Capsid protein mutants capable of increasing ability of aavs to infect ocular tissue and use thereof

Abstract

Capsid protein mutants capable of increasing the ability of AAVs to infect ocular tissue and the use thereof. A Cap mutant is obtained by replacing an amino acid sequence of an AAV capsid protein which is homologous to amino acids at positions D561 to R588 of a wild-type AAV2 capsid protein with one of the polypeptides as shown in SEQ ID NOs. 6-39. Some Cap mutants can infect a mouse retina from the RGC layer, cone and rod cells of INL and ONL, corneal limbus regions, trabecular meshwork, RPE and a small amount of RPE cells by administration via intravitreous injection, can solve the problem that it is difficult for the conventional serotype AAV to infect the cells in the posterior layer of the retina due to insufficient penetrability, and is expected to become a potential carrier for the treatment of glaucoma. Experimental data show that the abilities of some Cap mutants to infect and penetrate the retina of non-human primates are optimized, thereby providing a new tool for developing gene therapy preparations for ophthalmic diseases or constructing animal models related to ophthalmic diseases.

Description

Capsid protein mutants for improving the ability of AAV virus to infect eye tissue and their applications Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to capsid protein mutants that can enhance the ability of AAV viruses to infect ocular tissues and their applications. The present invention claims priority from the following Chinese patent application:

[0002] The entire contents of the above priority documents are incorporated herein by reference. Background Art

[0003] The genome of adeno-associated virus (AAV) consists of a single-stranded DNA fragment of approximately 4.7 kb, contained within a 20 nm diameter, icosahedral, non-enveloped viral capsid. It can be divided into three functional regions: two open reading frames (the Rep gene and the Cap gene) and inverted terminal repeats (ITRs). The Cap gene's open reading frame encodes three capsid proteins, VP1, VP2, and VP3, with molecular weights of 87, 73, and 61 kDa, respectively. These capsid proteins are required for complete viral assembly and play a crucial role in viral integration, replication, and assembly.

[0004] AAV is a widely used delivery vector for gene therapy. Its principle is to replace the sequence between the ITRs of the AAV genome with the target gene sequence through genetic engineering methods. This sequence is then delivered to the target cell via cell infection to achieve the purpose of gene therapy. Recombinant AAV has the characteristics of safety, efficiency, stability, persistence, specificity, and low integration, making it one of the main delivery methods in the field of gene therapy.

[0005] However, AAV still faces several challenges: First, the production process is complex, expensive, and production capacity is limited; second, high-dose administration may show neurotoxicity and hepatotoxicity, causing safety issues. Third, its ability to infect some tissue cells is weak. For example, it is difficult to infect the posterior retinal cells when administered intravitreally, and it is difficult to infect central nervous system cells when administered intravenously. Modifying and screening the AAV capsid protein to improve its targeting and transfection efficiency to specific tissues, in order to achieve a lower and safer administration dose, is an effective way to solve this problem.

[0006] Due to the existence of the blood-ocular barrier, the eye has a certain degree of immune exemption, and local administration is relatively safe. In addition, most fundus diseases are single-gene genetic diseases. Therefore, ophthalmology has become a hot area for gene therapy. 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 lower surgical risks, 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.

[0007] Previous studies have shown that partial amino acid replacement at sites D561 to R588 of the AAV2 capsid protein, or insertion of a polypeptide fragment of approximately 10 amino acids at sites 587 and 588, can enhance its ability to infect various tissues of the eye. Technical issues

[0008] Existing technologies have made significant modifications to the AAV capsid protein, mainly targeting the infection ability of the entire eye, cells in all layers of the retina, and the corneal limbus area, which needs further improvement. Technical Solutions

[0009] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a capsid protein mutant that improves the ability of AAV virus to infect eye tissue and its application.

[0010] The technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides: a capsid protein mutant that improves the ability of AAV virus to infect eye tissue, which is obtained by deleting the amino acid sequence of the AAV capsid protein that is homologous to amino acids D561 to R588 of the wild-type AAV2 virus capsid protein, and inserting one of the polypeptides shown in SEQ ID NO.6 to SEQ ID NO.39 at the deleted position.

[0012] In some examples, the AAV capsid protein is a capsid protein of an AAV2 virus or an AAV2 virus variant. In particular, the AAV2 virus is a wild-type AAV2 virus.

[0013] The amino acid sequence homologous to amino acids D561 to R588 of the wild-type AAV2 viral capsid protein can be determined based on existing methods, such as by comparative analysis of the amino acid sequences of AAV capsid proteins of different serotypes.

[0014] According to the insertion position analysis of the amino acid sequence, the amino acids inserted into the above-mentioned AAV virus capsid protein mutants will be displayed on the formed VP1, VP2 and VP3 proteins.

[0015] The second aspect of the present invention provides: a gene encoding the AAV virus capsid protein mutant described in the first aspect of the present invention.

[0016] In some examples, the gene is codon-optimized according to different expression systems.

[0017] The third aspect of the present invention provides: an expression vector that expresses the AAV virus capsid protein mutant described in the first aspect of the present invention, or contains the gene described in the second aspect of the present invention.

[0018] In some examples, the expression vector is selected from a plasmid or a viral vector. The expression vector can express the AAV viral capsid protein mutant described in the first aspect of the present invention alone, and / or work together with other auxiliary expression vectors to ultimately form an expression system from the AAV capsid to achieve the encapsulation of the target nucleic acid molecule.

[0019] In some examples, the viral vector is a recombinant AAV vector, which is obtained by inserting or replacing an AAV vector.

[0020] In some examples, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

[0021] In some examples, the expression vector further comprises a nucleic acid molecule encoding a functional gene product.

[0022] In some examples, the functional gene product is a factor that acts on ocular cells.

[0023] The fourth aspect of the present invention provides: a recombinant AAV virus, whose capsid protein is as described in the first aspect of the present invention, or obtained by shearing the capsid protein described in the first aspect of the present invention.

[0024] In some examples, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

[0025] The fifth aspect of the present invention provides: an AAV pseudovirus particle, comprising an outer shell and a gene encapsulated in the outer shell, wherein the constituent proteins of the outer shell include the capsid protein described in the first aspect of the present invention, or the outer shell is assembled from the capsid proteins VP1, VP2 and VP3 formed by the capsid protein mutant described in the first aspect of the present invention, or the outer shell is assembled from the capsid proteins VP1, VP2 and VP3 expressed by the gene encoding the gene described in the first aspect of the present invention.

