阅读说明：本技术 一种分离的核酸分子及其用途 (Isolated nucleic acid molecule and application thereof ) 是由 杨丽萍 陈邵宏 贾睿璇 张天赋 张凡 曾巧莉 和赛超 史天永 郝丹丹 姜尚伟 裴 于 2020-08-07 设计创作，主要内容包括：本申请涉及一种分离的核酸分子,其包括编码CAG启动子的核苷酸序列、编码第一蛋白的核苷酸序列、编码第二蛋白的核苷酸序列和编码BGH信号位点的核苷酸序列,其中所述第一蛋白和第二蛋白各自不同地选自下组：CYP4V2蛋白和RdCVF蛋白,其中编码所述CYP4V2蛋白的核苷酸序列包含SEQ ID NO:168的核苷酸序列,编码所述RdCVF蛋白的核苷酸序列包含SEQ ID NO:167的核苷酸序列。本申请还涉及所述核酸分子在制备用于治疗、缓解和/或预防与视网膜色素上皮(RPE)萎缩相关的疾病或病症的药物中的用途。(The present application relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a first protein, a nucleotide sequence encoding a second protein and a nucleotide sequence encoding a BGH signal site, wherein the first and second proteins are each differently selected from the group consisting of: CYP4V2 protein and RdCVF protein, wherein the nucleotide sequence for coding the CYP4V2 protein comprises a nucleotide sequence of SEQ ID NO:168, and the nucleotide sequence for coding the RdCVF protein comprises a nucleotide sequence of SEQ ID NO: 167. The application also relates to the use of said nucleic acid molecule for the preparation of a medicament for the treatment, alleviation and/or prevention of a disease or disorder associated with atrophy of the Retinal Pigment Epithelium (RPE).)

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a first protein, a nucleotide sequence encoding a second protein and a nucleotide sequence encoding a BGH signal site, wherein the first and second proteins are each differently selected from the group consisting of: CYP4V2 protein and RdCVF protein, wherein the nucleotide sequence for coding the CYP4V2 protein comprises a nucleotide sequence of SEQ ID NO:168, and the nucleotide sequence for coding the RdCVF protein comprises a nucleotide sequence of SEQ ID NO: 167.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises 1 or more nucleotide sequences encoding a CAG promoter comprising the nucleotide sequence of SEQ ID NO 165.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises more than 1 nucleotide sequence encoding a BGH signal site, said BGH signal site encoding nucleotide sequence comprising the nucleotide sequence of SEQ ID NO. 18.

4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence as set forth in any one of SEQ ID NO 174 and 178.

5. The nucleic acid molecule according to claim 1, wherein the nucleotide sequence encoding the signal peptide comprises the nucleotide sequence as set forth in any one of SEQ ID NO 169-173.

6. The nucleic acid molecule according to claim 1, which comprises the nucleotide sequence as set forth in any one of SEQ ID NO 156-161.

7. An injectable kit comprising a vector carrying the nucleic acid molecule of any one of claims 1-6 and a pharmaceutically acceptable excipient.

8. The kit of claim 7, wherein the pharmaceutically acceptable excipient is suitable for subretinal injection.

An AAV viral vector comprising the nucleic acid molecule of any one of claims 1-6.

10. Use of a viral vector carrying a nucleic acid molecule in the manufacture of a medicament for the treatment, alleviation and/or prevention of a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy, wherein the nucleic acid molecule comprises a nucleic acid molecule according to any one of claims 1-6.

11. The use of claim 10, wherein the disease or disorder comprises crystalline retinal degeneration.

Technical Field

The application relates to the field of biomedicine, in particular to an isolated nucleic acid molecule and application thereof.

Background

Crystalline retinal degeneration (BCD) is a rare disease of retinal degeneration whose symptoms mainly include crystals in the cornea (clear cover), fine, yellow or white crystalline deposits deposited in the light-sensitive tissues of the retina, and progressive atrophy of the retina, choroidal capillaries and choroid. The deposits can damage the retina, resulting in a gradual loss of vision. People with crystalline retinal degeneration typically begin to perceive vision problems at their teens or twenties, and the vision problems in each eye may worsen at different rates, even though the severity and progression of symptoms in the same household vary widely among individuals, but most patients become blind at their forties or fifties. It is estimated that 1 out of every 67,000 people worldwide suffer from crystalloid retinal degeneration, which is more common among east asians, especially china and japanese.

The existing research shows that BCD is autosomal recessive genetic disease and is caused by CYP4V2 gene mutation, and a plurality of gene mutation sites of CYP4V2 are found in the gene research of BCD patients at home and abroad at present. The CYP4V2 gene is one of the proteins in cytochrome P450 superfamily, and the protein coded by the CYP4V2 gene is involved in the process of fatty acid metabolism, and the CYP4V2 gene mutation in the crystal-like retinal degeneration is generally considered to destroy the function of the enzyme involved in the fatty acid metabolism, thereby influencing the lipolysis. However, it is unclear how CYP4V2 causes the specific signs and symptoms of BCD. The current treatment of BCD is mainly referred to a treatment method of Retinitis Pigmentosa (RP), and although the research on CYP4V2 gene mutation provides possibility for future gene therapy, no effective treatment method is available at present.

Disclosure of Invention

The present application provides an isolated nucleic acid molecule and uses thereof. The kit described herein has at least one beneficial effect selected from the group consisting of: 1) the expression level of CYP4V2 protein and/or RdCVF can be obviously improved; 2) effectively improving the function of retina; 3) prevention and/or treatment of BCD; 4) effectively reduces the lipid deposition of the RPE cells with the CYP4V2 gene mutation; 5) improving retinal function in a BCD patient; 6) improve the RPE cell morphology of BCD patients and maintain the RPE cell number. The present application also provides viral vectors comprising specific sequences and their use in the treatment, amelioration and/or prevention of diseases or disorders associated with atrophy of the Retinal Pigment Epithelium (RPE).

In one aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a first protein, a nucleotide sequence encoding a second protein and a nucleotide sequence encoding a BGH signal site, wherein the first and second proteins are each differently selected from the group consisting of: CYP4V2 protein and RdCVF protein, wherein the nucleotide sequence for coding the CYP4V2 protein comprises a nucleotide sequence of SEQ ID NO:168, and the nucleotide sequence for coding the RdCVF protein comprises a nucleotide sequence of SEQ ID NO: 167.

In certain embodiments, the nucleic acid molecule comprises 1 or more nucleotide sequences encoding a CAG promoter comprising the nucleotide sequence of SEQ ID No. 165.

In certain embodiments, the nucleic acid molecule comprises more than 1 nucleotide sequence encoding a BGH signal site, which comprises the nucleotide sequence of SEQ ID No. 18.

In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence set forth in any one of SEQ ID NO 174-178.

In certain embodiments, the nucleotide sequence encoding a signal peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO 169-173.

In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NO 156-161.

In another aspect, the present application provides a kit for injection comprising a vector carrying a nucleic acid molecule as described herein and a pharmaceutically acceptable excipient.

In certain embodiments, the pharmaceutically acceptable excipient is suitable for subretinal injection.

In another aspect, the present application provides an AAV viral vector comprising a nucleic acid molecule described herein.

In another aspect, the present application provides the use of a viral vector carrying a nucleic acid molecule in the manufacture of a medicament for the treatment, alleviation and/or prevention of a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy, wherein the nucleic acid molecule comprises a nucleic acid molecule as described herein.

In certain embodiments, the disease or disorder comprises crystalline retinal degeneration.

The present application provides the use of CYP4V2 and RdCVF in the manufacture of a medicament for the treatment, alleviation and/or prevention of diseases or disorders associated with atrophy of the Retinal Pigment Epithelium (RPE).

In certain embodiments, the disease or disorder comprises crystalline retinal degeneration.

In certain embodiments, the CYP4V2 is human CYP4V 2.

In certain embodiments, the CYP4V2 comprises the amino acid sequence set forth in any of SEQ ID NOS 76-82.

In certain embodiments, the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence set forth in any one of SEQ ID NOS 62-68.

In certain embodiments, the RdCVF is a human RdCVF.

In certain embodiments, the RdCVF comprises the amino acid sequence set forth in any one of SEQ ID NOs 83-89.

In certain embodiments, wherein the polynucleotide encoding RdCVF comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75.

In certain embodiments, the medicament comprises a polynucleotide encoding the CYP4V2 and a polynucleotide encoding the RdCVF.

In certain embodiments, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are on different vectors.

In certain embodiments, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are on the same vector.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector is a viral vector, wherein the viral vector comprises an AAV vector.

