Capsid protein mutants for improving AAV virus production and whole-eye infection ability and their applications

Abstract

The present invention belongs to the field of biotechnology, and discloses capsid protein mutants and applications for improving AAV virus yield and whole-eye infection ability. Capsid protein mutants are obtained by replacing amino acids 561 to 588 of wild-type AAV2 virus capsid protein with specific polypeptides. Capsid protein mutants of some examples of the present invention can effectively improve the whole-eye infection ability of AAV2, fundamentally solving the problem of AAV2 eye infection efficiency. Capsid protein mutants of some examples of the present invention have better yield. Capsid protein mutants of some examples of the present invention can be well applied to gene therapy of the eye or construction of eye disease models.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a capsid protein mutant capable of improving AAV virus production and whole-eye infection ability and an application thereof. Background Art

[0002] Adeno-associated virus (AAV) has the characteristics of low immunogenicity, tissue targeting, low integration, and long-term stable expression, making it the preferred vector for in vivo treatment in the field of gene therapy. It is widely used in the research of various diseases such as genetic diseases, cancer, and some chronic diseases. However, it still faces many challenges: First, the production process is complex, the cost is high, and the production capacity is limited; second, high-dose administration may show neurotoxicity and hepatotoxicity, causing safety issues. Third, the ability to infect some tissue cells is weak. For example, it is difficult to infect the posterior retinal cells by intravitreal administration, and it is difficult to infect central nervous cells by intravenous administration. Modification and screening of AAV capsid protein to improve its targeting and transfection efficiency to specific tissues, in order to achieve lower and safer administration doses, is an effective way to solve this problem.

[0003] Due to the existence of the blood-ocular barrier, the eye has a certain immune exemption, local administration is relatively safe, and fundus diseases are mostly single-gene genetic diseases, so ophthalmology has become a hot area for gene therapy. Different fundus diseases have different pathogenesis. When using AAV virus for gene therapy, the cell layers that need to be infected are different. There are two commonly used injection methods in clinical practice: intravitreal injection and subretinal injection. Subretinal injection has a better infection effect, especially for the RPE layer and outer nuclear layer, but this method is more invasive and can easily cause retinal detachment, which may lead to photoreceptor degeneration, visual function damage and glial cell proliferation. Intravitreal injection has a lower surgical risk, but conventional serotypes such as AAV2 and AAV5 can only infect the RGC layer, and have poor infection effects on cones, rods and RPE. Therefore, the development of AAV mutants that can improve the expression ability of the whole eye or can penetrate the RGC layer and inner nuclear layer to infect outer nuclear layer cells and RPE cells is of great significance to solving the problem of delivery vectors for ophthalmic gene therapy.

[0004] Previous studies have shown that mutations in the amino acid sites 561 to 588 of Cap2 can optimize its ability to infect mouse eye tissues, but the specific mechanism by which replacements can improve the AAV virus's ability to infect the entire eye remains unclear. Summary of the invention

[0005] The present invention aims to transform the AAV capsid protein to obtain an AAV virus mutant with higher yield and better whole-eye expression ability, and specifically provides a capsid protein mutant and application thereof that improves the AAV virus yield and whole-eye infection ability.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides: a capsid protein mutant that improves the ability of AAV virus to infect the entire eye, obtained by replacing amino acids 561 to 588 of the wild-type AAV2 virus capsid protein with any of the following polypeptides:

[0008] 1)DEEEIATVNPVATEQYGSVSTNLQRGNR

[0009] 2)DENEIATTNPVATEQYGWVSTNLQRGNR

[0010] 3)DEEEIVATNPVATEMYGVVSTNLQRGNR

[0011] 4) DEQEIAATNPVATEQYGSVSYNLQRINR

[0012] 5) DESEIATTNPVATEQYGVVSTNLQRGNR

[0013] 6) DEEEIRTTNPVATEQYGSVSTNLQRGNAAAQGYPPKPPAR

[0014] 7) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSQLGARGLSR

[0015] 8) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSARGGYTGLSR

[0016] 9) DEEEIRTTNPVATEQYGSVSTNLQRGNTGTSTFGASRGLSR

[0017] 10) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSVTFSGTQRGLSR

[0018] 11) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSALPSHLSGRGLSR

[0019] 12) DEEEIRTTNPVATEQYGSVSTNLQRGNTGLQATMGRGLSR

[0020] 13) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSLGTNPTRGLSR

[0021] 14) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSYVGQPPRGLSR

[0022] 15) DEEEIRTTNPVATEQYGSVSTNLQRGNTGRDKVGIQGLSR.

