Optimized CYP4V2 gene and uses thereof

Abstract

The present disclosure provides an optimized CYP4V2 gene and uses thereof relating to a polynucleotide encoding a CYP4V2 protein, the polynucleotide comprising a nucleotide sequence having 90% or greater identity to the nucleotide sequence set forth in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, or SEQ ID NO 9. The present disclosure also provides an expression vector comprising said gene, which can achieve efficient, sustained, and stable expression in the retinal pigment epithelium layer of the eye, effectively reducing the dose and possible side effects of gene therapy drugs for treating BCD, and improving therapeutic efficacy.

Description

[Technical field]

[0001] The present disclosure is in the field of gene therapy, and specifically relates to optimizing CYP4V2 gene expression for use in gene therapy targeting Vietti crystalline dystrophy (BCD). [Background technology]

[0002] Bietti crystalline dystrophy (BCD, human Mendelian disease OMIM210370) is a rare genetic autosomal recessive disorder caused by biallelic mutations in the CYP4V2 gene. The disease was first described in 1937 by Italian physician GB Vietti. The disease is characterized by the presence of numerous small shiny yellow-white crystal-like deposits in the posterior pole of the retina, atrophy of the retinal pigment epithelium (RPE), pigment coagulation, and choroidal sclerosis. Progression of the disease eventually leads to decreased visual acuity, night blindness, visual field defects, and impaired color vision. Onset of the disease occurs from puberty until age 30, but may occur after age 30. As the disease progresses, peripheral vision, central vision, or both decline, and eventually most patients become legally blind (Garcia-Garcia, Martinez-Rubio et al. “Current Perspectives in Bietti crystalline dystrophy.” Clin Ophthalmol 13: 1379-1399.).

[0003] BCD is caused by mutations in the CYP4V2 gene. This gene is located on the long arm of human chromosome 4 and is an important member of cytochrome P450 (family 4, subfamily IV, polypeptide 2), encoding a protein with 525 amino acids involved in fatty acid metabolism (Li, Jiao et al. (2004). "Bietti crystalline corneoretinal dystrophy is caused by mutations in the novel gene CYP4V2." Am J Hum Genet 74(5):817-826). CYP4V2 protein is present in the epithelial cells of the retina and cornea, and the enzyme is localized in the endoplasmic reticulum. This enzyme has typical CYP4 ω-hydroxylase activity for medium-chain saturated fatty acids. CYP4V2 is the only CYP4 present at significant levels in retinal cells, and may play an important role in polyunsaturated fatty acid metabolism in retinal cells. Mutations leading to catalytic deficiency of CYP4V2 inhibit the breakdown of ocular lipids, resulting in the formation of accumulations in the eyes of BCD patients (Hata, Ikeda et al. (2018). “Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes.” Proc Natl Acad Sci USA 115(15): 3936-3941; Zhang, Yan et al. (2020). “PSCs Reveal PUFA-Provoked Mitochondrial Stress as a Central Node Potentiating RPE Degeneration in Bietti's Crystalline Dystrophy.” Mol Ther 28(12): 2642-2661).In some patients, systemic lipid inclusions are also seen (Lai, Chu et al. (2010). “Alterations in serum fatty acid concentrations and desaturase activities in Bietti crystalline dystrophy unaffected by CYP4V2 genotypes.” Invest Ophthalmol Vis Sci 51(2):1092-1097).

[0004] BCD is estimated to affect 1 in 67,000 people. It is common in people of East Asian descent, especially those of Chinese and Japanese descent. It is estimated that there are 21,000 patients in China and approximately 5,000 in the United States. BCD symptoms are similar to those of other eye diseases that gradually damage the retina, so it may be underdiagnosed. There is currently no effective treatment available for BCD (Ng, Lai et al. (2016). “Genetics of Bietti Crystalline Dystrophy.” Asia Pac J Ophthalmol (Phila) 5(4): 245-252; Wang, Chen et al. (2021). “New compound heterozygous CYP4V2 mutations in bietti crystalline corneoretinal dystrophy.” Gene 790: 145698.).

[0005] Gene therapy strategies for BCD are currently under development. In a high-fat diet (HFD)-induced BCD mouse model, the human CYP4V2 gene carried by an adeno-associated virus (AAV) vector was expressed in the RPE layer by subretinal injection, effectively restoring several physiological and functional defects, conceptually demonstrating the efficacy of gene therapy for BCD disease (Qu, Wu et al. (2020). "Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy." Gene Ther 27 (7-8): 370-382).

[0006] AAV-mediated CYP4V2 gene therapy is currently in the early stages of research and development at several pharmaceutical companies. To date, no reports have demonstrated that it is possible to express CYP4V2 sufficiently and sustainably in vivo to achieve effective therapeutic effects. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure aims to provide a viral vector with optimized gene expression for efficient, sustained and stable expression of human CYP4V2 in the RPE layer of the retina of the eye, in order to solve the problems of the prior art, and for use in the treatment of BCD. [Means for solving the problem]

[0008] In one aspect, the disclosure provides a polynucleotide encoding a CYP4V2 protein comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity.

[0009] In some embodiments of the disclosure, the polynucleotide is depicted in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, or SEQ ID NO 9.

[0010] In some embodiments of the disclosure, the polynucleotide is that shown in SEQ ID NO:5.

[0011] In another aspect, the disclosure provides an expression cassette comprising the polynucleotide and a promoter operably linked to the polynucleotide.

[0012] In another aspect, the disclosure provides a vector comprising the polynucleotide of the above aspect, including said polynucleotide or said expression cassette.

[0013] In some embodiments of the disclosure, the polynucleotide encoding the CYP4V2 protein is operably linked to the expression control element.

[0014] In some embodiments of the present disclosure, the expression control element is one or more selected from transcriptional / translational control signals, origins of replication, promoters, enhancers, introns, poly A signals, ITRs, insulators, RNA processing signals, and elements that enhance mRNA and / or protein stability.

[0015] In another aspect, the disclosure provides a cell comprising the expression vector.

[0016] In another aspect, the disclosure provides a viral particle comprising the expression vector.

[0017] In another aspect, the present disclosure provides a pharmaceutical composition expressing a wild-type or codon-optimized CYP4V2 protein for the treatment of BCD comprising said polynucleotide, said expression cassette, said expression vector and / or said viral particle, and a pharma- ceutically acceptable carrier.

[0018] In another aspect, the disclosure provides use of said polynucleotide, said expression cassette, said expression vector, said viral particle, and / or said pharmaceutical composition in the preparation of a treatment for Vietti crystalline dystrophy (BCD).

[0019] In another aspect, the present disclosure provides a method for treating Vietti crystalline dystrophy (BCD), comprising administering to a subject an effective amount of the polynucleotide, the expression cassette, the expression vector, the viral particle, and / or the pharmaceutical composition.

