Use of CYP4V2 and RdCVF in pharmaceutical manufacturing

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, and more specifically to the use of CYP4V2 and RdCVF in the manufacture of pharmaceuticals. [Background technology]

[0002] Crystalline retinal degeneration (Bietti's crystalline dystrophy, BCD) is a rare retinal degeneration characterized by corneal crystals (a clear cover), fine, yellow, or white crystalline deposits in the photosensitive tissue of the retina, and progressive atrophy of the retina, choroidal capillaries, and choroid. The deposits damage the retina, leading to gradual vision loss. People with crystalline retinal degeneration typically begin experiencing vision impairment in their teens or twenties, and the vision loss in each eye may worsen at a different pace. The severity and progression of symptoms vary greatly from person to person, even within the same family, but most people become blind by the age of 40 or 50. It is estimated that 1 in 67,000 people worldwide have crystalline retinal degeneration, and it is more common in East Asians, particularly Chinese and Japanese.

[0003] Current research indicates that BCD is an autosomal recessive genetic disorder caused by mutations in the CYP4V2 gene. Numerous mutation sites in the CYP4V2 gene have already been discovered during genetic studies of BCD patients both domestically and internationally. The CYP4V2 gene is one of the proteins in the cytochrome P450 superfamily, and the CYP4V2 gene-coding protein participates in the process of fatty acid metabolism. It is thought that CYP4V2 gene mutations in crystalline retinal degeneration disrupt the function of enzymes participating in fatty acid metabolism, thereby affecting lipid breakdown. However, it remains unclear how CYP4V2 causes the specific signs and symptoms of BCD. Currently, BCD treatment mainly follows methods used to treat retinitis pigmentosa (RP), and while research into CYP4V2 gene mutations has offered possibilities for future gene therapy, there is currently no effective treatment. [Overview of the Initiative] [Means for solving the problem]

[0004] This application provides for the use of CYP4V2 and RdCVF in the manufacture of pharmaceuticals for the treatment, relief, and / or prevention of diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

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

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

[0007] In some embodiments, CYP4V2 includes the amino acid sequence shown in any of SEQ ID NO:76-82.

[0008] In some embodiments, the polynucleotide encoding CYP4V2 includes the nucleic acid sequence shown in one of SEQ ID NO: 62-68.

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

[0010] In some embodiments, RdCVF contains the amino acid sequence shown in any of SEQ ID NO: 83-89.

[0011] In some embodiments, the polynucleotide encoding the RdCVF includes a nucleic acid sequence shown in any of SEQ ID NO: 69-75.

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

[0013] In some embodiments, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are located in 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 includes a viral vector.

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

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

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

[0019] In some embodiments, the promoter includes a nucleotide sequence shown in any of SEQ ID NO: 1 to 12.

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

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

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

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

[0024] In some embodiments, the polynucleotide encoding the self-cleaving peptide comprises a nucleotide sequence shown in any of SEQ ID NO:22-25.

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

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

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

[0028] In some embodiments, the intron includes a nucleotide sequence shown in any of SEQ ID NO: 13-16.

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

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

[0031] In some embodiments, the vector includes a nucleotide sequence shown in any of SEQ ID NO:90-116.

[0032] The present invention also provides a combination of vectors for the treatment, mitigation and / or prevention of diseases or conditions associated with retinal pigment epithelium (RPE) atrophy, the combination of vectors 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 includes crystalline retinal degeneration.

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

[0035] In some embodiments, CYP4V2 includes the amino acid sequence shown in any of SEQ ID NO:76-82.

[0036] In some embodiments, the polynucleotide encoding CYP4V2 includes the nucleic acid sequence shown in one of SEQ ID NO: 62-68.

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

[0038] In some embodiments, RdCVF contains the amino acid sequence shown in any of SEQ ID NO: 83-89.

[0039] In some embodiments, the polynucleotide encoding RdCVF includes a nucleic acid sequence shown in any of SEQ ID NO: 69-75.

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

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

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

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

[0044] In some embodiments, the promoter includes a nucleotide sequence shown in one of SEQ ID NO:1 to 12.

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

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

[0048] In some embodiments, the first vector and / or the second vector further include introns.

[0049] In some embodiments, the intron includes a nucleotide sequence shown in any of SEQ ID NO: 13-16.

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

[0051] In some embodiments, the intron is located in 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 shown in any of SEQ ID NO:90-95, and / or the second vector comprises a nucleotide sequence shown in any of SEQ ID NO:96-100.

[0053] The present invention 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, CYP4V2 is human CYP4V2.

[0055] In some embodiments, CYP4V2 includes the amino acid sequence shown in any of SEQ ID NO:76-82.

[0056] In some embodiments, the polynucleotide encoding CYP4V2 includes the nucleic acid sequence shown in one of SEQ ID NO: 62-68.

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

[0058] In some embodiments, RdCVF contains the amino acid sequence shown in any of SEQ ID NO: 83-89.

[0059] In some embodiments, the polynucleotide encoding RdCVF includes a nucleic acid sequence shown in any of SEQ ID NO: 69-75.

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

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

[0062] In some embodiments, the promoter includes a nucleotide sequence shown in one of SEQ ID NO:1 to 12.

[0063] In some embodiments, the nucleic acid molecule includes 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, the polynucleotide encoding the self-cleaving peptide being located between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

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

[0066] In some embodiments, the polynucleotide encoding the self-cleaving peptide includes a nucleotide sequence shown in any of SEQ ID NO:22-25.

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

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

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

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

[0071] In some embodiments, the intron includes a nucleotide sequence shown in any of SEQ ID NO: 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 shown in any of SEQ ID NO: 101 to 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 includes an AAV vector.

[0077] This application further provides cells containing the nucleic acid molecule or vector described herein.

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

[0079] This application also provides the use of nucleic acid molecules, vectors, or cells described herein in the manufacture of a pharmaceutical product, which is used for the treatment, relief, and / or prevention of diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

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

[0081] Those skilled in the art will readily infer other aspects and advantages of the Application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the Application. As those skilled in the art will recognize, the content of the Application is such that those skilled in the art can modify the specific embodiments disclosed without departing from the spirit and scope of the invention. Accordingly, the accompanying drawings and descriptions of the Application are illustrative and not limiting.

[0082] The specific features of the invention described herein are as stated in the claims. The features and advantages of the invention described herein can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail herein. A general description of the accompanying drawings is as follows. [Brief explanation of the drawing]

[0083] [Figure 1] This invention demonstrates the effect of different CYP4V2 promoters on expression. [Figure 2] This invention demonstrates the expression effects of the CAG, EF1a, OPEFS, and EFS promoters. [Figure 3] This invention demonstrates the expression-enhancing effect of post-promoter-binding introns. [Figure 4] This invention demonstrates the expression-enhancing effect of post-promoter-binding introns. [Figure 5] This invention demonstrates the effect of selecting different PolyA signaling sites in the expression vector on the expression of the target gene. [Figure 6] This invention demonstrates the expression of the CYP4V2 and RdCVF gene expression vector in 293T cells. [Figure 7] This paper demonstrates the expression of the expression vector containing the CYP4V2 and RdCVF genes in ARPE-19 cells. [Figure 8] This image shows infection of the mouse retina by different serotypes of AAV virus in this invention, with double arrows indicating the expression range of the EGFP reporter gene. [Figure 9]The morphology of renal epithelial cells, iPSC cells, and RPE cells observed with a fluorescence microscope during the RPE cell production process described in this invention is shown. [Figure 10] Figure 10A shows the effect of viral titer on the death of RPE cells differentiated from human iPSCs in this application, Figures 10B and 10C show the effect of viral titer on the expression status of the inflammatory cytokines NLRP3 and TNF-α in RPE cells differentiated from human iPSCs, respectively, and Figure 10D shows the expression status of the target gene in RPE cells differentiated from human iPSCs under different viral titer conditions. [Figure 11] This shows the expression status of CYP4V2 and RdCVF when RPE cells differentiated from human iPSCs are infected with different viruses of this invention. [Figure 12] This study demonstrates the effects of different promoters and viral treatments containing the CYP4V2 gene on fundus crystal deposition in BCD mice. [Figure 13] This paper presents statistical results on the effects of viral treatments containing different promoters and the CYP4V2 gene on fundus crystal deposition in BCD mice. [Figure 14] The results of CYP4V2 immunofluorescence staining in BCD mice treated with viruses containing different promoters and the CYP4V2 gene are shown. [Figure 15] This paper demonstrates the effect on fundus crystal deposition in BCD mice after treatment with different doses of virus containing the CYP4V2 gene. [Figure 16] This paper presents statistical results on the effect of treating BCD mice with different doses of virus containing the CYP4V2 gene on the state of retinal crystal deposition. [Figure 17] This paper describes the effects on the expression levels of inflammatory cytokines TNF-α, IFN-γ, and NLRP3 after treating BCD mice with different doses of virus containing the CYP4V2 gene. [Figure 18] This paper demonstrates the effect of treating BCD mice with different viruses on the state of retinal crystal deposition. [Figure 19]This paper presents statistical results on the effect of treating BCD mice with different viruses on the state of retinal crystal deposition. [Figure 20] This paper demonstrates the effects on retinal function (electroretinography (ERG)) after treating BCD mice with different viruses. [Figure 21] This application shows the immunofluorescence staining results for CYP4V2 and RdCVF after treating BCD mice with different viruses. [Figure 22] This invention demonstrates the effects on the number and morphology of RPE cells after treating BCD mice with different viruses. [Figure 23] This paper presents statistical results on the effect of treating BCD mice with different viruses on the number of RPE cells. [Figure 24] This invention demonstrates the effect on retinal function (electroretinography (ERG)) after treating BCD mice with a different virus that does not contain introns after the promoter stage. [Figure 25] This paper demonstrates the effect on retinal function (electroretinography (ERG)) after treating BCD mice with viruses containing different p1 promoters. [Figure 26] This invention demonstrates the effect on retinal function (electroretinography (ERG)) after treating BCD mice with viruses that have different WPRE-SV40 poly A signaling sites. [Figure 27] This paper demonstrates the effect on retinal function (electroretinography (ERG)) after treating BCD mice with different viruses that do not contain signaling regions. [Modes for carrying out the invention]

[0084] The embodiments of the present invention will be described below using specific examples, but those skilled in the art will readily understand other advantages and effects of the present invention as disclosed herein.