[0023] In some examples of capsid protein mutants, the GenBank accession number of the wild-type AAV2 virus capsid protein amino acid sequence is YP_680426.1.

[0024] The second aspect of the present invention provides: a gene encoding the capsid protein mutant described in the first aspect of the present invention.

[0025] In some cases of genes, codon optimization was performed depending on the expression system.

[0026] The third aspect of the present invention provides: an expression system into which the gene described in the second aspect of the present invention is inserted.

[0027] In some examples of expression systems, the expression system is a recombinant AAV vector.

[0028] In some examples of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

[0029] The fourth aspect of the present invention provides: a composition comprising the expression system described in the third aspect of the present invention.

[0030] The fifth aspect of the present invention provides: use of the composition described in the fourth aspect of the present invention in the preparation of gene therapy preparations or transgenic preparations.

[0031] In some examples of applications, the gene therapy formulation is used to treat eye diseases.

[0032] In some application examples, the transgenic preparation is used to treat eye diseases or to construct disease animal models.

[0033] In some application examples, the animal is selected from mice, rats, cynomolgus monkeys, rhesus monkeys, etc.

[0034] In some application examples, the disease is an eye disease, particularly an eye hereditary disease.

[0035] The sixth aspect of the present invention provides: a method for constructing a transgenic animal model, comprising introducing the composition described in the fourth aspect of the present invention into the animal.

[0036] In some examples of the construction methods, the animal is selected from mice, rats, cynomolgus monkeys, rhesus monkeys, etc.

[0037] In some examples of the construction method, the transgenic animal model is a transgenic animal model for an eye disease.

[0038] In some examples of the construction methods, the eye disease is an eye genetic disease.

[0039] The beneficial effects of the present invention are:

[0040] The capsid protein mutants of some examples of the present invention can effectively improve the whole-eye infection ability of AAV2, and fundamentally solve the problem of AAV2 eye infection efficiency.

[0041] The capsid protein mutants of some examples of the present invention have better yields.

[0042] The capsid protein mutants of some examples of the present invention can be well applied to gene therapy of the eye or construction of eye disease models. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The agarose gel electrophoresis identification diagram of the mutant plasmid constructed in the test example of this invention.

[0044] Figure 2 This is the pAAV2-Rep2 / Cap2 packaging plasmid map in the examples of the present invention.

[0045] Figure 3 This is the pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid map in the examples of the present invention.

[0046] Figure 4 This is a map of the pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid constructed in the examples of the present invention.

[0047] Figure 5 This is the pAAV-CAG-Luciferase-HGH expression plasmid map of the virus packaging three-plasmid system in the embodiment of the present invention.

[0048] Figure 6 This is a map of the pHelper auxiliary plasmid in the three-plasmid system for virus packaging in an embodiment of the present invention.

[0049] Figure 7 The yield results of AAV2 wild type, 7M8 and AAV2 mutant prepared by the scheme in the test example of the present invention.

[0050] Figure 8The in vivo imaging results of the mouse eye infection effects of AAV2 wild type, 7M8 and some AAV2 mutants (AAV2-EYE-08 to EYE-22) prepared according to the scheme in the test example of the present invention.

[0051] Fig. 9 These are the chemiluminescence detection results of the mouse eye infection effects of the AAV2 wild type, 7M8 and AAV2 mutants (AAV2-EYE-08 to EYE-22) prepared in the scheme of the test example of the present invention.