[0026] In some embodiments, depending on the application, the gene encapsulated within the shell encodes a factor that acts on cells of the eye.

[0027] The sixth aspect of the present invention provides: a composition comprising the expression vector and an acceptable carrier described in the third aspect of the present invention, or the recombinant AAV virus described in the fourth aspect of the present invention, or the AAV pseudovirus particle described in the fifth aspect of the present invention.

[0028] The seventh aspect of the present invention provides: applications of the composition described in the sixth aspect of the present invention, including but not limited to 1) preparing gene therapy preparations or transgenic preparations targeting ocular tissue; 2) constructing transgenic animals targeting ocular tissue; 3) preparing drugs or transgenic vectors targeting ocular tissue.

[0029] The eighth aspect of the present invention provides: a transgenic method for treating eye diseases, comprising:

[0030] S1. Test the patient to confirm that he or she has an eye disease;

[0031] S2. Administer a therapeutic amount of a gene therapy preparation to the patient, wherein the gene therapy preparation is the recombinant AAV virus described in the fourth aspect of the present invention, and / or the AAV pseudovirus particles described in the fifth aspect of the present invention, and the nucleic acid molecules encapsulated by the recombinant AAV virus and the AAV pseudovirus particles express factors that can treat eye diseases. Beneficial effects

[0032] The capsid protein mutants of some embodiments of the present invention can effectively improve the ability of AAV virus to infect ocular tissues. AAV2-ROD-01, AAV2-ROD-02, AAV2-ROD-03, AAV2-ROD-05, AAV2-ROD-06, AAV2-ROD-07, AAV2-ROD-08, AAV2-ROD-09, AAV2-ROD-10, AAV2-ROD-11, AAV2-ROD-12, AAV2-ROD-15, AAV2-ROD-16, AAV2-ROD-17, AAV2-ROD-18, and AAV2-ROD-19, these 16 mutants, can infect the ganglion cell layer (RGC), inner nuclear layer (INL), and outer nuclear layer (ONL) of the mouse retina through intravitreal injection. Rod cells and a small amount of RPE cells can solve the problem that conventional serotypes have insufficient penetration ability to infect the posterior retinal cells; AAV2-EYE-08, AAV2-EYE-09, AAV2-EYE-10, AAV2-EYE-11, AAV2-EYE-12, AAV2-EYE-13, AAV2-EYE-14, AAV2-EYE-15, AAV2-EYE-16, AAV2-EYE-17, AAV2-EYE-18, AAV2-EYE-19, AAV2-EYE-20, AAV2-EYE-21, and AAV2-EYE-22 mutants can improve the infection ability of the entire eye, with a wide range of infection and a high overall expression level. The AAV2-A2HRE15 mutant can infect the limbus, trabecular meshwork, and RPE via intravitreal injection, making it a promising vector for the treatment of glaucoma. Compared to AAV2-WT, AAV2-NE01 and AAV2-NE02 have improved their ability to infect and penetrate the NHP (non-human primate) retina, effectively infecting the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), and the limbus. These variants address the issue of AAV2's ability to infect multiple ocular tissues from different perspectives, providing new tools for developing gene therapy agents for ophthalmic diseases or constructing animal models related to ophthalmic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1-2 are diagrams showing the EGFP expression effects of mice infected with AAV2 wild-type, AAV2-7M8, and AAV2-ROD-01 to AAV2-ROD-19 mutant viruses prepared by the scheme of the present invention in the test examples of the present invention for 3 weeks. Figure 1 is a fundus fluorescence imaging diagram, and Figure 2 is a quantitative statistical diagram of the fluorescence intensity of the fundus fluorescence imaging.

[0034] Figures 3-5 show the effects of retinal infection on mouse eye tissue after three weeks of infection with wild-type AAV2, AAV2-7M8, and AAV2 mutant viruses prepared according to the present invention. Figure 3 is a fluorescence photograph of a retinal frozen section, Figure 4 is a quantitative statistical graph of fluorescence intensity across the entire retinal region, and Figure 5 is a quantitative statistical graph of fluorescence intensity in the posterior retinal photoreceptor cells.

[0035] FIG6 is an alignment diagram of the amino acid sequences of Cap 561-588 of the AAV2 wild type, AAV2-7M8, and AAV2-ROD-01 to AAV2-ROD-19 mutant sequences prepared by the scheme of the present invention in the test examples of the present invention.

[0036] Figures 7-8 show the effect of luciferase expression in mouse eyes infected for three weeks with wild-type AAV2, AAV2-7M8, and mutant AAV2-EYE-08 to AAV2-EYE-22 viruses prepared according to the present invention's protocol. Figure 7 shows in vivo imaging after injection of a fluorescent substrate, and Figure 8 shows the results of chemiluminescence analysis of ocular tissue.

[0037] FIG9 is an alignment diagram of the amino acid sequences of Cap 561-588 of the AAV2 wild type, AAV2-7M8, and AAV2-EYE-08 to AAV2-EYE-229 mutant sequences prepared by the scheme of the present invention in the test examples of the present invention.

[0038] Figures 10-11 show the effects of ocular tissue infection in mice infected with wild-type AAV2 and AAV2-A2HRE15 mutant viruses prepared in accordance with the present invention for three weeks. Figure 10 is a fluorescence photograph of a frozen section of ocular tissue, and Figure 11 is a fluorescence photograph after immunofluorescence staining.

[0039] FIG12 is an alignment diagram of the amino acid sequences of Cap 561-588 of the AAV2 wild type and AAV2-A2HRE15 mutant sequences prepared by the scheme of the present invention in the test example of the present invention.

[0040] Figures 13-16 show the effects of ocular tissue infection in cynomolgus macaques infected with wild-type AAV2, AAV2-NE01, and AAV2-NE02 viruses prepared according to the present invention for three weeks. Figure 13 is a fluorescent photograph of the infection effect in the retinal region, Figure 14 is a fluorescent photograph of the infection effect in the limbal region, Figure 15 is a fluorescent photograph of the retinas infected with AAV2-EGFP and AAV2-NE01-EGFP after immunofluorescence staining, and Figure 16 is a fluorescent photograph of the retinas infected with AAV2-mCherry and AAV2-NE02-mCherry after immunofluorescence staining.

