Fusion type adeno-associated virus and application thereof

Abstract

The invention provides a fusion type adeno-associated virus AAV capsid protein, the fusion type adeno-associated virus comprises capsid protein peptide fragments of serotypes AAV1, AAV2, AAV6, AAV7 and AAV8, and further relates to nucleic acid for encoding the fusion type adeno-associated virus AAV capsid protein, a host cell, a carrier, AAV virus particles or a pharmaceutical composition, and application of the fusion type adeno-associated virus AAV capsid protein to preparation of a medicine for applying an effective amount of diseases to a subject in need of the fusion type adeno-associated virus AAV capsid protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a fusion adeno-associated virus and its application. Background Art

[0002] In 2012, the first AAV drug, Glybera, was approved in Europe. In the following decade, many AAV drugs were successfully launched. The launch of these drugs clearly demonstrated the safety and effectiveness of AAV-based treatment methods. Today, AAV vectors have achieved good results in clinical trials of in vivo gene therapy. The approval of multiple marketed drugs and ongoing clinical trials have proved that AAV vectors have become one of the main gene delivery tools for gene therapy. With the progress of research on the pathogenesis of monogenic hereditary neurological diseases, targets suitable for gene therapy have also emerged. The means of achieving disease treatment through AAV is generally to replace genes that have lost their functions or to silence genes with abnormally enhanced functions.

[0003] In in vivo gene therapy, genes are transferred in situ into organs, tissues and cells of a recipient organism, such as the nervous system, ear and eye. In vivo gene therapy by in situ introduction of the transgene into the eye has been used to treat ocular diseases. Examples of such diseases include retinitis pigmentosa, color blindness, age-related macular degeneration (AMD), Leber congenital amaurosis, Lieber hereditary optic neuropathy, early-onset retinal dystrophy and glaucoma.

[0004] Studies have shown that the tissue tropism and cell transformation efficiency of AAV vectors can be improved by changing the structure of AAV capsid protein. The modification of some structural proteins of the virus to change its performance has been carried out in this field, but how to make it produce significant benign effects is still a bottleneck in this field. It is known that AAV1, 2, 4, 5, 7, 8, and 9 serotype capsids can transduce retinal pigment epithelial cells or photoreceptor cells by subretinal cavity administration, while the serotype transduction efficiency of the above technologies is greatly reduced by vitreous cavity administration. The existing AAV2-based modified capsid serotype AAV2.7m8 can significantly improve the penetration of AAV2 capsid into the inner limiting membrane, but cannot effectively transduce the retinal pigment epithelial cell layer. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a new adeno-associated virus vector that can efficiently and specifically deliver genes in the retinal tissue of the eye for gene therapy. The present invention modifies the AAV capsid protein by DNA shuffling to obtain a new adeno-associated virus vector, which is injected into the eyeball of mice through the vitreous body. It is found that the virus vector has better infection characteristics than the existing vectors, and also has broad application value and market prospects in the analysis of the structure and function of ophthalmic cells, the establishment of disease models and gene therapy.

[0006] On the one hand, the present disclosure provides a fusion adeno-associated virus AAV capsid protein, characterized in that the capsid protein comprises a fusion peptide segment or one or more of the peptide segments of serotypes AAV1, AAV2, AAV6, AAV7 and AAV8 or a variant thereof.

[0007] In a specific embodiment, the fusion peptide segment comprises a first peptide segment, a second peptide segment, a third peptide segment, a fourth peptide segment and a fifth peptide segment connected in sequence; the first peptide segment comprises a peptide segment from AAV1, the second peptide segment comprises a peptide segment from AAV2 or AAV7, the third peptide segment comprises a peptide segment from AAV1, AAV2 or AAV8, the fourth peptide segment is from a peptide segment containing AAV1, and the fifth peptide segment is from a peptide segment containing AAV6.

[0008] In a specific embodiment, the first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 10 or SEQ ID No: 11, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 12, SEQ ID No: 13 or SEQ ID No: 14, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16; Preferably, The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 11, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 13, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16; or The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 10, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 12, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16; or The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 11, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 14, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16.

[0009] In a specific embodiment, the capsid protein includes: a) an amino acid sequence as shown in SEQ ID No: 17, SEQ ID No: 18 or SEQ ID No: 19; b) a polypeptide fragment having a sequence identity of more than 90% with SEQ ID No: 17, SEQ ID No: 18 or SEQ ID No: 19, and having the function of the amino acid sequence shown in SEQ ID No: 17, SEQ ID No: 18 or SEQ ID No: 19.

