Capsid protein mutant capable of improving whole-eye infectivity of aav virus and application thereof

Abstract

The application discloses a capsid protein mutant capable of improving the whole-eye infection ability of an AAV virus and an application thereof. The inventors have found that the whole-eye infection ability of an AAV virus can be effectively improved by using a specific peptide to replace amino acids at positions 561 to 588 of a wild-type AAV2 virus capsid protein, and the problem of the infection efficiency of AAV2 on eyes is fundamentally solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a capsid protein mutant that can enhance the whole-eye infection ability of AAV virus. Background Technology

[0002] Adeno-associated virus (AAV) is a commonly used vector for gene therapy delivery. Its principle involves replacing the ITR sequences within the AAV genome with the target gene sequence through genetic engineering. This target gene is then delivered to target cells via cell infection, achieving the gene therapy objective. Recombinant AAV possesses advantages such as safety, high efficiency, stability, persistence, specificity, and low integration, making it one of the primary delivery methods in gene therapy. However, in vivo injection into animals often requires high doses and high purity of AAV virus, and the high production cost of AAV remains a bottleneck in gene therapy.

[0003] As downstream gene therapy technologies mature, the limitations of upstream AAV production throughput become increasingly apparent. To address this, existing strategies focus on two main aspects: first, optimizing existing AAV production processes to increase yield; and second, modifying the AAV capsid protein to enhance its targeting and infectivity to specific tissues, thereby achieving lower and safer dosages. AAV production processes primarily aim to increase viral yield by optimizing plasmid ratios, transfection reagent ratios, cell culture conditions, and scaling up production, without involving modifications to the AAV itself. However, based on the structural characteristics of the AAV virus, its viral properties, such as tissue targeting, immunogenicity, and packaging yield, are mainly determined by its surface capsid protein. Previous studies have shown that mutations at amino acid sites 561 to 588 in Cap2 can optimize its infectivity in mouse eye tissues. However, the specific mutations that can improve the AAV virus's ability to infect the entire eye remain unknown. Summary of the Invention

[0004] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a capsid protein mutant that can improve the whole-eye infectivity of AAV virus.

[0005] The technical solution adopted in this invention is:

[0006] The first aspect of the present invention provides:

[0007] A capsid protein mutant that enhances the ability of AAV virus to infect the entire eye is obtained by replacing amino acids 561 to 588 of the wild-type AAV virus capsid protein with one of the following peptides:

[0008] 1) DEHEIKTTNPVATEGYGEVATNWQRGNR

[0009] 2) DEEEIRTTNPVATEQYGSVSTNLQRGNTGRSAGLGTGLSR

[0010] 3) DEQEIAATNPVATEQYGSVSTNLQRGNR

[0011] 4) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR

[0012] 5) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR

[0013] 6) DEEEIRTTNPVATEQYGSVSTNLQRGNTGPLLDGTKGLSR

[0014] 7) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSPIKDGTKGLSR.

[0015] In some instances of capsid protein mutants, the amino acid sequence of the wild-type AAV2 viral capsid protein is shown in SEQ ID NO.1.

[0016] A second aspect of the present invention provides:

[0017] A gene encoding a capsid protein mutant as described in the first aspect of the invention.

[0018] In some gene instances, codon optimization is performed based on the expression system.

[0019] A third aspect of the present invention provides:

[0020] An expression system having the gene described in the second aspect of the present invention inserted therein.

[0021] In some instances of expression systems, the expression system is a recombinant AAV vector.

[0022] In some instances of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 and their variants.

[0023] A fourth aspect of the present invention provides:

[0024] A composition comprising the expression system described in the third aspect of the present invention.

[0025] In some examples of compositions, excipients are also added.

[0026] A fifth aspect of the present invention provides:

[0027] The use of the composition described in the fourth aspect of this invention in the preparation of gene therapy formulations or transgenic formulations.

[0028] In some applications, it is used to prepare gene therapy formulations or transgenic formulations for targeting the eye.

[0029] A fifth aspect of the present invention provides:

[0030] A method for constructing a transgenic animal model includes introducing the composition described in the fourth aspect of the present invention into the animal.

[0031] In some examples of construction methods, this is used for the construction of transgenic animal models of eye diseases.