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

[0086] In this application, the term “isolated” generally refers to something artificially obtained from its natural state. If an “isolated” substance or component appears in nature, it may be due to a change in the natural environment in which it is found, or to the isolation of the substance from its natural environment, or both. For example, certain unisolated polynucleotides or polypeptides naturally exist within a living animal, and a high-purity version of the same polynucleotide or polypeptide isolated from this natural state is referred to as isolated. The term “isolated” does not exclude the presence of substances that are artificial or synthetic, or other impurities that do not affect the activity of the substance.

[0087] In this application, the term “isolated nucleic acid molecule” usually refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, or an analogue thereof isolated from its natural environment or artificially synthesized.

[0088] In this application, the term "CYP4V2" usually refers to a protein that is member 2 of subfamily V of the cytochrome P450 family. Cytochrome P450, also known as cytochrome P450 or CYP450, usually refers to a single family of iron heme proteins, belongs to the monooxygenase class, and participates in the metabolism of endogenous substances or exogenous substances, including pharmaceuticals and environmental compounds. Based on the degree of cognacy of their amino acid sequences, its members are divided into three classes: family, subfamily, and enzyme entity. The cytochrome P450 enzyme system can be abbreviated as CYP, where the family is represented by Arabic numerals, the subfamily by uppercase letters, and the enzyme entity by Arabic numerals, for example, CYP4V2 in this application. The human CYP4V2 gene (HGNC::23198) is 19.28kb in length, located at 4q35, has 11 exons, and plays a crucial 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 and other tissues. 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 called rod-derived cone survival factor and usually refers to a cleavage-type thioredoxin that lacks thiol oxidoreductase activity. RdCVF is a splice variant of the retinoid 1 (nucleoredoxin-like 1, Nxnl1) gene, and the additional splice product of this gene is RdCVFL, which is 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 gene-deficient mice can exhibit age-dependent loss of function in rod and cone photoreceptor cells and degenerative changes in cone photoreceptor cells, as well as hypersensitivity of rod and cone photoreceptor cells to oxidative stress (Cronin et al., 2010). Nxnl1 expression can be rod photoreceptor-dependent and is significantly reduced after rod photoreceptor death in retinitis pigmentosa (RP) (Delyfer et al., 2011; Reichman et al., 2010). RdCVF can protect cone cell function in several different genotype (Byme et al., 2015; Le'veillard et al., 2004; Yang et al., 2009) models of retinitis pigmentosa (RP). The RdCVF molecule is secreted extracellularly as a signal peptide that promotes glucose uptake by photoreceptor cells and maintains the survival of photoreceptor cells. The gene is conserved in humans, chimpanzees, rhesus monkeys, dogs, cattle, rats, mice, chickens, zebrafish, and frogs. In humans, Nxnl1 is also called Txnl6, and it is located at 19 p13.11 and contains two exons. Nxnl1 is universally expressed in human tissues such as the lens, retina, stomach, kidneys, heart, colon, and spleen, and is expressed at relatively high levels in the lens and retina.

[0090] In this application, the term "promoter" usually refers to a sequence of deoxyribonucleic acid (DNA) that causes a specific gene to be transcribed. Promoters are recognized by RNA polymerase and initiate RNA transcription and synthesis. In ribonucleic acid (RNA) synthesis, promoters interact with transcription factors that control gene transcription, controlling the timing and degree of gene expression (transcription) initiation. Promoters include a core promoter region and a control region, are located upstream of the gene transcription initiation site (5' direction of the DNA antisense strand) within the control sequence that controls gene expression, and do not have a compiling function themselves. They are classified into three types based on their mode of action and function: constitutive promoters (maintaining sustained activity in almost all tissues), specific promoters (tissue specificity or developmental stage specificity), and inductive promoters (controlled by external chemical or physical signals).

[0091] In this application, the term “operably ligated” generally refers to positioning the regulatory sequences necessary for the expression of an encoding sequence at appropriate locations relative to the encoding sequence in order to enable the expression of the encoding sequence. For example, when a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is operably ligated to the second nucleic acid sequence. In some embodiments, the encoding sequence and the sequences of transcriptional regulatory elements in the expression vector can be represented. The regulatory devices include promoters, augerants, and stop devices. The Nxnl1 gene is conserved in humans, chimpanzees, rhesus monkeys, dogs, cattle, rats, mice, chickens, zebrafish, and frogs. In some embodiments, the term “operably ligated” may further refer to the fact that the target gene is ligated within the vector such that the transcriptional and translational regulatory sequences within the vector exert the expected function of regulating the transcription and translation of the target gene.

[0092] In this application, the term "self-cleaving peptide" is also called 2A peptide (2A self-cleaving peptides), and is typically a peptide fragment of 18-22 amino acid residues that can induce self-splicing of recombinant proteins containing 2A peptides within cells. 2A peptides are usually derived from the 2A region of a viral genome. In genetic engineering, 2A peptides can split a peptide chain translated from a single open reading frame (ORF) into several independent peptide chains. In some embodiments, if it is necessary to express two proteins separately (for example, one protein entering the nucleus and the other being expressed in the cytoplasm), and it is desired to construct only one open reading frame on a vector, this can be achieved by inserting a single-step 2A peptide sequence into their coding regions. In some embodiments, if the fusion protein after two protein fusion is non-functional, a sequence encoding a single-step 2A peptide can be inserted between the coding regions of the two proteins, or the binding peptide can be replaced with a 2A peptide, separating the two proteins after translation and allowing them to fold independently, thereby potentially restoring the function of the two proteins. 2A peptides can include P2A, E2A, F2A, and T2A, all of which are named after their source virus. P2A, in particular, is derived from the 2A peptide of Porcine teschovirus.

[0093] In this application, the term "polyadenylation signal sequence," also known as a polyadenylation signal tail or Poly A tail, typically refers to a sequence of tens to hundreds of single adenylate chains added to the 3' end of transcribed mRNA. Polyadenylation usually occurs in the nucleus during the transcription of deoxyribonucleic acid (DNA) into ribonucleic acid (RNA) and the subsequent completion of this reaction by polyadenylate polymerase. In eukaryotes, polyadenylation is a mechanism that can interrupt mRNA molecules at their 3' end, and the polyadenylate sequence can protect mRNA from attack by nucleic acid exonucleases, all of which are crucial for mRNA export from the cell nucleus, translation, and stability.

[0094] In this application, the term "polyadenylation signal site" typically refers to a nucleotide sequence identified by polyadenylation-associated cleavage factors located at the 3' end of messenger RNA (mRNA). It is also typically an anterograde regulatory signal on mRNA. Generally, the tailing process (polyadenylation) begins after transcription is complete, and polyadenylation-associated cleavage factors add tens to hundreds of single adenylic acids 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 aforementioned polyadenylation-associated degradation factors include degradation / polyadenylate specificity factors (CPSF), degradation stimulants (CstF), degradation factor I (CFI), and degradation factor II (CFII). Polyadenylation signal sites typically contain AAUAAA sequences, but these vary among eukaryotic groups. For example, most human polyadenylation signal sites contain AAUAAA sequences, while this sequence is rare in plants and fungi.

[0095] In this application, the term "intron" may include DNA fragments removed from an RNA transcript by joining either end of a sequence (exon). Introns are typically considered interfering sequences within the protein-coding region of a gene and do not usually contain information represented by the proteins produced by the gene.

[0096] In this application, the term "vector" typically refers to a nucleic acid delivery vehicle into which a protein-coding polynucleotide is inserted, allowing for protein expression. Vectors express genes in host cells through transformation, transfection, or transfection into host cells. Examples of vectors include: plasmids; bacteriophages; cosplasmides; 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 single vector contains elements that control multiple expression types, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain replication initiation sites. Furthermore, vectors may contain, but are not limited to, components that assist in cell entry, such as viral particles, liposomes, and protein shells.

[0097] In this application, the term “viral vector” generally refers to a non-wild-type recombinant viral particle containing a recombinant viral genome packaged within a viral capsid, which functions as a gene delivery vector. The types of animal viruses used as vectors may include reverse transcriptase viruses (including slow viruses), adenoviruses, adeno-associated viruses (AAVs), herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papilloma vacuolar viruses (e.g., SV40).

[0098] In this application, the term "AAV vector" is also called an 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 Microviridae. The AAV genome can contain inverse terminal repeat sequences (ITRs) at both ends of the DNA strand and two open reading frames (ORFs). The open reading boxes can contain rep and cap. The rep consists of multiple duplicate genes encoding the Rep protein necessary for the AAV life cycle and contains duplicate nucleotide sequences encoding a capsid protein that can contain 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 from a latent state until the assistant virus (Kotin et al., 1990) is rescued. Generally, AAV is thought to be predominantly in inconsistent form. Due to its site-specific ability to integrate, its natural defects, and its low immunogenicity, AAV makes an ideal gene therapy vector. AAV has many common serotypes and more than 100 viral variants. In this application, AAV couplers, 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 known or mentioned variant of AAV, or any AAV or variant or mixture thereof that has not been discovered.

