Nucleic acids encoding rpgr and uses thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a nucleic acid encoding RPGR and application thereof. The present application provides a nucleic acid encoding RPGR, and provides a vector and an adeno-associated virus containing the nucleic acid. The nucleic acid is specially optimized so that the expression amount of RPGR is significantly improved, and thus the nucleic acid can be used for treating X-linked retinitis pigmentosa caused by RPGR mutation. Experiments show that an AAV-RPGR drug can significantly improve eye lesions of a defective mouse of X-linked retinitis pigmentosa caused by RPGR mutation.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to nucleic acids encoding RPGR and their applications. Background Technology

[0002] Retinitis pigmentosa (RP) is a phenotypic linkage group of inherited retinal dystrophys that causes a gradual decline in vision. The incidence of RP is between 1 in 4,000 and 1 in 3,000. Early symptoms of RP include decreased night vision and peripheral vision. As the disease progresses, central vision and color vision may also be affected. The age of onset of RP symptoms is variable, but it is usually between 10 and 30 years of age, and the rate of deterioration varies from person to person.

[0003] Retinitis pigmentosa (RP) is caused by mutations in one or more genes related to eye health and function. Of all the single genes causing RP, X-linked lesions due to defects in the retinitis pigmentosa GTPase regulatory gene (RPGR) are the most common. X-linked retinitis pigmentosa (XLRP) is considered the most severe form of retinitis pigmentosa. Approximately 70% of XLRP cases are caused by RPGR mutations. RPGR is located in the connective cilia of photoreceptor cells and plays a role in protein transport. More than 300 RPGR mutations have been identified. RPGR has multiple splice isoforms, with RPGR-ORF15 primarily expressed in the photoreceptor cells of the retina. RPGR gene mutations cause degeneration and deterioration of cone and rod cells in patients as early as childhood.

[0004] The RPGR gene is highly mutable, and these in vivo mutations increase the likelihood of disease. This mutable nature also makes it more difficult to clone the sequence encoding the RPGR protein into vectors for gene therapy. In fact, previously developed strategies for XLRP gene replacement therapy have also been hampered by these factors.

[0005] Therefore, developing a highly effective and specific drug is urgently needed to address the current need for treating X-linked retinitis pigmentosa caused by RPGR mutations. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a nucleic acid encoding RPGR and its application.

[0007] The nucleic acid encoding RPGR provided by this invention includes at least one of I) to IV):

[0008] I) Nucleic acids having the nucleotide sequence shown in SEQ ID NO:1;

[0009] II) Nucleic acids in which one or more nucleotides are replaced, deleted, or added to the fragment described in I);

[0010] III) The sequence of the nucleic acid described in I) has at least 90% homology and encodes the nucleic acid of RPGR;

[0011] IV) Nucleic acids that are partially or completely complementary to any one of I) to III).

[0012] In some embodiments, the nucleic acid sequence encoding RPGR is shown in SEQ ID NO:1.

[0013] The present invention also provides a recombinant vector comprising a backbone vector and the nucleic acid described in the present invention.

[0014] In this invention, the backbone vector of the recombinant vector is a viral vector. In some embodiments, the viral vector is selected from at least one of lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors; wherein the serotype of the adeno-associated virus vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2.7M8, or AAV2-TYF mutant. In some specific embodiments, its backbone vector contains an RK1 promoter and / or an SV40 intron. In embodiments of this invention, the recombinant vector includes a sequentially linked RK1 promoter, an SV40 intron, the nucleic acid shown in SEQ ID NO:1, and an SV40 poly(A) signal. Specifically, the recombinant vector includes a sequentially linked RK1 promoter, an SV40 intron, the nucleic acid shown in SEQ ID NO:1, and an SV40 poly(A) signal. The nucleic acid sequence of the RK1 promoter is shown in SEQ ID NO:4, and the nucleic acid sequence of the SV40 intron is shown in SEQ ID NO:5.

[0015] The present invention also provides a plasmid assembly comprising the recombinant vector, an auxiliary functional plasmid, and an accessory functional plasmid. The auxiliary functional plasmid is pAdHelper; the accessory functional plasmid is pAAV-r2c5.

[0016] This invention also provides a method for preparing adeno-associated virus expressing RPGR, comprising: transfecting the aforementioned plasmid combination into host cells, and purifying the resulting adeno-associated virus expressing RPGR. The host cells are 293 cells or 293T cells.

[0017] The adeno-associated virus expressing RPGR was prepared by the preparation method described in this invention.

[0018] The present invention relates to the use of the recombinant vector, plasmid combination, or adeno-associated virus described herein in the preparation of drugs for the prevention and treatment of eye diseases. In this invention, the eye disease is a disease caused by RPGR mutation. In some embodiments, the eye disease is retinitis pigmentosa. In some embodiments, the prevention and treatment include repairing retinal structure, increasing the number of photoreceptor cells, and / or improving eye function.

[0019] The present invention also provides a drug comprising the recombinant vector, plasmid combination, or adeno-associated virus as described in the present invention.

[0020] The medicament described in this invention also includes pharmaceutically acceptable carriers and excipients. In some embodiments, the excipients are nanocarriers and / or liposomes, and the dosage form of the medicament is an injection, wherein the content of the adeno-associated virus is 1 × 10⁻⁶. 9 ~1×10 16 One virus / mL, and in some embodiments, the adeno-associated virus content is 1 × 10⁻⁶. 12 ~1×10 14 Virus / mL. In some specific embodiments, the adeno-associated virus titer is 1 × 10⁻⁶. 13 vg / mL.

[0021] The administration methods of the drug described in this invention include subretinal injection, intravitreal injection, anterior chamber injection, or subconjunctival injection.

[0022] The drugs described in this invention also include other drugs with activity in improving retinitis pigmentosa.

[0023] The present invention also provides a method for preventing and treating retinitis pigmentosa, which involves administering the drug described in the present invention.

[0024] This invention provides a nucleic acid encoding RPGR and a vector and adeno-associated virus containing this nucleic acid. This encoding nucleic acid has been specifically optimized to significantly increase RPGR expression, thus enabling its use in the treatment of X-linked retinitis pigmentosa caused by RPGR mutations. Experiments show that the AAV-RPGR drug can significantly improve ocular lesions in mice with RPGR mutation-induced X-linked retinitis pigmentosa. Attached Figure Description

[0025] Figures 1A to 1D This is a comparison of the codon-optimized RPGRORF15 with the wild-type sequence. The differential codon sequences after optimization are bolded and underlined.

