CN112301057B - Recombinant adeno-associated virus vector and preparation method and application thereof - Google Patents

Abstract

The invention relates to a recombinant adeno-associated virus vector, a preparation method and application thereof. Specifically, the invention carries out targeted special optimization design on the XCT/SLC7A11 gene coding sequence, thereby obtaining a nucleotide sequence which is particularly suitable for efficiently expressing the XCT/SLC7A11 protein in cells of mammals (such as human beings), and constructing a recombinant AAV for expressing the XCT/SLC7A11 protein. The recombinant AAV of the invention has excellent infection effect in mouse cells, is stably expressed in cell nuclei, is a stable recombinant adeno-associated virus vector, can obviously reduce the apoptosis of retina outer nuclear layer cells and increase the survival rate of mouse photoreceptor cells, and therefore, can be used for preparing medicaments for treating the apoptosis of the retina detachment photoreceptor cell damage, and has wide market prospect.

Description

Recombinant adeno-associated virus vector and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant adeno-associated virus vector and a preparation method and application thereof.

Background

Retinal Detachment (RD) is a common pathological feature of Retinal choroidal diseases such as limbal Retinal detachment, age-related macular degeneration, diabetic retinopathy and the like. Retinal detachment, i.e., the separation of the retinal neuroepithelial layer from the RPE and choroid complexes, is clinically manifested as anterior ocular floaters, glistening sensation, defective and obstructed peripheral visual field, and when the retinal detachment extends and involves macula lutea, the vision of the patient is rapidly reduced. Because RPE and choroid complex are the main sources of oxygen, blood and energy substances of the outer retinal nerve fiber layer/photoreceptor cell, and the intracellular oxidative metabolism of the photoreceptor is active to maintain the normal visual chemical signal-electric signal conversion to form the visual function, after the retinal detachment occurs, the supply of oxygen and energy substances of the photoreceptor cell is interrupted, so that a large number of photoreceptor cells are rapidly apoptotic, and the key pathological basis of the photoreceptor cells to cause visual impairment is provided.

The pathological mechanism of photoreceptor apoptosis in RD is complex, and has genetic and environmental factors, but the exact molecular mechanism is not clear. Research finds that oxidative stress injury is an important pathological mechanism for the occurrence and development of photoreceptor cell injury after retinal detachment, and in recent years, research suggests that xCT/slc7a11 regulates the synthesis of intracellular GSH so as to maintain the intracellular redox balance possibly is an important antioxidant protection signal path of photoreceptor cells. xCT/slc7a11 is Na ⁺ The independent glutamate/cystine transporter is positioned on a cell membrane of a photoreceptor cell, and the expression of xCT/slc7a11 is regulated and controlled by a complex signal molecule network, including transcriptional regulation of Nrf2, REST and the like, and epigenetic regulation of H3K27 and the like.

Research shows that under the oxidative stress environment, xCT/slc7a11 synthesis is increased, and the synthesis is transferred and positioned on a photoreceptor cell membrane, and the function of 1:1 for reversely transferring glutamic acid and cystine is exerted, namely 1 glutamic acid is transferred to the outside of cells, and 1 cystine is transferred to the inside of the cells. Cystine transferred into cells is subjected to a series of enzymatic reactions, and finally GSH is generated under the catalysis of glutathione synthetase, so that the strong antioxidation effect is exerted, and the intracellular redox balance is maintained. An xCT/slc7a11 signal channel is in a silent state in normal photoreceptor cells, most of photoreceptor cells after retinal detachment cannot effectively activate the xCT/slc7a11 channel and finally die, and researches prove that the activation of the xCT/slc7a11 channel can promote the survival of the photoreceptor cells after retinal detachment, and the xCT/slc7a11 channel is very critical to the survival of the photoreceptor cells after retinal detachment. Therefore, in various photoreceptor cell injury and apoptosis diseases, enhancing the xCT/slc7a11 signal path in photoreceptor cells may be an effective therapeutic measure for preventing degeneration of the photoreceptor cells and promoting the survival of the photoreceptor cells.

However, there are many limitations and disadvantages in the current research for treating eye diseases using xCT/slc7a11 signaling pathway, and there is an urgent need in the art to develop methods and therapeutic agents for effectively treating retinal photoreceptor cell injury diseases.

Disclosure of Invention

The invention aims to provide a recombinant adeno-associated virus vector, a preparation method thereof and application of the recombinant adeno-associated virus vector in treating retinal photoreceptor cell injury diseases.

Specifically, the first objective of the present invention is to provide a recombinant adeno-associated virus vector, which addresses the deficiencies of the prior art.

The second purpose of the invention is to provide a preparation method of the recombinant adeno-associated virus vector.

The third purpose of the invention is to provide a pharmaceutical composition.

The fourth purpose of the invention is to provide an application of the recombinant adeno-associated virus vector.

The fifth purpose of the invention is to provide a method for treating the apoptosis of the retinal detachment photoreceptor cells.

In order to realize the first purpose, the invention adopts the technical scheme that:

a recombinant adeno-associated virus vector comprises an enhancer, a specific or non-specific promoter and a gene sequence for expressing xCT/slc7a11 protein, wherein the sequence of the xCT/slc7a11 protein is shown as SEQ ID NO. 2.

Preferably, the gene sequence expressing the xCT/slc7a11 protein is selected from the group consisting of nucleotide sequences having at least 96%, 97%, 98%, 99%, 100% homology with SEQ ID NO 1.

More preferably, the gene sequence expressing xCT/slc7a11 protein is shown as SEQ ID NO. 1.

Preferably, the enhancer/promoter is CMV selected from nucleotide sequences having at least 95% homology with SEQ ID NO. 4.

More preferably, the enhancer/promoter is CMV and has the sequence shown in SEQ ID NO 4.

Preferably, the adeno-associated virus may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV2-AAV3, aavrh.10, aavhhu.14, AAV3a/3b, aavrh32.33, aavhsc15, AAV-HSC17, aavhu.37, aavrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, aavm41, aav9.45, aav6 (Y445F/Y F), aav2.5t, AAV-HAE1/2, aavrlone 32/83, aavshhh 10, aav2 (Y- > F), AAV8 (Y733F), AAV2.15, AAV2.4, aa41, vrh 3.45, aavs2 or AAV5.

More preferably, the adeno-associated virus is AAV2.

More preferably, the adeno-associated virus is single-stranded AAV2.

Preferably, the recombinant adeno-associated viral vector further comprises a shortened chimeric intron sequence selected from the group consisting of nucleotide sequences having at least 90% homology to SEQ ID No. 5.

Preferably, the recombinant adeno-associated virus vector comprises a CMV enhancer/promoter, a gene sequence for expressing xCT/slc7a11, a beta-globin intron, hGHpA (human growth hormone poly (A) tail), L-ITR, R-ITR, ampicillin, and f1 ori.

More preferably, the gene sequence of the protein expressing xCT/slc7a11 is shown in SEQ ID NO. 1, the L-ITR sequence is shown in SEQ ID NO. 3, the CMV enhancer/promoter sequence is shown in SEQ ID NO.4, the beta-globin intron sequence is shown in SEQ ID NO. 5, the hGHpA sequence is shown in SEQ ID NO. 7, the R-ITR sequence is shown in SEQ ID NO. 8, the Ampicillin sequence is shown in SEQ ID NO. 9, and the f1 ori sequence is shown in SEQ ID NO. 11.

Preferably, the recombinant adeno-associated virus vector sequence comprises SEQ ID NO 10.

More preferably, the recombinant adeno-associated virus vector has a sequence shown in SEQ ID NO 12.

In order to achieve the second object, the invention adopts the technical scheme that:

a method for preparing the recombinant adeno-associated virus vector comprises the following steps:

1) Constructing a recombinant plasmid AAV _ xCT/slc7a11, wherein the main elements comprise a CMV enhancer/promoter and an xCT/slc7a11 gene sequence;

2) Co-transfecting an AAV capsid protein gene and a helper plasmid containing a gene capable of assisting AAV replication, an AAV _ xCT/slc7a11 plasmid and a HEK 293T cell to preliminarily form a recombinant adeno-associated virus vector;

3) After the recombinant adeno-associated virus vector is primarily purified by ioxol, the recombinant adeno-associated virus vector is further purified by a fast protein liquid chromatograph with 5ml-Hitrp Q sepharose as a filler through ion exchange chromatography;

4) Then eluting the agarose gel column by using NaCl with the pH =8.0 and the concentration of 215mM, and collecting the recombinant adeno-associated virus vector with the peak value;

5) After the collected liquid passes through a concentrator, eluting the concentrator by using 0.014% Tween 20 to concentrate the recombinant adeno-associated virus vector, digesting DNA (deoxyribonucleic acid) except virus particles in the concentrated liquid by using DNase I (deoxyribose nucleic acid), and determining the titer of the virus by using a real-time fluorescent quantitative PCR (polymerase chain reaction) method;

6) Finally, silver nitrate staining-SDS polyacrylamide gel electrophoresis is used to ensure that the recombinant adeno-associated virus vector particles are not polluted and do not contain endotoxin, and the recombinant adeno-associated virus vector particles are subpackaged at minus 80 ℃ for storage.