[0099] In this application, the term “cell” usually refers to a receptor for a nucleic acid molecule or vector, or to an existing single cell, cell line, or cell culture. A cell may contain the nucleic acid molecule described in this application or the vector described in the present invention. A cell may include offspring of a single cell. Due to spontaneous, accidental, or intentional mutations, the offspring may not necessarily be identical to the primordial mother cell (morphologically or genomically in terms of total DNA complement). A cell may include a cell transfected in vitro with the vector described in this application. A cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cell, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells. In some embodiments, the cell may be a mammalian cell. In some embodiments, the mammalian cell may be a HEK293T cell.

[0100] In this 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 may include a nucleic acid molecule described herein, a vector described herein and / or a cell described herein, and an optionally selected pharmaceutically acceptable adjuvant. In addition, such pharmaceutical composition may also include one or more (pharmaceutically effective) vectors, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are non-toxic to the recipient at the dose and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, cryogenic, and lyophilized compositions.

[0101] In this application, the term “prevention” generally means a combination administered prophylactically to a healthy subject to prevent the onset of a certain disease or condition. It may further include a combination of prophylactic administration to a patient in the pre-stage of an allergic disease of treatment. “Prevention” does not require 100% elimination of the possibility of the onset of the disease or disorder; in other words, “prevention” usually means that the likelihood of the onset of the disease or disorder is reduced in the presence of the combination of administrations.

[0102] In this application, the term “relief” means a reduction, decrease, or delay of a particular medical condition, disease, state, or phenotype. The medical condition, disease, state, or phenotype may include subjectively perceived symptoms such as pain, dizziness, or other physiological disturbances, or medically detectable indicators such as lesions detected by medical testing instruments.

[0103] In this application, the term “treatment” generally refers to a clinical intervention used in a clinicopathological process to alter the natural course of a treated individual or cell. This may include improvement of the condition, disappearance of a lesion, or improvement of the prognosis.

[0104] In this application, the term "retinal pigment epithelium (RPE)" usually refers to the layer of pigment cells attached to the outside of the retinal sensory nerves. The retinal pigment epithelium consists of a single layer of hexagonal cells densely packed with pigment granules. The retinal pigment epithelium (RPE) is tightly connected to the choroid below it and the retinal nerve cells above it, and its main functions include: controlling the fluid and nutrients in the subretinal space and performing the function of the blood-retinal barrier; regulating local structures through synthetic growth factors; absorbing light and regulating electrical balance; regenerating and synthesizing visual pigments; phagocytosis and digestion of the outer segments of photoreceptors; maintaining retinal attachment; and regeneration and repair after damage. The RPE is an important tissue that maintains photoreceptor function and is simultaneously affected by many lesions 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 known as retinal pigment lesions, usually refers to a hereditary ophthalmic disease. There are three modes of inheritance: autosomal recessive, dominant, and X-linked, and digenotypes and mitochondrial types also exist. Common early signs include night blindness, narrowing of the visual field, and loss of vision in the field of view that is clear in front but slightly deviated to the left or right. RP includes monocular primary retinitis pigmentosa, quadrant primary retinitis pigmentosa, central or paracentral primary retinitis pigmentosa, white punctate retinal degeneration due to achromatic retinitis pigmentosa, crystalline retinal degeneration, intravenous hypertrophic retinitis pigmentosa, parapigmentous retinitis pigmentosa of arterioles, and retinitis pigmentosa in Leber congenital amaurosis and other syndromes.

[0106] In this application, the term “crystalline retinal degeneration” usually refers to an autosomal recessive eye disease first described in 1937 by Italian ophthalmologist Dr. G.B. Bietti. Main symptoms include corneal crystals (a clear cover), small yellow or white crystalline deposits in the photosensitive tissue of the retina, and progressive atrophy of the retina, choroidal capillaries, and choroid. Crystalline retinal degeneration may include diseases resulting from mutations in the CYP4V2 gene.

[0107] In this application, the terms “including” or “including” generally mean including clearly defined features without excluding other elements.

[0108] In this application, the term "approximately" usually means a variation of 0.5% to 10% above a specified value, for example, a variation within the range 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] On the other hand, this application provides the use of CYP4V2 and RdCVF in the manufacture of a pharmaceutical product, which is used for the treatment, relief and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy.

[0110] In this application, the pharmaceutical may include a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF. For example, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF may be located in different vectors. For example, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF may be located in the same vector.

[0111] On the other hand, the present invention also provides a combination of vectors for the treatment, mitigation and / or prevention of diseases or conditions associated with retinal pigment epithelium (RPE) atrophy, comprising a first vector and a second vector, wherein the first vector may contain a polynucleotide encoding CYP4V2 and the second vector may contain a polynucleotide encoding RdCVF.

[0112] On the other hand, the present application provides an isolated nucleic acid molecule that can contain a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

[0113] On the other hand, the present application further provides cells that may contain a combination of nucleic acid molecules or vectors described herein.

[0114] The present application also provides a pharmaceutical composition which may include the nucleic acid molecule described herein, a combination of the vector described herein, and / or the cell described herein.

[0115] On the other hand, the present application also provides the use of the nucleic acid molecules, vector combinations, cells, and / or pharmaceutical compositions described herein in the manufacture of a pharmaceutical, which can be used for the prevention, mitigation, and / or treatment of diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0116] CYP4V2 In this application, CYP4V2 includes, but is not limited to, CYP4V2 or functional variants of humans, chimpanzees, gorillas, rhesus monkeys, dogs, cattle, mice, rats, chickens, fruit flies, nematodes, or frogs, and may also include proteins that are dysfunctional or proteins that encode gene mutations causing crystalline retinal degeneration.

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

[0118] For example, CYP4V2 may contain amino acid sequences shown in any of SEQ ID NO: 76-82.

[0119] For example, the CYP4V2 may include the amino acid sequence indicated by SEQ ID NO:76.

[0120] For example, the CYP4V2 can include an amino acid sequence having at least 90% identity with the amino acid sequences 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 the combined administration of the amino acid sequence and the RdCVF of this application can improve diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0121] In this application, the polynucleotide encoding CYP4V2 may include the nucleic acid sequence shown in any of SEQ ID NO:62-68.

[0122] For example, the polynucleotide encoding CYP4V2 may contain the nucleic acid sequence indicated by SEQ ID NO:62.

[0123] For example, the polynucleotide encoding CYP4V2 may include an amino acid sequence having at least 90% identity with the nucleic acid sequences shown in SEQ ID NO: 62-68, for example, any polynucleotide sequence having 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 this application to improve diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0124] RdCVF In this application, the RdCVF may be produced from rod cells and may contain proteins favorable to pyramidal cells. The RdCVF includes, but is not limited to, human, chimpanzee, gorilla, rhesus monkey, dog, cattle, rat, mouse, chicken, zebrafish, and frog RdCVF or functional variants thereof.

[0125] For example, RdCVF can contain human RdCVF.

[0126] For example, RdCVF may contain the amino acid sequence shown in any of SEQ ID NO: 83-89.

[0127] For example, the RdCVF may contain the amino acid sequence indicated by SEQ ID NO:83.

[0128] For example, the RdCVF may include any amino acid sequence that is at least 90% identical to the amino acid sequences shown in SEQ ID NO: 83-89, such as 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 and the CYP4V2 of this application may improve diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0129] In this application, the polynucleotide encoding the RdCVF may include the nucleic acid sequence shown in any of SEQ ID NO: 69 to 75.

[0130] For example, the polynucleotide encoding RdCVF may contain the nucleic acid sequence indicated by SEQ ID NO:69.

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

[0132] promoter In this application, the promoter may include an RPE cell-specific promoter, a retinal cell-specific promoter, a corneal cell-specific promoter, an ophthalmic cell-specific promoter, or a constitutive promoter. The promoter may also include a mammalian β-actin promoter or a viral promoter. The promoter may also include a CAG promoter (heterozygous CMV early enhancer / chicken β-actin promoter, also known as the CAGGS promoter, CB promoter, or CBA promoter), a human β-actin promoter, a small CBA (smCBA) promoter, a CBS promoter or CBh promoter, an extension factor 1α short (EFS) promoter, an extension factor 1α (EF-1α) promoter, a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, a VMD2 (also known as BEST1) promoter, an OPEFS promoter, a CYP4V2 self-promoter, a RPE65 promoter, or its heterogenes or derivatives.

[0133] For example, the promoter may include a CYP4V2-self promoter, which may include all or part of the 2000 bp nucleotide sequence upstream of the polynucleotide of the code CYP4V2.

[0134] For example, the promoter may include a nucleotide sequence shown in any of SEQ ID NO:1 to 12.

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

[0136] For example, the promoter may be OPEFS and may include the nucleic acid sequence indicated by SEQ ID NO:1.

[0137] For example, the promoter may be an EFS containing the nucleic acid sequence indicated by SEQ ID NO:2.

[0138] For example, the promoter may be EF1a containing the nucleic acid sequence indicated by SEQ ID NO:3.

[0139] For example, the promoter may be a CAG containing the nucleic acid sequence indicated by SEQ ID NO:4.

[0140] For example, the promoter may be RPE65 containing the nucleic acid sequence indicated by SEQ ID NO:5.

[0141] Polyadenylation signaling sites In this application, the polyadenylation signal site includes the SV40 signal site, the BGH signal site, the WPRE signal site, the WPRE-SV40 signal site, the WPRE-BGH signal site, or a derivative thereof.

