AAV2 variant and application thereof

Abstract

The present invention provides an AAV2 capsid protein variant comprising one or more modifications comprising the insertion of a peptide having a length of 8 amino acids between the 588 and 589 amino acids of the AAV2 capsid protein, and also relates to a polynucleotide, a host cell, a vector, an AAV virus particle or a pharmaceutical composition thereof, the AAV2 capsid protein variant comprising one or more modifications comprising the insertion of a peptide having a length of 8 amino acids between the 588 and 589 amino acids of the AAV2 capsid protein variant. And its use in preparing a medicament for administering an effective amount of a medicament for treating a disease to a subject in need thereof.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and in particular to adeno-associated virus variants and applications thereof. Background Art

[0002] There are more than 7,000 human diseases known so far, of which about 80% are caused by genetic problems. More than 300 million people in the world suffer from genetic diseases. Current chemotherapy, protein therapy, etc. can only treat less than 500 human diseases, and are usually ineffective against diseases caused by genetic problems, so new drugs and treatment plans need to be developed.

[0003] At present, gene therapy has become a research hotspot in the field of biomedicine. Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defects and abnormal genes to achieve the purpose of treatment. It also includes the application of technologies such as transgenics, that is, inserting exogenous genes into appropriate recipient cells of patients through gene transfer technology so that the products produced by exogenous genes can treat certain diseases. The most commonly used vectors in gene therapy include adenovirus, lentivirus, retrovirus and adeno-associated virus. Adeno-associated virus (AAV) was first discovered in laboratory adenovirus (AdV) preparations in the mid-1960s and was soon discovered in human tissues. Due to its good safety, wide range of host cells, low immunogenicity, and high efficiency and long-term expression of exogenous genes, it has become an important tool for gene delivery.

[0004] Today, AAV vectors have achieved good results in clinical trials of in vivo gene therapy, and multiple marketed drug approvals and ongoing clinical trials have proven that AAV vectors have become one of the main gene delivery tools for gene therapy. In 2012, Glybera was the first AAV therapy approved in Europe for the treatment of lipoprotein lipase deficiency. The launch of these drugs clearly demonstrates the safety and efficacy of AAV-based treatments. Since then, Luxturna for the treatment of Leber congenital amaurosis and Zolgensma for the treatment of spinal muscular atrophy (SMA) have been approved by the US Food and Drug Administration (FDA) in 2017 and 2019, respectively. Current clinical research focuses on the treatment of monogenic diseases by gene replacement, gene silencing or gene editing. In addition to being used to treat a variety of diseases such as cancer, blindness, immune and neuronal diseases, AAV is also expected to be used in vaccine development.

[0005] The main factors that make AAV a successful therapeutic gene delivery vector are its low immunogenicity and lack of pathogenicity. However, there are certain problems with existing AAV serotypes that affect its potential for gene therapy. First, although the existing serotypes provide a range of tissue tropisms to choose from, there are still some types of tissues that cannot be covered by the specificity of these serotypes in clinical applications. Second, although some specific serotypes of AAV can specifically infect target tissues, their gene delivery efficiency may be low, or they may have off-target effects and infect other tissues. Third, the pre-existing neutralizing antibodies against multiple AAV serotypes in the body will hinder AAV infection from the beginning or after multiple administrations. Finally, some AAV serotypes are more difficult to obtain high titers, purity, and stability in production than others.

[0006] Therefore, to achieve precise gene therapy, efficient and specific new recombinant adeno-associated virus vectors are needed. To overcome these problems, AAV capsid development has always been a very important technology in accelerating the acquisition of new AAV mutants and improving AAV performance.

[0007] Directed evolution (error-prone PCR, DNA shuffling, random peptide display and other technologies) is a powerful tool for modifying AAV capsids. In principle, directed evolution uses high-throughput technology to introduce mutations into genes and the proteins they encode, and simulates natural evolution to greatly accelerate protein diversification and selection processes. By modifying the capsid protein structure of AAV vectors to produce specific vectors that can efficiently target target organ receptors, the expression and toxic side effects of non-target tissues can be reduced, and the effective expression of target genes can be improved. The above methods have been used to screen new AAV mutants with high-efficiency and specific gene transfer efficiency on various natural serotypes of AAV, such as AAV2, AAV5 and AAV9, such as AAV-DJ, AAV7M8, AAVPHB, etc.