In order to achieve the third object, the invention adopts the technical scheme that:

a pharmaceutical composition for treating retinal detachment photoreceptor cell damage apoptosis comprises any one of the recombinant adeno-associated virus vectors and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition expresses xCT/slc7a11 protein.

In order to achieve the fourth object, the invention adopts the technical scheme that:

the application of the adeno-associated virus vector in preparing a medicament for treating retinal detachment photoreceptor cell damage apoptosis.

In order to achieve the fifth purpose, the invention adopts the technical scheme that:

a method for treating retinal detachment photoreceptor cell damage apoptosis, the adeno-associated viral vector as described in any one of the above is administered to an individual in need of treatment.

Preferably, the method of administration is intraocular injection.

More preferably, the intraocular injection is a subretinal injection or a vitreous cavity injection.

In a first aspect of the invention, there is provided an expression cassette having from the 5'-3' end a structure of formula I:

Z1-Z2-Z3-Z4 (I)

wherein each "-" is independently a bond or a nucleotide linking sequence;

z1 is an enhancer and/or a promoter;

z2 is nothing or an intron;

z3 is a nucleotide sequence for coding XCT/SLC7A11 protein; and

z4 is an optional hGHpA (human growth hormone poly (A) tail) sequence.

In another preferred embodiment, the nucleotide sequence is selected from the group consisting of:

(a) The nucleotide sequence is shown as SEQ ID NO. 1; and

(b) The nucleotide sequence has more than or equal to 95 percent of identity with the nucleotide sequence shown in SEQ ID NO. 1, preferably more than or equal to 98 percent, and more preferably more than or equal to 99 percent;

(c) A nucleotide sequence complementary to the nucleotide sequence of (a) or (b).

In another preferred embodiment, the nucleotide sequence comprises a DNA sequence, a cDNA sequence, or an mRNA sequence.

In another preferred embodiment, the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.

In another preferred embodiment, the nucleotide sequence comprises a nucleotide sequence that is fully complementary to SEQ ID No. 1.

In another preferred embodiment, the amino acid sequence of the XCT/SLC7A11 protein is as shown in SEQ ID No. 2.

In another preferred embodiment, the promoter comprises a tissue-specific promoter.

In another preferred embodiment, the enhancer and/or promoter is a CMV enhancer/promoter.

In another preferred embodiment, the nucleotide sequence of the CMV enhancer/promoter is 95% identical or more, preferably 98% or more, more preferably 99% or more to the nucleotide sequence shown in SEQ ID No. 4.

In another preferred embodiment, the nucleotide sequence of the CMV enhancer/promoter is as shown in SEQ ID No. 4.

In another preferred embodiment, the intron is a beta-globin intron.

In another preferred embodiment, the nucleotide sequence of the intron has 95% identity or more, preferably 98% or more, and more preferably 99% or more with the nucleotide sequence shown in SEQ ID No. 5.

In another preferred embodiment, the nucleotide sequence of the intron is shown in SEQ ID No. 5.

In another preferred embodiment, the nucleotide sequence of hGHpA has 95% identity or more, preferably 98% or more, and more preferably 99% or more with the nucleotide sequence shown in SEQ ID No. 7.

In another preferred embodiment, the nucleotide sequence of hGHpA is shown in SEQ ID NO. 7.

In another preferred embodiment, each nucleotide linker sequence is 0 to 30nt, preferably 1 to 15nt, in length.

In a second aspect of the invention, there is provided a vector comprising an expression cassette as described in the first aspect of the invention.

In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, enhancer, intron, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site.

In another preferred embodiment, the vector comprises a plasmid or a viral vector.

In another preferred embodiment, the vector comprises a DNA virus or a retroviral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), or a combination thereof. Preferably, the vector is an AAV vector.

In another preferred embodiment, the vector is an AAV vector comprising or inserted with a nucleotide sequence encoding the XCT/SLC7A11 protein.

In another preferred embodiment, the vector is for expressing the human XCT/SLC7A11 protein.

In a third aspect of the invention, there is provided an adeno-associated viral vector comprising an expression cassette as described in the first aspect of the invention.

In another preferred embodiment, the serotype of the adeno-associated virus is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a/3b, AAVvh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6 (Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y- > F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV 3.45, AAV2 or AAV5, or a combination thereof.

In another preferred embodiment, the adeno-associated virus is AAV2, preferably the adeno-associated virus is single-stranded AAV2.

In another preferred embodiment, the adeno-associated viral vector is used for treating ocular diseases.

In another preferred embodiment, the adeno-associated viral vector has the structure of formula II:

A1-Z1-Z2-Z3-Z4-A2-A3(I)

wherein each "-" is independently a bond or a nucleotide linking sequence;

a1 is an L-ITR sequence;

z1 is CMV enhancer/promoter;

z2 is a beta-globin intron;

z3 is a nucleotide sequence for coding XCT/SLC7A11 protein;

z4 is hGHpA sequence;

a2 is an R-ITR sequence;

a3 is an optional tag sequence; and

a4 is an optional f1 ori sequence.

In another preferred embodiment, the L-ITR sequence is as shown in SEQ ID NO. 3.

In another preferred embodiment, the R-ITR sequence is as shown in SEQ ID NO. 8.

In another preferred embodiment, the label comprises a resistance marker and a fluorescein marker.

In another preferred embodiment, the tag sequence is an Ampicillin sequence, and preferably, the tag sequence is shown in SEQ ID NO. 9.

In another preferred embodiment, the f1 ori sequence is as shown in SEQ ID No. 11.

In another preferred example, the sequence of the adeno-associated viral vector comprises the sequence shown in SEQ ID No.:10 (SEQ ID No.:10 is the nucleotide sequence of the vector into which the gene of interest is not inserted).

In another preferred embodiment, the sequence of the adeno-associated virus vector is shown in SEQ ID NO. 12 (SEQ ID NO. 12 is the nucleotide sequence of the vector after the target gene is inserted).

In a fourth aspect of the invention, there is provided a host cell comprising a vector according to the second aspect of the invention or an adeno-associated viral vector according to the third aspect of the invention, or having integrated into its chromosome an exogenous expression cassette according to the first aspect of the invention.

In another preferred embodiment, the host cell is a mammalian cell, including human and non-human mammals.

In another preferred embodiment, the host cell is selected from the group consisting of: HEK cells, photoreceptor cells (including cone cells and/or rods), other visual cells (such as bipolar cells, horizontal cells), (optic) nerve cells, or combinations thereof.

In another preferred embodiment, the host cell is selected from the group consisting of: rod cells, cone cells, light donating bipolar cells, light withdrawing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combinations thereof.

In a fifth aspect of the invention, there is provided the use of a vector according to the second aspect of the invention or an adeno-associated viral vector according to the third aspect of the invention for the preparation of a formulation or composition for the treatment of an ocular disease.

In another preferred embodiment, the formulation or composition is used for treating a retinal photoreceptor cell injury disease.

In another preferred embodiment, the formulation or composition is used to reduce apoptosis in the outer nuclear layer of the retina and/or increase photoreceptor cell survival.

In another preferred embodiment, the retinal photoreceptor cell injury disease is selected from the group consisting of: retinal detachment, hereditary retinitis pigmentosa, macular degeneration, diabetic retinopathy, or a combination thereof.

In a third aspect of the invention, there is provided a pharmaceutical formulation comprising (a) the vector of the second aspect of the invention or the adeno-associated viral vector of the third aspect of the invention, and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the dosage form of the pharmaceutical formulation is selected from the group consisting of: a lyophilized formulation, a liquid formulation, or a combination thereof.

In another preferred embodiment, the content of the carrier in the pharmaceutical preparation is 1 × 10 ⁹ -1×10 ¹⁶ Individual virus/ml, preferably 1X 10 ¹² -1×10 ¹³ One virus/ml.

In another preferred embodiment, the pharmaceutical formulation is for use in the treatment of an ocular disease.

In another preferred embodiment, the pharmaceutical formulation is for use in reducing apoptosis of the outer nuclear layer of the retina and/or increasing photoreceptor cell survival.

In another preferred embodiment, the ocular disease is a retinal photoreceptor cell injury disease.