In certain embodiments, the vector further comprises a promoter located 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

In certain embodiments, the vector further comprises a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

In certain embodiments, the promoter comprises a nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

In certain embodiments, the vector further comprises a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

In certain embodiments, the vector further comprises a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

In certain embodiments, the vector further comprises a polynucleotide encoding a self-cleaving peptide, the polynucleotide encoding a self-cleaving peptide being located between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

In certain embodiments, the self-cleaving peptide comprises P2A.

In certain embodiments, the polynucleotide encoding a self-cleaving peptide comprises a nucleotide sequence set forth in any one of SEQ ID NOs 22-25.

In certain embodiments, the vector comprises, in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding CYP4V2, the polynucleotide encoding a self-cleaving peptide, the polynucleotide encoding an RdCVF, and the polyadenylation signal site.

In certain embodiments, the vector comprises, in order in the 5 'to 3' direction: said promoter, said polynucleotide encoding CYP4V2, said polyadenylation signal site; alternatively, the vector comprises in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding RdCVF, the polyadenylation signal site.

In certain embodiments, the vector further comprises an intron.

In certain embodiments, the intron comprises a nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

In certain embodiments, the intron is located in the polynucleotide encoding CYP4V2, or, alternatively, is located 5' to the polynucleotide encoding CYP4V 2.

In certain embodiments, the intron is located in the RdCVF-encoding polynucleotide, or, alternatively, is located 5' to the RdCVF-encoding polynucleotide.

In certain embodiments, the vector comprises the nucleotide sequence set forth in any one of SEQ ID NOS 90-116.

The present application also provides a vector combination for treating, ameliorating and/or preventing a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy, comprising a first vector comprising a polynucleotide encoding CYP4V2 and a second vector comprising a polynucleotide encoding RdCVF.

In certain embodiments, the disease or disorder comprises crystalline retinal degeneration.

In certain embodiments, the CYP4V2 is human CYP4V 2.

In certain embodiments, the CYP4V2 comprises the amino acid sequence set forth in any of SEQ ID NOS 76-82.

In certain embodiments, the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence set forth in any one of SEQ ID NOS 62-68.

In certain embodiments, the RdCVF is a human RdCVF.

In certain embodiments, the RdCVF comprises the amino acid sequence set forth in any one of SEQ ID NOs 83-89.

In certain embodiments, the RdCVF-encoding polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector is a viral vector, wherein the viral vector comprises an AAV vector.

In certain embodiments, the first vector further comprises a promoter located 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

In certain embodiments, the second vector further comprises a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

In certain embodiments, the promoter comprises a nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

In certain embodiments, the first vector further comprises a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

In certain embodiments, the second vector further comprises a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

In certain embodiments, the first vector comprises, in order in the 5 'to 3' direction: said promoter, said polynucleotide encoding CYP4V2, said polyadenylation signal site; and/or, the second vector comprises in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding RdCVF, the polyadenylation signal site.

In certain embodiments, the first vector and/or the second vector further comprises an intron.

In certain embodiments, the intron comprises a nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

In certain embodiments, the intron is located in the polynucleotide encoding CYP4V2, or, alternatively, is located 5' to the polynucleotide encoding CYP4V 2.

In certain embodiments, the intron is located in the RdCVF-encoding polynucleotide, or, alternatively, is located 5' to the RdCVF-encoding polynucleotide.

In certain embodiments, the first vector comprises a nucleotide sequence set forth in any one of SEQ ID NOs 90-95; and/or the second vector comprises a nucleotide sequence shown in any one of SEQ ID NO 96-100.

The present application also provides one or more isolated nucleic acid molecules comprising a) a polynucleotide encoding CYP4V2 and b) a polynucleotide encoding RdCVF.

In certain embodiments, the CYP4V2 is human CYP4V 2.

In certain embodiments, the CYP4V2 comprises the amino acid sequence set forth in any of SEQ ID NOS 76-82.

In certain embodiments, the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence set forth in any one of SEQ ID NOS 62-68.

In certain embodiments, the RdCVF is a human RdCVF.

In certain embodiments, the RdCVF comprises the amino acid sequence set forth in any one of SEQ ID NOs 83-89.

In certain embodiments, the RdCVF-encoding polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75.

In certain embodiments, the isolated nucleic acid molecule further comprises a promoter located 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

In certain embodiments, the isolated nucleic acid molecule further comprises a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

In certain embodiments, the promoter comprises a nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

In certain embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

In certain embodiments, the isolated nucleic acid molecule further comprises a polynucleotide encoding a self-cleaving peptide positioned between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

In certain embodiments, the self-cleaving peptide comprises P2A.

In certain embodiments, the polynucleotide encoding a self-cleaving peptide comprises a nucleotide sequence set forth in any one of SEQ ID NOs 22-25.

In certain embodiments, the isolated nucleic acid molecule further comprises a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

In certain embodiments, the isolated nucleic acid molecule further comprises a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

In certain embodiments, the isolated nucleic acid molecule comprises, in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding CYP4V2, the polynucleotide encoding a self-cleaving peptide, the polynucleotide encoding an RdCVF, and the polyadenylation signal site.

In certain embodiments, the isolated nucleic acid molecule further comprises an intron.

In certain embodiments, the intron comprises a nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

In certain embodiments, the intron is located in the polynucleotide encoding CYP4V2, or, alternatively, 5' to the polynucleotide encoding CYP4V 2.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NO 101-115.

The present application also provides a vector comprising the isolated nucleic acid molecule described herein.

In certain embodiments, the vector is a viral vector.

In certain embodiments, the viral vector comprises an AAV vector.

The present application also provides a cell comprising a nucleic acid molecule described herein or a vector described herein.

The present application also provides a pharmaceutical composition comprising an isolated nucleic acid molecule described herein, a vector described herein, and/or a cell described herein.

The present application also provides the use of the nucleic acid molecules described herein, the vectors described herein, the cells described herein for the preparation of a medicament for the treatment, alleviation and/or prevention of a disease or disorder associated with atrophy of the Retinal Pigment Epithelium (RPE).

In certain embodiments, the disease or disorder comprises crystalline retinal degeneration.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the expression effect of the different CYP4V2 promoters in the present application.

FIG. 2 shows the expression effect of CAG, EF1a, OPEFS and EFS promoters in the present application.

FIGS. 3 and 4 show the expression enhancement of the intron linked after the promoter in the present application.

FIG. 5 shows the effect of selecting different PolyA signal sites on the expression of the gene of interest in the expression vectors of the present application.

FIG. 6 shows the expression of CYP4V2, an expression vector of RdCVF gene in 293T cells after insertion of a signal peptide into the RdCVF gene.

FIG. 7 shows the expression of the expression vector containing CYP4V2, RdCVF gene in 293T cells.

FIG. 8 shows the expression of the expression vector containing CYP4V2, RdCVF gene in ARPE-19 cells.

FIG. 9 shows the infection of mouse retinas by AAV viruses of different serotypes in the present application, and the double arrows indicate the expression range of EGFP reporter gene.

FIG. 10 shows the morphology of renal epithelial cells, IPSC cells, and RPE cells observed by fluorescence microscopy during the preparation of RPE cells in the present application.

Fig. 11A shows the effect of virus titer on the death of human iPSC-differentiated RPE cells, 11B and 11C show the effect of virus titer on the expression of inflammatory factors NLRP3 and TNF- α in human iPSC-differentiated RPE cells, respectively, and 11D shows the expression of the target gene in human iPSC-differentiated RPE cells under different virus titer conditions.

Fig. 12 shows the expression of CYP4V2, RdCVF after infection of human iPSC differentiated RPE cells by different viruses in this application.

FIG. 13 shows the effect of virus treatment of BCD mice containing different promoters and CYP4V2 genes on fundus crystalloid deposition in the present application.

FIG. 14 shows the statistical results of the effect of virus-treated BCD mice containing different promoters and CYP4V2 genes on fundus crystallography, as described herein.

FIG. 15 shows the results of immunofluorescent staining for CYP4V2 in BCD mice treated with the virus containing the different promoters and CYP4V2 gene of the present application.

FIG. 16 shows the effect of different doses of the virus comprising the CYP4V2 gene in the present application on fundus crystalloid deposition in BCD mice treated with the virus.

FIG. 17 shows the statistical results of the effect of different doses of the virus comprising the CYP4V2 gene on the deposition of fundal crystals after treatment of BCD mice with the virus according to the present application.

FIG. 18 shows the effect of different doses of the virus comprising the CYP4V2 gene on the expression levels of the inflammatory factors TNF- α, IFN- γ, NLRP3 in BCD mice treated with the virus of the present application.

