Aav-mediated gene editing treatment method for RPGR x-linked retinitis pigmentosa

Abstract

The present application relates to a vector comprising a Cas protein coding sequence and a sgRNA for specifically targeting a retinitis pigmentosa GTPase regulator (RPGR) gene. The present application also relates to a CRISPR-Cas system comprising the vector and a targeting vector, a cell, a pharmaceutical composition, a kit, and uses thereof in the treatment of retinitis pigmentosa caused by RPGR gene mutations.

Description

AAV-mediated gene editing treatment method for RPGR X-linked retinal degeneration TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an AAV vector-based gene editing treatment method for treating RPGR X-linked retinal degeneration. BACKGROUND

[0002] Retinitis pigmentosa (RP) is a hereditary blinding eye disease characterized by progressive and selective loss of photoreceptor cells and retinal pigment epithelial cells (RPE cells), and is one of the main causes of irreversible bilateral blindness in children and working-age people. RP is usually caused by gene mutations, and X-linked RP is early onset and rapidly progressive, which is a more severe type of RP (Talib M et al., 2019), and there is currently no effective treatment. X-linked RP accounts for 10-20% of total RP patients (Talib M et al., 2019), more than 70% of which is caused by RPGR gene mutations; thus, it is estimated that about 210-420 thousand RP patients are caused by RPGR gene mutations.

[0003] There are 10 transcripts of the RPGR gene, of which 5 transcripts can translate into proteins. The two most important transcripts are as follows: the basic transcript RPGR Ex1-19 and the retina-specific expression transcript RPGR ORF15. RPGR Ex1-19 has a total of 19 exons, a CDS of 2448 bp, and encodes a protein containing 816 amino acids, about 90 kDa. RPGR ORF15 contains exons 1-14 and an open reading frame 15 (ORF15), and the CDS has a total of 3459 bp, encoding a protein containing 1152 amino acids, about 200 kDa. The ORF15 region is composed of exon 15 and part of intron 15, with a special AG repeat sequence, encoding a glycine and glutamic acid-rich amino acid sequence of 567 amino acids. Both of the above two transcripts encode a structure similar to chromosome condensation regulatory protein 1 (RCC1) between N-terminal 54-367 amino acids (exons 3-10); the C-terminal is rich in basic amino acids, also known as the basic functional domain. The ORF15 region of the RPGR gene is a high mutation site, and more than 60% of RPGR mutations are located in the ORF15 region (Vervoort R et al., 2000).

[0004] Adeno-associated virus (AAV) vectors are one of the most widely used vectors in gene therapy research. Internationally, there are three gene therapy drugs using rAAV as a carrier on the market, respectively Glybera (AAV1-LPL, approved by EMEA in 2012), Luxturna (AAV2-RPE65, approved by FDA in 2017) and Zolgensma (AAV9-SMN1, approved by FDA in 2019) of UniQure company, Spark company and AveXis company. In addition, as of August 2021, there are 218 items of intervention clinical research using recombinant AAV as a gene drug delivery vector on the Clinical-trails website (https: / / clinicaltrails.gov / ). AAV has become one of the most attractive vectors in the pharmaceutical field due to its non-pathogenicity, low immunogenicity, long expression time and numerous serotypes.

[0005] In February 2020, Nat Med reported the results of the RPGR gene replacement therapy clinical trial conducted by the University of Oxford, but the clinical treatment effect was very limited (Cehajic-Kapetanovic J et al., 2020). The reason is that the mutation hotspot of RPGR gene is located in the last exon ORF15 region, which accounts for more than 75% of the total mutation of RPGR gene. ORF15 region has a special AG repeat sequence, which is prone to mutation. At the same time, as the last exon, mutation in ORF15 region will not cause its mRNA to be degraded through the NMD pathway, and will translate into truncated abnormal proteins to cause disease (Schlegel J et al., 2019). Moreover, the fine control of RPGR protein expression is also crucial. The exogenous gene carried in replacement therapy is not subject to endogenous regulation, and the expression level is difficult to control. Overexpression of RPGR is more severe than gene knockout phenotype (Wright RN et al., 2011). In addition, RPGR gene has complex post-transcriptional processing, and the exogenous target gene packaged by AAV lacks intron sequence, which is prone to splicing errors (Megaw RD et al., 2015). In summary, for the mutation hotspot ORF15 region of RPGR gene, gene replacement therapy is not the best solution, and a better gene therapy method needs to be found.

[0006] Gene editing is a group of technologies used to modify the genome of an organism, which uses endonuclease to generate double-strand breaks at specific locations in the genome, and the repair of the break point through endogenous DNA repair process can be modified to obtain the desired genotype. CRISPR / Cas9 is widely used in cell line modification, disease animal model establishment and gene therapy due to its simple target design, easy operation, low cost and high editing efficiency (Frederiksen HR et al., 2019; Shrock E and Guell M, 2017; Karimian A et al., 2019).

[0007] Through the AAV viral vector, the mutation of the ORF15 region of the human RPGR gene mutation hotspot, the application of CRISPR / Cas9 technology, the exogenous introduction of Cas9 protein, and the provision of repair templates, the precise repair of the ORF15 region of the RPGR gene can be realized. SUMMARY

[0008] Early studies show that RPGR-ORF15 is mainly expressed in the CC part of photoreceptor cells, mainly affecting the transport of opsin, and the currently researched gene therapy drugs all use photoreceptor cell-specific promoters, but clinical studies show that the treatment effect is limited. Based on the above research basis, the inventors explored the potential function of RPGR protein in RPE cells in human iPSC-induced RPE cells and humanized mouse models, and unexpectedly found that the RPGR-ORF15 transcript was also expressed in RPE cells. In addition, the mutated RPGR-ORF15 can cause abnormal RPE phagocytosis. Based on the research conclusion, the inventors further screened and optimized the structure of the gene editing treatment drug for xl-RP (X-linked RP) caused by RPGR gene mutation by selecting a broad-spectrum promoter or a non-retina-specific promoter, thereby obtaining an improved gene editing treatment method for treating RPGR X-linked retinal degeneration based on CRISPR / Cas9 technology using AAV as a carrier.

[0009] Therefore, according to one aspect of the present application, a vector is provided, comprising: (1) a nucleotide sequence encoding a Cas protein and a first promoter operably linked thereto, wherein the first promoter is a broad-spectrum promoter or a non-retina-specific promoter; and (2) a nucleotide sequence encoding a gRNA and a second promoter operably linked thereto, wherein the gRNA targets or specifically binds to a gene (RPGR gene) encoding a retinal pigment degeneration GTPase regulator or a fragment thereof.

[0010] In some embodiments, the first promoter is a mammalian constitutive promoter, preferably selected from the group consisting of elongation factor 1 alpha short (EFS) promoter, elongation factor 1 alpha (EF1a) promoter, RK promoter, CMV promoter, CAG promoter, human beta-actin promoter, small CBA (smCBA) promoter, CBS promoter or CBh promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, OPEFS promoter and SV40 promoter.

[0011] In some embodiments, the first promoter is an EFS promoter.

[0012] In some embodiments, an intron sequence is added after the first promoter.

[0013] In some embodiments, the first promoter is an EFS promoter (SEQ ID NO: 68), enRK promoter (SEQ ID NO: 69), EFS-intronl promoter (SEQ ID NO: 70), EFS-intron2 promoter (SEQ ID NO: 71), CMV promoter (SEQ ID NO: 72) or smCAG promoter (SEQ ID NO: 73). Preferably, the first promoter is an EFS promoter (SEQ ID NO: 68), EFS-intronl promoter (SEQ ID NO: 70) or EFS-intron2 promoter (SEQ ID NO: 71).

[0014] In some embodiments, the Cas protein comprises a Cas9 protein.

[0015] In some embodiments, the Cas9 protein comprises SaCas9, SpCas9, SauriCas9, KKH-SaCas9, SlugCas9 or SlugCas9-HF.

[0016] In some embodiments, the gRNA targets or specifically binds to a portion of intron 14 sequence of the RPGR gene, preferably the intron 14 comprises the nucleotide sequence set forth in SEQ ID NO: 75.

[0017] In some embodiments, the gRNA is a single guide RNA (sgRNA), the nucleotide sequence encoding the gRNA preferably comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 22, more preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 8. In some embodiments, the gRNA is a single guide RNA (sgRNA), preferably comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76 to 97, more preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 77, SEQ ID NO: 81 or SEQ ID NO: 83, most preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 83.

[0018] In some embodiments, the second promoter is a U6 promoter, preferably the U6 promoter comprises a nucleotide sequence as set forth in SEQ ID NO: 74.

[0019] In some embodiments, the vector comprises, in the 5' to 3' direction, the following elements in order: an inverted terminal repeat (ITR), a first promoter, a Kozak sequence, a nuclear import signal, a nucleotide sequence encoding a Cas protein, a nuclear import signal, a transcription terminator sequence, a second promoter, a nucleotide sequence encoding a gRNA, a gRNA scaffold sequence and an inverted terminal repeat (ITR).

