Gene therapy vector for ameliorating eye disease and use thereof

Abstract

The present invention relates to the technical field of biomedicine, and relates to a gene therapy vector for ameliorating an eye disease and a use thereof. Specifically, targeted optimization design and screening were carried out on an hRs1 gene coding sequence and a promoter sequence thereof, an rAAV viral vector (rAAV2-hSyn-Kozak-hRS1-bGHpA) expressing the normal human RS1 protein was constructed, and the effectiveness thereof was proven in an Rs1-KO mouse model. Experimental results show that the hRs1 gene, driven by the hSyn promoter, can be highly expressed in a mammal (e.g., human) retinal ganglion cell (RGC), and X-linked retinoschisis can be effectively treated.

Description

Gene therapy vector for improving eye diseases and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on January 26, 2024, with application number 202410117677.7 and invention name “Gene therapy vector for improving eye diseases and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a gene therapy vector for improving eye diseases and applications thereof. Background Art

[0003] X-linked retinoschisis (XLRS) is an X-linked recessive genetic disorder caused by mutations in the retinoschisin 1 (Rs1) gene or loss of protein function. The cause is not fully understood. With an incidence of up to 1 in 5,000, XLRS is the most common monogenic macular dystrophy in males, with approximately hundreds of thousands of patients in China. XLRS patients typically experience progressive visual impairment during school age, and in later stages of the disease can develop vitreous hemorrhage, retinal detachment, and, in severe cases, blindness. Currently, there is no effective treatment for XLRS. Conventional treatments primarily consist of follow-up observation, conservative treatment, and surgical treatment of retinal detachment complications. However, these methods have very limited effects on improving vision and are not ideal in terms of therapeutic effect.

[0004] Adeno-associated virus (AAV) vectors are one of the most promising gene therapy vectors currently available. They offer advantages such as good safety, low immunogenicity, and stable expression. They are widely used in ophthalmology research to treat inherited retinal diseases (IRDs). Gene therapy for monogenic diseases using AAV vectors has begun to meet expectations, with successful treatments for a variety of genetic defect diseases over the past few years, and therapeutic drugs have been released to the market. Recently, AAV-mediated gene therapy has been shown to be safe and effective in Leber congenital amaurosis type 2, restoring partial vision in patients with mutations in the retinal pigment epithelium (RPE) 65 gene. Currently, eight AAV-related gene therapy drugs have been approved for marketing, including Glybera (for the treatment of lipoprotein lipase deficiency), Luxturna (for the treatment of hereditary optic neuritis), Zolgensma (for the treatment of spinal muscular atrophy), Upstaza (for the treatment of aromatic L-amino acid decarboxylase deficiency), Roctavian (for the treatment of hemophilia A), Hemgenix (for the treatment of hemophilia B), Elevidys (for the treatment of Duchenne muscular dystrophy), and BEQVEZ (for the treatment of moderate to severe hemophilia B). Therefore, AAV-based gene therapy is also a very promising treatment option for XLRS.

[0005] Although there is currently no effective treatment for XLRS, the occurrence of XLRS has a clear genetic mutation basis, so this field is in urgent need of developing gene therapy methods and therapeutic drugs that can effectively treat XLRS. In the prior art, most gene therapies for XLRS target photoreceptors and bipolar cells, but there are mutant Mut-RS1 protein monomers in the retinal cells of XLRS patients. WT and Mut co-assemble to form heterologous RS1 octamers that are secreted outside the cells, making it impossible for RS1 protein to perform its normal function, resulting in poor clinical effects. Studies have shown that retinal ganglion cells (RGC) are the only output neurons that transmit visual information from the retina to the brain. During the development and progression of XLRS disease, photoreceptor cells and bipolar cells are severely affected, while RGC has not been found to have obvious pathological changes in the development and progression of XLRS disease. Targeting RGC can achieve long-term stable expression. In addition, due to the severe retinal splitting and severe retinal pathological damage in XLRS patients, intravitreal injection (IVT) is a more suitable intraocular drug administration method for XLRS patients with minimal retinal damage. The retinal ganglion cell layer (GCL) is the cell layer closest to the vitreous cavity in the retina and is the cell layer most directly infected by IVT administration. Therefore, RGC is the most suitable target cell for IVT. Therefore, in the development of rAAV-based gene therapy drugs, it is particularly necessary to use a specific promoter that can target RGC cells to drive the expression of the target gene, thereby increasing the specific high-level expression of the target gene in RGC cells and reducing the immune response. Therefore, there is an urgent need for clinically efficient and more effective gene therapy drugs that can improve X-linked retinoschisis.

[0006] Summary of the Invention

[0007] In view of this, the present invention provides a gene therapy vector for improving eye diseases and its application, which overcomes the technical defects of the prior art such as low hRs1 gene expression efficiency and poor therapeutic effect.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides an expression cassette comprising an hSyn promoter and an hRs1 gene.

[0010] In some specific embodiments of the present invention, the hRs1 gene has:

[0011] (I), the nucleotide sequence shown in SEQ ID No. 1; or

[0012] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0013] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0014] (IV) A nucleotide sequence having at least 95% sequence identity with the nucleotide sequence described in any one of (I) to (III); preferably a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence described in any one of (I) to (III); more preferably a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence described in any one of (I) to (III).

[0015] In some specific embodiments of the present invention, the hSyn promoter has:

[0016] (i) the nucleotide sequence shown in SEQ ID No. 3; or

[0017] (ii) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (i) but differs from the nucleotide sequence shown in (i) due to the degeneracy of the genetic code; or

[0018] (iii) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (i) or (ii), and having the same or similar functions as the nucleotide sequence shown in (i) or (ii); or

[0019] (iv) A nucleotide sequence having at least 95% sequence identity with the nucleotide sequence described in any one of (i) to (iii); preferably, a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence described in any one of (i) to (iii); more preferably, a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence described in any one of (i) to (iii).

[0020] In a second aspect, the present invention further provides use of the expression cassette in any of the following items:

[0021] (I), expressing hRS1 protein; or

[0022] (II) Application in constructing a recombinant vector expressing hRS1 protein.

[0023] In some specific embodiments of the present invention, the targets for expressing the hRS1 protein include but are not limited to retinal ganglion cells.

[0024] In a third aspect, the present invention also provides a recombinant vector, including but not limited to the expression cassette.