FIG. 19 shows the effect of different viruses on the deposition of fundal crystals after treatment of BCD mice in this application.

FIG. 20 shows the statistical results of the effect of different viruses on the deposition of fundal crystals after treatment of BCD mice in this application.

FIG. 21 shows the effect on retinal function (electroretinogram (ERG)) after treatment of BCD mice with different viruses in this application.

FIG. 22 shows the results of immunofluorescent staining for CYP4V2, RdCVF following treatment of BCD mice with different viruses as described herein.

FIG. 23 shows the effect of different viruses on RPE cell number and morphology after treatment of BCD mice in the present application.

FIG. 24 shows the statistical results of the effect on the number of RPE cells after treatment of BCD mice with different viruses in the present application.

FIG. 25 shows the effect on retinal function (electroretinogram (ERG)) of BCD mice treated with different viruses containing no intron after the promoter according to the present application.

FIG. 26 shows the effect of the p1 promoter on retinal function (electroretinogram (ERG)) after treatment of BCD mice with different viruses in this application.

FIG. 27 shows the effect of different viruses at the WPRE-SV40 poly A signal site on retinal function (electroretinogram (ERG)) after treatment of BCD mice in the present application.

FIG. 28 shows the effect on retinal function (electroretinogram (ERG)) of BCD mice treated with different viruses that did not contain a signal site as described herein.

FIG. 29 shows the effect on retinal function (electroretinogram (ERG)) after treatment of BCD mice with different viruses that increase the signal peptide of the present application.

FIGS. 30-31 show schematic structural diagrams of nucleic acid molecules described herein.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

The present application is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized synthetically.

In the present application, the term "CYP 4V 2" generally refers to a protein that is member 2 of the cytochrome P450 family 4 subfamily V. Cytochrome P450, also known as cytochromeP450 or CYP450, generally refers to a family of heme proteins, belonging to the class of monooxygenases, involved in the metabolism of endogenous substances or exogenous substances including drugs, environmental compounds. According to the degree of homology of amino acid sequences, the members thereof are sequentially divided into three classes, namely families, subfamilies and enzyme individuals. The cytochrome P450 enzyme system may be abbreviated CYP, wherein families are indicated by arabic numerals, subfamilies are indicated by capital english letters, and enzyme individuals are indicated by arabic numerals, such as CYP4V2 in this application. The human CYP4V2 gene (HGNC: 23198) is 19.28kb in length, is located at 4q35, has 11 exons, and plays an important role in fatty acid metabolism (Kumar S., Bioinformation, 2011,7: 360-. CYP4V2 is expressed in almost all tissues, but at high levels in the retina and retinal pigment epithelium, and at slightly lower levels in the cornea, tissues, mutations in the CYP4V2 gene may lead to BCD (Li et al, Am J Hum Genet.74: 817-826, 2004).

In the present application, the term "RdCVF", also known as Rod-derived cone growth factor (Rod dervetcon survivor factor), generally refers to a truncated thioredoxin-like protein that lacks thiol oxidoreductase activity. RdCVF is a nucleo-like redox protein 1 (nucleoredoxin-like 1,Nxnl1) A splice variant of a gene whose additional splice product is rdcvsl, an active thioredoxin that protects its binding partner, the microtubule-associated protein TAU, from oxidation and aggregation (Elachouri et al, 2015 and Fridlich et al, 2009).Nxnl1Mice with gene deletions can exhibit age-dependent loss of rod and cone function and cone degeneration, as well as hypersensitivity of rods and cones to oxidative stress (Cronin et al, 2010).Nxnl1Can be rod-dependent, which is significantly reduced following rod death in retinal pigment degeneration (RP) (Delyfer et al, 2011; Reichman et al, 2010). RdCVF can protect cone cell function in several different genotype retinal pigment degeneration (RP) models (Byrne et al, 2015; Le')veillard et al, 2004; Yang et al, 2009). The RdCVF molecules can be secreted to the outside of cells as signal peptides, so that glucose absorption of the photoreceptor cells is promoted, and the survival of the photoreceptor cells is maintained.Nxnl1Genes are conserved in humans, chimpanzees, rhesus monkeys, dogs, cows, rats, mice, chickens, zebrafish, and frogs. In humansNxnl1Also known asTxnl6，Nxnl1At 19p13.11, two exons were included.Nxnl1Can be commonly expressed in human tissues including lens, retina, stomach, kidney, heart, colon and spleen, and has relatively higher expression level in lens and retina.

In the present application, the term "promoter" generally refers to a sequence of deoxyribonucleic acid (DNA) that enables transcription of a particular gene. The promoter is recognized by RNA polymerase and transcription to synthesize RNA begins. In ribonucleic acid (RNA) synthesis, promoters may interact with transcription factors that regulate gene transcription, controlling the timing of initiation and the extent of expression of a gene (transcription). The promoter comprises a core promoter region and a regulatory region, is located upstream (5' direction of DNA antisense strand) of the transcription initiation site of a gene in a regulatory sequence controlling the expression of the gene, and has no coding function per se. The method is divided into three categories according to the action mode and the function: constitutive promoters (which maintain sustained activity in most or all tissues), specific promoters (tissue-specific or developmental stage-specific), and inducible promoters (which are regulated by external chemical or physical signals).

In the present application, the term "operably linked" generally refers to the placement of the regulatory sequences necessary for the expression of a coding sequence in an appropriate position relative to the coding sequence in order to achieve expression of the coding sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. In certain embodiments, the arrangement of coding sequences and transcriptional control elements in an expression vector may be expressed. The control elements may include promoters, enhancers and termination elements. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. In certain embodiments, "operably linked" may also refer to the linkage of a gene of interest into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the gene of interest.

In the present application, the term "self-cleaving peptides", also known as 2A peptides (2A self-cleaving peptides), generally refers to a class of peptide fragments 18-22 amino acid residues in length that induce self-cleavage of recombinant proteins containing the 2A peptide within cells. The 2A peptide is typically derived from the 2A region of the viral genome. In genetic engineering, the 2A peptide can divide a peptide chain translated from one Open Reading Frame (ORF) into several independent peptide chains. In certain embodiments, if it is desired to express the two proteins separately (e.g., one protein into the nucleus and the other protein in the cytoplasm), it may be desirable to construct only one open reading frame in the vector, by inserting a 2A peptide sequence in their coding region. In some embodiments, if the fusion protein is not functional after the fusion of the two proteins, a sequence encoding the 2A peptide can be inserted between the coding regions of the two proteins, or the linker peptide can be replaced by the 2A peptide, so that the two proteins can be separated and independently folded after the translation is completed, thereby providing the possibility of restoring the function of the two proteins. 2A peptides may include P2A, E2A, F2A, T2A, all of which are named for the virus of origin. Wherein P2A is derived from the 2A peptide of Porcine teschovirus (Porcine teschovirus).

In the present application, the term "polyadenylation sequence", also known as polyadenylation tail, PolyA tail, generally refers to a single strand of several tens to several hundreds of adenylic acid added to the 3' end of the transcribed mRNA. Polyadenylation generally occurs during and after the transcription of deoxyribonucleic acid (DNA) into ribonucleic acid (RNA) within the nucleus of the cell, and is usually accomplished by polyadenylic acid polymerase. In eukaryotes, polyadenylation is a mechanism by which the mRNA molecule is interrupted at its 3' end, and the polyadenylation sequence protects the mRNA from exonuclease attack and is important for nuclear export, translation, and stability of the mRNA.

In the present application, the term "polyadenylation signal site" generally refers to a sequence of bases located 3' to messenger RNA (mRNA) that is recognized by polyadenylation-associated cleavage factors. And is also typically a cis-regulatory signal on the mRNA. In general, the process of tailing (i.e., polyadenylation) begins after transcription has terminated, with polyadenylation-associated cleavage factors followed by several tens to hundreds of single adenylates in the 3' UTR of the mRNA under the control of polyadenylation signal sites. Common tailed signals include SV40, BGH, HSV, TK signals, and the like. The polyadenylation-associated lytic factor may include lytic/polyadenylation specific factor (CPSF), lytic stimulatory factor (CstF), lytic factor i (cfi), lytic factor ii (cfii). The polyadenylation signal site may typically comprise an AAUAAA sequence, but differs between eukaryotic groups. For example, most human polyadenylation signal sites contain an AAUAAA sequence, but this sequence is less common in plants and fungi.

