Personalized approaches for gene therapy

Abstract

Compositions and methods of treating, arresting, rescuing, ameliorating, preventing or slowing progression of a disease associated with oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS) and / or (ii) an aldehyde are described.

Description

[0001] F&R Ref No.: 43219-0011WO1 PERSONALIZED APPROACHES FOR GENE THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Application No.63 / 671,689 filed on July 15, 2024, and U.S. Application No.63 / 676,023 filed on July 26, 2024. TECHNICAL FIELD This disclosure generally relates to personalized approaches for gene therapy. In addition, it relates to the use of CYP4V2 to reduce oxidative stress or lipid peroxidation in cells and to treat diseases associated with oxidative stress or lipid peroxidation. BACKGROUND Gene therapy has become a powerful tool for treating genetic disorders since its first FDA approval in 2017. Interspecies differences between animals and humans are one limitation of animal-based testing that happens in preclinical trials. Additionally, animal models cannot factor in genotype differences among human patients. Importantly, individual differences such as genotypes, genetic polymorphisms, and individual cell surface receptor expression levels can have a significant impact on gene therapy transduction and treatment efficacy. It is essential to consider individual patient responses in gene therapy development to maximize the clinical benefits. One of the barriers to moving forward a clinical trial is the variation in response to therapy from individual patients. Personalized medicine approaches are one such way to overcome this barrier, as patient-specific cell lines provide a platform to tailor personalized treatments. Divergent genotypes may cause different disease expressivity, and individual genetic features (i.e., gene modifiers and the receptor genes that affect transfection / transduction) may lead to variable treatment responses. SUMMARY Patient-specific induced pluripotent stem cell (iPSC)-derived cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. F&R Ref No.: 43219-0011WO1 Bietti Crystalline Dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from BCD patients to evaluate adeno-associated virus (AAV)-mediated gene augmentation therapy strategies. We found that BCD iRPEs were vulnerable to blue light- induced oxidative stress, and cellular phenotype can be quantified using three robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) and cell death rate. Additionally, we demonstrated that AAV-mediated CYP4V2 gene therapy can significantly reduce light-induced cell death in BCD iRPEs. This proof-of-concept study showed that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD and to reduce abnormally high levels of polyunsaturated fatty acids (PUFAs), ROS and 4-HNE in BCD patients’ iRPEs. Further, we observed significant variability in cellular phenotypes among BCD iRPE subjects of divergent mutations, which outlined genotype- phenotype correlations in BCD patient-specific cell disease models. Our results revealed that patient-specific iRPEs retained personalized responses to AAV-mediated gene therapy. Therefore, this approach advanced BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity. ROS and lipid peroxidation are harmful to cells, which may cause cell damage, degeneration or death. By reducing ROS or lipid peroxidation, CYP4V2 can prevent, treat, or rescue cell damage, degeneration or death and associated diseases. In some embodiments, CYP4V2 is introduced into the target cells through a gene expression vector. In some embodiments, CYP4V2 is introduced into the target cells by protein delivery. In one aspect, methods of treating, arresting, rescuing, ameliorating, preventing or slowing progression of a disease associated with oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS), or (ii) an aldehyde in a human subject are provided. Such methods typically include delivering to the human subject’s organ, tissue or cell affected by the disease, a therapeutically effective amount of a vector comprising a nucleic acid molecule comprising a sequence encoding a CYP4V2 protein, wherein said organ, tissue or cell of the human subject is transduced with the vector, whereby said disease is treated, arrested or prevented in the human subject. F&R Ref No.: 43219-0011WO1 In another aspect, methods of reducing, rescuing, preventing, treating, ameliorating, or slowing progression of, the death, dysfunction, degeneration, atrophy or dystrophy of a cell associated with oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS), or (ii) an aldehyde are provided. Such methods typically include delivering to the cell, a therapeutically effective amount of a vector comprising a nucleic acid molecule comprising a sequence encoding a CYP4V2 protein, wherein the cell is transduced with the vector. In some embodiments, the oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS), or (ii) an aldehyde is induced or aggravated by light. In some embodiments, the cell is a retinal cell, a retinal pigment epithelium (RPE) cell, or a photoreceptor. In yet another aspect, methods of treating, arresting, rescuing, ameliorating, preventing or slowing progression of a disease associated with light-induced or light- aggravated retinal damage, atrophy, dystrophy or degeneration in a human subject are provided. Such methods typically include delivering to the human subject’s retina, a therapeutically effective amount of a vector comprising a nucleic acid molecule comprising a sequence encoding a CYP4V2 protein, whereby said disease is treated, arrested or prevented in the human subject, wherein the disease associated with light-induced or light-aggravated retinal damage, atrophy, dystrophy or degeneration in a human subject is attributed to oxidative stress, lipid peroxidation and / or abnormally high level of (i) a polyunsaturated fatty acid (PUFA), (ii) reactive oxygen species (ROS) and / or (iii) an aldehyde. In some embodiments, the PUFA is an omega-6 PUFA or an omega-3 PUFA. In some embodiments, the PUFA is arachidonic acid (AA) or docosahexaenoic acid (DHA). In some embodiments, the aldehyde is 4-hydroxynonenal (4-HNE) or 4-hydroxy-2-hexenal (4-HHE). In some embodiments, the CYP4V2 protein is the human CYP4V2 protein (SEQ ID NO: 1) or its non-pathological polymorphism or variant. In some embodiments, the CYP4V2 protein comprises up to two (2) amino acid differences to the human CYP4V2 protein of SEQ ID NO:1 or comprises the following amino acid difference as compared to the human CYP4V2 protein of SEQ ID NO: 1: Gln259Lys, Leu22Val, Met123Val, or Glu275Lys, optionally, wherein the CYP4V2 protein is selected from any one of SEQ ID NO: 4, 6, 7 or 8. F&R Ref No.: 43219-0011WO1 In some embodiments, the vector is a viral vector, a plasmid, a non-viral vector, or a hybrid thereof. In some embodiments, the vector is a naked nucleic acid, liposome, dendrimer, nanoparticle, polymer, polyplexes, or lipid-polymer system. In some embodiments, the nanoparticle is a liposome nanoparticle, solid lipid nanoparticle, or liposome protamine / DNA lipoplex (LPD). In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a herpes simplex virus (HSV) vector, a baculovirus vector, a sendai virus vector, and a retrovirus vector. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the vector comprises a promoter and at least one additional regulatory element operably linked to the sequence encoding the CYP4V2 protein. In some embodiments, the promoter is a retinal pigment epithelium (RPE) cell- specific promoter, a photoreceptor-specific promoter, a retinal cell-specific promoter, a corneal cell-specific promoter, an ocular cell-specific promoter, a cell-specific promoter, a tissue-specific promoter, a constitutive promoter, a ubiquitous promoter, a regulated promoter, an inducible promoter, or a derivative, hybrid or combination thereof. In some embodiments, the promoter is a CAG promoter, a CBA promoter, a CMV promoter, an EF-1 alpha promoter, a EFS promoter, or a derivative or variant thereof. In some embodiments, the CAG promoter is a hybrid CMV early enhancer / Chicken beta actin promoter. It is known in the art that a promoter and other regulatory elements, e.g., the CAG promoter, may have multiple versions. In some embodiments, the promoter comprises or consists of a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO: 9 or 23. In some embodiments, the regulatory element is a polyadenylation (polyA) signal. In some embodiments, the polyA signal is a bovine growth hormone (bGH) PolyA, a SV40 PolyA, SV40 late polyA, or small PolyA (SPA), or a variant thereof. In some embodiments, the regulatory element is a Kozak sequence. In some embodiments, the regulatory element is an enhancer. In some embodiments, the enhancer is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof (exemplary sequence shown in SEQ ID NO: 10). F&R Ref No.: 43219-0011WO1 In some embodiments, the rAAV vector comprises: a VP1, VP2, or VP3 capsid protein selected from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or clade of AAV, or hybrids, variants or derivatives thereof, or a 5′ AAV inverted terminal repeat (ITR) or a 3′ AAV ITR selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or clade of AAV, or mutations, chimeras, variants or fusions thereof. In some embodiments, the vector is a chimeric AAV, a shuffled AAV, or a capsid- modified AAV. In some embodiments, the rAAV vector is a pseudotyped AAV, or a hybrid AAV. In some embodiments, the vector is selected from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 2, AAV2 (Y444F+Y500F+Y730F), AAV2 / 1, AAV2 / 9, AAV2 / 8(Y733F), AAV2 / 6, AAV2 / 4, AAV2 / 7, AAV5, AAV2, AAV8, AAV1, AAV9, AAV6, AAV10, AAV3, AAV4, AAV7, AAV11, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV204, AAV-PHP.B, rh10, and a hybrid, a derivative or variant thereof, or wherein the rAAV vector is a single- stranded AAV vector or a self-complementary AAV (scAAV) vector. In some embodiments, the nucleic acid sequence encoding the CYP4V2 protein shares at least 60%, at least 70%, at least 75%, at least 77%, at least 78%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any one of SEQ ID NO: 2, 3 or 5. In some embodiments, the sequence encoding the CYP4V2 protein is a codon-optimized sequence. In some embodiments, the nucleic acid sequence encoding the CYP4V2 protein is a cDNA sequence. In some embodiments, any part of the nucleic acid molecule comprised within the vector shares at least 43% sequence identity with the entirety of any one of the CYP4V2 expression cassette sequences in SEQ ID NOs: 24 to 29 or at least 43% sequence identity with the entirety of any one of the following sequences: nucleotide (nt) 237 – nt 3579 of SEQ ID NO 24; nt 166 – nt 3515 of SEQ ID NO 25; nt 130 – nt 2097 of SEQ ID NO 26; nt 166 – nt 3515 of SEQ ID NO 27; nt 166 – nt 3515 of SEQ ID NO 28; or nt 166 – nt 2736 of SEQ ID NO 29. It is known in the art that by using a different promoter, adding or deleting an optional regulatory element (e.g., enhancer), a different PolyA, a different CYP4V2 cDNA, different linker sequences, and / or other different regulatory elements / sequences, the nucleic acid molecule comprised within the vector can share very low sequence identity. For F&R Ref No.: 43219-0011WO1 example, SEQ ID NO: 25 and SEQ ID NO: 26 only shares 43% sequence identity. Similarly, nt 166 – nt 3515 of SEQ ID NO 25 only shares 43% sequence identity with nt 130 – nt 2097 of SEQ ID NO 26. In some embodiments, the disease is an ocular disease, a retinal disease or a disease associated with the retinal pigment epithelium (RPE). In one embodiment, the disease is Bietti Crystalline Dystrophy (BCD). In some embodiments, an rAAV vector comprising a capsid protein of SEQ ID NO: 30, 31 or 32 and a nucleic acid molecule comprising a sequence encoding a CYp4V2 protein is provided. In some embodiments, such rAAV vector is used in the methods described above. In one embodiment, such vector is used for treating BCD. In some embodiments, the sequence encoding the CYP4V2 protein is operably linked to a promoter. In some embodiments, the promoter comprises or consists of a nucleic acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO: 9 or 23. In some embodiments, any part of the nucleic acid molecule comprised within the vector shares at least 43% sequence identity with any one of the CYP4V2 expression cassette sequences in SEQ ID NOs: 24 to 29 or at least 43% sequence identity with any one of the following sequences: nucleotide (nt) 237 – nt 3579 of SEQ ID NO 24; nt 166 – nt 3515 of SEQ ID NO 25; nt 130 – nt 2097 of SEQ ID NO 26; nt 166 – nt 3515 of SEQ ID NO 27; nt 166 – nt 3515 of SEQ ID NO 28; or nt 166 – nt 2736 of SEQ ID NO 29. In some embodiments, the sequence encoding the CYP4V2 protein shares at least 60%, at least 70%, at least 77%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 2, 3 or 5. In some embodiments, the capsid protein of such vector is encoded by a nucleic acid molecule sharing at least 60% sequence identity with SEQ ID NO: 33. In one embodiments, such vector is used in subretinal administration. In another embodiment, such vector is delivered to the retina via intravitreal administration. In some embodiments, such vector is used to transduce or treat the photoreceptors or RPE cells. In some embodiments, such vector is used to treat a disease associated with the death, degeneration, dysfunction, dystrophy or atrophy of the RPE or photoreceptors. F&R Ref No.: 43219-0011WO1 In yet another aspect, methods of determining the optimal AAV vector and / or the optimal dose of the AAV vector in AAV-mediated gene therapy treatment for a human subject are provided. Such methods typically include generating iPS-derived cells from the human subject, wherein the iPS-derived cell is the cell type targeted for gene therapy treatment; contacting the iPS-derived cells with a plurality of different AAV vectors and / or a plurality of different doses of the AAV vectors; determining the treatment effect on the iPS-derived cells of the plurality of different AAV vectors and / or the plurality of different doses of the AAV vectors, thereby determining the optimal AAV vector and / or the optimal dose of the AAV vector for the human subject. In some embodiments, the iPS-derived cells are iPS-RPE (iRPE) cells or iPS- photoreceptor cells. As used herein, non-pathological means non-pathogenic. A non-pathological variant means a non-pathogenic variant. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. DESCRIPTION OF DRAWINGS FIG.1 shows that BCD iRPEs are susceptible to shortwave light (blue light) induced- oxidative stress and this phenotype can be quantified by monitoring ROS, 4-HNE and cell death rate. (1A) Schematic showing the establishment of the patient-specific iPSC-derived- cell-based BCD disease platform. Scale bar: 50 µm. (1B) Immunoblot showing CYP4V2 expression levels are lower in iRPE cells from all six BCD patients compared with iRPEs from WT donors. GAPDH serves as a loading control. (1C left): Scheme of blue light induced oxidative stress application: 430 nm blue light exposure. (1C right): Quantification F&R Ref No.: 43219-0011WO1 chart of ROS, 4-HNE level, cell death rate of iRPE from WT donors and BCD patients. (1D) Relative fold change of ROS, 4-HNE and cell death rate in WT and BCD iRPEs. Data are presented as mean ± SD, n=6 biological replicates in all groups, except n=4 biological replicates in WT group of 4-HNE, *P<0.05, **P<0.01, ***P<0.001. FIG.2 shows that significant cellular phenotype differences can be observed in iRPEs of six individual BCD subjects with divergent mutations in CYP4V2 gene. Violin plot of comparison of oxidative stress induced (2A) ROS level, (2B) 4-HNE concentration and (2C) cell death rate of iRPEs of six individual BCD subjects, n=6-13, significance in ROS and 4- HNE chart, is calculated by one-way ANOVA with Tukey’s multiple comparisons tests, *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001; significance in cell death rate chart is calculated by Welch ANOVA test with multiple comparisons, *P<0.05. FIG.3 shows personalized preference to different AAV treatment strategies and distinct outcome measurements of therapeutic efficacy from individual BCD iRPE subjects. (3A) Schematic of AAV gene augmentation therapy strategies, which indicates the timing and dosage of the AAV application. High dosage equals AAV MOI of 1x105(vg / cell), and low dosage equals AAV MOI of 1x104(vg / cell). (3B-3D) Comparison of AAV therapeutic efficacy from different vectors and dosages (indicated by color legend) as measured by (3B) ROS levels, (3C) 4-HNE concentration and (3D) cell death rate under AAV treatment plan