[0026] Figure 2The diagram shows the AAV-RPGR vector: A is a schematic diagram of the codon-optimized RPGRORF15 plasmid vector, which contains AAV2 5'ITR, RK1 promoter, SV40 intron, codon-optimized RPGR ORF15, SV40 polyA sequence, and AAV2 3'ITR; B is a schematic diagram of the wild-type RPGR ORF15 plasmid vector, which contains AAV2 5'ITR, RK1 promoter, SV40 intron, wild-type RPGR ORF15, SV40 polyA sequence, and AAV2 3'ITR.

[0027] Figure 3 The cloning and restriction enzyme digestion verification of the AAV-RPGR vector are shown. A represents the transformation of the optimized AAV-RPGR ORF15 plasmid into the Stbl3 *E. coli* strain; B represents the transformation of the wild-type AAV-RPGR ORF15 plasmid into the Stbl3 *E. coli* strain; C represents the extraction of plasmids from single clones and identification by double digestion with HindIII and XhoI, verifying the transformation efficiency and sequence integrity of the codon-optimized vector compared to the wild-type vector; 1-5: Restriction enzyme digestion results of the codon-optimized plasmid; M: 10kb Marker; 6-10: Restriction enzyme digestion results of the wild-type plasmid.

[0028] Figure 4 The expression of the optimized RPGR sequence-encoded protein in vitro was verified. In A, the AAV-RPGR plasmid was transfected into the mouse cone cell line 661W. After 48 hours, the cells were lysed and the expression level of RPGR protein was detected. The difference in expression of opt1, opt2 and wild-type RPGR protein after codon optimization was detected. In B, 661W cells were infected with AAV5-RPGR-opt1 virus with different multiplici of infection (MOI = 1E4, 3E4, 1E5). After 72 hours, the cells were lysed and the expression level of RPGR mRNA was detected by qPCR.

[0029] Figure 5 This demonstrates the validation of AAV-mediated RPGRopt1 protein expression levels in mice;

[0030] Figure 6 This study compares the expression efficiency of RPGR in mice and observes the distribution of RPGR protein in the retina using immunofluorescence staining. In A, RPGR antibody is stained after retinal planarization to observe the distribution and density of RPGR positive signals in the retina, and in B, the overall proportion of RPGR positive cells in the retina is observed.

[0031] Figure 7 Displaying the tissue localization of RPGR in mice;

[0032] Figure 8This study demonstrates the therapeutic effect of AAV5-RPGRopt gene therapy on X-linked retinal pigment abnormalities caused by RPGR mutations. A shows the electroretinogram (ERG) analysis of the drug-treated and control eyes of RPGR knockout mice 18 months after injection. Under light conditions, progressively increasing light stimulation was applied, and the b-wave amplitude of each mouse (n=20) and control eye was recorded under different light stimulation intensities. B shows the ERG analysis of the drug-treated and control eyes of RPGR knockout mice 18 months after injection. Under dark conditions, progressively increasing light stimulation was applied, and the b-wave amplitude of each mouse (n=20) and control eye was recorded under different light stimulation intensities. C shows the quantitative analysis of the outer nuclear layer thickness (n=20) of the retina in the drug-treated and control eyes of RPGR knockout mice 18 months after injection. Detailed Implementation

[0033] This invention provides nucleic acids encoding RPGR and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0034]

[0035] Analysis and experimental screening showed that, compared to other optimized sequences, the nucleic acid shown in SEQ ID NO:1 exhibited increased transcriptional levels and significantly enhanced expression, thus providing a more significant therapeutic effect on retinitis pigmentosa. Wild-type or other optimized sequences were less effective than the nucleic acid shown in SEQ ID NO:1.

[0036] This invention utilizes adeno-associated virus (AAV) vectors for gene replacement therapy, which has proven effective in rescuing retinal function and structure in numerous animal models of retinal degeneration. A recombinant vector is provided in which the RPGRORF15 coding region is placed under the control of a tissue-specific promoter, and its sequence is codon-optimized, providing an active RPGR protein at near-wild-type levels for X-chromosome-linked retinitis pigmentosa caused by RPGR mutations. First, the constructed AAV-RPGR plasmid is transformed into *E. coli*, and after single-colony amplification, plasmid extraction, enzyme digestion, and sequencing are performed to verify the efficiency of RPGR sequence cloning and the integrity and consistency of the RPGR sequence. Second, the plasmid is transfected into the mouse cone cell line 661W, and the expression efficiency of the RPGR protein is higher than that of the wild-type protein. When the RPGR protein, controlled by the RK1 promoter, is packaged into a virus and used to infect 661W cells, its expression increases with increasing viral dose. Subsequently, C57 mice were injected subretinally with the drug to compare the in vivo expression efficiency of RPGR before and after optimization, and the correct localization and expression of RPGR protein in retinal tissue were detected. Finally, the efficacy of drug treatment in RPGR knockout mice was evaluated. Electroretinography analysis showed improved eye function in treated mice; OCT results showed that the outer nuclear layer thickness of the retinal tissue in treated eyes was significantly greater than that in untreated eyes, indicating a significant increase in the overall number of viable photoreceptor cells. In summary, in vitro experiments demonstrated the efficiency of the AAV5-RPGRopt1 drug, and in vivo mouse experiments demonstrated that AAV-mediated gene therapy can effectively treat X-linked retinitis pigmentosa caused by RPGR mutations.

[0037] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0038] Example 1: Cloning and Enzyme Digestion Verification of the AAV-RPGR Vector

[0039] The wild-type RPGRORF15 coding sequence underwent codon optimization, denoted as RPGRopt1 and RPGRopt2, respectively. The alignment of RPGRopt1 with the wild-type sequence is as follows: Figures 1A-1D And constructed the corresponding AAV vector plasmid ( Figure 2Plasmids containing codon-optimized RPGRORF15 and wild-type RPGR ORF15 sequences (200 ng / μL) were transformed into Escherichia coli Stbl3 strain and plated on LB agar plates supplemented with the corresponding antibiotics. After overnight incubation, the growth of single colonies was observed.

