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 Bietti crystalline dystrophy (BCD). [Background technology]

[0002] Bietti crystalline dystrophy (BCD, a human Mendelian disorder OMIM 210370) 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 Bietti. The disease is characterized by the presence of numerous small, shiny, yellowish-white crystalline deposits in the posterior pole of the retina, atrophy of the retinal pigment epithelium (RPE), pigment coagulation, and choroidal sclerosis. Progression of the disease ultimately leads to decreased visual acuity, night blindness, visual field defects, and color vision impairment. Onset of the disease occurs between adolescence and age 30, but may occur after age 30. As the disease progresses, peripheral vision, central vision, or both deteriorates, and ultimately 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, located on the long arm of human chromosome 4, is an important member of the cytochrome P450 (family 4, subfamily IV, polypeptide 2) family and encodes a 525-amino acid protein 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 retinal and corneal epithelial cells, and the enzyme is localized in the endoplasmic reticulum. This enzyme possesses typical CYP4 ω-hydroxylase activity toward 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 that lead to catalytic deficiencies in CYP4V2 inhibit the breakdown of ocular lipids, resulting in the formation of deposits 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).Some patients also have systemic lipid inclusions (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 approximately one in 67,000 people. It is most common in people of East Asian descent, particularly 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. Because the symptoms of BCD are similar to those of other eye diseases that gradually damage the retina, it may be underdiagnosed. Currently, there are no effective treatments 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 790:145-698).

[0005] Gene therapy strategies for BCD are currently under development. In a high-fat diet (HFD)-induced BCD mouse model, the human CYP4V2 gene delivered by an adeno-associated virus (AAV) vector was expressed in the RPE layer by subretinal injection. This effectively restored several physiological and functional deficits, demonstrating the conceptual effectiveness of gene therapy for BCD (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 sufficient and sustained expression of CYP4V2 in vivo is possible to achieve effective therapeutic effects. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure aims to solve the problems of the prior art by providing a viral vector with optimized gene expression that efficiently, continuously, and stably expresses human CYP4V2 in the RPE layer of the retina of the eye and can be used to treat BCD. [Means for solving the problem]

[0008] In one aspect, the disclosure provides a polynucleotide encoding a CYP4V2 protein 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 comprising a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably comprising a nucleotide sequence having 98% or 99% or more identity.

[0009] In some embodiments of the present disclosure, the polynucleotide is set forth 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 present 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 present disclosure provides a vector comprising the polynucleotide of the above aspect, or the polynucleotide comprising the expression cassette.

[0013] In some embodiments of the present 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 present disclosure provides a cell comprising the expression vector.

[0016] In another aspect, the present 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 the polynucleotide, the expression cassette, the expression vector and / or the viral particle, and a pharmaceutically acceptable carrier.

[0018] In another aspect, the present 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 treatment for Bietti crystalline dystrophy (BCD).

[0019] In another aspect, the present disclosure provides a method for treating Bietti 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: Step (1) of 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 explanation 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. [Figure 2] In vitro expression of the codon-optimized CYP4V2 opt gene in HEK293 cells transfected with the plasmid is shown. (A) is a representative image of a Western blot of CYP4V2 protein, and (B) is a Western blot quantification of CYP4V2 protein to detect the expression levels of CYP4V2 and GFP (plasmid transfection control) in HEK293 cells. Here, the WT or opt gene and GFP were expressed using the CAG-CYP4V2-WT / opt and CMV-EGFP double plasmids. Cell lysates were collected two days after transfection with equal amounts of plasmid. Equal amounts of total protein were separated by SDS-PAGE and then immunoblotted. CYP4V2 expression levels were normalized to GFP, and the ratio to the WT expression level was calculated. [Figure 3] In vitro expression of codon-optimized CYP4V2 in AAV2-transfected ARPE-19 cells is shown. (A) Representative image of Western blot for CYP4V2 protein. (B) Quantification of CYP4V2 protein by Western blot to detect expression levels of CYP4V2 and GAPDH (internal control) in ARPE-19 cells. WT or opt gene or EGFP control were transfected with equal amounts of AAV2 viral particles (MOI = 20,000). Cell lysates were collected 2 days after viral particle infection. Equal amounts of total protein were separated by SDS-PAGE and then immunoblotted. CYP4V2 expression levels were normalized to GAPDH and the ratio to WT expression levels was calculated (compared to WT expression levels: *, 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. Construction and phenotypic characterization of a HEK293 CYP4V2 mutant cell model are also shown. CYP4V2-mutated HEK293 cells exhibit defective cell phenotypes. (A) Schematic diagram of the CYP4V2 exon 5 mutation generated using the CRIPSR / Cas9 method. (B) Sanger sequencing results of the CYP4V2 gene target site in HEK293 exon 5 mutant cell clone 14 (5-C14). (C) Cell proliferation rates of HEK293 WT and mutant 5-C14 cells were evaluated after 6 days of culture (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 hours). (D) Representative image of protein blot. (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). [Figure 5]The in vitro efficacy of AAV2-CYP4V2 in a BCD cell model is shown. Construction of a CYP4V2 mutant ARPE-19 cell model, phenotypic characterization, and the effects of AAV2-CYP4V2 WT and the optimized gene opt18 on the phenotype of the ARPE-19 mutant cell model are shown. CYP4V2 exon 5 mutations were generated using the CRISPR / Cas9 method. (A) Sanger sequencing of the CYP4V2 gene target site in ARPE-19 cells. (B) Cell proliferation rates of WT and mutant 5-C13 cells were assessed over a 6-day culture period. ***, P<0.01; Student's test; n=6. (C) Cellular lipid deposition. WT, mutant 5-C13 cells, AAV2-CYP4V2 WT, or opt18 were infected at an MOI of 20,000. After arachidonic acid (AA) treatment, cells were stained for lipids and DAPI. Representative images are from a laser confocal microscope. 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]This figure shows the in vitro efficacy of AAV2-CYP4V2 in a BCD cell model. This figure shows the effect of AAV2-CYP4V2 WT and optimized genes on the phenotype of a HEK293 CYP4V2 mutant cell model. (A) Two days after infection with AAV2-CYP4V2 WT, opt18, or EGFP control at an MOI of 20,000, CYP4V2 protein expression levels were detected by Western blot. (B) After infection, cell proliferation was 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 on day 6 between wild-type HEK293 cells and HEK293 mutant cells infected with AAV2-CYP4V2 opt18 and EGFP control at an MOI of 20,000 (*, p<0.05; ****, p<0.001; n=6, student test). (EF) Expression of the autophagy marker LC3B-I / II was assessed in HEK293 mutant cells infected with AAV2-CYP4V2 WT opt18 and EGFP control at an MOI of 20,000, 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 following subretinal injection is shown. (A) Expression of AAV2-CYP4V2 wild-type and optimized genes in mouse eyes. Wild-type mouse eyes were injected with 1 μL of 5 × 10 12 vg / ml AAV2-CYP4V2 WT or opt recombinant viral particles via the subretinal space. Four weeks later, 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 image of Western blot for CYP4V2 protein. (B): Quantification of CYP4V2 protein by Western blot to detect the expression levels of CYP4V2 and GAPDH (internal control). CYP4V2 expression levels were normalized to GAPDH and the ratio to the WT expression level was calculated (compared to the WT expression level: *, p < 0.05; Student's test; n = 6–9). Std: purified recombinant human CYP4V2 protein standard sample. NC: uninjected mouse eye negative control. DETAILED DESCRIPTION OF THE INVENTION

