Use of CYP4v2 and rdcvf in medicament manufacture

Abstract

To provide use of CYP4V2 and RdCVF in medicament manufacture.SOLUTION: Disclosed are: the use of CYP4V2 and RdCVF in the manufacture of a medicament for treating, alleviating, and / or preventing a disease or disorder associated with retinal pigment epithelium (RPE) atrophy; an isolated nucleic acid molecule comprising a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF; an amino acid sequence encoded by the isolated nucleic acid molecule; a vector comprising the isolated nucleic acid molecule; and a cell comprising the nucleic acid or the vector; and use thereof in the manufacture of a medicament for treating, alleviating, and / or preventing a disease or disorder associated with retinal pigment epithelium (RPE) atrophy.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application relates to the biopharmaceutical field, specifically to the use of CYP4V2 and RdCVF in the manufacture of medicines. [Background technology]

[0002] Bietti's crystalline dystrophy (BCD) is a rare disease of retinal degeneration characterized by crystals in the cornea (transparent covering), fine, yellow, or white crystalline deposits in the light-sensitive tissues of the retina, and progressive atrophy of the retina, choriocapillaris, and choroid. The deposits damage the retina, causing gradual loss of vision. People with crystalline retinal degeneration typically begin to experience vision loss during their teens and twenties, and vision loss in each eye may worsen at different paces. The severity and progression of symptoms can vary greatly from individual to individual, even within the same family, but most people become blind by age 40 or 50. It is estimated that 1 in 67,000 people worldwide has crystalline retinal degeneration, and it is more common in East Asians, particularly Chinese and Japanese.

[0003] Current research has shown that BCD is an autosomal recessive genetic disease caused by mutations in the CYP4V2 gene. Many gene mutation sites in CYP4V2 have been found in the genetic research of BCD patients at home and abroad. The CYP4V2 gene is one of the proteins in the cytochrome P450 superfamily, and the protein encoded by the CYP4V2 gene is involved in the process of fatty acid metabolism. It is believed that CYP4V2 gene mutations in crystalline retinal degeneration disrupt the enzyme function involved in fatty acid metabolism, thereby affecting the breakdown of lipids. However, it is unclear how CYP4V2 causes the specific signs and symptoms of BCD. Currently, the treatment of BCD mainly refers to the treatment method for retinitis pigmentosa (RP), and although research into CYP4V2 gene mutations has provided the possibility for future gene therapy, there is still no effective treatment method at present. Summary of the Invention [Means for solving the problem]

[0004] The present application provides the use of CYP4V2 and RdCVF in the manufacture of a medicament for the treatment, amelioration and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0005] In some embodiments, the disease or condition comprises crystalline retinal degeneration.

[0006] In some embodiments, the CYP4V2 is human CYP4V2.

[0007] In some embodiments, CYP4V2 comprises an amino acid sequence set forth in any of SEQ ID NOs:76-82.

[0008] In some embodiments, the polynucleotide encoding CYP4V2 comprises a nucleic acid sequence set forth in any of SEQ ID NOs:62-68.

[0009] In some embodiments, the RdCVF is human RdCVF.

[0010] In some embodiments, the RdCVF comprises an amino acid sequence set forth in any of SEQ ID NOs:83-89.

[0011] In some embodiments, the polynucleotide encoding RdCVF comprises a nucleic acid sequence set forth in any of SEQ ID NOs:69-75.

[0012] In some embodiments, the medicament comprises a polynucleotide encoding the CYP4V2 and a polynucleotide encoding the RdCVF.

[0013] In some embodiments, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are located on different vectors.

[0014] In some embodiments, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are located on the same vector.

[0015] In some embodiments, the vector comprises a viral vector.

[0016] In some embodiments, the vector is a viral vector, and the viral vector comprises an AAV vector.

[0017] In some embodiments, the vector further comprises a promoter located at the 5' end of and operably linked to the polynucleotide encoding CYP4V2.

[0018] In some embodiments, the vector further comprises a promoter located at the 5' end of and operably linked to the polynucleotide encoding RdCVF.

[0019] In some embodiments, the promoter comprises a nucleotide sequence set forth in any of SEQ ID NOs:1-12.

[0020] In some embodiments, the vector further comprises a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide encoding CYP4V2.

[0021] In some embodiments, the vector further comprises a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide encoding RdCVF.

[0022] In some embodiments, the vector further comprises a polynucleotide encoding a self-shearing peptide, wherein the polynucleotide encoding the self-shearing peptide is located between the polynucleotide encoding the CYP4V2 and the polynucleotide encoding the RdCVF.

[0023] In some embodiments, the self-cleaving peptide comprises P2A.

[0024] In some embodiments, the polynucleotide encoding the self-cleaving peptide comprises a nucleotide sequence set forth in any of SEQ ID NOs:22-25.

[0025] In some embodiments, the vector comprises, in order from 5' to 3', the promoter, a polynucleotide encoding the CYP4V2, a polynucleotide encoding the self-cleaving peptide, a polynucleotide encoding the RdCVF, and the polyadenylation signal site.

[0026] In some embodiments, the vector comprises, in order from 5' to 3', the promoter, a polynucleotide encoding CYP4V2, and the polyadenylation signal site; or, the vector comprises, in order from 5' to 3', the promoter, a polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0027] In some embodiments, the vector further comprises an intron.

[0028] In some embodiments, the intron comprises a nucleotide sequence set forth in any of SEQ ID NOs:13-16.

[0029] In some embodiments, the intron is located within the polynucleotide encoding the CYP4V2 or at the 5' end of the polynucleotide encoding the CYP4V2.

[0030] In some embodiments, the intron is located within the polynucleotide encoding the RdCVF or at the 5' end of the polynucleotide encoding the RdCVF.

[0031] In some embodiments, the vector comprises a nucleotide sequence set forth in any of SEQ ID NOs:90-116.

[0032] The present application also provides a vector combination for the treatment, alleviation and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy, the vector combination comprising a first vector and a second vector, the first vector comprising a polynucleotide encoding CYP4V2, and the second vector comprising a polynucleotide encoding RdCVF.

[0033] In some embodiments, the disease or condition comprises crystalline retinal degeneration.

[0034] In some embodiments, the CYP4V2 is human CYP4V2.

[0035] In some embodiments, CYP4V2 comprises an amino acid sequence set forth in any of SEQ ID NOs:76-82.

[0036] In some embodiments, the polynucleotide encoding CYP4V2 comprises a nucleic acid sequence set forth in any of SEQ ID NOs:62-68.

[0037] In some embodiments, the RdCVF is human RdCVF.

[0038] In some embodiments, the RdCVF comprises an amino acid sequence set forth in any of SEQ ID NOs:83-89.

[0039] In some embodiments, the polynucleotide encoding RdCVF comprises a nucleic acid sequence set forth in any of SEQ ID NOs:69-75.

[0040] In some embodiments, the vector comprises a viral vector.

[0041] In some embodiments, the vector is a viral vector, and the viral vector comprises an AAV vector.

[0042] In some embodiments, the first vector further comprises a promoter located at the 5' end of and operably linked to the polynucleotide encoding CYP4V2.

[0043] In some embodiments, the second vector further comprises a promoter located at the 5' end of and operably linked to the polynucleotide encoding RdCVF.

[0044] In some embodiments, the promoter comprises a nucleotide sequence set forth in any of SEQ ID NOs:1-12.

[0045] In some embodiments, the first vector further comprises a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide encoding CYP4V2.

[0046] In some embodiments, the second vector further comprises a polyadenylation signal site, wherein the polyadenylation signal site is located at the 3' end of the polynucleotide encoding RdCVF.

[0047] In some embodiments, the first vector comprises, in order from 5' to 3', the promoter, a polynucleotide encoding CYP4V2, and the polyadenylation signal site; and / or the second vector comprises, in order from 5' to 3', the promoter, a polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0048] In some embodiments, the first vector and / or the second vector further comprises an intron.

[0049] In some embodiments, the intron comprises a nucleotide sequence set forth in any of SEQ ID NOs:13-16.

[0050] In some embodiments, the intron is located within the polynucleotide encoding the CYP4V2 or at the 5' end of the polynucleotide encoding the CYP4V2.

[0051] In some embodiments, the intron is located within the polynucleotide encoding the RdCVF or at the 5' end of the polynucleotide encoding the RdCVF.

[0052] In some embodiments, the first vector comprises a nucleotide sequence set forth in any of SEQ ID NOs:90-95, and / or the second vector comprises a nucleotide sequence set forth in any of SEQ ID NOs:96-100.

[0053] The present application also provides one or more isolated nucleic acid molecules, the nucleic acid molecules comprising: a) a polynucleotide encoding CYP4V2; and b) a polynucleotide encoding RdCVF.

[0054] In some embodiments, the CYP4V2 is human CYP4V2.

[0055] In some embodiments, CYP4V2 comprises an amino acid sequence set forth in any of SEQ ID NOs:76-82.

[0056] In some embodiments, the polynucleotide encoding CYP4V2 comprises a nucleic acid sequence set forth in any of SEQ ID NOs:62-68.

[0057] In some embodiments, the RdCVF is human RdCVF.

[0058] In some embodiments, the RdCVF comprises an amino acid sequence set forth in any of SEQ ID NOs:83-89.

[0059] In some embodiments, the polynucleotide encoding RdCVF comprises a nucleic acid sequence set forth in any of SEQ ID NOs:69-75.

[0060] In some embodiments, the nucleic acid molecule further comprises a promoter located at the 5' end of and operably linked to the polynucleotide encoding CYP4V2.

[0061] In some embodiments, the nucleic acid molecule further comprises a promoter located at the 5' end of and operably linked to the polynucleotide encoding the RdCVF.

[0062] In some embodiments, the promoter comprises a nucleotide sequence set forth in any of SEQ ID NOs:1-12.

[0063] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

[0064] In some embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotide encoding the self-cleaving peptide is located between the polynucleotide encoding the CYP4V2 and the polynucleotide encoding the RdCVF.

[0065] In some embodiments, the self-cleaving peptide comprises P2A.

[0066] In some embodiments, the polynucleotide encoding the self-cleaving peptide comprises a nucleotide sequence set forth in any of SEQ ID NOs:22-25.

[0067] In some embodiments, the nucleic acid molecule further comprises a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide encoding CYP4V2.

[0068] In some embodiments, the nucleic acid molecule further comprises a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide encoding RdCVF.

[0069] In some embodiments, the nucleic acid molecule comprises, in order from 5' to 3', the promoter, a polynucleotide encoding the CYP4V2, a polynucleotide encoding the self-cleaving peptide, a polynucleotide encoding the RdCVF, and the polyadenylation signal site.

[0070] In some embodiments, the nucleic acid molecule further comprises an intron.

[0071] In some embodiments, the intron comprises a nucleotide sequence set forth in any of SEQ ID NOs:13-16.

[0072] In some embodiments, the intron is located within the polynucleotide encoding CYP4V2 or at the 5' end of the polynucleotide encoding CYP4V2.

[0073] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any of SEQ ID NOs:101-115.

[0074] The present application further provides a vector comprising the isolated nucleic acid molecule described herein.

[0075] In some embodiments, the vector is a viral vector.

[0076] In some embodiments, the viral vector comprises an AAV vector.

[0077] The present application further provides a cell comprising the nucleic acid molecule described herein or the vector described herein.

[0078] The present application also provides a pharmaceutical composition, said pharmaceutical composition comprising said nucleic acid molecule of the present application, said vector of the present application and / or said cell of the present application.

[0079] The present application also provides the use of a nucleic acid molecule described herein, a vector described herein, or a cell described herein in the manufacture of a medicament, wherein the medicament is for the treatment, alleviation and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0080] In some embodiments, the disease or condition comprises crystalline retinal degeneration.

[0081] Those skilled in the art can easily ascertain other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Therefore, the accompanying drawings and the description of the present application are merely illustrative and not restrictive.