In a seventh aspect of the invention, there is provided a method of treatment comprising administering the vector of the second aspect of the invention or the adeno-associated viral vector of the third aspect of the invention to a subject in need thereof.

In another preferred embodiment, the adeno-associated viral vector is injected into the eye of a subject in need thereof.

In another preferred embodiment, the injection comprises a sub-retinal injection and a vitreous cavity injection.

In another preferred embodiment, the subject in need thereof includes humans and non-human mammals.

In another preferred embodiment, the method of treatment is a method of treating an ocular disease.

In an eighth aspect of the present invention, there is provided a method for preparing XCT/SLC7a11 protein, comprising culturing the host cell of the fourth aspect of the present invention, thereby obtaining XCT/SLC7a11 protein.

In a ninth aspect of the present invention, there is provided a method of preparing the vector of the second aspect of the present invention or the adeno-associated virus vector of the third aspect of the present invention, comprising the steps of:

1) Constructing a recombinant plasmid AAV _ xCT/slc7a11, wherein the main elements comprise a CMV enhancer/promoter and an xCT/slc7a11 gene sequence;

2) Co-transfecting an AAV capsid protein gene and a helper plasmid containing a gene capable of assisting AAV replication, an AAV _ xCT/slc7a11 plasmid and a HEK 293T cell to preliminarily form a recombinant adeno-associated virus vector;

3) After the recombinant adeno-associated virus vector is primarily purified by ioxol, the recombinant adeno-associated virus vector is further purified by a fast protein liquid chromatograph with 5ml-Hitrp Q sepharose as a filler through ion exchange chromatography;

4) Then eluting the agarose gel column by using NaCl with the pH =8.0 and the concentration of 215mM, and collecting the recombinant adeno-associated virus vector with the peak value;

5) After the collected liquid passes through a concentrator, eluting the concentrator by using Tween 20 containing 0.014% to concentrate the recombinant adeno-associated virus vector, digesting DNA (deoxyribonucleic acid) except virus particles in the concentrated liquid by using DNase I (deoxyribose nucleic acid), and determining the titer of the virus by using a real-time fluorescent quantitative PCR (polymerase chain reaction) method;

6) Finally, silver nitrate staining-SDS polyacrylamide gel electrophoresis is used for ensuring that the recombinant adeno-associated virus vector particles are not polluted and do not contain endotoxin, and the recombinant adeno-associated virus vector particles are subpackaged and stored at 80 ℃ below zero.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows a characteristic map of the viral vector AAV _ xCT/slc7a11 and the start-stop sites of the respective elements.

FIG. 2 shows immunofluorescent staining patterns of retinal sections two weeks after subretinal injection of AAV _ xCT/slc7a11 in C57BL/6J mice.

FIG. 3 shows immunofluorescent staining patterns of experimental mouse retinal spreads. Specifically, C57BL/6J mice were injected subretinally with AAV _ xCT/slc7a11 or AAV-GFP,2 weeks later, retinal detachment was caused by subretinal injection of HA, and TUNEL staining pattern of retinal sections 3 days after retinal detachment.

FIG. 4 shows a graph of apoptotic cell count and viability analysis results after TUNEL staining. Specifically, C57BL/6J mice were injected subretinally with AAV _ xCT/slc7a11 or AAV-GFP, and after 2 weeks, retinal detachment was caused by subretinal injection of HA, and TUNEL staining patterns of retinal sections 3 days after retinal detachment, which are graphically depicted as the results of their apoptotic cell counts and viability assays.

Detailed Description

The inventor carries out extensive and intensive research, carries out targeted special optimization design on the XCT/SLC7A11 gene coding sequence, thereby obtaining a nucleotide sequence which is particularly suitable for efficiently expressing the XCT/SLC7A11 protein in mammalian (such as human) cells and constructing a recombinant AAV for expressing the XCT/SLC7A11 protein. Experimental results show that the recombinant AAV has excellent infection effect in mouse cells, is stably expressed in cell nuclei, is a stable recombinant adeno-associated virus vector, can remarkably reduce apoptosis of outer nuclear layer cells of retina and increase survival rate of photoreceptor cells of mice, can be used for preparing medicaments for treating retinal detachment photoreceptor cell damage apoptosis, and has wide market prospect.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

As used herein, the terms "subject", "subject in need thereof" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

xCT/slc7a11

xCT/slc7a11 is Na ⁺ The independent glutamate/cystine transporter is positioned on RGCs cell membranes, and the expression of xCT/slc7a11 is regulated and controlled by a complex signal molecule network, including transcription regulation of Nrf2, REST and the like, epigenetic regulation of H3K27 and the like.

Research shows that under the oxidative stress environment, xCT/slc7a11 synthesis is increased, and the synthesis is transferred and positioned on RGCs cell membranes, and the function of 1:1 for reverse transfer of glutamic acid and cystine is exerted, namely 1 glutamic acid is transferred to the outside of cells, and 1 cystine is transferred to the inside of the cells. Cystine transferred into cells is subjected to a series of enzymatic reactions, and finally GSH is generated under the catalysis of glutathione synthetase, so that the strong antioxidation effect is exerted, and the intracellular redox balance is maintained. The xCT/slc7a11 signaling pathway is in a silent state in normal RGCs, most RGCs cannot effectively activate the xCT/slc7a11 pathway after axon injury and finally die, and researches prove that the activation of the xCT/slc7a11 pathway can promote the survival of the RGCs after the axon injury, and the xCT/slc7a11 pathway is key to the survival of the RGCs after the axon injury. Thus, enhancement of the xCT/slc7a11 signaling pathway in RGCs in various optic neurodegenerative diseases may be an effective therapeutic measure to prevent RGC degeneration, promote RGC survival and axon regeneration.

Nucleic acid coding sequences

The invention provides an optimized XCT/SLC7A11 coding sequence, which is shown as SEQ ID NO:1 is shown. Researches show that the optimized XCT/SLC7A11 coding sequence (SEQ ID No.: 1) has higher XCT/SLC7A11 protein expression efficiency, can more effectively reduce the apoptosis of the outer nuclear layer cells of the retina and/or increase the survival rate of photoreceptor cells, and effectively prevent and/or treat the retinal detachment.

The nucleotide sequence of the nucleic acid for coding the XCT/SLC7A11 protein is shown as SEQ ID NO. 1. In another preferred embodiment, the nucleotide sequence has 95% identity or more, preferably 98% or more, and more preferably 99% or more to the nucleotide sequence shown in SEQ ID No. 1. .

The polynucleotide of the present invention may be in the form of DNA or RNA. In another preferred embodiment, the nucleotide is DNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The nucleotide sequence of the invention codes an amino acid sequence shown in SEQ ID NO. 2.

The NCBI reference sequence number of the XCT/SLC7A11 protein is NP-055146, and the amino acid sequence is shown as SEQ ID NO. 2.

The nucleic acid sequence may be DNA, RNA, cDNA or PNA. The nucleic acid sequence may be genomic, recombinant or synthetic. The nucleic acid sequence may be isolated or purified. The nucleic acid sequence may be single-stranded or double-stranded. Preferably, the nucleic acid sequence will encode an XCT/SLC7a11 protein as described herein. Nucleic acid sequences can be derived by Cloning, for example using standard Molecular Cloning techniques including restriction, ligation, gel electrophoresis, for example as described in Sambrook et al Molecular Cloning: A Laboratory Manual, cold Spring harbor Laboratory Press). The nucleic acid sequence may be isolated, for example, using PCR techniques. Isolation means the isolation of a nucleic acid sequence from any impurities and from other nucleic acid sequences and/or proteins that are naturally found in association with the nucleic acid sequence in its source. Preferably, it will also be free of cellular material, culture medium, or other chemicals from the purification/production process. The nucleic acid sequence may be synthetic, for example produced by direct chemical synthesis. The nucleic acid sequence may be provided as naked nucleic acid or may be provided complexed with a protein or lipid.

The full-length nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and to genetically engineered host cells with the vector or polypeptide coding sequences of the invention. The polynucleotide, vector or host cell may be isolated.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

In a preferred embodiment of the invention, the nucleotide sequence is as shown in SEQ ID No. 1.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The invention also relates to a vector containing the polynucleotide of the invention, a host cell produced by genetic engineering by using the vector or the protein coding sequence of the invention, and a method for expressing XCT/SLC7A11 protein by using the host cell through a recombinant technology.

Host cells (e.g., mammalian cells) expressing the XCT/SLC7A11 protein of the invention can be obtained using the polynucleotide sequences of the invention by conventional recombinant DNA techniques. Generally comprising the steps of: the polynucleotide according to the first aspect of the invention or the vector according to the third aspect of the invention or the adeno-associated viral vector according to the fourth aspect of the invention is transduced into a host cell.

Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a polypeptide of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the polypeptide.

The host cell may be a prokaryotic cell, or a lower eukaryotic cell, or a higher eukaryotic cell, such as a mammalian cell (including human and non-human mammals). Representative examples are: CHO, NS0, COS7, or 293 cells. In a preferred embodiment of the present invention, 293T cells, photoreceptor cells (including cone cells and/or rod cells), other visual cells (e.g., binodal cells), and neural cells are selected as host cells. In another preferred embodiment, the host cell is selected from the group consisting of: rod cells, cone cells, light donating bipolar cells, light withdrawing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combinations thereof.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The transformant obtained can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

Adeno-associated virus

Due to the characteristics of Adeno-associated virus (AAV) that it is smaller than other viral vectors, it is not pathogenic, and can transfect dividing and non-dividing cells, gene therapy methods based on AAV vectors for ocular diseases, especially for hereditary retinal degenerative diseases, have received much attention.

Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family parvoviridae, is the single-stranded DNA-deficient virus of the simplest structure currently found, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are crucial for replication and packaging of viruses. The cap gene encodes the viral capsid protein, and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells.

The recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, is considered to be one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (divided and non-divided cells), low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy and vaccine research in the world. Over 10 years of research, the biological properties of recombinant adeno-associated viruses have been well understood, and many data have been accumulated on the application effects of recombinant adeno-associated viruses in various cells, tissues and in vivo experiments. In medical research, rAAV is used in the study of gene therapy for a variety of diseases (including in vivo, in vitro experiments); meanwhile, the gene transfer vector is used as a characteristic gene transfer vector and is widely applied to the aspects of gene function research, disease model construction, gene knock-out mouse preparation and the like.

In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of cells that they infect in a stable and site-specific manner. They are able to infect a large series of cells without any effect on cell growth, morphology or differentiation and they do not appear to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. AAV contains an Inverted Terminal Repeat (ITR) region of about 145 bases at each end, which serves as the viral origin of replication. The remainder of the genome is divided into two important regions with encapsidation functions: the left part of the genome comprising the rep gene involved in viral replication and viral gene expression; and the right part of the genome comprising the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying the support can be found, for example, in U.S. patent nos. 6566118, 6989264, and 6995006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of vectors derived from AAV for in vitro and in vivo gene transfer has been described (see, e.g., international patent application publication Nos. WO91/18088 and WO93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535 and 5,139,941, and European patent No.0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs to transport the gene of interest in vitro (into cultured cells) or in vivo (directly into the organism). Replication-defective recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): plasmids containing the nucleic acid sequence of interest flanked by two AAV Inverted Terminal Repeat (ITR) regions, and plasmids carrying AAV encapsidation genes (rep and cap genes). The AAV recombinants produced are then purified by standard techniques.

In some embodiments, the recombinant vector is encapsidated into a virion (e.g., an AAV virion including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV 16). Accordingly, the disclosure includes recombinant viral particles (recombinant as they comprise a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No.6,596,535.

Expression vectors and host cells

The invention also provides an expression vector for XCT/SLC7A11 protein, which contains the optimized XCT/SLC7A11 coding sequence.

With the sequence information provided, the skilled artisan can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

Preferably, the nucleic acid sequence encoding the XCT/SLC7a11 protein is provided as a vector, preferably an expression vector. Preferably, it may be provided as a gene therapy vector, preferably suitable for transduction and expression in retinal target cells. The vector may be viral or non-viral (e.g., a plasmid). Viral vectors include those derived from adenovirus, adeno-associated virus (AAV), including mutated forms, retroviruses, lentiviruses, herpes viruses, vaccinia virus, MMLV, gaLV, simian Immunodeficiency Virus (SIV), HIV, poxviruses, and SV40. Preferably, the viral vector is replication defective, although it is envisaged that it may be replication deficient, replication capable or conditionally replicating. Viral vectors can generally remain extrachromosomal without integrating into the genome of the target retinal cell. A preferred viral vector for introducing a nucleic acid sequence encoding the XCT/SLC7A11 protein into a target cell of the retina is an AAV vector, such as a self-complementary adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 through AAV serotype 12) or modified versions of any of these serotypes, including AAV 4YF and AAV 7m8 vectors.

In another preferred embodiment, the adeno-associated virus used in the present invention is AAV2, preferably said adeno-associated virus is single-stranded AAV2.

The viral vector may be modified to delete any non-essential sequences. For example, in AAV, the virus may be modified to delete all or part of the IX, ela and/or Elb genes. Replication is very inefficient for wild type AAV without the presence of helper viruses such as adenovirus. For recombinant adeno-associated viruses, preferably, the replication and capsid genes are provided in trans (in the pRep/Cap plasmid), and only the 2 ITRs of the AAV genome are retained and packaged into virions, while the desired adenoviral genes are provided by the adenovirus or another plasmid. Similar modifications can be made to lentiviral vectors.

Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids may be complexed with agents to facilitate uptake of the viral vector by the target cell. Such agents include polycationic agents. Alternatively, a delivery system such as a liposome-based delivery system may be used. The vector for use in the present invention is preferably suitable for use in vivo or in vitro, and preferably for use in humans.

The vector will preferably comprise one or more regulatory sequences to direct expression of the nucleic acid sequence in a target cell of the retina. Regulatory sequences may include promoters, introns, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to a nucleic acid sequence. The vector may also include a selectable marker, for example, to determine expression of the vector in a growth system (e.g., bacterial cells) or in retinal target cells.

By "operably linked" is meant that the nucleic acid sequences are functionally related to the sequences to which they are operably linked such that they are linked in a manner such that they affect the expression or function of each other. For example, a nucleic acid sequence operably linked to a promoter will have an expression pattern that is affected by the promoter.

Promoters mediate the expression of nucleic acid sequences to which they are linked. Promoters may be constitutive or may be inducible. The promoter may direct ubiquitous expression in internal retinal cells, or neuron-specific expression. In the latter case, the promoter may direct cell type specific expression, for example, to an optic ganglion cell. Suitable promoters will be known to those skilled in the art. For example, suitable promoters may be selected from the group consisting of L7, thy-1, restorer protein, calbindin, human CMV, GAD-67, chicken beta actin, hSyn, grm6 enhancer SV40 fusion proteins. Targeting can be achieved using cell-specific promoters, e.g., grm6-SV40 for selective targeting to optic nerve cells. The Grm6 promoter is a fusion of the 200 base pair enhancer sequence of the Grm6 gene, which encodes a metabotropic glutamate receptor mGluR6 specific for optic nerve cells, and the SV40 eukaryotic promoter. Preferred sources of the Grm6 gene are mouse and human. Ubiquitous expression can be achieved using pan-neuronal promoters, examples of which are known and available in the art. One such example is CAG. The CAG promoter is a fusion of the CMV early enhancer and the chicken beta actin promoter.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV 40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebutan-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

A number of expression vectors can be used to express the XCT/SLC7A11 protein in mammalian cells (preferably human, more preferably human optic nerve cells or photoreceptor cells). The present invention preferably uses adeno-associated virus as an expression vector.

The invention also provides a construction method of the recombinant application adeno-associated virus vector of the XCT/SLC7A11 optimized coding sequence, and the method can quickly and simply construct the recombinant adeno-associated virus vector carrying the XCT/SLC7A11 optimized coding sequence and package the recombinant adeno-associated virus vector to obtain the complex defect adeno-associated virus vector.

In another preferred embodiment, the sequence of the adeno-associated viral vector carrying the optimized coding sequence XCT/SLC7A11 according to the present invention is shown in SEQ ID NO. 12. Wherein, the gene sequence of the protein expressing xCT/slc7a11 is shown as SEQ ID NO. 1, the L-ITR sequence is shown as SEQ ID NO. 3, the CMV enhancer/promoter sequence is shown as SEQ ID NO.4, the beta-globin intron sequence is shown as SEQ ID NO. 5, the hGHpA sequence is shown as SEQ ID NO. 7, the R-ITR sequence is shown as SEQ ID NO. 8, the Ampicillin sequence is shown as SEQ ID NO. 9, and the f1 ori sequence is shown as SEQ ID NO. 11.

