Capsid protein mutants that enhance the infectivity of AAV to ocular tissues and their applications

Abstract

The present invention belongs to the field of biotechnology, and discloses a capsid protein mutant that improves the infectivity of AAV to eye tissues and its application. By inserting AAVRFDGTERAA and RQYSDAVRAE at amino acid positions 587 and 588 corresponding to the wild-type AAV2 virus capsid protein, the capsid protein mutants AAV2-NE01 and AAV2-NE02 are obtained. The virus yields of AAV2-NE01 and AAV2-NE02 are 1.96 times and 1.20 times that of AAV2-WT. After intravitreal injection, they can preferably infect various layers of cells from the ganglion cell layer (GCL), inner nuclear layer (INL) to outer nuclear layer (ONL) of the NHP retina and the trabecular meshwork region, with a high infection positive rate, strong fluorescence intensity, strong retinal penetration ability, and can infect the nuclear part and inner segment of the optic nerve. It has good application prospects and is expected to be developed into an AAV vector for treating eye diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to capsid protein mutants AAV2-NE01 and AAV2-NE02 that can improve the infectivity of AAV virus to non-human primate (NHP) eye tissues and their applications. Background Art

[0002] The genome of adeno-associated virus (AAV) is a single-stranded DNA fragment of about 4.7 kb, which is contained in an icosahedral non-enveloped virus capsid with a diameter of 20 nm. It can be divided into three functional regions: two open reading frames (Rep gene, Cap gene) and inverted terminal repeats (ITR). Among them, the open reading frame of the Cap gene encodes three capsid proteins VP1, VP2, and VP3, with molecular weights of 87, 73, and 61 kDa respectively. They are the capsid proteins required for assembling into complete viruses and play important roles in virus integration, replication, and assembly.

[0003] AAV is a delivery vector widely used in gene therapy. Its principle is to replace the sequence between the ITRs of the AAV genome with the target gene sequence by genetic engineering methods, and transfer it to the target cells through cell infection to achieve the purpose of gene therapy. Based on the characteristics of recombinant AAV such as safety, high efficiency, stability, persistence, specificity, and low integrativity, AAV has become one of the main delivery means in the field of gene therapy.

[0004] However, AAV still faces multiple challenges: First, the production process is complex, the cost is expensive, and the production capacity is limited; Second, high-dose administration may show neurotoxicity and hepatotoxicity, causing safety problems. Third, the infectivity to some tissue cells is weak. For example, it is difficult to infect the posterior layer cells of the retina by intravitreal injection, and it is difficult to infect central nerve cells by intravenous injection. Modifying and screening the AAV capsid protein to improve its targeting and transfection efficiency to specific tissues, with the expectation of achieving lower and safer dosing, is an effective way to solve this problem.

[0005] Due to the existence of the blood-eye barrier, the eye has a certain immune privilege, and local drug administration is relatively safe. Moreover, most fundus diseases are monogenic genetic diseases. Therefore, ophthalmology has become a hot field for gene therapy. The commonly used injection methods in clinical practice are intravitreal injection and subretinal injection. Subretinal injection has a better infection effect, especially on the RPE layer and the outer nuclear layer. However, this method is highly invasive and prone to retinal detachment, which may lead to photoreceptor degeneration, visual function damage, and gliosis. Intravitreal injection has a lower surgical risk, but conventional serotypes such as AAV2 and AAV5 can only infect the RGC layer and have a poor infection effect on cone cells, rod cells, and RPE. Therefore, developing AAV mutants that can improve the whole-eye expression ability or penetrate the RGC layer and the inner nuclear layer to infect outer nuclear layer cells and RPE cells is of great significance for solving the problem of delivery vectors in ophthalmic gene therapy.

[0006] Previous studies have shown that inserting a polypeptide fragment between positions 587 and 588 of Cap can optimize its infectivity to cynomolgus monkey eye tissues. The prior patent applications CN 116836237A and CN 117143203A have disclosed some capsid protein mutants, and the data show that different fragment insertions have different effects. However, whether the ability of AAV virus to infect the retinal tissues of NHP can be improved by this method remains to be further studied. Summary of the Invention

[0007] 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 infectivity of AAV to eye tissues and its application.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In the first aspect of the present invention, there is provided: a capsid protein mutant that improves the infectivity of AAV to eye tissues, which is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the AAV capsid protein, and the insertion site is homologous to the amino acids at positions 587 and 588 of the wild-type AAV2 virus capsid protein.