[0020] In some embodiments, the transcription terminator sequence is a bGH poly(A) or a SV40 poly(A), preferably a bGH poly(A).

[0021] In some embodiments, the vector comprises a viral vector.

[0022] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector.

[0023] In some embodiments, the adeno-associated virus vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and any AAV variant or mixture, preferably an AAV8 vector.

[0024] According to another aspect of the application, there is provided a CRISPR-Cas system (or composition or kit) comprising a vector according to the application.

[0025] In some embodiments, the system (or composition or kit) further comprises a targeting vector, wherein preferably the targeting vector comprises, in the 5' to 3' end direction, in order, a RPGR gene intron 14 partial sequence, a wild type or codon-optimized RPGR gene ORF15 region sequence, and a transcription terminator sequence, preferably a bGH PolyA.

[0026] In some embodiments, the 5' and 3' ends of the vector have introduced thereto target sites corresponding to reverse sgRNAs.

[0027] In some embodiments, the system (or composition or kit) further comprises a tag, such as a fusion tag or a resistance tag, preferably a GFP fusion tag, a HA fusion tag, or a puromycin resistance tag.

[0028] In some embodiments, the codon-optimized RPGR gene ORF15 region sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO: 45.

[0029] In some embodiments, the RPGR gene intron 14 partial sequence is selected from the group consisting of SEQ ID NOs: 56-58.

[0030] In some embodiments, the bGH PolyA transcription terminator sequence comprises the nucleotide sequence set forth in SEQ ID NO: 62.

[0031] In some embodiments, in the system (or composition or kit):

[0032] the RPGR gene intron 14 partial sequence has the nucleotide sequence set forth in SEQ ID NO: 56, and the nucleotide sequence encoding the sgRNA has the nucleotide sequence set forth in SEQ ID NO: 77;

[0033] the RPGR gene intron 14 partial sequence has the nucleotide sequence set forth in SEQ ID NO: 57, and the nucleotide sequence encoding the sgRNA has the nucleotide sequence set forth in SEQ ID NO: 81; or

[0034] the RPGR gene intron 14 partial sequence has the nucleotide sequence set forth in SEQ ID NO: 58, and the nucleotide sequence encoding the sgRNA has the nucleotide sequence set forth in SEQ ID NO: 83.

[0035] In some embodiments, the targeting vector is a HITI (homology-independent targeted integration) donor vector, preferably an AAV2 / 8 type vector.

[0036] In some preferred embodiments, in said vector or CRISPR-Cas system, the Cas protein is saCas9; the first promoter is an EFS promoter (SEQ ID NO: 68), an enRK promoter (SEQ ID NO: 69), an EFS-intronl promoter (SEQ ID NO: 70), an EFS-intron2 promoter (SEQ ID NO: 71), a CMV promoter (SEQ ID NO: 72), or a smCAG promoter (SEQ ID NO: 73), preferably an EFS-intronl promoter (SEQ ID NO: 70) or an EFS-intron2 promoter (SEQ ID NO: 71); the nucleotide sequence encoding the gRNA preferably comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 22, more preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 8, most preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 8; and / or the second promoter is a U6 promoter, preferably the U6 promoter comprises a nucleotide sequence as set forth in SEQ ID NO: 74.

[0037] In some particularly preferred embodiments, in said vector or CRISPR-Cas system, the Cas protein is saCas9; the first promoter is an EFS promoter (SEQ ID NO: 68), an EFS-intronl promoter (SEQ ID NO: 70), or an EFS-intron2 promoter (SEQ ID NO: 71); the nucleotide sequence encoding the gRNA comprises a nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 8, most preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 8; and / or the second promoter is a U6 promoter (SEQ ID NO: 74).

[0038] In one particularly preferred embodiment, in said vector or CRISPR-Cas system, the Cas protein is saCas9; the first promoter is an EFS promoter (SEQ ID NO: 68); the nucleotide sequence encoding the gRNA comprises a nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 8, most preferably comprises a nucleotide sequence as set forth in SEQ ID NO: 8; and / or the second promoter is a U6 promoter (SEQ ID NO: 74).

[0039] In a particularly preferred embodiment, in said vector or CRISPR-Cas system, the Cas protein is saCas9; the first promoter is the EFS-intronl promoter (SEQ ID NO: 70); the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably the nucleotide sequence set forth in SEQ ID NO: 8; and / or the second promoter is the U6 promoter (SEQ ID NO: 74).

[0040] In a particularly preferred embodiment, in said vector or CRISPR-Cas system, the Cas protein is saCas9; the first promoter is the EFS-intronl promoter (SEQ ID NO: 70); the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably the nucleotide sequence set forth in SEQ ID NO: 8; and / or the second promoter is the U6 promoter (SEQ ID NO: 74).

[0041] According to another aspect of the application, there is provided a cell comprising a vector according to the application or a system according to the application.

[0042] According to another aspect of the application, there is provided a pharmaceutical composition comprising a vector according to the application, a system according to the application, and / or a cell according to the application.

[0043] According to another aspect of the application, there is provided a kit comprising a vector according to the application, a system according to the application, and / or a cell according to the application.

[0044] According to another aspect of the application, there is provided the use of a vector according to the application, a system according to the application, and / or a cell according to the application for the manufacture of a medicament for the treatment of a disease caused by a mutation in the RPGR gene, in particular in the ORF15 region.

[0045] According to another aspect of the present application, there is provided a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a vector according to the present application, a system according to the present application, a cell according to the present application, and / or a pharmaceutical or pharmaceutical composition according to the present application, preferably wherein the disease is a disease caused by a mutation in the RPGR gene, in particular in the ORF15 region. In some embodiments, the subject comprises a mammal, such as a human, a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a rodent such as a mouse or a rat, for example.

[0046] In some embodiments, the disease comprises retinitis pigmentosa.

[0047] In some embodiments, the disease comprises X-linked retinitis pigmentosa.

[0048] In some embodiments, the pharmaceutical is formulated in a form suitable for injection.

[0049] In some embodiments, the pharmaceutical is formulated in a form suitable for subretinal cavity injection or intravitreal injection.

[0050] The present application is further optimized based on the previous invention of the gene repair method targeting human RPGR intron 14, and sgRNAs more suitable for clinical application are screened, and the promoter and vector structure are also screened. The previous research results show that the RPGR sequence can be effectively edited in vitro by the CRISPR / Cas9 gene editing method, and the optimized ORF15 sequence is correctly inserted, but the effectiveness of the method is not further studied. The present application provides a vector structure screened, which can effectively insert the optimized RPGR ORF15 sequence and ensure correct splicing of RPGR-ORF15 mRNA and effective expression of repair protein. In addition, by comparing different vector structures, the vector molecule screened can effectively improve the retinal structure and functional abnormalities of the humanized RPGR mutant mouse model. BRIEF DESCRIPTION OF DRAWINGS

[0051] The above features and advantages of the present application will be more apparent from the following detailed description in conjunction with the accompanying drawings, in which:

[0052] Figure 1. T7E1 experiment to detect the editing efficiency of target sites 1-10 in HEK293A cells.

[0053] Figure 2. Schematic diagram of targeting vectors. Four different types of vectors were constructed according to the experimental requirements, and different marker fragments were added after the ORF15 region. The 5' and 3' ends of each vector introduce the target site corresponding to the reverse sgRNA, which can be recognized and cut by the sgRNA-SaCas9 complex.

[0054] Figure 3. PCR amplification detects the insertion of target sequence into the corresponding site in the genome. A. Schematic diagram of primer design and PCR amplification of target bands; B. P1 and P2 primer pairs amplify the sequence on both sides of the upstream integration site, and the target band is 917 bp in size; C. P3 and P4 primer pairs amplify the sequence on both sides of the downstream integration site, and the target bands are 828 bp, 608 bp and 542 bp in size after editing of target points 2, 6 and 8, respectively.

[0055] Figure 4. After TA cloning of the PCR amplification bands (bands corresponding to B and C in Figure 3), Sanger sequencing analyzes the sequence information formed after editing of the target site. The results show that the targeting vector is correctly inserted into the target site.

[0056] Figure 5. The insertion of the targeting sequence into the target site does not affect the transcription of the RPGR gene. A. Two main transcripts of the RPGR gene and primer design; B. After extracting RNA, cDNA is obtained by reverse transcription, and then PCR amplification is used to detect the ORF15-specific transcript; C. Sanger sequencing of the PCR amplification fragment confirms that the RPGR-ORF15-OPT transcript is normal.