[0020] In another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (1) designing an sgRNA targeting the CYP4V2 gene; Step (2) of constructing the sgRNA obtained in step (1) into a Cas9-sgRNA vector to obtain a Cas9-sgRNA plasmid; and Step (3) of introducing the Cas9-sgRNA plasmid obtained in step (2) into cells to obtain cells having a CYP4V2 gene mutation; The present invention provides a method for constructing a BCD cell model having a CYP4V2 mutation, comprising: [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram of the structure of the recombinant AAV (rAAV) vector CYP4V2 expression cassette of the present disclosure. [Diagram 2] In vitro expression of codon-optimized CYP4V2 opt gene in plasmid-transfected HEK293 cells is shown. Here, (A) is a representative image of Western blot of CYP4V2 protein, and (B) is the quantification by Western blot of CYP4V2 protein to detect the expression levels of CYP4V2 and GFP (plasmid transfection control) in HEK293 cells. Here, WT or opt gene and GFP were expressed using CAG-CYP4V2-WT / opt and CMV-EGFP double plasmids. Cell lysates were harvested 2 days after transfection with equal amounts of plasmids. Equal amounts of total protein were separated by SDS-PAGE, followed by immunoblotting. The expression levels of CYP4V2 were normalized to GFP, and the ratio to the WT expression level was calculated. [Diagram 3] In vitro expression of codon-optimized CYP4V2 in AAV2-transfected ARPE-19 cells is shown. Here, (A) is a representative image of Western blot of CYP4V2 protein, and (B) is quantification by Western blot of CYP4V2 protein to detect the expression levels of CYP4V2 and GAPDH (internal control) in ARPE-19 cells, where WT or opt gene or EGFP control were mediated by equal amounts of AAV2 viral particles (MOI=20,000). Cell lysates were harvested 2 days after viral particle infection. Equal amounts of total proteins were separated by SDS-PAGE, followed by immunoblotting. The expression level of CYP4V2 was normalized with GAPDH, and the ratio to the WT expression level was calculated (compared to the WT expression level: *, p<0.05; **, p<0.01; n=3, student test). [Figure 4]The in vitro efficacy of AAV2-CYP4V2 in a BCD cell model is shown. The construction and phenotype identification of a HEK293 CYP4V2 mutant cell model are shown. CYP4V2 mutant HEK293 cells exhibit defective cell phenotypes. Here, (A) is a schematic diagram of CYP4V2 exon 5 mutations generated using the CRIPSR / Cas9 method. (B) is the Sanger sequencing result of the CYP4V2 gene target site in HEK293 exon 5 mutant cell clone 14 (5-C14). (C) is the evaluation of cell proliferation rate in 6-day culture of HEK293 WT and mutant 5-C14 cells (compared to WT: **, p<0.01; ***, p<0.005; student test; n=4). (D-E) Evaluation of the autophagy marker LC3B-I / II in HEK293 WT and mutant cells treated with or without bafilomycin-A1 (Baf, 100 nM, 2 h). (D) Representative images of protein blots. (E) Quantification of LC3B-II protein levels, normalized to GAPDH and calculated as a ratio to that in WT cells without Baf (NT) (*, p<0.05; ns: not significant; student test; n=3). [Diagram 5]The in vitro efficacy of AAV2-CYP4V2 in a BCD cell model is shown. The construction of a CYP4V2 mutant ARPE-19 cell model, identification of the phenotype, and the effect of AAV2-CYP4V2 WT and optimized gene opt18 on the phenotype of the ARPE-19 mutant cell model are shown. The CYP4V2 exon 5 mutation was generated using the CRISPR / Cas9 method. Here, (A) is Sanger sequencing of the CYP4V2 gene target site in ARPE-19 cells. (B) is the evaluation of the cell proliferation rate of WT and mutant 5-C13 cells over a 6-day culture period. ***, P<0.01; student test; n =6. (C) is the deposition of cellular lipids. WT, mutant 5-C13 cells, AAV2-CYP4V2 WT or opt18 were infected into 5-C13 cells at an MOI of 20,000. After arachidonic acid (AA) treatment, cells were stained for lipids and DAPI. Representative images of laser confocal microscopy. Scale bar: 25 μm. (D) Quantification of intracellular lipid particle deposition. After infection of mutant cells with AAV2-CYP4V WT and opt18, lipid particle deposition was significantly reduced. Opt18 was more effective at reducing lipid particle deposition than WT (***, p<0.005; ****, p<0.001; n=50, student test). (E) Electron micrographs of WT and mutant cells 5-C13 showing autophagic vacuoles (black arrows) and vacuoles (white arrows). Scale bar: 500 nm. [Figure 6]Figure 1 shows the in vitro efficacy of AAV2-CYP4V2 in a BCD cell model. Figure 2 shows the effect of AAV2-CYP4V2 WT and optimized genes on the phenotype of a HEK293 CYP4V2 mutant cell model. (A) CYP4V2 protein expression levels were detected by Western blot 2 days after cells were infected with AAV2-CYP4V2 WT or opt18 or EGFP control at an MOI of 20,000. (B) After infection, cell proliferation rates were assessed over a 6-day time course. (C) Cell proliferation in HEK293 mutant cells infected with AAV2-CYP4V2 opt18 or EGFP control at an MOI of 5,000 or 20,000 on day 6. (D) Comparison of cell proliferation in wild-type HEK293 cells and HEK293 mutant cells infected with AAV2-CYP4V2 opt18 and EGFP control at MOI 20000 on day 6 (*,p<0.05;****,p<0.001;n=6,student test). (EF) Autophagy marker LC3B-I / II was assessed in HEK293 mutant cells infected with AAV2-CYP4V2 WT opt18 and EGFP control at MOI 20000, treated with or without Baf. (E) Representative images of protein blots. (F) Quantification of LC3B-II protein levels, normalized to GAPDH and calculated as a ratio of EGFP NT cases (*,p<0.05;**,p<0.01;ns: not significant;student test;n=3). [Figure 7]In vivo expression of codon-optimized AAV-CYP4V2 in mouse eyes by subretinal injection. (A) Expression of AAV2-CYP4V2 wild-type and optimized genes in mouse eyes. Wild-type mouse eyes were injected with 1 μL of AAV2-CYP4V2 WT or opt recombinant viral particles at 5 × 1012 vg / ml through the subretinal space. After 4 weeks, ocular tissues were enucleated, and retinas were isolated, homogenized, and lysed. Equal amounts of total protein were separated by SDS-PAGE and immunoblotted. (A): Representative images of Western blots of CYP4V2 protein. (B): Quantification of CYP4V2 protein by Western blot to detect the expression levels of CYP4V2 and GAPDH (internal control). The expression level of CYP4V2 was normalized to GAPDH and the ratio to the WT expression level was calculated (compared to the WT expression level: *, p < 0.05; student test; n = 6-9). Std: purified recombinant human CYP4V2 protein standard sample. NC: uninjected mouse eye negative control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In this disclosure, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art, unless otherwise specified.In addition, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and common procedures widely used in the respective fields.In addition, in order to better understand this disclosure, the definitions and explanations of related terms are provided below.

[0023] As used herein, a reference to "about" a value or parameter includes (describes) an embodiment of that value or parameter itself. For example, a statement that refers to "about X" includes the statement of "X."

[0024] As used herein, the singular articles "a," "an," and "said" include plural referents unless specifically stated otherwise.

[0025] As used herein, "vector" refers to a recombinant plasmid or virus containing a nucleic acid to be introduced into a host cell (in vitro or in vivo).

[0026] The term "polynucleotide" or "nucleic acid" as used herein refers to nucleotides of any length in polymeric form, be they ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers of purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivative nucleotide bases. The backbone of a nucleic acid may contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a nucleic acid may contain synthetic subunits, such as polymers of aminophosphates, and thus may be oligodeoxynucleoside aminophosphates (P-NH2) or mixed aminophosphate-phosphodiester oligomers. Additionally, double-stranded nucleic acids can be obtained from single-stranded polynucleotide products that are chemically synthesized (by synthesizing the complementary strand under appropriate conditions and annealing the strands, or by synthesizing the complementary strand from scratch using DNA polymerase and an appropriate primer).

[0027] By "recombinant viral vector" is meant a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, and preferably two, inverted terminal repeats (ITRs).

[0028] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences of non-AAV origin) flanked by at least one, and preferably two, AAV inverted terminal repeats (ITRs). When present in a host cell infected with a suitable helper virus (or expressing suitable helper functions) and expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), the rAAV vector can be replicated and packaged into infectious viral particles. When the rAAV vector is integrated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the rAAV vector is called a "pro-vector" and can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes; they can be complexed with liposomes, encapsulated within liposomes, and in embodiments, encapsulated within viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV virus capsids to generate "recombinant adeno-associated viral particles (rAAV particles)". AAV helper functions (i.e., functions that allow AAV to replicate and be packaged by host cells) can be provided in any of a variety of forms, including but not limited to helper viruses or helper virus genes that help AAV replicate and package. Other AAV helper functions are known in the art.

[0029] By "rAAV virus" or "rAAV viral particle" is meant a viral particle consisting of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0030] "Heterologous" means derived from a genotypically different entity than the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (and can encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0031] When used in reference to viral titer, the term "genomic particles (gp)", "genomic equivalents" or "genomic copies" refers to the number of viral particles that contain a recombinant AAV DNA genome, regardless of whether they are infectious or functional. The number of genomic particles in a particular vector preparation can be measured by the methods described herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0032] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of particles of a recombinant AAV vector capable of infecting and replicating, as measured by an infectious center assay (also called a replication center assay), e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0033] The term "transduction unit (tu)" when used with respect to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product as measured in a functional assay, as described in the embodiments herein or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0034] "Inverted terminal repeat" or "ITR" sequences are a term well known in the art and refer to relatively short sequences in opposing orientation found at the ends of viral genomes.