[0040] Plasmid transformation of E. coli:

[0041] Mix the following reaction mixture thoroughly, place on ice for 20 min, then at room temperature for 10 min. Add 500 μL of antibiotic-free LB and incubate at 37°C and 200 rpm on a shaker for 40 min. Centrifuge at 5000 rpm for 3 min, discard 500 μL of supernatant, resuspend the bacterial pellet in the remaining liquid, and spread evenly on the corresponding antibiotic-resistant LB agar plates. Incubate the plates overnight at 37°C.

[0042] 5X KCM 10μL plasmid 5-20μL <![CDATA[ddH2O]]> Add to 50μL competent cells 50-100μL

[0043] like Figure 3 As shown, for the same concentration of plasmid DNA, the number of single colonies on the optimized plasmid plate is higher than that on the wild-type plasmid plate. Figure 3 A in Figure 3 The B in the figure indicates that the cloning efficiency of the codon-optimized RPGR ORF15 sequence is higher than that of the wild type.

[0044] Small-scale extraction of plasmid DNA:

[0045] 1. Pick a single colony and place it in an appropriate amount of LA liquid medium (the amount of antibiotic added depends on the plasmid resistance), and incubate overnight (16-20 h) at 37°C and 200 rpm with shaking.

[0046] 2. Aliquot the culture medium into 1.5mL EP tubes, 1mL per tube, centrifuge at 12000rpm for 3min, and discard the supernatant by inverting the EP tubes;

[0047] 3. Add 100 μL of solution I and vortex until the cells are fully suspended; add 200 μL of solution II and gently invert to mix until the liquid is clear and viscous; add 150 μL of solution III and quickly invert to mix, place on ice for 5 min, and then centrifuge at 12000 rpm for 10 min.

[0048] 4. Transfer the supernatant to a new EP tube, add 2 volumes of anhydrous ethanol to precipitate the DNA, invert and mix well, and incubate at -20°C for 30 min; centrifuge at 12000 rpm for 10 min, discard the supernatant, add 180 μL of 70% ethanol, centrifuge at 12000 rpm for 2-3 min, and discard the supernatant.

[0049] 5. Air dry the plasmid DNA at room temperature, dissolve it in TE buffer, and store at room temperature. For short-term storage, store at 4°C, and for long-term storage, store at -20°C.

[0050] Enzyme digestion verification of plasmid DNA:

[0051] Prepare the reaction solution according to the following system, incubate at 37℃ for 1-4 hours, and take an appropriate volume for electrophoresis detection.

[0052] 1X Buffer 2 plasmid DNA 1-2 Hind III 1 XhoI 1 <![CDATA[ddH2O]]> Add to 20μL

[0053] After double enzyme digestion of the plasmids, the band sizes were observed by electrophoresis. It was found that the optimized plasmid digestion results showed that RPGR ORF15 maintained its intact size, while the wild-type plasmid digestion results showed that RPGRORF15 failed to maintain its intact size. Figure 3 The result (C) indicates that the codon-optimized RPGRORF15 exhibits higher fidelity during the cloning process than the wild type.

[0054] Example 2: Expression of AAV-mediated RPGR in vitro and in mice

[0055] Plasmids containing codon-optimized RPGRORF15 sequences and wild-type RPGRORF15 sequences were transfected into 661W cells, respectively. After 48 hours, the cells were lysed, and the protein expression level was confirmed by Western blotting after extraction.

[0056] I. Cell transfection:

[0057] 1. The day before transfection, trypsin digests the cells and counts them. The cells are then plated to achieve a density of 90% on the day of transfection.

[0058] 2. For each well of cells, dilute the DNA with 50 μl of serum-free DMEM medium.

[0059] 3. Mix the diluted DNA and diluted LIPOFECTAMINE 2000 and incubate at room temperature for 20 minutes.

[0060] 4. Add the complex directly to each well, shake the culture plate, and mix gently.

[0061] 5. Incubate at 37°C and 5% CO2. After adding the complex to the cells for 24-72 hours, analyze the cell extract or perform in situ cell staining to detect reporter gene activity.

[0062] II. Viral infection of cells:

[0063] 1. Digest HEK293T cells and plate them in 6-well plates.

[0064] 2. After culturing overnight, take cells from a portion of the wells and infect them with the target virus and its control virus at an appropriate MOI.

[0065] 3. Cells were collected 36-48 hours after infection for subsequent experiments.

[0066] III. mRNA content determination by qPCR: Protein expression was detected by Western blotting after total RNA extraction, reverse transcription, and quantitative PCR.

[0067] 1. Protein sample preparation: Add PMSF to the lysis buffer at a ratio of 1:100 (prepare fresh for each use).

[0068] 2. Lyse cells using a strong lysis buffer.

[0069] 3. Protein concentration was determined using the BCA method.

[0070] 4. Electrophoresis

[0071] a. Prepare the appropriate separating gel (5ml / block) according to the size of the protein being tested, and wait for the separating gel to solidify.

[0072] b. Prepare 5% concentrated gel (2ml / block), fill the glass plate, and insert the comb.

[0073] c. Add 5 μl of pre-stained protein molecule marker SDS-PAGE to the sample well, and use 10 μl of 1x SDS-PAGE protein loading buffer to load the sample into the blank sample well next to the sample well.

[0074] 5. Transfer membrane

[0075] Place a wet pad on the white clamp for transfer, then place three sheets of wet filter paper stacked together on the pad. On the filter paper, place the wet PVDF membrane, gel, filter paper, pad, and black clamp in sequence. Place the assembled clamp into the electrophoresis tank containing transfer buffer and place the transfer tank in an ice bath for transfer.

[0076] 6. Enclosed

[0077] After transfer, rinse for 1-2 minutes, remove all buffer solution with a dropper, add 5% skim milk powder, and gently shake on a side-swinging shaker. Block at room temperature for 15-60 minutes. Wash with TBS washing buffer for 5 minutes. Repeat the washing process 3 times.