[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) the embodiment of that value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

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

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

[0026] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, either 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 composed 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 aminophosphate (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 recombinant AAV vectors, 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 containing 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 replicate and be packaged into infectious viral particles. When an 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 referred to as 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 and linear artificial chromosomes; they can be complexed with liposomes, encapsulated in liposomes, and in embodiments, can be encapsulated in viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV virus capsids to produce "recombinant adeno-associated viral particles (rAAV particles)". AAV helper functions (i.e., functions that allow AAV to be replicated and 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 from 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, when expressed, can encode a heterologous polypeptide). 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] The terms "genome particles (gp)," "genome equivalents," or "genome copies," when used in reference to viral titer, refer to the number of viral particles containing a recombinant AAV DNA genome, regardless of infectiousness or functionality. The number of genome 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 infection and replication, 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, such as those described in 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 opposite orientation found at the ends of viral genomes.

[0035] The term "AAV inverted terminal repeat (ITR)" is well known in the art and refers to a sequence of approximately 145 nucleotides present at both ends of a naturally occurring 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 (called the A, A', B, B', C, C', and D domains), allowing interstrand base pairing to occur within this ITR portion.

[0036] A "helper virus" for AAV refers to a virus that enables AAV (a defective microvirus) to replicate and be packaged by host cells. Various helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as cowpox. Adenoviruses encompass several different subclasses, but subclass C adenovirus type 5 (Ad5) is the most commonly used. Various adenoviruses of human, nonhuman mammalian, and avian origin are known and are available from depositories such as the American College of Cardiovascular Diseases (ATCC). Herpesvirus families available from depositories such as the ATCC include herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV).

[0037] "Percentage 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 those in the reference peptide or nucleic acid sequence after sequence alignment and gap introduction (if necessary, to achieve the maximum percent sequence identity, any conservative substitutions are not considered part of the sequence identity). Alignment to determine the percentage identity of amino acid sequences or nucleic acid sequences can be achieved in a variety of 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 appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. As used herein, the % amino acid sequence identity of a given amino acid sequence A with or relative to 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 relative to a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical 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 relative to a given nucleic acid sequence D (which may alternatively be referred to as a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity with or relative to 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 a match by the sequence alignment program in the program alignment of C and D, and Z is the total number of nucleotides in D. It is understood that if 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 result, including a clinical outcome (e.g., improvement of symptoms, achievement of a clinical endpoint, etc.). An effective amount can be administered in one or more doses. With respect to a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or slow 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 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 human.

[0040] As used herein, "treatment" refers to an approach used to obtain beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired 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 palliation of the disease state, and remission (whether partial or total) (whether detectable or undetectable). "Treatment" can also mean prolonging 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 present disclosure, the polynucleotide is set forth 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 present disclosure provides an expression vector comprising the polynucleotide or the expression cassette.