[0035] "AAV inverted terminal repeat (ITR)" sequence is a well-known term in the art for a sequence of about 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be in either of two interchangeable orientations, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides further contain several short self-complementary domains (referred to as A, A', B, B', C, C', D domains), allowing interstrand base pairing to occur within this ITR portion.

[0036] A "helper virus" for AAV refers to a virus that allows AAV (which is a defective microvirus) to replicate and be packaged by a host cell. Various helper viruses have been identified, including adenoviruses, herpes viruses, and poxviruses such as cowpox virus. Adenoviruses encompass several different subclasses, but adenovirus type 5 (Ad5) of subclass C is the most commonly used. Various adenoviruses of human, non-human mammalian, and avian origin are known and are available from depositories such as the ATCC. The herpesvirus family available from depositories such as the ATCC includes herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), pseudorabies viruses (PRV), and others.

[0037] "Percentage of sequence identity (%)" to a reference peptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference peptide or nucleic acid sequence after sequence alignment and gap introduction (if necessary, to achieve the maximum sequence identity percentage, any conservative substitutions are not considered as part of the sequence identity). Alignment to determine the percentage of identity of amino acid or nucleic acid sequences can be achieved in various ways in the art, for example, by using commonly available computer software programs, including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software, as described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, table 7.7.1. A preferred alignment software is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. In this specification, the % amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (which can alternatively be referred to as an amino acid sequence A having or containing a certain % amino acid sequence identity with or against a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as matching by the sequence alignment program in the program alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A.As used herein, the % nucleic acid sequence identity of a given nucleic acid sequence C with or against a given nucleic acid sequence D (which may alternatively be referred to as a given nucleic acid sequence C having or containing a certain % nucleic acid sequence identity with or against a given nucleic acid sequence D) is calculated as follows: 100 multiplied by the fraction W / Z, where W is the number of nucleotides scored as matching by the sequence alignment program in the program alignment of C and D, and Z is the total number of nucleotides in D. Here, it is understood that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D is not equal to the % nucleic acid sequence identity of D to C.

[0038] An "effective amount" is an amount sufficient to affect a beneficial or desired outcome, including a clinical outcome (e.g., amelioration of symptoms, achievement of a clinical endpoint, etc.). An effective amount can be administered in one or more separate doses. In the context of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay the progression of the disease. For example, an effective amount of rAAV particles expresses an expected amount of a heterologous nucleic acid, such as a therapeutic peptide or a therapeutic nucleic acid.

[0039] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0040] As used herein, "treatment" refers to a method used to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or expected clinical results include, but are not limited to, amelioration of symptoms, reduction in the extent of disease, stabilization of disease (e.g., not worsening), prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or total) (whether detectable or undetectable). "Treatment" may also refer to prolonged survival compared to expected survival in the absence of treatment.

[0041] In one aspect, the disclosure provides a polynucleotide encoding a CYP4V2 protein comprising a nucleotide sequence having 90% or greater identity to the nucleotide sequence set forth in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, or SEQ ID NO 9, Preferably, polynucleotides encoding CYP4V2 proteins are provided that contain nucleotide sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably contain nucleotide sequences with 98% or 99% or more identity.

[0042] In some embodiments of the disclosure, the polynucleotide is depicted in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, or SEQ ID NO 9.

[0043] In another aspect, the disclosure provides an expression cassette comprising the polynucleotide and a promoter operably linked to the polynucleotide.

[0044] In another aspect, the disclosure provides an expression vector comprising said polynucleotide or said expression cassette.

[0045] In some embodiments of the disclosure, the polynucleotide encoding the CYP4V2 protein is operably linked to the expression control element.

[0046] In some embodiments of the present disclosure, the expression control element is one or more selected from transcriptional / translational control signals, origins of replication, promoters, enhancers, introns, poly A signals, ITRs, insulators, RNA processing signals, and elements that enhance mRNA and / or protein stability.

[0047] In some embodiments of the present disclosure, the expression vector comprises an origin of replication; preferably, the origin of replication sequence is selected from f1 phage ori, RK2oriV, pUC ori, and pSC101ori.

[0048] In some embodiments of the present disclosure, the expression vector further comprises a 5' ITR; preferably, the 5' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO 1, preferably, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably, 98% or 99% or more identity, and more preferably, the polynucleotide is that set forth in SEQ ID NO 1.

[0049] In some embodiments of the present disclosure, the expression vector further comprises a 3' ITR, preferably the 3' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO 11, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and more preferably the polynucleotide is set forth in SEQ ID NO 11.

[0050] In some embodiments of the present disclosure, the expression vector further comprises an enhancer, preferably the enhancer is a CMV enhancer, more preferably the enhancer comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO 2, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and more preferably the polynucleotide is set forth in SEQ ID NO 2.

[0051] In some embodiments of the present disclosure, the expression vector further comprises a promoter. In some embodiments of the present disclosure, the promoter is a specific or non-specific promoter. In some embodiments of the present disclosure, the promoter is selected from a CBA promoter, a CMV promoter, an SV40 promoter, an hPGK promoter, and a TRE3GS promoter. In some embodiments of the present disclosure, the promoter is a CBA promoter, and preferably, the CBA promoter comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 3, preferably, comprises a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably, comprises a nucleotide sequence having 98% or 99% or more identity. In some embodiments of the present disclosure, the polynucleotide is that shown in SEQ ID NO 3. In some embodiments of the present disclosure, the promoter is an inducible promoter, and preferably, the inducible system comprises one or more of a tetracycline-regulated promoter, an alcohol-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, a pathogenicity-regulated promoter, a temperature / heat-inducible promoter, a light-regulated promoter, and an IPTG-inducible system. In some embodiments of the present disclosure, the tetracycline-regulated promoter is selected from a Tet on promoter, a Tet off promoter, and a Tet Activator promoter. In some embodiments of the present disclosure, the alcohol-regulated promoter is selected from an alcohol dehydrogenase I (alcA) gene promoter, a promoter responsive to alcohol transactivator protein (AlcR). In some embodiments of the present disclosure, the steroid-regulated promoter is selected from a rat glucocorticoid receptor promoter, a human estrogen receptor promoter, a moth ecdysteroid receptor promoter, a retinoid promoter, and a thyroid receptor superfamily promoter.In some embodiments of the present disclosure, the metal-regulated promoter is selected from yeast, mouse and human metallothionein promoters. In some embodiments of the present disclosure, the pathogenicity-regulated promoter is selected from salicylic acid-regulated promoter, ethylene-regulated promoter and benzothiadiazole-regulated (BTH) promoter. In some embodiments of the present disclosure, the temperature / heat-inducible promoter is selected from HSP-70 promoter, HSP-90 promoter and soybean heat shock promoter. In some embodiments of the present disclosure, the light-regulated promoter is a light-responsive promoter in plant cells.

[0052] In some embodiments of the present disclosure, the expression vector further comprises an exon and an intron. In some embodiments of the present disclosure, the exon and intron are the first exon and the first intron of chicken β-actin gene. In some embodiments of the present disclosure, the exon and intron comprise a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 4, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity. In some embodiments of the present disclosure, the polynucleotide is that shown in SEQ ID NO 4.

[0053] In some embodiments of the present disclosure, the expression vector further comprises a poly A signal. In some embodiments of the present disclosure, the poly A signal is bovine growth hormone poly A (BGH poly A), short poly A, SV40 poly A, and / or human beta bead protein poly A. In some embodiments of the present disclosure, the poly A signal comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 10, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide string having 98% or 99% or more identity. In some embodiments of the present disclosure, the polynucleotide is shown in SEQ ID NO 10.

[0054] In some embodiments of the disclosure, the expression vector comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, or SEQ ID NO 16, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or 99% or more identity. In some embodiments of the disclosure, the polynucleotide is set forth in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, or SEQ ID NO 16.

[0055] In some embodiments of the present disclosure, the vector comprises a post-transcriptional regulatory element. In some embodiments of the present disclosure, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).

[0056] In some embodiments of the present disclosure, the vector further comprises a gene encoding a marker, which is one or more selected from an antibiotic resistance protein, a toxin resistance protein, a colored or fluorescent or luminescent protein, and a protein that mediates enhanced cell growth and / or gene amplification.

[0057] In some embodiments of the disclosure, the antibiotic is selected from ampicillin, neomycin, G418, puromycin, and plastidin.

[0058] In some embodiments of the present disclosure, the toxin is selected from anthrax toxin and diphtheria toxin.