[0025] In some specific embodiments of the present invention, the recombinant vector further comprises a Kozak sequence and / or a polyadenylation sequence; the polyadenylation sequence includes but is not limited to SV40 polyA, bGH polyA, hGH polyA, rbGlob polyA; or the regulatory sequence includes but is not limited to a 5' enhancer sequence and / or a 3' enhancer sequence and / or a WPRE sequence.

[0026] In some specific embodiments of the present invention, the Kozak sequence has:

[0027] (I), the nucleotide sequence shown in SEQ ID No. 2; or

[0028] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0029] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0030] (IV) A nucleotide sequence having at least 95% sequence identity with the nucleotide sequence described in any one of (I) to (III); preferably a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence described in any one of (I) to (III); more preferably a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence described in any one of (I) to (III).

[0031] In some specific embodiments of the present invention, the polyadenylation sequence is bGHpolyA, which has:

[0032] (i) the nucleotide sequence shown in SEQ ID No. 4; or

[0033] (ii) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (i) but differs from the nucleotide sequence shown in (i) due to the degeneracy of the genetic code; or

[0034] (iii) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (i) or (ii), and having the same or similar functions as the nucleotide sequence shown in (i) or (ii); or

[0035] (iv) A nucleotide sequence having at least 95% sequence identity with the nucleotide sequence described in any one of (i) to (iii); preferably, a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence described in any one of (i) to (iii); more preferably, a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence described in any one of (i) to (iii).

[0036] In some specific embodiments of the present invention, the Kozak sequence is placed at the 5′ end of the hRs1 gene; preferably, there are no extra nucleotides between the Kozak sequence and the hRs1 gene.

[0037] In some specific embodiments of the present invention, the recombinant vector comprises the following elements in sequence: a 5' terminal inverted repeat sequence, the hSyn promoter, the Kozak sequence, the hRs1 gene, a bGH polyA signal sequence and a 3' terminal inverted repeat sequence.

[0038] In some specific embodiments of the present invention, the sources of the backbone vector of the recombinant vector include but are not limited to plants, animals, bacteria, fungi, phages or viruses;

[0039] The backbone vector includes, but is not limited to, one or more of an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, or a lentiviral vector;

[0040] Preferably, the backbone vector is an adeno-associated virus (AAV) vector;

[0041] More preferably, the serotype of the adeno-associated virus (AAV) vector includes but is not limited to: one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh.10, preferably AAV2 capsid.

[0042] In a fourth aspect, the present invention also provides a plasmid combination, comprising the recombinant vector and a helper plasmid for virus packaging.

[0043] In some specific embodiments of the present invention, the helper plasmid for virus packaging in the plasmid combination includes but is not limited to Helper plasmid or Rep-Cap2 plasmid.

[0044] In a fifth aspect, the present invention further provides a host:

[0045] (I), integrated with the expression cassette; or

[0046] (II), transfecting or transforming the recombinant vector; or

[0047] (III), transfecting or transforming the plasmid combination.

[0048] In some embodiments of the present invention, the host includes but is not limited to prokaryotes or animal cells;

[0049] The prokaryotes include but are not limited to Escherichia coli;

[0050] The animal cells include, but are not limited to, one or more of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9, or FD-CHOS.

[0051] In a sixth aspect, the present invention further provides a virus particle prepared by any of the following:

[0052] (i), the expression cassette; or

[0053] (ii), the recombinant vector; or

[0054] (iii), the plasmid combination; or

[0055] (iv) the host.

[0056] In a seventh aspect, the present invention further provides the use of any of the following in the preparation of a vaccine and / or drug for preventing, improving and / or treating X-linked retinoschisis;

[0057] (I), the expression cassette; or

[0058] (II), the recombinant vector; or

[0059] (III), the plasmid combination; or

[0060] (IV), the host; or

[0061] (V) the viral particles.

[0062] In some specific embodiments of the present invention, the targets of the vaccine and / or drug include but are not limited to retinal ganglion cells.

[0063] In some specific embodiments of the present invention, the amount of the virus particles is 1×10 9 GC / eye~1×10 13 GC / eye.

[0064] In an eighth aspect, the present invention further provides a vaccine comprising any of the following items and acceptable adjuvants or adjuvants:

[0065] (I), the expression cassette; or

[0066] (II), the recombinant vector; or

[0067] (III), the plasmid combination; or

[0068] (IV), the host; or

[0069] (V) the viral particles.

[0070] In a ninth aspect, the present invention further provides a drug comprising any of the following items and a pharmaceutically acceptable excipient or adjuvant:

[0071] (I), the expression cassette; or

[0072] (II), the recombinant vector; or

[0073] (III), the plasmid combination; or

[0074] (IV), the host; or

[0075] (V) the viral particles.

[0076] In some specific embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of a buffer, an osmotic pressure regulator, an antibacterial agent, or a preservative.

[0077] In some specific embodiments of the present invention, the dosage form of the drug includes but is not limited to injection or eye drops;

[0078] The injection includes injection solution or injection powder;

[0079] Preferably, the injection is one or more of a vitreous cavity injection, a subretinal space injection or a suprachoroidal space injection.

[0080] In a tenth aspect, the present invention also provides a drug combination comprising the drug and any other active ingredients.

[0081] In an eleventh aspect, the present invention further provides a method for preventing, ameliorating and / or treating X-linked retinoschisis, comprising administering any of the following:

[0082] (i) the vaccine; or

[0083] (ii), the drug; or

[0084] (iii) the drug combination.

[0085] In some embodiments of the present invention, the method of administration includes but is not limited to injection;

[0086] The injection includes one or more of intravitreal injection, subretinal injection or suprachoroidal injection.

[0087] In some specific embodiments of the present invention, the amount of injection is 1×10 9 GC / eye~1×10 13 GC / eye.

[0088] In some specific embodiments of the present invention, the targets of the method include but are not limited to retinal ganglion cells.