[0082] Specific features of the invention according to the present application are set forth in the appended claims. A better understanding of the features and advantages of the invention according to the present application can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is as follows: [Brief description of the drawings]

[0083] [Figure 1] 1 shows the expression effect of different CYP4V2 promoters in the present application. [Diagram 2] 1 shows the expression effects of CAG, EF1a, OPEFS and EFS promoters in the present application. [Diagram 3] 1 shows the expression enhancing effect of the promoter-post-binding intron in the present application. [Figure 4] 1 shows the expression enhancing effect of the promoter-post-binding intron in the present application. [Diagram 5] 1 shows the effect of the selection of different PolyA signal sites in the expression vectors of the present application on the expression of a gene of interest. [Figure 6] 1 shows the expression of the expression vector containing the CYP4V2 and RdCVF genes of the present invention in 293T cells. [Figure 7] 1 shows the expression of the expression vector containing the CYP4V2 and RdCVF genes of the present invention in ARPE-19 cells. [Figure 8] FIG. 1 shows infection of mouse retina with different serotypes of AAV viruses in this study, with the double arrow indicating the range of expression of the EGFP reporter gene. [Figure 9]The morphology of renal epithelial cells, IPSC cells, and RPE cells observed under a fluorescent microscope during the production of RPE cells according to the present invention is shown. [Figure 10] 10A shows the effect of the virus titer in this application on the death of RPE cells differentiated from human iPSCs, 10B and 10C show the effect of the virus titer on the expression of inflammatory cytokines NLRP3 and TNF-α in RPE cells differentiated from human iPSCs, and 10D shows the expression of target genes in RPE cells differentiated from human iPSCs under different virus titer conditions. [Figure 11] The expression status of CYP4V2 and RdCVF is shown when RPE cells differentiated from human iPSCs are infected with different viruses of the present application. [Figure 12] 1 shows the effect of treatment with viruses containing different promoters and the CYP4V2 gene on fundus crystal deposition in BCD mice. [Figure 13] Statistical results of the effect of treatment with viruses containing different promoters and the CYP4V2 gene on fundus crystal deposition in BCD mice are shown. [Figure 14] 1 shows the results of immunofluorescence staining of CYP4V2 in BCD mice treated with viruses containing different promoters and CYP4V2 genes. [Figure 15] The present application shows the effect on ocular crystal deposition in BCD mice after treatment with different doses of the virus containing the CYP4V2 gene. [Figure 16] The present application presents statistical results of the effects on ocular crystal deposition status after treatment of BCD mice with different doses of a virus containing the CYP4V2 gene. [Figure 17] Herein, we show the effects on the expression levels of inflammatory cytokines TNF-α, IFN-γ, and NLRP3 after treatment of BCD mice with different doses of the virus containing the CYP4V2 gene. [Figure 18] Here, we show the effect on fundus crystal deposition in BCD mice after treatment with different viruses. [Figure 19]Herein, the statistical results of the effect on the ocular fundus crystal deposition status after treatment of BCD mice with different viruses are shown. [Figure 20] Here we show the effect on retinal function (electroretinography (ERG)) after treatment of BCD mice with the different viruses. [Figure 21] Herein, we present the results of immunofluorescence staining of CYP4V2 and RdCVF after treatment of BCD mice with different viruses. [Figure 22] Here we show the effect on RPE cell number and morphology after treatment of BCD mice with different viruses. [Figure 23] Herein, we present the statistical results of the effect on RPE cell numbers after treatment of BCD mice with different viruses. [Figure 24] Here we show the effect on retinal function (electroretinography (ERG)) after treatment of BCD mice with different viruses that do not contain an intron after the promoter. [Diagram 25] Here we show the effect on retinal function (electroretinography (ERG)) after treatment of BCD mice with viruses with different p1 promoters. [Figure 26] In this application, the effect on retinal function (electroretinography (ERG)) after treatment of BCD mice with viruses having different WPRE-SV40 poly A signal sites is shown. [Figure 27] Here we show the effect on retinal function (electroretinography (ERG)) after treatment of BCD mice with different viruses that do not contain a signal site. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] In the following, the embodiments of the present invention will be described using specific specific embodiments, but those skilled in the art can easily understand other advantages and effects of the present invention from the disclosure in this application.

[0085] The present application is further described below: In the present invention, unless otherwise specified, the scientific and technical terms used in the present application have the meanings that can be commonly understood by those skilled in the art. Furthermore, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the respective fields. In addition, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0086] In this application, the term "isolated" generally refers to something that has been artificially obtained from a natural state. If a substance or component occurs in nature that is "isolated," then there may have been a change in the natural environment in which it was placed, or the substance may have been isolated from its natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide may naturally occur in a living animal, and the same polynucleotide or polypeptide may be said to be isolated in high purity from its natural state. The term "isolated" does not exclude the presence of artificial or synthetic contaminants, or other impurities that do not affect the activity of the substance.

[0087] As used herein, the term "isolated nucleic acid molecule" generally refers to an isolated form of any length of nucleotide, deoxyribonucleotide, or ribonucleotide, or an analogue that has been isolated from its natural environment or artificially synthesized.

[0088] In this application, the term "CYP4V2" generally refers to a protein that is a member 2 of cytochrome P450 family 4 subfamily V. Cytochrome P450, also called cytochrome P450 or CYP450, generally refers to a family of iron hemoproteins, which belong to a type of monooxygenase and participate in the metabolism of endogenous substances or exogenous substances, including medicines and environmental compounds. According to the degree of homology of amino acid sequence, its members are divided into three classes: family, subfamily and enzyme individual. Cytochrome P450 enzyme system can be abbreviated as CYP, in which family is represented by Arabic numerals, subfamily is represented by capital letters, and enzyme individual is represented by Arabic numerals, for example, CYP4V2 in this application. The human CYP4V2 gene (HGNC::23198) is 19.28 kb long, located at 4q35, has 11 exons, and plays an important role in fatty acid metabolism (Kumar S., Bioinformation, 2011, 7:360-365). CYP4V2 is expressed in almost all tissues, but is expressed at high levels in the retina and retinal pigment epithelium, and at somewhat lower levels in the cornea, tissue, while mutations in the CYP4V2 gene can cause BCD (Li et al., Am J Hum Genet. 74:817-826, 2004).

[0089] In this application, the term "RdCVF" is also referred to as rod derived cone survival factor, which generally refers to a truncated thioredoxin lacking thiol oxidoreductase activity. RdCVF is a splice variant of the retinoid 1 (nucleoredoxin-like 1, Nxnl1) gene, the additional splice product of which is RdCVFL, an active thioredoxin that protects its binding ligand, the microtubule-associated protein TAU, from oxidation and aggregation (Elachouri et al., 2015 and Fridlich et al., 2009). Nxnl1 knockout mice can show age-dependent loss of rod and cone function and degenerative changes in cone cells, as well as hypersensitive response of rod and cone cells to oxidative stress (Cronin et al., 2010). Nxnl1 expression can be rod-dependent and is significantly decreased after rod cell death in RP (Delyfer et al., 2011; Reichman et al., 2010). RdCVF can protect cone cell function in several different genotypes (Byme et al., 2015; Le'veillard et al., 2004; Yang et al., 2009) of RP models. RdCVF molecule is secreted extracellularly as a signal peptide that promotes glucose uptake by photoreceptor cells and maintains photoreceptor cell survival. The 1 gene is conserved in humans, chimpanzees, rhesus monkeys, dogs, cows, rats, mice, chickens, zebrafish, and frogs. In humans, Nxnl1 is also called Txnl6, and Nxnl1 is located at 19 p13.11 and contains two exons. Nxnl1 is ubiquitously expressed in human tissues, including the lens, retina, stomach, kidney, heart, colon, and spleen, with relatively high levels expressed in the lens and retina.

[0090] In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that causes transcription of a specific gene. Promoters are recognized by RNA polymerase and initiate RNA transcription synthesis. In ribonucleic acid (RNA) synthesis, promoters interact with transcription factors that control gene transcription, and control the timing and level of gene expression (transcription). Promoters include a core promoter region and a control region, and are located upstream of the gene transcription start site (5' direction of the DNA antisense strand) in the control sequence that controls gene expression, and do not have a compilation function themselves. Based on their mode of action and function, they are divided into three types: constitutive promoters (which maintain continuous activity in most or all tissues), specific promoters (tissue-specific or developmental-specific), and inducible promoters (which are controlled by external chemical or physical signals).

[0091] In this application, the term "operably linked" generally refers to the placement of control sequences necessary for the expression of a coding sequence in an appropriate position relative to the coding sequence to allow expression of the coding sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. In some embodiments, it can refer to a sequence of coding sequences and transcriptional control elements in an expression vector. The control devices include a promoter, an enhancer, and a termination device. The Nxnl1 gene is conserved in humans, chimpanzees, rhesus monkeys, dogs, cows, rats, mice, chickens, zebrafish, and frogs. In some embodiments, the term "operably linked" can also refer to a gene of interest being linked into a vector such that the transcriptional and translational control sequences in the vector perform their expected function of regulating the transcription and translation of the gene of interest.

[0092] In this application, the term "self-cleaving peptides", also referred to as 2A peptides, is a peptide fragment usually of 18 to 22 amino acid residues, which can induce self-splicing of recombinant proteins containing 2A peptides in cells. 2A peptides are usually derived from the 2A region of a virus genome. In genetic engineering, 2A peptides can split a peptide chain translated from one open reading frame (ORF) into several independent peptide chains. In some embodiments, when two proteins need to be expressed separately (e.g., one protein needs to enter the nucleus and the other needs to be expressed in the cytoplasm), and only one open reading frame needs to be constructed on a vector, this can be achieved by inserting a single 2A peptide sequence into their coding regions. In some embodiments, if the fusion protein after the fusion of two proteins does not function, a sequence encoding a single 2A peptide can be inserted in the middle of the coding regions of the two proteins, or the binding peptide can be replaced with the 2A peptide, which allows the two proteins to be separated after translation completion and folded independently, thereby providing the possibility of restoring the function of the two proteins. 2A peptides can include P2A, E2A, F2A, and T2A, all named after their virus of origin, of which P2A is derived from the 2A peptide of Porcine teschovirus.

[0093] In this application, the term "polyadenylation signal sequence", also known as polyadenylation signal tail or Poly A tail, usually refers to the addition of tens to hundreds of single adenylate chains to the 3' end of transcribed mRNA. Polyadenylation usually occurs in the nucleus during the process of transcribing deoxyribonucleic acid (DNA) into ribonucleic acid (RNA) and completing the reaction with polyadenylate polymerase. In eukaryotes, polyadenylation is a mechanism that can terminate mRNA molecules at their 3' ends, and the polyadenylate sequence can protect mRNA from the attack of nucleic acid exonucleases, and is all very important for the export, translation and stability of mRNA from the cell nucleus.

[0094] In this application, the term "polyadenylation signal site" refers to a base sequence recognized by polyadenylation-associated cleavage factors, which are usually located at the 3' end of messenger RNA (mRNA). It is also usually an anterograde regulatory signal on mRNA. Generally, the tailing process (polyadenylation) begins after the end of transcription, and polyadenylation-associated cleavage factors add tens to hundreds of single adenylates after the 3'UTR of mRNA under the control of the polyadenylation signal site. Common tail signals include SV40, BGH, HSV, and TK signals. The polyadenylation-associated degradation factors include degradation / polyadenylate specificity factor (CPSF), degradation stimulation factor (CstF), degradation factor I (CFI), and degradation factor II (CFII). Polyadenylation signal sites can typically contain an AAUAAA sequence, but vary among eukaryotic groups. For example, most human polyadenylation signal sites contain an AAUAAA sequence, but this sequence is rare in plants and fungi.

[0095] As used herein, the term "intron" includes DNA segments removed from an RNA transcript by splicing together sequences (exons) at either end. Introns are typically considered to be interfering sequences within the protein-coding region of a gene and do not normally contain information that is expressed by the protein produced by the gene.

[0096] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and in which the protein can be expressed. A vector expresses a gene in a host cell by transformation, transformation or transfection into a host cell. Examples of vectors include: plasmids; bacteriophages; coplasmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-derived artificial chromosomes (PACs); phages such as lambda phages and M13 phages, and viral vectors. A vector contains various expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain a replication origin. A vector may also contain components that aid in cell entry, such as, but not limited to, viral particles, liposomes, and protein shells.

[0097] In this application, the term "viral vector" generally refers to a non-wild type recombinant viral particle that functions as a gene delivery vector and contains a recombinant viral genome packaged within a viral capsid. Types of animal viruses used as vectors can include reverse transcriptase viruses (including slow viruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papilloma-vacuolar viruses (e.g., SV40).

[0098] In this application, the term "AAV vector" is also called adeno-associated virus vector, and usually refers to the adenovirus itself or its derivatives. Adeno-associated virus (AAV) is a single-stranded DNA virus that usually belongs to the family Minute Virus. The AAV genome can contain inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs). The open reading boxes can include rep and cap. Rep consists of multiple overlapping genes that code for the Rep protein required for the life cycle of AAV, and contains overlapping nucleotide sequences that code for capsid proteins that can include VP1, VP2 and VP3. The capsid proteins interact to form a capsid. In the absence of an assistant virus, AAV can integrate its genome into a specific locus (AAVS site) on human chromosome 19 until rescue of the assistant virus (Kotin et al., 1990) from the latent state. AAV is generally considered to be predominantly in a mismatched form. The site-specific integration ability of AAV, its natural defects and its low immunogenicity make it an ideal gene therapy vector. There are many common serotypes of AAV, with over 100 different viral variants. In this application, the AAV coupler, ITRs and other selected AAV components are selected from any AAV, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, any variant of AAV known or referenced, or an undiscovered AAV or variant or mixture thereof.

[0099] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that is or is already a recipient of a nucleic acid molecule or vector. The cell can contain a nucleic acid molecule described herein or a vector described in the present invention. The cell can include the progeny of a single cell. The progeny may not necessarily be identical (morphological or genomic of the total DNA cross-complement) to the primordial mother cell due to spontaneous, accidental, or deliberate mutation. The cell can include cells transfected in vitro with a vector described in this application. The cell can be a bacterial cell (e.g., E. coli), yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293T cell.

[0100] In the present application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, such as a human. For example, a pharmaceutical composition described herein can include a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally a pharma- ceutical acceptable adjuvant. In addition, the pharmaceutical composition can also include a suitable formulation of one or more (pharma- ceutical effective) vectors, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of a composition are non-toxic to a recipient at the dosages and concentrations used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0101] In this application, the term "prevention" generally refers to a combination administered prophylactically to a healthy subject to prevent the occurrence of a disease or condition. It may further include a combination administered prophylactically to a patient in a pre-stage of the allergic disease to be treated. "Prevention" does not necessarily eliminate 100% the possibility of the occurrence of a disease or disorder; in other words, "prevention" usually means that the possibility of the occurrence of a disease or disorder is reduced in the presence of the administration combination.

[0102] As used herein, the term "ameliorate" refers to the reduction, narrowing, or slowing of a particular condition, disease, condition, or phenotype, which may include a condition, disease, condition, or phenotype that is subjectively felt by a subject, such as pain, dizziness, or other physiological disturbance, or a medically detectable indicator, such as a focal condition detected by a medical testing procedure.

[0103] As used herein, the term "treatment" generally refers to a clinical intervention used to alter the natural history of a treated individual or cell in a clinical pathological process, which may include amelioration of disease state, disappearance of lesions, or improvement of prognosis.