[0059] In some embodiments of the present disclosure, the colored or fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, and luciferase. In some embodiments of the present disclosure, the protein that mediates enhanced cell proliferation and / or gene amplification is dihydrofolate reductase (DHFR).

[0060] In some embodiments of the disclosure, the expression vector is selected from a plasmid, a cosmid, a viral vector, an RNA vector, or a linear or circular DNA or RNA molecule.

[0061] In some embodiments of the disclosure, the plasmid is selected from pCI, puc57, pcDNA3, pSG5, pJ603, and pCMV.

[0062] In some embodiments of the present disclosure, the viral vector is selected from retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-stranded RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-stranded RNA viruses, such as small RNA viruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., cowpox virus, fowlpox virus, and canarypox virus), Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, baculoviruses, and hepatitis viruses.

[0063] In some embodiments of the present disclosure, the retrovirus is selected from avian leukocytosis hyperplasia virus, mammalian type C, type B, type D virus, HTLV-BLV aggregate, lentivirus, and foamy virus.

[0064] In some embodiments of the present disclosure, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, and ovine demyelinating leukoencephalitis lentivirus.

[0065] In some embodiments of the present disclosure, the expression vector is an adeno-associated virus.

[0066] In some embodiments of the present disclosure, the adeno-associated virus is selected from AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

[0067] In some embodiments of the present disclosure, the expression vector further comprises a truncated chimeric intron and a Kozak initiation sequence.

[0068] In some embodiments of the present disclosure, the truncated chimeric intron is shown in SEQ ID NO:4.

[0069] In some embodiments of the disclosure, the Kozak initiation sequence is shown in SEQ ID NO:17.

[0070] In another aspect, the disclosure provides a viral particle comprising the expression vector.

[0071] In another aspect, the disclosure provides a pharmaceutical composition expressing a wild-type or codon-optimized CYP4V2 protein for the treatment of BCD comprising any of the polynucleotides, expression cassettes, expression vectors and / or viral particles, and a pharma- ceutically acceptable carrier.

[0072] In another aspect, the disclosure provides use of the polynucleotide, the expression cassette, the expression vector, the viral particle, and / or the pharmaceutical composition in the preparation of a method for treating Bietti crystalline dystrophy (BCD).

[0073] In another aspect, the disclosure provides a method of treating Bietti crystalline dystrophy (BCD) comprising administering to a subject an effective amount of said polynucleotide, said expression cassette, said expression vector, said viral particle, and / or said pharmaceutical composition.

[0074] In another aspect, the present disclosure provides: (1) designing an sgRNA targeting the CYP4V2 gene; Step (2) of constructing the sgRNA obtained in step (1) into a Cas9-sgRNA vector to obtain a Cas9-sgRNA plasmid; and (3) introducing the Cas9-sgRNA plasmid obtained in the step (2) into a cell to obtain a cell having a CYP4V2 gene mutation; The present invention provides a method for constructing a BCD cell model having a CYP4V2 mutation, comprising:

[0075] In some embodiments of the present disclosure, the method further comprises a step (4) of screening for cells having a CYP4V2 gene mutation.

[0076] In some embodiments of the present disclosure, the method further comprises a step (5) of identifying the CYP4V2 gene mutant cell.

[0077] In some embodiments of the present disclosure, said identifying is selected from identifying cell proliferation, identifying altered autophagy, and identifying lipid particle deposition.

[0078] In some embodiments of the disclosure, the sgRNA is one or more selected from SEQ ID NOs: 18-21.

[0079] The present disclosure provides an adeno-associated virus vector expressing a codon-optimized CYP4V2 gene for efficient, sustained and stable expression in the retinal pigment epithelium of the eye. The present disclosure can effectively reduce the dose and possible side effects of gene therapy drugs used to treat BCD, and improve the therapeutic effect.

[0080] Although for clarity and conciseness of description, features are described herein as part of several identical or separate embodiments, it will be understood that the scope of the disclosure may encompass several embodiments having all or a partial combination of the described features.

[0081] The present disclosure will now be described in more detail with reference to specific examples, which are illustrative and not limiting of the present disclosure.

[0082] Materials and methods CYP4V2 expression cassettes and AAV vectors All DNA sequences used in this study were synthesized by GenScript. Codon optimization was improved using the GenSmart codon optimization tool. The AAV vector-mediated CYP4V2 expression cassette is shown in Figure 1. This expression cassette sequence was cloned into a shuttle plasmid vector to obtain a shuttle plasmid containing the expression of CYP4V2 target genes mediated by AAV vectors.

[0083] AAV Vector Production and Purification Methods AAV vectors were produced by adopting a three-plasmid system, i.e., a shuttle plasmid containing the CYP4V2 target gene, a pRepCap plasmid containing the repcap gene of the AAV vector, and an auxiliary plasmid pHelper, which were co-transfected into HEK293 cells using PEI as a transfection reagent to package the AAV viral vector by recombination. The cells were harvested 48-72 hours after transfection, and the harvested solution was purified to obtain recombinant AAV viral vectors of a certain purity. The purification method is as follows:

[0084] First, the harvest liquid is pretreated. The HEK293 cells are completely lysed to release the AAV viral vectors in the cells, and nuclease is added to digest the released nucleic acids. After digestion is complete, deep filtration is performed to remove large molecular impurities and cell debris, and the filtrate after deep filtration is secondarily filtered to obtain a clear liquid for affinity chromatography sampling.

[0085] Affinity chromatography utilizes the specific adsorption of ligands and proteins to capture the AAV viral vector in the harvest fluid and remove most of the process-related impurities to achieve the effect of concentration and impurity removal. The collected eluate is mixed homogeneously and neutralized with neutralization buffer, and then stored in a sterile reservoir bottle as a sample for anion chromatography.

[0086] Anion chromatography utilizes the difference in isoelectric points of different components to separate solid and empty shell AAV viruses, while simultaneously removing remaining impurities. The eluate is collected in a new sterile reservoir bottle, and the buffer is replaced by a pharmaceutical stable buffer by ultrafiltration, reducing the virus titer to approximately 5 × 10 12 Concentrate to 1000 mg / mL and finally sterile filter and dispense into aliquots.

[0087] Quantification of AAV vector reservoir titers After AAV virus purification is completed, the viral content needs to be measured, and the genome titer is the most classical standard for characterizing the physical titer of AAV. Designing a primer probe targeting the genome sequence of rAAV and then performing Q-PCR is the most common method for measuring the genome titer.

[0088] Considering that the codon optimization of the ORF coding frame in this disclosure involves screening of multiple vector structures, in order to ensure the stability and accuracy of quantification between different vector structures, a sequence common to the vectors is selected to design a primer probe. The CMV enhancer sequence is a part of the CAG promoter, and in the process of codon optimization, this part becomes a common part of different vector structures, so a primer probe is designed for this sequence.

[0089] In the process of detecting genome titer, a standard curve must be established first, and the positive standard plasmid is diluted with sample dilutions at 2 × 10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2Dilute to copies / μL and use it as a template for the standard curve. The standard curve needs to control linearity and amplification efficiency, and generally requires R2>0.99 and amplification efficiency between 90%-110%. Then, dilute the pre-treated rAAV sample and perform QPCR detection to ensure that the detection Ct value of the sample is within the range of the standard curve. The Ct value of the sample is substituted into the standard curve to calculate the genome titer of the rAAV sample and label the content of the product.

[0090] In vitro cell plasmid transfection experiments HEK293 cells were digested 1 day prior and plated in a 6-well plate at 7.0 × 10 5 The cells were inoculated at 1000 cells / well. After overnight culture, plasmid transfection was performed. The plasmid and transfection reagent were mixed in the following amounts: 125 μL of opti-MEM medium (Gibco, 31985-070), 2 μg of CYP4V2-opt / WT expression plasmid, 200 ng of CMV-EGFP plasmid, and 5 μL of P3000 were added to a 1.5 mL centrifuge tube and mixed uniformly to obtain a plasmid tube; 125 μL of opti-MEM medium and 5 μL of Lipo 3000 (Thermo, L3000015) were added to another 1.5 mL centrifuge tube, mixed uniformly, then added to the plasmid tube and incubated at room temperature for 15 minutes. This mixture was slowly added dropwise to the HEK293 cells in a 6-well plate, followed by CO2 injection. 2 The cells were then cultured in a constant temperature incubator for 48 hours.