[0010] In some examples of the capsid protein mutant, it is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the capsid protein of an AAV2 variant, and the insertion site is homologous to the amino acids at positions 587 and 588 of the wild-type AAV2 virus capsid protein.

[0011] In some examples of the capsid protein mutant, it is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE between the amino acids at positions 587 and 588 of the wild-type AAV2 capsid protein.

[0012] In some examples of capsid protein mutants, the Genbank accession number of the amino acid sequence of the wild-type AAV2 capsid protein is YP_680426.1.

[0013] The second aspect of the present invention provides: a gene encoding the capsid protein mutant described in the first aspect of the present invention.

[0014] In some examples of genes, codon optimization is performed according to the expression system.

[0015] The third aspect of the present invention provides: an expression vector that expresses the capsid protein mutant described in the first aspect of the present invention, or into which the gene described in the second aspect of the present invention is inserted.

[0016] In some examples of expression vectors, the expression vector is one of a plasmid and a recombinant AAV vector.

[0017] In some examples of expression vectors, the AAV is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 and its variants.

[0018] In some examples of expression vectors, it further includes a gene encoding a functional gene product.

[0019] The fourth aspect of the present invention provides: an AAV virus particle, the capsid of which is assembled by capsid proteins VP1, VP2, and VP3 formed by the capsid protein mutant described in the first aspect of the present invention, or assembled by capsid proteins VP1, VP2, and VP3 encoded by the gene described in the second aspect of the present invention.

[0020] The fifth aspect of the present invention provides: a composition comprising the expression vector described in the third aspect of the present invention, or the AAV virus particle described in the fourth aspect of the present invention.

[0021] The sixth aspect of the present invention provides: the application of the composition described in the fifth aspect of the present invention, and the application includes:

[0022] Preparing a gene therapy preparation targeting eye tissues;

[0023] Constructing a transgenic model animal for eye tissue diseases;

[0024] Preparing a drug or a transgenic vector targeting eye tissues.

[0025] The beneficial effects of the present invention are:

[0026] In some examples of the present invention, the capsid protein mutant and the complete AAV capsid protein assembled with other capsid structural protein subunits can effectively improve the infectivity of AAV virus to eye tissues. Brief Description of the Drawings

[0027] Figure 1 It is a graph showing the production results of the adeno-associated virus variant prepared by the inventive solution in the test example of the present invention and the wild-type adeno-associated virus of the control group.

[0028] Figure 2 It is a retinal infection effect diagram of wild-type AAV2, AAV2-NE01 and AAV2-NE02 viruses of the present invention in the embodiment of the present invention infecting the eye tissue of cynomolgus monkeys for 3W.

[0029] Figure 3 It is a trabecular meshwork infection effect diagram of wild-type and AAV2-NE01 and AAV2-NE02 viruses of the present invention in the embodiment of the present invention infecting the eye tissue of cynomolgus monkeys for 3W.

[0030] Figure 4 It is a retinal immunofluorescence staining diagram of AAV2-NE01 of the present invention in the embodiment of the present invention infecting the eye tissue of cynomolgus monkeys for 3W.

[0031] Figure 5 It is a retinal immunofluorescence staining diagram of AAV2-NE02 of the present invention in the embodiment of the present invention infecting the eye tissue of cynomolgus monkeys for 3W.

[0032] Figure 6 It is a sequence alignment diagram of the Cap587-588 sequences of wild-type AAV2, AAV2-NE01 and AAV2-NE02 sequences of the present invention in the embodiment of the present invention. Detailed Description of the Invention

[0033] In the first aspect of the present invention, there is provided: a capsid protein mutant that improves the infectivity of AAV to eye tissue, which is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the AAV capsid protein, and the insertion site is homologous to the amino acids at positions 587 and 588 of the capsid protein of wild-type AAV2 virus.

[0034] In some examples of the capsid protein mutant, it is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the capsid protein of the AAV2 variant, and the insertion site is homologous to the amino acids at positions 587 and 588 of the capsid protein of wild-type AAV2 virus.