[0104] In this application, the term "retinal pigment epithelium (RPE)" refers to a layer of pigment cells that is usually attached to the outer side of the retina. The retinal pigment epithelium consists of a single layer of hexagonal cells, with dense pigment granules inside. The retinal pigment epithelium (RPE) is tightly connected to the choroid below and the retinal neurons above it, and its main functions include: controlling the fluid and nutrients in the subretinal space and acting as a blood-retinal barrier; regulating the local structure by synthesizing growth factors; absorbing light and adjusting the electrical balance; regenerating and synthesizing visual pigments; phagocytosis and digestion of the outer segments of photoreceptors; maintaining retinal attachment; and regenerating and repairing after injury. The RPE is an important tissue that maintains photoreceptor function and is simultaneously affected by many pathologies of the choroid and retina.

[0105] In this application, the term "retinal pigment epithelium (RPE) atrophy" usually refers to degenerative changes in the retinal pigment epithelium (RPE) that indicate cell death or dysfunction. Age-related macular degeneration or retinitis pigmentosa (RP) is often accompanied by atrophy of the retinal pigment epithelium. Retinitis pigmentosa (RP), also called retinal pigment disease, usually refers to a genetic ophthalmic disease. It is inherited in three ways: autosomal recessive, dominant, and X-linked, as well as digenic and mitochondrial. Common early signs include night blindness, narrowing of the visual field, and loss of vision in the frontal view and slightly deviated vision to the left and right. RP includes unicular primary retinitis pigmentosa, quadrant primary retinitis pigmentosa, central or paracentral primary retinitis pigmentosa, white punctate retinal degeneration with amelanotic retinitis pigmentosa, crystalline retinal degeneration, intravenous hypertrophic retinitis pigmentosa, arteriolar parapigment epithelial sparing retinitis pigmentosa, retinitis pigmentosa in Leber congenital amaurosis and other syndromes.

[0106] In this application, the term "crystalline retinal degeneration" generally refers to an autosomal recessive eye disease first described in 1937 by Italian ophthalmologist Dr. GB Bietti. Primary symptoms include crystals in the cornea (transparent covering), small yellow or white crystalline deposits in the light-sensitive tissue of the retina, and progressive atrophy of the retina, choriocapillaris, and choroid. Crystalline retinal degeneration may include diseases caused by mutations in the CYP4V2 gene.

[0107] In this application, the term "comprises" or "comprises" generally means the inclusion of specifically identified features without the exclusion of other elements.

[0108] As used herein, the term "about" generally refers to a variation of 0.5% to 10% above the specified value, for example, a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0109] Meanwhile, the present application provides the use of CYP4V2 and RdCVF in the manufacture of a medicament, said medicament being for the treatment, alleviation and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0110] In the present application, the medicine can comprise the polynucleotide encoding the CYP4V2 and the polynucleotide encoding the RdCVF.For example, the polynucleotide encoding the CYP4V2 and the polynucleotide encoding the RdCVF are located in different vectors.For example, the polynucleotide encoding the CYP4V2 and the polynucleotide encoding the RdCVF are located in the same vector.

[0111] Meanwhile, the present application also provides a combination of vectors for the treatment, alleviation and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy, comprising a first vector and a second vector, wherein the first vector may comprise a polynucleotide encoding CYP4V2, and the second vector may comprise a polynucleotide encoding RdCVF.

[0112] In contrast, the present application provides an isolated nucleic acid molecule that can include a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

[0113] However, the present application further provides cells that can contain any combination of the nucleic acid molecules described herein or the vectors described herein.

[0114] The present application also provides pharmaceutical compositions, which can comprise the nucleic acid molecules described herein, the vector combinations described herein, and / or the cells described herein.

[0115] However, the present application also provides the use of the nucleic acid molecules described herein, the combination of vectors described herein, the cells described herein and / or the pharmaceutical compositions described herein in the manufacture of a medicament, which can be used for the prevention, alleviation and / or treatment of a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0116] CYP4V2 In this application, CYP4V2 includes, but is not limited to, CYP4V2 from humans, chimpanzees, gorillas, rhesus monkeys, dogs, cows, mice, rats, chickens, fruit flies, nematodes or frogs or functional variants thereof, and can include proteins having dysfunction or encoding genetic mutations that cause crystalline retinal degeneration.

[0117] For example, CYP4V2 can include human CYP4V2.

[0118] For example, CYP4V2 can include an amino acid sequence set forth in any of SEQ ID NOs:76-82.

[0119] For example, the CYP4V2 can comprise the amino acid sequence shown in SEQ ID NO:76.

[0120] For example, the CYP4V2 can include an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:76-82, for example, any amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, and combined administration of the amino acid sequence with the RdCVF of the present application can improve a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0121] In the present application, a polynucleotide encoding CYP4V2 can include a nucleic acid sequence set forth in any of SEQ ID NOs:62-68.

[0122] For example, a polynucleotide encoding CYP4V2 can include the nucleic acid sequence shown in SEQ ID NO:62.

[0123] For example, the polynucleotide encoding the CYP4V2 can include any polynucleotide sequence having an amino acid sequence at least 90% identity to the nucleic acid sequence shown in SEQ ID NO:62-68, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, and the polypeptide encoded by the nucleotide sequence can be used in combination with the RdCVF of the present application to improve diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0124] RdCVF In the present application, the RdCVF may comprise a protein produced from rod cells and favoring cone cells, including, but not limited to, human, chimpanzee, gorilla, rhesus monkey, dog, cow, rat, mouse, chicken, zebrafish and frog RdCVF or functional variants thereof.

[0125] For example, the RdCVF can include human RdCVF.

[0126] For example, RdCVF can include an amino acid sequence shown in any of SEQ ID NOs:83-89.

[0127] For example, the RdCVF may comprise the amino acid sequence shown in SEQ ID NO:83.

[0128] For example, the RdCVF can include an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:83-89, for example, any amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the combined use of the amino acid sequence with the CYP4V2 of the present application can ameliorate a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0129] In the present application, the polynucleotide encoding the RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75.

[0130] For example, a polynucleotide encoding RdCVF can include the nucleic acid sequence shown in SEQ ID NO:69.

[0131] For example, the polynucleotide encoding the RdCVF can include any polynucleotide sequence having an amino acid sequence at least 90% identical to the nucleic acid sequence shown in SEQ ID NO:69-75, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the combined use of the polypeptide encoded by the nucleotide sequence with the CYP4V2 of the present application can ameliorate diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0132] promoter In the present application, the promoter can include an RPE cell-specific promoter, a retinal cell-specific promoter, a corneal cell-specific promoter, an ocular cell-specific promoter, or a constitutive promoter. The promoter can also include a mammalian β-actin promoter or a viral promoter. The promoter can also include a CAG promoter (heterozygous CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoter, CB promoter, or CBA promoter), a human β-actin promoter, a small CBA (smCBA) promoter, a CBS promoter, or a CBh promoter, an elongation factor 1 alpha short (EFS) promoter, an elongation factor 1 alpha (EF-1α) promoter, a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, a VMD2 (also called BEST1) promoter, an OPEFS promoter, a CYP4V2 itself promoter, an RPE65 promoter, or a heterologous or derivative thereof.

[0133] For example, the promoter can include the CYP4V2 promoter itself, which can include all or part of the 2000 bp nucleotide sequence upstream of the polynucleotide encoding CYP4V2.

[0134] For example, the promoter can include a nucleotide sequence shown in any of SEQ ID NOs:1-12.

[0135] For example, the CYP4V2 autologous promoter may include a CYP4V2-Pf promoter, a CYP4V2-P1 promoter, a CYP4V2-P2 promoter, a CYP4V2-P3 promoter, a CYP4V2-P4 promoter, a CYP4V2-P5 promoter, or a CYP4V2-P6 promoter. The CYP4V2-Pf promoter may comprise a nucleic acid sequence represented by SEQ ID NO:6, the CYP4V2 promoter may comprise a nucleic acid sequence represented by CYP4V2-Pf:7, the CYP4V2-P1 promoter may comprise a nucleic acid sequence represented by CYP4V2-P2:8, the CYP4V2-P3 promoter may comprise a nucleic acid sequence represented by CYP4V2-P4:9, the CYP4V2-P5 promoter may comprise a nucleic acid sequence represented by CYP4V2-P6:10, the CYP4V2-P5 promoter may comprise a nucleic acid sequence represented by SEQ ID NO:11, and the CYP4V2-P1 promoter may comprise a nucleic acid sequence represented by SEQ ID NO:12.

[0136] For example, the promoter may be OPEFS and comprises the nucleic acid sequence shown in SEQ ID NO:1.

[0137] For example, the promoter may be an EFS comprising the nucleic acid sequence shown in SEQ ID NO:2.

[0138] For example, the promoter may be EF1a, which comprises the nucleic acid sequence shown in SEQ ID NO:3.

[0139] For example, the promoter may be CAG, which comprises the nucleic acid sequence shown in SEQ ID NO:4.

[0140] For example, the promoter may be RPE65, which comprises the nucleic acid sequence shown in SEQ ID NO:5.

[0141] Polyadenylation signal site In the present application, the polyadenylation signal region includes the SV40 signal region, the BGH signal region, the WPRE signal region, the WPRE-SV40 signal region, the WPRE-BGH signal region, or derivatives thereof.

[0142] For example, the polyadenylation signal site is recognized by a polyadenylation-associated degradation factor to produce SV40 polyadenylate sequence, BGH signal polyadenylate sequence, HSV signal polyadenylate sequence, TK signal polyadenylate sequence, WPRE signal polyadenylate sequence, and the like.

[0143] For example, the polyadenylation signal site can include the sequence AAUAAA.

[0144] For example, the polyadenylation signal site can include the nucleic acid sequences represented by SEQ ID NOs:17-21.

[0145] Self-cleaving peptides In the present application, the polynucleotide encoding the self-cleaving peptide is located between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

[0146] For example, the self-cleaving peptide can include T2A, P2A, E2A, or F2A.

[0147] For example, the self-cleaving peptide can include P2A.

[0148] For example, the self-cleaving peptide can include an amino acid sequence set forth in any of SEQ ID NOs:26-29.

[0149] For example, a polynucleotide encoding the self-cleaving peptide can comprise a nucleic acid sequence set forth in any of SEQ ID NOs:22-25.

[0150] For example, the N-terminus of the splicing peptide may be linked to a GSG (Gly-Ser-Gly, glycine, serine, glycine) sequence.

[0151] Introns In the present application, the intron can be located at the 5' end of the gene of interest or in the nucleotide sequence of the gene of interest. The gene of interest can include a polynucleotide encoding CYP4V2 or a polynucleotide encoding RdCVF. The intron can enhance the expression of the gene of interest. For example, the intron can include a human β-globin intron, an SV40 intron, a heterosynthetic CBA / MVM intron, or other artificially synthesized introns.

[0152] For example, the intron comprises a nucleotide sequence shown in any one of SEQ ID NOs:13-16.

[0153] Isolated Nucleic Acid Molecules In the present application, the isolated nucleic acid molecule can include, in order from the 5' to the 3' end, a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

[0154] For example, the isolated nucleic acid molecule may comprise, in order from the 5' end to the 3' end, a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF, and the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68, and the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75.

[0155] In the present application, the isolated nucleic acid molecule may comprise, in order from the 5' end to the 3' end, a polynucleotide encoding the RdCVF and a polynucleotide encoding the CYP4V2.

[0156] For example, the isolated nucleic acid molecule may comprise, in order from the 5' end to the 3' end, a polynucleotide encoding the RdCVF and a polynucleotide encoding the CYP4V2, and the polynucleotide encoding the RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75, and the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68.

[0157] In the present application, the isolated nucleic acid molecule can also include the promoter.

[0158] For example, the promoter is located at the 5' end of the polynucleotide encoding the CYP4V2 and is operably linked to the polynucleotide encoding the CYP4V2.

[0159] For example, the promoter is located at the 5' end of the polynucleotide encoding the RdCVF and is operably linked to the polynucleotide encoding the RdCVF.

[0160] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, the promoter, a polynucleotide encoding the CYP4V2, and a polynucleotide encoding the RdCVF.

[0161] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, the promoter, a polynucleotide encoding the RdCVF, and a polynucleotide encoding the CYP4V2.

[0162] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs:1 to 12, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs:69 to 75, and the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs:62 to 68.

[0163] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, the promoter, a polynucleotide encoding the CYP4V2, the promoter, and a polynucleotide encoding the RdCVF.

[0164] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, the promoter, a polynucleotide encoding the CYP4V2, the promoter, and a polynucleotide encoding the RdCVF.

[0165] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs:1 to 12, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs:62 to 68, and the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs:69 to 75.

[0166] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, the promoter, a polynucleotide encoding the RdCVF, the promoter, and a polynucleotide encoding the CYP4V2.

[0167] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs:1 to 12, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs:69 to 75, and the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs:62 to 68.

[0168] In the present application, the isolated nucleic acid molecule can also include a polynucleotide encoding the self-cleaving peptide.

[0169] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, a promoter, a polynucleotide encoding CYP4V2, a polynucleotide encoding the self-shearing peptide, and a polynucleotide encoding RdCVF.

[0170] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, and the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75.

[0171] For example, the isolated nucleic acid molecule can comprise, in order from the 5' to the 3' end, a promoter, a polynucleotide encoding RdCVF, a polynucleotide encoding the self-shearing peptide, and a polynucleotide encoding CYP4V2.

[0172] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, and the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68.

[0173] For example, the isolated nucleic acid molecule can comprise a nucleic acid sequence set forth in any of SEQ ID NOs:101-105.

[0174] In the present application, the isolated nucleic acid molecule may also contain the polyadenylation signal site, which is located at the 3' end of the polynucleotide encoding CYP4V2.

[0175] In the present application, the isolated nucleic acid molecule may also contain a polyadenylation signal site, which is located at the 3' end of the coding RdCVF polynucleotide.

[0176] For example, the isolated nucleic acid molecule may comprise, in order from the 5' to 3' direction, the promoter, a polynucleotide encoding the CYP4V2, a polynucleotide encoding the self-cleaving peptide, a polynucleotide encoding the RdCVF, and the polyadenylation signal site.

[0177] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68, and the polyadenylation signal site may comprise a nucleotide sequence shown in any of SEQ ID NOs: 17 to 21.