according to (3A), respectively. In (3B), (3C) and (3D), respectively, left chart presents the outcome comparison of iRPE from grouped BCD subjects of each AAV treatment strategy, and the right chart presents outcome measurement comparison of iRPE from individual BCD subjects of each AAV treatment strategy. All data are presented as mean ± SD, in (3B), (3C) and (3D) for group comparison, n=6 biological repeats, except n=4 biological replicates in WT group of 4-HNE; for individual comparison, each subject, n=6-23, significance calculated by t-test, *P<0.05, **P<0.01, ***P<0.001. FIG.4 shows distinct AAV transduction rates from individual BCD iRPE subject. (4A) Immunoblot results of CYP4V2 expression levels in individual BCD subject iRPE lines in response to each AAV treatment strategy. (4B) Gating criteria for quantification of GFP- positive iRPE cells. (4C) Comparison of transduction rate between AAV2 and AAV5 from individual iRPE subjects. All data are presented as mean ± SD. n=4 technical repeats, significance is calculated by t-test, *P<0.05, **P<0.01, ***P<0.001. (4D) Comparison of F&R Ref No.: 43219-0011WO1 transduction rate of AAV2 and AAV5. All data are presented as mean ± SD. n=9 biological repeats. Significance is calculated by t-test, P>0.05. FIG.5 shows higher AAV efficacy in homozygous iRPEs carrying deletion mutation than iRPE of homozygous missense mutations. (5A) Correlation of AAV transduction rate and cell death rate of iRPEs with homozygous mutation in CYP4V2 gene. P1, P4, P5 and P6 are BCD iRPE of homozygous mutations. P1, carries homozygous 17-base pair deletion mutation, while P4, P5 and P6 carry three distinct homozygous missense mutation respectively. For each chart, red linear regression lines contain 4 samples of BCD-P1, P4, P5 and P6, while black linear regression lines contain 3 samples of BCD-P4, P5 and P6. R- squares are shown within each chart. (5B) Oxidative-induced cell death rate after AAV2 and AAV5 treatment, respectively, among iRPEs from homozygous mutation BCD subjects. Changes of (5B) ROS, (5C) 4-HNE and (5D) cell death rate measurements comparison due to AAV2 and AAV5 treatment, normalized by transduction rate, respectively, among iRPEs from homozygous mutation BCD subjects. In (5B) to (5E), data are presented as mean ± SD, n= 6-23. Significance is calculated by one-way ANOVA with multiple comparison, **P<0.005, ***P<0.0005, ****P<0.0001. FIG.6 shows cell fate validation and CYP4V2 expression in BCD iRPE. (6A) Representative immunofluorescence images of pluripotency markers in established BCD patient-specific iPSC lines. Red signals represent NANOG and green signals represent Tra-1- 60. Scale bar: 50 µm. (6B) Karyotype result of all six BCD patient-specific iPSC lines. All the BCD iPSC lines have normal chromosome karyotype. (6C) Light microscopy images of BCD iRPE cells. All BCD iRPE present classic human RPE cells morphology, including pigment, hexagonal shape and tight junction between cells. Scale bar: 50 µm. (6D) Immunoblot analysis of mature human RPE marker RPE65 (65 kDa) and CRALBP (36 kDa) in iRPE cells from all six BCD patients. GAPDH serves as the loading control. (6E) Immunoblot of CYP4V2 expression in multi-clones of each BCD individual iRPE samples. GAPDH serves as a loading control. FIG.7 shows that AAV-CYP4V2 reduced major omega-6 and omega-3 PUFAs levels in CYP4V2 patient iRPE cell lines. (7A) Untargeted lipidomic quantification results show that medium-chain poly-unsaturated fatty acid accumulated in BCD iRPEs. Arachidonic acid (AA) had the highest levels compared to other detected PUFAs. Both omega-3 (n3) and F&R Ref No.: 43219-0011WO1 omega-6 (n6) PUFA of 20-Carbon and 22-Carbon are significantly higher in BCD iRPEs compared with WTs. (7B) Lipidomic quantification shows AAVs can reduce major accumulated PUFA levels in BCD iRPE cells. iRPE treated with AAV2 and AAV5 had the lowest levels of PUFAs among all tested serotypes of AAVs compared to untreated BCD cells. Data presented as mean ± SD, n=5-8, ***P<0.001, **P<0.01, *P<0.05. FIG.8 shows CRISPR-mediated genetic repair of the c.802-8_810del17insGC mutation in the CYP4V2 gene and rescued cellular phenotypes in the genetically repaired P1 isogenic iRPE cells. (8A) Upper scheme image showed PAM and gRNA binding site of CYP4V2 gene. The CRISPR guide RNA and Cas9 protein were provided in a ribonucleoprotein (RNP) complex. Lower image of dideoxy sequencing of BCD Patient 1 iPSCs and the corresponding isogenic control iPSC line, indicating a repair of the parental homozygous mutations in isogenic iPSC line. Top panel labeled with WT as CYP4V2 reference sequencing. (8B left): Representative brightfield images showed typical human RPE cellular morphology of isogenic control iRPEs. Scale bar: 20 µm. (8B middle): expression of mature RPE markers RPE65 and CRALBP in isogenic control iRPE. (8B right): CYP4V2 protein expression in iRPEs from WT donor, BCD-P1, and CRISPR repaired BCD-P1 isogenic line. (8C) Representative fluorescence microscopy images of propidium iodide (red) labeled iRPE cell death status after blue light exposure, cell line labeled within each image, red signal indicated dead cells. Scale bar: 20 µm. (8D) Quantification of ROS, 4- HNE level, cells death rate in iRPEs from WT donor, BCD-P1 and BCD-P1 isogenic control line. Data are presented as mean ± SD, n=4-16, significance calculated by t-test, ***P<0.001. FIG.9 shows that AAV2-null and AAV5-null failed to show therapeutics effect. (9A left chart): AAV2-null failed to mitigate the increased ROS level caused by blue light exposure in BCD- iPREs. (9A right chart): ROS relatively changes before and after blue light exposure in BCD iRPEs and AAV2-null treated BCD iRPEs are similar. (9B left chart): AAV2-null failed to reduce ROS level in BCD iRPEs compared with AAV2-CYP4V2. (9B right chart): AAV2-null and AAV5-null failed to rescue cell death caused by blue light exposure in BCD-iRPEs. All data presented as mean ± SD, n=4, ***P<0.001, *P<0.05. FIG.10 shows normalized outcome measurements fold changes of ROS, 4-HNE and cell death rate from different AAV treatment. Outcome values of each biomarker, (10A) F&R Ref No.: 43219-0011WO1 ROS, (10B) 4-HNE, (10C) cell death rate, are normalized against the values of blue light exposure without any treatment. All data presented as mean ± SD, n=6-23. FIG.11 shows in vitro culture time of BCD iRPE affects AAV transduction rate but does not affect the personalized AAV serotype preference. (11A) Representative gating images of BCD-P1 at 6-month culturing (upper panel) and 12-month culturing (lower panel). Left panel images represent all the iRPE cells tested by FACS. Compared with 6-month cultured iRPE, longer culturing time (12-month) iRPE cells present various on cell size, more complexity of the cytoplasm, and more cell debris in the entire population. Right panel images represent the gating of GFP positive iRPE cells, which reflects the transduction rate of AAV2.12-month cultured iRPE have lower AAV transduction rate than 6-month cultured iRPE cells. SSC, side scatter (reflecting complexity); FSC, forward scatter (reflecting size). (11B) Quantification result of AAV2 and AAV5 transduction rate of 6-month culture iRPE and 12-month cultured iRPE, respectively. Data presented as average ± SD, significance is calculated by t-test, n=4, P*<0.05, P**<0.01. (11C) Trending of AAV2 and AAV5 transduction rate changing according to culturing time increasing, respectively. (11D) Changing of transduction rate according to culturing time is not altering the personalized AAV serotype preference. Data presented as mean ± SD, significance is calculated by t-test, n=4, P>0.05. FIG.12 shows images of processing cell viability quantification. (12A upper picture): live iRPE cells labeled by Calcein AM present green fluorescent signal; (12A lower picture): dead iRPE cells labeled by Propidium Iodide (PI) present red fluorescent signal. Scale bar: 20 µm. (12B). Binary images (converted from fluorescent images) with counted particles of iRPE from WT and BCD patient after blue light exposure. Sample names labeled within each image. Scale bar: 50 µm. DETAILED DESCRIPTION Patient-specific induced pluripotent stem cell (iPSC) allow treatments to be tested on patient cells before they are enrolled into trials. In recent years, patient-specific cell lines (i.e., ex vivo tissues and organoids) have