[0057] Figure 6. The targeting vector can correctly repair the patient's iPSC DNA sequence. A. P1 and P2 primer pairs amplify the sequence on both sides of the upstream integration site, and the target band is 917 bp in size; P3 and P4 primer pairs amplify the sequence on both sides of the downstream integration site, and the target bands are 828 bp, 608 bp and 542 bp in size after editing of target points 2, 6 and 8, respectively; B. After TA cloning of the PCR amplification bands (bands corresponding to A in Figure 6), Sanger sequencing analyzes the sequence information formed after editing of the target site.

[0058] Figure 7. Schematic diagram of the structure of the RPGR gene editing treatment vector.

[0059] Figure 8. ERG detects changes in retinal function of mice in different administration groups (data from left to right for each coordinate (point): control vector, sgRNA-2, sgRNA-6 and sgRNA-8).

[0060] Figure 9. PCR detects the insertion of DNA into the retinal cells of mice in different administration groups.

[0061] Figure 10. RT-PCR and q-RT-PCR detect changes in the expression level of RPGR-ORF15 mRNA in mouse retinal cells.

[0062] Figure 11. Detection of RPGR transcript expression in iPSC-RPE cells.

[0063] Figure 12. Detection of phagocytic function of iPSC-RPE cells.

[0064] Figure 13. ERG detected the effect of different promoter vectors on the retinal function of model mice (the data of each coordinate (point) from left to right are: control, EFS, hGRK1 and enRK, respectively).

[0065] Figure 14. Q-RT-PCR detected the effect of different promoter vectors on the expression level of RPGR ORF15 mRNA in the retinas of model mice (the data of each coordinate (point) from left to right are: control, EFS, hGRK1 and enRK, respectively).

[0066] Figure 15. ERG detected the effect of different spectral promoter vectors on the retinal function of model mice 6 months after administration (the data of each coordinate (point) from left to right are: control, EFS-intronl, EFS-intron2, CMV and smCAG, respectively).

[0067] Figure 16. ERG detected the effect of different element combination vectors on the retinal function of model mice (the data of each coordinate (point) from left to right are: control, EFS-Cas9-sg8, EFS-Cas9, enRK-Cas9-sg8 and enRK-Cas9, respectively).

[0068] Figure 17. Q-RT-PCR detected the effect of different element combination vectors on the expression level of RPGR ORF15 mRNA in the retinas of model mice (the data of each coordinate (point) from left to right are: control, EFS-Cas9-sgRNA, EFS-Cas9, enRK-Cas9-sgRNA and enRK-Cas9, respectively). DETAILED DESCRIPTION

[0069] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in the art, it is particularly recommended that the skilled person refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0070] Definitions of terms

[0071] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably and generally refer to polymeric forms of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of a polynucleotide can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation to an labeling component.

[0072] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host, to transfer an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-described functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0073] In the present application, the term "viral vector" generally refers to a non- wild type recombinant viral particle that functions as a gene delivery vector and comprises a recombinant viral genome packaged within a viral capsid. The animal viral species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV), herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papova viruses (e.g., SV40).

[0074] In the present application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables transcription of a particular gene. The promoter can be recognized by RNA polymerase and initiates the transcription to synthesize RNA. In the synthesis of ribonucleic acid (RNA), the promoter can interact with transcription factors that regulate gene transcription, control the initiation time of gene expression (transcription) and the degree of expression. The promoter includes a core promoter region and a regulatory region, which is located in the regulatory sequence that controls gene expression, upstream of the transcription start site of the gene (5' direction of the DNA anti-sense strand), and has no coding function itself. According to its mode of action and function, it is divided into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue-specific or development stage-specific), and inducible promoters (regulated by external chemical or physical signals).

[0075] In the present application, the term "broad spectrum promoter" or "non-retina-specific promoter" refers to a promoter that can initiate expression in all tissues or is not limited to expression in the retina, and is a promoter that is opposite to retina-specific promoters such as hGRK1, RPE65, and VMD2. The "broad spectrum promoter" or "non-retina-specific promoter" described in the present application includes, but is not limited to, the elongation factor 1 alpha short (EFS) promoter, the elongation factor 1 alpha (EF1 alpha) promoter, the RK promoter, the CMV promoter, the CAG promoter, the human beta-actin promoter, the small CBA (smCBA) promoter, the CBS promoter or CBh promoter, the PGK promoter, the UBC promoter, the GUSB promoter, the UCOE promoter, the OPEFS promoter, and the SV40 promoter.

[0076] In the present application, the term "operably linked" generally refers to the placement of regulatory sequences necessary for the expression of a coding sequence in the appropriate position relative to the coding sequence so as to effect expression of the coding sequence. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. In certain embodiments, it can indicate the arrangement of a coding sequence and transcription control elements in an expression vector. The control elements can include promoters, enhancers, and termination elements. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. In certain embodiments, "operably linked" can also mean that a gene of interest is linked to a vector, so that the transcription and translation control sequences within the vector perform their intended function of regulating the transcription and translation of the gene of interest.

[0077] In the present application, the term "cell" can generally be or have been a single cell, a cell line, or a cell culture that is the recipient of a nucleic acid molecule or a vector. The cell can include a nucleic acid molecule described in the present application or a vector described in the present application. The cell can include progeny of a single cell. The progeny can not necessarily be identical to the original parent cell (in morphology of total DNA complement or on the genome) due to natural, accidental, or deliberate mutation. The cell can include a cell transfected in vitro with a vector described in the present application. The cell can be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell / HEK293A cell, a COS-1 cell, an NSO cell, or a myeloma cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293T cell.

[0078] In the present application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, a human patient. For example, a pharmaceutical composition described in the present application, which can include a vector described in the present application and / or a cell described in the present application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition can also include one or more suitable formulations of (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable ingredients of the composition can be non-toxic to the recipient at the used dose and concentration. The pharmaceutical composition of the present application includes but is not limited to liquid, frozen and lyophilized compositions.

[0079] In the present application, the term "treatment" generally refers to a clinical intervention aimed at altering the natural course of a treated individual or cell in a clinical pathology process. It can include improving the disease state, eliminating the lesion, or improved prognosis.

[0080] In the present application, the term "subject" generally refers to any subject for which diagnosis, treatment or therapy is desired. For example, in the present application, the RPGR gene of a subject in need thereof includes a pathological mutation in the ORF15 region (particularly a mutation that causes inherited retinitis pigmentosa or X-linked retinitis pigmentosa). In certain cases, the subject can include a mammal. For example, the subject can include but is not limited to a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, or a monkey. In certain cases, the subject can include a human. In certain cases, the subject can include an East Asian.

[0081] In the present application, the term "retinitis pigmentosa" generally refers to a genetic, blinding eye disease characterized by progressive, selective loss of retinal photoreceptor cells (rods and cones) and retinal pigment epithelium (RPE). The inheritance pattern of RP can include autosomal recessive (arRP), autosomal dominant (adRP), and X-linked (xlRP), with xlRP being the earliest onset and most severe. Clinical manifestations of RP can include night blindness, progressive visual field loss, central vision loss with macular involvement, and eventual blindness. Major fundus changes in RP include equatorial retinal pigment disorder with bone cell-like pigmentation, which gradually progresses to the posterior pole and ora serrata, with RPE, photoreceptor, and choriocapillary layers atrophy, large choroidal vessels visible through the atrophy, a blue-gray retina, retinal arteriole attenuation, and waxy atrophy of the optic disc. Methods to assess retinal function and morphology can include best corrected visual acuity (BCVA), fundus autofluorescence, visual field testing, electroretinography (ERG), fundus color photography, optical coherence tomography (OCT), and fluorescein angiography (FFA).

[0082] In the present application, the term "X-linked retinitis pigmentosa" generally refers to X-linked retinitis pigmentosa, also known as xlRP. Approximately 70-75% of xlRP is currently attributed to mutations in the RPGR gene, with over 75% of RPGR mutations located in the ORF15 region of the last exon of the RPGR ORF15 subtypes. Clinical signs of the xlRP include, but are not limited to, peripheral vision loss, central (reading) vision loss, night vision loss, loss of color perception, decreased visual acuity, decreased photoreceptor cell function, and pigment changes.

[0083] In the present application, the term "retinitis pigmentosa GTPase regulator" is encoded by the RPGR gene, which is usually a protein with a series of RCC1-like domains (RLD). The "gene encoding retinitis pigmentosa GTPase regulator" can also be referred to as the "RPGR gene" herein. The "retinitis pigmentosa GTPase regulator" can include the full-length gene itself or a functional fragment thereof. The retinitis pigmentosa GTPase regulator can be derived from any mammal that naturally expresses the RPGR gene or its homologs, such as primates (e.g., humans), rodents (e.g., mice, rats). The "RPGR gene" can encode a variety of different isoforms of spliced transcripts, which can include all spliced forms, transcripts and / or functional variants thereof herein. For example, human RPGR isoforms can include isoform A, isoform C, isoform D, isoform E, isoform F, isoform G, isoform I and isoform J. Isoform A and isoform C are full-length human RPGR isoforms. For example, the nucleotide sequence of exemplary isoform A can be found at NCBI Accession No. NM_000328.3, and the amino acid sequence can be found at NCBI Accession No. NP_000319.1, and the nucleotide sequence of exemplary isoform C can be found at NCBI Accession No. NM_001034853.2, and the amino acid sequence can be found at NCBI Accession No. NP_001030025.1.