[0032] The beneficial effects of this invention are:

[0033] Some examples of capsid protein mutants of the present invention can effectively improve the whole-eye infection ability of AAV virus, fundamentally solving the problem of AAV2's efficiency in infecting the eye. Attached Figure Description

[0034] Figure 1 This is an agarose gel electrophoresis image of the mutant plasmid constructed in this test example.

[0035] Figure 2 The image shows the agarose gel electrophoresis identification of the expression plasmid pAAV-CAG-EGFP-Barcod-HGH constructed in this test example.

[0036] Figure 3 This is a pAAV2-Rep2 / Cap2 packaging plasmid map from an embodiment of the present invention.

[0037] Figure 4 This is a diagram of the pAAV2-7M8-Rep2 / Cap2-7M8 packaging plasmid in an embodiment of the present invention.

[0038] Figure 5 This is a map of the pAAV2-Mut-Rep2 / Cap2-Mut packaging plasmid constructed in an embodiment of the present invention.

[0039] Figure 6 This is a map of the pAAV-CAG-EGFP-Barcod-HGH expression plasmid of the viral packaging three-plasmid system in this embodiment of the invention.

[0040] Figure 7 This is a pHelper auxiliary plasmid map of the virus packaging three-plasmid system in an embodiment of the present invention.

[0041] Figure 8 The results show the whole-eye infection performance of the adeno-phase variant virus and the wild-type virus prepared according to the scheme in the test example of this invention.

[0042] Figure 9 This is a sequence alignment diagram of the wild type, 7M8, and AAV2-EYE-01 to EYE-07 amino acids in an embodiment of the present invention. Detailed Implementation

[0043] The first aspect of the present invention provides:

[0044] A capsid protein mutant that enhances the ability of AAV virus to infect the entire eye is obtained by replacing amino acids 561 to 588 of the wild-type AAV virus capsid protein with one of the following peptides:

[0045] 1) DEHEIKTTNPVATEGYGEVATNWQRGNR

[0046] 2) DEEEIRTTNPVATEQYGSVSTNLQRGNTGRSAGLGTGLSR

[0047] 3) DEQEIAATNPVATEQYGSVSTNLQRGNR

[0048] 4) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR

[0049] 5) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR

[0050] 6) DEEEIRTTNPVATEQYGSVSTNLQRGNTGPLLDGTKGLSR

[0051] 7) DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSPIKDGTKGLSR.

[0052] Wild-type AAV virus capsid proteins have similar structures and, after the same modifications, have the same or similar functions.

[0053] In some examples of capsid protein mutants, the amino acid sequence of the wild-type AAV2 virus capsid protein is shown in SEQ ID NO.1. Experimental data show that modifying the wild-type AAV2 virus capsid protein can effectively improve the AAV virus's ability to infect the entire eye.

[0054] A second aspect of the present invention provides:

[0055] A gene encoding a capsid protein mutant as described in the first aspect of the invention.

[0056] In some gene instances, codon optimization is performed based on the expression system.

[0057] A third aspect of the present invention provides:

[0058] An expression system having the gene described in the second aspect of the present invention inserted therein.

[0059] In some instances of expression systems, the expression system is a recombinant AAV vector.

[0060] In some instances of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 and their variants.

[0061] A fourth aspect of the present invention provides:

[0062] A composition comprising the expression system described in the third aspect of the present invention.

[0063] In some examples of the compositions, excipients are also added. Excipients are commonly used in AAV expression systems.

[0064] A fifth aspect of the present invention provides:

[0065] The use of the composition described in the fourth aspect of this invention in the preparation of gene therapy formulations or transgenic formulations.

[0066] In some applications, it is used to prepare gene therapy formulations or transgenic formulations for targeting the eye.

[0067] A fifth aspect of the present invention provides:

[0068] A method for constructing a transgenic animal model includes introducing the composition described in the fourth aspect of the present invention into the animal.

[0069] In some examples of construction methods, this is used for the construction of transgenic animal models of eye diseases.

[0070] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Example 1

[0071] This embodiment provides multiple adeno-associated virus variants (AAV2-EYE-01 to AAV2-EYE-07) based on AI design and previous research. The preparation of these adeno-associated virus variants includes the following steps:

[0072] 1. Plasmid construction

[0073] The nucleic acid sequences corresponding to the amino acid sequences AAV2-EYE-01 to EYE-07 (as shown in Table 1) were synthesized by Genewiz Biotechnology Co., Ltd. The synthesized sequences were cloned into the pAAV2-Rep2 / Cap2 plasmid, and the Cap2 sequences 561 to 588 were replaced with mutant sequences to construct the pAAV-mutant plasmid vector. The ligation product was transformed into competent E. coli Stbl3 cells, and single colonies were picked to extract plasmids for sequencing and enzyme digestion verification. The results were identified by agarose gel electrophoresis. Figure 1 As shown, the mutant plasmid was successfully constructed.