been used to model certain genetic disorders. Moreover, patient iPSCs may be used to fill the gaps in modeling diseases with no suitable animal models and generate preclinical proof-of-concept efficacy data to support IND F&R Ref No.: 43219-0011WO1 application for clinical trials. Our results provide an example of using patient-specific iPSC- based cellular models to screen for individualized optimal vectors and dosing, which can be referenced in personalized gene therapy. Furthermore, our results suggest that adeno- associated virus (AAV)-mediated gene therapy human clinical trials might benefit from testing on patient-derived cells to prioritize inclusion into gene supplementation Phase II trials. Bietti crystalline dystrophy (BCD, also known as Bietti crystalline corneoretinal dystrophy; OMIM 210370) is an autosomal recessive retinal degenerative disorder caused by mutations in the CYP4V2 gene. Clinically, BCD is associated with retinal pigment epithelium (RPE) cell death, and BCD patients’ onset age and phenotype manifestations are highly variable. Genetically, over 100 CYP4V2 mutations have been reported. Given the wide genotype and phenotype variability among patients, BCD is an ideal disease for studying patient individual differences. Moreover, an in-depth worldwide CYP4V2 mutation carrier frequency and BCD genetic prevalence study estimated that BCD may affect about 67,000 individuals worldwide and revealed that the most common CYP4V2 mutations are different among various populations. These factors underpin the importance of establishing BCD patient-specific iPSC-based models of different ethnic backgrounds with distinct mutations to generate highly representative results and assess individual variations. In recent years, researchers began to use iPSC-derived retinal pigment epithelium (iRPE) cells to study BCD and to test drug candidates, including gene therapies. However, these studies did not research individual patient differences due to the limited number of patient samples and mutations. Further, no study to date has tested different AAV serotypes or doses. CYP4V2 (Cytochrome P450, Family 4, Subfamily V, Polypeptide 2, (OMIM 608614), synonym: CYP4AH1) is one of the proteins in the cytochrome P450 superfamily and a member of the heme thiolate cytochrome P450 subfamily 4 (CYP4). Human CYP4V2 non-pathological polymorphisms and variants can be found in scientific and medical literature, as well as in online database, including without limitation, LOVD (databases.lovd.nl / shared / genes / CYP4V2 on the World Wide Web), OMIM (omim.org / allelicVariant / 608614 on the World Wide Web), ClinVar (ncbi.nlm.nih.gov / clinvar?term=608614[MIM] on the World Wide Web) and gnomAD (on the World Wide Web: gnomad.broadinstitute.org / ) F&R Ref No.: 43219-0011WO1 In this study, we established iRPE cell lines from six BCD patients with three different ethnic origins harboring distinct CYP4V2 mutations. Interestingly, these iRPE cell lines exhibited individual differences in phenotypes, including the clinically significant phenotype of RPE cell death. Importantly, our iRPE from BCD patients successfully overcame the limitations observed in Cyp4v3- / -mouse models (Cyp4v3 is the murine ortholog of human CYP4V2). Our iRPE cultures mimic cell death seen in BCD patients. It should be noted that no RPE cell death has been reported in Cyp4v3- / -mouse model. Due to the absence of appropriate Cyp4v3- / -mouse models, the mechanisms underlying RPE death in BCD remain unclear. In addition, we tested different AAV-CYP4V2 vector serotypes and dosages in these iRPE cell lines. Our results revealed that the AAV vectors achieved different treatment efficacy in different patients’ iRPE cell lines. These results explained the variability at the cellular level in gene therapy efficacy among patients and showed the ability of patient cells based “disease-on-dish” can contribute to developing precision medicine approach in future gene therapies. To successfully translate an in vitro study into a clinical trial, two key points should be kept in mind when conducting the bench-to-bedside research: first, the patient cell-based disease model should recapitulate the disease features from patients, and second, outcome(s) which can be measured in patient cell-based disease models, need to be identified. Clinically, BCD is associated with RPE atrophy. The eye is the light-sensing organ and a key function of the RPE is light absorption. Light is also a source of oxidative stress to the retina, which should be a key factor to be considered when modelling a retina disease in vitro. However, none of the previously published research on BCD iRPE has considered the impact of photodamage on BCD iRPE. Furthermore, no prior studies have investigated whether BCD gene therapy or other drug candidates can mitigate light-induced RPE cell death. Blue light, pervasive in our environment from both sunlight and artificial sources such as office lighting and electronic devices (TVs, computer monitors, smartphones, notebooks, and tablets), poses a significant risk. Our study reveals that blue light exposure significantly induces lethal oxidative stress and increases the cell death rate in iRPE cells from BCD patients compared to those from wild-type individuals. These findings underscore the critical role of light-induced retinal damage in the development of BCD. Furthermore, F&R Ref No.: 43219-0011WO1 this in vitro phenotype in BCD patient iRPE cells can be accurately measured through cell viability assays, establishing the level of cell death as a strong biomarker and a clinically relevant indicator for evaluating the efficacy of potential treatments, including gene therapy. This insight should guide ophthalmologists to carefully consider the implications of prescribing intensive light exposure tests, such as retinal autofluorescence, for BCD patients. Additionally, our research has shown that gene therapy, specifically AAV-mediated therapy, can significantly mitigate light-induced cell death in BCD iRPE cells. This constitutes the first proof-of-concept study indicating that AAV-CYP4V2 gene therapy could be an effective treatment for light-induced RPE damage in BCD. To understand the connection between biochemical abnormalities (such as unusually high levels of PUFAs) and the clinical manifestations (notably, increased cell death) in iRPE cells of BCD patients, we focused on arachidonic acid (AA, C20:4, omega-6), the predominant PUFA accumulated in BCD iRPE cells (FIG.7A). Our objective was to identify a biomarker directly linked to iRPE cell death. We hypothesized that exposure to blue light could cause the energy from short-wavelength light to interact with the numerous double bonds in the accumulated PUFAs within BCD iRPE cells, leading to lipid peroxidation. The resulting peroxidation products of these PUFAs might be the primary cause of cell death in BCD RPE cells. We considered two highly reactive aldehydes produced by PUFA degradation as potential culprits: 4-hydroxy-2-hexenal (4-HHE) (from omega-3 PUFAs) and 4-HNE (from omega-6 PUFAs). Given the significant accumulation of AA (an omega-6 PUFA) in BCD iRPE cells, we measured the levels of 4-HNE after blue light exposure and found abnormally high concentrations correlating with cell death in BCD RPE cells. Consequently, 4-HNE levels can serve as a measurable molecular biomarker to assess the effectiveness of various therapeutic approaches in cell-based disease models specific to BCD patients. Our study fills the gaps in other BCD patient iRPE studies by examining individual differences in phenotype and response to different AAV-mediated gene therapy treatment strategies among patient iRPEs from a diverse pool of six BCD patients of three ethnic origins harboring distinct CYP4V2 mutations. No more than three BCD patients’ iRPEs have been studied and analyzed in any BCD research previously. In previous BCD studies, all the studied subjects were from the single ethnic origin of east Asian (either Chinese or Japanese). F&R Ref No.: 43219-0011WO1 To date, over 100 CYP4V2 mutations have been reported, CYP4V2 mutation carrier frequency study revealed that the prevalence of BCD may have been underestimated, and the most common CYP4V2 mutations are different among various populations. Here, our research timely reported results from iRPEs of six individual BCD subjects of