[0052] Fig.10 It is an amino acid sequence alignment diagram of AAV2 wild type, 7M8 and AAV2 mutants (AAV2-EYE-08 to EYE-22) according to the embodiments of the present invention. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels. Example

[0054] This embodiment provides a variety of adeno-associated virus variants (AAV2-EYE-08 to AAV2-EYE-22, AAV2-Mut201 to AAV2-Mut213) based on AI design and previous studies. The preparation of the adeno-associated virus variants includes the following steps:

[0055] Table 1 Amino acid sequence information of AAV2 mutants with substitutions at positions D561 to R588 in Cap2

[0056]

[0057] 1. Plasmid construction

[0058] The nucleic acid sequences corresponding to the amino acid sequences of AAV2-EYE-08~EYE-22 and AAV2-Mut201~AAV2-Mut213 (as shown in Table 1) were synthesized (GenScript Biotechnology) and cloned into the pAAV2-Rep2 / Cap2 plasmid by a seamless method. The sequences of Cap2 561 to 588 of wild-type AAV2 were replaced with mutant sequences to construct the pAAV-mutant plasmid vector. The ligation products were transformed into competent Escherichia coli Stbl3, and single colonies were picked for expansion and plasmid extraction for sequencing verification and restriction enzyme digestion verification. The sequencing verification was consistent with the theoretical sequence, and the results were identified by agarose gel electrophoresis. Figure 1 As shown, the mutant plasmid was successfully constructed.

[0059] 2. AAV2-WT, 7M8 and AAV mutant virus packaging

[0060] AAV2-WT, 7M8 or AAV2-mutant packaging plasmids were co-transfected with pAAV-CAG-Luciferase-hGH expression plasmid and pHelper helper plasmid into suspension HEK293T cells.

[0061] Virus packaging includes the following steps:

[0062] A. Cell culture

[0063] (1) Take out a tube of suspended 293T cells from the liquid nitrogen tank, put it in a 37℃ water bath, shake it quickly to thaw the cells, add it to a centrifuge tube containing 5ml Wayne 293 serum-free medium, centrifuge it at 200×g for 5min, discard the supernatant, resuspend it in 10ml Wayne 293 serum-free medium, place it in a 125ml triangular shake flask, add Wayne 293 serum-free medium to 20ml, and culture it in a shaking incubator at 120rpm, 37℃, and 5% CO2;

[0064] (2) After 48-72 hours of culture, the cell density reaches 2-3E+6 cells / ml, and the cells can be subcultured;

[0065] (3) Take out the seed cells to the clean bench, shake well and then pipette about 500 μl of sample, take 20 μl of sample and add 20 μl of trypan blue to mix, pipette 20 μl of the mixture into the cell counting plate, read the data three times on the automatic cell counter, and take the average value as the cell density;

[0066] (4) Take an appropriate amount of cells, dilute them to 0.6-0.65E+6 cells / ml with fresh Wayne 293 serum-free medium, and culture them in a 500 ml triangular shake flask with a culture volume of 25 ml per flask; culture them in a shaking CO2 cell culture incubator under the following conditions: 120 rpm, 37°C, and 5% CO2.

[0067] B. Cell transfection

[0068] (1) Cultivate the above cells for 48 hours, and the cell density reaches 3-3.8E+6 cells / ml, and the cells can be transfected;

[0069] (2) For each bottle of cells, add packaging plasmid, pHelper, and pAAV-CAG-Luciferase--hGH (100 μg in total) to 4 mL of DMEM medium and mix thoroughly to prepare the "plasmid dilution solution".

[0070] (3) Take another 1 mL of DMEM medium and add 300 μg of PEI to mix well, which is the "PEI dilution solution";

[0071] (4) Pour the "PEI diluent" into the "plasmid diluent", mix quickly, and let stand at room temperature for 25 minutes to form a transfection complex; add the transfection complex dropwise into the cell solution while gently shaking;

[0072] (5) Place the culture flask in a shaking CO2 cell culture incubator and culture under the following conditions: 120 rpm, 37°C, and 5% CO2.

[0073] C. Virus Harvest

[0074] (1) After transfection, cells were cultured for 72 hours and virus harvesting was started.