[0078] 7. Primary antibody incubation

[0079] Dilute an appropriate amount of primary antibody with 5% skim milk powder / PBS + 2% BSA according to the specified ratio, and incubate overnight at 4°C with gentle shaking, or at room temperature on a side-shaking shaker for 2 hours. Wash after incubation.

[0080] 8. Secondary antibody incubation

[0081] Add the diluted secondary antibody and incubate on a side-shaking incubator at room temperature for 40 minutes to 1 hour. Wash after incubation.

[0082] 9. Protein detection

[0083] To detect proteins using ECL reagents, mix 1 ml of each reagent and drop the mixture onto the protein membrane surface. Incubate in the dark for 1-2 minutes. Use tweezers to neatly arrange the protein membrane on a plastic sheet and expose it to light using a gel imaging system.

[0084] like Figure 4 As shown, the expression level of codon-optimized RPGRopt1 protein was higher than that of wild-type and optimized sequence RPGRopt2 (…). Figure 4 In the A section, the in vitro expression level of the optimized RPGRopt1 protein was increased by 3.6 times compared with the wild-type sequence (Table 1).

[0085] Table 1. Relative gray values ​​of protein bands

[0086] RPGRopt1 4.6 RPGRopt2 1.1 RPGRwt 1

[0087] The optimized AAV-RPGRopt1 vector was used to package AAV5 serotype virus. 661W cells were infected with different multiples of infection (MOIs). After 72 hours, cells were lysed and RNA was extracted. qPCR was used to confirm mRNA expression levels. The results showed that the expression level of RPGRopt1 mRNA in vitro increased with increasing viral dose, exhibiting a significant dose-dependent effect. Figure 4 (See B in Table 2).

[0088] Table 2. Expression levels of RPGR mRNA in 661W cells after infection with different doses of virus.

[0089] PBS 1 AAV-GFP 0.89 AAV-RPGRopt1(1E4) 310.84 AAV-RPGRopt1(3E4) 1046.95 AAV-RPGRopt1(1E5) 2779.96

[0090] IV. Virus packaging and viral drug injection into mice:

[0091] 1. HEK293T cells with a polymerization degree of 90% or higher were transferred to discs at a ratio of 1:3.

[0092] 2. About 1-2 hours before plasmid transformation, switch to serum-free medium and use transfection reagent to transform the target gene plasmid and helper plasmid into HEK293T.

[0093] 3. Replace with fresh serum-free culture medium 24 hours after plasmid transformation.

[0094] 4. Harvest the virus 72 hours after transfection. With the culture medium attached, aspirate the cells and centrifuge; then harvest the culture medium supernatant and cell pellet separately. Precipitate the virus in the culture medium supernatant using PEG8000, and collect the virus pellet after incubation overnight.

[0095] 5. The virus mixture was purified by density gradient centrifugation with iodixanol, and then concentrated by ultrafiltration.

[0096] 6. Construct humanized RPGR knockout mice.

[0097] 7. Prepare 1×10 13 AAV-RPGRopt1 drug at a concentration of vg / ml was diluted to different doses.

[0098] 8. Administer 1 μl / eye of AAV-RPGRopt1 drug and PBS subretinally into the retinal region of mice.

[0099] The AAV5-RPGRopt1 antiviral drug was administered subretinally to the eyes of 6-8 week old C57 mice. Four weeks after administration, the retina was harvested, and the protein in the tissue was extracted for Western blotting to observe the expression of RPGR in the mouse retinal tissue.

[0100] like Figure 5 As shown, RPGR protein expression was detected in the treated eyes of knockout mice, while the corresponding protein expression was not detected in the control eye tissues, and the protein expression level also showed a linear relationship with the drug dose.

[0101] V. Immunofluorescence staining:

[0102] 1. Wash the slices or samples twice with PBS, 5 minutes each time.

[0103] 2. Discard the PBS, add 200 μl of 1% Triton to each well and punch for 15 min. After punching, wash three times with 0.05% Triton.

[0104] 3. Add 30 μl of Beyotime blocking solution to each sample and let it stand at room temperature for 2 hours to block.

[0105] 4. Add 30 μl of the appropriate concentration of primary antibody (1:200) to each sample. Place the entire sealing film in a humidified chamber and incubate overnight at 4°C.

[0106] 5. Wash three times with PBS containing 0.05% Triton, each time for 5 minutes.

[0107] 6. Add 40-50 μl of a suitable concentration of secondary antibody (1:1000) to each sample. Place the entire sealing film in a humidified chamber and let it stand at room temperature for 1 hour.

[0108] 7. Wash three times with PBS containing 0.05% Triton.

[0109] 8. Add 30 μl of DAPI staining solution to each sample and stain for 15 min.

[0110] 9. Wash three times with PBS.

[0111] Add 15 μl of anti-fluorescence quenching mounting solution to the coverslip, carefully seal the coverslip onto the slide, and then seal the slide with nail polish.

[0112] like Figure 6 As shown, retinal patch results indicate that the optimized RPGRopt1 drug has a larger distribution area on the retina, and the density of positive signal per unit area is higher than that of wild-type RPGR drug and the optimized sequence RPGRopt2 (…). Figure 6 The "A" in the figure indicates higher protein abundance. Calculations showed that the wild-type drug accounted for 7.82% of retinal expression, the optimized drug RPGRopt2 accounted for 15.7%, and the optimized drug RPGRopt1 accounted for 36.3%. Figure 6 (See B in Table 3).

[0113] Table 3. Percentage of positively expressed cells in the whole retina

[0114] RPGRwt 17.7％ 44.2％ 7.82％ RPGRopt1 67.8％ 53.5％ 36.3％ RPGRopt2 34.6％ 45.6％ 15.7％

[0115] like Figure 7 As shown, the sectioning results indicate that the RPGR protein (red fluorescent signal, indicated by the arrow) can be correctly localized in the intracellular segment (IS) of photoreceptor cells, and the optimized drug opt1 has a stronger signal than the optimized drug opt2 and the unoptimized drug.

[0116] The above results indicate that the AAV-RPGRopt1 viral drug can correctly express the protein in vivo, and the optimized drug has a significantly improved expression efficiency in vivo compared with the unoptimized drug.

[0117] Example 3: AAV-RPGRopt1 gene therapy improves ocular function and repairs retinal structure in RPGR knockout mice.