[0089] The present invention uses the hSyn promoter to specifically target the retinal ganglion cell layer. Compared to photoreceptor / bipolar cells, ganglion cells undergo less apoptosis and have a more stable morphology during disease progression. Consequently, they are able to produce more functional homo-octamers of hRS1, achieving better therapeutic effects. This invention provides a theoretical basis for developing gene therapy for X-linked retinoschisis, expanding the range of clinical treatment options and possessing significant application and promotional value. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0091] Figure 1 shows the construction of a recombinant adeno-associated virus plasmid in an embodiment of the present invention; Figure A is a plasmid map of pAAV2-hSyn-Kozak-hRS1-bGHpA; Figure B is an electrophoresis diagram for plasmid enzyme digestion identification;

[0092] Figure 2 shows the expression of hRS1 protein in retinal cells verified by plasmid transfection of pAAV2-hSyn-Kozak-hRS1-bGHpA and pAAV2-CBA-hRS1-bGHpA plasmids in an embodiment of the present invention; wherein, A shows the expression of hRS1 in RGC and 661W cells verified by ELISA after transfection of the two plasmids; B shows the expression of hRS1 in RGC-5 and 661W cells verified by Western blot after infection of the two viruses;

[0093] Note: CBA-hRS1: pAAV2-CBA-hRS1-bGHpA group; hSyn-hRS1: pAAV2-hSyn-Kozak-hRS1-bGHpA group;

[0094] Figure 3 shows the results of cesium chloride density gradient centrifugation of adeno-associated virus and the detection of virus purity in an embodiment of the present invention; wherein A is cesium chloride density gradient centrifugation; B is silver staining to verify the virus capsid; C is agarose to verify the virus genome;

[0095] FIG4 shows the expression of rAAV2-hSyn-Kozak-hRS1-bGHpA virus in the eyes of Rs1-KO mice after intravitreal injection of the present invention; A is the absolute quantification result of the mouse eye genome extracted 2 weeks after injection by qPCR; B is the relative quantification result of the mouse eye mRNA extracted 2 weeks after injection by real-time PCR;

[0096] Note: rAAV2-hSyn-hRS1: rAAV2-hSyn-Kozak-hRS1-bGHpA group;

[0097] FIG5 shows that Western blot was used to verify that the expression of hRS1 protein can be detected in the mouse eyeball by rAAV2-hSyn-Kozak-hRS1-bGHpA virus in an embodiment of the present invention;

[0098] Note: rAAV2-hSyn-hRS1: rAAV2-hSyn-Kozak-hRS1-bGHpA group;

[0099] Figure 6 shows the expression of rAAV2-hSyn-Kozak-hRS1-bGHpA virus in the retina of the Rs1-KO mouse model 2 weeks after intravitreal injection of the present invention. The expression of hRS1 (red fluorescence) was mainly detected in the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL) and outer plexiform layer (OPL) of the retina of the Rs1-KO mouse;

[0100] Note: rAAV2-hSyn-hRS1: rAAV2-hSyn-Kozak-hRS1-bGHpA group;

[0101] Figure 7 shows the retinal structure evaluation of Rs1-KO mice 2 weeks after rAAV2-hSyn-Kozak-hRS1-bGHpA administration in an embodiment of the present invention. Compared with the drug-treated group, the inner and outer segments of photoreceptors in the Rs1-KO and Rs1-KO+NC groups were significantly shortened, and the retinoschisis cavity was significantly enlarged, indicating that rAAV2-hSyn-Kozak-hRS1-bGHpA virus administration delayed the progression of retinoschisis.

[0102] Note: rAAV2-hSyn-hRS1: rAAV2-hSyn-Kozak-hRS1-bGHpA group. DETAILED DESCRIPTION

[0103] The present invention discloses gene therapy vectors and their applications for improving ocular diseases. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0104] The present invention provides an adeno-associated virus vector specifically targeting retinal ganglion cells (RGC) and application thereof in preparing a drug for improving X-linked retinoschisis.

[0105] The present invention provides a recombinant adeno-associated viral vector for treating X-linked retinoschisis, wherein the recombinant adeno-associated viral vector comprises an expression cassette for tandem co-expression of an hSyn promoter and an hRs1 gene;

[0106] Preferably, the above-mentioned recombinant adeno-associated virus vector is adeno-associated virus type 2 or any mutant thereof.

[0107] Preferably, the above-mentioned recombinant adeno-associated virus type 2 contains the hRs1 gene, the nucleotide sequence of which is the sequence of SEQ ID: 1.

[0108] Preferably, the recombinant adeno-associated virus type 2 comprises a Kozak sequence, and its nucleotide sequence is the sequence of SEQ ID NO: 2.

[0109] Preferably, there are no redundant nucleotides between the Kozak sequence and the nucleotide sequence expressing the hRS1 protein.

[0110] Preferably, the above-mentioned recombinant adeno-associated virus type 2 comprises an hSyn promoter, the nucleotide sequence of which is the sequence of SEQ ID NO: 3.

[0111] Preferably, the recombinant adeno-associated virus type 2 comprises a polyadenylation sequence bGHpolyA, the nucleotide sequence of which is the sequence of SEQ ID NO: 4.

[0112] In some embodiments, the recombinant viral vector includes the following elements in sequence: a 5′ terminal inverted repeat sequence, a promoter sequence, a Kozak sequence, a sequence encoding the hRs1 gene, a bGHpolyA signal sequence, and a 3′ terminal inverted repeat sequence. The sources of the vector backbone described in the present invention include plants, animals, bacteria, fungi, phages, or viruses, but the present invention is not limited to this. The viral vectors include: adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors or lentivirus vectors, etc. The present invention uses an adeno-associated virus vector as a skeleton to express the target protein. In some specific embodiments, the backbone vector of the recombinant vector is pAAV2.

[0113] The present invention also provides a plasmid combination, which includes the recombinant vector as described above and a helper plasmid for virus packaging. In the present invention, the helper plasmid for virus packaging includes a Helper plasmid and a Rep-Cap2 plasmid.

[0114] The present invention also provides a host, the genome of which is integrated with the aforementioned nucleic acid; or is transfected or transformed with the aforementioned recombinant vector or the aforementioned plasmid combination.

[0115] The host described in the present invention is used for the preservation or amplification of the nucleic acid described in the present invention, and can also be used for the construction and expression of adeno-associated viruses, which is not limited by the present invention. In the present invention, the host is a prokaryotic organism or an animal cell. The prokaryotic organism includes but is not limited to Escherichia coli, and the animal cell is selected from at least one of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9 and FD-CHOS. In some specific embodiments of the present invention, HEK293T cells are used as the host for virus packaging.

[0116] The recombinant adeno-associated virus construction steps of the present invention include: constructing a pAAV2-hSyn-Kozak-hRS1-bGHpA recombinant plasmid; verifying the expression of the hRS1 target gene by in vitro cell plasmid transfection; and packaging the AAV2-hSyn-Kozak-hRS1-bGHpA virus.