[0008] Compared with the injection of the inferior cavity, intravitreal injection has the advantages of convenient operation, patient comfort, and low surgical risks. Therefore, this method of administration has been favored by many researchers and clinicians for the treatment of various retinal diseases. The wild-type AAVs that have been discovered so far are difficult to cross the inner limiting membrane by intravitreal injection to achieve the effect of infecting the RPE and outer nuclear layer. Wild-type AAV2 can only infect ganglia and inner nuclear layer cells through intravitreal injection, and cannot infect the mouse RPE and outer nuclear layer and outer nuclear layer cells. Summary of the invention

[0009] On the one hand, the present disclosure provides an AAV2 capsid protein variant, which comprises one or more modifications, wherein the modifications include inserting 8 consecutive random peptides between amino acids 588 and 589 of the parent AAV2 capsid protein, and the random peptides comprise an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 or an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

[0010] In one embodiment, the sequence of the parent AAV2 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO:1.

[0011] In one embodiment, the random peptide is selected from the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

[0012] In one embodiment, the AAV2 capsid protein variant comprises an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or a sequence that is 75% or more identical to SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

[0013] The present disclosure also provides an isolated polynucleotide comprising a nucleic acid sequence encoding the AAV2 capsid protein variant as described above.

[0014] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 or a nucleic acid sequence that is 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.

[0015] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide as described above.

[0016] The present disclosure provides a host cell, comprising the isolated polynucleotide as described above.

[0017] The present disclosure provides a recombinant AAV virus particle comprising a variant or an isolated polynucleotide as described above.

[0018] In one embodiment, the recombinant AAV virus particle comprises a functional gene product, which is a polypeptide.

[0019] The present disclosure also provides a pharmaceutical composition comprising the recombinant AAV virus particles as described above and a pharmaceutically acceptable excipient.

[0020] The present disclosure also provides a recombinant AAV vector, which comprises a nucleic acid sequence encoding 1) an AAV2 capsid protein variant as described above or an isolated polynucleotide as described above; 2) 5' and 3' inverted terminal repeat sequences (5' ITR and 3' ITR); 3) a nucleic acid sequence encoding a functional gene product; and 4) a regulatory sequence that directs the expression of the gene product in target cells.

[0021] The present disclosure also provides use of the AAV2 capsid protein variant, isolated polynucleotide, vector, host cell, recombinant AAV virus particle, pharmaceutical composition or recombinant AAV vector as described above in the preparation of a drug for administering an effective amount of a disease-treating drug to a subject in need thereof.

[0022] In one embodiment, the disease is selected from one or more of hearing impairment diseases, ophthalmic diseases, inflammation, tumors, metabolic diseases, pain, and neurodegenerative inflammatory diseases.

[0023] In one embodiment, the ophthalmic disease refers to retinal damage disease in adult individuals, preferably, the ophthalmic disease is selected from RPE layer and outer nuclear layer related diseases, photoreceptor cell related diseases, cell damage related diseases, related diseases caused by genetic defects, related diseases caused by acquired factors and related diseases caused by aging. In a specific embodiment, the ophthalmic disease is RPE and outer nuclear layer related diseases, such as dry AMD, wet AMD or choroidal neovascularization (CNV); such as can be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), cystoid macular edema (CME), epiretinal membrane (ERM) and macular hole; myopia-related choroidal neovascularization, vascular streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), atrophic lesions of retinal pigment epithelial cells (RPE), hypertrophy of retinal pigment epithelial cells (RPE) and retinal pigment epithelial cells (RPE) The invention relates to one or more of the following conditions: corneal edema, retinal vein occlusion, chorioretinal vein occlusion, macular edema; corneal angiogenesis, pterygium conjunctiva, subretinal edema and intraretinal edema due to hypoxia; or macular edema due to retinal vein occlusion, retinitis pigmentosa, Stargardt's disease, glaucoma, inflammatory diseases, cataracts, refractory abnormalities, keratoconus, retinopathy of prematurity, angiogenesis of the anterior part of the eye, corneal angiogenesis after keratitis, corneal transplantation or corneal formation; congenital amaurosis (LCA), pigmentary retinal degeneration, Stargardt disease caused by gene mutations related to RPE and outer nuclear layer cells.