[0041] FIG17 is an alignment diagram of the amino acid sequences of Cap 561-588 of the AAV2 wild type, AAV2-NE01, and AAV2-NE02 mutant sequences prepared by the scheme of the present invention in the test examples of the present invention. Modes for Carrying Out the Invention

[0042] As used herein, "vector" refers to a macromolecule or macromolecular complex that contains or is associated with a polynucleotide molecule and can be used to mediate delivery of the polynucleotide molecule to a cell. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

[0043] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise specified, the term encompasses all subtypes and naturally occurring and recombinant forms. AAV variants generally refer to mutations in one or several amino acid sites based on a wild serotype of AAV, such as AAV2, AAV9, etc., such as deletion mutations, point mutations, insertion mutations, etc. The biological effects of AAV variants, such as virus yield, infectivity, immunogenicity, etc., are somewhat different from those of the original AAV. The newly mutated amino acid sequence itself may have specific effects. For example, the common YF mutation can reduce the possibility of AAV being degraded by the proteasome and improve the infection effect of AAV (see Zhong L, Li B, Mah CS, et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses[J]. Proceedings of the National Academy of Sciences, 2008, 105(22): 7827-7832.). Moreover, this characteristic is effective in different serotypes such as AAV2, AAV8, and AAV9 (see Petrs-Silva H, Dinculescu A, Li Q, et al. High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors[J]. Molecular therapy, 2009, 17(3): 463-471.). The specificity of AAV vectors is mainly determined by their capsid proteins. The same capsid proteins often exhibit the same or similar functions in different AAV vectors. Capsid proteins can also self-assemble with other AAV structural proteins to form a complete capsid and encapsulate genes.

[0044] "AAV virion" or "AAV viral particle" or "AAV viral vector" or "AAV vector particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and the encapsidated polynucleotide AAV vector. Thus, the production of an AAV vector particle necessarily includes the production of an AAV vector, as such vector is contained within the AAV vector particle.

[0045] " gene " refers to the polynucleotide that can encode specific gene product after transcription and sometimes translation containing at least one open reading frame.Term " gene " or " coding sequence " refers to the nucleotide sequence in vitro or in vivo that gene product is encoded.In some cases, gene is made up of coding sequence or is made up of coding sequence basically, and described coding sequence is the sequence that gene product is encoded.In other cases, gene comprises other non-coding sequence.For example, gene may include or may not include the zone before and after coding region, for example 5 ' untranslated region (5 ' UTR) or " leader " sequence or 3 ' UTR or " tail " sequence and each coding segment (exon) between insertion sequence (intron).

[0046] As used herein, "expression vector" encompasses vectors, including but not limited to plasmids, minicircles, viral vectors, liposomes, etc. known in the art, and is used to achieve expression of gene products in intended target cells, wherein the vector includes a polynucleotide encoding the gene product of interest.

[0047] The sequences, proteins and fragments of the present invention can be produced by any suitable method, including recombinant production, chemical synthesis or other synthetic methods. Such production methods are within the knowledge of those skilled in the art and are not intended to limit the present invention.

[0048] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the embodiments described 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 those skilled in the art without creative work are 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.

[0049] Example 1

[0050] This example constructs a library based on the previously described amino acid substitutions at positions D561 to R588 and peptide insertions at positions 587 to 588. Through multiple rounds of directed evolution, sequences with superior ocular and retinal infectivity and penetration were screened from NGS sequencing data. These mutants include AAV2-ROD-01 to AAV2-ROD-19, AAV2-EYE-08 to AAV2-EYE-22, AAV2-A2HRE15, AAV2-NE01, and AAV2-NE02. The sequence information for the different AAV2 mutants with amino acid substitutions at positions D561 to R588 in the Cap protein is shown in Table 1.

[0051] Table 1. Amino acid sequence information of AAV2 mutants with substitutions at positions D561 to R588 of the Cap protein

[0052] The preparation of the adeno-associated virus variant comprises the following steps:

[0053] 1. Plasmid construction

[0054] Through Jinweizhi Biotechnology Co., Ltd., nucleic acid sequences corresponding to amino acid sequences such as AAV2-ROD-01 to AAV2-ROD-19, AAV2-EYE-08 to AAV2-EYE-22, AAV2-A2HRE15, AAV2-NE01, and AAV2-NE02 (as shown in Table 1) were synthesized, and the synthesized sequences were cloned into the pAAV2-WT plasmid by seamless cloning, and the sequence of sites 561 to 588 of the Cap protein was replaced with the mutant sequence shown in Table 1 to construct the pAAV2-mutant plasmid vector; the ligation product was transformed into Escherichia coli competent cell stbl3, single colonies were picked for culture, and the plasmid was extracted for sequencing and enzyme digestion verification. The mutant plasmid was successfully constructed as confirmed by agarose gel electrophoresis.

[0055] 2. AAV2-WT, AAV2-7M8 and other AAV2-mutant virus packaging

[0056] The pAAV2-WT or pAAV2-mutant packaging plasmid, along with the pAAV-CAG-EGFP (or pAAV-CAG-Luciferase or pAAV-CAG-mcherry) expression plasmid and the pHelper helper plasmid, were co-transfected into suspension HEK293T cells. After 72 hours of incubation, the transfected cells were lysed and the virus was harvested. The virus was purified by iodixanol density gradient centrifugation, which involved the following steps:

[0057] A. Cell culture

[0058] (1) Take out a tube of suspended 293T cells from the liquid nitrogen tank, place it in a 37°C water bath, shake rapidly to thaw the cells, add it to a centrifuge tube containing 5 mL of Wayne 293 serum-free medium, centrifuge at 200×g for 5 min, discard the supernatant, resuspend it in 10 mL of Wayne 293 serum-free medium, place it in a 125 mL triangular shake flask, add Wayne 293 serum-free medium to 20 mL, and culture it in a shaking incubator at 120 rpm, 37°C, and 5% CO2;

[0059] (2) After 48-72 hours of culture, the cell density reaches 2-3E+6 cells / mL and the cells can be subcultured;

[0060] (3) Take out the seed cells and place them on a clean bench. After shaking, take 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 and add it to the cell counting plate. Read the data three times on an automatic cell counter. The average value is the cell density.