Gene therapy vector

The gene therapy vector in the present invention is a viral expression vector, according to the present invention, the viral expression vector is an adeno-associated virus (AAV) vector such as an AAV vector selected from the group consisting of serotype AAV1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a chimeric AAV derived therefrom, e.g., AAV2-AAV3, aavrh.10, aavhu.14, AAV3a/3b, aavrh32.33, aavhsc15, AAV-HSC17, aavhhu.37, aavrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, aa41, aav9.45, aav6 (Y445F/Y731F), aav2.5t, AAV-HAE1/2, AAV clone 32/83, aavshh10, aav2 (Y- > AAV), AAV8 (Y-vrf), aavrf, aav2.5F, aav5, AAV2.5, AAV2, AAV clone 32/83, AAV tissues are more preferably of interest for AAV tissues transduction. Upon transfection, AAV elicits only a mild immune response (if any) in the host. In a preferred embodiment of the invention, the gene therapy vector is an AAV serotype 2 or 5 vector. In a further preferred embodiment, the gene therapy vector is an AAV2 vector.

The AAV vector of the invention is a single-stranded AAV, and a recombinant viral vector can be produced according to standard techniques. For example, recombinant adeno-associated virus vectors can be transmitted in human 293 cells (which provide for trans E1A and E1B characteristics) to achieve a distribution at 10 ⁷ -10 ¹³ Titers in the range of individual virus particles/mL. Prior to in vivo use, the viral vectors may be desalted by gel filtration methods (such as agarose columns) and purified by subsequent filtration. Purification reduces potential deleterious effects in the host of the administration vehicle. The administered virus is substantially free of wild-type and replication-competent viruses. The purity of the virus can be demonstrated by a suitable method, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining.

Suitable dosages for human AAV are in the range of about 1X 10 ¹⁰ -1×10 ¹⁴ Individual viral particles.

The gene therapy vector can be administered by intraocular injection, and can be administered by subretinal injection or intravitreal injection.

The invention also provides a host cell for expressing XCT/SLC7A11 protein. Preferably, the host cell is a mammalian cell (preferably a human, more preferably a human optic nerve cell or a photoreceptor cell) and increases the expression level of the XCT/SLC7A11 protein.

Formulations and compositions

The present invention provides a formulation or composition comprising (a) a vector according to the third aspect of the present invention or an adeno-associated viral vector according to the fourth aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the pharmaceutical preparation is used for treating eye diseases, and preferably, the pharmaceutical preparation is used for treating retinal photoreceptor cell injury diseases.

For convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an administration device for injection (e.g., a needle for injection), in which the pharmaceutical composition may be contained in an amount administered at one time. The administration device for injection may be contained in a cartridge for convenient storage and use. The transportation requires placing a tiny container filled with the drug suspension in dry ice. It should be stored in a refrigerator at-80 deg.C.

The kit or kit of the present invention may further comprise instructions for use, which will facilitate the use of the kit or kit in a proper manner by those skilled in the art.

The "active ingredient" in the pharmaceutical composition of the present invention refers to a vector of the present invention, such as a viral vector (including adeno-associated viral vectors). The "active ingredients", formulations and/or compositions of the present invention are useful for treating ocular diseases. "safe and effective amount" means: the amount of active ingredient is sufficient to significantly ameliorate the condition or symptom without causing serious side effects. "pharmaceutically acceptable carrier or excipient (excipient)" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.

The composition may be a liquid or a solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

In the present invention, the vector may be administered to the eye by subretinal or intravitreal administration. In either mode of administration, preferably, the carrier is provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), gelatin, talc, gelatin, calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), and the likeAlcohols, etc.), emulsifiers (e.g.

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The nucleic acid or fusion nucleic acid for coding XCT/SLC7A11 provided by the invention can be used for producing XCT/SLC7A11 protein in vitro or in vivo, and a preparation containing the XCT/SLC7A11 optimized coding sequence can be applied to preparing medicaments for treating eye diseases.

The optimized nucleic acid for coding the human XCT/SLC7A11 protein has higher expression level, so that more XCT/SLC7A11 proteins are translated, and compared with the prior art, the optimized nucleic acid for coding the human XCT/SLC7A11 protein expresses more XCT/SLC7A11 proteins, and the retinal photoreceptor cell injury disease can be better treated.

Method of treatment

The present invention provides a method of treating a retinal photoreceptor cell injury disorder comprising introducing into the eye a vector comprising an optimized sequence encoding XCT/SLC7a 11. The method may comprise administering the nucleic acid vector subretinally or intravitreally to an inner retinal cell of the eye.

The present invention provides a nucleic acid vector for use in a method of treating retinal degeneration by providing photoreceptor function to a cell, the nucleic acid vector comprising an optimized sequence encoding XCT/SLC7a 11. The compositions of the invention may be administered alone or in combination with (e.g. formulated in the same pharmaceutical composition as) other therapeutic agents.

As used herein, treating a disease means administering a nucleic acid or vector as described herein to ameliorate or alleviate one or more symptoms of the disease, including reducing apoptosis of outer nuclear layer cells of the retina and/or increasing photoreceptor cell survival, among others.

The methods of the invention comprise introducing into the vitreous cavity of the eye a nucleic acid sequence encoding the XCT/SLC7A11 protein. Preferably, the method comprises contacting the cell with a vector (preferably a virus, more preferably an adeno-associated virus) comprising a nucleic acid sequence encoding the XCT/SLC7A11 protein. Preferably, the cell is a retinal cell, preferably a cone cell, a rod cell, a light donating bipolar cell, a light withdrawing bipolar cell, a horizontal cell, a ganglion cell and/or an amacrine cell.

When the nucleic acid sequence and the one or more enzymes are provided in multiple (two or more) doses, the doses may be separated by a suitable time interval, for example 30 seconds to several hours or 1 day or more.

Each dose may contain an effective amount of the nucleic acid sequence or viral vector. An effective amount of the nucleic acid sequence or viral vector can be 1X 10 per treatment regimen ⁹ -1×10 ¹⁶ The range of viruses.

The present invention is based on targeting an optimized nucleic acid sequence encoding XCT/SLC7A11 to retinal cells to compensate for degeneration of photoreceptor cells in the retina. The cell to which the nucleic acid sequence is targeted is a cell of the retina, which is living and capable of expressing the foreign nucleic acid sequence. Retinal cells are cells of the retina, which are neural or neuronal cells and are capable of becoming excited and transmitting electrical signals. Preferably, the target retinal cell will be able to generate an electrical signal and initiate a signaling cascade resulting in the transmission of a signal to the optic nerve. Preferably, the target retinal cell is an inner retinal cell. The target cells may be rods or cones, and/or may be non-photoreceptor cells (i.e., retinal cells that are not responsive to light in their native form). The target retinal cell may comprise one or more cell types selected from the group consisting of: rod cells, cone cells, light donating bipolar cells, light withdrawing bipolar cells, level cells, ganglion cells, miller cells, and/or amacrine cells.

Thus, when the target retinal cell is a retinal-targeting light-donating bipolar cell, a light-withdrawing bipolar cell, a horizontal cell, a ganglion cell, and/or an amacrine cell, expression of the nucleic acid encoding XCT/SLC7a11 may be referred to as ectopic expression. Thus, the present invention includes within its scope a method for ectopically expressing a nucleic acid sequence encoding XCT/SLC7A11 in a non-photoreceptor cell. Such ectopic expression has the effect of providing photoreceptor cell function to cells through the expression of heterologous XCT/SLC7A11 protein therein. This serves to increase the photosensibility of the retina where degeneration is observed.

The horizontal cells are inner retinal cells, involved in signal processing and feedback to the photoreceptor cells; bipolar cells are inner retinal cells and communicate between rods/cones and amacrine and/or ganglion cells; amacrine cells are found in the inner retina and allow communication between the photoreceptor pathways and ganglion cells; the ganglion cells are the innermost retinal cells, which transmit signals from the photoreceptor cells to the optic nerve.

Reference herein to a cell includes the progeny of the cell. Preferably, the modification of the cells according to the invention also takes place in subsequent generations of the transformed host cell. The progeny cell may not be identical to the original targeted cell, but preferably will also exhibit non-native XCT/SLC7a11 expression.

The main advantages of the invention include:

(a) The invention optimizes the human xCT/slc7a11 gene sequence, has higher expression efficiency and is suitable for expressing in the body of mammals;

(b) The invention selects proper vectors and related elements, constructs the recombinant adeno-associated virus vector AAV _ xCT/slc7a11, has excellent infection effect, can be stably expressed in cell nucleus, and is a stable recombinant adeno-associated virus vector.