[0010] The present disclosure also provides a nucleic acid encoding the fusion-type adeno-associated virus AAV capsid protein as described above.

[0011] In one embodiment, the nucleic acid is selected from SEQ ID NOs: 28-30 or a nucleic acid sequence having greater than 75% identity with SEQ ID NOs: 28-30.

[0012] In another aspect, the present disclosure provides a vector comprising the nucleic acid as described above.

[0013] The present disclosure provides a host cell, wherein the host cell comprises the fusion-type adeno-associated virus AAV capsid protein, nucleic acid or vector as described above.

[0014] The present disclosure provides a fusion adeno-associated virus, characterized in that the capsid structure of the fusion adeno-associated virus contains the fusion adeno-associated virus AAV capsid protein as described above.

[0015] In one embodiment, the fusion adeno-associated virus further includes a heterologous nucleotide sequence encoding a target product; preferably, the target product is a nucleic acid or a protein; further, the nucleic acid includes but is not limited to a small guide RNA and an interfering RNA.

[0016] The present disclosure also provides a pharmaceutical composition, which comprises the fusion-type adeno-associated virus as described above and a pharmaceutically acceptable excipient.

[0017] The present disclosure also provides a recombinant AAV vector, which comprises 1) a nucleic acid or vector encoding as described above; 2) AAV 5' and 3' inverted terminal repeats (ITRs) (5' ITR and 3' ITR), 3) an engineered nucleic acid sequence encoding a functional gene product, and 4) a regulatory sequence directing the expression of the gene product in a target cell.

[0018] The present disclosure also provides the use of the fusion adeno-associated virus AAV capsid protein, nucleic acid, vector, host cell, fusion adeno-associated virus, pharmaceutical composition or recombinant AAV vector as described above in the preparation of a drug for administering an effective amount of a disease treatment drug to a subject in need thereof.

[0019] 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.

[0020] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present disclosure can be more fully understood with reference to the following drawings.

[0022] Figure 1 shows the structural schematic diagrams and vector information of AAV-XM03, AAV-XM04 and AAV-XM05. Figure 1A shows the structural schematic diagram of AAV-XM03, Figure 1B shows the vector information of AAV-XM04; Figure 1C shows the vector information of AAV-XM05.

[0023] Figure 2 The expression distribution of AAV-XM03, AAV-XM04, and AAV-XM05 in the mouse retina is shown. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In the pharmaceutical composition provided by the present invention, the fusion adeno-associated virus can be a single active ingredient, or it can be combined with one or more other active components useful for the treatment of hearing loss, ophthalmic diseases, inflammation, tumors, metabolic diseases, pain, and neurodegenerative inflammatory diseases to form a combined preparation. The other active components can be other various drugs that can be used to treat hearing loss, ophthalmic diseases, inflammation, tumors, metabolic diseases, pain, and neurodegenerative inflammatory diseases. The content of the active ingredient in the composition is generally a safe and effective amount, which should be adjustable by a person skilled in the art. For example, the dosage of the active ingredient of the fusion adeno-associated virus and the pharmaceutical composition generally depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound as an active ingredient generally can be 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0028] Host cell As representative examples of suitable host cells, mammalian cells (such as CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (such as yeast), insect cells (such as Sf9), etc. can be cited. Those skilled in the art can select a suitable host according to the teachings of this article. Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell can be a cultured cell or a primary cell, that is, directly isolated from an organism (such as a human). The host cell can be an adherent cell or a suspended cell, that is, a cell grown in the form of a suspension.

[0029] 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.

[0030] In certain embodiments, the fusion adeno-associated virus AAV capsid protein, nucleic acid, vector, host cell, fusion adeno-associated virus, pharmaceutical composition or recombinant AAV vector is used in an animal model or a cell model.

[0031] 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.

[0032] 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.

[0033] Example 1: Acquisition of novel adeno-associated disease antigens AAV-XM03, AAV-XM04 and AAV-XM05 (1) Construction of DNA family shuffling library The capsid sequences of 13 AAV serotypes from AAV1 to 13 were used as parents for DNA family shuffling. The parental capsid sequences were mixed in an equimolar ratio to a total of 4 μg for DNA family shuffling and treated with 0.04 U of DNase I at 25°C for 30 s to randomly break the complete parental capsid sequence into fragments of different lengths. DNA fragments of 100-500 bp in size were recovered and purified, and 500 ng was taken for primer-free PCR to extend them into complete chimeric capsid sequences. After amplifying enough chimeric capsid sequences, the chimeric capsid sequences were recombined into the AAV2 Rep ( 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) ) gene and ITR library vector. The recombinant product was 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 of culture medium and cultured at 37°C overnight. The plasmid extracted the next day was the DNA family shuffling library plasmid.