[0117] More specifically, the construction method includes:

[0118] S1. Construction of recombinant plasmid pAAV2-hSyn-Kozak-hRS1-bGHpA, the steps are as follows:

[0119] (1) Gene synthesis hSyn promoter fragment, using BglⅡ and BsrG I restriction endonucleases to double digest, after digestion at 37 ℃ overnight, using gel recovery purification kit to recover; (2) Using BglⅡ and BsrG I restriction endonucleases to double digest vector: empty vector, after digestion at 37 ℃ overnight, using gel recovery purification kit to recover the vector; (3) The vector and insert fragment were connected by T4 at a molar ratio of 1:3, and the transformation plate was cultured at 37 ℃ overnight; (4) Single clone overnight shake bacteria were picked (with kanamycin added), plasmids were extracted using a plasmid miniprep kit, and plasmids were double digested with BsrG I and EcoRV restriction endonucleases for identification. After successful identification, the plasmids were sent to a third-party company for sequencing. If the sequence alignment was successful, it means that the plasmid construction was successful.

[0120] Cells were transfected with S2, pAAV2-hSyn-Kozak-hRS1-bGHpA, and pAAV2-CBA-hRS1-bGHpA plasmids. RGC cells and 661W cells were passaged into 10 cm dishes and transfected after stable cell growth. Transfection method: Label 1.5 mL EP tubes. In tube A, add 10 μg of the target plasmid, add 480 μL of Opti-MEM, mix well, and incubate at room temperature for 5 minutes. In tube B, add 20 μg of PEI, add 480 μL of Opti-MEM, mix well. The liquid in tube A was added to tube B, mixed well, and incubated at room temperature for 15 minutes. After 15 minutes, gently mix the liquid and add it to a 10 cm dish. Incubate in a 37°C incubator for 2-3 days, then harvest the cells for ELISA and Western blot analysis. AAV2-hSyn-Kozak-hRS1-bGHpA and AAV2-CBA-hRS1-bGHpA viruses expressed hRS1 protein in vitro.

[0121] S3. Virus packaging and purification.

[0122] It also includes verifying that the AAV2-hSyn-Kozak-hRS1-bGHpA virus expresses hRS1 protein; verifying the expression of AAV2-hSyn-Kozak-hRS1-bGHpA virus in the eyes of the Rs1-KO mouse animal model through in vivo experiments; and evaluating the retinal structure of Rs1-KO mice after administration of AAV2-hSyn-Kozak-hRS1-bGHpA virus.

[0123] The present invention also provides a drug for improving X-linked retinoschisis, comprising the recombinant adeno-associated virus of the present invention.

[0124] The drug described in the present invention is a novel gene therapy drug for X-linked retinoschisis based on AAV vector. The drug is carried by AAV2 vector with hRs1 gene expression cassette and contains hSyn promoter, which further improves the targeting and expression level of the target gene in retinal ganglion cells.

[0125] The drug of the present invention includes the recombinant adeno-associated virus as described above and also includes pharmaceutically acceptable excipients. In some embodiments, the total amount of the recombinant adeno-associated virus is 1×10 9 GC / eye-1×10 13 GC / eye. Pharmaceutically acceptable excipients include, but are not limited to, buffers, osmotic pressure regulators, antimicrobial agents, or preservatives. In some embodiments, the drug of the present invention is in the form of an injection, including an injection solution or an injection powder, which is not limited in the present invention. The injection is an intravitreal injection, a subretinal injection, or a suprachoroidal injection.

[0126] The present invention also provides a method for improving X-linked retinoschisis, which comprises administering the drug of the present invention. In the present invention, the administration method comprises injection, and the injection comprises intravitreal injection, subretinal injection or suprachoroidal injection. The injection amount is 1×10 9 GC / eye-1×10 13 GC / eye.

[0127] Advantages and beneficial effects of the present invention:

[0128] The present invention uses the hSyn promoter to specifically target the retinal ganglion cell layer. Compared to photoreceptor / bipolar cells, ganglion cells undergo less apoptosis and have a more stable morphology during disease progression. Consequently, they are able to produce more functional homo-octamer hRS1 protein, achieving better therapeutic effects. This invention provides a theoretical basis for developing gene therapy for X-linked retinoschisis, expanding the range of clinical treatment options and possessing significant application and promotional value.

[0129] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0130] In the present invention, the nucleotide sequence of the gene expressing the hRS1 protein is as follows:

[0131] The nucleotide sequence of the Kozak sequence is as follows:

[0132] The nucleotide sequence of the specific promoter hSyn that can target retinal ganglion cells is as follows:

[0133] The nucleotide sequence of the polyadenylation sequence bGHpolyA is as follows:

[0134] The present invention will be further described below in conjunction with the embodiments:

[0135] Example 1

[0136] The recombinant plasmid pAAV2-hSyn-Kozak-hRS1-bGHpA was constructed (the genome diagram is shown in Figure 1A) by the following steps:

[0137] (1) Gene synthesis hSyn-Kozak-hRS1 fragment was double-digested with BglⅡ and BsrG I restriction endonucleases. After digestion at 37℃ overnight, it was recovered using a gel recovery and purification kit.

[0138] (2) Use BglⅡ and BsrG I restriction endonucleases to double-digest the empty vector. After digestion at 37℃ overnight, use a gel recovery and purification kit to recover the vector.

[0139] (3) The vector and the synthetic fragment (hSyn-Kozak-hRS1 fragment) were ligated by T4 at a molar ratio of 1:3, and the transformation plate was cultured at 37°C overnight.

[0140] (4) Single clones were selected and shaken overnight (with kanamycin added). Plasmids were extracted using a plasmid extraction kit. The plasmids were double-digested with BsrG I and EcoR V restriction enzymes for identification. After successful identification, the plasmids were sent to a third-party company for sequencing. Successful sequence alignment indicates successful plasmid construction. Figure 1, B, shows the constructed plasmid map and enzyme digestion identification results.

[0141] Example 2

[0142] The cells were transfected with pAAV2-hSyn-Kozak-hRS1-bGHpA and pAAV2-CBA-hRS1-bGHpA plasmids. RGC-5 cells and 661W cells were transferred to 10 cm dishes and transfected after the cells grew stably.

[0143] Transfection method: Take 1.5mL EP tubes and mark them. Tube A: add 10μg of target plasmid, add 480μL Opti-MEM, mix well, and let it stand at room temperature for 5 minutes; Tube B: add 20μg PEI, add 480μL Opti-MEM, mix well; add the liquid in tube A to tube B, mix well, and let it stand at room temperature for 15 minutes. After 15 minutes, gently mix the liquid and add it to a 10cm dish. Place it in a 37℃ incubator and culture for 2-3 days. Collect the cells for Western blot analysis.