[0045] In some embodiments of the present 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 comprising a nucleotide sequence having 90% or greater identity to the nucleotide sequence set forth in SEQ ID NO 11, preferably comprising a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity, more preferably comprising a nucleotide sequence having 98% or greater identity, and more preferably comprising a nucleotide sequence having 99% or greater identity, and more preferably wherein the polynucleotide is that 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 that 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 set forth in SEQ ID NO: 3, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity. In some embodiments of the present disclosure, the polynucleotide is that set forth 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 and 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 a salicylic acid-regulated promoter, an ethylene-regulated promoter, and a benzothiadiazole-regulated (BTH) promoter. In some embodiments of the present disclosure, the temperature / heat-inducible promoter is selected from an HSP-70 promoter, an HSP-90 promoter, and a 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 first intron of a 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 set forth in SEQ ID NO 4, 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 present disclosure, the polynucleotide is set forth 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 set forth in SEQ ID NO 10, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity. In some embodiments of the present disclosure, the polynucleotide is set forth in SEQ ID NO 10.

[0054] In some embodiments of the present disclosure, the expression vector comprises a nucleotide sequence having 90% or greater 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 greater identity, and more preferably a nucleotide sequence having 98% or 99% or greater identity. In some embodiments of the present 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, fluorescent, or luminescent protein, and a protein that mediates enhanced cell growth and / or gene amplification.

[0057] In some embodiments of the present 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 protein, fluorescent protein, 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 present 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 present 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-strand 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-strand RNA viruses such as small RNA viruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus 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 leukocyte hyperplasia sarcoma, mammalian type C, type B, type D viruses, HTLV-BLV aggregates, lentiviruses, and foamy viruses.

[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 set forth in SEQ ID NO:4.

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

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

[0071] 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 any of the polynucleotides, expression cassettes, expression vectors, and / or viral particles, and a pharmaceutically acceptable carrier.

[0072] In another aspect, the present 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 present disclosure provides a method for treating Bietti 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.

[0074] In another aspect, the present disclosure provides: Step (1) of 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:

[0075] In some embodiments of the present disclosure, the method further comprises a step (4) of screening for cells with 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, the identifying is selected from identifying cell proliferation, identifying altered autophagy, and identifying lipid particle deposition.

[0078] In some embodiments of the present 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, which can effectively reduce the dose and potential side effects of gene therapy drugs used to treat BCD, thereby improving therapeutic efficacy.

[0080] Although, for clarity and conciseness of description, features are described herein as part of several embodiments, either the same or separate, it will be understood that the scope of the disclosure may encompass several embodiments having all or any 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 with respect to the present disclosure.

[0082] Experimental materials and methods CYP4V2 expression cassette and AAV vector 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 AAV vector-mediated expression of the CYP4V2 target gene.

[0083] AAV vector production and purification methods AAV vectors were produced using a three-plasmid system: a shuttle plasmid containing the CYP4V2 target gene, the pRepCap plasmid containing the AAV vector repcap gene, and the auxiliary plasmid pHelper. These were co-transfected into HEK293 cells using PEI as a transfection reagent, and the AAV viral vector was packaged by recombination. Cells were harvested 48-72 hours post-transfection, and the harvest solution was purified to obtain recombinant AAV viral vectors of a certain purity. The purification method is as follows.

[0084] First, the harvest solution was pretreated. HEK293 cells were completely lysed to release the intracellular AAV viral vector, and nuclease was added to digest the released nucleic acids. After digestion was complete, deep filtration was performed to remove large molecular impurities and cellular debris. The filtrate was then subjected to secondary filtration to obtain a clear solution for affinity chromatography sampling.

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

[0086] Anion chromatography utilizes the difference in the 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 ultrafiltration is performed to replace the buffer with a pharmaceutical-stable buffer, reducing the virus titer to approximately 5 x 10 12 Concentrate to 1000 mg / mL and finally sterile filter and dispense.

[0087] Quantification of AAV vector reservoir titer After AAV virus purification is complete, the viral content must be measured, and the genome titer is the most classic criterion for characterizing the physical titer of AAV. The most common method for measuring genome titer is to design a primer probe targeting the rAAV genome sequence followed by Q-PCR.

[0088] Considering that the codon optimization of the ORF coding frame in this disclosure involves screening multiple vector structures, primer probes are designed based on sequences common to the vectors to ensure the stability and accuracy of quantification across different vector structures. The CMV enhancer sequence is part of the CAG promoter, and since this portion is common to different vector structures during the codon optimization process, primer probes are designed for this sequence.

[0089] In the process of detecting genome titer, a standard curve must first be established, and the positive standard plasmid is diluted to 2 × 10 with the sample dilution. 7 , 2 × 10 6 , 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , 2 × 10 2The sample was diluted to copies / µL and used as a template for a standard curve. The standard curve must control linearity and amplification efficiency; typically, an R2 of >0.99 and an amplification efficiency of 90%-110% are required. The pre-treated rAAV sample was then diluted and subjected to QPCR detection to ensure that the sample's detection Ct value was within the range of the standard curve. The sample's Ct value was then substituted into the standard curve to calculate the genome titer of the rAAV sample and label the product content.

[0090] In vitro cell plasmid transfection experiments HEK293 cells were digested 1 day before and plated in a 6-well plate at 7.0 × 10 5 Cells were seeded 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 thoroughly to obtain the 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 and mixed thoroughly. The mixture was then added to the plasmid tube and incubated at room temperature for 15 minutes. This mixture was slowly added dropwise to HEK293 cells in a 6-well plate and subsequently cultured in a CO2 incubator for 48 hours.