[0118] Examples 1-2 confirmed the correct expression of the RK1 promoter-controlled codon-optimized RPGRORF15 coding sequence in vitro and in vivo. To further demonstrate the therapeutic effect of AAV5-RPGRopt1 gene therapy on X-linked retinitis pigmentosa caused by RPGR mutation, in vivo experiments were conducted using an RPGR knockout mouse model. The improvement of ocular lesions in mice was observed 18 months after injection.

[0119] First, we used electroretinography (ERG) analysis to evaluate the function of the drug-treated eye and the control eye.

[0120] The methods for virus packaging and viral drug injection into mice are the same as described above.

[0121] I. Electroretinogram Analysis:

[0122] 1. Mice were anesthetized and their pupils were dilated. 2.5% hydroxypropyl methylcellulose solution containing electrodes was instilled into the eyes, and corneal potential responses were recorded.

[0123] 2. To acclimate mice to overnight darkness under dark adaptation conditions, use an LED light source to provide -2 to +3 logsc cd.s / m 2 Brief flashes of light of varying intensity were used to record dark-adapted ERGs. Responses were recorded at intervals of 3 to 60 seconds, depending on the intensity of the stimulus.

[0124] 3. Under light adaptation conditions, LED lights provide -0.5 to +2 logsc cd.s / m 2 A brief flash of light of intense intensity was applied, and the light-adapted ERG was recorded, with the response recorded at 2-minute intervals.

[0125] like Figure 8 As shown, under illumination conditions, as the intensity of light stimulation increased, the amplitude of the b-wave in the treated eye gradually increased compared to the control eye. Figure 8 (A in Table 4); Under dark conditions, the b-wave amplitude of the treated eye was significantly higher than that of the control eye at different light stimulation intensities (P<0.5), and close to that of the wild type. Figure 8 (See B in Table 5), which shows that the drug treatment has a significant effect on improving eye function.

[0126] Table 4. Amplitude of b-wave in mouse ERG ocular ERG under light conditions.

[0127]

[0128] Table 5. Amplitude of b-wave in mouse ERG ocular ERG under dark conditions.

[0129]

[0130] Subsequently, the mouse's eye tissue was used for OCT analysis.

[0131] II. Mouse OCT Detection

[0132] 1. Animal preparation

[0133] After anesthetizing the animal, instill pupil dilation solution and keep it in place for 2-3 minutes. Then, gently wipe away the pupil dilation solution with a cotton swab and apply carbomer gel to keep the animal's eyes moist.

[0134] When examining the left eye, place the mouse slightly to the right of the center of the platform in front of the camera, and adjust the mouse's position so that the left eye is directly facing the camera lens; similarly, when examining the right eye, place the mouse slightly to the left of the center of the platform in front of the camera, and adjust the mouse's position so that the left eye is directly facing the camera lens.

[0135] 2. Image Acquisition

[0136] In this experiment, two modes were selected: IR and IR+OCT.

[0137] Select IR mode (30° lens), locate the central region of the mouse's optic disc, acquire and save the image.

[0138] 3. Image analysis of the outer kernel layer

[0139] Mark the layers whose thickness you want to measure with two horizontal red lines. Move the green vertical line to positions of 45°, 90°, 135°, 225°, 270° and 315°, and record the numbers displayed on the right side of the green vertical line.

[0140] The thickness of this layer in the retinas of different mice was collected into an Excel file, the average value was calculated, and the data were analyzed.

[0141] Quantitative analysis of the outer nuclear layer thickness showed that the outer nuclear layer thickness in the treated eye was significantly greater than that in the control eye. Figure 8 The results in C (Table 6) indicate that more photoreceptor cells were preserved in the retina of the treated eye after drug injection, while the structure of the retina was basically maintained.

[0142] Table 6. Thickness of the outer nuclear layer (ONL) of the mouse retina

[0143]

[0144] In summary, the AAV-RPGRopt1 gene therapy drug, by providing a normally functioning protein in RPGR knockout mice, compensates for the adverse effects of gene deletion on ocular lesions. We have demonstrated the therapeutic effect of the AAV-RPGRopt1 gene therapy drug on X-linked retinitis pigmentosa caused by RPGR mutations, laying the foundation for further clinical application development.