[0091] In vitro cell virus infection experiments ARPE-19 cells were digested 1 day before and plated in a 6-well plate at 3.0 × 10 5After culturing for 24 hours, an AAV2 virus infection experiment was performed. The cells in one well were digested and counted, and the required number of virus Vg was calculated according to the infection parameter of 20,000 MOI, and the required amount of virus was diluted to 1 mL with Opti-MEM medium. Then, the cell culture medium of the 6-well plate inoculated overnight was completely aspirated, and 1 mL of the diluted virus liquid was added, followed by CO 2 The cells were cultured in a constant temperature incubator. After 4 hours, 1 mL of DMEM complete medium (DMEM + 10% FBS + 1% double antibiotic medium (Hyclone, SV30010)) was added and incubated in a CO 2 The cells were then cultured in a constant temperature incubator for 48 hours.

[0092] Construction of BCD cell model with CYP4V2 mutation The BCD cell model with CYP4V2 mutation was constructed by the CRISPR-Cas9 method as follows: HEK293 cells or ARPE-19 cells were digested 1 day prior to the start of the experiment, and 7.0 × 10 cells were plated on a 6-well plate. 5 The cells were inoculated at 1000x1000 cells / well, and after 24 hours, the plasmid transfection experiment was performed. To prepare the dilution of the plasmid sample, 125 μL of opti-MEM medium, 2 μg of Cas9-sgRNA plasmid (addgene, #58766), and 5 μL of P3000 (Exon7-sgRNA1-HDR repair construct supplemented with 3 μg of CYP4V2-ssDNA) were added to a 1.5 mL centrifuge tube, mixed uniformly, and to prepare the dilution of Lipo 3000 (Thermo, L3000015), 125 μL of opti-MEM medium and 5 μL of Lipo 3000 were added to a 1.5 mL centrifuge tube, mixed uniformly, and then added to the plasmid sample dilution tube, mixed uniformly, and incubated at room temperature for 15 minutes. The mixture was slowly dripped onto the cells to be transfected in a 6-well plate, gently shaken to mix well, and then incubated at room temperature for 15 minutes. 2The cells were placed in a constant temperature incubator and cultured. After 48 hours of transfection, fluorescent photographs were taken to confirm the transfection efficiency, and then the cells in the 6-well plate were digested, DMEM complete medium containing 5 μg / mL puromycin was added, and pressure culture was performed. During this time, DMEM-Full medium containing 5 μg / mL puromycin was replaced daily as the medium. After 72 hours of culture, the cell culture medium in the 6-well plate was discarded, and DMEM medium without puromycin was added and cultured. After 48 hours of culture, the cells were digested and counted, and the cells were sorted and cultured at 1 cell / well in a 96-well plate (containing 200 μL DMEM + 20% FBS + 1% double antibiotic medium). After 2 to 3 weeks of culture, the growth of the clone formation spot was observed, and the clone formation spot was gradually expanded from the 96-well plate to the 24-well plate to the 6-well plate. After the cells filled the 6-well plate, some cells were taken to extract the cellular genomic DNA (Tiangen, DP304-03), which was amplified by PCR, and the gel was cut and collected for verification by Sanger sequencing, and the genotype of the monoclonal cells was analyzed using SeqMan software. The remaining cells were expanded and frozen according to the experimental needs.

[0093] Western blot analysis 200 μL of RIPA lysis solution (Beyotime Biotech. Inc., P0013B) containing protease inhibitors was added to each well of the 6-well plate in which the cells were inoculated and cultured, and the cells were completely lysed on ice for 5 minutes. The cell lysate was collected in a 1.5 mL centrifuge tube, placed on ice for 30 minutes, and then centrifuged at 4°C and 15,000 g for 20 minutes to collect the supernatant. The protein sample was diluted 10-fold with PBS and mixed thoroughly. Protein was quantified using a BCA protein quantification kit (Pierce TMThe analysis was performed using a BCA Protein Detection Kit (Thermo Fisher, 23225). 5x SDS loading buffer was added to the protein sample and mixed thoroughly. 30μg of protein sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk (prepared in PBS) for 1 hour at room temperature, the antibody was incubated and detected by ECL. Imaging analysis was performed using a Bio-Rad ChemiDocTM Touch Imaging System.

[0094] Cell proliferation analysis CYP4V2 mutant and wild-type cells were digested and counted. The cell suspension with adjusted cell density was mixed thoroughly and inoculated into 96-well plates with 4 replicate wells per cell. For the in vitro efficacy assay, virus infection was performed 24 hours after plate seeding. CCK-8 assay was performed 48 hours after plate seeding, and the assay days were designated as Day 2, Day 3, Day 4, Day 5, and Day 6, respectively. On the day of the assay, the total number of wells to be analyzed was counted and an appropriate amount of DMEM medium containing CCK-8 reagent was prepared. CCK-8 reagent (Dojindo, CK04) and DMEM medium were prepared in a ratio of 1:10, and 100 μL of DMEM medium containing CCK-8 reagent was added to each well, and the wells were cultured in a cell culture incubator at 37 °C for 1.5 hours, after which the wells were read at 450 nm using a multimode reader (BioTek, SYNERGY / LX). The data were statistically analyzed using Graphpad Prism software.

[0095] Autophagosome functional assay Wild-type, mutant, and virus-infected cells were treated with medium containing 100 nM Bafilomycin-A1 (Baf) or drug-free medium for 2 h, and intracellular proteins were extracted and the LC3B-II / GAPDH ratio was analyzed by WB assay to determine whether intracellular autophagic flux was inhibited.

[0096] Lipid deposition staining Cells were treated with 160μM arachidonic acid (AA) for 4 consecutive days and fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were stained with 3μM BODIPY 493 / 503 for 20 minutes at 37℃. Counterstained with 5μg / mL DAPI for 10 minutes. Photographs were taken with a laser confocal microscope. 50 cells were selected for each experimental group, and the area of ​​intracellular lipid deposition particles and the number of lipid deposition particles per unit cell area were analyzed with Image pro plus software.

[0097] Mouse subretinal space injection After the mouse was anesthetized, the pupil was sufficiently dilated, and under the direct vision of a surgical microscope dedicated to ophthalmology, the sclera was punctured with a 30 1 / 2 gauge disposable sharp needle outside the corneal scleral edge so as not to damage the iris and lens, and the needle was inserted into the sclera along the puncture opening using a microinjector with a 33 gauge flat needle, and after bypassing the lens and reaching the vitreous body, the needle was gradually inserted into the potential retinal space between the retinal nerve layer and the retinal pigment epithelium (RPE) layer, and injected while slowly pushing in, with an injection volume of 1 μL. 0.1% sodium fluorescein dye (safe concentration) was added to the injection carrier suspension to make it easier to observe the success of the injection and the degree of retinal detachment. 2.5% hydroxypropyl methylcellulose was instilled during the injection so that the fundus could be observed at any time. If the round retinal bulge is clearly visible under the surgical microscope and green color is visible under the retinal bulge, it is proven that the injection was successful. After a certain time, the bubble disappears and the local retinal bulge becomes flat. If no retinal swelling or green color was observed beneath the retina, or if complications such as retinal hemorrhage were observed during surgery, another mouse was selected for injection again. After surgery, 1% atropine eye ointment and tetracycline-cortisone eye ointment were applied to reduce inflammatory responses and prevent infection, and the procedure was repeated three times every other day.

[0098] Mouse retina sample extraction Mouse eye tissue was collected, and after removing the cornea and lens, 100 μL of ATL solution (QIAGEN, 19076) was added and homogenized for 2 minutes. For DNA sample extraction, 10 μL of the homogenate was aspirated, 10 μL of ATL solution and 2 μL of proteinase K (QIAGEN, 19133) were added and mixed well, 20 μL of AL solution (QIAGEN, 19075) was added and mixed well, and then incubated at 56 ° C for 10 minutes to obtain a sample for DNA detection. For protein extraction, 50 μL of the remaining homogenate was aspirated, 5 μL of 10 × RIPA lysate (CST) and protease inhibitor (Biyun Tian) were added and mixed well, and dissolved on ice for 30 minutes, centrifuged at 4 ° C and 15,000 g for 30 minutes, the supernatant was collected, and protein was quantified and subjected to WB assay.