[0091] In vitro cell virus infection experiments ARPE-19 cells were digested one day before and plated in a 6-well plate at 3.0 × 10 5Cells were inoculated at 1000 cells / well. After 24 hours of culture, an AAV2 virus infection experiment was performed. Cells from one well were digested and counted. The required number of virus Vg was calculated based on the infection parameter of 20,000 MOI, and the required amount of virus was diluted to 1 mL with Opti-MEM medium. Next, the cell culture medium from the overnight inoculated 6-well plate was completely aspirated, and 1 mL of the diluted virus solution was added. The plate was then cultured in a CO2 incubator. After 4 hours, 1 mL of DMEM complete medium (DMEM + 10% FBS + 1% double antibiotic medium (Hyclone, SV30010)) was added, and the plate was placed in a CO2 incubator and cultured for 48 hours.

[0092] Construction of a BCD cell model with CYP4V2 mutation The CYP4V2 mutation-introduced BCD cell model was constructed using the CRISPR-Cas9 method as follows: HEK293 or ARPE-19 cells were digested 1 day prior to the initiation of ... 5Cells were seeded at 1000x1000x1000 cells / well. 24 hours later, plasmid transfection experiments were performed. Plasmid sample dilutions were prepared by adding 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) to a 1.5 mL centrifuge tube and mixing thoroughly. Lipo 3000 (Thermo, L3000015) dilutions were prepared by adding 125 μL of opti-MEM medium and 5 μL of Lipo 3000 to a 1.5 mL centrifuge tube and mixing thoroughly. The mixture was then added to the plasmid sample dilution tube, mixed thoroughly, and incubated at room temperature for 15 minutes. The mixture was slowly added dropwise to the cells to be transfected in a 6-well plate, gently shaken to mix thoroughly, and then placed in a CO2 incubator for incubation. 48 hours after transfection, fluorescent photographs were taken to confirm transfection efficiency. Then, cells in 6-well plates were digested, added to DMEM-Full medium containing 5 μg / mL puromycin, and cultured under pressure. During this time, DMEM-Full medium containing 5 μg / mL puromycin was added daily. After 72 hours of culture, the cell culture medium in the 6-well plates was discarded and replaced with DMEM-Full medium without puromycin. After 48 hours of culture, cells were digested and counted. Cells were sorted and cultured at 1 cell / well in 96-well plates (containing 200 μL DMEM + 20% FBS + 1% double antibiotic medium). After 2–3 weeks of culture, the growth of clonal spots was observed, and the clonal spots gradually expanded from the 96-well plate to the 24-well plate and then to the 6-well plate. After the cells filled the 6-well plate, some cells were harvested and cellular genomic DNA (Tiangen, DP304-03) was extracted and amplified by PCR. The DNA was then excised from the gel and analyzed by Sanger sequencing. The genotype of the monoclonal cells was analyzed using SeqMan software. The remaining cells were expanded and cryopreserved according to experimental needs.

[0093] Western blot analysis To each well of a 6-well plate containing inoculated cells, 200 μL of RIPA lysis solution (Beyotime Biotech. Inc., P0013B) containing protease inhibitors was added, 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 15,000 g for 20 minutes at 4°C. The supernatant was collected. The protein sample was diluted 10-fold with PBS and mixed thoroughly. Protein was quantified using a BCA protein quantification kit (Pierce TM The 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. 30 μg 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 ChemiDoc™ Touch Imaging System.

[0094] Cell proliferation analysis CYP4V2 mutant and wild-type cells were digested and counted. The cell suspension was thoroughly mixed and seeded into a 96-well plate with four replicate wells per cell. For the in vitro efficacy assay, virus infection was performed 24 hours after plating. CCK-8 assays were performed 48 hours after plating, and the assay days were designated 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 Laboratories, CK04) and DMEM medium were mixed at a 1:10 ratio. 100 μL of DMEM medium containing CCK-8 reagent was added to each well. After 1.5 hours of incubation in a 37°C cell culture incubator, the cells were read at 450 nm using a multimode reader (BioTek, SYNERGY / LX). Data were statistically analyzed using Graphpad Prism software.

[0095] Autophagosome function assay Wild-type, mutant, and virus-infected cells were treated with medium containing 100 nM Bafilomycin-A1 (Baf) or drug-free medium for 2 hours, and intracellular proteins were extracted. 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 160 μM arachidonic acid (AA) for four consecutive days and then fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were stained with 3 μM BODIPY 493 / 503 for 20 minutes at 37°C. They were counterstained with 5 μg / mL DAPI for 10 minutes. Images were taken using a laser confocal microscope. Fifty cells were selected for each experimental group, and the area of ​​intracellular lipid deposit particles and the number of lipid deposit particles per cell unit area were analyzed using Image Pro Plus software.