In the present application, the term "signal peptide" generally refers to a peptide chain that can direct the transfer of a synthetic protein to the secretory pathway. The signal peptide may be about 5 to about 30 amino acids in length. The N-terminus of the signal peptide may comprise a basic amino acid and/or a hydrophobic amino acid. The signal peptide may comprise a negatively charged C-terminus, which may serve as a cleavage site for the signal peptide. When the synthesized protein enters the lumen of the endoplasmic reticulum, the signal peptide can be cleaved by the action of a signal peptidase.

In the present application, the term "intron" may include a segment of DNA that is transcribed, but is removed from an RNA transcript by splicing together either end of the sequence (exon). Introns are generally considered to be interfering sequences within the protein-coding region of a gene and generally do not contain the information represented by the protein produced by the gene.

In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage and viral vector. A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In the present application, the term "viral vector" is generally intended to refer to a non-wild-type recombinant viral particle that serves as a gene delivery vector and comprises a recombinant viral genome packaged within a viral capsid. Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyomavirus vacuolium (e.g., SV 40).

In the present application, the term "AAV vector", also referred to as an adeno-associated viral vector, generally refers to the adenovirus itself or a derivative thereof. Adeno-associated virus (AAV) generally refers to a class of single-stranded DNA viruses belonging to the genus dependovirus, the family parvoviridae. The AAV genome may comprise Inverted Terminal Repeats (ITRs) and two Open Reading Frames (ORFs) at both ends of a DNA strand. The open reading frame may compriserepAndcap。repconsisting of a plurality of overlapping genes encoding Rep proteins required by the life cycle of AAV,capcomprising overlapping nucleotide sequences encoding capsid proteins, which may include VP1, VP2, and VP 3. The capsid proteins interact to form the capsid. In the absence of helper virus, AAV can integrate its genome into a particular site on human chromosome 19 (the AAVS site) until it is rescued from latency by helper virus (Kotin et al, 1990). AAV is generally considered to be predominantly in an unintegrated form. Site-specific integration ability of AAV, its natural defect and its immunityThe low immunogenicity makes it an ideal vector for gene therapy. AAV has many common serotypes, 100 virus variants. In the present application, the AAV capsid, 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, variants of any known or mentioned AAV or AAV yet to be discovered or variants or mixtures thereof.

In the present application, the term "cell" can generally be or has been nucleic acid molecules or carrier recipients of single cells, cell lines or cell cultures. The cell may comprise a nucleic acid molecule as described herein or a vector as described herein. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may comprise cells transfected in vitro with a vector described herein. The cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293T cell.

In the present application, the term "pharmaceutical composition" generally refers to compositions that are suitable for administration to a patient, e.g., a human patient. For example, a pharmaceutical composition described herein, which may comprise a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition may be non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "kit" generally refers to a packaged product comprising a vector as described herein. The kit may comprise a container holding the kit components. The container may contain instructions and/or dosing regimens and/or instructions for the vectors described herein. The container may comprise a container with a lid.

In the present application, the term "prevention" generally refers to prophylactic administration of a combination to a healthy subject to prevent the occurrence of a certain disease or disorder. It may also comprise prophylactic administration of the combination to a patient at a pre-stage of the allergic disease to be treated. "preventing" does not require 100% elimination of the likelihood of the occurrence of a disease or condition, in other words "preventing" generally means that the likelihood of the occurrence of a disease or condition is reduced in the presence of the administered combination.

In this application, the term "alleviating" refers to reducing, diminishing or delaying a condition, disease, disorder or phenotype. The condition, disease, disorder or phenotype may include subjective perception by the subject, such as pain, dizziness or other physiological disorder, or medically detectable indication, such as a disease condition detected by medical testing means.

In the present application, the term "treatment" generally refers to clinical intervention to alter the natural course of the treated individual or cell in the course of clinical pathology. May include improving the disease state, eliminating the lesion, or improving prognosis.

In this application, the term "Retinal Pigment Epithelium (RPE)" generally refers to a layer of pigmented cells that lie next to the outside of the sensory nerve of the retina. The retinal pigment epithelium consists of a monolayer of hexagonal cells containing a dense population of pigment particles. The Retinal Pigment Epithelium (RPE), which is closely associated with the underlying choroid and the overlying retinal nerve cells, may have major functions including: control the fluid and nutrients in the subretinal space, functioning as a blood-retinal barrier; synthesizing growth factors to adjust local structure; absorbing light, and adjusting electric balance; regeneration and synthesis of visual pigment; phagocytosis and digestion of photoreceptor outer segments; maintaining the attachment of the retina; regeneration and repair after injury. RPE is generally considered to be an important tissue for maintaining photoreceptor function, and is also affected by many pathologies of the choroid and retina.

In the present application, the term "Retinal Pigment Epithelium (RPE) atrophy" generally refers to degenerative changes in the Retinal Pigment Epithelium (RPE) that manifest as cell death or dysfunction. Age-related macular degeneration or retinal pigment degeneration (RP) is usually accompanied by atrophy of the retinal pigment epithelium. Retinitis Pigmentosa (RP), also known as Retinitis Pigmentosa, is a generic ophthalmic disorder. The inheritance modes of the gene are autosomal recessive, dominant and X-linked, and the gene is also inherited by double genes and mitochondria. The common initial symptoms are night blindness, narrow visual field, and the vision can see the scenery right in front but not the slightly left or right visual field, and then the vision will disappear gradually. RP may include primary pigmentary degeneration of the single eye, quadrant, central or paracentric, leucovorin, crystalloid, varicose, arteriolar peripigmentary, periarteritic, preserved retinal pigmentary degeneration, Leber congenital amaurosis, and retinary degeneration in other syndromes.

In the present application, the term "crystalline retinal degeneration" generally refers to a class of autosomal recessive inherited eye diseases first described by the italian ophthalmologist GBBietti, doctor in 1937. The main symptoms include crystals in the cornea (clear cover), fine, yellow or white crystalline deposits deposited in the light-sensitive tissues of the retina, and progressive atrophy of the retina, choroidal capillaries and choroid. Crystalline retinal degeneration may include diseases caused by mutations in the CYP4V2 gene.

In the present application, the term "comprising" or "comprises" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 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% above or below the stated value.

In one aspect, the application provides the use of CYP4V2 and RdCVF in the manufacture of a medicament for the treatment, alleviation and/or prevention of a disease or disorder associated with atrophy of the Retinal Pigment Epithelium (RPE).

In the present application, the medicament may comprise a polynucleotide encoding the CYP4V2 and a polynucleotide encoding the RdCVF. For example, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are located on different vectors. For example, the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are on the same vector.

In another aspect, the present application also provides a vector combination for treating, ameliorating and/or preventing a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy, comprising a first vector that can comprise a polynucleotide encoding CYP4V2, and a second vector that can comprise a polynucleotide encoding RdCVF.

In another aspect, the present application also provides an isolated nucleic acid molecule which may comprise a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

In another aspect, the present application also provides a cell, which may comprise a nucleic acid molecule described herein or a vector combination described herein.

Also provided are pharmaceutical compositions that can comprise the nucleic acid molecules described herein, the vector combinations described herein, and/or the cells described herein.

In another aspect, the present application also provides the use of a nucleic acid molecule described herein, a vector combination described herein, a cell described herein and/or a pharmaceutical composition described herein for the preparation of a medicament that can be used for the prevention, alleviation and/or treatment of a disease or disorder associated with atrophy of the Retinal Pigment Epithelium (RPE).

CYP4V2

In the present application, CYP4V2 may comprise a class of proteins, the dysfunction of which or mutations in genes encoding them, which may lead to crystal-like retinal degeneration, including but not limited to CYP4V2 or functional variants thereof from humans, chimpanzees, gorillas, rhesus monkeys, dogs, cows, mice, rats, chickens, drosophila, nematodes or frogs.

For example, the CYP4V2 may comprise human CYP4V 2.

For example, the CYP4V2 may comprise the amino acid sequence set forth in any of SEQ ID NOS: 76-82.

For example, the CYP4V2 may comprise the amino acid sequence set forth in SEQ ID NO: 76.

For example, the CYP4V2 may comprise an amino acid sequence that is at least 90% homologous, e.g., any one of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, to the amino acid sequence set forth in SEQ ID NOS: 76-82, and the amino acid sequence administered in combination with an RdCVF as described herein is capable of ameliorating a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy.

In the present application, the polynucleotide encoding CYP4V2 may comprise the nucleic acid sequence set forth in any one of SEQ ID NOS: 62-68.

For example, the polynucleotide encoding CYP4V2 may comprise the nucleic acid sequence set forth in SEQ ID NO: 62.