[0178] In the present application, the nucleic acid molecule to be isolated may include, in the 5' to 3' direction, the promoter, a polynucleotide encoding the RdCVF, a polynucleotide encoding the self-shearing peptide, a polynucleotide encoding the CYP4V2, and the polyadenylation signal site.

[0179] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68, and the polyadenylation signal site may comprise a nucleotide sequence shown in any of SEQ ID NOs: 17 to 21.

[0180] For example, the isolated nucleic acid molecule can comprise a nucleic acid sequence set forth in any of SEQ ID NOs:106-108.

[0181] In the present application, the isolated nucleic acid molecule may also include the intron.

[0182] For example, the intron may comprise a nucleotide sequence set forth in any of SEQ ID NOs:13-16.

[0183] For example, the intron is located in the polynucleotide encoding the CYP4V2 or is located at the 5' end of the polynucleotide encoding the CYP4V2.

[0184] For example, the isolated nucleic acid molecule may comprise, in order from 5' to 3', the promoter, the intron, a polynucleotide encoding the CYP4V2, a polynucleotide encoding the self-cleaving peptide, and a polynucleotide encoding the RdCVF.

[0185] For example, the isolated nucleic acid molecule may comprise, in the 5' to 3' direction, the promoter, a polynucleotide encoding the CYP4V2 into which the intron has been inserted, a polynucleotide encoding the self-cleaving peptide, and a polynucleotide encoding the RdCVF.

[0186] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the intron may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 13 to 16, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, and the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75.

[0187] For example, the nucleic acid molecule to be isolated comprises, in the 5' to 3' direction, the promoter, the intron, a polynucleotide encoding CYP4V2, a polynucleotide encoding the self-cleaving peptide, a polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0188] For example, the nucleic acid molecule to be isolated may include, in the 5' to 3' direction, the promoter, a polynucleotide encoding the CYP4V2 into which the intron is inserted, a polynucleotide encoding the self-shearing peptide, a polynucleotide encoding the RdCVF, and the polyadenylation signal site.

[0189] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the intron may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 13 to 16, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75, and the polyadenylation signal site may comprise a nucleotide sequence shown in any of SEQ ID NOs: 17 to 21.

[0190] For example, the polynucleotide into which the intron encoding CYP4V2 is inserted can comprise the nucleic acid sequence set forth in SEQ ID NO:116.

[0191] For example, the intron is located in the polynucleotide encoding the RdCVF or is located at the 5' end of the polynucleotide encoding the RdCVF.

[0192] For example, the isolated nucleic acid molecule may comprise, in order from 5' to 3', the promoter, the intron, a polynucleotide encoding the RdCVF, a polynucleotide encoding the self-cleaving peptide, and a polynucleotide encoding the CYP4V2.

[0193] For example, the isolated nucleic acid molecule may comprise, in the 5' to 3' direction, the promoter, a polynucleotide encoding the RdCVF into which the intron has been inserted, a polynucleotide encoding the self-cleaving peptide, and a polynucleotide encoding the CYP4V2.

[0194] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 1 to 12, the intron may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 13 to 16, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 69 to 75, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 22 to 25, and the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs: 62 to 68.

[0195] For example, the nucleic acid molecule to be isolated comprises, in the 5' to 3' direction, the promoter, the intron, a polynucleotide encoding the RdCVF, a polynucleotide encoding the self-cleaving peptide, a polynucleotide encoding the CYP4V2, and the polyadenylation signal site.

[0196] For example, the nucleic acid molecule to be isolated may include, in the 5' to 3' direction, the promoter, a polynucleotide encoding the RdCVF into which the intron is inserted, a polynucleotide encoding the self-shearing peptide, a polynucleotide encoding the CYP4V2, and the polyadenylation signal site.

[0197] For example, the promoter may comprise a nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 12, the intron may comprise a nucleic acid sequence shown in any one of SEQ ID NOs: 13 to 16, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any one of SEQ ID NOs: 69 to 75, the polynucleotide encoding the self-cleaving peptide may comprise a nucleic acid sequence shown in any one of SEQ ID NOs: 22 to 25, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any one of SEQ ID NOs: 62 to 68, and the polyadenylation signal site may comprise a nucleotide sequence shown in any one of SEQ ID NOs: 17 to 21.

[0198] For example, the isolated nucleic acid molecule can comprise a nucleotide sequence set forth in any of SEQ ID NOs:109-115.

[0199] vector In the present application, the vectors can include plasmids, phages, cosplasmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and viral vectors.

[0200] For example, the viral vector can include reverse transcriptase viruses (including slow viruses), adenoviruses, adeno-associated viruses (AAV vectors), herpes viruses (such as herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and human papilloma viruses (e.g., SV40).

[0201] For example, the adeno-associated virus vector (AAV vector) gene can include an inverted terminal repeat (ITR), an open reading frame (ORF), which can include a polynucleotide encoding a Rep protein, and can also include a polynucleotide encoding a capsid.

[0202] For example, an adeno-associated viral vector (AAV vector) can include a recombinant adeno-associated viral vector (rAAV vector).

[0203] For example, the capsid, ITRs and other selected AAV components in the recombinant adeno-associated viral vector may be selected from any AAV, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, DJ, DJ / 8, Rh10.

[0204] For example, the AAV vector may be any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, DJ, DJ / 8, and Rh10.

[0205] For example, the AAV vector is an ocular tissue-tropic AAV vector, such as AAV2, AAV3, AAV4, AAV5, AAV8, DJ / 8 or any rAAV vector.

[0206] For example, the AAV vector can be AAV2 / 2, AAV2 / 5, AAV2 / 8, or AAV2 / 9.

[0207] For example, viral vectors can include pAAV-RC5-Amp, RC8-cap, AAV2 / 8, AAV-helper-Amp, and AAV-helper.

[0208] For example, the viral vector can comprise a nucleotide sequence set forth in any of SEQ ID NOs:133-137.

[0209] For example, the vector can contain a nucleotide sequence shown in any of SEQ ID NOs:90-115.

[0210] For example, the vector can include a nucleotide sequence shown in any of SEQ ID NOs:117-121.

[0211] For example, the vector can include a nucleotide sequence set forth in any of SEQ ID NOs:122-132.

[0212] As used herein, the vector can comprise an isolated nucleic acid molecule as described herein.

[0213] For example, the vector can include a nucleotide sequence set forth in any of SEQ ID NOs:101-115.

[0214] In the present application, the vector may comprise a polynucleotide encoding the CYP4V2 or a polynucleotide encoding the RdCVF.

[0215] For example, the vector can further comprise a promoter located 5' to and operably linked to the polynucleotide encoding CYP4V2.

[0216] For example, the vector can further comprise a promoter located at the 5' end of and operably linked to the polynucleotide encoding the RdCVF.

[0217] For example, the vector also contains a polyadenylation signal site located at the 3' end of the polynucleotide encoding CYP4V2.

[0218] For example, the vector can comprise, in order from 5' to 3', the promoter, the polynucleotide encoding CYP4V2, and the polyadenylation signal site.

[0219] For example, the vector may comprise, in order from the 5' to 3' direction: a promoter sequence shown in any of SEQ ID NOs: 1 to 12, a polynucleotide sequence encoding CYP4V2 shown in any of SEQ ID NOs: 62 to 68, and a polyadenylation signal site sequence shown in any of SEQ ID NOs: 17 to 21.

[0220] For example, the vector can include a nucleotide sequence shown in any of SEQ ID NOs:90-92.

[0221] For example, the vector also contains a polyadenylation signal site, which is located at the 3' end of the coding polynucleotide RdCVF.

[0222] For example, the vector may comprise, in the 5' to 3' direction, the following: the promoter, the polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0223] For example, the vector may comprise, in order from 5' to 3', a promoter sequence as set forth in any of SEQ ID NOs:1-12, a polynucleotide sequence encoding RdCVF as set forth in any of SEQ ID NOs:69-75, and a polyadenylation signal site sequence as set forth in any of SEQ ID NOs:17-21.

[0224] For example, the vector can include a nucleic acid sequence set forth in any of SEQ ID NOs:96-98.

[0225] For example, the vector may also include an intron.

[0226] For example, the intron is located in the polynucleotide encoding the CYP4V2 or at the 5' end of the polynucleotide encoding the CYP4V2.

[0227] For example, the intron is located in the polynucleotide encoding the RdCVF or at the 5' end of the polynucleotide encoding the RdCVF.

[0228] For example, the vector may comprise, in the 5' to 3' direction, the following in order: the promoter, the intron, the polynucleotide encoding CYP4V2, or the polynucleotide encoding RdCVF.

[0229] For example, the vector may comprise, in 5' to 3' direction, the promoter, the polynucleotide encoding the CYP4V2 into which the intron is inserted, or the polynucleotide encoding the RdCVF into which the intron is inserted.

[0230] For example, the vector may comprise, in order from the 5' to 3' direction: the promoter, the intron, the polynucleotide encoding CYP4V2 or the polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0231] For example, the vector may comprise, in order from 5' to 3', the promoter, the polynucleotide encoding CYP4V2 into which the intron is inserted or the polynucleotide encoding RdCVF into which the intron is inserted, and the polyadenylation signal site.

[0232] For example, the promoter may comprise a nucleic acid sequence shown in any of SEQ ID NOs:1 to 12, the intron may comprise a nucleic acid sequence shown in any of SEQ ID NOs:13 to 16, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence shown in any of SEQ ID NOs:62 to 68, the polynucleotide encoding RdCVF may comprise a nucleic acid sequence shown in any of SEQ ID NOs:69 to 75, and the polyadenylation signal site may comprise a nucleotide sequence shown in any of SEQ ID NOs:17 to 21.

[0233] For example, the polynucleotide into which the intron encoding CYP4V2 is inserted can comprise the nucleic acid sequence set forth in SEQ ID NO:116.

[0234] For example, the vector can comprise a nucleotide sequence set forth in any of SEQ ID NOs:93-95 and 99-100.

[0235] Vector combination The present application also provides a combination of vectors for the treatment, alleviation and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy, comprising a first vector and a second vector, wherein the first vector may comprise a polynucleotide encoding CYP4V2, and the second vector may comprise a polynucleotide encoding RdCVF.

[0236] For example, the first vector can further comprise a promoter located at the 5' end of and operably linked to the polynucleotide encoding CYP4V2.

[0237] For example, the first vector may include, in the 5' to 3' direction, a promoter sequence such as that shown in any of SEQ ID NOs:1 to 12, and a polynucleotide sequence encoding CYP4V2 such as that shown in any of SEQ ID NOs:62 to 68, sequentially.

[0238] For example, the first vector can include the polynucleotide sequence shown in SEQ ID NO:117.

[0239] For example, the second vector can further comprise a promoter located at the 5' end of and operably linked to the polynucleotide encoding RdCVF.

[0240] For example, the second vector can include the polynucleotide sequence shown in SEQ ID NO:120.

[0241] For example, the first vector may contain, in the 5' to 3' direction, a promoter sequence shown in any of SEQ ID NOs:1 to 12, and a polynucleotide sequence encoding RdCVF shown in any of SEQ ID NOs:69 to 75, in that order.

[0242] For example, the first vector also contains a polyadenylation signal site located at the 3' end of the polynucleotide encoding CYP4V2.

[0243] For example, the second vector also contains a polyadenylation signal site, which is located at the 3' end of the coding polynucleotide RdCVF.

[0244] For example, the first vector may comprise, in order from the 5' to 3' direction: the promoter, the polynucleotide encoding CYP4V2, the polyadenylation signal site; and / or the second vector may comprise, in order from the 5' to 3' direction: the promoter, the polynucleotide encoding RdCVF, the polyadenylation signal site.

[0245] For example, the first vector may comprise, in order from the 5' to 3' direction: a promoter sequence as shown in any of SEQ ID NOs:1-12, a polynucleotide sequence encoding CYP4V2 as shown in any of SEQ ID NOs:62-68, and a polyadenylation signal site sequence as shown in any of SEQ ID NOs:17-21.

[0246] For example, the first vector may contain a nucleotide sequence shown in any of SEQ ID NOs:90 to 92.

[0247] For example, the second vector may comprise, in order from the 5' to 3' direction: a promoter sequence as set forth in any of SEQ ID NOs: 1-12, a polynucleotide sequence encoding RdCVF as set forth in any of SEQ ID NOs: 69-75, and a polyadenylation signal site sequence as set forth in any of SEQ ID NOs: 17-21.

[0248] For example, the second vector may comprise a nucleotide sequence shown in any of SEQ ID NOs:96-98.

[0249] For example, the first vector and / or the second vector may also include an intron.

[0250] For example, the intron is located in the polynucleotide encoding the CYP4V2 or at the 5' end of the polynucleotide encoding the CYP4V2.

[0251] For example, the intron is located in the polynucleotide encoding the RdCVF or at the 5' end of the polynucleotide encoding the RdCVF.

[0252] For example, the first vector may comprise a nucleotide sequence shown in any one of SEQ ID NOs: 93 to 95. And / or, the second vector may comprise a nucleotide sequence shown in any one of SEQ ID NOs: 99 to 100.

[0253] For example, the first vector may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 118 to 119, and / or the second vector may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 121.

[0254] cell The present application further provides cells that can include a combination of the nucleic acid molecules described herein or the vectors described herein.

[0255] For example, the cell is one in which the nucleic acid molecule is expressed.

[0256] For example, the cells may include the progeny of a single cell, which may not necessarily be identical (morphologically or genomically in terms of the total DNA cross-complement) to the primordial mother cell.

[0257] For example, the cells may further comprise cells transfected in vitro with a vector according to the invention.

[0258] For example, the cells can include bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells, 293T cells.

[0259] For example, the cells are cells from a patient with crystalline retinal degeneration.

[0260] For example, the cells can include somatic cells or stem cells.