different ethnic origins with distinct CYP4V2 mutations common in each of East Asian, European or South Asian populations. Among the six BCD subjects, distinct mutations caused various expression levels of CYP4V2 protein in BCD iRPEs (FIG.1B). Moreover, after exposure to blue light, the individual ROS, 4-HNE and cell death levels among all the six BCD patient iRPEs were variable and significantly different. The high cell death rate observed in the iRPE cell lines BCD-P1, P4, P5 and P6 (FIG.2), are consistent with prior studies which reported P1’s mutation c.802-8_810del17insGC (deletion of Exon 7) as a severe mutation, and P4’s mutation homozygous p.Arg400Cys and P6’s mutation homozygous p.Met66Arg to be associated with early onset. P5’s mutation p.Ile111Thr is considered to be deleterious but patients harboring homozygous p.Ile111Thr have shown wide phenotype variability, which suggests environmental or epigenetic factors may affect disease progression. Next, we investigated if the personalized AAV-mediated gene augmentation treatment approach is preferable for individual BCD subjects, namely, we tested that if grouped BCD samples favor the same AAV serotype and dosage. We grouped the six individual BCD cellular disease models under each AAV treatment strategy to evaluate the overall therapeutic efficacy. The results showed that the high dose of AAV2 is most effective at treating this inherited retinal disorder (FIGs.3B, 3C and 3D left charts). In homozygous BCD iRPEs, all the four cellular models (BCD-P1, BCD-P4, BCD-P5 and BCD-P6) in this study only responded significantly to high-dose treatments (FIGs.3C-3E). We also observed that BCD-P1 and BCD-P5 responded the best to a high dose of AAV5, while BCD-P4 and BCD-P6 responded the best to the high dose of AAV2 (FIGs.3B, 3C and 3D right and left charts). In the iRPEs of BCD-P2 and BCD-P3, the high dose of AAV2 and the high dose of AAV5 had similar rescue effects. However, BCD-P2 responded best to the high dose of AAV5, while BCD-P3 responded best to the high dose of AAV2 (FIGs.3B, 3C and 3D right and left charts). Notably, iRPEs of BCD-P2, responded the most favorably to all tested treatment strategies. The average cell death rates of AAV-treated iRPEs of BCD-P2 from each AAV-treatment plan were similar, independent of dosage. Our findings demonstrate the F&R Ref No.: 43219-0011WO1 importance of analyzing individual responses to treatment as they may differ from the aggregate response to treatment. Additionally, there are also personalized response to AAV vector serotypes among BCD patient-specific iRPE cellular models. To further validate the individual preference for various AAV vectors, we assessed the AAV transduction efficiency in the iRPE cells of each subject. Using CYP4V2 immunoblots and AAV-eGFP (for serotypes AAV2 and AAV5), we confirmed the personalized responses of BCD patient- specific iRPE cells to different AAV vectors (FIG.4). These findings indicate that patient- specific stem cell-derived cellular models, such as iRPEs, hold the potential to be utilized to screen the most effective gene therapy vector for an individual patient. This approach supports the development of precision gene therapy, taking advantage of the response of each patient for specific AAV vectors at the cellular level. Among all four homozygous BCD subjects, BCD-P1 carries the most common mutation in CYP4V2 gene: a 17-base deletion in exon 7, while BCD-P4, P5 and P6 carry 3 distinct missense mutations, respectively. Oxidative stress-induced ROS, 4-HNE concentration and cell death rates in all four homozygous BCD patient iRPE cells were similar (FIG.2). Interestingly, compared to the other three homozygous models (BCD-P4, BCD-P5 and BCD-P6), the oxidative stress-induced phenotype in BCD-P1’s iRPE was more readily reversed by AAV-mediated gene augmentation therapy (FIG.5). Even though the AAV transduction rate of BCD-P1 was lower than the other three homozygous BCD iRPEs (FIG.4C), cell death in BCD-P1 iRPEs was rescued the most by AAV treatment compared to the other homozygous BCD iRPE cell lines. In the AAV2 treatment, BCD-P1 had a greater reduction in all the three outcome biomarkers compared to the other three homozygous BCD iRPE subjects (BCD-P4, P5, P6) (FIG.5). As autosomal recessive genetic disorders, usually the null phenotype is expected to be found in cellular or molecular level. However, scientists have found in CNGB3 associated recessive achromatopsia, certain disease associated mutations caused “gain-of-function” alterations. In our study, homozygous missense mutations in BCD-P4, BCD-P5 and BCD-P6 may lead to “gain-of-function” phenotypes by interference with the multimerization of transgenic CYP4V2. In contrast, the homozygous deletion mutation in BCD-P1 caused a “loss-of-function” null phenotype, that may be more easily corrected by gene augmentation therapies. F&R Ref No.: 43219-0011WO1 Currently, gene therapy is increasingly acknowledged by the Centers for Medicare & Medicaid Services (CMS) as a promising method for treating monogenic disorders. One of the most significant challenges that scientists and physicians face is the variability in responses from individual subjects, often referred to as “chemical individuality”. Despite this, the prevalent strategy for developing gene augmentation therapies still relies on a “one vector” and “one dose” approach for all patients diagnosed with the same condition. Our study revealed that the diseased tissues of individual patients respond differently to AAV vectors. This diversity leads to varied patient responses to specific gene therapy treatments, including the use of different vectors and doses. However, our study faces limitations, primarily due to the limited number of BCD patient cell lines analyzed (six individuals). Ideally, further research should expand the collection and analysis of iRPE cells from a broader cohort of BCD patients, even though amassing a large sample size is a well-known challenge in the study of rare diseases. Furthermore, to investigate if the phenotype differences and distinct response among patients to a specific AAV treatment strategy is due to mutation differences or other individual variability among patients, future studies should enroll not only patients of divergent mutations, but also multiple patients for each specific mutation, which may be feasible for more common disorders. Finally, considering the systemic immune response and other in vivo features relating the communication among different tissues, the in vitro testing on single type of cells cannot completely reflected the actual in vivo environment. The differences we observed in our in vitro transduction studies suggest the need to better understand the variability of individuals within the monogenic inherited retinal disorders. Taken together, adopting a personalized medicine approach to determine the most effective therapeutic vector and dosage for each individual could be crucial for enhancing treatment outcomes, even before an Investigational New Drug (IND) application for phase 1 trials. Our findings support the method of first testing candidate vectors on cellular models derived from the patient's stem cells before potential translation of the treatment strategy to patients. Hence, clinical trials could be improved by recruiting patients whose cells have undergone testing in culture and have demonstrated favorable responses to specific candidate vectors. Optimization of vector and dosage in patient cells should improve the efficacy of F&R Ref No.: 43219-0011WO1 gene therapies for genetic disorders, validating the principle and core value of precision medicine: designing the right dose of the right vector for the right person. It is known in the art that it is common for a regulatory sequence or a hybrid regulatory sequence to have multiple versions and have more than one names. For example, various promoters, enhancers and polyA signals have multiple versions, including without limitation, the CMV promoter, EF1α promoter, the WPRE enhancer, and the SV40 polyA signal. The CAG promoter has multiple alternative names including without limitation, the CBA promoter, CB promoter or CAGGS promoter. In addition, it is also known the in art that a regulatory sequence can be shortened, modified or combined with other sequences to generate a derivative or variant, e.g., the CAG (a / k / a CBA, CB or CAGGS) promoter is a hybrid of CMV immediate early enhancer, chicken beta actin promoter and rabbit beta- globin gene, the smCBA promoter is a truncated CAG promoter. the CBSB promoter is a shortened CAG promoter, differing by about 152 bp at the 5′ end of the CMV immediate early enhancer. Furthermore, a regulatory sequence can be termed differently, e.g., a post- transcriptional regulatory element such as HPRE or WPRE can also be referred to as an enhancer. Any regulatory sequences described herein contemplate all variations, derivatives and / or hybrids of such regulatory sequence. Any exemplary sequence provided herein relating to a regulatory sequence is exemplary in nature and does not limit the definition or scope of such regulatory sequence to the one shown in the exemplary sequence. The full-length CAG promoter is about 1.7 Kb, see SEQ ID NO: 9 for exemplary sequence. It should be understood that a variant of the CAG promoter can also be used. For example, a shorter version (0.9-1 kb) of the full-length CAG promoter and includes the cytomegalovirus (CMV) enhancer and the Chicken beta actin promoter can also be used (see SEQ ID NO: 23 for exemplary sequence of a short version of the CAG promoter). The CAG promoter is sometimes also referred to as the CB promoter or CBA promoter. It is known in the field that alternative codons can be used to encode the same amino acid. Algorithms, software and tools for designing codon-optimized nucleic acid sequences are also available. Hence, nucleic acid sequences encoding the same protein can share very low sequence identity, e.g., below 40%, below 50%, below 60%, below 70%, below 80%, below 90% or below 95% sequence identity. F&R Ref No.: 43219-0011WO1 For example, SEQ ID NO 2 and 3, both encoding the human CYP4V2 protein (SEQ ID NO: 1), only share 77% sequence identity. By using different promoters or variants thereof, different polyA, and / or different nucleic acid sequences encoding the CYP4V2 protein, different AAV ITRs, including or excluding an enhancer or other regulatory elements, two CYP4V2 expression cassettes can share very low sequence identity. For example, SEQ ID NO: 25 and 26 only share 43% sequence identity. Similarly, from the start of the promoter sequence to the stop codon of the CYp4V2 encoding sequence, the shared sequence identity between two constructs can be very low. For example, nucleotide (nt) 166 – nt 3515 of SEQ ID NO 25 and nt 130 – nt 2097 of SEQ ID NO 26 only share 43% sequence identity. As used herein, "operably linked" means that a promoter and / or other regulatory element(s) are positioned relative to a nucleic acid coding sequence in such a way as to direct, influence or regulate expression of the nucleic acid coding sequence. A regulatory element can be "operably linked" with a nucleic acid coding sequence in the same vector or in a different vector. One or more regulatory sequences operably linked to a nucleic acid coding sequence can be contiguous and / or can act in trans or at a distance to direct, influence or regulate expression of the nucleic acid coding sequence. Among the regulatory sequences, a promoter is essential, while some other regulatory sequences such enhancers, introns and terminators can be beneficial but are optional. The term “effective amount” or “effective dosage” or "therapeutically effective amount" refers to an amount of a compound (e.g., a vector) sufficient and / or suitable to effect treatment when administered to a subject in need of such treatment. The effective amount will vary depending upon the number of cells targeted for treatment, the specific activity of the therapeutic agent being used, the severity of the patient’s disease state, and the age, physical condition, existence of other disease states, and nutritional status of the subject. Additionally, route of administration, delivery device, and other medication and / or treatment the patient may be receiving will affect the determination of the effective amount of the therapeutic agent to administer. For example, the therapeutically effective amount of the vector can range between about 102vector genome (vg) per cell to about 107vg per cell multiplied by the number of cells targeted for treatment. For subretinal or intravitreal F&R Ref No.: 43219-0011WO1 administration for treating a retinal disease, the therapeutically effective amount of the vector can range between about 108vg to about 1013vg per eye. Routes of administration, surgical techniques and delivery devices used in gene therapy administration is known in the field. For example, for retinal gene therapy, delivery methods include but not limited to subretinal or intravitreal administration can be used. In accordance with the present invention, there may be employed molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims. EXAMPLES Example 1—Generation of Induced Pluripotent Stem Cell (iPSC) Derived Retinal Pigment Epithelium (RPE) Cells (iRPEs) from BCD Patients and Wild Type (WT) Individuals Skin fibroblast cells from six BCD patients and their age and sex matched WT control donors were each plated and cultured in a 12-well plate until the cells became adherent and approximately 70%-80% confluent. The culture medium was then removed and the cells were transfected with CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit, (A16517, Life Technologies) as previously described (32-34). iPSC differentiation started at passage 3 to 6 for all iPSC lines of BCD patients and healthy controls according to previously published protocols (35). In brief, iPSC colonies were cultured to confluence in 6-well culture dishes (Costar, CORNING) pre-treated with 1:50 diluted Matrigel (CORNING, 356230) in differentiation medium consisting of Knock- Out (KO) DMEM (Thermo Fisher Scientific, 10829018), 15% KO serum replacement (Thermo Fisher Scientific, 10829028), 1% non-essential amino acids (Thermo Fisher Scientific, 11140050), 2 mmol / L glutamine (Thermo Fisher Scientific, 35050061), 50 U / ml penicillin-streptomycin (Thermo Fisher Scientific, 10378016), and 10 mmol / L nicotinamide (Sigma-Aldrich, N0636) for the first 14 days. During the 15th to 28th days of differentiation, medium was supplemented with 100 ng / ml human Activin-A (PeproTech, 120-14). From day 29, Activin-A was removed until differentiation was completed. After 8–10 weeks, pigmented clusters formed and were manually picked and plated on Matrigel-coated dishes. F&R Ref No.: 43219-0011WO1 Those cells were maintained in RPE culture medium, MEM alpha modification(Sigma- Aldrich, M-4526)-based medium, which contains N1 supplement (5 ml per 500 ml medium), taurine (125 mg per 500 ml medium), hydrocortisone (10 μg per 500 ml medium), triiodo- thyronin (0.0065 μg per 500 ml medium) (all from Sigma-Aldrich), 2 mmol / L glutamine, 50 U / ml penicillin-streptomycin, 1% non-essential amino acids and 5% fetal bovine serum (all from GIBCO) and cultured for another 6-8 weeks to allow them to form a functional monolayer or longer periods as specified below before testing for functional assays (35, 36). The RPE cells differentiated from BCD patients’ iPSCs were observed under light microscopy and distinct RPE pigment and hexagonal cell shapes were seen (FIG.6). In addition to morphological distinctions, iPSC-derived RPE cells from BCD patients also were validated by the presence of mature RPE-specific markers, RPE65, CRALBP. Example 2—Construction and Production of Recombinant Adeno-Associated Virus (AAV) Vectors AAV vectors encoding human CYP4V2 protein (SEQ ID NO:1) (AAV-CYP4V2) vectors tested in this study were provided by Reflection Biotechnologies and custom made by Vector Biolabs and the Viral Vector Core of Andelyn Biosciences. A human CYP4V2 cDNA (SEQ ID NO:2) or a codon-optimized cDNA (SEQ ID NO:3) (ordered from GenScript by Reflection Biotechnologies) encoding human CYP4V2 protein (NP_997235.3) was packaged in the AAV-CYP4V2 vectors. See Table 1 for detailed information of AAV vectors used in this study. The AAV2-CYP4V2 and AAV5-CYP4V2 vectors used in generating the immunoblots, ROS, 4-HNE and cell viability results shown in this paper were manufactured by Andelyn Biosciences. AAV-CYP4V2 vector productions were accomplished using a 3-plasmid transfection method in HEK 293 cells (37). AAV2-null and AAV5-null vectors (provided by Reflection Biotechnologies and produced