[0061] (4) Take an appropriate amount of cells and dilute them to 0.6-0.65E+6 cells / mL with fresh Wayne 293 serum-free medium. Place the cells in a 500 mL shake flask with a culture volume of 25 mL per flask. Incubate in a shaking CO2 cell incubator at 120 rpm, 37°C, and 5% CO2.

[0062] B. Cell transfection

[0063] (1) The cells are cultured for 48 hours until the cell density reaches 3-3.8E+6 cells / mL, and the cells can be transfected;

[0064] (2) For each flask of cells, add 100 μg of packaging plasmid, pHelper, and pAAV-CAG-EGFP (or pAAV-CAG-Mcherry) to 4 mL of DMEM medium in sequence and mix thoroughly to prepare the "plasmid dilution solution."

[0065] (3) Take another 1 mL of DMEM medium, add 300 μg of PEI and mix well to make the "PEI dilution solution";

[0066] (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 to the cell solution while gently shaking;

[0067] (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.

[0068] C. Virus Harvest

[0069] (1) After transfection, cells were cultured for 72 hours and virus harvesting was started;

[0070] (2) Collect the cell suspension from each bottle into a centrifuge bottle and centrifuge at 1000 g for 5 min. Collect the supernatant into a new centrifuge bottle. Add 0.245 mL of 50% PEG8000 solution (containing 0.5 mol / L NaCl) per mL of supernatant, mix thoroughly, and let stand at 2-8°C overnight.

[0071] (3) Add an appropriate amount of 0.5% Triton X-100 cell lysis buffer (containing universal nuclease) to the cell pellet and treat at 37°C for 1-2 hours. Then add 1 / 10 volume of 5 mol / L NaCl, mix thoroughly, and centrifuge at 3000g for 15 minutes at 4°C. Collect the supernatant, which is the "crude virus extract", and temporarily store it at 2-8°C.

[0072] (4) The supernatant after PEG8000 precipitation was centrifuged at 4°C and 3000g for 15 min, the supernatant was discarded, and the supernatant was resuspended with the "viral crude extract" in step (3) for later use.

[0073] D. Virus Purification

[0074] 1) Take an Ultra-Clear centrifuge tube and add 0.5 mL of 60% iodixanol, 2 mL of 40% iodixanol, 1.5 mL of 25% iodixanol, 1.5 mL of 15% iodixanol, the collected virus suspension, and finally the cell lysate to the bottom for balancing;

[0075] 2) Ultracentrifugation at 10°C, 230,000 g, 8 ascent and 9 ascent for 18 h;

[0076] 3) Take an ultrafiltration tube and soak the filter membrane with 1 mL of PBS buffer;

[0077] 4) Carefully extract the intermediate layer between 40% iodixanol and 60% iodixanol from the ultracentrifuge tube using a pipette, avoiding protein, and transfer to an ultrafiltration tube.

[0078] 5) Add an appropriate amount of PBS buffer, pipette evenly, centrifuge at 4500 g for 3-5 minutes, and repeat this step 5-7 times to remove the iodixanol.

[0079] 6) Dilute the crude virus product from step 5 to 8 mL and repeat steps 1 to 5;

[0080] 7) Add 1 mL of PBS buffer and pipette 40-50 times to form a virus suspension. Transfer to an EP tube.

[0081] 8) Use a 5 mL syringe to draw the virus suspension from the EP tube and filter it through a 0.22 μm filter. Collect 20 μL of the virus liquid as a test sample and aliquot it into 100 μL tubes to obtain the adeno-associated virus variant.

[0082] 3. Virus titer detection

[0083] (1) Viral lysis

[0084] 1) Take 20 μL of the virus samples of Example 1 and the comparative example respectively;

[0085] 2) Add 1 μL each of 10% SDS, 0.5 mol / L EDTA, and proteinase K and mix well;

[0086] 3) Incubate at 56°C for 1 hour in a thermomixer, then at 90°C for 10 minutes;

[0087] 4) Take 10 μL of virus lysate and dilute it 10-fold to 10,000-fold to prepare the virus dilution solution for later use.

[0088] (2) Preparation of standard samples for standard curve

[0089] Take 2×10 12 The plasmid standard of 100 copies / mL was diluted with ultrapure water ddH2O in 6 gradients as the template of the standard 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.

[0090] (3) Absolute quantitative qPCR

[0091] 1) In a 0.2 mL PCR tube, prepare the following reaction system, performing three replicates for each virus dilution: 10 μL of 2× qPCR Mix;

[0092] 0.2 μL each of forward and reverse primers; 5 μL virus dilution solution; 4.2 μL ddH2O;

[0093] Amplification primers:

[0094] Forward primer 5′-GGAACCCCTAGTGATGGAGTT-3′ (SEQ ID NO. 2);

[0095] Reverse primer 5′-CGGCCTCAGTGAGCGA-3′ (SEQ ID NO. 3);

[0096] 2) qPCR amplification conditions

[0097] Pre-denaturation: 95°C, 2 min;

[0098] 40× cycles: 95°C, 15 s; 60°C, 60 s;

[0099] 3) qPCR data processing: Virus titer = dilution factor × viral gene array copy number.

[0100] Comparative Example 1

[0101] 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 between them is that the sequence of the adeno-associated virus capsid protein adopts the wild-type AAV2 capsid protein sequence.

[0102] Comparative Example 2

[0103] This comparative example provides an AAV2 adeno-associated virus mutant (AAV2-7M8) with strong ocular tissue targeting 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.1).

[0104] Test Example 1: Mouse retinal infection effect test

[0105] (1) Intravitreal virus injection

[0106] The CAG-EGFP virus samples prepared in the Example and Comparative Example were diluted to 2E+9 vg / μl and injected into the vitreous cavity of C57BL-6J mice, with 1.5 μl injected into each eye (total virus volume of 3E+9 vg). Three mice were injected with each virus. The specific steps are as follows:

[0107] 1) Before anesthesia is injected, an ophthalmologist will perform an ocular examination (slit lamp) to observe the ocular surface. If any abnormality is found, the patient will be removed to ensure that there is no ocular lesion.