[0261] For example, the cells can include retinal cells, corneal cells, choroidal cells, lens cells, neural cells, RPE cells, stem cells, and the stem cells can include induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), adult stem cells, or any cells derived from stem cells.

[0262] For example, the retinal cells, corneal cells, choroidal cells, lens cells, neural cells, and RPE cells can be induced to differentiate from the stem cells.

[0263] For example, the cells can include ARPE-19 cells, human iPSC-derived RPE cells.

[0264] Pharmaceutical Compositions The present application also provides pharmaceutical compositions, which can comprise the nucleic acid molecules described herein, the vector combinations described herein, and / or the cells described herein.

[0265] For example, the pharmaceutical composition may also optionally include a pharma- ceutically acceptable adjuvant.

[0266] For example, the pharmaceutical compositions may also contain suitable formulations of one or more (pharmaceutical effective) vectors, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives.

[0267] For example, acceptable components of the composition are non-toxic to recipients at the dosages and concentrations employed.

[0268] For example, such pharmaceutical compositions include, but are not limited to, liquid, frozen and lyophilized compositions.

[0269] For example, such pharma- ceutically acceptable excipients include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration and are typically safe, non-toxic, and biologically or otherwise undesirable.

[0270] For example, the pharmaceutical compositions include parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intraocular administration or by injection directly into tissue.

[0271] For example, the pharmaceutical composition may be administered to a patient or subject by infusion, infusion or injection.

[0272] For example, the pharmaceutical composition may be used continuously (or continuously).

[0273] For example, non-stop (or continuous) administration can be achieved by a small pump system worn by the patient to meter the therapeutic agent into the patient's body, as described in WO2015 / 036583.

[0274] In the present application, the subject can include humans and non-human animals, for example, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0275] Diseases or conditions associated with retinal pigment epithelium (RPE) atrophy In the present application, the disease or condition associated with retinal pigment epithelium (RPE) atrophy can include age-related macular degeneration or retinitis pigmentosa (RP).

[0276] For example, the retinitis pigmentosa includes unicular primary retinitis pigmentosa, quadrant primary retinitis pigmentosa, central or paracentral primary retinitis pigmentosa, apigmentary retinitis pigmentosa, white punctate retinal degeneration, crystalline retinal degeneration, venous lipopigmentary retinitis pigmentosa, arteriolar parapigment epithelium-sparing retinitis pigmentosa, Leber's congenital amaurosis and other syndromes of retinitis pigmentosa.

[0277] For example, the retinitis pigmentosa can include crystalline retinal degeneration.

[0278] For example, crystalline retinal degeneration can include diseases caused by mutations in the CYP4V2 gene.

[0279] For example, CYP4V2 gene mutations include, but are not limited to, missense mutations, duplication errors, splice site errors, frameshifts, base deletions or insertions, nonsense mutations, polymorphisms (e.g., single nucleotide polymorphisms), premature termination, CYP4V2 partial or whole gene deletions, and CYP4V2 gene mutations associated with unidentified crystalline retinal degeneration.

[0280] For example, the CYP4V2 gene mutation may include a mutation shown in Table 1.

[0281] [Table A-1] [Table A-2] [Table A-3]

[0282] The CYP4V2 gene variants shown in Table 1 are taken from the database https: / / www.ncbi.nlm.nih.gov / clinvar / ?term=CYP4V2[gene].

[0283] Without intending to be limited by any theory, the following embodiments are not intended to limit the scope of the invention of the present application, but are intended to illustrate the nucleic acid molecules, production methods, and uses of the present application. The embodiments do not include detailed descriptions of conventional methods, such as how to construct vectors and plasmids, how to insert genes encoding proteins into such vectors and plasmids, or how to introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T.(1989)Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. All chemicals not described can be purchased through normal commercial channels. EXAMPLES

[0284] Example 1. Validation of promoter strength by expression of CYP4V2 (1) The full-length CYP4V2 promoter sequence was 2000 bp upstream of the coding region of human genome CYP4V2 (HGNC:23198) and was synthesized by Suzhou Jinweizhi Company.

[0285] (2) Primer sequences of promoters CYP4V2-Pf, CYP4V2-P1, CYP4V2-P2, CYP4V2-P3, CYP4V2-P4, CYP4V2-P5, and CYP4V2-P6 of different lengths were designed, and PCR amplification was performed using the nucleic acid molecule obtained in step (1) as a template, and the amplified products were recovered by gel electrophoresis. The cited sequences are shown in SEQ ID NO: 30 to 37.

[0286] [Table B]

[0287] (3) The pAV-CAG-CYP4V2-P2A-EGFP vector (purchased from Shandong Weizhen Biotechnology Co., Ltd.) was amplified from both ends of the CAG promoter, and the amplified products were collected by gel electrophoresis. The sequences are shown in SEQ ID NO: 38-39. The PCR reaction system (50 μl) was as follows:

[0288] [Table C]

[0289] (4) The different CYP4V2 promoter sequence (e.g., SEQ ID NO:6-12) obtained in step (2) is homologously ligated into the linear pAV-CAG-CYP4V2-P2A-EGFP vector obtained in step (3) to replace the CAG promoter in the original vector.

[0290] The coupling system (performed on ice) is as follows:

[0291] [Table D]

[0292] The ligated system was placed in a water bath at 50° C. for 40 minutes, and 5 μl of the reaction solution was transformed into transStbl3 E. coli competent cells, and the bacterial sequence was measured and the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0293] (5) On the first day, 293T cells (ATCC, CRL-3216) were split into 35 mm cell culture dishes, and from the second day to about 70%, the transfection system was prepared: 2 μg of the plasmid obtained in step (4) was added to 100 μl serum-free DMEM, 3 μl PEI was added, mixed evenly, and left to stand for 20 minutes. The obtained transfection system was added to the cell medium, shaken evenly, placed in a CO2 culture box, and the liquid was replaced after 6 h or overnight.

[0294] (6) RIPA lysis solution (Beijing Prile C1053-100) was used to lyse the cells after 48 hours, and protein gel electrophoresis was performed. Western blot was used to detect the expression of CYP4V2. Antibodies used were: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), and goat-anti-rabbit (Abclonal, AS014).

[0295] As a result, as shown in Figure 1, the CYP4V2-PF promoter, CYP4V2-P1 promoter, CYP4V2-P2 promoter, CYP4V2-P3 promoter, CYP4V2-P4 promoter, CYP4V2-P5 promoter, and CYP4V2-P6 promoters can all achieve the expression of CYP4V2, among which the CYP4V2-PF promoter, CYP4V2-P3 promoter, CYP4V2-P4 promoter, and CYP4V2-P5 promoter have relatively good expression effects, and the CYP4V2-P5 sequence is shorter and dominant.

[0296] Example 2. Verifying promoter strength using EGFP as a reporter gene (1) Promoters EF1a, EFS, and OPEFS were amplified by PCR. The EF1a primer sequence is shown in SEQ ID NO: 40-41, and the EFS primer sequence is shown in SEQ ID NO: 42-43. The OPEFS promoter was based on the general promoter EFS, and the primers were designed to convert EFS to OPEFS using two-way PCR. The primers used in the first PCR reaction were EFS forward primer (e.g., SEQ ID NO: 42) and OPEFS-overlapR1 (e.g., SEQ ID NO: 44), and the primers used in the second PCR reaction were EFS forward primer (shown in SEQ ID NO: 42) and OPEFS-overlapR2 (e.g., SEQ ID NO: 45). The amplified products were recovered by gel electrophoresis. The PCR reaction system (50 μl) was as follows:

[0297] [Table E]

[0298] (2) Homologous recombination of the amplification product obtained in step (1) (promoter OPEFS, EFS, EF1a nucleic acid molecule, sequences shown in SEQ ID NO: 1-3) is ligated to the linear pAV-CAG-CYP4V2-P2A-EGFP obtained in step (3) of Example 1 to obtain pAV-EF1a-CYP4V2-P2A-EGFP, pAV-EFS-CYP4V2-P2A-EGFP, and pAV-OPEFS-CYP4V2-P2A-EGFP. The ligation system (performed on ice) is as follows:

[0299] [Table F]

[0300] The ligated system was placed in a water bath at 50° C. for 40 minutes, and 5 μl of the reaction solution was transformed into transStbl3 E. coli competent cells, and the bacterial sequence was measured and the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0301] (3) According to the method of Example 1, step (5), pAV-CAG-CYP4V2-P2A-EGFP, pAV-EF1a-CYP4V2-P2A-EGFP, pAV-EFS-CYP4V2-P2A-EGFP and pAV-OPEFS-CYP4V2-P2A-EGFP plasmids were transfected into 293T cells (ATCC, CRL-3216) pre-plated in a 6-well plate, respectively, using PEI (Polysciences 24765-1), and the cell density was about 70%.

[0302] (4) After 24 hours, the fluorescent signal was observed using a fluorescence microscope (Life AMF4305).

[0303] The results are shown in Figure 2. CAG, EF1a, and OPEFS all have better expression effects than EFS, among which the OPEFS sequence is shorter and more advantageous.

[0304] Example 3. Verification of the enhancing effect of intron (I) An intron was cloned into the 5' non-coding region of the gene.

[0305] (1) P5-intron clone. Primers were designed based on the CYP4V2-P5 promoter sequence and the human β-globin intron, and the P5-intron was obtained by modifying the primers using overlap PCR. 1) PCR amplification was performed using the CYP4V2-P5 promoter sequence as a template and the CYP4V2-P5 forward primer (SEQ ID NO: 46, CYP4V2-P5F) and the CYP4V2-P5(in) reverse primer (SEQ ID NO: 47); 2) PCR amplification was performed using the human β-globin intron as a template and the β-globin intron (c) (SEQ ID NO: 48) and the β-globin intron reverse primer (SEQ ID NO: 49); 3) The amplified products were collected, and the target products obtained in 1) and 2) were used as a common template to perform PCR amplification using the CYP4V2-P5 forward primer (SEQ ID NO: 46, CYP4V2-P5F) and the β-CYP4V2-P5 intron reverse primer SEQ ID NO. PCR amplification was performed according to the system and conditions of Example 2, step (1-49). The amplified P5-intron target product was collected.

[0306] (2) The amplified P5-intron sequence is sequenced and then ligated by homologous recombination to the linear PAV-CAG-CYP4V2-P2A-EGFP obtained in step (3) of Example 1 to obtain pAV-p5-intron-CYP4V2-P2A-EGFP. The ligation system (performed on ice) is as follows:

[0307] [Table G]

[0308] (3) The ligated system was left in a water bath at 50°C for 40 minutes, and 5 μl of the reaction solution was taken and transformed into transStbl3 E. coli competent cells, and the bacterial sequence was measured and the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0309] (II) An intron was cloned into the gene coding region.

[0310] (1) CYP4V2-CDSintron clone, in which the intron is located between the first and second exons of CYP4V2. Based on the CDS sequence of CYP4V2 and human β-globin intron, primers were designed and overlap PCR was used to obtain the CDSintron.

[0311] 1) Using the CDS sequence of CYP4V2 as a template, PCR amplification was performed using primers CYP4V2-Fpr / CYP4V2-E1-R (sequences shown in SEQ ID NOs:50 and 51, respectively) and CYP4V2-E2-F / CYP4V2-R (sequences shown in SEQ ID NOs:52 and 53, respectively) to obtain PCR products CYP4V2-E1 and CYP4V2-E2-11, respectively.

[0312] 2) The β-globin intron was used as a template for PCR amplification using primers Intron-F / Intron-R (sequences shown in SEQ ID NOs: 54 and 55, respectively).

[0313] 3) The amplified products were collected, and 1) the obtained target product CYP4V2-E1 and 2) the obtained target product were used as a common template to perform PCR amplification using the CYP4V2-CDS forward primer CYP4V2-Fpr (sequence shown in CYP4V2-R:50) and the β-globin intron reverse primer intron-R (sequence shown in CYP4V2-E2:55), and the PCR amplification was performed under the system and conditions of Example 2, step (1). The amplified CYP4V2-E1-intron target product was collected.

[0314] 4) PCR amplification was performed using the desired product CYP4V2-E1-intron obtained in 3) and the desired products CYP4V2-E2 to 11 obtained in 1) as a common template, and the CYP4V2-CDS forward primer CYP4V2-Fpr and the CYP4V2-CDS reverse primer CYP4V2-R. The PCR amplification was performed under the system and conditions of Example 2, step (1).

[0315] (2) The amplified CDS intron sequences were sequenced.

[0316] (3) Enzyme digestion of pAV-p5-CYP4V2-P2A-EGFP was performed at 37℃ for 2h to obtain a linear vector, which was then recovered by gel electrophoresis. The enzyme system is as follows:

[0317] [Table H]

[0318] (4) CYP4V2-CDSintron homologous recombination was ligated into linear pAV-p5-CYP4V2-P2A-EGFP to obtain pAV-p5-CDSintron-CYP4V2-P2A-EGFP. The ligation system (performed on ice) is as follows:

[0319] [Table I]

[0320] (5) The ligated system was left in a water bath at 50°C for 40 minutes, and 5 μl of the reaction solution was taken and transformed into transStbl3 E. coli competent cells, and the bacterial sequence was measured and the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0321] (III) Verification of the enhancing effect when introns are located in the 5' non-coding region and the coding region of a gene (1) pAV-p5-CYP4V2-P2A-EGFP, pAV-p5-intron-CYP4V2-P2A-EGFP, and pAV-p5-CDSintron-CYP4V2-P2A-EGFP were transfected into 293T cells (ATCC, CRL-3216) pre-plated in a 6-well plate using PEI (Polysciences 24765-1) by the method of Example 1, step (5), and the cell density was about 70%.

[0322] (2) After 24 hours, the fluorescent signal was observed using a fluorescence microscope (Life AMF4305).