[0084] Isoform RPGR ex1-19 derived from exon 1 to exon 19, corresponding to isoform A above) and RPGR ORF15 derived from exon 1 to a portion of intron 15, corresponding to isoform C above) are two widely expressed isoforms of RPGR. Among them, RPGR ORF15 terminates before exon 16 to exon 19, the termination portion of RPGR ORF15 may be referred to as ORF15, which is also referred to as "RPGR ORF15" herein. RPGR ORF15 isoform is essential for normal rod and cone function in the retina and is mainly expressed in photoreceptor cells.

[0085] In the present application, the term "targeting vector" generally refers to a vector comprising a nucleic acid molecule comprising wild-type or codon-optimized human RPGR ORF15 nucleotide as described herein. The targeting vector can be used to introduce the nucleic acid molecule into a cell.

[0086] In the present application, the term "administering" can introduce cells and / or vectors into a subject, or a desired site of a subject, by a method or route. The cells and / or vectors can express nucleic acid molecules of the present application (e.g., sequences encoding gRNAs and / or gRNAs) at the desired site (e.g., site of injury or repair), thereby producing a desired effect. Cells (or their differentiated progeny) and / or vectors can be administered by any suitable route that can deliver the cells (or their differentiated progeny) and / or vectors to a desired site in a subject, and at least a portion of the implanted cells (or cellular components) and / or vectors remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, a few days, up to several years, or even consistent with the life span of the patient. In certain instances, the administration includes injection. For example, the vectors can be administered by a systemic route of administration, such as intraperitoneal or intravenous routes. For example, the administration can include subretinal cavity injection.

[0087] In the present application, the term "comprising" generally means including, but not excluding other elements.

[0088] In the present application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence that has some degree of homology to a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be equivalent to "identity". A homologous sequence can include an amino acid sequence that can be at least 70%, 75%, 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Generally, a homolog will contain the same active sites, etc. as the subject amino acid sequence. Homology can be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functions) or homology can be expressed in terms of sequence identity. In the present application, a reference to a sequence having a percentage identity to any of the SEQ ID NOs of the amino acid sequences or nucleotide sequences means a sequence having the stated percentage identity over the entire length of the referenced SEQ ID NO.

[0089] To determine sequence identity, sequence alignments can be performed, which can be done in various ways appreciated by those skilled in the art, e.g., using the BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, etc. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared.

[0090] CRISPR / Cas system

[0091] In the present application, the term "CRISPR / Cas system" or "CRISPR-Cas system" generally refers to a nuclease system composed of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (i.e., Cas proteins) that is capable of cleaving almost all genomic sequences adjacent to a protospacer-adjacent motif (PAM) in eukaryotic cells. "CRISPR / Cas system" can be used to refer collectively to transcripts involving CRISPR-associated ("Cas") genes, as well as other elements involved in their expression or in directing their activity, which can include sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions thereof), tracr-mate sequences (encompassing "direct repeats" and processed partial direct repeats in the context of endogenous CRISPR / Cas systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR / Cas systems), or other sequences and transcripts from CRISPR loci. Five types of CRISPR systems have been identified (e.g., Type I, Type II, Type III, Type U, and Type V).

[0092] In the present application, the term "Cas protein" also referred to as "CRISPR-associated protein" generally refers to a class of enzymes that are complementary to CRISPR sequences, capable of using CRISPR sequences as guides to recognize and cleave specific DNA strands. Non-limiting examples of Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and / or their homologs, or modified versions thereof. In some embodiments, the Cas protein is a Cas9 protein.

[0093] In the present application, the term "Cas9 protein" or "Cas9 nuclease", also known as Csn1 or Csx12, generally refers to a class of proteins in type II CRISPR / Cas system that are involved in both crRNA biosynthesis and destruction of invading DNA. Cas9 proteins generally include a RuvC nuclease domain and a HNH nuclease domain, which cut two different strands of a double-stranded DNA molecule, respectively. Cas9 proteins have been described in different bacterial species such as S. thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and S. Pyogenes (Deltcheva, Chylinski et al. 2011). For example, Streptococcus pyogenes Cas9 protein, the amino acid sequence of which can be found in SwissProt database accession number Q99ZW2; Neisseria meningitides Cas9 protein, the amino acid sequence of which can be found in UniProt database number A1IQ68; Streptococcus thermophilus Cas9 protein, the amino acid sequence of which can be found in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, the amino acid sequence of which can be found in UniProt database number J7RUA5.

[0094] The CRISPR / Cas system can include a number of short repeat sequences, referred to as "repeats". When expressed, the repeat sequences can form secondary structures (e.g. hairpins) and / or comprise unstructured single-stranded sequences. The repeat sequences usually occur in clusters and often diverge between species due to evolution. These repeat sequences are regularly spaced with unique intervening sequences referred to as "spacers", thereby forming a repeat-spacer-repeat locus structure. The spacers are identical or have high homology to known foreign invader sequences. The spacer-repeat units encode a crispRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. The crRNA comprises a "seed" or spacer sequence that targets a target nucleic acid (naturally occurring form in prokaryotes, the spacer sequence targets a foreign invader nucleic acid). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0095] The CRISPR / Cas system can also include a polynucleotide sequence encoding a CRISPR-associated protein (Cas protein). Cas genes encode nucleases involved in the biogenesis and interference stages of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures.

[0096] In nature, crRNA biogenesis in Type II CRISPR systems requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified by endogenous RNase III and then hybridizes to the crRNA repeat sequences in the pre-crRNA. Endogenous RNase III can be recruited to cleave the pre-crRNA. The cleaved crRNAs can be trimmed by exonucleases to produce mature crRNA forms (e.g., 5' end trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex can direct the complex to a target nucleic acid that can hybridize to the crRNA. Hybridization of the crRNA to the target nucleic acid can activate the Cas9 to cleave the target nucleic acid. The target nucleic acid in Type II CRISPR systems is referred to as a protospacer adjacent motif (PAM). In fact, the PAM is essential to promote binding of the site-directed polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) can be further subdivided into Type II-A (CASS4) and Type II-B (CASS4a). CRISPR / Cas9 systems useful for RNA-programmable gene editing can be found in Jinek et al., Science, 337(6096):816-821 (2012). International Patent Application Publication No. WO 2013 / 176772 provides numerous examples and applications of CRISPR / Cas endonuclease systems useful for site-specific gene editing.

[0097] gRNA

[0098] In the present application, the term "sequence encoding a gRNA" generally refers to a DNA sequence from which the gRNA can be obtained by transcription.

[0099] The gRNAs described herein can bind to a sequence in a target nucleic acid of interest. Nucleic acids (or portions thereof) that target a genome can interact with a target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The gRNAs described herein can be single guide RNAs (sgRNAs), and the nucleotide sequence of the sgRNAs can vary depending on the sequence of the target nucleic acid of interest.

[0100] In the CRISPR / Cas system of the present application, the gRNA sequence can be designed to hybridize to a target nucleic acid proximal to a PAM sequence recognizable by the Cas protein used in the system. The gRNA can be perfectly matched or mismatched to the target sequence. Cas proteins generally all have a specific PAM sequence that can be recognized in the target DNA.

[0101] For example, the Cas9 protein can be from S. pyogenes, which recognizes a PAM comprising the sequence 5'-NRG-3' in the target nucleic acid, wherein R comprises A or G, and wherein N can be any nucleotide. For another example, the Cas9 protein can be from Staphylococcus aureus, which recognizes a PAM comprising the sequence 5'-NNGRR(T)-3' in the target nucleic acid, wherein R comprises A or G, and wherein N can be any nucleotide. In some more specific cases, the PAM sequence recognized by SaCas9 can comprise 5'-NNGRR-3', wherein R comprises A or G, and wherein N can be any nucleotide. A mutant of SaCas9, KKH-SaCas9 (PAM sequence: 5'-NNNRRT-3'), can successfully generate high efficiency mutations in the host. A miniaturized Cas9 protein, such as SauriCas9 (Hu Z, Wang S, Zhang C, Gao N, Li M, et al. (2020) A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope. PLoS Biol 18: e3000686), SlugCas9, or SlugCas9-HF (Hu Z, Zhang C, Wang S, Gao S, Wei J, et al. (2021) Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity. Nucleic Acids Res 49: 4008-4019) can also be used.