(c) The recombinant adeno-associated virus vector can obviously reduce the apoptosis of the outer nuclear layer cells of the retina and increase the survival rate of the photoreceptor cells of mice, so that the recombinant adeno-associated virus vector can be used for preparing the medicine for treating the damage and the apoptosis of the photoreceptor cells after the retina is detached.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

EXAMPLE 1 construction and isolation and purification of recombinant adeno-associated Virus vectors

Gene sequences

SEQ ID NO 1 shows the sequence xCT/slc7a11,

SEQ ID NO. 2 shows the amino acid sequence xCT/slc7a11,

shown in SEQ ID NO 3 is the Left ITR sequence,

SEQ ID NO.4 shows the CMV sequence,

SEQ ID NO. 5 shows a human beta-globin intron sequence,

shown in SEQ ID NO 6 is the MCS sequence,

SEQ ID NO. 7 shows the hGHpA sequence,

SEQ ID NO 8 shows the Right ITR sequence,

shown in SEQ ID NO 9 is the Ampicillin Resistance sequence,

SEQ ID NO 10 shows the complete nucleotide sequence of the empty vector

SEQ ID NO 11 shows the f1 ori sequence,

SEQ ID NO 12 shows the nucleotide sequence of the recombinant vector.

The construction, characteristic map and start-stop sites of each element of the plasmid AAV _ xCT/slc7a11 are shown in figure 1, the main elements comprise a CMV enhancer/promoter (SEQ ID NO: 4) and an xCT/slc7a11 sequence (SEQ ID NO: 1), the CMV enhancer can enhance the expression of a transgene, a target gene is followed by hGHpA (SEQ ID NO: 7), an inverted Terminal Repeat (TR) is arranged at two sides of an expression cassette, namely, a virus vector comprises L-ITR (SEQ ID NO: 3) and R-ITR (SEQ ID NO: 8), and the virus vector further comprises Ampicillin (SEQ ID NO: 9) and f1 ori (SEQ ID NO: 11).

Viral vectors are obtained by plasmid cotransfection methods. Co-transfecting an auxiliary plasmid containing an AAV2 coat protein gene and a gene which can help AAV to replicate, an AAV _ xCT/slc7a11 plasmid and an HEK 293T cell to preliminarily form a recombinant adeno-associated virus vector; after preliminary purification with ioxobutanol, further purification by ion exchange chromatography on a fast protein liquid chromatograph using 5ml-Hitrp Q sepharose as packing using an instrument of Pharmacia AKTA FPLC system (Amersham Biosciences, piscataway, N.J.); then eluting the agarose gel column by using NaCl with pH8.0 and 215mM, and collecting the recombinant adeno-associated virus vector with the peak value; after the collected liquid passed through a concentrator (100K concentrator, millipore), the recombinant adeno-associated virus vector was concentrated by rinsing the concentrator with Tween 20 containing 0.014%; then digesting DNA except the virus particles by DNase I, and determining the titer of the virus by a real-time fluorescent quantitative PCR method; finally, silver nitrate staining-SDS polyacrylamide gel electrophoresis is used to ensure that the recombinant adeno-associated virus vector particles are not polluted and do not contain endotoxin, and the recombinant adeno-associated virus vector particles are subpackaged at minus 80 ℃ for storage.

Example 2 treatment of retinal detachment model mice with recombinant adeno-associated viral vectors

1 materials of the experiment

1.1 Experimental animals

Normal C57BL/6J mice were purchased from Shanghai slyke laboratory animals, inc. with a light cycle of 12h light-12 h dark, with free food intake and free water consumption, and all animal studies were conducted strictly in accordance with the Experimental animal management regulations issued by the national Committee for science and technology.

1.2 Experimental reagent and consumables

Physiological saline (Zhejiang Tianrui pharmaceutical Co., ltd.), a disposable syringe needle (Becton Dickinson and Company, USA), xCT antibody (Novus Biologicals), TUNEL (Roche Co., ltd.), anti-goat anti-rabbit IgG (Jackson Immuno) goat serum (Sigma), polyethylene glycol octyl phenyl ether (Triton X-100) (Sigma), and paraformaldehyde (Sigma).

1.3 Experimental instruments

Ophthalmic experimental operating microscope (Nikon corporation, japan); ophthalmic microscopy instruments (suzhou mingren medical instruments ltd); microsyrinths (Hamilton, usa); confocal microscopy (come, germany).

2 construction of mouse retinal detachment model

Two eyes of the eye-expanding mouse with the Tupidicamine eye drops are twice, the interval between every two eyes is 10min, and the mouse is subjected to intraperitoneal injection; dripping surface anesthesia eye drops on the anesthetized mouse cornea, and placing the mouse under a microscope; cutting a temporo-lateral conjunctiva, performing scleral puncture by a 34G needle at a position 3mm behind a corner scleral edge to form a full-layer scleral incision, and withdrawing the needle after a needle tip inclined plane completely enters a vitreous cavity when a patient looks straight under a mirror; the glass needle head connected with the micro-syringe enters the vitreous cavity through the sclera incision, vitreous humor is slowly extracted under direct vision, the retina gradually bulges, and the glass needle is rapidly extracted after being separated from the pigment epithelium layer. A34G needle enters the space between the retina and the pigment epithelium from the scleral incision, the needle is confirmed to be positioned under the retina but not under the choroid or in the vitreous cavity under direct vision, 0.01ml of sodium hyaluronate is slowly injected to the retina, and the retina is further raised in a grayish white color to form 3-4 retinal bulges. Observing the formation of retinal detachment under a microscope, wherein the height and the range of the retinal detachment exceed 80% of the whole retina, dripping biological tissue glue to the scleral incision for sealing, and finally spreading the dianbizuye ointment on the corneal surfaces of the eyes of the mouse. The mice were revived.

3 treatment method

3.1 subretinal injection

The 34G needle is used for inserting needle and puncturing at the position close to the junction of the retina edge and the corneosclera, when the front end of the needle enters the retina, injection is carried out, and the needle inserting angle and the needle inserting depth need to be paid attention to during injection, so that the injury to the lens or other intraocular tissues is avoided.

3.2 tissue treatment

Animals were perfused systemically first with PBS and then sequentially with 4% Paraformaldehyde (PFA) at a perfusion rate of 5 mL/min. After the eyeball and optic nerve were removed, the obtained material was immersed in a 4-vol% PFA solution for about 2 hours. The tissue should be stored in 0.1M PBS at 4 ℃ before staining; the tissue was soaked overnight in a 30% sucrose solution before cryosectioning.

4 adeno-associated virus infection and detection expressing xCT/slc7a11 protein

After two weeks of successful model creation mouse subretinal injection of AAV _ xCT/slc7a11, pretreatment of the eyeball was performed according to the aforementioned tissue treatment method, then the eyeball was gripped by a pair of tweezers and lifted upward, conjunctiva and muscle tissues around the eyeball were cut with scissors, the eyeball tissue was kept intact, the cornea, iris and crystalline lens were cut out to make an eyecup, and the eyecup was immersed overnight in a 30% sucrose solution to prepare a 10 μm thick tissue section.

And (3) immunofluorescence staining: selecting retinas, sealing in 20% goat serum solution for 1h, incubating the secondary antibody overnight, washing with antibody xCT and PBS for three times (10 min each time), incubating the secondary antibody for 1h, washing with PBS for three times (10 min each time), and sealing; the retinal structure changes were observed using a confocal microscope and photographed.

The results are as follows:

two weeks after the mice were injected with AAV _ xCT/slc7a11, immunofluorescence staining of retinal sections was performed, and a large amount of AAV _ xCT/slc7a11 was observed, localized on cell membrane and expressed around DAPI, showing that AAV _ xCT/slc7a11 had excellent infection effect (FIG. 2).

5 photoreceptor apoptosis rate detection

8 weeks old mice single side eye subretinal injection AAV _ xCT/slc7a11 (experimental group), AAV _ GFP (control group), 2 weeks later to make retinal detachment model, retinal detachment 3 days later, according to the tissue processing method processing eyeball and retina, immunofluorescence staining, using confocal microscope to observe retinal structure change and take pictures, each eyeball selecting 5 visual field counting TUNEL positive cells (proportional bar: 20 μm), and calculating photoreceptor cell survival rate.

The results are as follows:

grouping	Number of TUNEL
		Blank group	1.12±1.74
Model set	30.25±1.29
		Control group	32.16±1.35
Experimental group	12.95±1.17

As can be seen from the above table, the number of TUNEL positive cells in the model group, the control group and the experimental group was increased and statistically different (P < 0.05) compared to the blank group, the number of TUNEL positive cells in the control group was not statistically different (P > 0.05) compared to the model group, and the number of TUNEL positive cells in the experimental group was significantly decreased and statistically different (P < 0.05) compared to the control group.

Quantitative analysis proves that the photoreceptor cells reach the apoptosis peak in 3 days after the retinal detachment, and the thickness of the photoreceptor cell layer is obviously reduced (a model group and a control group); in contrast, mice injected with AAV _ xCT/slc7a11 showed no significant apoptosis, indicating that AAV _ xCT/slc7a11 has protective effect on photoreceptor cells (fig. 3, fig. 4).