[0074] The Barcode41-Barcode49 nucleic acid sequences (as shown in Table 2) were synthesized using gene technology (Genewiz Biotechnology Co., Ltd.). The synthesized sequences were cloned into the pAAV-CAG-EGFP-hGH plasmid. A 15bp nucleic acid sequence was inserted between the EGFP protein stop codon TAA and hGH-polyA to construct the pAAV-CAG-EGFP-Barcode-hGH plasmid. The ligation product was transformed, single colonies were picked, and the plasmid was extracted for sequencing and enzyme digestion verification. Agarose gel electrophoresis was used for identification. If... Figure 2 As shown, the Barcode plasmid was successfully constructed.

[0075] 2. AAV2-WT, AAV mutant - Barcode virus database packaging

[0076] The AAV2-WT or AAV2- mutant packaging plasmid, along with the pAAV-CAG-EGFP-Barcode-hGH expression plasmid and the pHelper helper plasmid, were co-transfected into suspension HEK293T cells. The packaging plasmids correspond one-to-one with the unique barcode sequence of the pAAV-CAG-EGFP-Barcode-hGH expression plasmid, as shown in Table 3. The maps of the AAV2-WT, AAV2- mutant, pAAV-CAG-EGFP-Barcode-hGH, and pHelper plasmids are shown below. Figures 3-7 As shown.

[0077] Virus packaging includes the following steps:

[0078] A. Cell Culture

[0079] (1) Take out a suspension of 293T cells from the liquid nitrogen tank, place it in a 37℃ water bath, shake it rapidly to thaw the cells, add it to a centrifuge tube containing 5ml Wayne 293 serum-free medium, centrifuge at 200×g for 5min, discard the supernatant, resuspend it in 10ml Wayne 293 serum-free medium, place it in a 125ml Erlenmeyer flask, add Wayne 293 serum-free medium to 20ml, and culture at 120rpm, 37℃, 5% CO2.

[0080] (2) After culturing for 48-72 hours, the cell density reaches 2-3E+6 cells / ml, and the cells can be passaged.

[0081] (3) Take out the seed cells to the clean bench, shake well and then take about 500µl of sample. Take 20µl of sample and add 20µl of trypan blue and mix well. Take 20µl of the mixture and add it to the cell counting chamber. Read the data 3 times in the automatic cell counter and take the average value as the cell density.

[0082] (4) Take an appropriate amount of cells, dilute them to 0.6-0.65E+6 cells / ml with fresh Wayne 293 serum-free culture medium, and place them in 125ml Erlenmeyer flasks for culture, with each flask containing 25ml of culture volume. Culture in a shaking CO2 cell culture incubator under the following conditions: 120rpm, 37℃, 5% CO2.

[0083] B. Cell transfection

[0084] (1) After culturing the above cells for 48 hours, the cell density reaches 3-3.8E+6 cells / ml, and the cells can be transfected. Transfect one flask of cells with each packaging plasmid;

[0085] (2) For each bottle of cells, add the packaging plasmid, pHelper, and pAAV-CAG-EGFP--Barcode-hGH in sequence to 1 mL of DMEM medium, mix thoroughly, and this is the "plasmid dilution solution";

[0086] (3) Take another 1 mL of DMEM culture medium, add 112.5 μg of PEI and mix well to make "PEI dilution solution";

[0087] (4) Pour the “PEI dilution buffer” into the “plasmid dilution buffer”, mix quickly, and let stand at room temperature for 25 minutes to form a transfection complex; add the transfection complex dropwise into the cell slurry while gently shaking.

[0088] (5) Place the culture flask in a shaking CO2 cell culture incubator and culture under the following conditions: 120 rpm, 37°C, 5% CO2.