For example, the polynucleotide encoding CYP4V2 may comprise an amino acid sequence that is at least 90% homologous, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, to the nucleic acid sequence set forth in SEQ ID NOS: 62-68, and the polypeptide encoded by such a nucleotide sequence when administered in combination with an RdCVF as described herein may be capable of ameliorating a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy.

RdCVF

In the present application, the RdCVF may comprise a protein produced by rod cells and favoring cone cells. The RdCVF includes, but is not limited to, RdCVF of humans, chimpanzees, gorillas, rhesus monkeys, dogs, cows, rats, mice, chickens, zebrafish, and frogs, or functional variants thereof.

For example, the RdCVF may comprise a human RdCVF.

For example, the RdCVF can comprise the amino acid sequence shown in any one of SEQ ID NOs 83-89.

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

For example, the RdCVF can comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NOs 83-89, e.g., any one of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and the amino acid sequence can ameliorate a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy when administered in combination with CYP4V2 described herein.

In the present application, the polynucleotide encoding RdCVF may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 69 to 75.

For example, the polynucleotide encoding RdCVF may comprise the nucleic acid sequence shown in SEQ ID NO: 69.

For example, the polynucleotide encoding RdCVF may comprise an amino acid sequence that is at least 90% homologous, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, to the nucleic acid sequence set forth in SEQ ID NOs 69-75, and the polypeptide encoded by the nucleotide sequence when administered in combination with CYP4V2 described herein is capable of ameliorating a disease or disorder associated with atrophy of the Retinal Pigment Epithelium (RPE).

Promoters

In the present application, the promoter may comprise an RPE cell-specific promoter, a retinal cell-specific promoter, a corneal cell-specific promoter, an ocular cell-specific promoter, or a constitutive promoter. The promoter may also comprise a mammalian beta actin promoter or a viral promoter. The promoter may also comprise a CAG promoter (hybrid CMV early enhancer/chicken β actin promoter, also known as CAGGS promoter, CB promoter or CBA promoter), human β actin promoter, small CBA (smcba) promoter, CBs promoter or CBh promoter, elongation factor 1 α short (EFS) promoter, elongation factor 1 α (EF-1 α) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 (also known as BEST 1) promoter, OPEFS promoter, CYP4V2 self promoter, RPE65 promoter or hybrids or derivatives thereof.

For example, the promoter may comprise the CYP4V2 self promoter, and the CYP4V2 self promoter may comprise all or part of the nucleotide sequence 2000bp upstream of the polynucleotide encoding CYP4V 2.

For example, the promoter may comprise a nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

For example, the CYP4V2 self promoter may include CYP4V2-Pf promoter, CYP4V2-P1 promoter, CYP4V2-P2 promoter, CYP4V2-P3 promoter, CYP4V2-P4 promoter, CYP4V2-P5 promoter, CYP4V2-P6 promoter; the CYP4V2-Pf promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 6, the CYP4V2-P1 promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 7, the CYP4V2-P2 promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 8, the CYP4V2-P3 promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 9, the CYP4V2-P4 promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 10, the CYP4V2-P5 promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 11, and the CYP4V2-P6 promoter can comprise a nucleic acid sequence shown in SEQ ID NO. 12.

For example, the promoter may be an OPEFS comprising the nucleic acid sequence shown in SEQ ID NO. 1.

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

For example, the promoter can be EF1a, comprising the nucleic acid sequence shown in SEQ ID NO. 3.

For example, the promoter may be CAG comprising the nucleic acid sequence shown in SEQ ID NO. 4.

For example, the promoter may be RPE65, comprising the nucleic acid sequence shown in SEQ ID NO. 5.

Polyadenylation signal site

In the present application, the polyadenylation signal site may comprise the SV40 signal site, the BGH signal site, the WPRE-SV40 signal site, the WPRE-BGH signal site or derivatives thereof.

For example, the polyadenylation signal site is recognized by polyadenylation-associated cleavage factors to produce the SV40 polyadenylation sequence, the BGH signal polyadenylation sequence, the HSV signal polyadenylation sequence, the TK signal polyadenylation sequence, the WPRE signal polyadenylation sequence, and the like.

For example, the polyadenylation signal site may comprise an AAUAAA sequence.

For example, the polyadenylation signal site may comprise the nucleic acid sequence shown in SEQ ID NOS: 17-21.

Self-cleaving peptides

In the present application, the polynucleotide encoding a self-cleaving peptide is located between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

For example, the self-cleaving peptide may comprise T2A, P2A, E2A, or F2A.

For example, the self-cleaving peptide may comprise P2A.

For example, the self-cleaving peptide may comprise an amino acid sequence set forth in any one of SEQ ID NOs 26-29.

For example, the polynucleotide encoding a self-cleaving peptide may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 22-25.

For example, the N-terminus of the cleavage peptide may also be linked to a GSG (Gly-Ser-Gly, Ser, Gly) sequence.

Intron

In the present application, the intron may be located at the 5' end of the target gene or in the nucleotide sequence of the target gene. The gene of interest may comprise a polynucleotide encoding CYP4V2 or a polynucleotide encoding RdCVF. The intron can enhance the expression of the gene of interest. For example, the intron can comprise a human β -globin intron, an SV40 intron, a hybrid CBA/MVM intron, or other synthetic introns.

For example, the intron comprises a nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

Separated from each otherNucleic acid molecules

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

For example, the isolated nucleic acid molecule may comprise the polynucleotide encoding CYP4V2, the polynucleotide encoding RdCVF, the polynucleotide encoding CYP4V2, the nucleic acid sequence set forth in any one of SEQ ID NOs 62 to 68, and the polynucleotide encoding RdCVF, in that order from 5 'to 3' of the nucleic acid molecule.

In the present application, the isolated nucleic acid molecule may comprise, from 5 'to 3', the polynucleotide encoding the RdCVF, the polynucleotide encoding CYP4V2, in that order.

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

In the present application, the isolated nucleic acid molecule may further comprise the promoter.

For example, the promoter is located 5' to the polynucleotide encoding CYP4V2 and is operably linked to the polynucleotide encoding CYP4V 2.

For example, the promoter is located 5' to the RdCVF-encoding polynucleotide and is operably linked to the RdCVF-encoding polynucleotide.

For example, the isolated nucleic acid molecule may comprise the promoter, the polynucleotide encoding CYP4V2, and the polynucleotide encoding RdCVF, in that order, from 5 'to 3'.

For example, the isolated nucleic acid molecule may comprise, from 5 'to 3', the promoter, the polynucleotide encoding RdCVF, and the polynucleotide encoding CYP4V2, in that order.

For example, the promoter may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 1-12, the polynucleotide encoding RdCVF may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75, and the polynucleotide encoding CYP4V2 may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 62-68.

For example, the isolated nucleic acid molecule may comprise, from 5 'to 3', the promoter, the polynucleotide encoding CYP4V2, the promoter, and the polynucleotide encoding RdCVF, in that order.

For example, the isolated nucleic acid molecule may comprise, from 5 'to 3', the promoter, the polynucleotide encoding CYP4V2, the promoter, and the polynucleotide encoding RdCVF, in that order.

For example, the promoter may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 1-12, the polynucleotide encoding CYP4V2 may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 62-68, and the polynucleotide encoding RdCVF may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75.

For example, the isolated nucleic acid molecule may comprise, from 5 'to 3', the promoter, the polynucleotide encoding RdCVF, the promoter, the polynucleotide encoding CYP4V2, in that order.

For example, the promoter may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 1-12, the polynucleotide encoding RdCVF may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75, and the polynucleotide encoding CYP4V2 may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 62-68.

In the present application, the isolated nucleic acid molecule may further comprise a polynucleotide encoding the self-cleaving peptide.

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

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

For example, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a promoter, a polynucleotide encoding RdCVF, a polynucleotide encoding a self-cleaving peptide, a polynucleotide encoding CYP4V 2.

For example, the promoter may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 1-12, the polynucleotide encoding an RdCVF may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75, the polynucleotide encoding a self-cleaving peptide may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 22-25, and the polynucleotide encoding CYP4V2 may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs 62-68.

For example, the isolated nucleic acid molecule may comprise the nucleic acid sequence as set forth in any one of SEQ ID NO 101-105.

In the present application, the isolated nucleic acid molecule may further comprise the polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

In the present application, the isolated nucleic acid molecule may further comprise a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

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

For example, the promoter may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 1-12, the polynucleotide encoding an RdCVF may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 69-75, the polynucleotide encoding a self-cleaving peptide may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 22-25, the polynucleotide encoding CYP4V2 may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 62-68, and the polyadenylation signal site may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 17-21.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', the promoter, the polynucleotide encoding RdCVF, the polynucleotide encoding a self-cleaving peptide, the polynucleotide encoding CYP4V2, and the polyadenylation signal site.