The experiments show that the recombinant adeno-associated virus AAV _ xCT/slc7a11 can obviously reduce apoptosis of mouse photoreceptor cells and effectively treat retinal detachment photoreceptor cell injury.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> first-person hospital in Shanghai City

<120> recombinant adeno-associated virus vector, preparation method and application thereof

<130> P2019-0851

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1506

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

atggtcagaa agcctgttgt gtccaccatc tccaaaggag gttacctgca gggaaatgtt 60

aacgggaggc tgccttccct gggcaacaag gagccacctg ggcaggagaa agtgcagctg 120

aagaggaaag tcactttact gaggggagtc tccattatca ttggcaccat cattggagca 180

ggaatcttca tctctcctaa gggcgtgctc cagaacacgg gcagcgtggg catgtctctg 240

accatctgga cggtgtgtgg ggtcctgtca ctatttggag ctttgtctta tgctgaattg 300

ggaacaacta taaagaaatc tggaggtcat tacacatata ttttggaagt ctttggtcca 360

ttaccagctt ttgtacgagt ctgggtggaa ctcctcataa tacgccctgc agctactgct 420

gtgatatccc tggcatttgg acgctacatt ctggaaccat tttttattca atgtgaaatc 480

cctgaacttg cgatcaagct cattacagct gtgggcataa ctgtagtgat ggtcctaaat 540

agcatgagtg tcagctggag cgcccggatc cagattttct taaccttttg caagctcaca 600

gcaattctga taattatagt ccctggagtt atgcagctaa ttaaaggtca aacgcagaac 660

tttaaagacg ccttttcagg aagagattca agtattacgc ggttgccact ggctttttat 720

tatggaatgt atgcatatgc tggctggttt tacctcaact ttgttactga agaagtagaa 780

aaccctgaaa aaaccattcc ccttgcaata tgtatatcca tggccattgt caccattggc 840

tatgtgctga caaatgtggc ctactttacg accattaatg ctgaggagct gctgctttca 900

aatgcagtgg cagtgacctt ttctgagcgg ctactgggaa atttctcatt agcagttccg 960

atctttgttg ccctctcctg ctttggctcc atgaacggtg gtgtgtttgc tgtctccagg 1020

ttattctatg ttgcgtctcg agagggtcac cttccagaaa tcctctccat gattcatgtc 1080

cgcaagcaca ctcctctacc agctgttatt gttttgcacc ctttgacaat gataatgctc 1140

ttctctggag acctcgacag tcttttgaat ttcctcagtt ttgccaggtg gctttttatt 1200

gggctggcag ttgctgggct gatttatctt cgatacaaat gcccagatat gcatcgtcct 1260

ttcaaggtgc cactgttcat cccagctttg ttttccttca catgcctctt catggttgcc 1320

ctttccctct attcggaccc atttagtaca gggattggct tcgtcatcac tctgactgga 1380

gtccctgcgt attatctctt tattatatgg gacaagaaac ccaggtggtt tagaataatg 1440

tcggagaaaa taaccagaac attacaaata atactggaag ttgtaccaga agaagataag 1500

ttatga 1506

<210> 2

<211> 506

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Met Val Arg Lys Pro Val Val Ser Thr Ile Ser Lys Gly Gly Tyr Leu

1 5 10 15

Gln Gly Asn Val Asn Gly Arg Leu Pro Ser Leu Gly Asn Lys Glu Pro

20 25 30

Pro Gly Gln Glu Lys Val Gln Leu Lys Arg Lys Val Thr Leu Leu Arg

35 40 45

Gly Val Ser Ile Ile Ile Gly Thr Ile Ile Gly Ala Gly Ile Phe Ile

50 55 60

Ser Pro Lys Gly Val Leu Gln Asn Thr Gly Ser Val Gly Met Ser Leu

65 70 75 80

Thr Ile Trp Thr Val Cys Gly Val Leu Ser Leu Phe Gly Ala Leu Ser

85 90 95

Tyr Ala Glu Leu Gly Thr Thr Ile Lys Lys Ser Gly Gly His Tyr Thr

100 105 110

Tyr Ile Leu Glu Val Phe Gly Pro Leu Pro Ala Phe Val Arg Val Trp

115 120 125

Val Glu Leu Leu Ile Ile Arg Pro Ala Ala Thr Ala Val Ile Ser Leu

130 135 140

Ala Phe Gly Arg Tyr Ile Leu Glu Pro Phe Phe Ile Gln Cys Glu Ile

145 150 155 160

Pro Glu Leu Ala Ile Lys Leu Ile Thr Ala Val Gly Ile Thr Val Val

165 170 175

Met Val Leu Asn Ser Met Ser Val Ser Trp Ser Ala Arg Ile Gln Ile

180 185 190

Phe Leu Thr Phe Cys Lys Leu Thr Ala Ile Leu Ile Ile Ile Val Pro

195 200 205

Gly Val Met Gln Leu Ile Lys Gly Gln Thr Gln Asn Phe Lys Asp Ala

210 215 220

Phe Ser Gly Arg Asp Ser Ser Ile Thr Arg Leu Pro Leu Ala Phe Tyr

225 230 235 240

Tyr Gly Met Tyr Ala Tyr Ala Gly Trp Phe Tyr Leu Asn Phe Val Thr

245 250 255

Glu Glu Val Glu Asn Pro Glu Lys Thr Ile Pro Leu Ala Ile Cys Ile

260 265 270

Ser Met Ala Ile Val Thr Ile Gly Tyr Val Leu Thr Asn Val Ala Tyr

275 280 285

Phe Thr Thr Ile Asn Ala Glu Glu Leu Leu Leu Ser Asn Ala Val Ala

290 295 300

Val Thr Phe Ser Glu Arg Leu Leu Gly Asn Phe Ser Leu Ala Val Pro

305 310 315 320

Ile Phe Val Ala Leu Ser Cys Phe Gly Ser Met Asn Gly Gly Val Phe

325 330 335

Ala Val Ser Arg Leu Phe Tyr Val Ala Ser Arg Glu Gly His Leu Pro

340 345 350

Glu Ile Leu Ser Met Ile His Val Arg Lys His Thr Pro Leu Pro Ala

355 360 365

Val Ile Val Leu His Pro Leu Thr Met Ile Met Leu Phe Ser Gly Asp

370 375 380

Leu Asp Ser Leu Leu Asn Phe Leu Ser Phe Ala Arg Trp Leu Phe Ile

385 390 395 400

Gly Leu Ala Val Ala Gly Leu Ile Tyr Leu Arg Tyr Lys Cys Pro Asp

405 410 415

Met His Arg Pro Phe Lys Val Pro Leu Phe Ile Pro Ala Leu Phe Ser

420 425 430

Phe Thr Cys Leu Phe Met Val Ala Leu Ser Leu Tyr Ser Asp Pro Phe

435 440 445

Ser Thr Gly Ile Gly Phe Val Ile Thr Leu Thr Gly Val Pro Ala Tyr

450 455 460

Tyr Leu Phe Ile Ile Trp Asp Lys Lys Pro Arg Trp Phe Arg Ile Met

465 470 475 480

Ser Val Thr Glu Asn Thr Lys Cys Arg Leu Ile Asn Thr Pro Leu Phe

485 490 495

Lys Glu Arg Val Pro Arg Val Asp Tyr Pro

500 505

<210> 3

<211> 141

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc t 141

<210> 4

<211> 663

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

acgcgtggaa ttcgctagtt attaatagta atcaattacg gggtcattag ttcatagccc 60

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgt caatagggac 180

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240

agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300

gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360

agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420

gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 540

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag 600

atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc 660

agc 663

<210> 5

<211> 493

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gattcgaatc ccggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 60

gtaagtaccg cctatagagt ctataggccc acaaaaaatg ctttcttctt ttaatatact 120

tttttgttta tcttatttct aatactttcc ctaatctctt tctttcaggg caataatgat 180

acaatgtatc atgcctcttt gcaccattct aaagaataac agtgataatt tctgggttaa 240

ggcaatagca atatttctgc atataaatat ttctgcatat aaattgtaac tgatgtaaga 300

ggtttcatat tgctaatagc agctacaatc cagctaccat tctgctttta ttttatggtt 360

gggataaggc tggattattc tgagtccaag ctaggccctt ttgctaatca tgttcatacc 420

tcttatcttc ctcccacagc tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt 480