[0099] Vector genome copy number analysis 5 μL of DNA assay sample was taken, 45 μL of sample diluent (5 μL of Pluronic F68 (Gibco, 24040032) and 2 μL of tRNA (Ambion, AM7119) were dissolved in 1 mL of RNAase-free water) was added, mixed, and then 5 μL of diluent was aspirated, 45 μL of sample diluent was added and mixed to obtain a 100-fold dilution of the DNA sample to be tested. A standard curve was plotted using a linearized and absolutely quantified plasmid (pAAV-CMV-EGFP) as a standard. Prepare qPCR reaction solution according to the following reaction system: Taqman PCR Mix, upstream primer (10 μM), downstream primer (10 μM), probe (10 μM), DNA template. The qPCR reaction was performed according to the following reaction conditions: 50 °C, 2 min; 95 °C, 10 min; 95 °C, 15 sec; 60 °C, 30 sec, 40 cycles; 37 °C, 2 sec. After the reaction was completed, a standard curve was prepared from the Ct values ​​of the standard substances, and the Vg number of each test sample was calculated by linear regression.

[0100] Mouse OCT examination Optical coherence tomography (OCT) examination: The anesthetized animals were placed on a lifting platform, and the pupils were dilated with compound tropicamide eye drops, and then topical anesthesia was administered with proparacaine hydrochloride eye drops. The eyes were aligned and the pupils were enlarged. Medical carbomer eye drop gel was applied to the test cornea, and the light source of the ophthalmic ultramicroscopic imaging system was adjusted to focus, and the focal position of the lens was aligned with the retina, and an optical coherence tomography (OCT) examination was performed. Both eyes of each experimental animal were photographed. After the experiment, both eyes were washed with saline and instilled with levofloxacin eye drops to prevent infection.

[0101] Mouse ERG assay The subject mice were kept in a dark room for 3 days, and electroretinogram (ERG) waveform collection was performed on the mice that reached the experimental time point in the dark room. After anesthetizing the mice to enlarge the pupils, 0.25% hydroxypropyl methylcellulose solution was instilled into both eyes to protect the cornea, the mice were placed on an operating table with a heating pad, a red wire electrode was inserted under the skin epidermis in the center of the mouse's glabella, and a black ground electrode was inserted under the skin epidermis of the mouse's tail. ERG image collection was performed using a whole retina electrooculogram Ganzfeld ERG system (Micron IV, Phoenix Research Laboratories, Inc.), and the average amplitude of a-wave and b-wave for each stimulus intensity was obtained by automatic analysis using the software of the system, and statistical analysis was performed. The waveform of the electroretinogram can reflect the conduction function of retinal neurons, and the measured waveform mainly consists of negative a-wave and positive b-wave, the a-wave reflects the function of retinal primary neurons, i.e., photoreceptor cells, and the b-wave reflects the function of retinal secondary neurons, i.e., bipolar cells.

[0102] Working Example Example 1: Construction, isolation and purification of adenovirus vector 1.1 Construction of adenovirus vectors The structure of the CYP4V2 expression cassette is shown in Figure 1. The CYP4V2 expression cassette includes, from the 5' end to the 3' end, 5'ITR, CMV enhancer, CBA promoter, CBA exon 1 & intron 1, Kozak sequence, target gene: wild-type human CYP4V2 gene hCYP4V2 WT or optimized CYP4V2 gene (hCYP4V2 opt, BGH polyA) and 3'ITR. Here, The nucleotide sequence of the 5'ITR is shown in SEQ ID NO: 1. CMV enhancer is a cytomegalovirus (CMV) enhancer element, the nucleotide sequence of which is set forth in SEQ ID NO: 2; The CBA promoter is the chicken β-actin gene promoter, the nucleotide sequence of which is shown in SEQ ID NO: 3. CBA exon 1 & intron 1 are the first exon and first intron of the chicken β-actin gene, the nucleotide sequence of which is shown in SEQ ID NO 4; The hCYP4V2WT gene is derived from the wild-type human CYP4V2 gene (GeneID:285440), the gene sequence of which is shown in SEQ ID NO 9, and the NCBI accession number of the wild-type hCYP4V2WT protein it encodes is NP_997235.3.

[0103] The optimized CYP4V2 genes hCYP4V2 opt are codon-optimized genes opt18, opt7, opt8 and opt6 encoding the wild-type hCYP4V2WT protein, and their nucleotide sequences are SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, respectively.

[0104] BGH poly A is the bovine growth hormone polyadenylation signal, the nucleotide sequence of which is shown in SEQ ID NO:10.

[0105] The nucleotide sequence of the 3'ITR is shown in SEQ ID NO:11.

[0106] The Kozak sequence was inserted in front of the CYP4V2 cDNA sequence, the sequence of which is shown in SEQ ID NO 17 (GCCACC).

[0107] An expression cassette containing the wild-type human CYP4V2 gene, hCYP4V2WT, was constructed as the plasmid pAAV2-CYP4V2 WT.

[0108] An expression cassette containing the optimized CYP4V2 gene was constructed as a plasmid pAAV2-CYP4V2 opt, in which SEQ ID NO 12 shows the nucleotide sequence of the expression cassette containing the codon-optimized gene opt6, SEQ ID NO 13 shows the nucleotide sequence of the expression cassette containing the codon-optimized gene opt7, SEQ ID NO 14 shows the nucleotide sequence of the expression cassette containing the codon-optimized gene opt8, SEQ ID NO 15 shows the nucleotide sequence of the expression cassette containing the codon-optimized gene opt18, and SEQ ID NO 16 shows the nucleotide sequence of the expression cassette containing CYP4V2 WT.

[0109] The CYP4V2 expression cassette can be packaged into a vector having a capsid from any AAV serotype or a hybrid or mutant thereof in a rAAV.

[0110] The viral vector was obtained by the plasmid cotransfection method. HEK293T cells were cotransfected with a helper plasmid containing the AAV2 capsid protein gene and a gene capable of assisting AAV replication, and the shuttle plasmid pAAV2-CYP4V2 WT or plasmid pAAV2-CYP4V2 opt, which contains a CYP4V2 target gene expression cassette, to form a recombinant adeno-associated viral vector.

[0111] 1.2 Isolation and purification of adenovirus vectors AAV vectors were produced using a three-plasmid system, i.e., a shuttle plasmid containing the CYP4V2 target gene, a pRepCap plasmid containing the repcap gene of the AAV vector, and a helper plasmid, pHelper, which were co-transfected into HEK293 cells using PEI as a transfection reagent to package the AAV viral vector by recombination. Harvesting was performed 48-72 hours after transfection, and the harvested solution was purified by affinity chromatography, further purified by anion chromatography, concentrated by ultrafiltration, and replaced with buffer. The genome titer of the purified recombinant AAV viral vector was measured, sterile filtered, and dispensed for use.

[0112] Example 2: In vitro expression assay of codon-optimized CYP4V2 opt In this example, the in vitro expression level of codon-optimized CYP4V2 opt was evaluated in two cell lines, HEK293 and ARPE-19, where the target gene was introduced into the cells by plasmid transfection in the HEK293 cell line and infection with AAV virus in the ARPE-19 cell line.

[0113] HEK293 cells were transiently transfected with the plasmid AAV2-CYP4V2 WT expressing CYP4V2 WT or the plasmid AAV2-CYP4V2 opt expressing CYP4V2 opt, and protein expression in cell lysates was evaluated by Western blot analysis (Figure 2). The expression levels of the codon-optimized CYP4V2 genes opt6, opt7, opt8, and opt18 (SEQ ID NOs 8,6,7,5) in HEK293 cells were all significantly higher than that of the wild-type human CYP4V2 gene (WT).

[0114] In ARPE-19, protein expression in cell lysates after infection with plasmid AAV2-CYP4V2 WT or plasmid AAV2-CYP4V2 opt was assessed by Western blot analysis (Fig. 3 ).

[0115] As can be seen from Figures 2 and 3, the codon-optimized CYP4V2 opt genes, especially opt18, showed significantly higher expression levels compared to the wild-type human CYP4V2 WT gene.

[0116] Example 3: Construction and identification of BCD cell model To evaluate the effectiveness of AAV2-CYP4V2 WT and opt genes in BCD gene therapy, in this example, we used the CRISPR-Cas9 method to construct cell models in which CYP4V2 mutations were introduced into two cell lines, HEK293 and ARPE-19 (Figures 4 and 5).