[0024] Beneficial Effects The adeno-associated virus variants XM06 / XM07 / XM08 of the present invention and the wild-type AAV2 exhibit different infection characteristics using the vitreous injection technique. The variant capsid can efficiently infect the RPE and photoreceptor cells in the outer layer of the retina, thus resolving the limitation that AAV2 cannot infect. The application of the variant in the prevention and / or treatment of ophthalmic diseases effectively overcomes the various shortcomings of the prior art and has a high industrial utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present disclosure can be more fully understood with reference to the following drawings.

[0026] Figure 1 shows the structural diagrams and carrier information of XM06, XM07 and XM08. Figure 1A shows the structural diagram of XM06, Figure 1B shows the carrier information of XM07, and Figure 1C shows the carrier information of XM08.

[0027] Figure 2The differences in extracellular and intracellular production of AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, AAV-XM08-CAG-EGFP and AAV2 are shown.

[0028] Figure 3 The expression distribution of AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, and AAV-XM08-CAG-EGFP in the mouse retina is shown.

[0029] Figure 4 The infection efficiency of AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, and AAV-XM08-CAG-EGFP viruses on photoreceptor cells is shown. DETAILED DESCRIPTION

[0030] The following description of the present disclosure is intended only to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0031] Throughout this disclosure, unless the context requires otherwise, the word "comprising" should be understood to imply the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to the elements indicated by ... in the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required and mandatory, and no other elements may be present. "Consisting essentially of" means including any elements indicated by ..., and is limited to other elements that do not interfere with or contribute to the activity or action of the listed elements specified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required and mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0032] Drug composition The present disclosure also provides a pharmaceutical composition comprising a polypeptide complex or a bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" indicates that the specified carrier, medium, diluent, excipient and / or salt are generally chemically and / or physically compatible with the other ingredients constituting the dosage form, and are physiologically compatible with the recipient thereof. "Pharmaceutically acceptable excipient" refers to other ingredients in a pharmaceutical dosage form other than the active ingredient, which have acceptable biological activity and are non-toxic to the subject. The pharmaceutically acceptable excipients used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / formulating agents, multivalent sequestration or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0033] As used herein, the term "subject" includes any human or non-human mammal. The term "non-human mammal" includes all vertebrates such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably.

[0034] In certain embodiments, the AAV capsid protein variant, isolated polynucleotide, vector, host cell, recombinant AAV virus particle, pharmaceutical composition or recombinant AAV vector is used in an animal model or a cell model.

[0035] In some embodiments, the disease is selected from one or more of hearing impairment diseases, ophthalmic diseases, inflammation, tumors, metabolic diseases, pain, and neurodegenerative inflammatory diseases. In one embodiment, the ophthalmic disease refers to retinal damage diseases in adult individuals, and preferably, the ophthalmic disease is selected from RPE layer and outer nuclear layer related diseases, photoreceptor cell related diseases, cell damage related diseases, related diseases caused by gene defects, related diseases caused by acquired factors, and related diseases caused by aging. In a specific embodiment, the ophthalmic disease is a disease associated with the RPE and outer nuclear layer, such as dry AMD, wet AMD or choroidal neovascularization (CNV); such as can be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), cystoid macular edema (CME), epiretinal membrane (ERM) and macular hole; myopia-related choroidal neovascularization, angioid streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), atrophic lesions of retinal pigment epithelial cells (RPE), hypertrophy of retinal pigment epithelial cells (RPE); The invention relates to one or more of the following conditions: corneal edema, retinal vein occlusion, chorioretinal vein occlusion, macular edema; corneal angiogenesis, pterygium conjunctiva, subretinal edema and intraretinal edema due to hypoxia; or macular edema due to retinal vein occlusion, retinitis pigmentosa, Stargardt's disease, glaucoma, inflammatory diseases, cataracts, refractory abnormalities, keratoconus, retinopathy of prematurity, angiogenesis of the anterior part of the eye, corneal angiogenesis after keratitis, corneal transplantation or corneal formation; congenital amaurosis (LCA), pigmentary retinal degeneration, Stargardt disease caused by gene mutations related to RPE and outer nuclear layer cells.