[0323] (3) RIPA lysis solution (Beijing Prile C1053-100) was used to lyse the cells after 48 hours, and protein gel electrophoresis was performed. Western blot was used to detect the expression of CYP4V2. Antibodies used were: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), and goat-anti-rabbit (Abclonal, AS014).

[0324] As a result, as shown in Fig. 3 and Fig. 4, expression was enhanced when the 5' non-coding region and coding region of the gene were inserted into the human β-globin intron.

[0325] Example 4. Selection of Poly A signal site (1) The SV40 of the pAV-OPEFS-CYP4V2-P2A-EGFP vector obtained in embodiment 2 was replaced with BGH, WPRE, WPRE-SV40, and WPRE-BGH, as shown in SEQ ID NO:19-21, to obtain pAV-OPEFS-CYP4V2-P2A-EGFP-BGH, pAV-OPEFS-CYP4V2-P2A-EGFP-WPRE, pAV-OPEFS-CYP4V2-P2A-EGFP-WPRE-SV40, and pAV-OPEFS-CYP4V2-P2A-EGFP-WPRE-BGH. Beijing Optimiko was responsible for gene synthesis and subcloning of this part.

[0326] (2) The vector obtained in step (1) was sequenced and verified, and then the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0327] (3) Using the method of Example 1, step (5), the plasmids obtained in step (2) of this Example were transfected into 293T cells (ATCC, CRL-3216) pre-plated in a 6-well plate using PEI (Polysciences 24765-1), and the cell density was about 70%.

[0328] (4) After 24 hours, the fluorescent signal was observed using a fluorescence microscope (Life AMF4305).

[0329] (5) RIPA lysis solution (Beijing Prile C1053-100) was used to lyse the cells after 48 hours, and protein gel electrophoresis was performed. Western blot was used to detect the expression of CYP4V2. Antibodies used were: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), and goat-anti-rabbit (Abclonal, AS014).

[0330] The results are shown in Figure 5. Here, M (mock) is the blank control, S is SV40, B is BGH, W is WPRE, WS is WPRE-SV40, and WB is WPRE-BGH. CYP4V2 expression was detected in each experimental group compared to the control group.

[0331] Example 5. Construction of expression vectors (1) Construction of pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH (Calluna resistance). The ambenzyl resistance gene in pAV-OPEFS-CYP4V2-P2A-EGFP-BGH was replaced with the Calluna resistance gene, and Jin Weizhi of Suzhou was responsible for the construction of the vector. Jin Weizhi of Suzhou was responsible for the synthesis of the relevant genes and the construction of the vector (Calluna resistance) for pAV-OPEFS-CYP4V2-BGH (expression sequence shown in SEQ ID NO:90), pAV-OPEFS-RdCVF-BGH (expression sequence shown in SEQ ID NO:96), and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH (expression sequence shown in SEQ ID NO:106).

[0332] (2) Construction of pAV-p5-intron-CYP4V2-BGH (expression sequence shown in SEQ ID NO: 93), pAV-p5-intron-RdCVF-BGH (expression sequence shown in SEQ ID NO: 99), pAV-p5-intron-CYP4V2-P2A-RdCVF-BGH (expression sequence shown in SEQ ID NO: 113) (Calluna resistance). pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH (Calluna resistance) were digested with enzymes at 37°C for 2 hours to obtain linear vectors, which were then recovered by gel electrophoresis. The enzyme system is as follows:

[0333] [Table J]

[0334] (3) The P5-intron fragment was obtained by PCR amplification from pAV-p5-intron-CYP4V2-P2A-EGFP and recovered by gel electrophoresis. The forward primer was SEQ ID NO:46 and the reverse primer was SEQ ID NO:55.

[0335] (4) Ligate the enzyme-cleaved recovered vector with the linear fragment that requires recombination with it. The ligation system (performed on ice) is as follows:

[0336] [Table K]

[0337] The ligated system was placed in a water bath at 50° C. for 40 minutes, and 5 μl of the reaction solution was transformed into transStbl3 E. coli competent cells, and the bacterial sequence was measured and the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0338] Example 6. Examination of the expression status of expression vectors in 293T cells (1) The pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH plasmids obtained by the modification in Example 5, step (1) were transfected into 293T cells (ATCC, CRL-3216) that had been spread in advance on a 6-well plate using PEI (Polysciences 24765-1) by the method in Example 1, step (5), to a cell density of about 70%.

[0339] (2) The cell culture supernatant (sup) was collected after 48 hours, and the cells were washed with PBS. The cells (lys) were lysed using RIPA lysis solution (Beijing Prile C1053-100) and the expression of CYP4V2 and / or RdCVF in the supernatant or cell lysate (lys) of each group was examined by Western blotting. Antibodies used: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), goat-anti-rabbit (Abclonal, AS014), RdCVF Antibody (anti-CYP4V2, Atlas).

[0340] The results are shown in Figure 6. Here, M (mock) was the blank control, C was the OPEFS-CYP4V2 group, CR was the OPEFS-CYP4V2-P2A-RdCVF group, and R was the OPEFS-RdCVF group. There was no expression of CYP4V2 or RdCVF in the lysate (lys) and cell culture supernatant (sup) of the blank control group and M group cells. There was expression of CYP4V2 in the pAV-OPEFS-CYP4V2-BGH transfected cell lysate, there was expression of RdCVF in the pAV-OPEFS-RdCVF-BGH transfected cell supernatant and lysate, there was expression of CYP4V2 in the pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH transfected cell lysate, and at the same time, there was expression of RdCVF in the cell lysate and supernatant.

[0341] Example 7. Examination of the expression status of expression vectors in ARPE-19 cells (1) The pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH plasmids obtained by the modification in Example 5, step (1) were transfected into ARPE-19 cells (ATCC, CRL-2302) that had been spread in advance on a 6-well plate using PEI (Polysciences 24765-1) by the method in Example 1, step (5), to a cell density of about 70%.

[0342] (2) The cell culture supernatant (sup) was collected after 48 hours, and the cells were washed with PBS, and then the cells (lys) were lysed using RIPA lysis solution (Beijing Prile C1053-100) and the expression of CYP4V2 and / or RdCVF in the supernatant or cell lysate (lys) of each group was examined by Western blot. Antibodies used: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), goat-anti-rabbit (Abclonal, AS014), TXNL6 Antibody (anti-CYP4V2).

[0343] The results are shown in Figure 7. Here, M (mock) was the blank control, C was the OPEFS-CYP4V2 group, CR was the OPEFS-CYP4V2-P2A-RdCVF group, and R was the OPEFS-RdCVF group. There was no expression of CYP4V2 or RdCVF in the lysate and cell culture supernatant of the blank control group and mock group cells. There was expression of CYP4V2 in the pAV-OPEFS-CYP4V2-BGH transfected cell lysate, there was expression of RdCVF in the pAV-OPEFS-RdCVF-BGH transfected cell supernatant and cell lysate, there was expression of CYP4V2 in the pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH transfected cell lysate, and at the same time there was expression of RdCVF in the supernatant and cell lysate.

[0344] Example 8. Viral Vectors (I) Selection of AAV serotype: Subretinal injections of AAV2 / 2, AAV2 / 5, AAV2 / 8, and AAV2 / 9 serotype viruses (purchased from Shandong Weizhen Biotechnology Co., Ltd.) containing GFP reporter were performed in wild-type mice. One month later, retinal histomorphological observations were performed on sections. Results As shown in Figure 8, the double arrow indicates the expression range of the EGFP reporter gene, and the AAV2 / 8 serotype with relatively good retinal preference was selected.

[0345] AAV2 / 8 vector construction: (1) The vector was modified based on pAAV-RC5-Amp (purchased from Beijing Xi Mei Jie Technology Co., Ltd., the vector sequence is shown below: SEQ ID NO: 133). PCR was used to amplify the pAAV-RC5 sequence except Amp, and the primers used were RC5-F / RC5-R as shown in SEQ ID NO: 56-57.

[0346] (2) The Kana portion was amplified from pEGFP-N1 by PCR using primers Kana-F / Kana-R as shown in SEQ ID NO:58-59.

[0347] (3) The two types of DNA fragments obtained by the above PCR were combined using homologous recombination to obtain pAAV-RC5-Kana.

[0348] (4) Using pAAV-RC5-Kana as a base, the pAAV-Kana sequence other than RC5-cap was amplified using PCR, and the primers used were RC5-Kana-F / RC5-Kana-R, as shown in SEQ ID NO:60-61.

[0349] (5) The gene synthesized RC8-cap sequence (Beijing Optimus, the sequence is shown in SEQ ID NO:134) and the pAAV-Kana linear vector obtained in step (4) were used to obtain pAAV-RC8-Kana (i.e., AAV2 / 8) by homologous recombination, the vector sequence of which is shown in SEQ ID NO:135.

[0350] (II) AAV-helper selection: A general-purpose AAV-helper auxiliary plasmid was selected, and the resistance was changed from ammonia to callus. The specific modification process is as follows:

[0351] 1) pAAV-helper-Amp (purchased from Beijing Xi Mei Jie Technology Co., Ltd., vector sequence is shown below: SEQ ID NO: 136) was used as a base for modification. The pAAV-helper sequence other than Amp was amplified by PCR, and the primers used were RC5-F / RC5-R, as shown in SEQ ID NO: 56-57.

[0352] 2) The Kana portion was obtained by amplified from pEGFP-N1 by PCR using primers Kana-F / Kana-R, which are shown in SEQ ID NO:58-59.

[0353] 3) The two types of DNA fragments obtained by the above PCR were subjected to homologous recombination to obtain pAAV-helper-Kana (i.e., AAV-helper) vector sequence, which is shown in SEQ ID NO:137.

[0354] Example 9. Virus packaging and purification (I) Virus packaging (1) Day 0: Cell inoculation (number of inoculations: 1 x 10 7 ), 293T cells were seeded into a 15 cm petri dish.

[0355] (2) Day 1: When the volume of 293T cells was 80% to 90%, the medium was replaced with 20 ml of complete medium (Gibco, C11965500BT) containing 10% serum (Shanghai Gibco, FSP500), and plasmid transfection was performed. For the specific transfection system, see Table 5.

[0356] [Table L]

[0357] (3) The expression vectors in Table 5 are expression vectors constructed according to the method of Example 5. (3) Day 2: 12-18 hours after transfection, the medium was replaced with 30 ml of fresh complete medium.

[0358] (4) Day 3: 48 hours after transfection, replace with 30 ml of fresh complete medium.

[0359] (5) Day 4: 72 h after transfection, 293T cells were digested with pancreatic enzymes in the usual way, then collected in a 50 ml centrifuge tube, washed twice with PBS, centrifuged at 1200 rpm for 5 minutes, aspirated the PBS, and stored in a -80°C refrigerator.

[0360] (II) Virus purification 1) The AAV293T cells obtained in step (5) of this Example were thawed at 15°C to 25°C.

[0361] 2) The cells were resuspended in 2 ml of cell lysis solution (150 mM NaCl, 50 mM Tris, pH 8.5) per 15 cm dish (three 15 cm dishes were taken for each 50 ml centrifuge tube, and 6 ml of cell lysis solution was prepared for each of the three dishes).

[0362] 3) Chloroform in a volume equivalent to half the volume of the cell lysis solution (3 ml) was added to the centrifuge tube, the opening of the tube was clamped, and the tube was placed horizontally on a rocking bed at 37° C. and shaken at 250 rpm for 30 minutes.

[0363] 4) 1 / 4 volume (1.5 ml) of 5M sodium chloride was added to the aqueous phase and mixed uniformly. The mixture was transferred to a high-speed centrifuge tube and centrifuged at 11,000 rpm for 25 minutes.

[0364] 5) The upper aqueous phase was removed, and the difficult-to-aspirate part at the boundary was transferred to a 1.5 ml centrifuge tube and centrifuged at 12,000 rpm for 30 seconds. The supernatant was combined, and nuclease (Benzonase) was added to the upper aqueous phase to a final concentration of 50 U / ml, deoxycholic acid was added to a final concentration of 0.4%, and final concentrations of 10 mM MgCl2, 0.5 mM CaCl2, 5 IU / ml Turbo DNase I, and 25 ug / ml RNaseA (retention solution concentration 10 mg / ml) were added.

[0365] 6) 37°C water bath for 30 minutes.

[0366] 7) 50% PEG8000 was added in a volume of 1 / 4 (2 ml) of the aqueous phase, shaken, and centrifuged on ice for 1 hour at 11,000 rpm for 25 minutes.

[0367] 8) The supernatant was aspirated and discarded, and the mixture was centrifuged again for 1 minute to remove the remaining supernatant.

[0368] 9) Add PBS according to the demand for the final lysis volume, spray the suspension, and transfer to a 1.5 ml centrifuge tube. Add a small volume of transfer fluid (<200 μl) during transfer to avoid virus loss due to adhesion of the precipitate.

[0369] 10) 500 μl of chloroform was added, shaken, and centrifuged at 12,000 rpm for 20 minutes.

[0370] 11) The supernatant was aspirated under sterile conditions and stored in aliquots at -80°C.

[0371] Example 10. Measurement of viral titer (I) Formulation of the pAAV-EGFP preparation 1. Bell Gradient: 1x10 8 Copy / 5μl, 1x10 7 Copy / 5μl, 1x10 6 copy / 5 μl, 1x10 5 Copy / 5μl, 1x10 4 Copy / 5μl, 1x10 3 Copies / μl, 1x10 2 copies / 5μl.

[0372] 2. Dilution Procedure: a. The stock solution was diluted 10 times and quantified using Nano, and subsequent dilutions were performed based on the quantification results. b. Quantified samples were serially diluted to 1 ng / μl. c. 1ng / μl sample 1x10 8 Diluted to copies / μl. d. Dilution solution 1x10 8 Dilute 5 times to 1x10 copies / μl 8 Copies / 5 μl. e. Dilute 10 times to 1x10 2 Copies / 5 μl.

[0373] (II) Sample processing and dilution A 50 U / ml buffer was prepared using 1.17 μl DNase 1 + 1 ml 1x buffer (TransGen Biotech, GD-201-01).