[0108] 2) After the mice were anesthetized, tropicamide was instilled into the eyeball to dilate the pupil, and then tetracaine hydrochloride was instilled into the eyeball for surface anesthesia;

[0109] 3) Use a microsyringe to draw up an appropriate amount of virus for later use. Place the anesthetized mouse on the operating table and administer lidocaine hydrochloride for local anesthesia to the eyes. Hold the head with the left hand to partially protrude the eyeball. Hold the microsyringe in the right hand and insert it into the vitreous cavity from the posterior edge of the cornea. Insert the needle tip vertically first, then tilt it and slowly push the drug in. After the injection is complete, slowly withdraw the needle.

[0110] (2) Fluorescent ophthalmoscopy

[0111] Three weeks after viral injection, mice were anesthetized, their eyes dilated, and transferred to an observation platform for optical coherence tomography (OCT) imaging using a Micron IV small animal retinal imaging system. Acquisition software generated brightfield fundus images and fluorescence channel fundus images of corresponding locations. Image parameters were: Gain (dB) -10dB, Frame Rate (fps) -2fps, Exposure -Auto.

[0112] (3) Preparation of eyeball frozen sections and observation of fluorescence signals

[0113] Select mice with strong fundus fluorescence signals, sacrifice them, remove their eyeballs, prepare frozen sections, and observe the expression of GFP fluorescence signals in various regions of the eye tissue as follows:

[0114] 1) Dissect fresh eyeball tissue and immediately place it in tissue fixative for more than 24 hours;

[0115] 2) After removing the tissue from the fixative, trim the surrounding tissue flat, place the trimmed tissue block in a 10% sucrose solution in a 4°C refrigerator for dehydration, and after the tissue block sinks to the bottom, transfer it to a 20% sucrose solution in a 4°C refrigerator for dehydration. After the tissue block sinks to the bottom, transfer it to a 30% sucrose solution in a 4°C refrigerator for dehydration and sink to the bottom;

[0116] 3) Take out the dehydrated tissue and dry the surface moisture with filter paper. Place it in an embedding frame with the cut surface facing up. Add OCT embedding medium (avoid bubbles). Place the embedding frame in a -20℃ refrigerator and freeze for half an hour. Then transfer it to a -80℃ refrigerator for storage.

[0117] 4) Before sectioning, remove the OCT embedded block from -80℃ and place it in a freezing microtome to equilibrate the temperature for 30 minutes. Load the sample and slice according to the thickness required by the experiment. Label the remaining slices and store them at -20℃ for later use.

[0118] 5) Add DAPI prepared in a certain proportion to the slices, incubate at room temperature in the dark for 3 minutes, place the slides in PBS and wash them 3 times, each time for 5 minutes, and use anti-fluorescence quenching mounting medium for sealing;

[0119] 6) Use a fluorescence microscope to observe and collect pictures.

[0120] The present invention scheme injects AAV2-WT, AAV2-7M8 and some variant viruses into the vitreous cavity of mice, and after 3W of virus injection, takes photos of live fluorescence. Select some embodiments and comparative examples, as shown in Figure 1, it can be seen from the figure that the expression of multiple preferred variant viruses such as AAV2-ROD-01, AAV2-ROD-02, AAV2-ROD-03 in the whole eye is significantly stronger than AAV2-WT virus. The fluorescence intensity is analyzed by ImageJ software, and AAV2 is normalized as a control to obtain the multiples of other variant fundus fluorescence signals relative to AAV2. As shown in Figure 2, 16 variant GFP expression intensities are stronger than AAV2-WT in the selected mutants, among which AAV2-ROD-05, AAV2-ROD-09, AAV2-ROD-16 and AAV2-ROD-19 are the strongest, about 10 times that of AAV2-WT. The EGFP expression intensity of comparative example 2 (AAV2-7M8) is close to that of AAV2-WT virus.

[0121] In order to explore the infection effect on various layers of the retina, the paraffin sections of the eyeballs were stained with DAPI, and the results of fluorescence microscopy showed that after the variant virus was injected into the vitreous cavity, it could infect cells in all layers of the retina from RGC to PRC, while AAV2-WT mainly infected RGC layer cells. The variant virus infection area and the ability to infect the posterior layer of the retina were stronger than AAV2-WT. As shown in Figure 3, some examples and comparative examples were selected. The infection range of multiple preferred variants such as AAV2-ROD-01, AAV2-ROD-02, and AAV2-ROD-03 was greater than that of AAV2-WT and 7M8. From the perspective of infection level, AAV2-WT mainly infects RGC layer cells, and 7M8 has a certain penetration ability and can penetrate into the posterior layer of the retina to infect a small number of PRC cells. Several preferred variants such as AAV2-ROD-01, AAV2-ROD-02, and AAV2-ROD-03 can infect cells in all layers of the retina from RGC to PRC, among which AAV2-ROD-05 has the best infection rate and expression effect on PRC cells.

[0122] The retinal area and the posterior retinal area were selected separately by ImageJ software, and the fluorescence intensity of the corresponding area was counted. The retinal area was normalized with AAV2-WT as the control, and the multiples of the expression of other variants in the whole retina relative to AAV2-WT were obtained. The results are shown in Figure 4. AAV2-ROD-10 has the strongest whole retinal infection ability, which is 10 times that of AAV2-WT, and the other variants are 3 to 5.5 times that of AAV2-WT. The posterior retinal area was normalized with AAV2-7M8 as the control, and the multiples of the penetration of other variants relative to 7M8 were obtained. The results are shown in Figure 5. AAV2-WT cannot infect the posterior retinal cells, AAV2-ROD-05 has the strongest penetration ability, which is 2.1 times that of AAV2-7M8, and the other variants are 1.1 to 1.7 times that of AAV2-7M8. It is proved that the adeno-associated virus variants prepared by the technology of the present invention have significantly improved the infection ability of the whole retina and the posterior retina.