[0089] C. Virus Harvest

[0090] (1) After transfection, the cells were cultured for another 72 hours before virus harvesting began;

[0091] (2) Mix the cell suspensions from each bottle and collect them in a centrifuge bottle. Centrifuge at 1000g for 5 min. Collect the supernatant in a new centrifuge bottle. Add 0.245 mL of 50% PEG8000 solution (containing 0.5 mol / L NaCl) to each ml of supernatant. Mix thoroughly and let stand overnight at 2-8℃.

[0092] (3) Add an appropriate amount of 0.5% Triton X-100 cell lysis buffer (containing totipotent nuclease) to the cell pellet, treat at 37 ℃ for 1-2 h, then add 1 / 10 volume of 5 mol / L NaCl, mix thoroughly, centrifuge at 4 ℃ and 3000g for 15 min, collect the supernatant, which is the "crude virus extract", and temporarily store at 2-8 ℃;

[0093] (4) Centrifuge the supernatant after PEG8000 concentration at 4°C and 3000g for 15 min, discard the supernatant, and resuspend it in the “crude virus extract” in step (3) for later use.

[0094] D. Virus purification

[0095] (1) Take an ultracentrifuge tube and slowly add 15 ml of "crude virus extract", 9 ml of 15% iodixanol, 6 ml of 25% iodixanol, 5 ml of 40% iodixanol and 5 ml of 60% iodixanol to each ultracentrifuge tube from the bottom using a stainless steel needle; after leveling, seal the tube with a heat sealer.

[0096] (2) Centrifuge at 340,000 × g (57,500 rpm) for 2 h at 16 °C;

[0097] (3) Carefully draw 4-5 ml of the 40% Iodixanol-60% Iodixanol interface layer (i.e., virus solution) from the ultracentrifuge tube using a syringe, avoiding aspirating the white film layer;

[0098] (4) Take an ultrafiltration tube, rinse the filter membrane with 4 mL of PBS buffer, and pour the virus solution collected in step 3) into the ultrafiltration tube;

[0099] (5) Add an appropriate amount of PBS buffer and mix well. Centrifuge at 3000g for 3-5 min. Repeat this step 5-7 times until iodixanol is removed.

[0100] (6) Add 1 mL of PBS buffer and pipette for 40-50 times to form a virus suspension, then transfer it to an EP tube;

[0101] (7) Use a 1mL syringe to draw the virus suspension from the EP tube and filter it through a 0.22μm filter. Collect 20μL of virus solution as a test sample. Then, dispense the remaining virus into 100μL tubes to obtain the AAV-CAG-EGFP-Barcode virus library.

[0102] 3. Virus yield detection

[0103] A. Viral lysis

[0104] (1) Take 20 μL of the Barcode virus library sample, add 1 μL each of 10% SDS, 0.5 mol / L EDTA and proteinase K, and mix well;

[0105] (2) Incubate at 56°C for 1 hour in a constant temperature mixer, and then incubate at 90°C for 10 minutes;

[0106] (3) Take 10 μL of virus lysis buffer and dilute it 10 times to 1000 times to obtain "virus diluent" for later use.

[0107] B. PCR amplification

[0108] The inserted Bacode sequence was amplified by PCR using the "virus dilution buffer" from step (1) as a DNA template. The PCR system was as follows:

[0109] (1) PCR amplification primers:

[0110] Forward primer 5'- GAGTTCGTGACCGCCGCC-3' (SEQ ID NO.21);

[0111] Reverse primer 5'-TTTATTAGGACAAGGCTGGT-3' (SEQ ID NO.22).

[0112] (2) The PCR amplification system is shown in Table 4.

[0113] (3) PCR amplification conditions are

[0114] Pre-denaturation: 98℃, 3 min;

[0115] 25 cycles: 98℃, 15s; 60℃, 20s; 72℃, 24s.

[0116] Extended time: 72℃, 2 minutes.

[0117] C. Gel recovery of PCR products

[0118] The PCR products were subjected to 2% agarose gel electrophoresis and observed using a gel imaging system. The gel pieces with the correct bands were cut out and the gels were recovered using a gel recovery kit.