[0145] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Wuhan NewFos Biotechnology Co., Ltd. <120> Nucleic Acid Encoding RPGR and Uses Thereof <130> MP22012736 <160> 5 <170> SIPOSequence Listing 1.0 <210> 1 <211> 3456 <212> DNA <213> Artificial Sequence <400> 1 atgagagaac ccgaggaact gatgccagat tcaggggctg tctttacctt cggcaagtct 60 aagtttgctg aaaacaatcc tggaaagttc tggtttaaaa acgacgtccc cgtgcacctg 120 tcttgtggcg atgaacattc cgccgtggtc acggggaaca acaagttgta tatgtttggt 180 tccaataact ggggccagtt gggcctcggc tcaaagagcg ctataagcaa acctacatgc 240 gtgaaagccc tcaagcctga aaaagtcaag ttggcagcct gcggacggaa ccacaccctt 300 gtgtcaactg agggcggcaa cgtttacgcc acgggtggaa ataacgaggg ccagcttggg 360 ctcggcgata cagaggagcg aaatactttc catgttatat ccttttttac atctgaacac 420 aagattaagc aattgagtgc cggaagcaat acatcagcag ccctgacaga ggatggtaga 480 ttgtttatgt ggggggataa ttccgagggt cagatcggac tgaagaatgt gagcaatgtg 540 tgtgtacccc agcaggtgac tatcggcaaa ccagtctctt ggatctcctg cgggtactat 600 cattctgcct ttgtgaccac ggacggcgaa ctgtatgttt tcggagagcc agaaaatgga 660 aaactgggtc tgcccaatca gctgttgggc aatcacagaa ccccgcagtt ggtgagtgaa 720 attccagaga aggtaataca ggtggcgtgc ggtggcgagc ataccgttgt tctgaccgag 780 aatgcagtgt atacgtttgg gctcggtcaa ttcggacaac tcggactggg aaccttcctg 840 tttgaaacga gcgagcccaa agtgattgag aacataagag atcagacaat cagctatatc 900 tcttgcggcg aaaaccatac cgcactgatc acagatatcg gtctcatgta cacttttggc 960 gatgggcgcc atggaaaatt ggggctgggc ctggaaaatt tcactaatca cttcataccg 1020 actctgtgca gtaattttct tcgatttatt gtaaaacttg tggcatgtgg cggctgccac 1080 atggtcgttt tcgctgcacc acacaggggc gtcgctaaag aaattgaatt tgacgagatc 1140 aacgacacct gtctttccgt ggcgacattc cttccctact caagcctcac atctggaaat 1200 gtgctccaga ggacactctc agctcgaatg agacgcaggg aaagagagcg aagcccagac 1260 agcttttcta tgagacgcac acttcctccc atcgagggga ctttggggact tagtgcttgt 1320. tttctgccta attctgtctt tccgagatgt agcgagagga acctccagga atccgtgttg 1380 tccgagcagg atctgatgca gccagaagag cctgattacc tgctcgatga gatgactaag gaggctgaga tcgataatag ttccaccgtg gaatccctcg gcgagaccac tgacattctc aatatgacac atatcatgtc cctgaactcc aatgaaaagt cactgaagct ctctccagtg caaaaacaga agaagcagca gacaatcgga gagctgactc aagacaccgc cctgacaga aatgacgaca gcgacgaata cgaagagatg tcagagatga aggagggcaa ggcatgtaaa 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. gatgaagagg tggaaatccc tgaggaaaag gagggggctg aggactccaa ggggaatggg attgaggagc aggaggttga ggcaaacgaa gaaaacgtta aggtccatgg gggcagaaag gagaaaactg agattctttc agatgacctg actgataagg ctgaggtttc cgaggaaa gccaaaagcg taggggaggc tgaggatggc cccgaagga gaggtgatgg cacatgtga gagggcagca gtggtgccga acactggcag gacgggagagagaaggg ggaaaggat 2040 aagggggcgcg gagagatgga aagacctgga gaagggaga aggaactcgc cgaaaaggaa 2100 gagtggaaaa agcgggatgg agagagcag gaacaaaagg aaagggagaca gggacaccag 2160 aaggagcaa atcaggaaat ggagaagggt ggtgaagg agcacggaga gggtgagaa 2220 gaaagggcg atagggaa ggagaggag aaagaaggcg agggaaaga ggagggaaa 2280 ggggaggagg ttgaagggga gagggaaaag gagagggcg aaagaaagaa agaagagcgg 2340 gctggcaaag aggaaaaagg cgaagaagaa ggagatcagg gcgaagggga gggaagaagag 2400 actgaggga gaggggaga gaggagggagagggtgaag tggaggggg agaggttgaa 2460 gagggcaagg gcgagcggga agagaagaa gagaggggag agagaagaa ggagaagggg 2520 gaggggaaag aagagaagg cgaaggggaa gaagagaag gggaaggtaa gggtgaagag 2580 gaaggcgagg agggggaagg cgaaggaa ggagaagagg gtgaaggtga aggcgaagaa 2640 gaaaggtg aaggagaggg cgagaagaa ggagaggcg aaggcgaaga agaagaggc 2700 gagggagggagggagggagggggggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2760 Gagagagggagggagggagggagggagggagggagggagggagggagggagggagg 2820 gggaagatg gcgaggggcga gggaggg gggaagggg gggaagg gggaagg 2880 gagggaag gtgaggtga gagggagggagggagggagggagggggggagggagggg 2940 ggcgaagaag agaggggtga gaggaagg gagggagg agggagg agaggcgaa 3000 wag wag wag wag wag wag wag wag wag 3060 gttgagggagg aagttgagggg cgagagggagagagggagagagagagagagagagagagaa 3120 Gaggagggagggggggagggagggaggggggaaataggcgg aaatagggagg 3180 gaggaagg aggagggggg taagtatcag gagaactggcg aggaaaa cgaaggcag 3240 gacggcgaag agtacaaaa agtgagtaaa attaaggct ccgtgaagta cggtaaacat 3300 aagacctacc agaaaaagtc cgttacaaat acacaaggta acgghagga acagcgatct 3360 aagatgcccg tccagtcaa acgactcctg aagaatgggc ctagtgggag caaaaaatttc 3420 tggaataatg tcctccctca ctatctggaa ctcaag 3456 <210> 2 <211> 3456 <212> DNA <213> Human (homo sapiens) <400> 2 atgagggagc cggaagagct gatgcccgat tcgggtgctg tgtttacatt tgggaaaagt 60 aaatttgctg aaaataatcc cggtaaattc tggtttaaaa atgatgtccc tgtacatctt 120 tcatgtggag atgaacattc tgctgttgtt accggaaata ataaacttta catgtttggc 180 agtaacaact ggggtcagtt aggattagga tcaaagtcag ccatcagcaa gccaacatgt 240 gtcaaagctc taaaacctga aaaagtgaaa ttagctgcct gtggaaggaa ccacaccctg 300 gtgtcaacag aaggaggcaa tgtatatgca actggtggaa ataatgaagg acagttgggg 360 cttggtgaca ccgaagaaag aaacactttt catgtaatta gcttttttac atccgagcat 420 aagattaagc agctgtctgc tggatctaat acttcagctg ccctaactga ggatggaaga 480 ctttttatgt ggggtgacaa ttccgaaggg caaattggtt taaaaaatgt aagtaatgtc 540 tgtgtccctc agcaagtgac cattgggaaa cctgtctcct ggatctcttg tggatattac 600 cattcagctt ttgtaacaac agatggtgag ctatatgtgt ttggagaacc tgagaatggg 660 aagttaggtc ttcccaatca gctcctgggc aatcacagaa caccccagct ggtgtctgaa 720 attccggaga aggtgatcca agtagcctgt ggtggagagc atactgtggt tctcacggag 780 aatgctgtgt atacctttgg gctgggacaa tttggtcagc tgggtcttgg cacttttctt 840 tttgaaactt cagaacccaa agtcattgag aatattaggg atcaaacaat aagttatatt 900 tcttgtggag aaaatcacac agctttgata acagatatcg gccttatgta tacttttgga 960 gatggtcgcc acggaaaatt aggacttgga ctggagaatt ttaccaatca cttcattcct 1020 actttgtgct ctaatttttt gaggtttata gttaaattgg ttgcttgtgg tggatgtcac 1080 atggtagttt ttgctgctcc tcatcgtggt gtggcaaaag aaattgaatt cgatgaaata 1140 aatgatactt gcttatctgt ggcgactttt ctgccgtata gcagtttaac ctcaggaaat 1200 gtactgcaga ggactctatc agcacgtatg cggcgaagag agagggagag gtctccagat 1260 tctttttcaa tgaggagaac actacctcca atagaaggga ctcttggcct ttctgcttgt 1320 tttctcccca attcagtctt tccacgatgt tctgagagaa acctccaaga gagtgtctta 1380 tctgaacagg acctgca gccagaggaa ccagattatt tgctagatga atgaccaaa 1440 gaagcagaga tagatattc ttcaactgta gaagccttg gagaaactac tgatatctta 1500 aacatgacac acatcatgag cctgaattcc aatgaaaagt cattaaaatt atcaccagtt 1560 cagaaaaaaaaaaaaaaaatggg gaactgacgc aggatacagc tcttactgaa 1620 aacgatgata gtgatgaata tgaagaatg tcagaatga aagagggaa agcatgtaaa 1680 cacatgtgt cacaagggat ttcatgacg cagccagcta cgactatcga agcattttca 1740 Gaggaggagagggagatccc