[0035] The homology with the amino acids at positions 587 and 588 of the capsid protein of wild-type AAV2 virus can be determined based on existing methods, such as by comparative analysis of the amino acid sequences of AAV capsid proteins of different serotypes.

[0036] In some examples of the capsid protein mutant, it is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE between the amino acids at positions 587 and 588 of the wild-type AAV2 capsid protein.

[0037] In some examples of capsid protein mutants, the Genbank accession number of the amino acid sequence of the wild-type AAV2 capsid protein is YP_680426.1.

[0038] The second aspect of the present invention provides: a gene encoding the capsid protein mutant described in the first aspect of the present invention.

[0039] In some examples of genes, codon optimization is performed according to the expression system.

[0040] The third aspect of the present invention provides: an expression vector that expresses the capsid protein mutant described in the first aspect of the present invention, or into which the gene described in the second aspect of the present invention is inserted.

[0041] In some examples of expression vectors, the expression vector is one of a plasmid and a recombinant AAV vector.

[0042] In some examples of expression vectors, the AAV is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 and its variants.

[0043] In some examples of expression vectors, it further includes a gene encoding a functional gene product. The gene product preferably acts on eye tissues.

[0044] The fourth aspect of the present invention provides: an AAV virus particle, the capsid of which is assembled by capsid proteins VP1, VP2, and VP3 formed by the capsid protein mutant described in the first aspect of the present invention, or assembled by capsid proteins VP1, VP2, and VP3 encoded by the gene described in the second aspect of the present invention.

[0045] The fifth aspect of the present invention provides: a composition comprising the expression vector described in the third aspect of the present invention, or the AAV virus particle described in the fourth aspect of the present invention.

[0046] The sixth aspect of the present invention provides: the application of the composition described in the fifth aspect of the present invention, and the application includes:

[0047] Preparing a gene therapy preparation targeting eye tissues;

[0048] Constructing a transgenic model animal for eye tissue diseases;

[0049] Preparing a drug or a transgenic vector targeting eye tissues.

[0050] In some examples of applications, the animal is a non-human primate mammal, including but not limited to macaques, cynomolgus monkeys, Yunnan snub-nosed monkeys, flat-top monkeys, Assamese macaques, red-faced monkeys, marmosets, and slow lorises.

[0051] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with the embodiments 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, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall 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, are all reagents and materials that can be obtained from commercial channels. Embodiment

[0052] Based on the previous rounds of directed evolution of a 10 - 12 amino acid polypeptide insertion library, the top 30 sequences with the optimal comprehensive performance of retinal infection ability and yield are screened from the NGS sequencing data, and a variety of adeno-associated virus variants AAV2-NE01, AAV2-NE02, AAV2-PN001 to AAV2-PN028 are provided. The preparation of the adeno-associated virus variants includes the following steps:

[0053] 1. Plasmid construction

[0054] Through GenScript Biotech Corporation, nucleic acid sequences corresponding to amino acid sequences such as AAV2-NE01, AAV2-NE02, and AAV2-PN001 to AAV2-PN028 (as shown in Table 1) are synthesized, and the synthesized sequences are cloned into the pAAV2-WT plasmid by seamless cloning. The mutant sequences shown in Table 1 are inserted at the 587 - 588 sites of the Cap protein to construct a pAAV2-mutant plasmid vector; the ligation product is transformed into Escherichia coli competent stbl3, single colonies are picked for culture, and plasmids are extracted for sequencing and restriction enzyme digestion verification. The mutant plasmids are successfully constructed as identified by agarose gel electrophoresis.

[0055] Table 1. Information on amino acid sequences inserted between N587 and R588 of the Cap protein of AAV2 mutants

[0056]

[0057] 2. Packaging of mutant viruses such as AAV2-WT, AAV2-NE01, and AAV2-NE02

[0058] The pAAV2-WT or AAV2-mutant packaging plasmid, together with the pAAV-CAG-EGFP (or pAAV-CAG-Mcherry) expression plasmid and the pHelper helper plasmid, are co-transfected into suspension HEK293T cells. After culturing the transfected cells for 72 hours, the cells are lysed to harvest the virus, and the virus is purified by iodixanol density gradient centrifugation, which mainly includes the following steps:

[0059] A. Cell culture

[0060] (1) Take out a vial of suspended 293T cells from the liquid nitrogen tank, place it in a 37 °C water bath, shake it quickly to thaw the cells, add them to a centrifuge tube containing 5 mL of Wayne 293 serum-free medium, centrifuge at 200×g for 5 min, discard the supernatant, resuspend in 10 mL of Wayne 293 serum-free medium, place in a 125 mL Erlenmeyer flask, supplement with Wayne 293 serum-free medium to 20 mL, and culture in a shaking incubator at 120 rpm, 37 °C, 5% CO2;

[0061] (2) After culturing for 48 - 72 hours, when the cell density reaches 2 - 3E+6 cell / mL, the cells can be passaged;

[0062] (3) Take out the seed cells to the laminar flow hood, shake well and aspirate about 500 μL of the sample. Add 20 μL of the sample to 20 μL of trypan blue and mix well. Aspirate 20 μL of the mixture and add it to the cell counting chamber. Read the data 3 times on the automatic cell counter, and take the average value as the cell density;

[0063] (4) Take an appropriate amount of cells, dilute them with fresh Wayne 293 serum-free medium to 0.6 - 0.65E+6 cell / mL, place in a 500 mL Erlenmeyer flask for culture, and the culture volume per flask is 25 mL. Culture in a shaking CO2 cell incubator, and the culture conditions are: 120 rpm, 37 °C, 5% CO2.

[0064] B. Cell transfection

[0065] (1) Culture the above cells for 48 h. When the cell density reaches 3 - 3.8E+6 cell / mL, the cells can be transfected;

[0066] (2) For each flask of cells, add the packaging plasmid, pHelper, and pAAV-CAG-EGFP (or pAAV-CAG-Mcherry) to 4 mL of DMEM medium in sequence, with a total of 100 μg, and mix well to obtain the "plasmid dilution solution";

[0067] (3) Take another 1 mL of DMEM medium, add 300 μg of PEI and mix well to obtain the "PEI dilution solution";

[0068] (4) Pour the "PEI diluent" into the "plasmid diluent", mix well quickly, and let it stand at room temperature for 25 minutes to form a transfection complex; add the transfection complex dropwise to the cell suspension while gently shaking.

[0069] (5) Place the culture flask in an oscillating CO2 cell culture incubator for cultivation. The cultivation conditions are: 120 rpm, 37 °C, 5% CO2.

[0070] C. Virus Harvest

[0071] (1) Continue to culture the transfected cells for 72 hours and start virus harvest.

[0072] (2) Collect the cell suspensions in each bottle into a centrifuge bottle, 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) to each mL of the supernatant, mix well, and let it stand overnight at 2 - 8 °C.

[0073] (3) Add an appropriate amount of 0.5% TritonX - 100 cell lysis buffer (containing universal nuclease) to the cell pellet, incubate at 37 °C for 1 - 2 h, then add 1 / 10 volume of 5 mol / L NaCl, mix well, centrifuge at 4 °C and 3000 g for 15 min, collect the supernatant, which is the "crude virus extract" and store it temporarily at 2 - 8 °C.

[0074] (4) Centrifuge the supernatant after PEG8000 precipitation at 4 °C and 3000 g for 15 min, discard the supernatant, and resuspend it with the "crude virus extract" in step (3) for standby.

[0075] D. Virus Purification

[0076] 1) Take an Ultra - Clear centrifuge tube and successively 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 balance with cell lysate.

[0077] 2) Ultra - centrifuge at 10 °C and 230000 g, with a ramp of 8 and a deceleration of 9 for 18 h.

[0078] 3) Take an ultrafiltration tube and moisten the filter membrane with 1 mL of PBS buffer.

[0079] 4) Carefully aspirate the middle layer of 40% - 60% Iodixanol in the ultra - centrifuge tube with a pipette gun, avoiding sucking the protein, and transfer it to the ultrafiltration tube.

[0080] 5) Add an appropriate amount of PBS buffer and pipette gently to mix evenly. Centrifuge at 4500 g for 3 - 5 min, and repeat this step 5 - 7 times until iodixanol is removed.

[0081] 6) Dilute the crude virus product in step 5 to 8 mL, and repeat steps 1 to 5.

[0082] 6) Add 1 mL of PBS buffer and pipette 40 - 50 times to form a virus suspension, and transfer it to an EP tube.