[0036] Example In order to enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0037] Example 1: Construction of random peptide library plasmid A random peptide primer was used to insert 8 consecutive random peptide fragments between the 588th and 589th positions of the AAV2 capsid protein to amplify part of the AAV2 capsid protein sequence, and the remaining capsid protein sequence and REP sequence were amplified simultaneously. pITR was digested with Mlu I and EcoRV ( Tong, D.et al.Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques.Protein&Cell14, 69–73,doi:10.1093 / procel / pwac020 (2022)) plasmid, and obtain random peptide recombinant products by homologous recombination. The recombinant products were transformed into DH10B competent cells by electroporation, and part of the bacterial solution was plated. The colonies grown on the plate were counted and identified by sequencing to determine their diversity. The remaining bacterial solution was inoculated into 500 ml SOC liquid culture medium and cultured at 37°C overnight. The plasmid extracted the next day was the random peptide library plasmid.

[0038] Example 2: Packaging of random peptide library viruses The obtained random peptide library plasmid and adenovirus helper plasmid pHelper were co-transfected into HEK-293T cells in appropriate amounts. The pHelper plasmid ( Tong, D. et al. Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques. Protein&Cell 14, 69–73, doi:10.1093 / procel / pwac020 (2022) ). Each 15 cm dish of cells was transfected with 30 μg of random peptide library plasmid. The culture medium was replaced 24 hours after transfection, and the cells and supernatant culture medium were collected 96 hours later. The cells were lysed with 500 mM Tris (trishydroxymethylaminomethane) buffer (pH 8.0) to release AAV, and residual nucleic acids were treated with a full-strength nuclease at a final concentration of 40 U / ml. After centrifugation, polyethylene glycol 8000 and 500 mM sodium chloride were added to the supernatant containing the virus at a final concentration of 8%, and the virus was precipitated at 4°C overnight. After centrifugation, the precipitate was resuspended in 2 mM Mg 2+ , 3% polysorbate 20, and 0.0001% poloxamer 188 in phosphate buffer. After centrifugation to remove impurities, the virus suspension was purified by ultracentrifugation using iodixanol density gradient solutions (15%, 25%, 40%, and 60%), and then the virus titer was determined by qPCR using AAV2 Rep gene-specific primers REP-F / REP-R.

[0039] The specific steps for virus titer detection are as follows: (1) Preparation of standard samples for standard curve Take 100 ng / μL of the standard plasmid and add ddHO. 2 O was diluted to 20 μL and mixed thoroughly to obtain a 10 ng / μL standard plasmid. It was diluted in 10 series gradients, with a total of 8 concentration gradients, namely 1×100 0 ~1×10 -8 ng / μL.

[0040] (2) Absolute quantitative qPCR a) Take 0.2 mL fluorescent quantitative PCR 8-tube strips and cap, prepare the following reaction system, 3 tubes for each virus: 2 × qPCR Mix 5 μL, forward and reverse primers 0.5 μL each, 4 μL virus dilution sample.

[0041] b) Amplification primers: REP-F (SED ID NO: 15) REP-R (SED ID NO: 16) PCR amplification: Pre-denaturation: 95°C, 6 min; 40 × cycles: 95°C, 10 s; 60°C, 30 s.

[0042] c) Data processing: Virus titer = dilution multiple × viral genome copy number.

[0043] Table 1. Sequence information

[0044] Example 3: In vivo screening A novel adeno-associated virus that can efficiently infect RPE and outer nuclear layer cells was screened by intravitreal injection into both eyes of C57BL / 6J mice.

[0045] About 6E+9vg to 6E+10vg of library virus was injected into the eyes of C57BL / 6J mice (SPF grade 7-8 weeks male, purchased from Beijing Weitong Lihua Experimental Animal Breeding Co., Ltd.) through the vitreous cavity. After 21 days of injection, the mice were euthanized and the retina, RPE and outer nuclear layer cells were collected. At this time, the AAV in the library virus that can infect retinal cells enters the cells, and the AAV that fails to infect cells is cleared by the immune system and circulatory system. Therefore, the present invention can recover the capsid gene of the AAV that infects the cells from the genomic DNA of the tissue cells. The tissue genomic DNA is extracted using a DNA extraction kit (Biyuntian DP304), and the capsid gene of AAV is recovered from the genomic DNA using the PCR method to complete an in vivo screening of the AAV library. The fragment obtained by PCR is cloned into the library vector to obtain the next round of library plasmids, which are packaged into viruses again. The in vivo screening process can be repeated. After 3 to 5 rounds of screening, the capsid gene of the recovered AAV is subjected to third-generation sequencing. In this way, we obtained some information about AAV mutant capsid genes enriched in retinal cells, among which some mutant AAVs with high abundance efficiently transduced retinal cells. Among them, XM06, XM07, and XM08 were obtained as high-abundance mutants. The schematic diagram of their structure is shown in Figure 1, and the sequence is shown in Table 2.