gaggagagggagggagggaggcaaagaagaaaagggaa 1800 attagaggagc agaggtaga agcaatgag gaaatgtga aggtgcatgg aggaaggaag 1860 gagaaaacag agatcctac agatgacctt agahaaag cagaggtgag tgaggcag 1920 gcaaaatcag tgggaagc agaggatggg cctgaaggta gagggatgg aacctgtgag 1980 gaggtagtt caggaaga acactggca gatgaggaga gggagaaggg gggaagagac 2040 aagggtagag gagaatga gaggchagg gagggagaga aggtagc gagaagaa 2100 gatgaga agagggatgg ggagagcag gagcaaagg agaggca gggccatcag 2160 Noooooooooooooooooooooooooooooooooooo 2220 Gaggagggagggagggagggagggagggagggagggagggagagagagagagagagagagaga 2280 gggaaag tggagggaga acgtgaaaag gaggaagg gaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa has Gcgggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2400 ahaggaggga gagggagga aaaaaaaaaaaaaaaaaaaaaaaaaaggzag outside 2460 gagggagggagggagggagggagggagggagggagggagggagggagggg 2520 gagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2580 gagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2640 Gagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggg 2700 gagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2760 gggagggg agggagggggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2820 gggagggag gagagggagggagggagggagggagggagggagggagggagggagggagggag 2880 gagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 2940 wag wag wag wag wag wag wag wag wag wag wag 3000 gagagagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggg 3060 gtgaagggg gagggagggagggagggagggagggagggagggagggagggagggagggagg 3120 Gaggagggagggagggagggagggagggaaaaaaaaahgaaaaaaaahgaaaaaaaahgaaa 3180 gaggagggggagggagggagggagggagggagggagggagggagggagg 3240 gatgagagg agtacaaaa agtgagacaa aaaaggat ctgtgaaata tggcaacat 3300 aaaaacatac aaaaaaagtc agttactac acacagggaa atgggaaga gcagaggtcc 3360 aaaatgccag tccagtcaa acgacttta aaaaacggggc catcaggttc caaaagttc 3420 tggaataatg tattaccaca tgacttgga tgag 3456 <210> 3 <211> 3456 <212> DNA <213> Artificial Sequence <400> 3 atgagggagc ccgaggagct gatgccagac tctggggcag tgttcacctt cggtaagagt 60 aagtttgctg aaaacaatcc cggaaagttc tggtttaaaa acgacgtacc cgttcatttg 120 tcttgcggag atgagcacag cgctgtagta acaggcaata acaagttgta tatgtttgga 180 agcaataatt ggggccaact tgggctggga agtaaatccg caatttctaa gcctacttgc 240 gtaaaagcat tgaagccgga aaaagtaaaa cttgcggcgt gcgggcgcaa tcacactctc 300 gtctccacag agggtggaaa tgtatatgca accggaggga ataacgaggg ccagctgggc 360 cttggcgata cagaagagcg aaataccttt catgtaatat ctttttttac ctccgaacat 420 aagatcaagc agctctctgc cggcagcaac acatccgctg cactcacaga ggacggtcga 480 ctcttcatgt ggggggacaa ttccgaaggg cagatagggc tcaagaatgt atcaaacgtg 540 tgtgtaccgc agcaggttac tatcggcaag cccgtgtctt ggatcagctg cggctattac 600 catagcgctt tcgtaactac ggatggagag ctctatgtat ttggcgaacc tgaaaatggc 660 aaactcggcc tccctaacca gttgctgggc aatcatagaa cccctcaatt ggtcagcgaa 720 ataccagaaa aagtaattca ggtagcgtgc ggtggggaac atacagtggt acttacggaa 780 aacgctgtat acacttttgg gcttggacag ttcggtcaac tgggacttgg gacatttctt 840 tttgaaacgt cagagcccaa ggttatgag aatatacggg accaaacaat atcttacatc 900 agttgcggag aaaaccatac ggctctgatt actgatatcg gtctgatgta taccttcggg 960 gatggcaggc acggaaaact cggattgggt ctggagaact tcaccaacca cttcattcct 1020 accttgtgca gcaatttcct ccggttcatc gtaaaactcg tcgcttgtgg cggatgccat 1080 atggttgtat tcgccgctcc ccatcgaggt gtggctaaag aaattgagtt tgacgaaata 1140 aacgatacat gtctttctgt tgcgaccttc ctcccgtatt ctagtttgac gagtggtaac 1200 gtgctccaaa gaacgttgtc agcaagaatg agacgacgag agcgcgaaag atccccagat 1260 tcattctcaa tgcggaggac actcccgcca attgaaggaa cacttgggct ctccgcatgc 1320 tttcttccaa atagcgtctt tccacgatgc tcagagagga acctccaaga aagtgttctc 1380 agcgagcaag accttatgca accagagga cctgattatc tgcttgatga aatgactaag gaggcggaga ttgacaattc tagcaccgta gagtccttgg gcgagacaac cgacatcctt aatatgaccc atataatgtc actgaactca aacgaaaagt cactgaagct gtcccctgta 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620 aacgacgatt ctgatgagta cgaagagatg tccgaaatga aggaggaa agcatgtaag cagcacgtgt cacaaggtat ctttatgacc cacacgcga ccactattga agccttttca 1740. gatgaggagg tcgagatacc gaggagaaa gagggtgcag gaggatagtaa gggtaacggg atcgaggagc aggaggtaga agcaaatgaa gaaaatgtaa aggtgcatgg cggtcggaaa 1920. 1920. 1920. 1920. 1920. 1920. 1920. 1920. 1920. 1920 gcgaaatcag tcggtgaggc ggaggacggt ccggaagggc ggggagacgg tacttgcgag gaagggtcca gtggtgccga gcattggcaa gatgaagaac gagagaaagg cgagaaagat aaagggagag gagaatgga gagaccgggg gaggggaga aagaactcgc aaaaaaga gaatggaaaa aaagagatgg tgaagagcaa gaacaaaaag agagggagca aggtcaccag 2160 aaagagcgaa atcaagagat ggaagagggg ggtgaagaag aacatgggaa aggagagaa 2220 gaaaggggg accgcgagga ggagaagaa aaagaagggg agggcaaaga ggaggcgag 2280 2340 gccggcaaag aagagaaggg cgaggaaag ggagatcagg gcgaaggcga ggagaggag 2400 accgagggaa gagggcgag aaaggagaa ggggagaag tcgaaggcgg agaagtaggaa 2460 gaggggaag gggagcgaga ggagaagaa gaagaagggg agggaagaa agaagaaggt 2520 gagggtgagg aaggaagg ggaagcgaa gaagagagg gggaaggaaa gggggaagag 2580 gagggcgaag aaggcgaagg tgaaagaag ggagaagagg gcgaggggga gggcgaggag 2640 gggggggg aaggcgaggg aggggaggg ggggagggcg aggggagga ggaagaagga 2700 gagggtgaag gcgagggaga gggcgagggc gaaggcgaag aaagaagg ggaaggaaaa 2760 ggagaaag aaggggagga aggggagggt gaggggggagg aaggaagg ggaagggag 2820 ggtgaggatg gtgagggaga gggaagagaa gaagagggag aatgggaagg cgaagaggag 2880 gaggggaag gagaaggcga agagaaagga gagggtgagg gagagaaggg cgaagggag 2940 ggtgaggagg aagagggcga gggagaagga gaggaagagg aagggagga agagggaaa 3000 gaaaggtg agggggaaga ggaaggtgaa gggagggcg aggaggagga agagggcgaa 3060 3120 gaaagggg aagaaaga aaaagaggga gaaggggagg aaaacaggcg caatagggag 3180 gaagagagg aggagaagg tagtatcag gagactggcg aagaagagaa tgagagacaa 3240 gatggtgagg agtacaaaaa agtatctaag attaaggggt cagttaagta cgggaagcac 3300 aagacctacc agaagaaatc cgtcaccaac actcaaggta atggcaagga gcaacgctca 3360 aagatgccgg tccaaagtaa gcggctgctc aaaaacgggc cgtcaggttc taagaagttc 3420 3456 <210> 4 <211> 292 <212> DNA <213> Artificial Sequence <400> 4 gggccccaga agcctggtgg ttgtttgtcc ttctcagggg aaaagtgagg cggccccttg 60 gaggaagggg ccgggcagaa tgatctaatc ggattccaag cagctcaggg gattgtcttt 120 ttctagcacc ttcttgccac tcctaagcgt cctccgtgac cccggctggg atttcgcctg 180 gtgctgtgtc agccccggtc tcccaggggc ttcccagtgg tccccaggaa ccctcgacag 240 ggcccggtct ctctcgtcca gcaagggcag ggacgggcca caggccaagg gc 292 <210> 5 <211> 97 <212> DNA <213> Artificial Sequence <400> 5 gtaagtttag tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa 60 agaactgctc ctcagtggat gttgccttta cttctag 97