[0119] D. NGS sequencing

[0120] The gel-recovered products were sent to BGI Genomics for NGS sequencing. The sequencing results were analyzed, including the readings and proportions of different barcode sequences. All sequencing results were standardized (RPM, as this sample is unaffected by gene length). Since each packaging plasmid corresponds one-to-one with a barcode expression plasmid, the RPM value of each barcode sequence in the barcode virus library is directly proportional to the yield of its corresponding packaging plasmid. RPM was used as the yield performance indicator for each mutant virus. Comparative Example 1

[0121] This comparative example provides a wild-type adeno-associated virus (AAV2-WT), which was prepared according to the method of Example 1, the only difference being that the adeno-associated virus capsid protein sequence adopts the wild-type AAV2 capsid protein sequence (SEQ ID NO.1). Comparative Example 2

[0122] This comparative example provides a retinal-targeting mutant of AAV2 (AAV2 / 7M8) that has been used in clinical trials. It was prepared in accordance with the method of Example 1, and the only difference is that the adeno-associated virus capsid protein sequence used is the mutant AAV2 / 7M8 capsid protein sequence (SEQ ID NO.2).

[0123] Test case

[0124] NHP Whole-Eye Infection Efficacy Test

[0125] The AAV-CAG-EGFP-Barcode virus library was injected into cynomolgus monkeys, whole eye tissue was collected, DNA was extracted, and the viral genome barcode region was amplified by PCR and sent for NGS testing. The infectivity of the virus to whole eye tissue was evaluated based on the readings of different barcode amplification products.

[0126] The specific steps are as follows:

[0127] A. Virus injection

[0128] After anesthetizing the cynomolgus monkeys, the AAV-CAG-EGFP-Barcode virus library was extracted and injected intravitreally, 100µl into each eye. The eyes were treated daily with Tobradex and Prednisone, and the condition of the eyes was observed.

[0129] B. Material sourcing

[0130] (1) Three weeks after the virus injection, the cynomolgus monkeys were euthanized, their eyeballs were removed and placed in pre-cooled DMEM medium on ice;

[0131] (2) Take a 10cm culture dish, pour in about 2ml of DMEM culture medium and place it on ice to remove the muscles and connective tissue around the eyes;

[0132] (3) The tip of the insulin syringe punctures the cornea, and Venus scissors cut along the boundary between the cornea and choroid (which is white), removing the cornea, iris and lens. The remaining tissue is used as the whole eye tissue and divided into 10 equal parts.

[0133] C. Genomic DNA extraction, PCR amplification, and gel recovery

[0134] (1) Place the whole eye tissue in a homogenizing tube, add zirconium beads and Buffer GA from the blood / cell / tissue genomic DNA extraction kit, homogenize at room temperature, 65 Hz for 30 s, stop for 30 s, and repeat 4 times;

[0135] (2) Extract genomic DNA from the tissue homogenate using a blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration using an ultra-micro UV spectrophotometer;

[0136] (3) Dilute the tissue DNA to 100 ng / ml and use it as a DNA template for PCR amplification, agarose gel electrophoresis, gel recovery and concentration determination. The specific experimental procedures and virus yield verification are the same.

[0137] D. NGS sequencing and analysis

[0138] The amplified products were sent to BGI Genomics for NGS sequencing. The sequencing results were analyzed to determine the readings and proportions of different barcode sequences; a higher proportion indicates a higher proportion of the corresponding mutant virus in the tissue. The test results were standardized (RPM, which is not affected by gene length) to eliminate the influence of sequencing depth on different samples. The RPM of the barcode sequence in the tissue was then divided by the RPM in the virus database (see virus yield detection in the examples) to obtain the tissue-targeting performance index of the corresponding mutant virus.

[0139] Three weeks after viral injection, samples were taken, genomic DNA was extracted, and NGS testing was performed. The results were as follows: Figure 8 As shown, the whole-eye infectivity of AAV2-7M8 is 4.3 times that of AAV2-WT. The whole-eye infectivity of the gland-associated mutants is 1.1 to 6.2 times that of AAV2-WT, with AAV2-EYE-04 exhibiting the strongest infectivity.

[0140] In summary, the adeno-associated virus variant prepared by the present invention significantly enhances the whole-eye infectivity of AAV2 virus compared to the wild type.

[0141] The sequence alignment results of the capsid protein of the adeno-associated virus variant are shown in the figure below. Figure 9 As shown. The difference between the adeno-associated virus variants EYE-01 to EYE-07 capsid protein and the wild-type AAV2 capsid protein is that the sequences of wild-type capsid protein D561 to R588 (shown in SEQ ID NO.3) are replaced with polypeptide sequences of 28 to 42 amino acids, where EYE-01 and EYE-03 are partial amino acid substitutions, and EYE-02, EYE-04, EYE-05, EYE-06, and EYE-07 are multiple amino acid insertions.