[0142] For example, the polyadenylation signaling site is identified by polyadenylation correlation decomposition factors, producing SV40 polyadenylate sequences, BGH signaling polyadenylate sequences, HSV signaling polyadenylate sequences, TK signaling polyadenylate sequences, WPRE signaling polyadenylate sequences, and the like.

[0143] For example, the polyadenylation signaling region can contain the sequence AAUAAA.

[0144] For example, the polyadenylation signaling region may include nucleic acid sequences indicated by SEQ ID NO: 17-21.

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

[0146] For example, self-cleaving peptides may include T2A, P2A, E2A, or F2A.

[0147] For example, self-cleaving peptides can contain P2A.

[0148] For example, self-cleaving peptides may contain amino acid sequences shown in any of SEQ ID NO:26-29.

[0149] For example, a polynucleotide encoding a self-cleaving peptide may contain nucleic acid sequences shown in any of SEQ ID NO:22-25.

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

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

[0152] For example, the intron contains a nucleotide sequence shown in any of SEQ ID NO:13~16.

[0153] Isolated nucleic acid molecules In this application, the isolated nucleic acid molecule may contain a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF, in order from the 5' end to the 3' end.

[0154] For example, the isolated nucleic acid molecule may contain, in order from the 5' end to the 3' end, a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF. The polynucleotide encoding CYP4V2 may contain a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, and the polynucleotide encoding RdCVF may contain a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75.

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

[0156] For example, the isolated nucleic acid molecule may contain, in order from the 5' end to the 3' end, a polynucleotide encoding RdCVF and a polynucleotide encoding CYP4V2. The polynucleotide encoding RdCVF may contain a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, and the polynucleotide encoding CYP4V2 may contain a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68.

[0157] In this application, the isolated nucleic acid molecule may also include the promoter.

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

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

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

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

[0162] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, and the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68.

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

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

[0165] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, and the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75.

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

[0167] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, and the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68.

[0168] In this application, the isolated nucleic acid molecule may also contain a polynucleotide encoding the self-cleaving peptide.

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

[0170] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, and the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75.

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

[0172] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, and the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68.

[0173] For example, the isolated nucleic acid molecule may contain a nucleic acid sequence indicated by SEQ ID NO: 101-105.

[0174] In this application, the nucleic acid molecule to be separated may also include the polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide of the code CYP4V2.

[0175] In this application, the nucleic acid molecule to be separated may also include a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide of the code RdCVF.

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

[0177] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, and the polyadenylation signal site may include a nucleotide sequence shown in any of SEQ ID NO: 17 to 21.

[0178] In this application, the nucleic acid molecule to be separated may include, in order from 5' to 3', the promoter, the polynucleotide encoding the RdCVF, the polynucleotide encoding the self-shear peptide, the polynucleotide encoding the CYP4V2, and the polyadenylation signal site.

[0179] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, and the polyadenylation signal site may include a nucleotide sequence shown in any of SEQ ID NO: 17 to 21.

[0180] For example, the isolated nucleic acid molecule may contain a nucleic acid sequence indicated by SEQ ID NO: 106-108.

[0181] In this application, the separated nucleic acid molecule may also contain the intron.

[0182] For example, the intron may include a nucleotide sequence shown in any of SEQ ID NO:13-16.

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

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

[0185] For example, the isolated nucleic acid molecule may include, in order from 5' to 3', the promoter, the polynucleotide encoding the CYP4V2 with the intron inserted, the polynucleotide encoding the self-cleaving peptide, and the polynucleotide encoding the RdCVF.

[0186] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the intron may include a nucleic acid sequence shown in any of SEQ ID NO: 13 to 16, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, and the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75.

[0187] For example, the nucleic acid molecule to be separated includes, in order from 5' to 3', the promoter, the intron, the polynucleotide encoding CYP4V2, the polynucleotide encoding the self-cleaving peptide, the polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0188] For example, the nucleic acid molecule to be separated may include, in order from 5' to 3', the promoter, the polynucleotide encoding the CYP4V2 that inserts the intron, the polynucleotide encoding the self-shearing peptide, the polynucleotide encoding the RdCVF, and the polyadenylation signal site.

[0189] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the intron may include a nucleic acid sequence shown in any of SEQ ID NO: 13 to 16, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, and the polyadenylation signal site may include a nucleotide sequence shown in any of SEQ ID NO: 17 to 21.

[0190] For example, the polynucleotide into which the intron encoding CYP4V2 is inserted may include the nucleic acid sequence described in SEQ ID NO:116.

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

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

[0194] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the intron may include a nucleic acid sequence shown in any of SEQ ID NO: 13 to 16, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, and the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68.

[0195] For example, the nucleic acid molecule to be separated includes, in order from 5' to 3', the promoter, the intron, the polynucleotide encoding the RdCVF, the polynucleotide encoding the self-cleaving peptide, the polynucleotide encoding the CYP4V2, and the polyadenylation signal site.

[0196] For example, the nucleic acid molecule to be separated may include, in order from 5' to 3', the promoter, the polynucleotide encoding the RdCVF that inserts the intron, the polynucleotide encoding the self-shearing peptide, the polynucleotide encoding CYP4V2, and the polyadenylation signal site.

[0197] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the intron may include a nucleic acid sequence shown in any of SEQ ID NO: 13 to 16, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, the polynucleotide encoding the self-cleaving peptide may include a nucleic acid sequence shown in any of SEQ ID NO: 22 to 25, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, and the polyadenylation signal site may include a nucleotide sequence shown in any of SEQ ID NO: 17 to 21.

[0198] For example, the isolated nucleic acid molecule may contain a nucleotide sequence shown in any of SEQ ID NO: 109-115.

[0199] vector In this application, the vector may include plasmids, phages, cosplasmides, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and viral vectors.

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

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

[0202] For example, an adeno-associated virus vector (AAV vector) may also include a recombinant adeno-associated virus vector (rAAV vector).

[0203] For example, the capsid, ITR, and other selected AAV components in a recombinant adeno-associated virus 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, and 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, or Rh10.

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

[0206] For example, an 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 may contain a nucleotide sequence shown in any of SEQ ID NO:133~137.

[0209] For example, the vector may also contain a nucleotide sequence shown in any of SEQ ID NO:90~115.

[0210] For example, the vector may also contain a nucleotide sequence shown in any of SEQ ID NO: 117 to 121.

[0211] For example, the vector may contain a nucleotide sequence shown in one of the SEQ ID NOs: 122-132.

[0212] In this application, the vector may include the isolated nucleic acid molecule described in this application.

[0213] For example, the vector may contain a nucleotide sequence shown in one of the SEQ ID NOs: 101-115.

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

[0215] For example, the vector may further include a promoter located at the 5' end of the polynucleotide encoding CYP4V2 and operably ligated to the polynucleotide encoding CYP4V2.

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

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

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

[0219] For example, the vector may include, in order from 5' to 3': a promoter sequence shown in any of SEQ ID NO: 1 to 12, a polynucleotide sequence encoding CYP4V2 shown in any of SEQ ID NO: 62 to 68, and a polyadenylation signal site sequence shown in any of SEQ ID NO: 17 to 21.

[0220] For example, the vector may contain a nucleotide sequence shown in any of SEQ ID NO:90~92.

[0221] For example, the vector also includes a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide of the code RdCVF.

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

[0223] For example, the vector may include, in order from 5' to 3': a promoter sequence as shown in any of SEQ ID NO: 1 to 12, a polynucleotide sequence encoding RdCVF as shown in any of SEQ ID NO: 69 to 75, and a polyadenylation signal site sequence as shown in any of SEQ ID NO: 17 to 21.

[0224] For example, the vector may contain nucleic acid sequences shown in SEQ ID NO: 96-98.

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

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

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

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

[0229] For example, the vector may include, in order from the 5' to the 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 include, in order from 5' to 3': the promoter, the intron, the polynucleotide encoding CYP4V2 or the polynucleotide encoding RdCVF, and the polyadenylation signal site.

[0231] For example, the vector may include, in order from 5' to 3': 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, and the polyadenylation signal site.

[0232] For example, the promoter may include a nucleic acid sequence shown in any of SEQ ID NO: 1 to 12, the intron may include a nucleic acid sequence shown in any of SEQ ID NO: 13 to 16, the polynucleotide encoding CYP4V2 may include a nucleic acid sequence shown in any of SEQ ID NO: 62 to 68, the polynucleotide encoding RdCVF may include a nucleic acid sequence shown in any of SEQ ID NO: 69 to 75, and the polyadenylation signal site may include a nucleotide sequence shown in any of SEQ ID NO: 17 to 21.

[0233] For example, the polynucleotide into which the intron encoding CYP4V2 is inserted may include the nucleic acid sequence described in SEQ ID NO:116.

[0234] For example, the vector may contain nucleotide sequences shown in SEQ ID NO: 93-95 and 99-100.

[0235] Combination of vectors The present invention also provides a combination of vectors for the treatment, mitigation and / or prevention of diseases or conditions associated with retinal pigment epithelium (RPE) atrophy, comprising a first vector and a second vector, wherein the first vector may contain a polynucleotide encoding CYP4V2 and the second vector may contain a polynucleotide encoding RdCVF.

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

[0237] For example, the first vector may sequentially include a promoter sequence as shown in any of SEQ ID NO:1 to 12 and a polynucleotide sequence encoding CYP4V2 as shown in any of SEQ ID NO:62 to 68, in the 5' to 3' direction.

[0238] For example, the first vector may contain the polynucleotide sequence indicated by SEQ ID NO:117.

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

[0240] For example, the second vector may contain the polynucleotide sequence indicated by SEQ ID NO:120.