[0083] 7) Aspirate 0.22 μm filter the virus suspension in the EP tube with a 5 mL syringe. After collecting 20 μL of the virus solution for testing samples, aliquot it into 100 μL per tube to obtain the adeno-associated virus variant.

[0084] Comparative Example

[0085] This comparative example provides a wild-type adeno-associated virus (AAV2-WT). The difference from the preparation method of the example is only that the sequence of the adeno-associated virus capsid protein uses the Cap sequence of wild-type AAV2 (Protein ID: YP_680426.1 SEQID NO.1).

[0086] Test Example

[0087] 1. Virus Titer Detection

[0088] (1) Virus Lysis

[0089] 1) Take 20 μL of the virus samples from Example 1 and the comparative example respectively.

[0090] 2) Add 1 μL of 10% SDS, 0.5 mol / L EDTA, and proteinase K respectively, and mix well.

[0091] 3) Incubate in a thermostatic mixer at 56 °C for 1 hour, and then incubate at 90 °C for 10 min.

[0092] 4) Take 10 μL of the virus lysate and dilute it 10-fold serially to 10000-fold to obtain the virus dilution, which is reserved for use.

[0093] (2) Preparation of Standards for Standard Curve

[0094] Take the plasmid standard of 2×10 12 copies / mL, and dilute it with ultrapure water ddH2O in proportion to 6 gradients as the templates of the standards 2×10 11 copies / mL, 2×10 10 copies / mL, 2×10 9 copies / mL, 2×10 8copies / mL, 2×10 7 copies / mL, 2×10 6 copies / mL.

[0095] (3)Absolute quantitative qPCR

[0096] 1) Take a 0.2 mL PCR tube and prepare the following reaction system, with 3 replicates for each virus dilution. 2× qPCR Mix 10 μL; 0.2 μL each of forward and reverse primers; 5 μL virus dilution; 4.2 μL ddH2O;

[0097] Amplification primers:

[0098] Forward primer 5’- GGAACCCCTAGTGATGGAGTT-3’ (SEQ ID NO.3);

[0099] Reverse primer 5’- CGGCCTCAGTGAGCGA-3’ (SEQ ID NO.4);

[0100] 2) qPCR amplification conditions

[0101] Pre-denaturation: 95°C, 2 min;

[0102] 40× cycles: 95°C, 15 s; 60°C, 60 s;

[0103] 3) qPCR data processing:

[0104] Virus titer = dilution factor * number of copies of virus gene array

[0105] Virus yield = virus titer * virus volume.

[0106] In the scheme of the present invention, pAAV2-WT and pAAV2 mutants are respectively packaged into viruses by the 3-plasmid system, and the suspension cell transient transfection method is used. After 72 hours, the cells are lysed to harvest the viruses, and the viruses are purified by iodixanol density gradient centrifugation. The virus yield is measured by the QPCR method. The experimental results are as Figure 1 shown. It can be seen from the figure that there are significant differences in the yields among different mutants. The virus yields of AAV2-NE01 and AAV2-NE02 are 1.96 times and 1.20 times that of AAV2-WT, respectively. The virus yields of the remaining 28 mutants are 0.04 times to 1.26 times that of AAV2-WT. Mutants AAV2-NE01, AAV2-NE02 and mutants with a yield not less than 80% of the AAV2-WT yield are selected for the in vivo infection ability test in cynomolgus monkeys.

[0107] 2. Test on the infection effect of cynomolgus monkey eye tissues

[0108] Inject AAV2-WT and AAV2-mutant viruses into cynomolgus monkeys. Three weeks later, remove the eyeballs and prepare frozen sections.

[0109] (1)Intravitreal injection

[0110] Dilute the samples of Example 1 and the comparative example to 1E+12 vg / µL respectively, and inject them intravitreally into cynomolgus monkeys. Inject 100 µL into each eye (the total virus amount is 1E+11 vg), and inject one cynomolgus monkey with each virus. The specific operation steps are as follows:

[0111] 1) Before injection anesthesia, observe the ocular surface condition by ophthalmic examination (slit lamp). If any abnormality is found, exclude it to ensure that there is no ocular lesion.

[0112] 2) After anesthetizing the cynomolgus monkey, instill tropicamide into the eye for pupil dilation. After mydriasis, instill tetracaine hydrochloride for surface anesthesia of the eyeball.