[0046] Table 2. Sequence information

[0047] Example 4: Construction of novel adeno-associated viruses AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, AAV-XM08-CAG-EGFP and AAV2-CAG-EGFP AAV-XM06, AAV-XM07, AAV-XM08 obtained from the above sequencing results were co-transfected with pITR and adenovirus helper plasmid pHelper in appropriate amounts into HEK-293T cells (pITR and adenovirus helper plasmid pHelper were all derived from Tong, D. et al. Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques. Protein&Cell 14, 69–73, doi:10.1093 / procel / pwac020 (2022) ). The medium was replaced 24 hours after transfection, and the cells and medium supernatant were collected 96 hours later. The cells were lysed with 500 mM Tris (trishydroxymethylaminomethane) buffer (pH 8.0) to release AAV, and residual nucleic acids were treated with universal nuclease at a final concentration of 40 U / ml. After centrifugation, polyethylene glycol 8000 and 500 mM sodium chloride were added to the virus-containing supernatant at a final concentration of 8%, and the virus was precipitated at 4°C overnight. After centrifugation, the precipitate was resuspended in 2 mM MgCl2 solution. 2+ , 3% polysorbate 20 and 0.0001% poloxamer 188 in phosphate buffer. After centrifugation to remove impurities, the virus suspension was purified by ultracentrifugation using iodixanol density gradient solution (15%, 25%, 40% and 60%). The final products were AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, AAV-XM08-CAG-EGFP and AAV2-CAG-EGFP. The specific primers forward primer 1 (SEQ ID NO: 17) and reverse primer 1 (SEQ ID NO: 18) were used for titer determination and placed at -80°C for use. By analyzing the titers of AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, AAV-XM08-CAG-EGFP and AAV2-CAG-EGFP in the supernatant and cells, it was found that the content of the new adeno-associated viruses AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP and AAV-XM08-CAG-EGFP in the supernatant was higher than that of the parent AAV2, among which AAV-XM07-CAG-EGFP had significant differences. Figure 2As shown in the figure, the virus content in the cells is lower than that of the parent AAV2. The extracellular secretion of the new adeno-associated virus is significantly higher than that of the parent strain. If it is used in future commercial production, the virus in the supernatant can meet the production requirements, thereby avoiding the complex step of cell lysis and eliminating more process impurities caused by cell lysis. This will reduce the preparation process cost of AAV virus vectors and improve the quality of virus vectors.

[0048] Example 5: In vivo verification of novel adeno-associated viruses AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, and AAV-XM08-CAG-EGFP in the visual system AAV2 containing 6E+9vg and AAV-XM06 / XM07 / XM08-CAG-EGFP were injected into the eyes of C57BL / 6J mice (SPF grade 7-8 weeks male, purchased from Beijing Weitong Lihua Experimental Animal Breeding Co., Ltd.) through the vitreous cavity. After the virus was fully expressed for 21 days, the mice were killed by cervical dislocation, and the eyeballs were collected and fixed in 4% paraformaldehyde at 4°C for 12 hours, washed with PBS 4-5 times, and the excess muscle tissue on the scleral surface was removed. After the eyeballs were cut along the cornea-sclera margin, the anterior segment structures such as the cornea and lens were removed, and the retina-choroid-sclera eye cup structure was retained. It was placed in 30% sucrose for dehydration, and the completely dehydrated retina-choroid-sclera eye cup structure was frozen and sectioned at 10 μm. The sections were washed with PBS 5-6 times and then sealed with a DAPI-containing sealing medium, and then imaged by fluorescence microscopy. The results of in vivo verification are shown in the following figure. Figure 3 As shown. This figure shows a retinal section injected with AAV2-EGFP and XM06-EGFP, XM07-EGFP, and XM08-EGFP viruses. Green shows the fluorescent protein expressed by viral infection, and blue shows the cell nucleus. The results show that AAV2-EGFP has a high infection in the ganglion cell layer and the inner nuclear layer, and no infection in the RPE layer and the outer nuclear layer. XM06-EGFP, XM07-EGFP, and XM08-EGFP have a high infection in the retinal RPE layer, among which XM07-EGFP has a high infection in the outer nuclear layer, and XM07-EGFP and XM08-EGFP have a small amount of infection in the outer nuclear layer; the number of EGFP-positive photoreceptor cells was counted, and the results are as shown Figure 4 As shown, the infection efficiency of AAV-XM06-CAG-EGFP, AAV-XM07-CAG-EGFP, and AAV-XM08-CAG-EGFP viruses in photoreceptor cells is higher than 80%, indicating that XM06, XM07, and XM08 can be used for ophthalmic gene therapy.