[0241] For example, the first vector may, in the 5' to 3' direction, sequentially include a promoter sequence indicated by one of SEQ ID NO: 1 to 12, and a polynucleotide sequence encoding an RdCVF indicated by one of SEQ ID NO: 69 to 75.

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

[0243] For example, the second vector also includes a polyadenylation signal site, the polyadenylation signal site being located at the 3' end of the polynucleotide of the code RdCVF.

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

[0245] For example, the first vector may include, in order from 5' to 3': a promoter sequence as shown in any of SEQ ID NO: 1 to 12, a polynucleotide sequence encoding CYP4V2 as shown in any of SEQ ID NO: 62 to 68, and a polyadenylation signal site sequence as shown in any of SEQ ID NO: 17 to 21.

[0246] For example, the first vector may contain a nucleotide sequence shown in any of SEQ ID NO:90~92.

[0247] For example, the second vector may sequentially include the following in the 5' to 3' direction: a promoter sequence shown in any of the SEQ ID NOs: 1 to 12, a polynucleotide sequence encoding RdCVF shown in any of the SEQ ID NOs: 69 to 75, and a polyadenylation signal site sequence shown in any of the SEQ ID NOs: 17 to 21.

[0248] For example, the second vector may contain a nucleotide sequence shown in any of SEQ ID NO:96~98.

[0249] For example, the first vector and / or the second vector may also contain introns.

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

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

[0252] For example, the first vector may contain a nucleotide sequence shown in any of SEQ ID NO: 93 to 95, and / or the second vector may contain a nucleotide sequence shown in any of SEQ ID NO: 99 to 100.

[0253] For example, the first vector may contain a nucleotide sequence shown in any of SEQ ID NO: 118-119, and / or the second vector may contain a nucleotide sequence shown in any of SEQ ID NO: 121.

[0254] cell The present application further provides cells that may contain the nucleic acid molecules described herein or combinations of the vectors described herein.

[0255] For example, the cell is a cell that expresses the nucleic acid molecule within it.

[0256] For example, the cells may include single-cell offspring. The offspring do not necessarily have to be identical to the primordial mother cell (morphologically or genomically, based on total DNA complementation).

[0257] For example, the cells may further include cells transfected in vitro with the vector described in the present invention.

[0258] For example, the cells may include bacterial cells (e.g., Escherichia 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 in question are from a patient with crystalline retinal degeneration.

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

[0261] For example, the cells can include retinal cells, corneal cells, choroidal cells, lens cells, nerve cells, RPE cells, and 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, nerve cells, and RPE cells can be induced to differentiate from the stem cells.

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

[0264] Pharmaceutical composition The present application also provides a pharmaceutical composition, which can include the nucleic acid molecules described in the present application, the combination of the vectors described in the present application, and / or the cells described in the present application.

[0265] For example, the pharmaceutical composition can also optionally include pharmaceutically acceptable adjuvants.

[0266] For example, the pharmaceutical composition can also include appropriate formulations of one or more (pharmaceutically effective) vectors, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives.

[0267] For example, the acceptable components of the composition are non-toxic to the recipient at the dosage and concentration used.

[0268] For example, the pharmaceutical composition includes, but is not limited to, liquids, frozen and lyophilized compositions.

[0269] For example, the pharmaceutically acceptable adjuvants include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration, and are usually safe, non-toxic, and not otherwise undesirable biologically.

[0270] For example, the pharmaceutical composition may be administered extragastrointestinally, transdermally, intracavitarially, intraarterially, intrathecally and / or intraocularly, or by direct injection into tissue.

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

[0272] For example, the pharmaceutical composition can be used without interruption (or continuously).

[0273] For example, non-stop (or continuous) administration can be performed by a small pump system worn by the patient to measure the amount of medication flowing into the patient's body, as described in W02015 / 036583.

[0274] In this application, the subjects may include humans and non-human animals. For example, the subjects may include, but are not limited to, cats, dogs, horses, pigs, dairy cows, sheep, rabbits, mice, rats, or monkeys.

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

[0276] For example, the aforementioned retinitis pigmentosa includes monocular primary retinitis pigmentosa, quadrant primary retinitis pigmentosa, central or paracentral primary retinitis pigmentosa, achromatic retinitis pigmentosa, punctate retinal degeneration, crystalline retinal degeneration, venous fatty pigment retinitis pigmentosa, parachromophylliteal retinitis pigmentosa, Leber congenital amaurosis, and retinitis pigmentosa of other syndromes.

[0277] For example, the aforementioned retinitis pigmentosa may 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, replication errors, splice site errors, frameshifts, base deletions or insertions, nonsense mutations, polymorphisms (e.g., single nucleotide polymorphisms), premature termination, partial or complete gene deletions of CYP4V2, and CYP4V2 gene mutations associated with unidentified crystalline retinopathy.

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

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

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

[0283] Not intended to be limited by any theory, the following embodiments are intended to illustrate the nucleic acid molecules, methods of preparation, and uses of the present invention, and do not limit the scope of the present invention. The embodiments do not include detailed descriptions of conventional methods, such as methods for constructing vectors and plasmids, methods for inserting protein-coding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous 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 mentioned can be purchased through normal commercial channels. [Examples]

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

[0285] (2) Primer sequences for 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 molecules obtained in step (1) as templates. The amplified products were recovered by gel electrophoresis. Reference sequences are shown in SEQ ID NO:30~37.

[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 recovered by gel electrophoresis. The referenced sequences are shown in SEQ ID NO:38~39. The PCR reaction system (50 μl) is as follows.

[0288] [Table C]

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

[0290] The linked systems (conducted on ice) are as follows:

[0291] [Table D]

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

[0293] (5) On day 1, 293T cells (ATCC, CRL-3216) were divided into 35 mm cell culture dishes, and from day 2 until about 70%, the transfection system was prepared: 2 μg of the plasmid obtained in step (4) was added to 100 μl of serum-free DMEM, 3 μl of PEI was added, and the mixture was homogeneously mixed and allowed to stand for 20 minutes. The resulting transfection system was added to the cell medium, mixed evenly by shaking, placed in a CO2 culture box, and the liquid was changed after 6 hours or overnight.

[0294] (6) After 48 hours, cells were lysed with RIPA lysis solution (Beijing Priele C1053-100), and protein gel electrophoresis was performed. Western blot detected CYP4V2 expression. Antibodies used: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), 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 promoter were all able to achieve CYP4V2 expression. Among these, the CYP4V2-PF promoter, CYP4V2-P3 promoter, CYP4V2-P4 promoter, and CYP4V2-P5 promoter showed relatively good expression effects, while the CYP4V2-P5 sequence was even shorter and dominant.

[0296] Example 2. Verify promoter strength using EGFP as a reporter gene. (1) Promoters EF1a, EFS, and OPEFS were amplified by PCR. The EF1a primer sequences are shown in SEQ ID NO: 40-41, and the EFS primer sequences are shown in SEQ ID NO: 42-43. The OPEFS promoter was based on the general-purpose promoter EFS, and the primers were designed by modifying EFS to OPEFS using two-wheel PCR. The primers used in the first PCR reaction were the 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 the 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) is as follows.

[0297] [Table E]

[0298] (2) The homologous recombination of the amplification products obtained in step (1) (promoter OPEFS, EFS, EF1a nucleic acid molecules, sequences shown in SEQ ID NO: 1~3) is linked 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 linking system (performed on ice) is as follows.

[0299] [Table F]

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

[0301] (3) Following the method of Step (5) of Example 1, 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-laid in a 6-well plate using PEI (Polysciences24765-1), with a cell density of approximately 70%.

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

[0303] As shown in Figure 2, CAG, EF1a, and OPEFS all showed relatively better expression effects compared to EFS, and among them, OPEFS had a relatively short sequence and was even more advantageous.

[0304] Example 3. Verification of the intron enhancement effect (I) Introns were cloned into the 5' non-coding region of the gene.

[0305] (1) A clone of the P5-intron. Based on the CYP4V2-P5 promoter sequence and human β-globin introns, primers were designed and modified by overlap PCR to obtain the P5-intron. 1) PCR amplification was performed using the CYP4V2-P5 promoter sequence as a template, with 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, with 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 recovered, and the target products obtained in 1) and 2) were used as a common template for PCR amplification using the CYP4V2-P5 forward primer (SEQ ID NO: 46, CYP4V2-P5F) and the β-CYP4V2-P5 intron reverse primer. PCR amplification was performed using the system and conditions of Step 2 (1 49). The amplified P5-intron target product was recovered.

[0306] (2) The amplified P5-intron sequence is sequenced, and then homologous recombination is ligated 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 coupling system was left in a 50°C water bath for 40 minutes, 5 μl of the reaction solution was taken and converted into competent cells of transStbl3 Escherichia coli, the bacterial sequence was measured, and the plasmid was extracted using an extraction kit (Omega, D6915-04).

[0309] (II) Introns were cloned into gene coding regions.

[0310] (1) This is a clone of CYP4V2-CDSintron, in which the intron is located between the first and second exons of CYP4V2. Based on the CDS sequence of CYP4V2 and the human β-globin intron, primers were designed and modified using overlap PCR 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 NO: 50 and 51, respectively) and CYP4V2-E2-F / CYP4V2-R (sequences shown in SEQ ID NO: 52 and 53, respectively), yielding PCR products CYP4V2-E1 and CYP4V2-E2~11, respectively.

[0312] 2) Using β-globin introns as a template, PCR amplification was performed using primers Intron-F / Intron-R (sequences shown in SEQ ID NO: 54 and 55, respectively).

[0313] 3) The amplified products were each recovered, and PCR amplification was performed using the obtained target product CYP4V2-E1 (1) and the obtained target product (2) as a common template, using the 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). PCR amplification was performed using the system and conditions of Step (1) of Example 2. The amplified CYP4V2-E1-intron target product was recovered.