[0097] Mouse subretinal space injection After general anesthesia, the mouse was thoroughly dilated. Under direct vision using an ophthalmology operating microscope, the sclera was punctured with a 30.5-gauge disposable sharp needle just outside the corneoscleral limbus, avoiding damaging the iris and lens. Using a microinjector equipped with a 33-gauge flat needle, the needle was inserted along the puncture hole into the sclera, bypassing the lens and reaching the vitreous. The needle was then gradually inserted into the potential retinal space between the retinal neural layer and the retinal pigment epithelium (RPE) layer, slowly pushing in to inject a 1 μL volume. 0.1% sodium fluorescein dye (safe concentration) was added to the injection carrier suspension to facilitate monitoring of the success of the injection and the extent of retinal detachment. During the injection, 2.5% hydroxypropyl methylcellulose was instilled to allow for easy fundus observation. A round retinal bulge was clearly visible under the operating microscope, and a green color beneath the retinal bulge confirmed successful injection. After a certain period of time, the bubble disappeared and the local retinal bulge flattened. If no retinal swelling or underlying green color was observed, or if complications such as retinal hemorrhage were observed during surgery, another mouse was selected for injection. After surgery, 1% atropine eye ointment and tetracycline-cortisone eye ointment were applied to reduce inflammation and prevent infection. This procedure was repeated three times every other day.

[0098] Mouse eye retina sample extraction Mouse ocular tissue was collected, and the cornea and lens were removed. 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, mixed thoroughly, and 20 μL of AL solution (QIAGEN, 19075) was added, mixed thoroughly, and incubated at 56°C for 10 minutes to obtain DNA samples. 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, mixed thoroughly, and lysed on ice for 30 minutes. The supernatant was collected, and protein was quantified for WB assay.

[0099] Vector genome copy number analysis 5 μL of DNA assay sample was taken, and 45 μL of sample diluent (5 μL of Pluronic F68 (Gibco, 24040032) and 2 μL of tRNA (Ambion, AM7119) dissolved in 1 mL of RNAase-free water) was added. After mixing, 5 μL of diluent was aspirated, and 45 μL of sample diluent was added and mixed to obtain a 100-fold dilution of the test DNA sample. A standard curve was plotted using a linearized, absolutely quantified plasmid (pAAV-CMV-EGFP) as a standard. qPCR reaction mixture was prepared according to the following reaction system: Taqman PCR Mix, upstream primer (10 μM), downstream primer (10 μM), probe (10 μM), and DNA template. qPCR reaction conditions were as follows: 50°C for 2 minutes; 95°C for 10 minutes; 95°C for 15 seconds; 60°C for 30 seconds, 40 cycles; and 37°C for 2 seconds. After the reaction was completed, a standard curve was created 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 platform, and their pupils were dilated with compound tropicamide eye drops. Proparacaine hydrochloride eye drops were used for topical anesthesia. The eyes were aligned and the pupils were dilated. Medical carbomer eye drops were applied to the test cornea. The light source of the ophthalmic ultramicroscopic imaging system was adjusted to focus the lens, and the focal point of the lens was aligned with the retina. Optical coherence tomography (OCT) examination was then 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 Mice were kept in a dark room for 3 days, and electroretinogram (ERG) waveforms were collected in a dark room after reaching the experimental time point. After anesthetizing and dilating the pupils, 0.25% hydroxypropyl methylcellulose solution was instilled into both eyes to protect the corneas. The mice were then placed on a heated operating table. A red wire electrode was inserted subcutaneously in the center of the eyebrow, and a black ground electrode was inserted subcutaneously in the tail. ERG images were collected using a Ganzfeld ERG system (Micron IV, Phoenix Research Laboratories, Inc.). Mean a- and b-wave amplitudes for each stimulus intensity were calculated and statistically analyzed using the system's software. The electroretinogram waveform reflected the conduction function of retinal neurons. The measured waveform consisted mainly of a negative a-wave and a positive b-wave. The a-wave reflected the function of primary retinal neurons, i.e., photoreceptor cells, and the b-wave reflected the function of secondary retinal neurons, i.e., bipolar cells.

[0102] 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 contains, from the 5' end to the 3' end, a 5'ITR, a CMV enhancer, a CBA promoter, a CBA exon 1 & intron 1, a Kozak sequence, a target gene: the wild-type human CYP4V2 gene hCYP4V2 WT or an optimized CYP4V2 gene (hCYP4V2 opt, BGH polyA), and a 3'ITR. 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, and their nucleotide sequences are 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 that encode 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 before 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 the plasmid pAAV2-CYP4V2 opt, where SEQ ID NO: 12 represents the nucleotide sequence of the expression cassette containing the codon-optimized gene opt6, SEQ ID NO: 13 represents the nucleotide sequence of the expression cassette containing the codon-optimized gene opt7, SEQ ID NO: 14 represents the nucleotide sequence of the expression cassette containing the codon-optimized gene opt8, SEQ ID NO: 15 represents the nucleotide sequence of the expression cassette containing the codon-optimized gene opt18, and SEQ ID NO: 16 represents 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 variant thereof in an rAAV.

[0110] The viral vectors were 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 supporting AAV replication, and the shuttle plasmid pAAV2-CYP4V2 WT or the plasmid pAAV2-CYP4V2 opt, which contains a CYP4V2 target gene expression cassette, to generate recombinant adeno-associated viral vectors.