[0113] 3) Aspirate an appropriate amount of virus with a microsyringe for standby. Place the anesthetized cynomolgus monkey on the operating table, give local anesthesia to the eye with lidocaine hydrochloride, hold the head with the left hand to make the eyeball protrude partially, and insert the microsyringe into the vitreous cavity from the posterior edge of the cornea with the right hand. First, insert the needle vertically, then tilt it, and slowly push the drug. After injection, slowly withdraw the needle.

[0114] (2)Sampling, preparation of frozen sections and observation of fluorescence signals

[0115] Three weeks after virus injection, euthanize the cynomolgus monkey, remove the eyeballs, prepare frozen sections, and observe the expression of GFP fluorescence signals in each region of the eye tissue. The steps are as follows:

[0116] 1) Dissect the fresh eyeball tissue and immediately immerse it in the tissue fixative for fixation for 24 - 48 hours.

[0117] 2) After taking the tissue out of the fixative, remove the redundant surrounding tissue, cut it with a sharp blade according to the experimental requirements, remove the lens and vitreous inside, and dehydrate the trimmed eyeball tissue block in 10% sucrose - 20% sucrose - 30% sucrose solution in a 4℃ refrigerator.

[0118] 3) Take out the dehydrated tissue, dry the surface moisture with filter paper, place it face up in the embedding frame, drop OCT embedding agent (avoiding air bubbles), put the embedding frame into a -20℃ refrigerator for freezing for half an hour, and then transfer it to an -80℃ refrigerator for storage.

[0119] 4) Before sectioning, take out the OCT embedding block from -80℃ and place it in the freezing microtome box to balance the temperature for 30 minutes. Load the sample and section it. Cut and retain the slices according to the experimental requirements. Mark the retained slices with labels and store them at -20℃ for standby.

[0120] 5), Drop the prepared DAPI in a certain proportion on the sections, incubate in the dark at room temperature for 3 min, place the slides in PBS and wash 3 times, 5 min each time, and mount the slides using an anti-fluorescence quenching mounting medium;

[0121] 6), Collect images before observation using a fluorescence microscope.

[0122] (3) Immunofluorescence staining

[0123] After labeling the frozen sections with Anti-Rhodopsin antibody respectively, observe the co-localization of GFP or Mcherry with photoreceptor cells under a fluorescence microscope. The specific steps are as follows:

[0124] 1) Take out the frozen sections from the -20 °C refrigerator and let them return to room temperature and dry. If it is an unfixed sample, fix it with tissue fixative for 15 min and then rinse with running water;

[0125] 2) Place the sections in the repair solution, boil in a pressure cooker with internal inflation for 3 min, and after natural cooling, place the slides in PBS and wash 3 times, 5 min each time;

[0126] 3), Put the sections into 0.3% Triton X-100 solution, incubate in the dark at room temperature for 10 min, place the slides in PBS and wash 3 times, 5 min each time;

[0127] 4), Drop blocking serum in the immunohistochemical circle to evenly cover the tissue and block at room temperature for 60 min;

[0128] 5), Gently shake off the blocking solution, drop the primary antibody (Anti-Rhodopsin antibody abcam, ab221664, 1:500) prepared in a certain proportion on the sections, place the sections flat in a wet box and incubate overnight at 4 °C or incubate for 1 hour at room temperature;

[0129] 6), Place the slides in PBS and wash 3 times, 5 min each time. After the sections are slightly dried, drop the secondary antibody (fluorescently labeled) corresponding to the primary antibody in the circle to cover the tissue and incubate at room temperature for 60 min. Place the slides in PBS and wash 3 times, 5 min each time. Place the slides in PBS and wash 3 times, 5 min each time;

[0130] 7), Drop the prepared DAPI in a certain proportion on the sections, incubate in the dark at room temperature for 3 min, place the slides in PBS and wash 3 times, 5 min each time;

[0131] 8), Mount the slides using an anti-fluorescence quenching mounting medium;

[0132] 9), Observe the fluorescence signal under a fluorescence microscope.