[0314] Using the target product CYP4V2-E1-intron obtained in 4)3) and the target products CYP4V2-E2~11 obtained in 1) as a common template, PCR amplification was performed using the CYP4V2-CDS forward primer CYP4V2-Fpr and the CYP4V2-CDS reverse primer CYP4V2-R. PCR amplification was carried out using the system and conditions of Step (1) of Example 2.

[0315] (2) The amplified CDSintron sequence was sequenced.

[0316] (3) The pAV-p5-CYP4V2-P2A-EGFP enzyme was digested at 37°C for 2 hours to obtain a linear vector, which was recovered by gel electrophoresis. The enzyme system is as follows:

[0317] [Table H]

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

[0319] [Table I]

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

[0321] (III) Verification of the enhancement effect when introns are located in the gene's 5' non-coding region and coding region. (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-packed in a 6-well plate using PEI (Polysciences 24765-1) in the method of Step (5) of Example 1, and the cell density was approximately 70%.

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

[0323] (3) After 48 hours, cells were lysed with RIPA lysis solution (Beijing Priele C1053-100), and protein gel electrophoresis was performed. Western blot detected CYP4V2 expression. Antibodies used: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), goat-anti-rabbit (Abclonal, AS014).

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

[0325] Example 4. Selection of Poly A signal site (1) In the pAV-OPEFS-CYP4V2-P2A-EGFP vector obtained in Embodiment 2, SV40 was replaced with BGH, WPRE, WPRE-SV40, and WPRE-BGH, respectively, 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. The gene synthesis and subcloning of this portion were carried out by Beijing Optimiko.

[0326] (2) After sequencing and verifying the vector obtained in step (1), plasmids were extracted using an extraction kit (Omega, D6915-04).

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

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

[0329] (5) After 48 hours, cells were lysed with RIPA lysis solution (Beijing Priele C1053-100), and protein gel electrophoresis was performed. Western blot detected CYP4V2 expression. Antibodies used: anti-CYP4V2 (Atlas, HPA029122), anti-actin (Abclonal, AC026), 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. In each experimental group, CYP4V2 expression was detected compared to the control group.

[0331] Example 5. Construction of an expression vector (1) Construction of pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH (Karna resistance). The ambensyl resistance gene in pAV-OPEFS-CYP4V2-P2A-EGFP-BGH was replaced with a Karna resistance gene, and Suzhou Jin Weizhi was in charge of vector modification. 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), Suzhou Jin Weizhi synthesized the related genes and modified the vector (Karna resistance).

[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), and pAV-p5-intron-CYP4V2-P2A-RdCVF-BGH (expression order shown in SEQ ID NO:113) (Karna-resistant). pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH (Karna-resistant) were enzymatically cleaved at 37°C for 2 hours to obtain linear vectors, which were recovered by gel electrophoresis. The enzyme system is as follows.

[0333] [Table J]

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

[0335] (4) The enzyme-cleaved recovered vector and the linear fragment that requires recombination with it are linked together, and the linking system (performed on ice) is as follows:

[0336] [Table K]

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

[0338] Example 6.293 Examination of expression status of expression vector in T cells (1) The pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH plasmids obtained in Step (1) of Example 5 were transfected into 293T cells (ATCC, CRL-3216) that had been pre-loaded into a 6-well plate using PEI (Polysciences 24765-1) in the manner of Step (5) of Example 1, with a cell density of approximately 70%.

[0339] (2) Cell culture supernatant (sup) was collected after 48 hours, the cells were washed with PBS, and the cells (lys) were lysed using RIPA lysate (Beijing Priele C1053-100). Western blot was used to examine the expression of CYP4V2 and / or RdCVF in the supernatant or cell lysate (lys) of each group. 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. In the blank control group, there was no expression of CYP4V2 or RdCVF in the lysate (lys) and cell culture supernatant (sup) of M group cells. In the pAV-OPEFS-CYP4V2-BGH transfected cell lysate, there was expression of CYP4V2, and in the cell supernatant and lysate of pAV-OPEFS-RdCVF-BGH transfected cells, there was expression of RdCVF, and in the pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH transfected cell lysate, there was expression of CYP4V2, and simultaneously, there was expression of RdCVF in the cell lysate and supernatant.

[0341] Example 7. Examination of expression status of expression vector in ARPE-19 cells. (1) The pAV-OPEFS-CYP4V2-BGH, pAV-OPEFS-RdCVF-BGH, and pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH plasmids obtained in Step (1) of Example 5 were transfected into ARPE-19 cells (ATCC, CRL-2302) that had been pre-loaded into a 6-well plate using PEI (Polysciences 24765-1) in the manner of Step (5) of Example 1, with a cell density of approximately 70%.

[0342] (2) Cell culture supernatant (sup) was collected after 48 hours, the cells were washed with PBS, and the cells (lys) were lysed using RIPA lysate (Beijing Priele C1053-100). Western blot was performed, and the expression of CYP4V2 and / or RdCVF in the supernatant or cell lysate (lys) of each group was examined. 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. No CYP4V2 or RdCVF expression was observed in the lysates and cell culture supernatants of the blank control and mock groups. CYP4V2 was expressed in the lysates of pAV-OPEFS-CYP4V2-BGH transfected cells, and RdCVF was expressed in the supernatant and lysates of pAV-OPEFS-RdCVF-BGH transfected cells. CYP4V2 was expressed in the lysates of pAV-OPEFS-CYP4V2-P2A-RdCVF-BGH transfected cells, and RdCVF was expressed in both the supernatant and lysates.

[0344] Example 8. Viral vector (I) Selection of AAV serotype: Wild-type mice were injected subretinally with AAV2 / 2, AAV2 / 5, AAV2 / 8, and AAV2 / 9 serotype viruses (purchased from Shandong Weizhen Biotechnology Co., Ltd.) containing a GFP reporter. Retinal tissue morphological observation was performed on sections one month later. As shown in Figure 8, the double-headed arrows indicate the expression range of the EGFP reporter gene, and the AAV2 / 8 serotype, which showed relatively good preference in the retina, was selected.

[0345] Building an AAV2 / 8 vector: (1) pAAV-RC5-Amp (purchased from Beijing Ximeijie Technology Co., Ltd., vector sequence shown below: SEQ ID NO: 133) was used as the base for modification. PCR amplified the pAAV-RC5 sequence other than 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 Kana-F / Kana-R primers, as shown in SEQ ID NO: 58-59.

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

[0348] (4) Based on pAAV-RC5-Kana, pAAV-Kana sequences other than RC5-cap were amplified using the PCR method, and the primers used were RC5-Kana-F / RC5-Kana-R, as shown in SEQ ID NO:60-61.

[0349] (5) Using the synthesized RC8-cap sequence (Beijing Optimus, sequence shown in SEQ ID NO:134) and the pAAV-RC8-Kana (i.e., AAV2 / 8) obtained by homologous recombination of the pAAV-Kana linear vector obtained in step (4), the vector sequence 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 carnauba. The specific modification process is as follows:

[0351] 1) The pAAV-helper-Amp (purchased from Beijing Ximeijie Technology Co., Ltd., with the vector sequence shown below: SEQ ID NO: 136) was modified. The pAAV-helper sequences other than the Amp were 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 amplification from pEGFP-N1 using PCR, with Kana-F / Kana-R primers used, indicated by SEQ ID NO: 58-59.

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

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

[0355] (2) Day 1: When the 293T cell volume is 80% to 90%, replace it with 20 ml of complete medium (Gibco, C11965500BT) containing 10% serum (Shanghai Gigay Co., Ltd., FSP500), and perform plasmid transfection. See Table 5 for the specific transfection system.

[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 hours after transfection, 293T cells were digested with pancreatic enzymes in the usual manner and collected in 50 ml centrifuge tubes, washed twice with PBS, centrifuged at 1200 rpm for 5 minutes, aspirated with PBS, and stored in a refrigerator at -80°C.

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

[0361] 2) Cells were resuspended in 2 ml of cell lysate (150 mM NaCl, 50 mM Tris, pH 8.5) per 15 cm petri dish (three cells from a 15 cm petri dish were taken into each 50 ml centrifuge tube, and 6 ml of cell lysate was prepared from these three cells).

[0362] 3) Add half the volume (3 ml) of chloroform to the cell lysate in the centrifuge tube, tighten the tube opening, and shake horizontally on a 37°C rocking bed at 250 rpm for 30 minutes.

[0363] 4) Add 1 / 4 volume (1.5 ml) of 5M sodium chloride to the aqueous phase and mix uniformly. Transfer to a high-speed centrifuge tube and centrifuge at 11000 rpm for 25 minutes.

[0364] 5) The upper aqueous phase was collected, the difficult-to-aspirate portion at the boundary was transferred to a 1.5 ml centrifuge tube, and the mixture was centrifuged at 12000 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 then 10 mM MgCl2, 0.5 mM CaCl2, 5 IU / ml Turbo DNase I, and 25 ug / ml RNase A (reservoir concentration 10 mg / ml) were added.

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

[0366] 7) Add 50% PEG8000 in volume 1 / 4 (2 ml) of the aqueous phase, shake well, and centrifuge on ice for 1 hour at 11000 rpm for 25 minutes.

[0367] 8) Aspirate and discard the supernatant, then centrifuge again for 1 minute to remove the remaining supernatant.

[0368] 9) PBS was added according to the final dissolution volume requirement, the suspension was blown in, and transferred to a 1.5 ml centrifuge tube. A small volume of transfer medium (<200 μl) was added during the transfer to avoid loss of virus due to precipitate adhesion.