Claims

1. The nucleic acid encoding RPGR, the sequence of which is shown in SEQ ID NO:

1.

2. A recombinant vector comprising a backbone vector and the nucleic acid as described in claim 1.

3. The recombinant vector according to claim 2, characterized in that, It is a viral vector; The viral vector is selected from at least one of lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors; wherein the serotype of the adeno-associated virus vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2.7M8, or AAV2-TYF mutant.

4. The recombinant vector according to claim 2 or 3, characterized in that, Its skeletal carrier contains the RK1 promoter and / or SV40 intron.

5. A plasmid combination, characterized in that, It includes the recombinant vector, auxiliary functional plasmid, and accessory functional plasmid as described in any one of claims 2 to 4.

6. The plasmid assembly according to claim 5, characterized in that, The auxiliary functional plasmid is pAdHelper; the accessory functional plasmid is pAAV-r2c5.

7. A method for preparing adeno-associated virus expressing RPGR, comprising: The plasmid combination described in claim 5 was transfected into host cells, and the resulting purified adeno-associated virus expressing RPGR was obtained.

8. The adeno-associated virus expressing RPGR prepared by the method of claim 7.

9. The use of the recombinant vector according to any one of claims 2 to 4, or the plasmid combination according to claim 5 or 6, or the adeno-associated virus according to claim 8 in the preparation of a medicament for treating retinitis pigmentosa.

10. The application according to claim 9, characterized in that, The treatment includes repairing retinal structures, increasing the number of photoreceptor cells, and / or improving eye function.

11. A drug, characterized in that, Includes the recombinant vector according to any one of claims 2 to 4, or the plasmid combination according to claim 5 or 6, or the adeno-associated virus according to claim 8.

12. The medicament according to claim 11, characterized in that, The dosage form is an injection, wherein the titer of the adeno-associated virus according to claim 8 is 1 × 10⁻⁶. 13 vg / mL.

13. The medicament according to claim 11 or 12, characterized in that, The administration methods include subretinal injection, intravitreal injection, anterior chamber injection, or subconjunctival injection.

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