[0123] The sequence alignment results of the adeno-associated virus variant capsid proteins are shown in Figure 6. The sequences of the adeno-associated virus variants ROD-01 to ROD-19 capsid proteins differ from those of the wild-type AAV2 capsid protein in that the sequences D561 to R588 of the wild-type capsid protein (shown in SEQ ID NO. 4) are replaced with polypeptide sequences of 28 to 42 amino acids. ROD-01 to ROD-07 represent partial amino acid substitutions, while ROD-08 to ROD-19 represent multiple amino acid insertions.

[0124] Test Example 2: Mouse Whole Eye Expression Ability Test

[0125] CAG-Luciferase viruses prepared in the Examples and Comparative Examples were diluted to 2E+9 vg / μl and injected intravitreally into Bab / c mice, with 1.5 μl injected into each eye (total virus volume of 3E+9 vg). Three mice were injected with each virus. Luciferase expression was assessed using in vivo imaging and chemiluminescence 3-4 weeks after injection.

[0126] (1) In vivo imaging detection

[0127] Three weeks after virus injection, D-luciferin potassium salt solution was injected intraperitoneally. Two minutes after the injection, the mouse was placed in the induction box of the anesthesia machine for isoflurane-induced anesthesia. When the mouse's breathing became stable and no longer showed significant movements, it was transferred to the breathing mask of the imaging device for maintenance of anesthesia. Imaging parameters were set and images were taken.

[0128] (2) Chemiluminescence detection

[0129] 1) Draw materials

[0130] Mice were sacrificed by cervical dislocation, the eyelids were opened, and the eyeballs were removed with forceps. The surrounding muscles and connective tissue were removed. The eyeballs were placed in 2 ml grinding tubes, one tube per eyeball, and stored on ice.

[0131] 2) Sample Pretreatment: Add 500 μL of 1x Cell Culture Lysis Reagent to a homogenate tube containing a single mouse eyeball and homogenize in a pre-chilled -65°C grinder. After homogenization, incubate at 4°C for 1–2 hours. Centrifuge at 15,000 rpm at 4°C for 3 minutes, and aspirate the supernatant.

[0132] 3) Color development

[0133] Take a black, opaque 96-well plate and add 20 μl of sample to each well. Draw out two to three replicates of each sample. Warm the substrate colorimetric solution at room temperature, away from light, and add 100 μl to each well. Immediately after adding the colorimetric solution, place the plate in a fluorescence microplate reader and read the fluorescence signal.

[0134] The present invention injected AAV2-WT, AAV2-7M8 and variant viruses into the vitreous cavity of mice. Three weeks later, in vivo imaging results showed that the Luciferase signals in the eyes of all mice injected with 15 preferred mutant viruses, including AAV2-EYE-08 to AAV2-EYE-22, were stronger than those of AAV2-WT and AAV2-7M8. Some selected examples and comparative examples are shown in Figure 7, indicating that the adeno-associated virus variants prepared by the present invention can improve the ability of AAV2 virus to infect the entire eye.

[0135] To more accurately quantify the expression effect, eyeballs were removed and homogenized for chemiluminescence detection. The results, as shown in Figure 8, showed that the expression levels of luciferase in the whole eye of mice injected with 15 preferred mutant viruses, including AAV2-EYE-08 to AAV2-EYE-22, were higher than those of AAV2-WT and AAV2-7M8, ranging from 1.23 to 8.4 times that of AAV2-WT. Among them, the expression levels in 15 groups were more than 5 times that of AAV2-WT, which is considered to have significantly improved whole-ocular expression capacity. Among them, AAV2-EYE-09 had the highest expression level. This shows that the adeno-associated virus variants prepared by the present invention can improve the whole-ocular expression capacity of AAV2 virus.

[0136] The sequence alignment results of the adeno-associated virus variant capsid proteins are shown in Figure 9. The sequences of the adeno-associated virus variant EYE-08 to EYE-22 capsid proteins differ from those of the wild-type AAV2 capsid protein in that the sequence of the wild-type capsid protein D561 to R588 (shown in SEQ ID NO. 4) is 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-22 are multiple amino acid insertions.

[0137] Test Example 3: Mouse eye tissue infection effect test

[0138] (1) Intravitreal virus injection

[0139] The CAG-EGFP viruses prepared in Example and Comparative Example 1 were diluted to 2E+9 vg / μl and injected intravitreally into C57BL-6J mice, with 1.5 μl injected into each eye (total virus volume of 3E+9 vg). Three mice were injected with each virus. The specific operation process and steps were the same as those in Test Example 1.

[0140] (2) Preparation of eyeball frozen sections and observation of fluorescence signals

[0141] The specific operation process and steps are the same as those in Test Example 1.

[0142] (3) Immunofluorescence staining

[0143] After the frozen sections were labeled with Anti-RPE65 antibody, the colocalization of GFP and RPE was observed under a fluorescence microscope. The specific steps were as follows:

[0144] 1) Remove frozen sections from -20°C freezer, return to room temperature, and dry. If the sample is unfixed, fix it with tissue fixative for 15 minutes and then rinse with running water.

[0145] 2) Place the slides in the repair solution, boil in a pressure cooker and purify with air for 3 minutes. After cooling naturally, place the slides in PBS and wash them three times, each time for 5 minutes.

[0146] 3) Place the sections in 0.3% Triton X-100 solution and incubate at room temperature in the dark for 10 minutes. Place the slides in PBS and wash three times, 5 minutes each time.

[0147] 4) Add blocking serum to the tissue culture circle to evenly cover the tissue and block at room temperature for 60 minutes;

[0148] 5) Gently shake off the blocking solution and add the primary antibody (Anti-RPE65 antibody, abcam, ab231782, 1:250) prepared in a certain ratio to the slices. Place the slices flat in a humidified chamber and incubate at 4°C overnight or at room temperature for 1 hour.

[0149] 6) Wash the slides in PBS three times, 5 minutes each time; after the sections are slightly dried, add the secondary antibody (fluorescently labeled) of the same species as the primary antibody to cover the tissue and incubate at room temperature for 60 minutes; wash the slides in PBS three times, 5 minutes each time; wash the slides in PBS three times, 5 minutes each time;

[0150] 7) Add DAPI prepared in a certain proportion on the slices, incubate at room temperature in the dark for 3 minutes, place the slides in PBS and wash three times, each time for 5 minutes;

[0151] 8) Use anti-fluorescence quenching mounting medium to seal the slides;

[0152] 9) Observe the fluorescence signal under a fluorescence microscope.