by Vector Biolabs) that do not express any transgene were used as negative control. AAV2-eGFP and AAV5-eGFP were purchased from Andelyn Biosciences by Reflection Biotechnologies. F&R Ref No.: 43219-0011WO1 Table 1. AAV-CYP4V2 vector list Serotype Promoter Transgene Packaged Other regulatory elements Manufacturer AAV2 CAG human CYP4V2 cDNA WPRE enhancer, (codon-optimized) bGH PolyA Vector Biolabs AAV5 CAG human CYP4V2 cDNA WPRE enhancer, bGH PolyA Vector Biolabs AAV5-op CAG human CYP4V2 cDNA WPRE enhancer, (codon-optimized) bGH PolyA Vector Biolabs scAAV1* EFS human CYP4V2 cDNA (codon-optimized) Small PolyA (SPA) Vector Biolabs scAAV5* EFS human CYP4V2 cDNA (codon-optimized) Small PolyA (SPA) Vector Biolabs scAAV9* EFS human CYP4V2 cDNA (codon-optimized) Small PolyA (SPA) Vector Biolabs AAV2 CAG human CYP4V2 cDNA WPRE enhancer, bGH PolyA Andelyn Biosciences AAV5 CAG human CYP4V2 cDNA WPRE enhancer, bGH PolyA Andelyn Biosciences * “sc” indicates self-complementary AAV Inverted terminal repeats (ITR) and regulatory element exemplary sequences (e.g., CAG promoter, EFS promoter, WPRE enhancer, bGH polyA, SPA) are provided in Example 22. Human CYP4V2 cDNA and human CYP4V2 cDNA (codon-optimized) sequences used in AAV.CYP4V2 vectors in table 1 are set forth in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. CYP4V2 expression cassette exemplary sequences used in the AAV.CYP4V2 vectors listed in Table 1 are provided in SEQ ID NOs: 24 to 26. Example 3—Transduction of AAV Vectors in BCD iRPEs iRPE cells derived from BCD patients were transfected with various AAV vectors described herein in serum-free RPE medium. After 1 day, the virus-containing medium was replaced with fresh serum-containing RPE medium to continue RPE culture. To assess the therapeutic effects of different dosages, different multiplicities of infection (MOI) were tested. For iRPE samples used in the untargeted lipidomic assay of free fatty acids, the AAV treatment was applied during the sixth week of culturing, then FFA were tested during the 4 weeks post-AAV transfection (10thweeks of culturing). For iRPE samples used in outcome measurement of ROS, 4-HNE and cell viability, AAV treatments were applied according to the treatment plan presented in FIG.3A. F&R Ref No.: 43219-0011WO1 Example 4—Establishment of BCD Isogenic Control Cell Lines using the CRISPR / Cas9 System BCD P1 isogenic control cell line which repaired the c.802-8_810del17insGC mutation in the CYP4V2 gene in BCD P1 iPSC was generated by CRISPR / Cas9 gene editing approach. DeskGen software was used to design the guide RNA. The guide RNA (Custom Alt-R®CRISPR-Cas9 sgRNA, Integrated DNA Technologies) and Cas9 protein (Catalog #: 1081060, Integrated DNA Technologies) in a ribonucleoprotein (RNP or protein-RNA) complex and the donor template (Integrated DNA Technologies) were used to correct / repair the c.802-8_810del17insGC mutation in the CYP4V2 gene in BCD patient P1 iPSC to create the isogenic control of P1. Two million iPSCs from BCD-P1 were electroporated with 30 pmol Cas9 protein + 270 pmol single-guide RNA + 10-20 μg single-stranded DNA oligonucleotides donor for a 100 μL final reaction volume (in reaction buffer from Lonza kit P3) in Lonza 4D Nucleofector (Lonza, V4XP-3032). Afterwards, iPSCs survived from nucleofection were sorted in single cell expansion, and then sequenced to identify the homologous genetically repaired iPSCs no longer harboring the c.802-8_810del17insGC mutation, which were then differentiated into iRPEs. Sequence information of gRNA, donor template and primers sequences used for amplifying the CYP4V2 homology-directed repair (HDR) site were summarized in Table 4. Table 4. Material information used in CRISPR / CAS9 mediated gene editing in BCD-P1 iPSCsgRNA sequenceUUCAUUGGCGUUCAUUUCAUSingle strand donor template sequenceTAGCATATTTTATAAGAAAATGTGTTAACTAGGGTGCATCCAAGTCCAAACAGAAGCATGTGATTATCATTC AAATCATACAGGTCATCGCTGAACGGGCtAATGAAA TGAACGCtAATGAAGACTGTAGAGGTGATGGCAGG GGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTT CTTGACTTGCTTTTAAGTGTPrimer of amplifying the CYP4V2 HDRForward: AGAGCCTATGTTGTCGAAATGTTGReverse: GCCTGTTCCCTTCGTCATCA Example 5—Immunoblots Cells were lysed in M-PER mammalian protein extraction reagent buffer (Cat# 78501; Pierce) containing proteinase inhibitors (Roche, 11836170001). Total protein was F&R Ref No.: 43219-0011WO1 quantified using a Bio-Rad protein reader. Protein samples (35 μg / sample per lane) were then separated on a 10% Tris–Cl gradient gel and electroblotted onto a nitrocellulose membrane. Membranes were incubated in blocking buffer for 1 hour at room temperature, washed 3 times in PBS + 0.1% Tween-20 for 10 minutes each, and then incubated with the primary antibody in blocking buffer overnight at 4°C. Primary antibodies against the following proteins were used for Western blots: CYP4V2 (rabbit polyclonal, 1:1500, Millipore Sigma, SAB1410565); RPE65 (mouse monoclonal, 1:7,500, Novus Biologicals, NB100-355); CRALBP (rabbit polyclonal, 1:10,000, Abcam, ab15051); and GAPDH (mouse monoclonal, 1:5,000, Abcam, ab9485). Anti-mouse and anti-rabbit secondary antibodies were obtained from Abcam (ab99697 and ab6728) and used at a concentration of 1:5,000 for CYP4V2 and 1:20,000 for the others. Example 6—Untargeted Free Fatty Acids Liquid Chromatography Mass Spectrometry (LC- MS) This assay was performed at the Columbia University Medical Center (CUMC) biomarker core facility. In brief, free fatty acids were chloroform-methanol extracted. After culturing for 10 weeks (both AAV-treated iRPEs and non-treated iRPEs), about 1 million iRPE cells were homogenized in 150 μL water, and 100 μL of homogenate were mixed with 3 mL chloroform:methanol (v / v = 2:1) containing internal standards (Palmitic acid-D31, C12 ceramide, C25 ceramide, C17 sphingosine, C17 sphinganine). The sample was vortexed well, and 0.5 mL of water was added to allow for phase separation. The mixture was vortexed again and centrifuged at 3,000 g for 10 minutes at 4°C. The lower organic phase was transferred to a clean glass tube using a Pasteur pipette. Two milliliters of chloroform were added to the residual aqueous phase, followed by vortex mixing and centrifugation to extract any remaining lipids. The lower organic phases were pooled and evaporated under nitrogen at 37°C. The extracted lipids were reconstituted in 50 μL methanol-acetonitrile (v / v = 1:1) and transferred to liquid chromatography autosampler vials for injection. All assays were performed on a Waters Xevo TQ MS ACQUITY UPLC system (Waters Corporation). Free fatty acids were eluted using a 100-mm Waters ACQUITY UPLC HSS C18 column and monitored using the negatively selected ion recording (SIR) method. F&R Ref No.: 43219-0011WO1 Example 7—Blue Light Exposure iRPE cells were seeded in 96-well black / clear bottom plate. After designated time points, they were exposed to 430±20 nm (blue) light at 1.5 mW / cm2for 25 mins for iRPE cultured in 96-well black / clear bottom plate in PBS (+) containing 10 μg / ml glucose. The same seeding density was used for all cell lines. After blue light exposure, treated cells were fed with fresh RPE medium and recovered in the incubator of 5% CO2 and 37°C overnight, then, all the cells were processed for outcome measurements. Example 8—Reactive Oxygen Species (ROS) Assay To observe the effect of blue light exposure on ROS / superoxide levels (a measurement of oxidative stress) on iRPE cells, we probed ROS on iRPE samples of WT and BCD patients (both untreated and AAV-treated) using the ROS Detection Cell-Based Assay Kit (DHE) (Cayman Chemical, Item No.601290). In brief, RPE cells cultured in black / clear bottom 96-well plate (Thermo Scientific™, Catalog number: 165305) were loaded with 10 μM Superoxide Detection Reagent, a cell-permeable probe that reacts with superoxide to produce a red fluorescent product. Next, the RPE samples were divided into two groups. Group 1 was exposed to blue light for 25 mins while group 2 was kept at room temperature without exposure to blue light. Then all samples were washed by ROS washing buffer carefully for 3 times. The plate was read using a fluorescent reader (Molecular Devices, SpectraMax®iD3) at an excitation wavelength of 485 nm and an emission wavelength of 600 nm. Example 9—Lipid Peroxidation (4-HNE) Assay Total protein from 5 x 106iRPE cells per testing sample were extracted and