeno-associated virus vector of claim 6 or 7 in gene delivery, preparation of gene delivery reagents, establishment of disease models, or preparation of gene therapy drugs, wherein the application is not for the purpose of disease diagnosis and treatment.

10. The application as described in claim 9, characterized in that, The gene delivery includes delivering a target gene encoding a target protein and / or functional RNA to a target location; the disease model includes a central nervous system-related disease model; and the gene therapy drug includes a gene therapy drug for central nervous system-related diseases.

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