[0153] DAPI staining of frozen sections of the eyeballs shows results in Figure 10. After intravitreal injection, the AAV2-A2HRE15 virus is able to infect the corneal limbus, trabecular meshwork, and RPE, with a high infection rate and strong fluorescence intensity. It can also infect a small number of cells in all retinal layers, from RGCs to PRCs. AAV2-WT, on the other hand, primarily infects the RGC layer, having superior infectivity to AAV2-A2HRE15 in this layer, but has little to no infection at the corneal limbus, trabecular meshwork, or RPE. This demonstrates that the adeno-associated virus variant AAV2-A2HRE15 prepared according to the present invention has significantly improved infectivity at the corneal limbus, trabecular meshwork, and RPE.

[0154] To further confirm that AAV2-A2HRE15 can effectively express RPE cells, frozen sections of the eyeball were labeled and stained with Anti-RPE65 (RPE-specific antibody). The results are shown in Figure 11. Part of the GFP fluorescence signal can overlap and co-localize with the red fluorescence signal (Anti-RPE65), proving that GFP is expressed in RPE cells.

[0155] The sequence alignment results of the adeno-associated virus variant capsid protein sequence AAV2-A2HRE15 and the wild-type AAV2 capsid protein are shown in Figure 12. The sequence of the adeno-associated virus variant capsid protein AAV2-A2HRE15 differs from that of the wild-type AAV2 capsid protein (protein ID: YP_680426.1) in that amino acids 561-588 of the wild-type AAV2 virus capsid protein are replaced by the polypeptide sequence DEQEIATTNPVATEQYGEAATNLQRANR, and the specific six mutation sites are: E563Q, R566A, S576E, V578A, S579A, and G586A.

[0156] Test Example 4: NHP eye tissue infection effect test

[0157] CAG-EGFP or CAG-mCherry viruses prepared from AAV2-WT and AAV2-mutant strains were injected into cynomolgus macaques. Three weeks later, eyeballs were removed and frozen sections were prepared. Fluorescence signals were directly observed under a microscope. To further determine the level of infected retinal cells, the retinas were immunofluorescently stained and observed under a microscope.

[0158] (1) Intravitreal virus injection

[0159] The samples of Example 1 and the comparative example were diluted to 1E+12 vg / μL and injected into the vitreous cavity of cynomolgus monkeys, with 100 μL injected into each eye (total virus volume of 1E+11 vg). One cynomolgus monkey was injected with each virus. The specific steps are as follows:

[0160] 1) Before anesthesia is injected, an ophthalmologist will perform an ocular examination (slit lamp) to observe the ocular surface. If any abnormality is found, the patient will be removed to ensure that there is no ocular lesion.

[0161] 2) After anesthetizing the cynomolgus monkeys, tropicamide was instilled into the eyeball to dilate the pupil, and then tetracaine hydrochloride was instilled into the eyeball for surface anesthesia;

[0162] 3) Use a microsyringe to draw up an appropriate amount of virus for later use. Place the anesthetized cynomolgus monkey on the operating table and administer lidocaine hydrochloride for local anesthesia to the eyes. Press the head with the left hand to partially protrude the eyeball. Hold the microsyringe in the right hand and insert it into the vitreous cavity from the posterior edge of the cornea. Insert the needle tip vertically first, then tilt it, and slowly advance the drug. After the injection is complete, slowly withdraw the needle.

[0163] (2) Sampling, frozen section preparation, and fluorescence signal observation

[0164] Three weeks after virus injection, the cynomolgus monkeys were euthanized, their eyeballs were removed, and frozen sections were prepared to observe the expression of GFP fluorescence signals in various areas of the eye tissue. The steps are as follows:

[0165] 1) Dissect the fresh eyeball tissue and immediately fix it in tissue fixative for 24-48 hours;

[0166] 2) After removing the tissue from the fixative, remove the surrounding tissue and excess tissue, cut it open with a sharp blade according to the experimental requirements, remove the lens and vitreous body inside, and dehydrate the trimmed eye tissue block in 10% sucrose-20% sucrose-30% sucrose solution in a 4°C refrigerator;

[0167] 3) Take out the dehydrated tissue and dry the surface moisture with filter paper. Place it in an embedding frame with the cut surface facing up. Add OCT embedding medium (avoid bubbles). Place the embedding frame in a -20℃ refrigerator and freeze for half an hour. Then transfer it to a -80℃ refrigerator for storage.

[0168] 4) Before sectioning, remove the OCT embedded block from -80℃ and place it in a freezing microtome to equilibrate the temperature for 30 minutes. Load the sample and slice according to the thickness required by the experiment. Label the remaining slices and store them at -20℃ for later use.

[0169] 5) Add DAPI prepared in a certain proportion to the slices, incubate at room temperature in the dark for 3 minutes, place the slides in PBS and wash them 3 times, each time for 5 minutes, and use anti-fluorescence quenching mounting medium for sealing;

[0170] 6) Collect images before observation using a fluorescence microscope.

[0171] (3) Immunofluorescence staining

[0172] After the frozen sections were labeled with anti-Rhodopsin antibodies, the co-localization of GFP or mCherry with photoreceptors was observed under a fluorescence microscope. The specific steps are as follows:

[0173] 1) Remove the frozen sections from the -20°C freezer, return them to room temperature, and dry them. If the samples are unfixed, fix them with tissue fixative for 15 minutes and then rinse with running water.

[0174] 2) Place the slides in the repair solution, boil in a pressure cooker and purify with air for 3 minutes. After cooling naturally, place the slides in PBS and wash them three times, each time for 5 minutes.

[0175] 3) Place the sections in 0.3% Triton X-100 solution and incubate at room temperature in the dark for 10 minutes. Place the slides in PBS and wash three times, 5 minutes each time.

[0176] 4) Add blocking serum to the tissue culture circle to evenly cover the tissue and block at room temperature for 60 minutes;

[0177] 5) Gently shake off the blocking solution and add the primary antibody prepared in a certain ratio (Anti-Rhodopsin antibody abcam, ab221664, 1:500) to the slices. Place the slices flat in a humidified chamber and incubate at 4°C overnight or at room temperature for 1 hour.