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

For example, the isolated nucleic acid molecule may comprise the nucleic acid sequence as set forth in any one of SEQ ID NO 106 and 108.

In the present application, the isolated nucleic acid molecule may further comprise the intron.

For example, the intron can comprise a nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

For example, the intron is located in the polynucleotide encoding CYP4V2, or 5' to the polynucleotide encoding CYP4V 2.

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

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

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

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

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

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

For example, the polynucleotide encoding CYP4V2 having the intron inserted therein may comprise the nucleic acid sequence set forth in SEQ ID NO: 116.

For example, the intron is located in the RdCVF-encoding polynucleotide, or, alternatively, at the 5' end of the RdCVF-encoding polynucleotide.

For example, the isolated nucleic acid molecule may comprise in order from 5 'to 3' the promoter, the intron, the polynucleotide encoding RdCVF, the polynucleotide encoding a self-cleaving peptide, and the polynucleotide encoding CYP4V 2.

For example, the isolated nucleic acid molecule may comprise in order from 5 'to 3' the promoter, the polynucleotide encoding the RdCVF into which the intron is inserted, the polynucleotide encoding the self-cleaving peptide, and the polynucleotide encoding CYP4V 2.

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

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

For example, the isolated nucleic acid molecule may comprise in order from 5 'to 3' the promoter, the polynucleotide encoding the RdCVF into which the intron is inserted, the polynucleotide encoding the self-cleaving peptide, the polynucleotide encoding CYP4V2, and the polyadenylation signal site.

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

For example, the isolated nucleic acid molecule may comprise a nucleotide sequence as set forth in any one of SEQ ID NO 109-115.

For a schematic representation of the structure of the isolated nucleic acid molecule, see FIGS. 30 and 31, wherein SP represents the nucleotide sequence encoding the signal peptide.

In the isolated nucleic acid molecules of the present application, the nucleotide sequence encoding the CYP4V2 protein may include a kozak sequence (e.g., the kozak sequence may be included at the 5' end). And/or, wherein the nucleotide sequence encoding the RdCVF protein may comprise a kozak sequence (e.g., the kozak sequence may be comprised at the 5' end). Wherein, the kozak sequence can comprise the nucleotide sequence shown in SEQ ID NO. 166.

In the present application, the 5 'of the isolated nucleic acid molecule may further comprise a packaging signal 5' ITR, which may comprise the nucleotide sequence set forth in SEQ ID NO: 162.

In the present application, the 3 'of the isolated nucleic acid molecule may further comprise a packaging signal 3' ITR, which may comprise the nucleotide sequence set forth in SEQ ID NO: 163.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the RdCVF protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the CYP4V2 protein, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the RdCVF protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the RdCVF protein, and a nucleotide sequence encoding a CAG promoter.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the CYP4V2 protein, and a nucleotide sequence encoding a CAG promoter.

In the present application, the isolated nucleic acid molecule may comprise a nucleotide sequence encoding a signal peptide. For example, the signal peptide may comprise the amino acid sequence as set forth in any one of SEQ ID NO 174 and 178. For example, the nucleotide sequence encoding the signal peptide may comprise the nucleotide sequence set forth in any one of SEQ ID NO 169-173.

For example, the nucleotide encoding the signal peptide may comprise a kozak sequence (e.g., the kozak sequence may be comprised at the 5' end). Wherein, the kozak sequence can comprise the nucleotide sequence shown in SEQ ID NO. 166.

In the isolated nucleic acid molecules of the present application, the nucleotide sequence encoding the CYP4V2 protein may include a kozak sequence (e.g., the kozak sequence may be included at the 5' end). Wherein, the kozak sequence can comprise the nucleotide sequence shown as SEQ ID NO. 166.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding the RdCVF protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the CYP4V2 protein, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding the RdCVF protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, and a nucleotide sequence encoding a BGH signal site.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding the CYP4V2 protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a signal peptide, and a nucleotide sequence encoding a CAG promoter.

In the present application, the isolated nucleic acid molecule may comprise, in order from 5 'to 3', a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding the RdCVF protein, a nucleotide sequence encoding a BGH signal site, a nucleotide sequence encoding the CYP4V2 protein, and a nucleotide sequence encoding a CAG promoter.

In the present application, the isolated nucleic acid molecule may further comprise a nucleotide sequence encoding a vector backbone, which may be pAV-kanAR, for example. For example, the nucleotide sequence encoding the vector backbone may comprise the nucleotide sequence shown in SEQ ID NO. 164.

In the present application, the isolated nucleic acid molecule may comprise the nucleotide sequence as set forth in any one of SEQ ID NO: 156-161.

Carrier

In the present application, the vector may include a plasmid, a phagemid, a cosmid, an artificial chromosome such as a Yeast Artificial Chromosome (YAC), a Bacterial Artificial Chromosome (BAC), or an artificial chromosome (PAC) of P1 origin, a bacteriophage such as a lambda phage or M13 phage, and a viral vector.

For example, the viral vector may comprise a retrovirus (including lentivirus), an adenovirus, an adeno-associated virus (AAV vector), a herpes virus (e.g., herpes simplex virus), a poxvirus, a baculovirus, a papilloma virus, a papilloma polyomavirus (e.g., SV 40).

For example, the adeno-associated viral vector (AAV vector) gene can comprise Inverted Terminal Repeats (ITRs), an Open Reading Frame (ORF), which can include a polynucleotide encoding a Rep protein, and can also include a polynucleotide encoding a capsid.

For example, the adeno-associated viral vector (AAV vector) can also include a recombinant adeno-associated viral vector (rAAV vector).

For example, the capsid, ITRs and other selected AAV components in the recombinant adeno-associated viral vector can be selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, DJ/8, Rh10 variants of any known or mentioned AAV or yet to be discovered AAV or variants or mixtures thereof.

For example, the AAV vector may be any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, DJ/8, Rh 10.

For example, the AAV vector is an ocular tissue affinity AAV vector, e.g., AAV2, AAV3, AAV4, AAV5, AAV8, DJ/8, or any rAAV vector.

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

For example, the viral vector may comprise pAAV-RC5-Amp, RC8-cap, AAV2/8, AAV-helper-Amp, AAV-helper.

For example, the viral vector may comprise the nucleotide sequence set forth in any one of SEQ ID NO 133 and 137.

For example, the vector may further comprise a nucleotide sequence as set forth in any one of SEQ ID NOS 90-115.

For example, the vector may further comprise a nucleotide sequence as set forth in any one of SEQ ID NO 117 and 121.

For example, the vector may comprise a nucleotide sequence as set forth in any one of SEQ ID NO 122 and 132.

In the present application, the vector may comprise an isolated nucleic acid molecule as described herein.

For example, the vector may comprise a nucleotide sequence as set forth in any one of SEQ ID NO 101 and 115.

In the present application, the vector may comprise the polynucleotide encoding CYP4V2 or the polynucleotide encoding RdCVF.

For example, the vector may further comprise a promoter located 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

For example, the vector may further comprise a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

For example, the vector may further comprise a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

For example, the vector may comprise, in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding CYP4V2, the polyadenylation signal site.

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

For example, the vector may comprise the nucleotide sequence set forth in any one of SEQ ID NOs 90-92.

For example, the vector may further comprise a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

For example, the vector may comprise, in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding RdCVF, the polyadenylation signal site.

For example, the vector may comprise, in order in the 5 'to 3' direction: a promoter sequence shown in any one of SEQ ID NO. 1-12, a polynucleotide sequence shown in any one of SEQ ID NO. 69-75 for coding RdCVF and a polyadenylation signal site sequence shown in any one of SEQ ID NO. 17-21.

For example, the vector may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOS 96-98.

For example, the vector may also comprise an intron.

For example, the intron is located in the polynucleotide encoding CYP4V2, or is located 5' to the polynucleotide encoding CYP4V 2.

For example, the intron is located in the RdCVF-encoding polynucleotide, or, alternatively, is located 5' to the RdCVF-encoding polynucleotide.

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

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

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

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

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

For example, the polynucleotide encoding CYP4V2 having the intron inserted therein may comprise the nucleic acid sequence set forth in SEQ ID NO: 116.

For example, the vector may comprise the nucleotide sequence set forth in any one of SEQ ID NOs 93-95 and 99-100.

Carrier combination

The present application also provides a vector combination for treating, ameliorating and/or preventing a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy, comprising a first vector that can comprise a polynucleotide encoding CYP4V2, and a second vector that can comprise a polynucleotide encoding RdCVF.