[0111] 1.2 Isolation and purification of adenoviral vectors AAV vectors were produced using a three-plasmid system: a shuttle plasmid containing the CYP4V2 target gene, the pRepCap plasmid containing the AAV vector repcap gene, and the helper plasmid pHelper. These were co-transfected into HEK293 cells using PEI as a transfection reagent, and the AAV viral vector was packaged by recombination. The harvested solution was harvested 48-72 hours post-transfection, purified by affinity chromatography, further purified by anion chromatography, concentrated by ultrafiltration, and buffered. The genome titer of the purified recombinant AAV viral vector was measured, sterile filtered, and aliquoted 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. The target gene was introduced into the cells by plasmid transfection in the HEK293 cell line and by AAV virus infection in the ARPE-19 cell line.

[0113] HEK293 cells were transiently transfected with the CYP4V2 WT plasmid (AAV2-CYP4V2 WT) or the CYP4V2 opt plasmid (AAV2-CYP4V2 opt), and protein expression in cell lysates was assessed 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, and 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, particularly 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, 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 for the cell model is shown in Figure 4A. Exon5-sgRNA1 (the nucleotide sequence of which is shown in Table 1) was engineered near exon 5, and Exon7-sgRNA1, Exon7-sgRNA2, and Exon7-sgRNA3 (their nucleotide sequences are shown in Table 1) were engineered near exon 7. All four sgRNAs were constructed in the Cas9-sgRNA vector (Addgene, #58766) to obtain the Cas9-sgRNA plasmids. The Cas9-sgRNA plasmids were transiently transfected into two types of cells, HEK293 and ARPE-19. After 48 hours of transfection, puromycin was added to remove unsuccessfully transfected cells. The cells were then seeded into 96-well culture plates by limiting dilution and cultured. After 2–3 weeks, PCR was performed to identify the genotype of the monoclonal cells in the 96-well plates. The primer sequences used are detailed in Table 1. [Table 1]

[0118] After screening and amplifying the monoclonal cells, cell clones with the correct mutation were verified by gene sequencing. The results are shown in Figures 4B and 5A. The genotypes and protein product descriptions of the cell clones 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 aa 223-224 of the full-length sequence, while the exon 7 mutant cells have a frameshift mutation at aa 268-271, resulting in the expression of a truncated protein compared to the wild-type. [Table 2-1] [Table 2-2]

[0119] The phenotype of HEK293 cells carrying the CYP4V2 mutation was identified by examining cell proliferation and autophagosome function. It has been reported that RPE cells differentiated from iPSC cells from BCD patients exhibit reduced cell proliferation 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 exhibited significantly reduced cell proliferation compared with HEK293 WT cells. We assessed the protein levels of microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, in HEK293 WT and mutant cells. LC3-I is generated by C-terminal processing of the LC3 protein. Modification occurs during autophagosome formation, converting 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 LC3-II protein levels in WT cells but not in mutant cells. This indicated that autophagosomes accumulated in mutant cells, but autophagy flux was impaired. These observations indicated that CYP4V2 mutations cause a loss of normal cell function, resulting in reduced cell proliferation and defective autophagosome function in HEK293 cells (Figure 4C-E). Figure 4 shows the representative results of the identification of 5-C14 cells. Other cell clones also showed similar phenotypic results.

[0120] CYP4V2 mutations also induced changes in the phenotype of the ARPE-19 cell line, which has characteristics more closely resembling those of RPE cells. ARPE-19 cells are derived from human retinal pigment epithelial cells and express the RPE cell surface markers CRALBP and RPE-65 on their cell surface. Using CRISPR-Cas9 technology, CYP4V2-mutant ARPE-19 cells (Figure 5A) were obtained, 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 the homeostatic balance of retinal polyunsaturated fatty acids. In the CYP4V2 mutant ARPE-19 cell line, cells were continuously treated with 160 μM arachidonic acid (AA) for 4 days. After lipid staining with 5 μM BODIPY 493 / 503, lipid particle deposition was found to be significantly higher than in wild-type cells (Figures 5C-5D). Transmission electron microscopy revealed the presence of more autophagic vacuoles in the mutant cells (Figure 5E). These observations suggest that the CYP4V2 mutation causes defects in normal cell function, resulting in reduced cell proliferation, abnormal lipid metabolism, and impaired autophagosome function in ARPE-19 cells. Figure 5 shows the results of the identification of 5-C13 cells as a representative example.

[0121] Example 4: In vitro efficacy evaluation of codon-optimized AAV2-CYP4V2 opt in a 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] AAV2-mediated expression of CYP4V2 WT and opt in the HEK293 CYP4V2 mutant BCD cell model is shown in Figure 6A. Under the same viral infection dose, the expression level of infected CYP4V2 opt was higher than that of WT.

[0123] Expression of CYP4V2 WT and opt rescued the cell proliferation defect of mutant cells in a dose-dependent manner in the HEK293 BCD cell model (Fig. 6B-C). After functional restoration of mutant cells with CYP4V2 opt, the cell proliferation level was still lower than that of wild-type HEK293 cells, suggesting that expression of CYP4V2 opt did not overactivate the cells (Fig. 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 the mutant cells. Expression of AAV2-CYP4V2 WT or opt in the mutant cells did not significantly restore autophagosome accumulation compared to the EGFP control (without Baf treatment: NT condition). However, in mutant cells expressing AAV2-CYP4V2 WT or opt, autophagic flux was significantly increased under Bafilomycin-A1 (Baf) treatment, indicating partial restoration of autophagic function (Figure 6E-F).