[0144] Example 3 pAAV2-hSyn-Kozak-hRS1-bGHpA and

[0145] Expression of hRS1 protein in retinal cells transfected with pAAV2-CBA-hRS1-bGHpA in vitro

[0146] (1) qPCR detection of hRs1 mRNA expression

[0147] In order to detect whether pAAV2-hSyn-Kozak-hRS1-bGHpA and pAAV2-CBA-hRS1-bGHpA were successfully transcribed after transfection in Example 2, RNA was extracted from the cells and subjected to real-time fluorescence quantitative PCR for quantitative statistics using the relative quantitative method.

[0148] Total RNA was extracted from cells using TRIzol reagent, and RNA concentration was measured using a Nano Photometer N50 Touch. Different RNAs were diluted to 200 ng / ul and reverse transcribed into cDNA using PrimeScript RT Master Mix. Fluorescence quantification was then performed. Relative quantification (2- △△Ct ) to calculate the relative expression values.

[0149] The primer sequences are as follows:

[0150] hRS1-F5: 5′-CTCCTTGGACTGTATACCAGAATG-3′ (forward), as shown in SEQ ID NO: 5;

[0151] hRS1-R5: 5′-ACATACTGCTCCGGGTTAGA-3′ (reverse), as shown in SEQ ID NO: 6;

[0152] GAPDH: 5′-TGACTTCAACAGCGACACCCA-3′ (forward), as shown in SEQ ID NO: 7;

[0153] GAPDH: 5′-CACCCTGTTGCTGTAGCCAAA-3′ (reverse), as shown in SEQ ID NO: 8;

[0154] As shown in Figure 2A, hRs1 mRNA expression was detected in both RGC-5 and 661W cells after transfection with the constructed pAAV2-CBA-hRS1-bGHpA plasmid. Transfection with the pAAV2-hSyn-Kozak-hRS1-bGHpA plasmid specifically expressed hRs1 mRNA only in RGC-5 cells, whereas there was almost no expression in 661W cells, demonstrating statistical significance.

[0155] (2) Western blot detection of hRS1 protein expression

[0156] Prepare a 10% SDS-PAGE gel and add approximately 400-500 ml of 1× electrophoresis buffer to the electrophoresis tank. Then, sequentially add samples (CBA-hRS1 histones obtained by transfection with the pAAV2-CBA-hRS1-bGHpA plasmid, hSyn-hRS1 histones obtained by transfection with the pAAV2-hSyn-Kozak-hRS1-bGHpA plasmid, and NC histones extracted from untreated cells) to the sample wells. Turn on the power supply and perform electrophoresis in constant voltage mode (90V for stacking gel, 110V for separating gel). Stop electrophoresis when the gel reaches the bottom. Activate the PVDF membrane by immersing it in methanol. Discard the stacking gel and immerse the membrane in transfer buffer in the order of blackboard, fiber pad, thick filter paper, PAGE gel, PVDF membrane, thick filter paper, fiber pad, and whiteboard. Perform wet transfer in constant current mode (200 mA, 120 min). After transfer, block the membrane with 5% skim milk powder. Wash with 1× TBST for 10 minutes on a horizontal shaker. The PVDF membrane was completely submerged in primary antibody (RS1 polyclonal antibody (1:2000, 24430-1-AP, Proteintech, China) at a 1:3000 dilution) and incubated on a horizontal shaker for 120 minutes at room temperature. The membrane was washed three times with 1× TBST for 10 minutes each to completely remove any residual primary antibody. Diluted secondary antibody (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1:10,000, SA00001-2, Proteintech, China) at a 1:5000 dilution) was then added and incubated at room temperature for 60 minutes. The membrane was washed five times with 1× TBST for 5 minutes each and then imaged in a pre-chilled chemiluminescence analyzer.

[0157] As shown in Figure 2B, Western blot analysis confirmed that the expression of the approximately 25 kDa hRS1 protein was detectable in both RGC-5 and 661W cells after transfection of the constructed pAAV2-CBA-hRS1-bGHpA plasmid. Transfection of the pAAV2-hSyn-Kozak-hRS1-bGHpA plasmid specifically expressed the hRS1 protein only in RGC-5 cells but not in 661W cells.

[0158] Example 4 Virus packaging and purification

[0159] Take the rAAV2-hSyn-Kozak-hRS1-bGHpA virus preparation process as an example:

[0160] (1) Add 500 μg of plasmid to the cell factory. When the cells grow to 70-80%, transfect the plasmid and prepare MIX:

[0161] A: Take a 50 ml centrifuge tube and add the following plasmids: Helper plasmid, Rep-Cap2 plasmid, and pAAV2-hSyn-Kozak-hRS1-bGHpA plasmid prepared in Example 1 in equal molar ratios.

[0162] B: The concentration ratio of PEI to plasmid (a mixture of Helper plasmid, Rep-Cap2 plasmid and pAAV2-hSyn-Kozak-hRS1-bGHpA plasmid) is 2:1.

[0163] 2. Vortex A and B to mix, add B dropwise to A, mix by inverting, and let stand at room temperature for 15 minutes to MIX.

[0164] 3. Take out the cell factory, turn the centrifuge tube upside down twice before adding MIX, add it dropwise into the cell factory, and place it in the incubator for culture.

[0165] 4. On the second day after transfection, discard the supernatant and replace with an equal amount of serum-free culture medium.

[0166] (2) Collect viruses

[0167] 1. Collect the virus on the third day after changing the medium.

[0168] 2. Shake the cell factory vigorously to make the cells float, add them to a 50ml centrifuge tube, centrifuge at 1500g, 4℃ for 10min, discard the supernatant and retain the cells at the bottom.

[0169] 3. Add appropriate amount of Lysis buffer to the centrifuge tube, mix the cells and vortex.

[0170] 4. Pour liquid nitrogen into a suitable container, quickly freeze the centrifuge tube in liquid nitrogen for 10 minutes, place it in 37°C until completely thawed, vortex for 2 minutes, and then place it in liquid nitrogen again. Repeat this process three times.

[0171] 5. Centrifuge at 1500g, 4°C for 30 min.