[0178] 6) Wash the slides in PBS three times, 5 minutes each time. After the sections are slightly dried, add a secondary antibody (fluorescently labeled) of the same species as the primary antibody to cover the tissue and incubate at room temperature for 60 minutes. Wash the slides in PBS three times, 5 minutes each time. Wash the slides in PBS three times, 5 minutes each time.

[0179] 7) Add DAPI prepared in a certain proportion to the slices, incubate at room temperature in the dark for 3 minutes, place the slides in PBS and wash them 3 times, each time for 5 minutes;

[0180] 8) Use anti-fluorescence quenching mounting medium to seal the slides;

[0181] 9) Observe the fluorescence signal under a fluorescence microscope.

[0182] Three weeks after virus injection, frozen sections were prepared and DAPI staining was performed on the eyeball sections. The results of fluorescence microscopy observation are shown in Figures 2 and 3. After intravitreal injection, AAV2-NE01-CAG-EGFP (green) and AAV2-NE02-CAG-Mcherry (red) viruses were able to infect the NHP retina from the ganglion cell layer (GCL), inner nuclear layer (INL) to the outer nuclear layer (ONL) (Figure 13) and the limbal area (Figure 14). The infection positive rate was high and the fluorescence intensity was strong. The infection range and positive rate of AAV2-NE01 were slightly better than those of AAV2-NE02. AAV2-WT mainly infects RGC layer cells, but has no effect on other layers of the NHP retina and the trabecular meshwork. This proves that the adeno-associated virus variants AAV2-NE01 and AAV2-NE02 prepared by the present invention have significantly improved the ability to infect NHP eye tissues.

[0183] To further and more accurately locate the layers of retinal cells infected by AAV2-NE01 and AAV2-NE02, frozen sections of ocular tissue were labeled and stained with anti-Rhodopsin (a specific antibody for the outer segments of photoreceptors). The results are shown in Figures 15 and 16. It can be seen that the GFP and mcherry signals overlap with the weaker portions of the anti-Rhodopsin signal, while the stronger portions are closely connected. Rhodopsin is weakly expressed in the nuclei and inner segments of cone and rod cells and strongly expressed in the outer segments of cone and rod cells, further demonstrating that AAV2-NE01 and AAV2-NE02 have strong retinal penetration and can infect the nuclei and inner segments of photoreceptors.

[0184] [Corrected 27.03.2025 according to Rule 91] The sequence alignment results of the variant adeno-associated virus capsid protein sequences AAV2-NE01 (SEQ ID NO.: 38) and AAV2-NE02 (SEQ ID NO.: 39) with the wild-type AAV2 capsid protein are shown in Figure 17. The sequences of the variant adeno-associated virus capsid proteins AAV2-NE01 and AAV2-NE02 differ from those of the wild-type AAV2 capsid protein (protein ID: YP_680426.1) in that the AAVRFDGTERAA and RQYSDAVRAE polypeptide sequences are inserted at amino acids 587 and 588 of the wild-type AAV2 viral capsid protein, respectively.

[0185] In summary, the adeno-associated virus variant prepared by the scheme of the present invention can significantly enhance the ocular tissue targeting infection performance of AAV2 virus compared with the wild type.

[0186] The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in Genbank accession number YP_680426.1. The sequence of the adeno-associated virus variant 7M8 is shown in SEQ ID NO.1.

[0187] 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.

[0188] The amino acid sequence of AAV2 adeno-associated virus mutant capsid protein Cap2-7M8 is SEQ ID NO.1:

[0189] 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 that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A capsid protein mutant that improves the ability of AAV virus to infect eye tissues, characterized in that: The AAV capsid protein is obtained by deleting the amino acid sequence homologous to the amino acids D561 to R588 of the wild-type AAV2 virus capsid protein, and inserting one of the polypeptides shown in SEQ ID NO.6 to SEQ ID NO.39 at the deleted position.

2. The capsid protein mutant according to claim 1, characterized in that The AAV capsid protein is a capsid protein of AAV2 virus or an AAV2 virus variant.

3. A gene encoding the AAV virus capsid protein mutant according to claim 1 or 2.

4. An expression vector, characterized in that: It expresses the AAV virus capsid protein mutant described in claim 1 or 2, or contains the gene described in claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is selected from plasmid and viral vector.

6. The expression vector according to claim 5, characterized in that The viral vector is a recombinant AAV vector, and the recombinant AAV vector is obtained by inserting or replacing the AAV vector.

7. The expression vector according to claim 6, characterized in that The AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

8. The expression vector according to any one of claims 4 to 6, characterized in that The expression vector also includes a nucleic acid molecule encoding a functional gene product.

9. A recombinant AAV virus, characterized in that: The capsid protein is as described in claim 1 or 2, or is obtained by shearing the capsid protein as described in claim 1 or 2.

10. The recombinant AAV virus according to claim 9, characterized in that The AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

11. An AAV pseudovirion, comprising an outer shell and a gene encapsulated in the outer shell, characterized in that: The constituent proteins of the shell include a capsid protein as described in claim 1 or 2, or are assembled from capsid proteins VP1, VP2 and VP3 formed by the capsid protein mutants as described in claim 1 or 2, or are assembled from capsid proteins VP1, VP2 and VP3 formed by the gene encoding and expression as described in claim 3.

12. A composition comprising the expression vector according to any one of claims 4 to 8 and an acceptable carrier, or the recombinant AAV virus according to claim 9 or 10, or the AAV pseudovirus particle according to claim 11.

13. The use of the composition of claim 11, wherein the use is selected from: 1) preparing gene therapy preparations or transgenic preparations targeting eye tissues; 2) constructing transgenic animals targeting eye tissues; 3) preparing drugs or transgenic vectors targeting eye tissues.

14. A transgenic method for treating an eye disease, comprising: S1. Test the patient to confirm that he or she has an eye disease; S2. Administering a therapeutic amount of a gene therapy preparation to the patient, wherein the gene therapy preparation is the recombinant AAV virus as described in claim 9 or 10, and / or the AAV pseudovirus particles as described in claim 11, and the nucleic acid molecules encapsulated by the recombinant AAV virus and the AAV pseudovirus particles express factors that can treat eye diseases.

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