[0133] Three weeks after virus injection, frozen sections were prepared from the samples, and the eye sections were stained with DAPI. The results observed under a fluorescence microscope are as Figure 2 and Figure 3 shown. After intravitreal injection of AAV2-NE01-CAG-EGFP (green) and AAV2-NE02-CAG-Mcherry (red) viruses, they can infect various layers of cells in the NHP retina from the ganglion cell layer (GCL), inner nuclear layer (INL) to outer nuclear layer (ONL) well ( Figure 2 ), and the trabecular meshwork area ( Figure 3 ), with a high infection positive rate and strong fluorescence intensity. The infection range and positive rate of AAV2-NE01 are slightly better than those of AAV2-NE02. While AAV2-WT only infects a small number of cells in the RGC layer, but hardly infects other layers of the NHP retina and the trabecular meshwork, and other variants have no good infection effect. It is proved that the adeno-associated virus variants AAV2-NE01 and AAV2-NE02 prepared by the scheme of the present invention have significantly improved the infection ability of NHP eye tissues.

[0134] To further and more accurately locate the layers of retinal cells infected by AAV2-NE01 and AAV2-NE02, the frozen eye sections were labeled and stained with Anti-Rhodopsin (an antibody specific to the outer segment of photoreceptor cells). The results are as Figure 4 and Figure 5 shown. It can be seen that the GFP signal and mcherry signal overlap with the weaker part of the Anti-Rhodopsin signal and are closely connected to the stronger part. Rhodopsin has weak expression in the nuclear part and inner segment of cone and rod photoreceptor cells and strong expression in the outer segment of cone and rod photoreceptor cells, further proving that AAV2-NE01 and AAV2-NE02 have strong retinal penetration ability and can infect the nuclear part and inner segment of photoreceptor cells.

[0135] The sequence alignment results of the adeno-associated virus variant capsid proteins AAV2-NE01 (SEQ ID NO.: 1) and AAV2-NE02 (SEQ ID NO.: 2) and the wild-type AAV2 capsid protein are as Figure 6 shown. The differences between the sequences of the adeno-associated virus variant capsid proteins AAV2-NE01 and AAV2-NE02 and the wild-type AAV2 capsid protein (protein ID: YP_680426.1) are that AAVRFDGTERAA and RQYSDAVRAE polypeptide sequences are inserted at amino acid positions 587 and 588 of the wild-type AAV2 virus capsid protein respectively.

[0136] In summary, compared with the wild type, the adeno-associated virus variant prepared by the solution of the present invention can significantly enhance the cells in each layer of the NHP retina of AAV2 virus.

[0137] Amino acid sequence of the capsid protein Cap of adeno-associated virus variant AAV2-NE01, SEQ ID NO.1: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAVRFDGTERAA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL。

[0138] Amino acid sequence of the capsid protein Cap of adeno-associated virus variant AAV2-NE02, SEQ ID NO.2:

[0139] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN RQYSDAVRAE RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL。

[0140] The above is a further detailed description of the present invention and should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, any simple deduction or substitution without departing from the concept of the present invention falls within the protection scope of the present invention.

Claims

1. A capsid protein mutant that improves the ability of AAV to infect eye tissues, characterized in that: The polypeptide is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE between amino acids 587 and 588 of the wild-type AAV2 capsid protein, and the protein ID of the wild-type AAV2 capsid protein is YP_680426.

1.

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

3. An expression vector, characterized in that: It expresses the capsid protein mutant described in claim 1, or has the gene described in claim 2 inserted therein.

4. The expression vector according to claim 3, characterized in that The expression vector is a plasmid or a recombinant AAV vector.

5. The expression vector according to claim 4, characterized in that The AAV is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

6. The expression vector according to claim 4, characterized in that Also included are genes encoding functional gene products.

7. An AAV virus particle, characterized in that The outer shell is formed by assembling capsid proteins VP1, VP2 and VP3 formed by the capsid protein mutant according to claim 1, or by assembling capsid proteins VP1, VP2 and VP3 encoded by the gene according to claim 2.

8. A composition, characterized in that It comprises the expression vector according to any one of claims 3 to 6, or the AAV virus particle according to claim 7.

9. Use of the composition according to claim 8, characterized in that The applications include: Construct transgenic animal models of eye tissue diseases; Prepare drug carriers or transgenic carriers targeted to eye tissues.

10. Use of the composition, characterized in that The composition comprises the expression vector according to claim 6, and the application comprises: preparing a gene therapy preparation targeting eye tissue.

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