[0172] (3) PEG precipitation

[0173] 1. Take the supernatant and transfer it to a new 50ml centrifuge tube, add PEG8000, mix thoroughly by inversion, and precipitate at 4 degrees overnight.

[0174] 2. The next day, centrifuge at 1500g at 4°C for 30 minutes.

[0175] 3. Discard the supernatant.

[0176] 4. Dissolve the pellet in 2 ml of PBS.

[0177] (4) Cesium chloride density gradient centrifugation

[0178] After precipitation, the virus was concentrated by cesium chloride density gradient centrifugation, and finally the titer and purity of the virus were tested by qPCR, silver staining and genome identification. Figure 3 shows the results of two cesium chloride density gradient centrifugations and virus silver staining. As can be seen in Figure 3A, clear virus bands were obtained after 24 hours of cesium chloride density gradient centrifugation. The lower band 3 is the genome band within the complete virus, and the empty virus shell 1 does not contain the genome; the virus band was extracted and silver stained to detect the purity of the virus. As can be seen from Figure 3B, the three proteins are all viral capsid proteins. Figure 3C shows the genome identified by virus agarose electrophoresis, and the band position and size are correct. The above results show that low-impurity and high-purity viruses have been prepared.

[0179] Example 5 Expression of rAAV2-hSyn-Kozak-hRS1-bGHpA in the eyes of Rs1-KO mice

[0180] (1) qPCR detection of hRs1 DNA and hRs1 mRNA expression

[0181] In order to test whether the rAAV2-hSyn-Kozak-hRS1-bGHpA virus prepared in Example 4 can be successfully injected and transcribed into the eyeball of Rs1-KO mice, a single intravitreal injection of 3×10 10 Fourteen days after intravitreal injection of the virus, the genome and RNA of the mouse eyeballs were extracted and quantitatively analyzed by real-time fluorescence quantitative PCR using absolute and relative quantification methods.

[0182] Genomic DNA (gDNA) was extracted using the DNeasy Blood and Histology Kit and quantified by qPCR.

[0183] Total RNA was extracted from the eyeball using TRIzol reagent, and RNA concentration was measured using a Nano Photometer N50 Touch. Different RNAs were diluted to 200 ng / ul and reverse transcribed into cDNA using PrimeScript RT Master Mix. Fluorescence quantification was then performed. Relative quantification (2- △△Ct ) to calculate the relative expression values.

[0184] The primer sequences are as follows:

[0185] hRS1-F5: 5′-CTCCTTGGACTGTATACCAGAATG-3′ (forward), as shown in SEQ ID NO: 9;

[0186] hRS1-R5: 5′-ACATACTGCTCCGGGTTAGA-3′ (reverse), as shown in SEQ ID NO: 10;

[0187] GAPDH: 5′-TGACTTCAACAGCGACACCCA-3′ (forward), as shown in SEQ ID NO: 11;

[0188] GAPDH: 5′-CACCCTGTTGCTGTAGCCAAA-3′ (reverse), as shown in SEQ ID NO: 12.

[0189] The results are shown in Figure 4. Panel A shows the genome extracted from the eyes of Rs1-KO mice (Untreated), Rs1-KO mice (14 days after intravitreal injection of rAAV2-hSyn-Kozak-hRS1-bGHpA virus), and RAAV2-NC mice. Absolute quantification by qPCR shows that hRs1 DNA was significantly increased in the rAAV2-hSyn-hRS1 group compared to the Untreated and rAAV2-NC groups, with statistical significance. Panel B shows the RNA extracted from the eyes of Rs1-KO mice (Untreated), Rs1-hSyn-Kozak-hRS1-bGHpA virus, and rAAV2-NC mice (14 days after intravitreal injection of rAAV2-hSyn-Kozak-hRS1-bGHpA virus), with statistical significance by real-time PCR. Compared to the Untreated and rAAV2-NC groups, hRs1 mRNA was increased approximately 5000-fold in the rAAV2-hSyn-hRS1 group, with statistical significance.

[0190] (2) Western blot detection of hRS1 protein expression

[0191] In order to test whether the rAAV2-hSyn-Kozak-hRS1-bGHpA prepared in Example 4 can successfully express hRS1 protein in the eyes of Rs1-KO mice, a single intravitreal injection of 3×10 10 Fourteen days after intravitreal injection of rAAV2-hSyn-Kozak-hRS1-bGHpA and rAAV2-NC of GC, proteins were extracted from the mouse eyeballs for Western blot analysis.

[0192] Prepare a 10% SDS-PAGE gel and add approximately 400-500 ml of 1× electrophoresis buffer to the electrophoresis tank. Then, load the samples sequentially into the sample wells. Connect the power supply and run electrophoresis in constant voltage mode (90 V for stacking gel, 110 V for separating gel). Stop the run when the gel reaches the bottom. Activate the PVDF membrane by immersing it in methanol. Discard the stacking gel. Immerse the membrane in transfer buffer in the order of blackboard, fiber pad, thick filter paper, PAGE gel, PVDF membrane, thick filter paper, fiber pad, and whiteboard. Perform wet transfer in constant current mode (200 mA, 120 min). After transfer, block the membrane with 5% skim milk powder. Wash with 1× TBST on a horizontal shaker for 10 min. Completely submerge the PVDF membrane in primary antibody (1:3000 dilution), incubate on a horizontal shaker at room temperature for 120 min. Wash the membrane three times with 1× TBST (10 min each time) to completely remove any residual primary antibody. Then, add diluted secondary antibody (1:5000 dilution) and incubate at room temperature for 60 min. Wash the membrane five times with 1× TBST (5 min each time) and image in a pre-chilled chemiluminescence instrument.

[0193] As shown in FIG5 , Western blot confirmed that the rAAV2-hSyn-Kozak-hRS1-bGHpA virus could detect the expression of hRS1 in the mouse eyeball.

[0194] Example 6 Expression and Distribution of rAAV2-hSyn-Kozak-hRS1-bGHpA Virus in the Retina of the Rs1-KO Mouse Model

[0195] In order to test whether the rAAV2-hSyn-Kozak-hRS1-bGHpA virus prepared in Example 4 can successfully express hRS1 protein in the retina of Rs1-KO mice, each eye of the mice received a single intravitreal injection of 3×10 10GC rAAV2-hSyn-Kozak-hRS1-bGHpA and rAAV2-NC. Eyeballs were removed 14 days after intravitreal injection of viral vectors for immunofluorescence staining.