For example, the first vector may further comprise a promoter located 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

For example, the first vector may comprise, in order in the 5 'to 3' direction: a promoter sequence shown as any one of SEQ ID NO. 1-12, and a polynucleotide sequence shown as any one of SEQ ID NO. 62-68 and encoding CYP4V 2.

For example, the first vector may comprise the polynucleotide sequence shown in SEQ ID NO. 117.

For example, the second vector may further comprise a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

For example, the second vector may comprise the polynucleotide sequence shown in SEQ ID NO. 120.

For example, the first vector may comprise, in order in the 5 'to 3' direction: a promoter sequence shown as any one of SEQ ID NO. 1-12 and a polynucleotide sequence coding RdCVF shown as any one of SEQ ID NO. 69-75.

For example, the first vector may further comprise a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

For example, the second vector may further comprise a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

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

For example, the first vector may comprise, in order in the 5 'to 3' direction: a promoter sequence shown as SEQ ID NO. 1-12, a polynucleotide sequence shown as SEQ ID NO. 62-68 and encoding CYP4V2, and a polyadenylation signal site sequence shown as SEQ ID NO. 17-21.

For example, the first vector may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs 90-92.

For example, the second vector may comprise, in order in the 5 'to 3' direction: a promoter sequence shown as SEQ ID NO. 1-12, a polynucleotide sequence shown as SEQ ID NO. 69-75 and encoding the RdCVF, and a polyadenylation signal site sequence shown as SEQ ID NO. 17-21.

For example, the second vector may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs 96-98.

For example, the first vector and/or the second vector may further comprise an intron.

For example, the intron is located in the polynucleotide encoding CYP4V2, or is located 5' to the polynucleotide encoding CYP4V 2.

For example, the intron is located in the RdCVF-encoding polynucleotide, or, alternatively, is located 5' to the RdCVF-encoding polynucleotide.

For example, the first vector may comprise a nucleotide sequence set forth in any one of SEQ ID NOs 93-95; and/or the second vector may comprise a nucleotide sequence set forth in any one of SEQ ID NOS 99-100.

For example, the first vector may comprise the nucleotide sequence set forth in any one of SEQ ID NO 118-119; and/or the second vector may comprise a nucleotide sequence set forth in any one of SEQ ID nos. 121.

For example, the vector may comprise the nucleic acid sequence as set forth in any one of SEQ ID NO: 156 and 161.

Cells

The present application also provides a cell, which can comprise a nucleic acid molecule described herein or a vector combination described herein.

For example, the cell may be one in which the nucleic acid molecule is expressed.

For example, the cell may comprise progeny of a single cell. The progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original mother cell.

For example, the cell may further comprise a cell transfected in vitro with a vector of the invention.

For example, the cell may comprise a bacterial cell (e.g., e.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell, 293T cell.

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

For example, the cells may comprise somatic cells or stem cells.

For example, the cells may comprise retinal cells, corneal cells, choroidal cells, lens cells, neural cells, RPE cells, stem cells, which may comprise induced pluripotent stem cells (ipscs), Embryonic Stem Cells (ESCs), Mesenchymal Stem Cells (MSCs), adult stem cells, or any cells derived from stem cells.

For example, the retinal cells, corneal cells, choroidal cells, lens cells, neural cells, RPE cells may be induced to differentiate by the stem cells.

For example, the cells may comprise ARPE-19 cells, human iPSC-induced RPE cells.

Pharmaceutical composition

Also provided are pharmaceutical compositions that can comprise the nucleic acid molecules described herein, the vector combinations described herein, and/or the cells described herein.

For example, the pharmaceutical composition may further comprise an optional pharmaceutically acceptable adjuvant.

For example, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives.

For example, the acceptable ingredients of the composition are not toxic to the recipient at the dosages and concentrations employed.

For example, the pharmaceutical compositions include, but are not limited to, liquid, frozen, and lyophilized compositions.

For example, the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

For example, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intraocular administration or direct injection into tissue.

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

For example, the pharmaceutical composition may be administered without interruption (or continuously).

For example, the uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flow into the patient, as described in WO 2015/036583.

In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

The present application also provides a kit for injection comprising a vector carrying the isolated nucleic acid molecule described herein and a pharmaceutically acceptable excipient. For example, the vector may comprise the nucleic acid sequence as set forth in any one of SEQ ID NO: 156 and 161.

For example, the pharmaceutically acceptable excipient may be suitable for subretinal injection.

For example, the pharmaceutically acceptable excipient may include a mydriatic agent. The mydriatic agent may include atropine.

For example, the pharmaceutically acceptable excipient may include an anesthetic. The anesthetic may include ketamine and/or xylazine.

The pharmaceutically acceptable excipients may also include physiological saline and/or a disinfectant.

In the present application, the kit for injection may further include a syringe.

Diseases or disorders associated with atrophy of the Retinal Pigment Epithelium (RPE)

In the present application, the disease or disorder associated with atrophy of Retinal Pigment Epithelium (RPE) may include age-related macular degeneration or retinal pigment degeneration (RP).

For example, the retinal pigment degeneration may include primary retinal pigment degeneration of the single eye, primary retinal pigment degeneration of the quadrant, primary retinal pigment degeneration of the central or paracentric nature, retinitis pigmentosa albipunctate, crystalline retinitis, pigmentosa venosus, retinitis pigmentosa of the arteriolar parachroma, retinitis pigmentosa in Leber congenital amaurosis and other syndromes.

For example, the retinitis pigmentosa may include crystalloid retinal degeneration.

For example, the crystalline retinal degeneration may include a disease caused by a mutation in the CYP4V2 gene.

For example, the CYP4V2 gene mutations may 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 CYP4V2 gene deletions, and unidentified CYP4V2 gene variations associated with crystalline retinal degeneration.

For example, the CYP4V2 gene mutations may include the mutations shown in table 1:

TABLE 1 mutant part types of CYP4V2 Gene

The mutants of CYP4V2 gene shown in Table 1 can be obtained from a database

And (4) obtaining.

The present application also includes the following embodiments:

use of CYP4V2 and RdCVF in the manufacture of a medicament for the treatment, alleviation and/or prevention of a disease or condition associated with atrophy of the Retinal Pigment Epithelium (RPE).

2. The use according to embodiment 1, wherein the disease or condition comprises crystalline retinal degeneration.

3. The use according to any one of embodiments 1-2, wherein the CYP4V2 is human CYP4V 2.

4. The use according to any one of embodiments 1-3, wherein the CYP4V2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 76-82.

5. The use of any one of embodiments 1-4, wherein the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence set forth in any one of SEQ ID NOS 62-68.

6. The use according to any one of embodiments 1-5, wherein the RdCVF is a human RdCVF.

7. The use according to any one of embodiments 1-6, wherein the RdCVF comprises the amino acid sequence set forth in any one of SEQ ID NOs 83-89.

8. The use according to any one of embodiments 1 to 7, wherein the polynucleotide encoding RdCVF comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs 69 to 75.

9. The use according to any one of embodiments 1 to 8, wherein the medicament comprises a polynucleotide encoding the CYP4V2 and a polynucleotide encoding the RdCVF.

10. The use according to embodiments 1-9, wherein the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are on different vectors.

11. The use according to any one of embodiments 1 to 9, wherein the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF are on the same vector.

12. The use of any one of embodiments 10-11, wherein the vector comprises a viral vector.

13. The use of any one of embodiments 10-12, wherein the vector is a viral vector, wherein the viral vector comprises an AAV vector.

14. The use of any one of embodiments 10-13, wherein the vector further comprises a promoter located 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

15. The use according to any one of embodiments 10 to 14, wherein the vector further comprises a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

16. The use according to any one of embodiments 14-15, wherein the promoter comprises a nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

17. The use of any one of embodiments 10-16, wherein the vector further comprises a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

18. The use according to any one of embodiments 10 to 16, wherein the vector further comprises a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

19. The use according to any one of embodiments 10 to 18, wherein the vector further comprises a polynucleotide encoding a self-cleaving peptide located between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

20. The use of any one of embodiments 19, wherein the self-cleaving peptide comprises P2A.

21. The use according to any one of embodiments 19-20, wherein said polynucleotide encoding a self-cleaving peptide comprises a nucleotide sequence set forth in any one of seq id NOs 22-25.

22. The use according to any one of embodiments 10-21, wherein the vector comprises in the 5 'to 3' direction: the promoter, the polynucleotide encoding CYP4V2, the polynucleotide encoding a self-cleaving peptide, the polynucleotide encoding an RdCVF, and the polyadenylation signal site.