[0369] 10) Add 500 μl of chloroform, shake well, and centrifuge at 12000 rpm for 20 minutes.

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

[0371] Example 10. Measurement of viral titer (I) Preparation of the standard product pAAV-EGFP 1. Berg gradient: 1x10 8 copies / 5μl, 1x10 7 copies / 5μl, 1x10 6 copies / 5 μl, 1x10 5 copies / 5μl, 1x10 4 copies / 5μl, 1x10 3 copies / μl, 1x10 2 copies / 5μl.

[0372] 2. Dilution procedure: a. The stock solution was diluted 10-fold and quantified by nano, and subsequent dilutions were performed based on the quantification result. b. The quantified sample was serially diluted to 1 ng / μl. c. The 1 ng / μl sample was diluted to 1x10 8 copies / μl. d. The dilution of 1x10 8 copies / μl was diluted 5-fold to 1x10 8 copies / 5μl. e. Diluted 10-fold to 1x10 2 copies / 5μl.

[0373] (II) Treatment and dilution of the sample 1. 17 μl of DNase 1 + 1 ml of 1x buffer (TransGen Biotech, GD-201-01) at 50 U / ml was prepared.

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

[0375] 3. Mixed uniformly (vortexed for 5 seconds), left at 37 °C for 30 minutes on a PCR device; left at 75 °C for 15 minutes.

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

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

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

[0379] (III) Fluorescence quantitative PCR

[0380] [Table M]

[0381] [Table N]

[0382] [Table O]

[0383] Table 8 shows that the AAV-OPEFS-EGFP and AAV-OPEFS-CYP4V2 virus titers were 1.00E+13vg / ml and 3.06E+13vg / ml, respectively, indicating that they can be used in subsequent infection experiments. The titers of the other viruses used in the examples of this application were all measured using 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 Saibei, CA3102500), and amplified using the Urineasy urine cell amplification kit (Beijing Saibei CA3103200). Reprogramming experiments were performed on cells that reached a fusion degree of 70-80% after 3-4 generations. The reprogramming experiment was performed using the hiPSC reprogramming kit (Beijing Saibei, CA5002002) according to the procedure described in the kit instructions to obtain human IPSCs for the subsequent cell differentiation experiment.

[0385] (2) Preparation of RPE cells. RPE cells were prepared using the method described in the reference (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 25 vials of T. After 5 days, the medium was replaced with 6 ml of 20% serum substitution (Gibco, CAT#A3181502) (Gibco, CAT#10829018), and culture was continued for approximately 20 weeks in 6 ml of serum-free medium (Gibco, CAT#10829018) after 2 days. Oval-shaped, dark-colored mitotic cells, i.e., RPE cells, were obtained. Figure 9 shows renal epithelial cells, IPSC cells, and RPE cells observed under a fluorescence microscope (Life AMF4305) at 4x, 10x, and 10x magnification, respectively, under white light.

[0386] (3) Equal amounts of human iPSC differentiated RPE cells are subjected to a dose gradient (MOI=0, 1x10) of wrapping virus containing pAV-OPEFS-EGFP-BGH or pAV-OPEFS-CYP4V2-BGH. 3 , 1x10 4 , 1x10 5 They infected the patient using ).

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

[0388] As shown in Figures 10A-10D, with increasing pAV-OPEFS-EGFP-BGH viral titers, EGFP expression gradually increased, the expression levels of inflammatory cytokines TNF-α and NLRP3 gradually increased, and cell mortality gradually increased. With increasing pAV-OPEFS-CYP4V2-BGH viral titers, CYP4V2 expression increased, the expression levels of inflammatory cytokines TNF-α and NLRP3 increased, and cell mortality increased. Since high titer AAV viruses are cytotoxic to RPE cells differentiated from human iPSCs, 1x10⁻⁶ 4 The following dosages can be selected and used in subsequent experiments with RPE cells differentiated from human iPSCs.

[0389] Example 12. Effects of iPSC-differentiated RPE cells from BCD patients with different viral infections. (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); D: pAV-p5-intron-EGFP-BGH.

[0390] (2) MOI = 1 x 10 4 Equivalent doses of viruses A, B, C, D, and virus A + virus B (hereinafter referred to as group E) were used to infect RPE cells differentiated from iPSCs of BCD patients (genotype CYP4V2: c.802-8_810 del17bpinsGC homozygous mutation). 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 Ethics Committee of Peking University Third Hospital approved all aspects of this study and obtained informed consent for sample collection from the subjects (or their legal guardians). Patient urine samples were processed within one hour of collection using the method described in Example 11.

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

[0392] (4) Cells were collected and the culture supernatant (sup) was taken; after washing the cells with PBS, the cells were lysed with RIPA lysis solution (Beijing Priele C1053-100) to obtain cell lysate (lys), and the protein expression levels were tested and measured using Western. 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, which were not infected with the virus, 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 did express EGFP. CYP4V2 expression was detectable in the lysate of RPE cells differentiated from BCD patient iPSCs infected with virus group A, and RdCVF expression was detectable in the supernatant and lysate of RPE cells differentiated from BCD patient iPSCs infected with virus group B. CYP4V2 expression was detected in the lysate of RPE cells differentiated from BCD patient iPSCs infected with viruses group C and E, and RdCVF expression was simultaneously detected in the supernatant and lysate.

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

[0395] Example 13. Promoter selection for virally transfected 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-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 Bai'aosaitu Gene Biotechnology Co., Ltd.) were injected into the subretinal space at 1 month of age, and both promoter viruses were injected into 10 mice each. 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, the pupils of the mice were dilated with 1% atropine, and then 80 mg / kg ketamine + 8 mg / kg tolthiazine were injected intraperitoneally.

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

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

[0399] 4) A small hole is first made in the flat part of the ciliary body of the mouse eyeball using an insulin needle. After piercing the hole with the needle of a microsyringe, the needle enters the vitreous cavity of the mouse eyeball. At this time, an appropriate amount of 2% hydroxymethylcellulose is added to the mouse eyeball so that the fundus of the mouse eye can be seen under a microscope. The needle is then inserted under the retina on the opposite side, avoiding the lens, and the virus containing fluorescein sodium is slowly injected. The injection volume for each eye is 10, and the virus concentration is 1 x 10⁻¹⁶. 9 Then, they decided whether or not to inject fluorescein sodium into the subretinal space as an indicator.

[0400] After the surgery, the surface of the eyeball was washed with saline solution, and the mouse was placed in a cage to wait for it to wake up.

[0401] (2) Observe the crystalline deposition situation by taking fundus photographs, 1) Blank controls (KOs) were obtained, and mice were 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). The eyes of these mice were dilated with 1% atropine, and they were anesthetized by intraperitoneal injection of 80 mg / kg ketamine + 8 mg / kg tolthiazine A.

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

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

[0404] 4) After imaging, 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 containing different promoters (n=5). Figures 12 and 13 showed that fundus crystal deposition improved after injection of viruses containing different promoters.

[0406] (3) Morphological observation of retinal tissue: 1) Fixed dehydration: Mice were sacrificed with cervical vertebral dislocation, their eyeballs were removed, and the mouse eyeballs were placed in a 1.5 ml EP tube, with 1 ml of 4% paraformaldehyde added, and 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 and dehydrated, causing the eyeballs to sink.

[0407] 2) The mouse eyeballs were placed in a 1.5 ml EP tube containing cryoembryo embedding medium (OCT). The mouse's eyes were positioned with tweezers to face straight ahead, and the EP tube was placed over them. The tube was then placed in liquid nitrogen until completely frozen. After freezing, sections were made, and the sections were 7 μm thick. After fixing with acetone at 4°C for 10 minutes, the sections were stored at -80°C.

[0408] 3) Remove the slices, let them return to room temperature, and then wash them three times with PBS for 5 minutes each.

[0409] 4) Wipe the slide glass with a tissue-free area, add 40 μl of occlusion solution (5% donkey serum) to each section, and occlude at room temperature for 1 hour.

[0410] 5) Dilute one antibody with 5% donkey serum, add approximately 40 μl of antibody activator dropwise, completely cover mouse eye tissue, and leave overnight at 4°C. The primary antibody used in this experiment was CYP4V2 (1:50, purchased from Sigma).

[0411] 6) Remove the slide glass and wash it three times with PBS 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 activator was added to each eye tissue of the mouse. The eye tissue was then completely covered and incubated at room temperature for 1 hour.

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

[0414] 9) A fluorescent anti-quenching capsule was dropped onto a glass slide, covered with a coverslip, and stored in a light-shielded place at -20°C.

[0415] 10) Observations were made and photographs were taken using a Nikon A1 laser confocal microscope equipped with NIS-Elements C software.

[0416] Immunofluorescence results in Figure 14 show that BCD mice injected with both promoter A virus and promoter B virus were able to express CYP4V2 (double arrows in the figure indicate the range).

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

[0418] The crystal deposition status is as shown in the imaging results in Figure 15. Figure 16 shows the number of crystals in the fundus of BCD mice injected with 1E8vg and 1E9vg viruses, statistically displayed in the same area as BCD mice that were not injected with the virus. BCD mice injected with 1E8vg and 1E9vg viruses showed a decrease in the number of crystals deposited compared to BCD mice that were not injected with the virus, with the decrease being more pronounced in BCD mice injected with 1E9vg virus. BCD mice injected with 1E10vg virus did not show a significant improvement in crystal deposition and were severely damaged.

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

[0420] Figure 17 shows that injection of 1E10vg virus increased the expression of inflammatory cytokines TNF-α, IFN-γ, and NLRP3, explaining the toxic side effects in BCD mice at 1E10vg viral doses, with 1E9vg being better than 1E8vg. Therefore, follow-up experiments were selected at viral doses below 1E10vg.