[0102] The gRNAs for use in the CRISPR system described herein can be synthesized chemically, e.g., by high performance liquid chromatography. For example, two or more RNA molecules are linked together. Longer RNAs, such as those encoding Cas9, can be obtained by enzymatic reactions. In the art, various types of RNA modifications can be introduced during or after chemical and / or enzymatic synthesis of the RNA, e.g., modifications to enhance stability, reduce innate immune responses, and / or enhance other properties.

[0103] gRNA scaffold

[0104] The CRISPR / Cas9-based gene editing system includes at least one gRNA scaffold. The gRNA scaffold facilitates the binding of Cas9 to the gRNA and the endonuclease activity. The gRNA scaffold is a polynucleotide sequence immediately following the gRNA targeting sequence. The gRNA targeting sequence and the gRNA scaffold together form a polynucleotide.

[0105] Vector

[0106] Vectors are provided herein. The vectors described herein can comprise the nucleic acid molecules (e.g., sequences encoding gRNAs and / or gRNAs) described herein. Polynucleotides can be delivered by non-viral delivery vectors, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes.

[0107] The vectors can also be polynucleotide vectors, e.g., plasmids, cosmids, or transposons. Vectors suitable for use have been widely described and are well known in the art. Those skilled in the art will appreciate that vectors comprising the nucleic acid molecules described herein can also comprise additional sequences and elements that can be required for replication of the vector in prokaryotic and / or eukaryotic cells. For example, the vectors described herein can include a prokaryotic replicon, i.e., a nucleotide sequence that has the ability to direct the host's own replication and maintenance in a prokaryotic host cell (e.g., a bacterial host cell). Such replicons are well known in the art. In certain instances, the vectors can comprise a shuttle element, which makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In addition, the vectors can also include a gene that is capable of expressing a detectable marker (e.g., a drug resistance gene). The vectors can also have a reporter gene, e.g., a gene that encodes a fluorescent or other detectable protein.

[0108] In some cases, the vector can comprise a viral vector, for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus. Methods for producing viral vectors comprising a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are well known in the art and can be performed by one of skill in the art without undue experimentation. In other cases, the vector can be a recombinant AAV virion packaged with a nucleic acid molecule described herein. Methods of producing recombinant AAV can include introducing a nucleic acid molecule described herein into a packaging cell line, producing AAV helper functions, AAV cap and rep genes, and recovering the recombinant AAV from the supernatant of the packaging cell line. Various types of cells can be used as packaging cell lines. For example, packaging cell lines that can be used include, but are not limited to, HEK 293 cells, HeLa cells, and Vero cells.

[0109] In some cases, the vector can be an adenovirus-associated vector (AAV). In the present application, the term "adenovirus-associated vector" generally refers to vectors derived from naturally occurring and available adeno-associated viruses as well as artificial AAVs. The AAVs can include different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, as well as any AAV variants or mixtures. The AAV genome usually has terminal inverted repeats (ITRs) at both ends, and the term "ITR" or "terminal inverted repeat" refers to a segment of nucleic acid sequence present in AAV and / or recombinant AAV that can form a T-shaped palindromic structure required for completion of the AAV lytic and latent life cycle. Techniques for producing AAV vectors are standard in the art, which include providing a cell with a polynucleotide to be delivered, a rAAV genome, AAV rep and cap genes, and helper virus functions to be packaged into the rAAV genome. Production of AAV vectors generally requires the presence of the following components within a single cell (herein referred to as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (e.g., not within) the rAAV genome, and helper virus. The AAV rep and cap genes can be from any AAV serotype, or from a different AAV serotype than the ITRs of the AAV genome, including but not limited to the AAV serotypes described herein. The AAV vector in the present application can comprise a gRNA targeting a mutation site of the RHO gene.

[0110] In some cases, the sequence encoding the gRNA can be located in the same vector as the nucleic acid encoding the Cas9 protein. In other cases, the sequence encoding the gRNA can be located in a different vector from the nucleic acid encoding the Cas9 protein.

[0111] The AAV vectors of the present application can be from a variety of species. For example, the AAV can be an avian AAV, a bovine AAV, or a goat AAV. In certain embodiments, the vector is AAV8.

[0112] The methods of the present application can include generating packaging cells, i.e., generating a cell line that can be used to stably express all the necessary components of AAV. For example, an AAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from an AAV genome, and a plasmid (or multiple plasmids) with a selection marker such as a neomycin resistance gene are integrated into the genome of the cell. The AAV genome can have been introduced into a bacterial plasmid by methods such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. U.S.A., 79:2077-2081). The packaging cell line can then be infected with a helper virus (e.g., adenovirus). In addition to plasmids, adenovirus or baculovirus can also be used to introduce the AAV genome and / or rep and cap genes into the packaging cell.

[0113] HITI (homology-independent targeted integration) donor vector

[0114] In the present application, the term "HITI (homology-independent targeted integration) donor vector" refers to a vector used in the hiti (homology-independent targeted integration) technology to provide an exogenous gene to be inserted into a target genome. In the present application, the exogenous gene can be a wild-type or codon-optimized RPGR gene ORF15 region sequence for repairing the normal function of ORF15. In HITI, the cell usually selects the non-homologous end joining (NHEJ) repair mechanism to insert the exogenous gene in the HITI donor vector into the break site of the target genome. Compared with traditional homologous recombination technology, the HITI technology is more efficient and accurate. For more details about the HITI technology, see US20190225991A1.

[0115] The present application designs 22 gRNA sequences for intron 14 of human RPGR, constructs the above 22 gRNAs into AAV-SaCas9-U6 vectors to form AAV-SaCas9-U6-sgRNA editing vectors, transfects them into HEK293A cells, extracts genomic DNA after puromycin selection. The primers are designed near the target sites, and the R2, R6 and R8 sgRNA target sites are screened by T7E1 enzyme digestion method, and the cutting efficiencies are 49.1%, 22.6% and 81.5%, respectively.

[0116] Based on the screened sgRNA sequence, an AAV vector was used as a skeleton to construct a targeting vector containing a partial region of RPGR intron 14, a codon-optimized RPGR ORF15 region, a bGH PolyA transcription termination signal, and a tag. Reverse sgRNA binding sequences were inserted into the 5' and 3' ends of the targeting vector to improve the proportion of forward insertion of the targeting vector. The targeting vector and AAV-saCas9-U6-sgRNA vector were co-transfected into the HEK293A cell line, and after culture, genomic DNA was extracted for identification. It was found that they could all be correctly inserted into the host cell genome. After TA cloning and sequencing analysis of the proportion of cells with repaired targeting sequences, the results showed that in HEK293A cells, the proportion of cells with repaired targeting sequences was between 5-10%. RNA was extracted from the cells edited by the PuroR targeting vector, and PCR and sequencing showed that the insertion of the targeting sequence into the target site did not affect the mRNA splicing of the RPGR-ORF15 transcript.

[0117] To further verify the function of the above-mentioned vector in a disease model mouse, the inventors replaced the AAV-saCas9-U6-sgRNA vector promoter with a photoreceptor cell-specific promoter and packaged it with the targeting vector as AAV. The in vivo editing efficiency of different sgRNAs was further compared in the model mouse. The experimental results showed that sgRNA8 had a better editing efficiency, but the specific promoter hGRK1 did not show changes in the function of the model mouse retina. To further clarify whether the RPGR protein only affects the function of photoreceptor cells, the inventors confirmed the function of RPGR in RPE cells by comparing the morphological and phagocytic function changes of patient-derived iPSC-RPE and model mouse RPE cells. The experimental results showed that RPGR-ORF15 mutations could cause abnormal morphology of RPE cells, further affecting the phagocytic function of mouse RPE cells.

[0118] The inventors of the present application further optimize the vector structure of the aforementioned AAV-saCas9-U6-sgRNA vector with high editing efficiency, replace the Cas9 protein expression promoter with EFS and enRK, and package it into an AAV vector. To further confirm the effect of the gene editing drug in vivo, the virus is injected into the subretinal space of a humanized model mouse. Six months after administration, PCR and q-RT-PCR experimental results show that the EFS, hGRK1, and enRK promoters can correctly insert the optimized RPGR DNA sequence, and the repaired DNA sequence can correctly express the optimized RPGR mRNA. The ERG results show that the EFS promoter shows more significant amplitude recovery effect compared to the hGRK1 and enRK promoters. The above results show that the broad-spectrum EFS vector combination can effectively repair the mutant RPGR expression and has a retinal function recovery effect in vivo. On this basis, the inventors of the present application further optimize and compare other broad-spectrum promoters such as the addition of intron sequences after the EFS promoter to form EFS-intron1 and EFS-intron2, and compare the in vivo effects of CMV and smCAG promoters.