[0034] (2) Packaging of DNA Shuffling Library Viruses The obtained DNA shuffle 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 only 30 μg of DNA family shuffling library plasmid.

[0035] The culture medium was collected 24 hours after transfection, and the cells and 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 (SEQ ID NO: 1) and WPRE-R (SEQ ID NO: 2).

[0036] (3) In vivo screening By injecting into the vitreous cavity of mice eyes, new adeno-associated viruses that can efficiently infect the retina and retinal pigment epithelial cells were screened.

[0037] About 1E+10~11vg 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 3~5 rounds of screening, the capsid gene of the recovered AAV is subjected to third-generation sequencing. In this way, we obtained some information about the capsid genes of AAV mutants that are enriched in retinal cells, among which some mutant AAVs with high abundance can theoretically efficiently transduce retinal cells. Among them, AAV-XM03, AAV-XM04 and AAV-XM05 high-abundance mutants were obtained. The schematic diagram of their structure is shown in Figure 1, and the sequence is shown in Table 2.

[0038] The sequencing results showed that the capsid protein of AAV-XM03 was composed of peptides from AAV1, AAV6, and AAV7. The first peptide contained a peptide from AAV1, the second peptide contained a peptide from AAV7, the third peptide contained a peptide from AAV1, the fourth peptide contained a peptide from AAV1, and the fifth peptide contained a peptide from AAV6. The capsid protein of AAV-XM04 was composed of peptides from AAV1, AAV6, and AAV7. 2. AAV6 peptide segment is chimeric, and its first peptide segment contains a peptide segment from AAV1, the second peptide segment contains a peptide segment from AAV2, the third peptide segment contains a peptide segment from AAV2, the fourth peptide segment contains a peptide segment from AAV1, and the fifth peptide segment contains a peptide segment from AAV6; AAV-XM05 capsid protein is chimeric by peptide segments of AAV1, AAV6, AAV7, and AAV8, and its first peptide segment contains a peptide segment from AAV1, the second peptide segment contains a peptide segment from AAV7, the third peptide segment contains a peptide segment from AAV8, the fourth peptide segment contains a peptide segment from AAV1, and the fifth peptide segment contains a peptide segment from AAV6; and contains an S430I mutation.

[0039] Example 2: Construction of novel adeno-associated viruses AAV-XM03-CAG-EGFP, AAV-XM04-CAG-EGFP, AAV-XM05-CAG-EGFP The AAV-XM03, AAV-XM04, AAV-XM05 obtained from the above sequencing results were compared with pITR ( 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) ), adenovirus helper plasmid pHelper was co-transfected into HEK-293T cells in appropriate amounts. The culture medium was collected 24 hours after transfection, and the cells and culture medium were collected 96 hours later. The cells were lysed with 500 mMTris (Tris) 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 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 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 solution (15%, 25%, 40% and 60%). The final products were AAV-XM03-CAG-EGFP, AAV-XM04-CAG-EGFP and AAV-XM05-CAG-EGFP, and the titer was determined using specific primers forward primer 1 (SEQ ID NO: 3) and reverse primer 1 (SEQ ID NO: 4), and the suspension was placed at -80°C for use.

[0040] Table 1. Primer sequence information

[0041] Table 2. CAP capsid sequence information

[0042] Example 3: Transduction efficiency of adeno-associated virus AAV-XM03-CAG-EGFP / AAV-XM04-CAG-EGFP / AAV-XM05-CAG-EGFP in retinal tissue in vivo Approximately 6E+9 vg / eye of AAV-XM03-CAG-EGFP / AAV-XM04-CAG-EGFP / AAV-XM05-CAG-EGFP were injected into the aqueous humor of 7-week-old C57BL / 6J mice through the vitreous body to screen new adeno-associated viruses that can efficiently infect RPE and outer nuclear layer cells. After the virus was fully infected and expressed EGFP for 21 days, the mice were killed by cervical dislocation, the eyeballs were collected and fixed in 4% paraformaldehyde at 4°C for 12 hours, washed with PBS 4-5 times, the excess muscle tissue on the scleral surface was removed, the eyeballs were cut along the corneal-scleral margin, the anterior segment structures such as the cornea and lens were removed, the retina-choroid-sclera eye cup structure was retained, and it was placed in 30% sucrose for dehydration. The completely dehydrated retina-choroid-sclera complex was placed in the embedding agent for 5 to 10 minutes, allowing OCT to infiltrate the retina-choroid-sclera eye cup cavity, and placed on the repaired sample holder for embedding, and frozen sections were performed after complete quick freezing. It was imaged by laser confocal scanning microscopy, and the virus-infected cells expressed green fluorescent protein in their cell nuclei. Figure 2 This is a section of the mouse eyeball after intravitreal injection of AAV-XM03-CAG-EGFP / AAV-XM04-CAG-EGFP / AAV-XM05-CAG-EGFP viruses. AAV-XM03-CAG-EGFP / AAV-XM04-CAG-EGFP can efficiently infect the retinal RPE layer and photoreceptor cell layer, and AAV-XM05-CAG-EGFP can efficiently infect the retinal RPE layer, indicating that XM03, XM04, and XM05 can be used for ophthalmic gene therapy.