[0142] Table 1. Information on the amino acid sequences substituted at Cap2 D561 to R588 sites in the AAV2 mutant.

[0143] sequence name Sequence information SEQ ID NO. AAV2-WT DEEEIRTTNPVATEQYGSVSTNLQRGNR 3 AAV2-7M8 DEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPAR 4 AAV2-EYE-01 DEHEIKTTNPVATEGYGEVATNWQRGNR 5 AAV2-EYE-02 DEEEIRTTNPVATEQYGSVSTNLQRGNTGRSAGLGTGLSR 6 AAV2-EYE-03 DEQEIAATNPVATEQYGSVSTNLQRGNR 7 AAV2-EYE-04 DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR 8 AAV2-EYE-05 DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR 9 AAV2-EYE-06 DEEEIRTTNPVATEQYGSVSTNLQRGNTGPLLDGTKGLSR 10 AAV2-EYE-07 DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSPIKDGTKGLSR 11

[0144] Table 2. Nucleic acid sequences of pAAV-CAG-EGFP-Barcode-hGH plasmid.

[0145] sequence name Barcode sequence information SEQ ID NO. Barcode41 GAGCGTAATTGTGAG 12 Barcode42 GTCGACTTCATGGCA 13 Barcode43 GGGCCCTAGCGCGTG 14 Barcode44 CGTGACCCAGGAAGT 15 Barcode45 GACTTTGACATGTCA 16 Barcode46 GTCCCGACTAGGACT 17 Barcode47 GGCCACCGTGTGTGA 18 Barcode48 TTGGACTCACAGATG 19 Barcode49 CAATCCGGCGCGGGT 20

[0146] Table 3. Correspondence between packaging plasmids and expression plasmids

[0147] experimental group Packaging plasmids Expression plasmid 1 AAV2-WT pAAV-CAG-EGFP-Barcode41 2 AAV2-7M8 pAAV-CAG-EGFP-Barcode42 3 AAV2-EYE-01 pAAV-CAG-EGFP-Barcode43 4 AAV2-EYE-02 pAAV-CAG-EGFP-Barcode44 5 AAV2-EYE-03 pAAV-CAG-EGFP-Barcode45 6 AAV2-EYE-04 pAAV-CAG-EGFP-Barcode46 7 AAV2-EYE-05 pAAV-CAG-EGFP-Barcode47 8 AAV2-EYE-06 pAAV-CAG-EGFP-Barcode48 9 AAV2-EYE-07 pAAV-CAG-EGFP-Barcode49 10 AAV2-EYE-08 pAAV-CAG-EGFP-Barcode50 11 AAV2-EYE-09 pAAV-CAG-EGFP-Barcode51 12 AAV2-EYE-10 pAAV-CAG-EGFP-Barcode52

[0148] Table 4. PCR amplification system

[0149] Reagent Name Add volume Q5 High-Fidelity 2X Master Mix 25ul 10 µM forward primer (SEQ ID NO.21) 2.5ul 10 µM reverse primer (SEQ ID NO.22) 2.5ul Template DNA 10µl Nucleic acid-free water Add to 50ul

[0150] Related Cap2 protein sequence:

[0151] Wild-type adeno-associated virus capsid protein Cap2:

[0152] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(Genbank ID: YP_680426.1) (SEQ ID NO.1).

[0153] Adeno-associated virus variant 7M8 capsid protein Cap2:

[0154] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVTTTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO. 2).

[0155] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A capsid protein mutant that enhances the ability of AAV virus to infect the entire eye, characterized in that, The wild-type AAV2 virus capsid protein was obtained by replacing amino acids 561 to 588 of the wild-type AAV2 virus capsid protein with one of the following polypeptides, the amino acid sequence of which is shown in SEQ ID NO.

1. 1) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR 2) DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR.

2. A gene encoding the capsid protein mutant of claim 1.

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

4. An expression system having the gene of claim 2 or 3 inserted therein, wherein the expression system is a recombinant AAV vector and the AAV is AAV2.

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

6. Use of the composition of claim 5 in the preparation of gene therapy formulations.

7. Use of the composition of claim 5 in the preparation of genetically modified preparations.

8. A method for constructing a transgenic animal model, comprising introducing the composition of claim 5 into an animal, wherein the method is not intended for the treatment of a disease.

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