[0119] In addition, the inventors of the present application compared the editing efficiency and functional recovery effect of AAV-saCas9-U6-sgRNA vector and AAV-saCas9 vector without sgRNA expression frame based on EFS and enRK promoters in vivo. The comparison results show that the AAV-saCas9-U6-sgRNA vector shows slightly higher editing efficiency, but the retinal function detection results do not show significant differences between the two combinations.

[0120] The present application is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present application. The reagents and materials used in the following experiments are commercially available products unless otherwise specified.

[0121] Example

[0122] Example 1 Comparison and screening of sgRNA sequences with high editing efficiency and high repair accuracy

[0123] Twenty-two gRNAs were designed for human RPGR intron 14, namely gRNA-1 to gRNA-22, corresponding to target points R1 to R22 (SEQ ID NO: 1 to SEQ ID NO: 22), and the corresponding PAM sequences were SEQ ID NO: 23 to SEQ ID NO: 44. The sequences of gRNA-1 to gRNA-22 are shown in SEQ ID NO: 76 to SEQ ID NO: 97, respectively.

[0124] The above 22 gRNAs (the target points recognized by the gRNAs are numbered R1 to R22, respectively) were constructed into px601 vectors (addgene: #164139) to form AAV-SaCas9-U6-sgRNA editing vectors, and transfected into HEK293A cells, and after selection with puromycin, genomic DNA was extracted, and primers P1 and P4 were designed for PCR sequencing in the vicinity of the target point, and the sequences are shown in Table 1.

[0125] Table 1. PCR primer sequences

[0126] The PCR product was treated with T7E1 enzyme and the product was recovered (see CN 116334141A for the T7E1 enzyme detection method), and agarose gel electrophoresis was performed for quantification to analyze the mutation efficiency. Taking the gRNAs targeting R1-R10 as an example, the mutation efficiency is shown in Figure 1 and Table 2, which shows that the gRNAs of the application can effectively cleave the 14th intron of RPGR. The gRNAs binding to the target points R2, R6 and R8 were selected for the next step of vector design and screening.

[0127] Table 2. Mutation efficiency of exemplary sites

[0128] Example 2. Construction of human RPGR gene editing optimization vector

[0129] The targeting vector has an AAV backbone, and contains 3-4 DNA fragment components, which are the partial region of RPGR gene intron 14, the codon-optimized RPGR ORF15 nucleotide sequence, the bGH PolyA transcription termination signal, and the fusion tag GFP or puromycin resistance tag added for detection under certain conditions (as shown in Figure 2).

[0130] The partial region of RPGR gene intron 14 of the targeting vector was constructed for the target sequences R2, R6 and R8 in intron 14, and the sequences are as follows:

[0131] The 5' end sequence upstream of target point R2 (partial region of intron 14) is shown in SEQ ID NO. 56;

[0132] The 5' end sequence upstream of target point R6 (partial region of intron 14) is shown in SEQ ID NO. 57;

[0133] The 5' end sequence upstream of target point R8 (partial region of intron 14) is shown in SEQ ID NO. 58.

[0134] The nucleotide sequence of the human RPGR ORF15 codon-optimized sequence is shown in SEQ ID NO: 45.

[0135] The GFP fusion tag sequence is shown as SEQ ID NO. 59.

[0136] The puromycin resistance tag sequence is shown as SEQ ID NO. 60.

[0137] The HA fusion tag sequence is shown as SEQ ID NO. 61.

[0138] The 3' end bGH PolyA transcription terminator sequence is shown as SEQ ID NO. 62.

[0139] After splicing the above sequences by overlap extension PCR, primers were designed to introduce reverse sgRNA targeting sequences and enzyme cutting sites at the 5' end and 3' end respectively, and cloned into double enzyme-digested linearized px601 plasmid.

[0140] Example 3. Detection of gene editing repair at the DNA level in vitro

[0141] The above targeting vector and AAV-saCas9-U6-sgRNA vector were co-transfected into the HEK293A cell line, and after culture, genomic DNA was extracted for identification. The primer sequence for identification is shown in Table 3. The primer binding site and the amplified fragment are shown in Figure 3A.

[0142] Table 3. Primer sequences for identification

[0143] The experimental results show that:

[0144] (1) Upstream integration site identification (P1, P2 primer pair): The three candidate target sites R2, R6 and R8 can all amplify positive bands (Figure 3B).

[0145] (2) Downstream integration site PCR identification (P3, P4 primer pair): The three candidate target sites R2, R6 and R8 can all amplify positive bands (Figure 3C).

[0146] (3) After TA cloning and sequencing analysis of the upstream and downstream amplified fragments, it was found that the targeting region was correctly inserted into the host cell genome (Figure 4).

[0147] Example 4. Detection of the proportion of repair cells

[0148] The proportion of cells repaired after targeting sequence repair was analyzed by TA cloning and sequencing analysis of the bands amplified by the P1, P4 primer pair (Table 4), and the results showed that in HEK293A cells, the proportion of cells repaired after targeting sequence repair was between 5-10%.

[0149] Table 4. Insertion efficiency of targeting sequence at different sites in HEK293A cells

[0150] Example 5. Effect of targeting sequence integration into genome on RPGR gene transcription

[0151] The targeting vectors of Example 2 and AAV-saCas9-U6-sgRNA vectors containing sgRNAs against R2, R6, R8 respectively screened in Example 1 were co-transfected into HEK293A cell line respectively, and cells edited at DNA level of RPGR gene were obtained. RNA was extracted from cells edited by targeting vectors containing PuroR tag in Example 2, and the effect of targeting sequence insertion into genome on RPGR gene transcription was analyzed after reverse transcription, and primers for identification are shown in Table 5.

[0152] Table 5. Primer sequences for identification

[0153] The two major transcripts of RPGR gene and primer design are shown in Figure 5A. The E6 / 7 primer pair amplifies RPGR exon 1-19 and ORF15 transcript, the E14 / 15 primer pair specifically amplifies RPGR-ORF15 transcript, the E14 / 15-WT primer pair detects wild-type ORF15 transcript, and the E14 / 15-OPT specifically amplifies codon-optimized ORF15 transcript. PCR and sequencing showed that the targeting sequence insertion into the target site did not affect the mRNA splicing of RPGR-ORF15 transcript (Figures 5B and 5C).

[0154] Example 6. DNA repair verification of patient iPSC

[0155] To further determine the editing and DNA repair efficiency of sgRNAs against R2, R6, R8, urine epithelial cells of RP patient with RPGR-ORF15 mutation (c.2218G>T, p.E740* hemizygote) were collected (from Peking University Third People's Hospital, with signed informed consent) and iPSCs were generated using CytoTune TMiPS2.0 Sendai Reprogramming Kit (Invitrogen, A16517) was used to induce iPSC according to the instruction. The targeting vector of Example 2 and the AAV-saCas9-U6-sgRNA vector of Example 1 were co-transfected into the iPSC cell line, and the cell DNA was extracted after puro selection for PCR detection. The iPSC detection primers were consistent with Example 3. The experimental results showed that: (1) upstream integration site identification (P1, P2 primer pair): three candidate target sites R2, R6 and R8 can all amplify positive bands; downstream integration site PCR identification (P3, P4 primer pair): three candidate target sites R2, R6 and R8 can all amplify positive bands (Figure 6, A). (2) The editing effect was detected by TA cloning and sequencing analysis of the upstream and downstream amplification fragments, which showed that the targeting region was correctly inserted into the host cell genome (Figure 6, B).

[0156] Example 7. Comparison of in vivo efficacy of different sgRNAs

[0157] To further clarify the editing effect and efficacy of the candidate molecules in vivo, the inventors constructed an AAV-saCas9-U6-sgRNA vector expressing a photoreceptor cell-specific promoter hGRK1 (SEQ ID NO: 67) targeting R2, R6, and R8, respectively, and packaged it with the targeting vector into two AAV vectors (AAV-I and AAV-II). The vector mode diagram is shown in FIG. 7. The above two AAV vectors were mixed at a dose of 1E9 vg / eye of each virus and injected into the RPGR humanized mouse model (the model was constructed by replacing the 13th intron to the 14th intron of the mouse RPGR gene with the 13th intron to the 15th intron of the human RPGR gene on the basis of C57BL / 6, and the specific construction method is described in the patent: Retinal pigment degeneration animal model and its construction method and application, China patent application number: 2024100447521) eye through subretinal administration. Six months after administration, ERG eye function detection was performed, and after completion of the detection, the mice were sacrificed, and the mouse retinal cell DNA and RNA were collected to detect the DNA editing and RNA repair efficiency of the mouse retinal cells after administration. The ERG results are shown in FIG. 8. Compared with the control group, the administration groups showed no significant changes. The DNA repair results showed that positive target bands were detected at the P1 / P2 upstream integration site and the P3 / P4 downstream integration site in each administration group, indicating that the mouse retina DNA was correctly inserted. In addition, the expression level of mRNA after repair in the model mice was detected by RT-PCR and q-RT-PCR, and the experimental results are shown in FIG. 9. The mRNA band after repair was detected in each administration group. In addition, the q-RT-PCR results showed (FIG. 10) that the sgRNA8 administration group showed a more obvious decrease in the transcription level of the model mouse RPGR-ORF15-mut mRNA, and the expression level of the optimized RPGR-ORF15-opt mRNA was up-regulated more significantly. The above results indicated that sgRNA8 had a more optimal in vivo valence effect. The q-RT-PCR primer sequences are shown in Table 6.