[0196] The mice were euthanized, and the intact eyeballs were removed. The eyeball tissues were fixed in 4% paraformaldehyde and embedded for tissue sectioning.

[0197] Preheat the oven to 70°C and bake the tissue sections in the oven for 1 hour and 30 minutes; dewax the paraffin sections in dewaxing solution and gradient concentrations of anhydrous ethanol until they are hydrated:

[0198] Antigen retrieval: Place the tissue sections in a microwave oven in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0). After cooling naturally, place the slides in PBS (pH 7.4) and wash three times for 5 minutes each on a decolorizing shaker.

[0199] Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw circles around the tissue (to prevent the antibody from flowing away), dry the PBS, add 5% BSA, and block for 30 minutes.

[0200] Primary antibody: Gently shake off the blocking solution and add the primary antibody (mouse polyclonal RS1 antibody (H00006247-B01P, Novus Biologicals, USA)) prepared in a certain ratio (1:200) in PBS to the sections. Place the sections flat in a humidified chamber and incubate at 4°C overnight.

[0201] Secondary antibody (CY3 fluorescent labeled goat anti-mouse IgG (GB21303, Servicebio, China) 1:2000): Slides were washed three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each. After the sections were briefly dried, fluorescent secondary antibody corresponding to the primary antibody was added dropwise to cover the tissue within the circle and incubated at room temperature for 50 minutes in the dark.

[0202] Sealing: After the sections are slightly dried, they are sealed with anti-fluorescence quenching mounting medium (DAPI).

[0203] Microscopic examination and photography: Sections were observed and images were collected under a fluorescence confocal microscope. (DAPI excitation wavelength: 330-380 nm, emission wavelength: 420 nm, blue light; CY3 excitation wavelength: 510-560 nm, emission wavelength: 590 nm, red light.

[0204] The results are shown in Figure 6. 2 weeks after intravitreal injection of rAAV2-hSyn-Kozak-hRS1-bGHpA virus, hRS1 (red fluorescence) expression can be detected in the retinal ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL) and outer plexiform layer (OPL) of Rs1-KO mice, but only trace amounts of hRS1 are expressed in the outer nuclear layer (ONL) and inner segments (IS) and outer segments (OS) of photoreceptors, indicating that rAAV2-hSyn-Kozak-hRS1-bGHpA can be specifically expressed in the retina of Rs1-KO mice.

[0205] Example 7 Evaluation of Retinal Structure in Rs1-KO Mice after Administration of rAAV2-hSyn-Kozak-hRS1-bGHpA

[0206] In order to evaluate the recovery of retinal structure in Rs1-KO mice after administration of rAAV2-hSyn-Kozak-hRS1-bGHpA prepared in Example 4, Rs1-KO mice received a single intravitreal injection of 3×10 10 GC rAAV2-hSyn-Kozak-hRS1-bGHpA virus and rAAV2-NC. Eyeballs were removed and HE staining was performed 14 days after intravitreal injection of the vector.

[0207] Remove the eyeball. Euthanize the mouse and remove the intact eyeball. Fix the eyeball tissue in FAS fixative at room temperature for at least 24 hours.

[0208] Dehydration: Dehydrate the fixed eyeball tissue using a gradient of alcohol. Then transfer the eyeball tissue to a xylene solution for transparency.

[0209] Embedding: Embed the eyeball tissue in paraffin and trim it after it forms a wax block sample.

[0210] Slice. Place the paraffin block in a paraffin microtome and slice serially along the sagittal plane of the eyeball, down to the optic nerve. Slices should be approximately 4 μm thick. Flatten the slices onto glass slides and bake in a 60°C oven.

[0211] Dewaxing: Place the sections in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, and 75% alcohol for 5 minutes. Wash with tap water.

[0212] Hematoxylin staining: Stain the sections in hematoxylin solution for 3-5 minutes, then wash with water, differentiate, wash with water, re-bluish, and rinse.

[0213] Eosin staining: Dehydrate the sections in graded alcohol for 5 minutes each, then stain the sections in eosin staining solution for 5 minutes.

[0214] Mounting: Wash the sections in three jars of anhydrous ethanol, then place them in two jars of xylene to make them transparent, and finally mount them with neutral gum.

[0215] Image acquisition: Observe the HE-stained sections using an optical microscope and scan and photograph them.

[0216] As shown in Figure 7 , compared with the rAAV2-hSyn-hRS1 group, the inner segments (IS) and outer segments (OS) of the photoreceptors in the Untreated group and rAAV2-NC were significantly shortened, and the retinoschisis cavity was significantly enlarged, indicating that the administration of rAAV2-hSyn-Kozak-hRS1-bGHpA virus delayed the progression of retinoschisis.

[0217] The above describes in detail the gene therapy vectors for ameliorating ocular diseases and their applications provided by the present invention. This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above examples are intended only to facilitate understanding of the methods and core concepts of the present invention. It should be noted that those skilled in the art will be able to make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications fall within the scope of protection of the claims of the present invention.

[0218] Sequence Listing

Claims

1. Expression cassette, characterized in that, It includes the hSyn promoter and the hRs1 gene.

2. The expression cassette according to claim 1, characterized in that, The hRs1 gene has: (I) a nucleotide sequence as shown in SEQ ID No.1; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) a nucleotide sequence having at least 95% sequence homology with the nucleotide sequence described in any one of (I) to (III); preferably a nucleotide sequence having at least 98% sequence homology with the nucleotide sequence described in any one of (I) to (III); more preferably a nucleotide sequence having at least 99% sequence homology with the nucleotide sequence described in any one of (I) to (III).

3. The expression cassette according to claim 1 or 2, characterized in that, The hSyn promoter has: (i) a nucleotide sequence as shown in SEQ ID No.3; or (ii) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (i), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (iii) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (i) or (ii), and having the same or similar function as the nucleotide sequence shown in (i) or (ii); or (iv) a nucleotide sequence having at least 95% sequence homology with the nucleotide sequence described in any one of (i) to (iii); preferably a nucleotide sequence having at least 98% sequence homology with the nucleotide sequence described in any one of (i) to (iii); more preferably a nucleotide sequence having at least 99% sequence homology with the nucleotide sequence described in any one of (i) to (iii).

4. The application of the expression cassette according to any one of claims 1 to 3 in any of the following; (I) expressing the hRS1 protein; or (II) the application in constructing a recombinant vector for expressing the hRS1 protein.

5. The application according to claim 4, wherein The targets for expressing the hRS1 protein include but are not limited to retinal ganglion cells.