[0075] (2) Collect the cell suspension in each bottle into a centrifuge bottle, centrifuge at 1000g for 5 min, collect the supernatant into a new centrifuge bottle, add 0.245mL of 50% PEG8000 solution (containing 0.5mol / L NaCl) per ml of supernatant, mix thoroughly, and let stand at 2-8℃ overnight;

[0076] (3) Add an appropriate amount of 0.5% TritonX-100 cell lysis buffer (containing universal nuclease) to the cell pellet, treat at 37°C for 1-2h, then add 1 / 10 volume of 5 mol / L NaCl, mix thoroughly and centrifuge at 4°C and 3000g for 15min, collect the supernatant, which is the "viral crude extract" and temporarily store at 2-8°C;

[0077] (4) Centrifuge the supernatant after PEG8000 precipitation at 4°C and 3000g for 15 min, discard the supernatant, and resuspend it in the "viral crude extract" in step (3) for later use.

[0078] D. Virus purification

[0079] 1) Take an Ultra-Clear centrifuge tube, add 0.5mL 60% Iodixanol, 2mL 40% Iodixanol, 1.5mL 25% Iodixanol, 1.5mL 15% Iodixanol, the collected virus suspension, and finally the cell lysate to balance at the bottom;

[0080] 2) 10°C, 230,000 g, 8°C, 9°C ultracentrifugation for 18 h;

[0081] 3) Take an ultrafiltration tube and soak the filter membrane with 1 mL of PBS buffer;

[0082] 4) Use a pipette to carefully extract the middle layer of 40% Iodixanol-60% Iodixanol in the ultracentrifuge tube, avoiding protein, and transfer it to the ultrafiltration tube;

[0083] 5) Add an appropriate amount of PBS buffer and blow evenly, centrifuge at 4500g for 3-5 min, and repeat this step 5-7 times to remove Iodixanol;

[0084] 6) Add 1 mL of PBS buffer and pipette for 40-50 times to form a virus suspension, then transfer to an EP tube;

[0085] 7) Use a 5mL syringe to draw the virus suspension in the EP tube and filter it through a 0.22μm filter. Collect 20μL of the virus liquid as the test sample and dispense it into 100μL tubes to obtain the adeno-associated virus variant.

[0086] Comparative Example 1

[0087] This comparative example provides a wild-type adeno-associated virus (AAV2-WT), which is prepared according to the method of Example 1 with respect to the AAV2-mutant, the only difference being that the sequence of the adeno-associated virus capsid protein adopts the wild-type AAV2 capsid protein sequence (SEQ ID NO.1).

[0088] Comparative Example 2

[0089] This comparative example provides an AAV2 adeno-associated virus mutant (AAV2-7M8) with strong whole-eye expression ability that has been used in ophthalmic clinical trials. It is prepared according to the method of Example 1 with the AAV2- mutant, and the only difference is that the adeno-associated virus capsid protein sequence adopts the mutant AAV2-7M8 capsid protein sequence (SEQ ID NO.2).

[0090] Test Case

[0091] 1. Virus titer detection

[0092] (1) Virus lysis

[0093] 1) Take 20 μL of the virus samples of the embodiment and the comparative example respectively;

[0094] 2) Add 1 μL each of 10% SDS, 0.5 mol / L EDTA, and proteinase K and mix well;

[0095] 3) Incubate at 56°C for 1 hour in a thermomixer, then at 90°C for 10 minutes;

[0096] 4) Take 10ul of virus lysate and dilute it 10-fold to 10,000-fold to obtain virus dilution solution for later use.

[0097] (2) Preparation of standard samples for standard curve

[0098] The concentration is 2×10 12 The plasmid standard of 10 copies / mL was diluted 10-fold with ddH2O into 6 gradients as the standard curve template 2×10 11 copies / mL, 2×10 10 copies / mL, 2×10 9 copies / mL, 2×10 8 copies / mL, 2×10 7 copies / mL, 2×10 6 copies / mL.