[0043] Incorporated by Reference Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0044] 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 fusion adeno-associated virus AAV capsid protein, characterized in that: The capsid protein comprises a fusion peptide segment of one or more of the peptide segments of serotypes AAV1, AAV2, AAV6, AAV7 and AAV8 or a variant thereof.

2. The fusion adeno-associated virus AAV capsid protein according to claim 1, characterized in that The fusion peptide segment comprises a first peptide segment, a second peptide segment, a third peptide segment, a fourth peptide segment and a fifth peptide segment connected in sequence; the first peptide segment comprises a peptide segment from AAV1, the second peptide segment comprises a peptide segment from AAV2 or AAV7, the third peptide segment comprises a peptide segment from AAV1, AAV2 or AAV8, the fourth peptide segment is from a peptide segment containing AAV1, and the fifth peptide segment is from a peptide segment containing AAV6.

3. The fusion adeno-associated virus AAV capsid protein according to claim 1, characterized in that The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 10 or SEQ ID No: 11, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 12, SEQ ID No: 13 or SEQ ID No: 14, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16; Preferably, The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 11, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 13, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16; or The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 10, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 12, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No: 16; or The first peptide segment comprises the amino acid fragment shown in SEQ ID No: 9, the second peptide segment comprises the amino acid fragment shown in SEQ ID No: 11, the third peptide segment comprises the amino acid fragment shown in SEQ ID No: 14, the fourth peptide segment comprises the amino acid fragment shown in SEQ ID No: 15, and the fifth peptide segment comprises the amino acid fragment shown in SEQ ID No:

16.

4. The fusion adeno-associated virus AAV capsid protein according to claim 1, characterized in that The capsid protein comprises: a) the amino acid sequence shown in SEQ ID No: 17, SEQ ID No: 18 or SEQ ID No: 19; b) a polypeptide fragment having a sequence identity of more than 90% with SEQ ID No: 17, SEQ ID No: 18 or SEQ ID No: 19, and having the function of the amino acid sequence shown in SEQ ID No: 17, SEQ ID No: 18 or SEQ ID No:

19.

5. A nucleic acid, characterized in that The nucleic acid encodes the fusion adeno-associated virus AAV capsid protein according to any one of claims 1 to 4. A vector comprising the nucleic acid according to claim 5 .

7. A host cell comprising the fusion-type adeno-associated virus AAV capsid protein according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the vector according to claim 6.

8. A fusion adeno-associated virus, characterized in that: The capsid structure of the fusion adeno-associated virus contains the fusion adeno-associated virus AAV capsid protein according to any one of claims 1 to 4.

9. The fusion adeno-associated virus according to claim 8, characterized in that The fusion adeno-associated virus also includes a heterologous nucleotide sequence encoding a target product; preferably, the target product is a nucleic acid or a protein; further, the nucleic acid includes but is not limited to a small guide RNA and an interfering RNA. 10 . A pharmaceutical composition comprising the fusion-type adeno-associated virus according to claim 8 or 9 and a pharmaceutically acceptable excipient.

11. A recombinant AAV vector comprising 1) a nucleic acid as described in claim 5, or a vector as described in claim 6; 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 the fusion adeno-associated virus AAV capsid protein as described in any one of claims 1 to 4, the nucleic acid as described in claim 5, the vector as described in claim 6, the host cell as described in claim 7, the fusion adeno-associated virus as described in claim 8 or 9, the pharmaceutical composition as described in claim 10 or the recombinant AAV vector as described in claim 11 in the preparation of a drug for administering an effective amount of a disease treatment 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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