6. Recombinant vector, characterized in that, It includes but is not limited to the expression cassette according to any one of claims 1 to 3.

7. The recombinant vector according to claim 6, wherein The recombinant vector further includes a Kozak sequence, a polyadenylation sequence and / or a regulatory sequence; the polyadenylation sequence includes but is not limited to SV40 polyA, bGH polyA, hGH polyA, rbGlob polyA; or the regulatory sequence includes but is not limited to a 5'-end enhancer sequence and / or a 3'-end enhancer sequence and / or a WPRE sequence.

8. The recombinant vector according to claim 7, characterized in that, The Kozak sequence has: (I) a nucleotide sequence as shown in SEQ ID No.2; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having at least 95% sequence homology with the nucleotide sequence described in any one of (I) to (III); preferably a nucleotide sequence having at least 98% sequence homology with the nucleotide sequence described in any one of (I) to (III); more preferably a nucleotide sequence having at least 99% sequence homology with the nucleotide sequence described in any one of (I) to (III).

9. The recombinant vector according to claim 7, wherein, The polyadenylation sequence is bGHpolyA, which has: (i) A nucleotide sequence as shown in SEQ ID No. 4; or (ii) A nucleotide sequence encoding the same protein as the nucleotide sequence shown in (i), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (iii) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (i) or (ii), and having the same or similar function as the nucleotide sequence shown in (i) or (ii); or (iv) A nucleotide sequence having at least 95% sequence homology with the nucleotide sequence described in any one of (i) to (iii); preferably a nucleotide sequence having at least 98% sequence homology with the nucleotide sequence described in any one of (i) to (iii); more preferably a nucleotide sequence having at least 99% sequence homology with the nucleotide sequence described in any one of (i) to (iii).

10. The recombinant vector according to any one of claims 7 to 9, characterized in that, The Kozak sequence is placed at the 5′ end of the hRs1 gene; preferably, there are no extra nucleotides between the Kozak sequence and the hRs1 gene.

11. The recombinant vector according to claim 10, characterized in that, The recombinant vector sequentially includes the following elements: a 5′-terminal inverted repeat sequence, the hSyn promoter, the Kozak sequence, the hRs1 gene, the bGHpolyA signal sequence, and a 3′-terminal inverted repeat sequence.

12. The recombinant vector according to any one of claims 6 to 11, characterized in that, The source of the backbone vector of the recombinant vector includes, but is not limited to, plants, animals, bacteria, fungi, phages or viruses; The backbone vector includes, but is not limited to, one or more of an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector or a lentivirus vector; Preferably, the backbone vector is an adeno-associated virus vector; More preferably, the serotypes of the adeno-associated virus vector include, but are not limited to, one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh.10, preferably the AAV2 capsid.

13. A plasmid combination, characterized in that, Comprising the recombinant vector as described in any one of claims 6 to 12 and an auxiliary plasmid for virus packaging.

14. The plasmid combination according to claim 13, wherein The auxiliary plasmid for virus packaging includes, but is not limited to, a Helper plasmid or a Rep-Cap2 plasmid.

15. A host, characterized in that (I) Integrates an expression cassette as described in any one of claims 1 to 3; or (II), transfecting or transforming the recombinant vector according to any one of claims 6 to 12; or (III), transfecting or transforming the plasmid combination according to claim 13 or 14.

16. The host according to claim 15, wherein, The host includes but is not limited to prokaryotes or animal cells; The prokaryotes include but are not limited to Escherichia coli; The animal cells include but are not limited to one or more of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9 or FD-CHOS.

17. Virus particles prepared from any of the following; (i), the expression cassette according to any one of claims 1 to 3; or (ii), the recombinant vector according to any one of claims 6 to 12; or (iii), the plasmid combination according to claim 13 or 14; or (iv), the host according to claim 15 or 16.

18. Use of any of the following in the preparation of a vaccine and / or drug for preventing, ameliorating and / or treating X-linked retinoschisis; (I), the expression cassette according to any one of claims 1 to 3; or (II), the recombinant vector according to any one of claims 6 to 12; or (III), the plasmid combination according to claim 13 or 14; or (IV), the host according to claim 15 or 16; or (V), the virus particles according to claim 17.

19. The application according to claim 18, characterized in that, The targets of the vaccine and / or drug include but are not limited to retinal ganglion cells.

20. The application according to claim 18 or 19, characterized in that The dosage of the virus particles is 1×10 9 GC / eye ~ 1×10 13 GC / eye.

21. A vaccine, characterized in that, Comprising any of the following and acceptable excipients or adjuvants: (I), the expression cassette according to any one of claims 1 to 3; or (II), the recombinant vector according to any one of claims 6 to 12; or (III), the plasmid combination according to claim 13 or 14; or (IV), the host according to claim 15 or 16; or (V), the virus particles according to claim 17.

22. A drug, characterized in that, Comprising any of the following and pharmaceutically acceptable excipients or adjuvants: (I), the expression cassette according to any one of claims 1 to 3; or (II), the recombinant vector according to any one of claims 6 to 12; or (III), the plasmid combination according to claim 13 or 14; or (IV), the host according to claim 15 or 16; or (V), the virus particles according to claim 17.

23. The drug according to claim 22, wherein, The pharmaceutically acceptable excipients include but are not limited to one or more of buffers, osmotic pressure regulators, antibacterial agents or preservatives.

24. The medicament according to claim 22 or 23, characterized in that, The dosage forms of the drug include but are not limited to injections or eye drops; The injections include injection solutions or lyophilized powder for injection; Preferably, the injection is one or more of intravitreal injection, subretinal injection or suprachoroidal injection.

25. A pharmaceutical combination, characterized in that, Comprising the drug according to any one of claims 22 to 24 and any other active ingredient.

26. A method for preventing, improving and / or treating X-linked retinoschisis, characterized in that, Administering any of the following: (i), the vaccine according to claim 21; or (ii) The drug according to any one of claims 22 to 24; or (iii) The drug combination according to claim 25.

27. The method according to claim 26, wherein The administration method includes but is not limited to injection; The injection includes one or more of intravitreal injection, subretinal injection, suprachoroidal injection, or periorbital venous injection.

28. The method according to claim 27, wherein The amount injected is 1×10 9 GC / eye ~ 1×10 13 GC / eye.

29. The method according to claim 27 or 28, characterized in that, The action target of the method includes but is not limited to retinal ganglion cells.

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