[0158] Table 6. Primer sequences for mRNA expression detection

[0159] Example 8. RPGR exists in RPE cells with potential function

[0160] The existing studies show that the specific promoter shows limited therapeutic effect. In order to further clarify the function of RPGR in cells other than photoreceptor cells, the inventors first detected the expression of RPGR exon1-19 transcript and RPGR-ORF15 transcript in iPSC-induced retinal organoids (induction method, see reference 10) and RPE cells by q-RT-PCR, and the experimental results are shown in Figure 11. There is no significant difference in RPGR exon1-19 transcript compared with retinal organoids, and although the expression of ORF-15 transcript in RPE cells is lower than that in retinal organoids, it still shows its transcription level, indicating that RPGR-ORF15 has potential function in RPE cells. In addition, in order to further confirm its function in photoreceptor cells, the inventors compared the phagocytosis function of patient iPSC-RPE cells (obtained by inducing the iPSC cells of Example 6) and normal human iPSC-RPE by phagocytosis experiment (in the differentiated mature RPE cells, 1X fluorescent magnetic bead working solution prepared by using RPE differentiation medium was added, and it was cultured in a 37°C cell incubator overnight. The culture medium in the detection cells was aspirated, washed with DPBS for at least 5 times, and observed under a fluorescence microscope to ensure that the floating fluorescent magnetic beads were cleaned, RPE differentiation medium was added, and the image was collected under a fluorescence microscope). The experimental results are shown in Figure 12, and the patient RPE shows that the fluorescent phagocytosis signal is significantly weaker than that of normal human iPSC-RPE. The above results show that RPGR-ORF15 transcript is expressed in RPE cells, and in addition, the mutant RPGR-ORF15 will cause abnormal RPE phagocytosis function.

[0161] Example 9. EFS promoter shows better editing efficiency and functional recovery effect

[0162] Based on the above research, the inventors modified the promoters expressing Cas9 in the AAV-saCas9-U6-sgRNA8 vector combination used in Example 7 to shorter broad-spectrum promoters EFS (SEQ ID NO: 68) and enRK (SEQ ID NO: 69), respectively, and packaged the modified and unmodified vectors into AAV-I. The obtained AAV-I vectors were mixed with the corresponding AAV-II vectors, and the mixture was subretinally injected (1E9 vg / eye of each virus) into 3-month-old RPGR humanized mouse models. Six months after administration, the retinal function of the mice in each group was detected by ERG, and the retinal cell RNA of the mice was collected after the functional detection to detect the change in mRNA expression efficiency after repair of the model mice. The ERG results are shown in FIG. 13. Compared with the control group, the EFS group showed a significant increase in the amplitudes of the a wave and b wave of the ERG dark response, while the enRK and hGRK1 groups did not show an increase in the amplitudes. In addition, the q-RT-PCR detection results are shown in FIG. 14. Compared with the control group, the EFS group showed a significant decrease in the expression of RPGR-ORF15-mut and a significant increase in the expression of RPGR-ORF15-opt (ORF15-opt represents optimized codons of ORF15), and the change in mRNA in the EFS group was significantly higher than that in the enRK and hGRK1 groups. The above results show that the broad-spectrum promoter EFS shows more effective in vivo editing effect and functional recovery effect.

[0163] Example 10. In vivo efficacy detection of other broad-spectrum promoters

[0164] The promoters expressing Cas9 in the AAV-saCas9-U6-sgRNA8 vector combination were modified to EFS-intron1 (intron1 is added after the EFS promoter, SEQ ID NO: 70), EFS-intron2 (intron2 is added after the EFS promoter, SEQ ID NO: 71), CMV (SEQ ID NO: 72), and smCAG (SEQ ID NO: 73) promoters, respectively, and the modified vectors were packaged into AAV-I. The obtained AAV-I vectors were mixed with the corresponding AAV-II vectors in the previous examples and subretinally injected (1E9 vg / eye of each virus) into 3-month-old RPGR humanized mouse models. Six months after administration, the retinal function of the mice in each group was detected by ERG, and the experimental results are shown in FIG. 15. Compared with the control group, the four promoters in the promoter group showed a certain degree of recovery trend for the ERG amplitude of the model mice, but the EFS-intron1 and EFS-intron2 groups showed a more optimal treatment trend.

[0165] Example 11. Comparison of in vivo editing efficiency and functional recovery effect of different vector element combinations

[0166] The aforementioned RPGR gene editing vector molecule structure combination is shown in Figure 7, wherein the AAV-saCas9-U6-sgRNA vector packaged in AAV-I comprises a Cas9 protein expression frame and an sgRNA expression frame targeting RPGR intron 14. The inventors compared the in vivo efficacy of AAV-saCas9 combined with AAV-II after administration to the RPGR humanized mouse model, wherein the AAV-saCas9 comprises an sgRNA expression frame or the sgRNA expression frame is removed. The experimental steps are described in the foregoing examples. Six months after administration, the ERG detection results are shown in Figure 16, and no significant difference in ERG amplitude is observed between AAV-saCas9-U6-sgRNA and AAV-saCas9 expressed by the same promoter. The Q-RT-PCR detection results of mRNA expression levels after repair are shown in Figure 17, and the expression efficiency after repair by AAV-saCas9-U6-sgRNA is slightly higher than that of the AAV-saCas9 group.

[0167] In summary, the inventors screened sgRNA2, sgRNA6, sgRNA8 targeting human RPGR intron 14 and the corresponding targeting vectors, which can effectively repair the RPGR DNA sequence, and the targeting sequence inserted into the target site does not affect the mRNA splicing of RPGR-ORF15 transcript, while ensuring the normal expression of RPGR-ORF15 protein; at the same time, the iPSC experimental results show that the above-mentioned sgRNA can also effectively play a repair role in patient cells; further, the in vivo experimental results show that sgRNA2, sgRNA6, sgRNA8 also have good DNA editing function in animals, and sgRNA8 has the highest editing efficiency. In addition, due to the potential function of RPGR-ORF15 in RPE, the photoreceptor-specific promoter does not show good functional repair, and the optimization of the gene editing treatment structure molecule shows that the broad-spectrum promoter has better treatment effect than the specific promoter in effectively repairing the abnormal function of the mouse retina. Therefore, repairing RPGR-ORF15 by AAV vector carrying the above-optimized vector combination is a potential treatment method for X-linked retinal degeneration caused by RPGR mutation.

[0168] Those skilled in the art should understand that, although the present application has been specifically described with reference to the foregoing examples, the present application is not limited to these specific examples. Based on the methods and technical solutions taught by the present application, those skilled in the art can make appropriate modifications or improvements without departing from the essence of the present application, and the resulting equivalent embodiments are within the scope of the present application.

[0169] References

[0170] 1) Mays Talib, Mary J van Schooneveld, Alberta A Thiadens, Marta Fiocco, Jan Wijnholds, Ralph J Florijn, Nicoline E Schalij-Delfos, Maria M van Genderen, Hein Putter, Frans P M Cremers, Gislin Dagnelie, Jacoline B Ten Brink, Caroline C W Klaver, L Ingeborgh van den Born, Carel B Hoyng, Arthur A Bergen, Camiel J F Boon. Clinical and genetic characteristics of male patients with RPGR-associated retinal dystrophies: A Long-Term Follow-up Study. Retina. 2019 Jun; 39(6): 1186-1199.

[0171] 2) Vervoort R, Lennon A, Bird AC, Tulloch B, Axton R, Miano MG, Meindl A, Meitinger T, Ciccodicola A, Wright AF. Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa. Nat Genet. 2000 Aug; 25(4): 462-6. doi: 10.1038 / 78182.

[0172] 3) Cehajic-Kapetanovic J, Xue K, Martinez-Fernandez de la Camara C, Nanda A, Davies A, Wood LJ, Salvetti AP, Fischer MD, Aylward JW, Barnard AR, Jolly JK, Luo E, Lujan BJ, Ong T, Girach A, Black GCM, Gregori NZ, Davis JL, Rosa PR, Lotery AJ, Lam BL, Stanga PE, MacLaren RE. Initial results from a first-in-human gene therapy trial on X-linked retinitis pigmentosa caused by mutations in RPGR. Nat Med. 2020 Feb 24. doi: 10.1038 / s41591-020-0763-1.