tggcaaagaa ttg 493

<210> 6

<211> 76

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

atcgattgaa ttccccgggg atcctctaga gtcgacctgc agaagcttgc ctcgagcagc 60

gctgctcgag agatct 76

<210> 7

<211> 479

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc 60

cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt 120

ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga 180

caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt 240

ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt 300

tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac 360

ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac 420

cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtcctt 479

<210> 8

<211> 141

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgcag g 141

<210> 9

<211> 861

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60

gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120

cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180

gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240

cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300

gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360

tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420

ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480

gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540

cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600

tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660

tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720

cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780

acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840

tcactgatta agcattggta a 861

<210> 10

<211> 4654

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgtggaat tcgctagtta ttaatagtaa 180

tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 240

gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 300

tatgttccca tagtaacgtc aatagggact ttccattgac gtcaatgggt ggagtattta 360

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 420

gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 480

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 540

tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 600

cccattgacg tcaatgggag tttgttttgc accaaaatca acgggacttt ccaaaatgtc 660

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 720

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 780

acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc cggccgggaa 840

cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 900

tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat cttatttcta 960

atactttccc taatctcttt ctttcagggc aataatgata caatgtatca tgcctctttg 1020

caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa tatttctgca 1080

tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt gctaatagca 1140

gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct 1200

gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct 1260

cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgggattcga 1320

acatcgattg aattccccgg ggatcctcta gagtcgacct gcagaagctt gcctcgagca 1380

gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg 1440

ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca 1500

ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca 1560

aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga 1620

gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc 1680

tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt 1740

tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat 1800

ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg 1860

ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga 1920

acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 1980

gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 2040

gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 2100

ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 2160

gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2220

cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 2280

aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 2340

cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 2400

ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 2460

aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 2520

ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 2580

acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 2640

cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 2700

tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 2760

ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 2820

ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2880

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2940

taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3000

cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3060

aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3120

aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3180

tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3240

ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3300

catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3360

aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3420

ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 3480

gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 3540

aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3600

gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3660

gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3720

gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3780

gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3840

gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3900

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3960

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4020

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4080

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4140

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4200

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4260

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4320

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4380

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4440

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4500

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 4560

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4620

ttcctggcct tttgctggcc ttttgctcac atgt 4654

<210> 11

<211> 306

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 60

gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 120

aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt 180

cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 240

acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc 300

tattgg 306

<210> 12

<211> 6124

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgtggaat tcgctagtta ttaatagtaa 180

tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 240

gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 300

tatgttccca tagtaacgtc aatagggact ttccattgac gtcaatgggt ggagtattta 360

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 420

gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 480

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 540

tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 600

cccattgacg tcaatgggag tttgttttgc accaaaatca acgggacttt ccaaaatgtc 660

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 720

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 780

acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc cggccgggaa 840

cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 900

tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat cttatttcta 960

atactttccc taatctcttt ctttcagggc aataatgata caatgtatca tgcctctttg 1020

caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa tatttctgca 1080

tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt gctaatagca 1140

gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct 1200

gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct 1260

cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgggattcga 1320

acatcgattg aattccacca tgatggtcag aaagcctgtt gtgtccacca tctccaaagg 1380

aggttacctg cagggaaatg ttaacgggag gctgccttcc ctgggcaaca aggagccacc 1440

tgggcaggag aaagtgcagc tgaagaggaa agtcacttta ctgaggggag tctccattat 1500

cattggcacc atcattggag caggaatctt catctctcct aagggcgtgc tccagaacac 1560

gggcagcgtg ggcatgtctc tgaccatctg gacggtgtgt ggggtcctgt cactatttgg 1620

agctttgtct tatgctgaat tgggaacaac tataaagaaa tctggaggtc attacacata 1680

tattttggaa gtctttggtc cattaccagc ttttgtacga gtctgggtgg aactcctcat 1740

aatacgccct gcagctactg ctgtgatatc cctggcattt ggacgctaca ttctggaacc 1800

attttttatt caatgtgaaa tccctgaact tgcgatcaag ctcattacag ctgtgggcat 1860

aactgtagtg atggtcctaa atagcatgag tgtcagctgg agcgcccgga tccagatttt 1920

cttaaccttt tgcaagctca cagcaattct gataattata gtccctggag ttatgcagct 1980

aattaaaggt caaacgcaga actttaaaga cgccttttca ggaagagatt caagtattac 2040

gcggttgcca ctggcttttt attatggaat gtatgcatat gctggctggt tttacctcaa 2100

ctttgttact gaagaagtag aaaaccctga aaaaaccatt ccccttgcaa tatgtatatc 2160

catggccatt gtcaccattg gctatgtgct gacaaatgtg gcctacttta cgaccattaa 2220

tgctgaggag ctgctgcttt caaatgcagt ggcagtgacc ttttctgagc ggctactggg 2280

aaatttctca ttagcagttc cgatctttgt tgccctctcc tgctttggct ccatgaacgg 2340

tggtgtgttt gctgtctcca ggttattcta tgttgcgtct cgagagggtc accttccaga 2400

aatcctctcc atgattcatg tccgcaagca cactcctcta ccagctgtta ttgttttgca 2460

ccctttgaca atgataatgc tcttctctgg agacctcgac agtcttttga atttcctcag 2520

ttttgccagg tggcttttta ttgggctggc agttgctggg ctgatttatc ttcgatacaa 2580

atgcccagat atgcatcgtc ctttcaaggt gccactgttc atcccagctt tgttttcctt 2640

cacatgcctc ttcatggttg ccctttccct ctattcggac ccatttagta cagggattgg 2700

cttcgtcatc actctgactg gagtccctgc gtattatctc tttattatat gggacaagaa 2760

acccaggtgg tttagaataa tgtcggagaa aataaccaga acattacaaa taatactgga 2820

agttgtacca gaagaagata agttatgaca gcgctgctcg agagatctac gggtggcatc 2880

cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca 2940

gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata 3000

ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg 3060

cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa 3120

tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc 3180

aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca 3240

tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca 3300

aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg 3360

taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc 3420

tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt 3480

tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg 3540

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 3600

tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 3660

gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 3720

acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 3780

agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 3840

ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 3900

ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 3960

taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 4020

aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 4080

tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 4140

tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 4200

tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 4260

atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 4320

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 4380

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 4440

agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 4500

cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4560

gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4620

cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4680

tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4740

ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4800

ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 4860

atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 4920

cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 4980

atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5040

gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5100

cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 5160

tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5220

tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 5280

gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 5340

gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 5400

atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5460

atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5520

aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5580

aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 5640

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5700

ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5760

tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5820

ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5880

acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5940

gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6000

cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6060

aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6120

atgt 6124

Claims (12)

1. Use of an adeno-associated viral vector for preparing a preparation or composition for treating retinal photoreceptor cell injury, wherein the adeno-associated viral vector comprises an expression cassette having a structure of formula I from the 5'-3' end:

Z1-Z2-Z3-Z4(I)

wherein each "-" is independently a bond or a nucleotide linking sequence;

z1 is an enhancer and/or a promoter;

z2 is an intron;

z3 is a nucleotide sequence for coding XCT/SLC7A11 protein; and

z4 is an optional hGHpA (human growth hormone poly (A) tail) sequence,

and the adeno-associated virus vector can express XCT/SLC7A11 protein in retinal cells.

2. The use of claim 1, wherein the nucleotide sequence encoding the XCT/SLC7A11 protein is as set forth in SEQ ID No. 1.

3. The use of claim 1, wherein the promoter is a tissue specific promoter.

4. The use according to claim 1, wherein the enhancer and/or promoter is CMV enhancer/promoter.

5. The use of claim 1, wherein the intron is a β -globin intron.

6. The use of claim 5, wherein the nucleotide sequence of the intron is as set forth in SEQ ID No. 5.

7. The use according to claim 1, wherein the nucleotide sequence of hGHpA is as shown in SEQ ID No. 7.

8. The use of claim 1, wherein the adeno-associated virus is AAV2.

9. The use of claim 1, wherein the adeno-associated virus is single-stranded AAV2.

10. The use of claim 1, wherein the adeno-associated viral vector has the structure of formula II:

A1-Z1-Z2-Z3-Z4-A2-A3(II)

wherein each "-" is independently a bond or a nucleotide linking sequence;

a1 is an L-ITR sequence;

z1 is CMV enhancer/promoter;

z2 is a beta-globin intron;

z3 is a nucleotide sequence for coding XCT/SLC7A11 protein;

z4 is hGHpA sequence;

a2 is an R-ITR sequence;

a3 is an optional tag sequence; and

a4 is an optional f1 ori sequence.

11. The use of claim 1, wherein the adeno-associated viral vector has the sequence shown in SEQ ID No. 12.

12. The use of claim 1, wherein the retinal photoreceptor cell injury disorder is selected from the group consisting of: retinal detachment, hereditary retinitis pigmentosa, macular degeneration, diabetic retinopathy, or a combination thereof.

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