[0117] The construction strategy of the cell model is shown in Figure 4A. Exon5-sgRNA1 (the nucleotide sequence of which is shown in Table 1) was designed near exon 5, and Exon7-sgRNA1, Exon7-sgRNA2, and Exon7-sgRNA3 (their nucleotide sequences are shown in Table 1, respectively) were designed near exon 7, and all four types of sgRNAs were constructed in the Cas9-sgRNA vector (Addgene, #58766) to obtain Cas9-sgRNA plasmids. The Cas9-sgRNA plasmids were transiently transfected into two types of cells, HEK293 and ARPE-19, respectively, and 48 hours after transfection, puromycin was added to remove cells that had failed to be transfected, and then the cells were inoculated into a 96-well culture plate by limiting dilution, cultured, and after 2 to 3 weeks, PCR was performed to identify the genotype of the monoclonal cells in the 96-well plate. Details of the primer sequences used are shown in Table 1. [Table 1]

[0118] After screening and amplifying the monoclonal cells, the cell clones with the correct mutation were verified by gene sequencing, and the results are shown in Figures 4B and 5A. The genotypes of the cell clones and the description of the protein products are shown in Table 2. The full-length sequence of the wild-type CYP4V2 protein is shown in SEQ ID NO 27, and its nucleotide sequence is shown in SEQ ID NO 28. The CYP4V2 exon 5 mutant cells have a frameshift mutation at 223-224aa of the full-length sequence, and the exon 7 mutant cells have a frameshift mutation at 268-271aa, which express a more truncated protein than the wild-type. [Table 2-1] [Table 2-2]

[0119] The phenotype of HEK293 cells with CYP4V2 mutations was identified by examining cell proliferation and autophagosome function. It has been reported in the literature that RPE cells differentiated from iPSC cells of BCD patients have reduced cell proliferation rate and defective autophagosome function (Hata, M., et al. "Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes." Proc Natl Acad Sci USA 115(15):3936-3941). We found that HEK293 mutant cells had significantly reduced cell proliferation rate compared to HEK293 WT cells. We evaluated the protein level of microtubule-associated protein 1 light chain 3 (LC3), an autophagosomal marker, in HEK293 WT and mutant cells. LC3-I is generated by C-terminal processing of LC3 protein. Modification occurs during autophagosome formation, changing LC3-I to LC3-II. The results showed that the expression level of LC3-II protein in HEK293 mutant cells was higher than that in HEK293 WT cells. Bafilomycin-A1, a vacuolar H+-ATPase inhibitor, increased the LC3-II protein level in WT cells but did not increase the LC3-II protein level in mutant cells. This indicated that autophagosomes accumulated in mutant cells but autophagy flux was impaired. These observations indicated that CYP4V2 mutations caused a loss of normal cell function, resulting in reduced cell proliferation and defective autophagosome function in HEK293 cells (Figure 4C-E). Figure 4 showed the identification results of 5-C14 cells as a representative. Other cell clones also showed similar phenotypic results.

[0120] CYP4V2 mutations also caused changes in the cell phenotype in the ARPE-19 cell line, which has characteristics closer to RPE cells. ARPE-19 cells are derived from human retinal pigment epithelial cells, and expression of RPE cell surface markers CRALBP and RPE-65 is observed on the cell surface. CYP4V2 mutant ARPE-19 cells (Figure 5A) were obtained using CRISPR-Cas9 technology, and the cell proliferation rate of ARPE-19 mutant cells was significantly reduced compared to ARPE-19 WT cells (Figure 5B). The CYP4V2 gene encodes a hydroxylase involved in fatty acid metabolism in the ocular RPE cell layer, maintaining a homeostatic balance of retinal polyunsaturated fatty acids. For the CYP4V2 mutant ARPE-19 cell line, after the cells were continuously treated with 160 μM arachidonic acid (AA) for 4 days and lipid stained with 5 μM BODIPY 493 / 503, deposition of lipid particles was found to be significantly higher than that in wild-type cells (Figure 5C-5D). Observation by transmission electron microscopy showed that there were more autophagic vacuoles in the mutant cells (Figure 5E). These observations suggested that the CYP4V2 mutation caused a defect in the normal functions of the cells, leading to reduced cell proliferation, lipid metabolism abnormalities, and defective autophagosome function in ARPE-19 cells. Figure 5 shows the identification results of 5-C13 cells as a representative.

[0121] Example 4: In vitro efficacy evaluation of codon-optimized AAV2-CYP4V2 opt in BCD cell model The in vitro efficacy of codon-optimized CYP4V2 opt was evaluated in two BCD cell models harboring CYP4V2 mutations, HEK293 and ARPE-19, by infection with AAV virus.

[0122] The AAV2-mediated expression of CYP4V2 WT and opt in the HEK293 CYP4V2 mutant BCD cell model was shown in Figure 6A. Under the same amount of virus infection, the expression level of infected CYP4V2 opt was higher than that of WT.

[0123] Expression of CYP4V2 WT and opt dose-dependently rescued the cell proliferation rate defect of mutant cells in the HEK293 BCD cell model (Figure 6B-C). The cell proliferation level after functional rescue of mutant cells by CYP4V2 opt was still lower than that of wild-type HEK293 cells, suggesting that expression of CYP4V2 opt did not overactivate the cells (Figure 6D).

[0124] Consistent with the partial restoration of reduced cell proliferation, expression of CYP4V2 WT and opt also partially restored the defective autophagosome function in mutant cells. Expression of AAV2-CYP4V2 WT or opt in mutant cells did not significantly restore the accumulation of autophagosomes compared to EGFP control (without Baf treatment: NT condition). However, in mutant cells with expression of AAV2-CYP4V2 WT or opt, autophagic flux was significantly increased under Bafilomycin-A1 (Baf) treatment conditions, indicating that a part of the autophagic function was restored (Figure 6E-F).

[0125] Similarly, AAV2-mediated CYP4V2 WT and opt also showed therapeutic effects in the ARPE-19 CYP4V2 mutant BCD cell model. As shown in Figure 5C-5D, the lipid deposition particles in ARPE-19 mutant cells 5-C13 infected with AAV2-WT and AAV2-opt18 viruses were significantly reduced to the level of wild-type ARPE-19 cells compared to uninfected mutant cells. The lipid deposition reduction effect of the optimized gene opt18 was better than that of WT, with a statistically significant difference (Figure 5C-5D).

[0126] The results for CYP4V2 opt18 are shown as a representative example in Figures 5 and 6. Similar phenotypic results were obtained for the expression of other opt genes.

[0127] These results suggest that the phenotypic defects observed in the HEK293 and ARPE-19 BCD cell models are due to loss of function of the CYP4V2 gene. Restoring expression of the CYP4V2 gene can rescue these cellular functional defects and is therapeutic for BCD.

[0128] Example 5: In vivo expression assay of codon-optimized CYP4V2 opt Four-week-old wild-type C57BL / 6 mice were injected subretinal with 1 μL of 1.5 × 10 12 The mice were injected with 1000μg / mL of AAV2-CYP4V2 WT and opt recombinant viral particles, and ocular tissues were collected every 4 weeks after injection. The mouse ocular tissues were homogenized, and then RIPA lysis solution was added to dissolve the tissues, homogenized and lysed. Equal amounts of total protein were separated by SDS-PAGE, and then immunoblotted to quantify the protein, and the expression level of CYP4V2 was detected by Western. The gene expression level was analyzed by Western blot assay. The protein expression level of CYP4V2 opt gene after codon optimization was compared with WT. The results at 4 weeks after injection showed that the AAV2-CYP4V2 opt gene was expressed at a higher level than WT in mouse eyes (Figure 7).

[0129] Example 6: Phenotyping of high-fat diet CYP4V3 KO BCD mouse model Compared to patients with BCD, mice with a knockout (KO) model of the human CYP4V2 gene, orthologue CYP4V3, show fewer physiological and functional changes and the phenotype appears much later than in patients. Mice aged 6 months (corresponding to 20-30 years in humans) show sporadic lipid deposition in the eyes, whereas mice aged 12 months (corresponding to 50 years in humans and developing ocular blindness) show significant lipid deposition but no impairment of ocular ERG function or vision (Lockhart, CM, et al. Generation and characterization of a murine model of Bietti crystalline dystrophy. Invest Ophthalmol Vis Sci 55(9):5572-5581). After administration of a high fat diet (HFD), the onset of retinopathy is accelerated and aggregation occurs in the CYP4V3 KO mouse model (Qu, B., et al. Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy. Gene Ther 27(7-8):370-382). In this example, the mice were weaned after birth and then raised on an HFD, and fundus image observation, OCT, and ERG function tests were performed every 4 weeks from 4 weeks after birth.