[0374] 2. 5 μl of the sample obtained in step 11) of Example 9 was added to 95 μl of the sample (10-fold dilution) diluted with the buffer prepared in step 1.

[0375] 3. Mix uniformly (vortex for 5 seconds) and leave on PCR machine at 37°C for 30 minutes; leave at 75°C for 15 minutes.

[0376] 4. The reaction solution from step 3 was shaken, 50 μl of it was aspirated, and 450 M ribozyme-free water was added to prepare the first dilution gradient (200-fold dilution).

[0377] 5. 50 μl of the first gradient reaction solution that was uniformly mixed in step 4 was aspirated, and 450 μl of ribozyme-free water was added to prepare the second dilution gradient (2000-fold dilution).

[0378] 6. 50 μl of the first gradient reaction solution that was uniformly mixed in step 5 was aspirated, and 450 μl of ribozyme-free water was added to prepare the third dilution gradient (2000-fold dilution).

[0379] (III) Fluorescent quantitative PCR

[0380] [Table M]

[0381] [Table N]

[0382] [Table O]

[0383] As a result of the testing and measurement, the titers of AAV-OPEFS-EGFP and AAV-OPEFS-CYP4V2 viruses were 1.00E+13vg / ml and 3.06E+13vg / ml, respectively, as shown in Table 8, and they can be used in subsequent infection experiments. The titers of other viruses used in the examples of this application were all measured by the method of this example.

[0384] Example 11. Different doses of virus infect human iPSC-differentiated RPE cells (1) Preparation of human induced pluripotent stem cells (IPSCs). Renal epithelial cells were extracted from urine using the Urineasy urine cell isolation kit (Beijing Speier, CA3102500), and cultured using the Urineasy urine cell amplification kit (Beijing Speier, CA3103200). Reprogramming experiments were performed using cells that had reached 70-80% confluence after 3-4 generations. The reprogramming experiments were performed using the hiPSC reprogramming kit (Beijing Speier, CA5002002) according to the instructions in the kit's manual, and human IPSCs were obtained for the subsequent cell differentiation experiments.

[0385] (2) Preparation of RPE cells. RPE cells were prepared according to the method described in the literature (da Cruz, L., et al. (2018), Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration, NatBiotechnol 36(4):328-337.). 3*10 4 / cm 2 Human IPSCs were cultured in 4 ml of TESR-E8 medium (STEMCELL, CAT#05990, #05991) in a T25 bottle, and after 5 days, the medium was replaced with 6 ml of 20% serum replacement (Gibco, CAT#A3181502) (Gibco, CAT#10829018), and after 2 days, the medium was replaced with 6 ml serum-free medium (Gibco, CAT#10829018) for approximately 20 weeks. Oval, highly pigmented dividing cells, i.e., RPE cells, were obtained. Figure 9 shows renal epithelial cells, IPSC cells, and RPE cells observed under white light with a fluorescent microscope (Life AMF4305) at 4, 10, and 10x magnification, respectively.

[0386] (3) Equal amounts of human iPSC-differentiated RPE cells were incubated with wrapping virus containing pAV-OPEFS-EGFP-BGH or pAV-OPEFS-CYP4V2-BGH in a dose gradient (MOI = 0, 1x10 3 , 1x10 4 , 1x10 5 ) was used for infection.

[0387] (4) After 72 h, the cells were digested and each group was divided into three groups, one of which was analyzed for cell death by Cell Titer Glow (Promega, G9241) according to the instructions. The expression of CYP4V2 was detected by Western blot according to the method of step (2) of Example 6. One part was used to extract RNA, and RT-qPCR was used to detect the mRNA levels of inflammatory cytokines: NLRP3, TNF-α, IFN-γ. Among them, RNA was extracted using an RNA extraction kit (Tengen DP430) according to the instructions; cDNA was reverse transcribed using a reverse transcription kit (Takara, RR407A) according to the instructions. RT-qPCR was performed according to the method of step (water) of Example 10, and the sequences of RT-qPCR primers are shown in SEQ ID NO:138-145.

[0388] The results are shown in Figures 10A-10D. As the titer of pAV-OPEFS-EGFP-BGH virus increased, the expression of EGFP gradually increased, the expression levels of inflammatory cytokines TNF-α and NLRP3 gradually increased, and the cell mortality gradually increased. As the titer of pAV-OPEFS-CYP4V2-BGH virus increased, the expression of CYP4V2 increased, the expression levels of inflammatory cytokines TNF-α and NLRP3 increased, and the cell mortality increased. The high titer AAV virus had cytotoxicity against RPE cells differentiated from human iPSCs, so the cytotoxicity of 1x10 4 The following doses can be selected for subsequent experiments with RPE cells differentiated from human iPSCs:

[0389] Example 12. Effects of iPSC-differentiated RPE cells from BCD patients infected with different viruses (1) The virus was packaged according to the method of Example 7. A: pAV-p5-intron-CYP4V2-BGH (expression sequence shown in SEQ ID NO:93); B: pAV-p5-intron-RdCVF-BGH (expression sequence shown in SEQ ID NO:99); C: pAV-p5-intron-CYP4V2-P2A-RdCVF-BGH (expression sequence shown in SEQ ID NO:113); D: pAV-p5-intron-EGFP-BGH.

[0390] (2) MOI=1x10 4 Viruses A, B, C, and D of 1 / 2 MOI and virus A + virus B of 1 / 2 MOI (hereafter referred to as group E) were employed to infect iPSC-differentiated RPE cells of BCD patients (genotype CYP4V2: c.802-8_810 del17bpinsGC homozygous mutation) in equal doses, and the method for producing RPE cells is described in Example 11. The BCD patients were treated at the Department of Ophthalmology, Peking University Third Hospital from November 2018 to December 2018. The Peking University Third Hospital Ethics Committee approved all aspects of the study, and informed consent for sample collection was obtained from the subjects (or their legal guardians). The patients' urine samples were processed as described in Example 11 within 1 hour of collection.

[0391] (3) After 72 hours, the deposition of lipids was observed under a microscope.

[0392] (4) Cells were harvested and culture supernatant (sup) was collected; after washing the cells with PBS, the cells were lysed with RIPA lysis solution (Beijing Prile C1053-100) to obtain cell lysates (lys), and protein expression levels were examined and measured by Western spectrometry. Antibodies used: anti-CYP4V2 (Atlas, HPA029122), anti-β-actin (Abclonal, AC026), goat-anti-rabbit (Abclonal, AS014), TXNL6 Antibody (Atlas).

[0393] The results of protein expression are shown in Figure 11A. RPE cells differentiated from BCD patient iPSCs in the blank control M (mock) group without virus infection did not express CYP4V2 or RdCVF. RPE cells differentiated from BCD patient iPSCs infected with virus group D did not express CYP4V2 or RdCVF, but expressed EGFP. CYP4V2 expression was detectable in the lysate of RPE cells differentiated from BCD patient iPSCs infected with virus group A, RdCVF expression was detectable in the supernatant and cell lysate of RPE cells differentiated from BCD patient iPSCs infected with virus group B, and CYP4V2 expression was detectable in the lysate of RPE cells differentiated from BCD patient iPSCs infected with viruses group C and E, and RdCVF expression was also detectable in the cell supernatant and cell lysate.

[0394] As shown in Figure 11B, lipid deposition improved in groups A, C, and E compared to group M. Group D was not included in the comparison because EGFP expression and lipid staining fluorescence were mutually contagious.

[0395] Example 13. Promoter selection for virally transfected BCD mice (1) The virus was packaged according to the method of Example 7. A: pAV-p5-intron-CYP4V2-BGH (expression sequence shown in SEQ ID NO:93); B: pAV-OPEFS-CYP4V2-BGH (expression sequence shown in SEQ ID NO:90). Promoter A: p5-intron; Promoter B: OPEFS. BCD mice (Cyp 4 v 3- / - purchased from Beijing Baiaosaitu Gene Biology Technology Co., Ltd.) were injected into the subretinal space at the age of 1 month, and both promoter viruses were injected into 10 mice each, and the therapeutic vector (pAV-p5-intron-CYP4V2-BGH (expression sequence shown in SEQ ID NO:93) / pAV-OPEFS-CYP4V2-BGH (expression sequence shown in SEQ ID NO:90)) was injected into both eyes, and the mice were observed for 3 months after injection.

[0396] Subretinal injection method: 1) The experimental subjects were prepared. Mice were dilated with 1% atropine and then intraperitoneally injected with 80 mg / kg ketamine + 8 mg / kg tortiazine.

[0397] 2) After anesthesia, the pupils were dilated again with 1% atropine, the mouse was placed in front of the animal experiment platform of an ophthalmic surgical microscope (Topcon, OMS800), and 0.5% promecaine local anesthetic was instilled onto the mouse's eyeball.

[0398] 3) Sodium fluorescein stock solution was added to the virus at a concentration of sodium fluorescein:virus=100:1 and mixed with a pipette.

[0399] 4) A small hole is made in the flat part of the ciliary body of the mouse eye with an insulin needle, and the needle of a micro syringe is pierced through the small hole and then inserted into the vitreous cavity of the mouse eye. An appropriate amount of 2% hydroxymethylcellulose is added to the mouse eyeball at this time so that the fundus of the mouse can be seen under a microscope. The needle is then inserted into the subretinal space around the opposite side, avoiding the lens, and the virus with sodium fluorescein is slowly pushed in. The injection volume for each eye is 10, and the virus concentration is 1x10. 9 We then determined whether sodium fluorescein should be injected into the subretinal space as an indicator.

[0400] After surgery, the ocular surface was washed with saline, and the mouse was placed in a cage to wait for awakening.

[0401] (2) Observe the crystalline deposits by fundus photography. 1) A blank control (KO) was taken, and the eyes of the mice injected with promoter A: pAV-p5-intron-CYP4V2-BGH (expression sequence shown in SEQ ID NO:93) and promoter B: pAV-OPEFS-CYP4V2-BGH (expression sequence shown in SEQ ID NO:90) were dilated with 1% atropine and anesthetized with an intraperitoneal injection of 80 mg / kg ketamine + 8 mg / kg tortiazine A.

[0402] 2) After anesthesia, the mouse was placed on the experimental stand of a Micro III small animal retinal imaging system (Phoenix Research Laboratory, Micro III), and an appropriate amount of 2% hydroxymethylcellulose was dropped onto the mouse eyeball to improve the contact effect between the lens and the cornea.

[0403] 3) The experimental table was raised and rotated to adjust the position of the mouse's eyes, and the focal length and light intensity were adjusted to obtain images of the mouse's fundus, which were then photographed using Micro III software.

[0404] 4) After photography, quantitative analysis of crystal deposition was performed using GraphPad Prism software.

[0405] The results of fundus photography are shown in Figure 12. Figure 13 shows the statistical results of the number of fundus crystals when mice were injected with viruses with different promoters (n=5). Figures 12 and 13 showed that fundus crystal deposition was improved after injection of viruses with different promoters.

[0406] (3) Morphological observation of retinal tissue: 1) Fixation and dehydration: Mice were sacrificed by cervical dislocation, and the eyeballs were removed. The mouse eyeballs were placed in a 1.5 ml EP tube, 1 ml of 4% paraformaldehyde was added, and the eyeballs were left overnight at 4°C. After that, the eyeballs were placed in a 1.5 ml EP tube containing 1 ml of 30% sucrose solution to dehydrate them, causing the eyeballs to sink.

[0407] 2) The mouse eyeball was placed in a 1.5 ml EP tube containing OCT (Optical Cosmetic Density Transplant), and the mouse's eye was oriented with tweezers so that it was looking straight ahead. The EP tube was then placed over the eyeball, and the eyeball was placed in liquid nitrogen. After the eyeball was completely frozen, it was frozen and sectioned to a thickness of 7 μm. The eyeball was then fixed in acetone at 4°C for 10 minutes and stored at -80°C.

[0408] 3) The slices were removed, cooled to room temperature, and washed three times with PBS for 5 minutes each.

[0409] 4) Slides were wiped free of tissue and 40 μl of blocking solution (5% donkey serum) was dropped onto each section and blocked for 1 h at room temperature.

[0410] 5) The antibody was diluted with 5% donkey serum, and about 40 μl of the antibody working solution was added dropwise to completely cover the mouse eye tissue and left overnight at 4° C. The primary antibody used in this experiment was CYP4V2 (1:50, purchased from Sigma).

[0411] 6) Remove the slides and wash them with PBS three times, for 5 minutes each time.

[0412] 7) The secondary antibody (purchased from Thermo Fisher Scientific) was diluted 1:800 with PBS, and 40 μl of the secondary antibody working solution was dropped onto the left and right sides to completely cover the mouse eye tissue and incubated at room temperature for 1 hour.

[0413] 8) After washing three times with PBS, a diluted DAPI solution (1:5000) was added dropwise and incubated at room temperature for 15 minutes.

[0414] 9) A fluorescence quenching prevention capsule was dropped onto the slide glass, covered with a cover glass, and stored at -20°C in the dark.

[0415] 10) Observations and photographs were taken using a Nikon A 1 laser confocal microscope equipped with NIS-Elements C software.

[0416] The immunofluorescence results in Figure 14 showed that BCD mice injected with both promoter A virus and promoter B virus were able to express CYP4V2 (the double arrow in the figure indicates the range).

[0417] Example 14. Dose selection for virus-infected BCD mice (1) The viral load was verified according to the method of Example 13. BCD mice (Cyp 4 v 3- / -) were subretinal injected at 1 month of age, and the therapeutic vector pAV-OPEFS-CYP4V2-BGH (the expression sequence is shown in SEQ ID NO:90) was injected into both eyes, and 1E8vg, 1E9vg, and 1E10vg viruses were injected into each eye. The mice were observed for 3 months after injection.