[0421] Example 15. Effects of different virus transfections on BCD mice (I) Effects of promoter virus infection in p5-intron BCD mice (1) The viruses were packaged using 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. Viruses A, B, and C, and virus A+virus B (A and B were each 5E8vg) were administered at 1E9vg doses. The KO group was used as a blank control and remained untreated. Subretinal injection was performed according to the method in Example 13.

[0423] (3) Fundus photographs were taken using the method of Example 13 to observe the crystalline deposition status. Figure 18 shows the results of fundus photography of BCD mice after subretinal injection therapy using different viruses, showing crystal deposition. Figure 19 shows the statistical results (n=5) of the number of fundus crystals in BCD mice after subretinal injection therapy using different viruses. The results indicate that the decrease in the number of crystals was more significant in mice from virus A+B group and virus C group. No clear improvement was observed in the fundus crystal status of mice from virus B group.

[0424] (4) Somatic retinal function test: Electroretinography (ERG) 1) Mouse dark adaptation: The mice were allowed to adapt to darkness for at least 16 hours, after which all operations were performed under dark red light.

[0425] 2) Mouse anesthesia: 80 mg / kg ketamine + 8 mg / kg tolthiazine were administered by intraperitoneal injection.

[0426] 3) After anesthesia was completed, the mice's pupils were dilated with 1% atropine under dark red light illumination, and the mice were taped to the front of the Espion E2 animal experiment platform for visual electrophysiology, with both eyes aligned and fully exposed. A ground electrode needle was inserted at the base of the mouse's tail, and a reference electrode needle was inserted into the mouse's mandible. Two ring-shaped recording electrodes were placed in the electrode holders of the animal experiment platform, their angles adjusted so that each lightly contacted the central tip of the left and right corneas, and an appropriate amount of 2% hydroxymethylcellulose was added to improve the contact effect between the ring electrodes and the cornea.

[0427] 4) The mouse number and lunar phase information were entered into the Espion E2 computer system, and the program was executed. The intensity of the dark-adapted flash for each row was 0.003, 0.01, 0.1, 1, 3, 10, and 100 cd·s / m. 2 (Background luminance is 0 cd·s / m) 2 The stimulation interval was 15 seconds, and the average value of three ERG signals was recorded. The light-adapted flash intensity was 3, 10, 30, and 100 cd·s / m². 2 (Adjustment time 5 minutes, background light intensity 30 cd·s / m) 2 The stimulation interval was 15 seconds, and the average value of five ERG signals was recorded. After the program finished, the execution results were automatically saved, and the results were compiled using GraphPad Prism.

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

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

[0430] Figure 21 shows immunofluorescence staining of BCD mice after subretinal injection therapy with different viruses. The results showed that virus group A mice expressed CYP4V2 (indicated by double arrows), and virus group B mice expressed RdCVF (indicated by a single arrow). Expression of both CYP4V2 and RdCVF was detected in both virus group A+B and virus group C.

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

[0432] 2) The mouse eyeballs were transferred to an EP tube containing 1.5 ml of 4% paraformaldehyde, which had been fixed for 1 hour.

[0433] 3) Under a stereomicroscope (Olympus, SZ61-SET), the anterior segment of the mouse eyeball was removed, separating the neuroretinal layer from the RPE layer, and the RPE layer was cut into four sections.

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

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

[0436] 6) The RPE section was placed in one of the holes of a 96-perforated plate, phalloidin working solution diluted in PBS at a ratio of 1:200 was added, and the mixture was incubated at room temperature for 1 hour.

[0437] 7) The RPE was washed three times with PBS for 5 minutes each time, then spread flat on a glass slide, a small amount of the tablet was placed in it, the tablet was wrapped in a coverslip, and observed under a Nikon fluorescence microscope.

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

[0439] Figure 22 shows the results of phalloidin-labeled F-actin staining, which did not show a significant change in group B compared to the KO group. This indicates that the hexagonal morphology of RPE cells in virus A+B and C groups was more regular and tightly arranged than in the other three groups. Figure 23 statistically shows that the number of RPE cells was counted over the same area, and that the number of RPE cells in the same area for virus A+B and virus C groups was greater than that for virus A group (n=5).

[0440] Comprehensive observation of crystalline deposition, electroretinography (ERG), and RPE cell count and morphology revealed that RdCVF (group B) alone did not improve crystalline deposition, ERG, RPE cell count, or morphology in BCD mice. Co-expression of RdCVF and CYP4V2 (groups A+B and C) showed that sole expression of CYP4V2 significantly enhanced the improvement in crystalline deposition, ERG, RPE cell count, and morphology in BCD mice, indicating that co-expression of CYP4V2 and RdCVF had a clearer synergistic effect than sole expression of CYP4V2 or ERG.

[0441] (II) ERG effect in virus-infected BCD mice without post-promoter introns (1) The viruses were packaged using 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. Viruses A, B, and C, and virus A+virus B (A and B were each 5E8vg) were administered at 1E9vg doses. The KO group was used as a blank control and remained untreated. Subretinal injection was performed according to the method in Example 13.

[0443] (3) An electroretinogram (ERG) is 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 different viruses described above. The ERG waveform of the mice with virus group B showed no significant change, while the ERG waveform of the mice with virus group A, virus A+B, and virus group C all increased (p<0.05), and moreover, virus group A+B and virus group C were superior to virus group A (p<0.05).

[0444] (III) ERG effect in p1 promoter virus-infected BCD mice (1) The viruses were packaged using 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. Viruses A, B, and C, and virus A+virus B (A and B were each 5E8vg) were administered at 1E9vg doses. The KO group was used as a blank control and remained untreated. Subretinal injection was performed according to the method in Example 13.

[0446] (3) An electroretinogram (ERG) is 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 the different viruses described above. The ERG waveform of the mice with virus group B showed no significant change, while the ERG waveform of the mice with virus group A, virus A+B, and virus group C all increased (p<0.05), and moreover, virus group A+B and virus group C were superior to virus group A (p<0.05).

[0447] (IV) ERG effect in WPRE-SV40 poly A signaling site virus-infected BCD mice (1) The viruses were packaged using 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 shown in 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. Viruses A, B, and C, and virus A+virus B (A and B were each 5E8vg) were administered at 1E9vg doses. The KO group was used as a blank control and remained untreated. Subretinal injection was performed according to the method in Example 13.

[0449] (3) An electroretinogram (ERG) is 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 different viruses described above. The ERG waveform of the mice with virus group B showed no significant change, while the ERG waveform of the mice with virus group A, virus A+B, and virus group C all increased (p<0.05), and moreover, virus group A+B and virus group C were superior to virus group A (p<0.05).

[0450] (V) Efficacy of virus-infected BCD mice that do not contain signaling sites (1) The viruses were packaged using 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. Viruses A, B, and C, and virus A+virus B (A and B were each 5E8vg) were administered at 1E9vg doses. The KO group was used as a blank control and remained untreated. Subretinal injection was performed according to the method in 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 different viruses described above. Compared to the control, there was no significant change in the ERG waveform of the virus B group mice, while the ERG waveform of the virus A group, virus A+B group mice, and virus C group mice all increased, and the virus A+B group and virus C group were superior to the virus A group (p<0.05).

[0453] The details provided herein are for illustrative purposes and examples only and are not intended to limit the scope of the appended claims. Various modifications of the embodiments described herein will be obvious to those skilled in the art and remain within the scope of the appended claims and equivalent methods.

Claims

1. A vector comprising, in order from 5' to 3', a promoter, a polynucleotide encoding CYP4V2, and a polyadenylation signal site, wherein the promoter is operably ligated to the polynucleotide encoding CYP4V2 and is a CAG promoter. The CAG promoter includes the polynucleotide sequence shown in SEQ ID NO: 4, The amino acid sequence of CYP4V2 includes the amino acid sequence shown in SEQ ID NO:

76. The polyadenylation signal region includes the nucleic acid sequence shown in SEQ ID NO: 17 and does not include the WPRE signal region. vector.

2. A vector comprising, in order from 5' to 3', a promoter, a polynucleotide encoding CYP4V2, and a polyadenylation signal site, wherein the promoter is operably ligated to the polynucleotide encoding CYP4V2 and is a CAG promoter. The CAG promoter includes the polynucleotide sequence shown in SEQ ID NO: 4, The amino acid sequence of CYP4V2 includes the amino acid sequence shown in SEQ ID NO:

76. The polyadenylation signal region includes the nucleic acid sequence shown in SEQ ID NO:

17. The vector according to claim 1.

3. The vector according to claim 1 or 2, 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.

4. (1) to (8) below: (1) Plasmid vector, (2) A viral vector, (3) AAV virus vector, (4) The rAAV vector, (5) Ocular tissue affinity AAV vector, (6) One or more selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, DJ, DJ / 8, and Rh10. (7) One or more selected from the group consisting of AAV2 / 2, AAV2 / 5, AAV2 / 8, and AAV2 / 9, (8) AAV2 / 2 or AAV2 / 8, A vector according to any one of claims 1 to 3, which is any one of the above.

5. The vector according to claim 4, which is AAV2 / 8.

6. A cell comprising the vector according to any one of claims 1 to 5.

7. A pharmaceutical composition comprising a vector according to any one of claims 1 to 5 and / or a cell according to claim 6, and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7 for the treatment, relief, and / or prevention of a disease or condition associated with retinal pigment epithelium (RPE) atrophy in a subject.

9. The pharmaceutical composition according to claim 8, wherein the disease or condition is crystalline retinal degeneration.

10. The pharmaceutical composition according to claim 8, wherein the subject is a human being.

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