[0130] Example 7: In vivo efficacy evaluation of codon-optimized AAV2-CYP4V2 opt in BCD mouse model CYP4V3 KO BCD mice with high-fat diet-induced ocular fundus pathological changes were injected with 1 μL of 5e12vg / mL AAV2-GFP or CYP4V2 opt viral particles via subretinal injection. OCT and ERG functional tests were performed every 4 weeks after injection to compare the therapeutic effects of AAV2-GFP and CYP4V2 opt viral particles.

Claims

1. A polynucleotide encoding a CYP4V2 protein, comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, Preferably, the nucleotide sequence has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, More preferably, the polynucleotide is as set forth in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, More preferably, said polynucleotide is a polynucleotide encoding a CYP4V2 protein as shown in SEQ ID NO 5.

2. 10. An expression cassette comprising the polynucleotide of claim 1 and a promoter operably linked to the polynucleotide.

3. 10. An expression vector comprising the polynucleotide of claim 1 or the expression cassette of claim 2.

4. further comprising an expression control element, wherein the polynucleotide encoding the CYP4V2 protein is operably linked to the expression control element; Preferably, the expression control element is one or more selected from transcriptional / translational control signals, origins of replication, promoters, enhancers, introns, poly A signals, ITRs, insulators, RNA processing signals, and elements that enhance the stability of mRNA and / or protein; Preferably, the expression vector comprises an origin of replication, and preferably the sequence of the origin of replication is selected from f1 phage ori, RK2 oriV, pUC ori, and pSC101 ori; Preferably, said expression vector further comprises a 5' ITR, preferably said 5' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 1, preferably comprises a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably comprises a nucleotide sequence having 98% or 99% or more identity, more preferably said polynucleotide is that shown in SEQ ID NO 1, Preferably, said expression vector further comprises a 3' ITR, preferably said 3' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 11, preferably comprises a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably comprises a nucleotide sequence having 98% or 99% or more identity, more preferably said polynucleotide is shown in SEQ ID NO 11, Preferably, said expression vector further comprises an enhancer, preferably said enhancer is a CMV enhancer, more preferably said enhancer comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 2, preferably comprising a nucleotide string having 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, more preferably comprising a nucleotide sequence having 98% or more or 99% or more identity, more preferably said polynucleotide is that shown in SEQ ID NO 2, Preferably, said expression vector further comprises a promoter, preferably said promoter is a specific or non-specific promoter, preferably said promoter is selected from CBA promoter, CMV promoter, SV40 promoter, hPGK promoter and TRE3GS promoter, more preferably said promoter is a CBA promoter, preferably said CBA promoter comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 3, preferably comprises a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably comprises a nucleotide string having 98% or 99% or more identity, more preferably said polynucleotide comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 3, more preferably ... 3, preferably the promoter is an inducible promoter, preferably the inducible system comprises one or more of a tetracycline-regulated promoter, an alcohol-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, a pathogenicity-regulated promoter, a temperature / heat-inducible promoter, and a light-regulated promoter, an IPTG-inducible system, preferably the tetracycline-regulated promoter is selected from a Tet on promoter, a Tet off promoter, and a Tet Activator promoter, preferably the alcohol-regulated promoter is selected from an alcohol dehydrogenase I (alcA) gene promoter, a promoter responsive to alcohol transactivator protein (AlcR);Preferably, said steroid-regulated promoter is selected from rat glucocorticoid receptor promoter, human estrogen receptor promoter, moth ecdysteroid receptor promoter, retinoid promoter, and thyroid receptor superfamily promoter, preferably, said metal-regulated promoter is selected from yeast, mouse and human metallothionein promoters, preferably, said pathogenesis-regulated promoter is selected from salicylic acid-regulated promoter, ethylene-regulated promoter and benzothiadiazole-regulated (BTH) promoter, preferably, said temperature / heat-inducible promoter is selected from HSP-70 promoter, HSP-90 promoter and soybean heat shock promoter, preferably, said light-regulated promoter is a light-responsive promoter in plant cells; Preferably, said expression vector further comprises an exon and an intron, preferably said exon and intron are the first exon and first intron of chicken β-actin gene, preferably said exon and intron comprise a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 4, preferably comprise a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably comprise a nucleotide sequence having 98% or 99% or more identity, more preferably said polynucleotide is shown in SEQ ID NO 4, Preferably, said expression vector further comprises a polyA signal, preferably said polyA signal is bovine growth hormone poly A (BGH poly A), short poly A, SV40 poly A, and / or human β-bead protein poly A; preferably said polyA signal comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 10, preferably comprises a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably comprises a nucleotide sequence having 98% or 99% or more identity, more preferably said polynucleotide is shown in SEQ ID NO 10, Preferably, said expression vector comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16, preferably comprises a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably comprises a nucleotide sequence having 98% or 99% or more identity, more preferably said polynucleotide is as shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16, Preferably, the vector comprises a post-transcriptional regulatory element, preferably the post-transcriptional regulatory element is the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE); Preferably, said vector further comprises a gene encoding a marker, preferably said marker being one or more selected from an antibiotic resistance protein, a toxin resistance protein, a colored or fluorescent or luminescent protein, and a protein mediating enhanced cell growth and / or gene amplification, Preferably, the antibiotic is selected from ampicillin, neomycin, G418, puromycin and blasticidin; Preferably, the toxin is selected from anthrax toxin and diphtheria toxin; Preferably, said colored or fluorescent or luminescent protein is selected from green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein and fluoresceinase, and preferably, said protein mediating enhanced cell proliferation and / or gene amplification is dihydrofolate reductase (DHFR). The expression vector of claim 3.

5. the expression vector is selected from a plasmid, a cosmid, a viral vector, an RNA vector, or a linear or circular DNA or RNA molecule; Preferably, the plasmid is selected from pCI, puc57, pcDNA3, pSG5, pJ603 and pCMV; Preferably, the viral vector is selected from retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-stranded RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-stranded RNA viruses, such as small RNA viruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., cowpox virus, fowlpox virus, and canarypox virus), Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, baculoviruses, and hepatitis viruses; Preferably, said retrovirus is selected from avian leukocytosis, mammalian C, B, D viruses, HTLV-BLV aggregates, lentiviruses and foamy viruses; Preferably, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV and ovine demyelinating leukoencephalitis lentivirus. The expression vector of claim 4.

6. It is an adeno-associated virus, Preferably, said adeno-associated virus is selected from AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV; Preferably, the expression vector further comprises a truncated chimeric intron and a Kozak initiation sequence, Preferably, said truncated chimeric intron is shown in SEQ ID NO 4, Preferably, the Kozak initiation sequence is as shown in SEQ ID NO 17: The expression vector according to any one of claims 3 to 5.

7. A viral particle comprising a polynucleotide according to claim 1, an expression cassette according to claim 2 and / or an expression vector according to any one of claims 3 to 6.

8. A pharmaceutical composition for the treatment of BCD comprising a polynucleotide according to claim 1, an expression cassette according to claim 2, an expression vector according to any one of claims 3 to 6 and / or a viral particle according to claim 7, and a pharma- ceutical acceptable carrier, the pharmaceutical composition expresses a wild-type or codon-optimized CYP4V2 protein; Pharmaceutical compositions.

9. Use of a polynucleotide according to claim 1, an expression cassette according to claim 2, an expression vector according to any one of claims 3 to 6, a viral particle according to claim 7, and / or a pharmaceutical composition according to claim 8 in the preparation of a treatment for Bietti crystalline dystrophy (BCD).

10. (1) designing an sgRNA targeting the CYP4V2 gene; Step (2) of constructing the sgRNA obtained in step (1) into a Cas9-sgRNA vector to obtain a Cas9-sgRNA plasmid; Step (3) of introducing the Cas9-sgRNA plasmid obtained in step (2) into a cell to obtain a cell having a CYP4V2 gene mutation; A method for constructing a BCD cell model having a CYP4V2 mutation, comprising: Preferably, the method further comprises the step (4) of screening cells having a CYP4V2 gene mutation, Preferably, the method further comprises the step (5) of identifying the CYP4V2 gene mutant cell, Preferably, the sgRNA is selected from one or more of SEQ ID NOs 18 to 21; Preferably, the identification is selected from the identification of cell proliferation, the identification of autophagy changes, and the identification of lipid particle deposition. method.

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