[0125] Similarly, AAV2-mediated CYP4V2 WT and opt18 also demonstrated therapeutic efficacy in the ARPE-19 CYP4V2 mutant BCD cell model. As shown in Figures 5C-5D, lipid deposition in ARPE-19 mutant cells 5-C13 infected with AAV2-WT and AAV2-opt18 viruses was 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 statistically significant compared to WT (Figures 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 the loss of CYP4V2 gene function. Restoring CYP4V2 gene expression can reverse these cellular functional defects and has therapeutic potential 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 Mice were injected with 1000µg / mL of AAV2-CYP4V2 WT and opt recombinant viral particles. Ocular tissues were collected every 4 weeks after injection. The mouse ocular tissues were homogenized and then lysed in RIPA lysis solution. Equal amounts of total protein were separated by SDS-PAGE, followed by immunoblotting. Protein quantification was performed, and CYP4V2 expression levels were detected by Western blot analysis. Gene expression levels were analyzed by Western blot assay. Protein expression levels of the codon-optimized CYP4V2 opt gene were compared with those of the WT gene. Results at 4 weeks after injection showed that the AAV2-CYP4V2 opt gene was expressed at higher levels in mouse eyes than the WT gene (Figure 7).

[0129] Example 6: Phenotyping of a high-fat diet CYP4V3 KO BCD mouse model Compared to patients with BCD, mice lacking the human CYP4V2 gene orthologue, CYP4V3, exhibit fewer physiological and functional changes and the phenotype appears much later than in patients. Six-month-old mice (equivalent to 20–30 years of age in humans) exhibit sporadic lipid deposition in the eyes, whereas 12-month-old mice (equivalent to 50 years of age in humans and developing ocular blindness) exhibit significant lipid deposition but no impairment of ocular ERG function or visual acuity (Lockhart, C.M., 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 and aggregation were accelerated 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, mice were weaned after birth and then placed on an HFD. Fundus imaging, OCT, and ERG function tests were performed every four weeks from four weeks after birth.

[0130] Example 7: In vivo efficacy evaluation of codon-optimized AAV2-CYP4V2 opt in a BCD mouse model CYP4V3 KO BCD mice with high-fat diet-induced ocular fundus pathology were injected with 1 μL of 5e12 vg / mL AAV2-GFP or CYP4V2 opt viral particles via subretinal injection. Optical coherence tomography (OCT) and electroretinogram (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 having the nucleotide sequence shown in SEQ ID NO:

5.

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

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

4. The expression vector of claim 3 , further comprising an expression control element, wherein the polynucleotide encoding the CYP4V2 protein is operably linked to the expression control element.

5. The expression vector according to claim 4 , wherein the expression control element is one or more selected from a replication origin, a promoter, an enhancer, an intron, a polyA signal, an ITR, and an insulator.

6. The expression vector of claim 3 further comprising an origin of replication.

7. The expression vector according to claim 6 , wherein the sequence of the replication origin is selected from the group consisting of f1 phage ori, RK2 oriV, pUC ori, and pSC101 ori.

8. The expression vector of claim 3 further comprising a 5' ITR.

9. 9. The expression vector of claim 8, wherein the nucleotide sequence of the 5' ITR is as set forth in SEQ ID NO:

1.

10. The expression vector of claim 3 further comprising a 3' ITR.

11. 11. The expression vector of claim 10, wherein the nucleotide sequence of the 3' ITR is as shown in SEQ ID NO 11.

12. The expression vector of claim 3 further comprising an enhancer.

13. The expression vector of claim 12, wherein the enhancer is a CMV enhancer.

14. 14. The expression vector of claim 13, wherein the nucleotide sequence of the enhancer is as set forth in SEQ ID NO:

2.

15. The expression vector of claim 3 further comprising a promoter.

16. 16. The expression vector of claim 15, wherein the promoter is a specific or non-specific promoter.

17. 17. The expression vector of claim 16, wherein the promoter is selected from a CBA promoter, a CMV promoter, an SV40 promoter, an hPGK promoter, or a TRE3GS promoter.

18. 18. The expression vector of claim 17, wherein the promoter is a CBA promoter.

19. 19. The expression vector of claim 18, wherein the nucleotide sequence of the CBA promoter is as set forth in SEQ ID NO 3.

20. 16. The expression vector of claim 15, wherein the promoter is an inducible promoter.

21. 21. The expression vector of claim 20, wherein the inducible promoter is one or more selected from 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 promoter, and the pathogenicity-regulated promoter is selected from a salicylic acid-regulated promoter, an ethylene-regulated promoter, or a benzothiadiazole-regulated promoter.

22. 22. The expression vector of claim 21, wherein the tetracycline-regulated promoter is selected from a promoter that activates gene expression in the presence of tetracycline or a promoter that activates gene expression in the absence of tetracycline.

23. 22. The expression vector of claim 21, wherein the alcohol-regulated promoter is selected from the alcohol dehydrogenase I gene promoter and a promoter responsive to alcohol transactivator protein.

24. 22. The expression vector of claim 21, wherein 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, or a thyroid receptor superfamily promoter.

25. 22. The expression vector of claim 21, wherein the metal-regulated promoter is selected from yeast, mouse, or human metallothionein promoters.