[0173] 4) Schlegel J, Hoffmann J, D, Muller B, Gunther S, Zhang W, Janise A, C, Fuhler B, Neidhardt J, Khanna H, Lorenz B, Stieger K. Toward genome editing in X-linked RP - development of a mouse model with specific treatment relevant features. Transl Res. 2019 Jan;203:57-72. doi: 10.1016 / j.trsl.2018.08.006.

[0174] 5) Wright RN, Hong DH, Perkins B. Misexpression of the constitutive Rpgr(ex1-19) variant leads to severe photoreceptor degeneration. Invest Ophthalmol Vis Sci. 2011 Jul 15;52(8):5189-201.

[0175] 6) Megaw RD, Soares DC, Wright AF. RPGR: Its role in photoreceptor physiology, human disease, and future therapies. Exp Eye Res. 2015 Sep;138:32-41.

[0176] 7) Frederiksen HR, Holst B, Mau-Holzmann UA, Freude K, Schmid B. Generation of two isogenic iPSCs lines with either a heterozygous or a homozygous E280A mutation in the PSEN1 gene. Stem Cell Res. 2019 Feb 7;35:101403.

[0177] 8) Shrock E, Guell M. CRISPR in Animals and Animal Models. Prog Mol Biol Transl Sci. 2017;152:95-114.

[0178] 9) Karimian A, Azizian K, Parsian H, Rafieian S, Shafiei-Irannejad V, Kheyrollah M, Yousefi M, Majidinia M, Yousefi B. CRISPR / Cas9 technology as a potent molecular tool for gene therapy. J Cell Physiol. 2019 Jan 30. doi: 10.1002 / jcp.27972. Review.

[0179] 10) David E. Buchholz, Britney O. Pennington, Roxanne H. Croze, Cassidy R. Hinman, Peter J. Coffey, Dennis O. Clegg. Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium. STEM CELLS TRANSLATIONAL MEDICINE. 2013; 2: 384-393.

Claims

1. A vector comprising: (1) a nucleotide sequence encoding a Cas protein and operably linked to a first promoter, wherein the first promoter is a broad-spectrum promoter or a non-retina-specific promoter; and (2) a nucleotide sequence encoding a gRNA and operably linked to a second promoter, wherein the gRNA targets or specifically binds to a gene encoding a regulator of retinitis pigmentosa GTPase (RPGR gene) or a fragment thereof.

2. The vector of claim 1, wherein the first promoter is a mammalian constitutive promoter, preferably selected from the group consisting of an elongation factor 1 alpha short (EFS) promoter, an elongation factor 1 alpha (EF1a) promoter, an RK promoter, a CMV promoter, a CAG promoter, a human beta-actin promoter, a small CBA (smCBA) promoter, a CBS promoter or CBh promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, an OPEFS promoter, and an SV40 promoter.

3. The vector of claim 2, wherein the first promoter is an EFS promoter.

4. The vector of claim 2, wherein the first promoter is followed by an intron sequence.

5. The vector of claim 3 or 4, wherein the first promoter is an EFS promoter (SEQ ID NO: 68), an enRK promoter (SEQ ID NO: 69), an EFS-intronl promoter (SEQ ID NO: 70), an EFS-intron2 promoter (SEQ ID NO: 71), a CMV promoter (SEQ ID NO: 72), or a smCAG promoter (SEQ ID NO: 73).

6. The vector of claim 5, wherein the first promoter is an EFS promoter (SEQ ID NO: 68), an EFS-intronl promoter (SEQ ID NO: 70), or an EFS-intron2 promoter (SEQ ID NO: 71).

7. The vector of any one of claims 1 to 5, wherein the Cas protein comprises a Cas9 protein.

8. The vector of claim 7, wherein the Cas9 protein comprises a SaCas9, a SpCas9, a SauriCas9, a KKH-SaCas9, a SlugCas9, or a SlugCas9-HF.

9. The vector of any one of claims 1 to 8, wherein the gRNA targets or specifically binds to an intron 14 partial sequence of the RPGR gene, preferably the intron 14 comprises a nucleotide sequence set forth in SEQ ID NO:

75.

10. The vector of claim 9, wherein the gRNA is a single guide RNA (sgRNA), preferably comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76 to 97, more preferably comprising a nucleotide sequence set forth in SEQ ID NO: 77, SEQ ID NO: 81 or SEQ ID NO: 83, most preferably comprising a nucleotide sequence set forth in SEQ ID NO:

83.

11. The vector of any one of claims 1 to 10, wherein the second promoter is a U6 promoter, preferably the U6 promoter comprises a nucleotide sequence set forth in SEQ ID NO:

74.

12. The vector of any one of claims 1 to 11, wherein the Cas protein is saCas9; the first promoter is an EFS promoter (SEQ ID NO: 68), an EFS-intronl promoter (SEQ ID NO: 70) or an EFS-intron2 promoter (SEQ ID NO: 71); the nucleotide sequence encoding a gRNA comprises a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 8, preferably a nucleotide sequence set forth in SEQ ID NO: 8; and the second promoter is a U6 promoter (SEQ ID NO: 74).

13. The vector of any one of claims 1 to 12, wherein the vector comprises the following elements in order from 5’ to 3’ end direction: an inverted terminal repeat (ITR), a first promoter, a Kozak sequence, a nuclear import signal, a nucleotide sequence encoding a Cas protein, a nuclear import signal, a transcription terminator sequence, a second promoter, a nucleotide sequence encoding a gRNA, a gRNA scaffold sequence and an inverted terminal repeat (ITR).

14. The vector of claim 13, wherein the transcription terminator sequence is bGH poly(A) or SV40 poly(A), preferably bGH poly(A).

15. The vector of any one of claims 1 to 14, wherein the vector comprises a viral vector.

16. The vector of claim 15, wherein the viral vector is an adeno-associated virus (AAV) vector.

17. The vector of claim 16, wherein the adeno-associated virus vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and any AAV variant or mixture, preferably an AAV8 vector.

18. A CRISPR-Cas system comprising the vector of any one of claims 1 to 17.

19. The system of claim 18, further comprising a targeting vector, wherein preferably the targeting vector comprises, in the 5’ to 3’ end direction, a RPGR gene intron 14 partial sequence, a wild type or codon-optimized RPGR gene ORF15 region sequence, and a transcription terminator sequence, preferably a bGH PolyA.

20. The system of claim 19, wherein the 5’ and 3’ ends of the targeting vector introduce target sites corresponding to a reverse sgRNA, wherein the sgRNA is the sgRNA of claim 1.

21. The system of claim 18 or 19, wherein the targeting vector further comprises a tag, such as a fusion tag or a resistance tag, preferably a GFP fusion tag, a HA fusion tag, or a puromycin resistance tag.

22. The system of any one of claims 19-20, wherein the codon-optimized RPGR gene ORF15 region sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:

45.

23. The system of any one of claims 19-22, wherein the RPGR gene intron 14 partial sequence is selected from the group consisting of SEQ ID NOs: 56-58.

24. The system of any one of claims 19-23, wherein the bGH PolyA transcription termination sequence comprises the nucleotide sequence set forth in SEQ ID NO:

62.

25. The system of any one of claims 19-24, wherein: the RPGR gene intron 14 partial sequence has the nucleotide sequence set forth in SEQ ID NO: 56, and the sgRNA has the nucleotide sequence set forth in SEQ ID NO: 77; the RPGR gene intron 14 partial sequence has the nucleotide sequence set forth in SEQ ID NO: 57, and the sgRNA has the nucleotide sequence set forth in SEQ ID NO: 81; or the RPGR gene intron 14 partial sequence has the nucleotide sequence set forth in SEQ ID NO: 58, and the sgRNA has the nucleotide sequence set forth in SEQ ID NO:

83.

26. The system of any one of claims 19-25, wherein the targeting vector is a HITI (homology-independent targeted integration) donor vector, preferably an AAV2 / 8 type vector.

27. A cell comprising the vector of any one of claims 1-17 or the system of any one of claims 18-26.

28. A pharmaceutical composition comprising the vector of any one of claims 1-17, the system of any one of claims 18-26, and / or the cell of claim 27.

29. A kit comprising the vector of any one of claims 1-17, the system of any one of claims 18-26, and / or the cell of claim 27.

30. Use of the vector of any one of claims 1-17, the system of any one of claims 18-26, and / or the cell of claim 27 in the manufacture of a medicament for the treatment of a disease caused by a mutation in the RPGR gene, in particular in the ORF15 region.

31. The use of claim 30, wherein the disease comprises retinitis pigmentosa.

32. The use of claim 30 or 31, wherein the disease comprises X-linked retinitis pigmentosa.

33. The use of any one of claims 30 to 32, wherein the medicament is formulated in a form suitable for injection.

34. The use of claim 33, wherein the medicament is formulated in a form suitable for subretinal space injection or intravitreal injection.

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