[0099] (3) Quantitative qPCR

[0100] 1) Take a 0.2mL PCR tube and prepare the following reaction system: 3 replicates for each virus dilution; 2× qPCR Mix 10μL; 0.2μL each of forward and reverse primers; 5μL virus dilution; 4.2μL ddH2O;

[0101] Amplification Primers:

[0102] Forward primer 5′- GGAACCCCTAGTGATGGAGTT-3′ (SEQ ID NO. 33);

[0103] Reverse primer 5'- CGGCCTCAGTGAGCGA-3' (SEQ ID NO. 34);

[0104] 2) PCR amplification

[0105] Pre-denaturation: 95°C, 2 min;

[0106] 40× cycles: 95°C, 15 s; 60°C, 60 s;

[0107] 3) qPCR data processing: Virus titer = dilution factor * viral gene array copy number.

[0108] The present invention scheme transfects AAV2-WT virus packaging three-plasmid system (including pAAV2, pAAV-CAG-Luciferase, pHelper) and AAV2-mutant virus packaging three-plasmid system (including pAAV2-mutant, pAAV-CAG-Luciferase, pHelper) into HEK293T cells in equal amounts, and harvests the virus solution by cell lysis, purifies it by iodixanol ultracentrifugation, and evaluates its yield by QPCR detection of virus titer. Figure 2 This is the pAAV2-Rep2 / Cap2 packaging plasmid map in the examples of the present invention. Figure 3 This is the pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid map in the examples of the present invention. Figure 4 This is a map of the pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid constructed in the examples of the present invention. Figure 5 This is the pAAV-CAG-Luciferase-HGH expression plasmid map of the virus packaging three-plasmid system in the embodiment of the present invention. Figure 6 : is the pHelper auxiliary plasmid map of the virus packaging three-plasmid system in the embodiment of the present invention. Figure 7 As shown in the figure, it can be seen that 6 mutants have a yield lower than 0.5 times that of AAV2-WT, and are identified as low-yield variants and are not subject to in vitro verification; among the remaining 22 variants, only AAV2-EYE-12 variant has a yield lower than AAV2-WT, which is 0.84 times its yield, and 21 mutant virus yields (accounting for 75%) are higher than AAV2-WT, which are 1.26 to 4.36 times its yield, among which AAV2-EYE-20 variant has the highest yield.

[0109] 2. Mouse whole eye expression ability test

[0110] The samples of the embodiment and the comparative example were diluted to 2E+9vg / µl and injected into the vitreous cavity of Bab / c mice, 1.5µl was injected into each eye (the total amount of virus was 3E+9vg), and 3 mice were injected with each virus. The expression of Luciferase was detected by in vivo imaging and chemiluminescence 3-4 weeks after injection.

[0111] (1) In vivo imaging detection

[0112] Three weeks after virus injection, D-luciferin potassium salt solution was injected intraperitoneally. Two minutes after injection, the mice were placed in the induction box of the anesthesia machine for isoflurane-induced anesthesia. When the mice's breathing became stable and there were no more large movements, they were transferred to the breathing mask of the imaging device to maintain anesthesia. The imaging parameters were set and the images were taken.

[0113] The present invention injects AAV2-WT, AAV2-7M8 and variant viruses into the vitreous cavity of mice. The in vivo imaging results 3 weeks later show that the Luciferase signal in the eyes of all mice injected with variant viruses is stronger than that of AAV2-WT and AAV2-7M8. Some of the best embodiments and comparison examples are selected. Figure 8 This indicates that the adeno-associated virus variant prepared by the scheme of the present invention can improve the ability of AAV2 virus to infect the whole eye.

[0114] (2) Chemiluminescence detection

[0115] 1) Draw materials

[0116] The mice were killed by sciatic nerve dislocation, the eyelids were opened, the eyeballs were removed with forceps, and the muscles and connective tissues around the eyeballs were removed. The eyeballs were placed in 2 ml grinding tubes, one tube for each eyeball, and temporarily stored in an ice bath.