[0418] The crystal deposition status is shown in the photographic results in Figure 15. Figure 16 statistically shows the number of fundus crystallizations in BCD mice injected with 1E8vg and 1E9vg viruses in the same area as BCD mice not injected with the virus. BCD mice injected with 1E8vg and 1E9vg viruses showed a reduction in the number of crystal deposits compared to BCD mice not injected with the virus, and the reduction was more pronounced in BCD mice injected with 1E9vg virus. BCD mice injected with 1E10vg virus did not show significant improvement in crystal deposition and had severe damage.

[0419] (2) The RPE cell layer was peeled off, RNA was extracted, and the expression levels of inflammatory cytokines TNF-α, IFN-γ, and NLRP3 were measured. Extraction of RNA and reverse transcription was performed according to the method of step (4) of embodiment 11, and RT-qPCR was performed according to the method of step (3) of embodiment 10. The qPCR primer sequences are shown in SEQ ID NO:146-153.

[0420] Figure 17 shows that the expression of inflammatory cytokines TNF-α, IFN-γ, and NLRP3 was increased by injecting 1E10vg virus, and the viral dose of 1E10vg explained the toxic side effects in BCD mice, and 1E9vg was better than 1E8vg. Therefore, we chose to conduct follow-up experiments with viral doses of 1E10vg or less.

[0421] Example 15. Effects of different virus transfections in BCD mice (I) Effect of p5-intron promoter virus infection in BCD mice (1) The viruses were packaged according to the method of Example 7. A: pAV-p5-intron-CYP4V2-BGH (expression sequence shown in SEQ ID NO:93); B: pAV-p5-intron-RdCVF-BGH (expression sequence shown in SEQ ID NO:99); C: pAV-p5-intron-CYP4V2-P2A-RdCVF-BGH (expression sequence shown in SEQ ID NO:113).

[0422] (2) One-month-old BCD mice (Cyp4v3- / -) were divided into five groups: KO group, A group, B group, A+B group, and C group, and 1E9vg of virus A, B, C and virus A+virus B (5E8vg for A and B, respectively) were used, and the KO group was used as a blank control and was not treated. Subretinal injection was performed according to the method of Example 13.

[0423] (3) The crystalline deposits were observed by taking fundus photographs according to the method of Example 13. Figure 18 shows the crystalline deposits in the fundus photographs of BCD mice after subretinal injection therapy with different viruses. Figure 19 shows the statistical results (n=5) of the number of crystals in the fundus of BCD mice after subretinal injection therapy with different viruses. The results show that the reduction in the number of crystals in the virus A+B group and the virus C group was more significant. The fundus crystal situation in the virus B group mice showed no obvious improvement.

[0424] (4) Retinal function test: electroretinogram (ERG) 1) Mouse dark adaptation: Mice were dark adapted for at least 16 hours, after which all operations were performed under dim red light.

[0425] 2) Mouse anesthesia: 80 mg / kg ketamine + 8 mg / kg torthiazine was injected intraperitoneally.

[0426] 3) After the anesthesia was completed, the mouse's eyes were dilated with 1% atropine under dim red light illumination, and the mouse was fixed with tape in front of the animal experimental platform of the visual electrophysiological instrument Espion E2, with both eyes aligned and fully exposed. The ground electrode needle was inserted into the base of the mouse's tail, and the reference electrode needle was inserted into the lower jaw of the mouse. The two gold ring recording electrodes were clamped into the electrode holder of the animal experimental platform, and their angles were adjusted so that they were lightly touching the central tip of the cornea of ​​the left and right eyes, respectively. An appropriate amount of 2% hydroxymethylcellulose was added to improve the contact effect between the gold ring electrodes and the cornea.

[0427] 4) The mouse number and age were entered into the computer Espion E2 system, and the program was run. The intensities of the dark adaptation flash for each row were 0.003, 0.01, 0.1, 1, 3, 10, and 100 cd·s / m 2 (Background luminosity is 0 cd s / m 2 The interstimulus interval was 15 seconds, and the average of three ERG signals was recorded. The photopic flash intensities were 3, 10, 30, and 100 cd·s / m 2 (Adaptation time 5 minutes, background light level 30 cd·s / m 2 (The stimulation interval was 15 seconds, and the average of five ERG signals was recorded.) After the program was completed, the results were automatically saved and compiled using GraphPad Prism.

[0428] Figure 20 shows the ERG recording results of BCD mice treated with subretinal injection of different viruses. The results showed that the ERG amplitude of the mice in virus group B had no obvious change, while the ERG amplitude of the mice in virus group A, virus group A+B, and virus group C all increased (p<0.05), and the virus group A+B and virus group C were superior to the virus group A (p<0.05).

[0429] (5) The expression status of CYP4V2 and RdCVF in BCD mice was examined using the method described in step (3) of Example 13. RdCVF (1:50, H00115861-D01P).

[0430] Figure 21 shows immunofluorescence staining of BCD mice after subretinal injection treatment with different viruses. As a result, the virus A group mice expressed CYP4V2 (double arrow indicated area), the virus B group mice expressed RdCVF (single arrow indicated area), and the expression of CYP4V2 and RdCVF was detected in both the virus A+B group and the virus C group.

[0431] (6) Examination of RPE cell number and morphology by staining mouse ocular RPE sections 1) Pre-cooled PBS was placed in a 1.5 ml EP tube, and after sacrificing a mouse, the mouse eyeball was removed and immersed in PBS for 15 minutes.

[0432] 2) The mouse eyeball was transferred to an EP tube containing 1.5 ml of 4% paraformaldehyde that had been fixed for 1 hour.

[0433] 3) Under a stereomicroscope (Olympus, SZ61-SET), the anterior segment of the mouse eyeball was removed, the neural retina layer and the RPE layer were separated, and the RPE layer was cut into four pieces.

[0434] 4) The RPE sheet was washed three times in pre-chilled PBS for 5 min each.

[0435] 5) RPE sections were passed through 0.1% Triton for 20 min and then washed three times with PBS, for 5 min each time.

[0436] 6) RPE slices were placed in one well of a 96-well plate, and phalloidin working solution diluted 1:200 with PBS was added and incubated at room temperature for 1 h.

[0437] 7) After washing with PBS three times, 5 min each time, the RPE was laid flat on a glass slide and a small amount of tablet was added, then the tablet was wrapped with a cover glass and observed under a Nikon fluorescence microscope.

[0438] The results of phalloidin-labeled F-actin staining in Figure 22 show that the hexagonal morphology of RPE cells was more complete and densely arranged in BCD mice injected with promoter A virus and promoter B virus compared to BCD mice not injected with the virus.

[0439] In the phalloidin-labeled F-actin staining results in Figure 22, no significant changes were observed in group B compared to the KO group, and the hexagonal morphology of the RPE cells in the virus A+B and C groups was more regular and closely arranged than in the other three groups. Figure 23 statistically shows that the number of RPE cells was counted in the same area, and the number of RPE cells in the virus A+B and C groups in the same area was higher than that of the virus A group (n=5).

[0440] Comprehensive observation of crystalline deposits, electroretinogram (ERG), and RPE cell number and morphology showed that the sole expression of RdCVF (group B) did not improve the crystalline deposits, electroretinogram (ERG), and RPE cell number and morphology of BCD mice. Co-expression of RdCVF and CYP4V2 (groups A+B and C) significantly improved the crystalline deposits, electroretinogram (ERG), and RPE cell number and morphology of BCD mice compared with the sole expression of CYP4V2, indicating that co-expression of CYP4V2 and RdCVF had a more obvious synergistic effect than sole expression of CYP4V2 or ERG.

[0441] (II) ERG effects in BCD mice infected with a virus that does not contain a post-promoter intron (1) The viruses were packaged according to the method of Example 7. A: pAV-p5-CYP4V2-BGH (expression sequence shown in SEQ ID NO:91); B: pAV-p5-RdCVF-BGH (expression sequence shown in SEQ ID NO:97); C: pAV-p5-CYP4V2-P2A-RdCVF-BGH (expression sequence shown in SEQ ID NO:107).

[0442] (2) One-month-old BCD mice (Cyp4v3- / -) were divided into five groups: KO group, A group, B group, A+B group, and C group, and 1E9vg of virus A, B, C and virus A+virus B (5E8vg for A and B, respectively) were used, and the KO group was used as a blank control and was not treated. Subretinal injection was performed according to the method of Example 13.

[0443] (3) Electroretinography (ERG) was performed according to the method of step (4) of Example (1). Figure 24 shows the ERG recording results of BCD mice after subretinal injection treatment with the above-mentioned different viruses, and the ERG amplitude of the mice in virus B group did not change significantly, while the ERG amplitude of the mice in virus A group, virus A+B group, and virus C group all increased (p<0.05), and the virus A+B group and virus C group were superior to the virus A group (p<0.05).

[0444] (III) ERG effect in p1 promoter virus-infected BCD mice (1) The viruses were packaged according to the method of Example 7. A: pAV-p1-CYP4V2-BGH (expression sequence shown in SEQ ID NO:92); B: pAV-p1-RdCVF-BGH (expression sequence shown in SEQ ID NO:98); C: pAV-p1-CYP4V2-P2A-RdCVF-BGH (expression sequence shown in SEQ ID NO:108).

[0445] (2) One-month-old BCD mice (Cyp4v3- / -) were divided into five groups: KO group, A group, B group, A+B group, and C group, and 1E9vg of virus A, B, C and virus A+virus B (5E8vg for A and B, respectively) were used, and the KO group was used as a blank control and was not treated. Subretinal injection was performed according to the method of Example 13.

[0446] (3) Electroretinography (ERG) was performed according to the method of step (4) of Example (1). Figure 25 shows the ERG recording results of BCD mice after subretinal injection treatment with different viruses as mentioned above. The results show that the ERG amplitude of the mice in virus B group has no obvious change, while the ERG amplitude of the mice in virus A group, virus A+B group, and virus C group all increased (p<0.05), and the virus A+B group and virus C group were superior to the virus A group (p<0.05).

[0447] (IV) ERG effect in BCD mice infected with WPRE-SV40 poly A signal site virus (1) Viruses were packaged according to the method of Example 7. A: pAV-p5-intron-CYP4V2-WPRE-SV40 (expression sequence shown in SEQ ID NO:94); B: pAV-p5-intron-RdCVF-WPRE-SV40 (presentation sequence as SEQ ID NO: (Figure 100); C: pAV-p5-intron-CYP4V2-P2A-RdCVF-WPRE-SV40 (expression sequence shown in SEQ ID NO:114).

[0448] (2) One-month-old BCD mice (Cyp4v3- / -) were divided into five groups: KO group, A group, B group, A+B group, and C group, and 1E9vg of virus A, B, C and virus A+virus B (5E8vg for A and B, respectively) were used, and the KO group was used as a blank control and was not treated. Subretinal injection was performed according to the method of Example 13.

[0449] (3) Electroretinography (ERG) was performed according to the method of step (4) of Example (1). Figure 26 shows the ERG recording results of BCD mice after subretinal injection treatment with the above-mentioned different viruses, and the ERG amplitude of the mice in virus B group did not change significantly, while the ERG amplitude of the mice in virus A group, virus A+B group, and virus C group all increased (p<0.05), and the virus A+B group and virus C group were superior to the virus A group (p<0.05).

[0450] (V) Effect of BCD mice infected with a virus that does not contain a signal site (1) The viruses were packaged according to the method of Example 7. A: pAV-p5-CYP4V2 (shown in SEQ ID NO:117); B: pAV-p5-RdCVF (shown in SEQ ID NO:120); C: pAV-p5-CYP4V2-P2A-RdCV (shown in SEQ ID NO:122).

[0451] (2) One-month-old BCD mice (Cyp4v3- / -) were divided into five groups: KO group, A group, B group, A+B group, and C group, and 1E9vg of virus A, B, C and virus A+virus B (5E8vg for A and B, respectively) were used, and the KO group was used as a blank control and was not treated. Subretinal injection was performed according to the method of Example 13.

[0452] (3) Electroretinography (ERG) was performed according to the method of step (4) of Example (1). Figure 27 shows the ERG recording results of BCD mice after subretinal injection treatment with the above-mentioned different viruses. Compared with the control, the ERG amplitude of the mice in virus B group had no obvious change, while the ERG amplitude of the mice in virus A group, virus A+B group, and virus C group all increased, and the virus A+B group and virus C group were superior to the virus A group (p<0.05).

[0453] The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the embodiments described herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. A vector comprising, in order from the 5' to 3' direction, a promoter, a polynucleotide encoding CYP4V2, and a polyadenylation signal site, wherein the promoter is operably linked to the polynucleotide encoding CYP4V2 and is a CAG promoter.

2. The vector of claim 1 , wherein the CAG promoter comprises the polynucleotide sequence shown in SEQ ID NO:

4.

3. The vector of claim 1, wherein the amino acid sequence of CYP4V2 comprises the amino acid sequence shown in SEQ ID NO: 76-82.

4. 2. The vector of claim 1, wherein the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence shown in SEQ ID NO: 62 or a polynucleotide sequence having at least 95% identity thereto.

5. The vector of claim 1, wherein the polyadenylation signal site comprises a nucleic acid sequence shown in SEQ ID NO: 17-21.

6. The vector of claim 1 , which is AAV2 / 2, AAV2 / 5, AAV2 / 8, or AAV2 / 9.

7. The vector of claim 6, which is AAV2 / 8.

8. A cell comprising the vector according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising the vector according to any one of claims 1 to 7 and / or the cell according to claim 8, and a pharma- ceutically acceptable excipient.

10. A method for treating, alleviating and / or preventing a disease or condition associated with retinal pigment epithelium (RPE) atrophy, comprising administering to a subject a vector described in any one of claims 1 to 7, a cell described in claim 8, or a pharmaceutical composition described in claim 9.

11. The method of claim 10, wherein the disease or condition is crystalline retinal degeneration.

12. The method of claim 10 , wherein the subject is a human.

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