23. The use according to any one of embodiments 10-18, wherein the vector comprises in the 5 'to 3' direction: said promoter, said polynucleotide encoding CYP4V2, said polyadenylation signal site; alternatively, the vector comprises in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding RdCVF, the polyadenylation signal site.

24. The use of any one of embodiments 10-23, wherein the vector further comprises an intron.

25. The use according to embodiment 24, wherein the intron comprises a nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

26. The use of any one of embodiments 24-25, wherein the intron is located in the polynucleotide encoding CYP4V2, or, alternatively, is located 5' of the polynucleotide encoding CYP4V 2.

27. The use according to any one of embodiments 24 to 25, wherein the intron is located in the RdCVF-encoding polynucleotide or, alternatively, is located at the 5' end of the RdCVF-encoding polynucleotide.

28. The use according to any one of embodiments 1-27, wherein the vector comprises a nucleotide sequence set forth in any one of SEQ ID nos. 90-116.

29. A vector combination for use in treating, ameliorating and/or preventing a disease or disorder associated with Retinal Pigment Epithelium (RPE) atrophy, comprising a first vector comprising a polynucleotide encoding CYP4V2, and a second vector comprising a polynucleotide encoding RdCVF.

30. The vector combination according to embodiment 29, wherein the disease or disorder comprises crystalline retinal degeneration.

31. The vector combination according to any one of embodiments 29-30, wherein the CYP4V2 is human CYP4V 2.

32. The vector combination according to any one of embodiments 29-31, wherein the CYP4V2 comprises the amino acid sequence set forth in any one of SEQ id nos 76-82.

33. The vector combination according to any one of embodiments 29-32, wherein the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 62-69.

34. The vector combination according to any one of embodiments 29-33, wherein the RdCVF is a human RdCVF.

35. The vector combination according to any one of embodiments 29-34, wherein the RdCVF comprises the amino acid sequence set forth in any one of SEQ id nos 83-89.

36. The vector combination according to any one of embodiments 29-35, wherein the RdCVF-encoding polynucleotide comprises a nucleic acid sequence set forth in any one of SEQ ID NOs 69-75.

37. The vector combination of any one of embodiments 29-36, wherein the vector comprises a viral vector.

38. The vector combination according to any one of embodiments 29-37, wherein the vector is a viral vector, wherein the viral vector comprises an AAV vector.

39. The vector combination of any one of embodiments 29-38, wherein the first vector further comprises a promoter 5' to the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

40. The vector combination according to any one of embodiments 29-39, wherein the second vector further comprises a promoter located 5' to the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

41. The vector combination according to any one of embodiments 39-40, wherein the promoter comprises the nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

42. The vector combination of any one of embodiments 29-41, wherein the first vector further comprises a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

43. The vector combination according to any one of embodiments 29-42, wherein the second vector further comprises a polyadenylation signal site located 3' to the RdCVF-encoding polynucleotide.

44. The vector combination of any one of embodiments 29-43, wherein the first vector comprises in order in the 5 'to 3' direction: said promoter, said polynucleotide encoding CYP4V2, said polyadenylation signal site; and/or, the second vector comprises in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding RdCVF, the polyadenylation signal site.

45. The vector combination of any one of embodiments 29-44, wherein the first vector and/or the second vector further comprises an intron.

46. The vector combination of embodiment 45, wherein the intron comprises a nucleotide sequence set forth in any one of SEQ ID NOs 13-16.

47. The vector combination of any one of embodiments 29-46, wherein the intron is located in the polynucleotide encoding CYP4V2, or is located 5' of the polynucleotide encoding CYP4V 2.

48. The vector combination according to any one of embodiments 29-47, wherein the intron is located in the RdCVF-encoding polynucleotide or, alternatively, is located at the 5' end of the RdCVF-encoding polynucleotide.

49. The vector combination according to any one of embodiments 29-48, wherein the first vector comprises the nucleotide sequence set forth in any one of SEQ ID NOs 90-95; and/or the second vector comprises a nucleotide sequence shown in any one of SEQ ID NO 96-100.

50. An isolated nucleic acid molecule or molecules comprising a) a polynucleotide encoding CYP4V2 and b) a polynucleotide encoding RdCVF.

51. The isolated nucleic acid molecule of embodiment 50, wherein the CYP4V2 is human CYP4V 2.

52. The isolated nucleic acid molecule according to any one of embodiments 50-51, wherein the CYP4V2 comprises the amino acid sequence set forth in any one of SEQ ID NOS 76-82.

53. The isolated nucleic acid molecule according to any one of embodiments 50-52, wherein the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence set forth in any one of SEQ ID NOS 62-68.

54. The isolated nucleic acid molecule according to any one of embodiments 50-53, wherein the RdCVF is human RdCVF.

55. The isolated nucleic acid molecule according to any one of embodiments 50-54, wherein the RdCVF comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83-89.

56. The isolated nucleic acid molecule according to any one of embodiments 50-55, wherein the polynucleotide encoding RdCVF comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 69-75.

57. The isolated nucleic acid molecule according to any one of embodiments 50-56, further comprising a promoter located 5' of the polynucleotide encoding CYP4V2 and operably linked to the polynucleotide encoding CYP4V 2.

58. The isolated nucleic acid molecule according to any one of embodiments 50-57, further comprising a promoter located 5' of the RdCVF-encoding polynucleotide and operably linked to the RdCVF-encoding polynucleotide.

59. The isolated nucleic acid molecule according to any one of embodiments 57-58, wherein the promoter comprises the nucleotide sequence set forth in any one of SEQ ID NOs 1-12.

60. An isolated nucleic acid molecule according to any one of embodiments 50-59, comprising a polynucleotide encoding CYP4V2 and a polynucleotide encoding RdCVF.

61. The isolated nucleic acid molecule according to embodiments 50-60, further comprising a polynucleotide encoding a self-cleaving peptide positioned between the polynucleotide encoding CYP4V2 and the polynucleotide encoding RdCVF.

62. The isolated nucleic acid molecule of embodiment 61, wherein the self-cleaving peptide comprises P2A.

63. The isolated nucleic acid molecule according to any one of embodiments 61-62, wherein the polynucleotide encoding a self-cleaving peptide comprises a nucleotide sequence set forth in any one of SEQ ID NOs 22-25.

64. The isolated nucleic acid molecule according to any one of embodiments 50-63, further comprising a polyadenylation signal site located 3' to the polynucleotide encoding CYP4V 2.

65. The isolated nucleic acid molecule according to any one of embodiments 50-64, further comprising a polyadenylation signal site located 3' to the RdCVF encoding polynucleotide.

66. The isolated nucleic acid molecule according to any one of embodiments 50-65, comprising in order in the 5 'to 3' direction: the promoter, the polynucleotide encoding CYP4V2, the polynucleotide encoding a self-cleaving peptide, the polynucleotide encoding an RdCVF, and the polyadenylation signal site.

67. The isolated nucleic acid molecule of any one of embodiments 50-66, further comprising an intron.

68. The isolated nucleic acid molecule of embodiment 67, wherein the intron comprises the nucleotide sequence set forth in any one of SEQ ID NOS 13-16.

69. The isolated nucleic acid molecule according to any one of embodiments 67-68, wherein said intron is located in the polynucleotide encoding CYP4V2, or, alternatively, is located 5' to the polynucleotide encoding CYP4V 2.

70. The isolated nucleic acid molecule according to any one of embodiments 50-69, comprising the nucleotide sequence as set forth in any one of SEQ ID NO 101-115.

71. A vector comprising the isolated nucleic acid molecule of any one of embodiments 50-70.

72. The vector of embodiment 71, which is a viral vector.

73. The vector of any one of embodiments 71-72, which is a viral vector, and the viral vector comprises an AAV vector.

74. A cell comprising the isolated nucleic acid molecule according to any one of embodiments 50-70 or the vector combination according to any one of embodiments 29-49.

75. A pharmaceutical composition comprising the isolated nucleic acid molecule of any one of embodiments 50-70, the vector combination of any one of embodiments 29-49, or the cell of embodiment 74.

76. Use of the isolated nucleic acid molecule of any one of embodiments 50-70, the vector combination of any one of embodiments 29-49, or the cell of embodiment 74 in the manufacture of a medicament for treating, ameliorating, and/or preventing a disease or disorder associated with atrophy of the Retinal Pigment Epithelium (RPE).

77. The use according to embodiment 76, wherein the disease or condition comprises crystalline retinal degeneration.

Without wishing to be bound by any theory, the following examples are only for illustrating the nucleic acid molecules, the preparation method, the use, etc. in the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring harbor Laboratory Press. The chemicals not identified are commercially available from conventional sources.