[0117] 2) Sample pretreatment

[0118] Add 500ul of 1xCell Culture Lysis Reagent to the homogenate tube containing the mouse eyeball (single eyeball) and homogenize in a grinder precooled to -65°C. After homogenization, incubate at 4°C for 1-2h, centrifuge at 15000r / min, 4°C for 3min, and aspirate the supernatant.

[0119] 3) Color rendering

[0120] Take a black opaque 96-well plate, add 20ul sample to each well, 2-3 replicates for each sample, take out the substrate colorimetric solution that has been warmed up at room temperature away from light, and add 100ul to each well. After adding the colorimetric solution, immediately put it into the ELISA reader for detection and read the fluorescence signal value.

[0121] The present invention injected AAV2-WT, AAV2-7M8 and mutant viruses into the vitreous cavity of mice. In order to more accurately quantify the expression effect, the eyeballs were removed and homogenized for chemiluminescence detection after 3 weeks. The results are as follows: Fig. 9As shown, 16 groups (accounting for 76.2%) of all mice injected with mutant viruses had higher Luciferase expression levels in the whole eye than AAV2-WT and AAV2-7M8, which were 1.23 to 8.4 times that of AAV2-WT. Among them, 15 groups had expression levels that were more than 5 times that of AAV2-WT, which was considered to have significantly improved whole-eye expression ability, among which AAV2-EYE-09 had the highest expression level. This indicates that the adeno-associated virus variant prepared by the scheme of the present invention can improve the whole-eye expression ability of the AAV2 virus.

[0122] The sequence alignment results of adeno-associated virus variant capsid proteins are shown in the figure Fig.10 The difference between the sequences of the adeno-associated virus variants EYE-08 to EYE-22 capsid proteins and the wild-type AAV2 capsid proteins is that the sequences of the wild-type capsid proteins D561 to R588 (shown in SEQ ID NO.3) are replaced with a polypeptide sequence of 28 to 42 amino acids. Among them, EYE-08 to EYE-12 are partial amino acid replacements, and EYE-13 to EYE-13 are multiple amino acid insertions.

[0123] The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO.1, and the sequence of the adeno-associated virus variant 7M8 is shown in SEQ ID NO.2.

[0124] In summary, the adeno-associated virus variant prepared by the scheme of the present invention can significantly enhance the whole-eye infection ability of AAV2 virus compared with the wild type.

[0125] The amino acid sequence of wild-type adeno-associated virus capsid protein Cap2 is SEQ ID NO.1: (protein ID: YP_680426.1).

[0126] The amino acid sequence of AAV2 adeno-associated virus mutant capsid protein Cap2-7M8 is SEQ ID NO.2:

[0127] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVF QAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRV T TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSY TFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LALGETTRPA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKS I NVDFTVDTNGVYSEPRPIGTRYLTRNL.

[0128] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A capsid protein mutant that improves the ability of AAV virus to infect the entire eye, characterized in that: The wild-type AAV2 virus capsid protein is obtained by replacing amino acids 561 to 588 of the wild-type AAV2 virus capsid protein with any of the following polypeptides, wherein the wild-type AAV2 virus capsid protein amino acid sequence has a GenBank number of YP_680426.1: 1)DEEEIATVNPVATEQYGSVSTNLQRGNR 2)DENEIATTNPVATEQYGWVSTNLQRGNR 3)DEEEIVATNPVATEMYGVVSTNLQRGNR 4) DEQEIAATNPVATEQYGSVSYNLQRINR 5) DESEIATTNPVATEQYGVVSTNLQRGNR.

2. A gene encoding the capsid protein mutant according to claim 1.

3. The gene according to claim 2, characterized in that Codon optimization was performed according to the expression system.

4. An expression system, into which the gene according to claim 2 or 3 is inserted, the expression system is a recombinant AAV vector, and the AAV is selected from any one of AAV2 and its variants.

5. A composition, characterized in that Comprising the expression system described in claim 4.

6. Use of the composition according to claim 5 in the preparation of gene therapy preparations or transgenic preparations.

7. A method for constructing a transgenic animal model, comprising introducing the composition of claim 5 into the animal.

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