26. 22. The expression vector of claim 21, wherein the temperature / heat-inducible promoter is selected from the group consisting of an HSP-70 promoter, an HSP-90 promoter, and a soybean heat shock promoter.

27. The expression vector of claim 21 , wherein the light-regulated promoter is a light-responsive promoter in plant cells.

28. The expression vector of claim 3 further comprising an exon and an intron.

29. 29. The expression vector of claim 28, wherein the exon and intron are the first exon and first intron of a chicken β-actin gene.

30. 30. The expression vector of claim 29, wherein the nucleotide sequences of the exons and introns are as set forth in SEQ ID NO:

4.

31. The expression vector of claim 3 further comprising a polyA signal.

32. 32. The expression vector of claim 31, wherein the polyA signal is bovine growth hormone polyA or SV40 polyA.

33. 33. The expression vector of claim 32, wherein the nucleotide sequence of the polyA signal is as set forth in SEQ ID NO:

10.

34. 4. The expression vector of claim 3, wherein the nucleotide sequence is as set forth in SEQ ID NO 15.

35. The expression vector of claim 3 further comprising a post-transcriptional regulatory element.

36. 36. The expression vector of claim 35, wherein the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element.

37. The expression vector of claim 3 , further comprising a gene encoding a marker.

38. 38. The expression vector of claim 37, wherein the marker is one or more selected from antibiotic resistance proteins, colored or fluorescent proteins.

39. 39. The expression vector of claim 38, wherein the antibiotic is selected from ampicillin, neomycin, G418, puromycin, or blasticidin.

40. 39. The expression vector of claim 38, wherein the colored or fluorescent protein is selected from green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, or luciferase.

41. 38. The expression vector of claim 37, wherein the marker is dihydrofolate reductase.

42. The expression vector of claim 4 , wherein the expression vector is a plasmid.

43. The expression vector of claim 4 , wherein the expression vector is a cosmid.

44. The expression vector of claim 4 , wherein the expression vector is a viral vector.

45. The expression vector of claim 4, wherein the expression vector is linear or circular DNA.

46. 43. The expression vector of claim 42, wherein the plasmid is selected from pCI, puc57, pSG5, pJ603, or pCMV.

47. 45. The expression vector of claim 44, wherein the viral vector is selected from adenovirus, adeno-associated virus, herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, cytomegalovirus, cowpox virus, fowlpox virus, canarypox virus, papovavirus, hepadnavirus, or baculovirus.

48. 48. The expression vector of claim 47, which is an adeno-associated virus.

49. 49. The expression vector of claim 48, wherein 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, and AAV type 10.

50. 4. The expression vector of claim 3, further comprising a truncated chimeric intron and a Kozak sequence, wherein the nucleotide sequence of said truncated chimeric intron is as set forth in SEQ ID NO:

4.

51. 51. The expression vector of claim 50, wherein the nucleotide sequence of the Kozak sequence is as set forth in SEQ ID NO 17.

52. A viral particle comprising the polynucleotide of claim 1 or the expression cassette of claim 2.

53. A viral particle comprising the expression vector of claim 3.

54. 10. A pharmaceutical composition for the treatment of Bietti crystalline dystrophy, comprising the polynucleotide of claim 1 or the expression cassette of claim 2, and a pharmaceutically acceptable carrier, the pharmaceutical composition expresses a codon-optimized CYP4V2 protein; Pharmaceutical compositions.

55. A pharmaceutical composition for treating Bietti crystalline dystrophy, comprising the expression vector of claim 3 and a pharmaceutically acceptable carrier, the pharmaceutical composition expresses a codon-optimized CYP4V2 protein; Pharmaceutical compositions.

56. 53. A pharmaceutical composition for the treatment of Bietti crystalline dystrophy, comprising the viral particle of claim 52 and a pharmaceutically acceptable carrier, the pharmaceutical composition expresses a codon-optimized CYP4V2 protein; Pharmaceutical compositions.

57. 54. A pharmaceutical composition for the treatment of Bietti crystalline dystrophy, comprising the viral particle of claim 53 and a pharmaceutically acceptable carrier, the pharmaceutical composition expresses a codon-optimized CYP4V2 protein; Pharmaceutical compositions.

58. Use of the polynucleotide of claim 1 or the expression cassette of claim 2 in the preparation of a medicament for treating Bietti crystalline dystrophy (BCD).

59. Use of the expression vector of claim 3 in the preparation of a medicament for treating Bietti crystalline dystrophy.

60. 53. Use of the viral particle of claim 52 in the preparation of a medicament for treating Bietti crystalline dystrophy.

61. 54. Use of the viral particle of claim 53 in the preparation of a medicament for treating Bietti crystalline dystrophy.

62. 55. Use of the pharmaceutical composition of claim 54 in the preparation of a medicament for treating Bietti crystalline dystrophy.

63. 56. Use of the pharmaceutical composition of claim 55 in the preparation of a medicament for treating Bietti crystalline dystrophy.

64. 57. Use of the pharmaceutical composition of claim 56 in the preparation of a medicament for treating Bietti crystalline dystrophy.

65. 58. Use of the pharmaceutical composition of claim 57 in the preparation of a medicament for treating Bietti crystalline dystrophy.

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