[0049] Incorporated by Reference Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0050] Equivalence The present disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than by the above description, and all changes within the equivalent meaning and scope of the claims are intended to be covered therein.

Claims

1. A variant of an AAV2 capsid protein, comprising one or more modifications, wherein the modifications comprise inserting a peptide of 8 amino acids between amino acids 588 and 589 of a parent AAV2 capsid protein, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, or an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:

6.

2. The variant of claim 1, wherein the sequence of the parent AAV2 capsid protein comprises the amino acid sequence as described in SEQ ID NO:

1.

3. An isolated polynucleotide comprising a nucleic acid sequence encoding the AAV2 capsid protein variant according to claim 1 or 2.

4. The isolated polynucleotide of claim 3, comprising a nucleic acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 or having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO:

14.

5. A vector comprising the isolated polynucleotide according to claim 3 or 4.

6. A host cell comprising the isolated polynucleotide according to claim 3 or 4.

7. A recombinant AAV virus particle comprising the variant of claim 1 or 2 or the isolated polynucleotide of claim 3 or 4.

8. The recombinant AAV virus particle of claim 7, comprising a functional gene product of interest.

9. The recombinant AAV virus particle of claim 7 or 8, wherein the functional target gene product is a polypeptide.

10. A pharmaceutical composition comprising the recombinant AAV virus particle according to claim 9 and a pharmaceutically acceptable excipient.

11. A recombinant AAV vector comprising 1) a nucleic acid sequence encoding the AAV2 capsid protein variant as described in claim 1 or 2 or an isolated polynucleotide as described in claim 3 or 4; 2) 5' and 3' inverted terminal repeat sequences (5' ITR and 3' ITR); 3) a nucleic acid sequence encoding a functional gene product; and 4) a regulatory sequence that directs the expression of the gene product in a target cell.

12. Use of a variant of the AAV2 capsid protein as claimed in claim 1 or 4, an isolated polynucleotide as claimed in claim 3 or 4, a vector as claimed in claim 5, a host cell as claimed in claim 6, a recombinant AAV virus particle as claimed in any one of claims 7 to 9, a pharmaceutical composition as claimed in claim 10, or a recombinant AAV vector as claimed in claim 11 in the preparation of a medicament for administering an effective amount of a disease to a subject in need thereof.

13. The use according to claim 12, wherein the disease is selected from one or more of hearing impairment diseases, ophthalmic diseases, inflammation, tumors, metabolic diseases, pain, and neurodegenerative inflammatory diseases.

14. The use as claimed in claim 13, wherein the ophthalmic disease refers to retinal damage diseases in adult individuals, and preferably, the ophthalmic disease is selected from RPE layer and outer nuclear layer related diseases, photoreceptor cell related diseases, cell damage related diseases, related diseases caused by gene defects, related diseases caused by acquired factors and related diseases caused by aging.

15. purposes as claimed in claim 13, described ophthalmic disease is RPE and outer nuclear layer related disease, such as dry AMD, wet AMD or choroidal neovascularization (CNV); Such as can be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), cystoid macular edema (CME), epiretinal membrane (ERM) and macular hole; Choroidal neovascularization, angioid streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), atrophic lesions of retinal pigment epithelial cells (RPE), retinal pigment epithelial cells (RPE) related to myopia hypertrophic lesions of the retinal plexus, retinal vein occlusion, chorioretinal vein occlusion, macular edema; corneal angiogenesis, pterygium conjunctiva, subretinal edema and intraretinal edema caused by hypoxia; or macular edema caused by retinal vein occlusion, retinitis pigmentosa, Stargardt's disease, glaucoma, inflammatory diseases, cataracts, refractory abnormalities, keratoconus, retinopathy of prematurity, angiogenesis of the anterior part of the eye, corneal angiogenesis after keratitis, corneal transplantation or corneal formation; congenital amaurosis (LCA), pigmentary retinal degeneration, Stargardt disease caused by gene mutations related to RPE and outer nuclear layer cells.

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