normalized as described above. Samples were tested by Lipid Peroxidation (4-HNE) Assay Kit (Abcam, ab238538) for detection and quantification of 4-hydroxynonenal (4-HNE)- protein adducts. Assays were performed according to the protocol provided by the kit manufacturer. F&R Ref No.: 43219-0011WO1 Example 10—Cell Viability Assay Live / healthy iRPE cells were labeled by cell-permeant dye Calcein AM (Thermo Fisher Scientific, Catalog no: C3099) at a final concentration of 3 μmol / ml PBS(+) and dead / sick cells were labeled by Propidium Iodide (PI) (Thermo Fisher Scientific, Catalog no.: P3566) at a final concentration of 2 μg / ml PBS(+) at room temperature for 1 hour. Since PI is DNA-binding and it is not permeant to live cells, it is commonly used to detect dead cells. Then after washing with PBS (-), cellular fluorescent levels were observed, and photos were taken using an inverted fluorescent microscope (Nikon Eclipse Ts2R) at 20-times magnification. Dead / live cell numbers were calculated after photos were processed by ImageJ (Fiji). For quantification of the results, all images were processed using Image J (representative processing image shown in FIG.12) as follows: 1. Open image: image type 8 bit, table: red / green 2. FFT bandpass filter range 3-40 pixels, tolerance of direction: 5% 3. Threshold adjust by default and watershed applied. 4. Analyze particles by size from 100 (red / dead cells) or 300 (green / live cells) to infinity (pixels2) Example 11—Flow Cytometry iRPE were dissociated with 0.05% trypsin for 20 mins (Thermo Fisher Scientific, Catalog No: 25300054) and resuspended in HBSS (Thermo Fisher Scientific Catalog No: 14025092) with 1 µg / ml DAPI. Green fluorescent positive cells were detected by fluorescent channel FITC. DAPI positive cells were detected by fluorescent channel Brilliant Violet 421. Data were acquired on cell sorter Sony MA900 and analyzed with NovoExpress Version 1.5.6. Example 12—Statistics Statistical analyses were performed by GraphPad Prism. An unpaired Student’s t-test was used for comparison between any 2 groups analyzed. One-way ANOVA with Tukey’s test and Wlech ANOVA with multiple comparison were used for the comparison of more than 2 groups. P-values of less than 0.05 were considered statistically significant. All data are presented as the mean (average) ± standard deviation (SD). F&R Ref No.: 43219-0011WO1 Example 13— Sex as a biological variable. Our study contained samples from both human male and females. In all reported data, sex was not considered as a biological variable and findings for both sexes were similar. Example 14—Data Availability The exact results reading of all the 3 outcome measurements, ROS, 4-HNE and cell death rate from all 6 BCD individual subjects’ iRPE under different treatment groups (including no treatment) are shown in Table 2. Exact transduction rate percentage of AAV2 and AAV5 on each sample (6 individual BCD iRPE samples and 3 individual WT control iRPE samples) is summarized in Table 3. Table 2. Therapeutic outcomes of Individual BCD patient cell-based model from different AAV-CYP4V2 treatment strategies Treatment Outcomes Samples No AAV2 high AAV2 low AAV5 high AAV5 low treatment dosage dosage dosage dosage BCD-P1 103683 81566 81193 77522 93817 BCD-P2 84990 72474 71814 66016 68739 ROS BCD-P3 81068 55349 59164 57922 63862 reading BCD-P4 107352 80756 98409 87142 102033 BCD-P5 95155 75942 81437 71174 80479 BCD-P6 99182 62628 81688 70541 99111 BCD-P1 116.25 54.98 64.40 44.31 83.83 BCD-P2 76.88 55.91 67.45 71.36 73.03 4-HNE BCD-P3 65.19 45.65 52.32 62.19 63.99 ug / ml BCD-P4 119.51 68.69 89.82 62.38 90.85 BCD-P5 118.89 88.02 91.69 74.96 99.87 BCD-P6 104.38 68.50 77.41 65.91 74.47 BCD-P1 23.28% 7.55% 11.16% 8.13% 20.33% BCD-P2 17.24% 10.78% 11.85% 8.07% 12.77% Cell BCD-P3 15.65% 8.51% 13.42% 10.95% 15.65% death% BCD-P4 25.56% 14.70% 28.27% 15.50% 26.73% BCD-P5 20.47% 13.81% 20.70% 13.24% 18.77% BCD-P6 24.14% 11.57% 21.99% 16.50% 24.40% F&R Ref No.: 43219-0011WO1 Table 3. eGFP positive rate of individual iRPE subjects after AAV-eGFP transduction Serotype Subject BCD-P1 BCD-P2 BCD-P3 BCD-P4 BCD-P5 BCD-P6 WT1 WT2 WT3 AAV2 13.66% 22.91% 11.68% 20.10% 46.07% 32.39% 45.71% 50.22% 4.67% AAV5 25.04% 57.16% 25.43% 8.24% 60.45% 22.61% 77.59% 76.84% 29.55% Example 15—Building BCD Cellular Disease Platform with a Diverse Pool of BCD Patient- Specific iRPE Cell Lines In this study, we built a pool of iPSC lines from six unrelated BCD patients of three ethnicities with distinct CYP4V2 mutations (referred to as BCD-P1 to BCD-P6 in the following text) and differentiated them into iRPE cells (FIG.1A). These cell lines harbor the common CYP4V2 mutations found in East Asian (c.802-8_810del17insGC, c.992A>C and c.1091-2A>G) (9, 17, 18), European (c.1198C>T and c.332T>C) (9, 17, 19), and South Asian populations of BCD patients (c.197T>G) (7, 9). These patient cell lines contain both homozygous (BCD-P1, P4, P5 and P6) and compound heterozygous (BCD-P2 and P3) CYP4V2 mutations with a total of seven different mutations affecting 6 out of 11 exons of CYP4V2 gene. The cell lines include different mutation types (indel, missense, and splice acceptor variant) (Table 5). After the patient-specific iPSCs were differentiated into RPE cell fate (FIG.6A-6D), we cultured the iRPEs for 6-8 weeks to allow them to reach a fully mature state. We then probed the CYP4V2 expression levels in these iRPEs. In all six BCD iRPE cell lines, expression levels of CYP4V2 were lower than in wild type (WT) iRPE cells (FIG.1B). Besides, we also have probed CYP4V2 expression level in multi-clones derived iRPE cells from the same BCD subject to avoid intra-individual variations, and no difference had been observed in iRPEs derived from different clones of the same BCD subject (FIG. 6E).

[0002] F&R Ref No.: 43219-0011WO1 Table 5. BCD Patient List Subject Sex Age Race Mutation Affected exon BCD-P1 M 42 East Asian Chinese c.802-8_810del17insGC homozygous 7 BCD-P2 F 35 East Asian c.219 T>A, c.992A>C, compound Chinese heterozygous 2, 8 BCD-P3 F 47 East Asian c.992 A>C, c.1091-2A>G, compound Chinese heterozygous 8, 9 BCD-P4 F 50 White c.1198C>T, homozygous 9 BCD-P5 F 47 White c.332T>C, homozygous 3 BCD-P6 F 42 South Asian Indian c.197T>G, homozygous 1

[0003] F&R Ref No.: 43219-0011WO1 Cytochrome P450 family 4 (CYP4) enzymes are traditionally associated with endogenous fatty acid metabolism (20). A previous study suggested that the CYP4V2 protein may be involved in the omega-hydroxylation of polyunsaturated fatty acids (PUFAs) and mutant CYP4V2 may result in abnormal accumulation of omega-3 and omega-6 PUFAs such as docosahexaenoic acid (DHA) and arachidonic acid (AA) in HepG2 cells (21). To probe the PUFAs levels in our BCD patient-specific iRPEs, untargeted lipidomics of free fatty acids (FFAs) was performed on multiple individual WT iRPE cell lines (iRPE cell lines from 6-8 individual healthy donors) and BCD iRPE cell lines (5-6 individual iRPE lines from two BCD patients, BCD-P1 and BCD-P2, 2-3 iRPE lines from 2-3 individual iPSC clones of each BCD subject). Nearly all tested medium-chain PUFAs were significantly higher in BCD iRPEs (FIG.7A) than in WT control iRPEs (**P<0.01 and ***P<0.001). Among all probed items, the omega-6 PUFA AA was the major accumulated PUFA in BCD iRPE cells. Omega-3 and omega-6 PUFAs of 20-Carbon and 22-Carbon are significantly higher in BCD iRPEs compared with WTs. Example 16—BCD iRPE Cellular Models are More Vulnerable to Blue Light-Induced Oxidative Stress and Lipid Peroxidation, and In Vitro Disease Phenotypes are Quantifiable We sought to investigate how the accumulated PUFAs cause cell death in BCD RPE. Double bonds in PUFAs are target substrates to propagate oxidative stress (22) and will degrade into aldehydes, which are highly reactive species that participate in the cellular pathways that may lead to apoptosis. In BCD iRPEs, omega-6 PUFA abnormally accumulates, and the major end-product of omega-6 PUFA from peroxidation was aldehyde 4-hydroxynonenal (4-HNE), which altered cell signaling and directly caused cell death (23). Here, to recapitulate the RPE cell death phenotype and determine if the terminal product of omega-6 PUFA correlates with BCD iRPE death in vitro, we exposed BCD (six iRPE cell lines from six individual BCD patients) and WT iRPE cells (multiple iRPE cell lines from 4-6 individual healthy donors) to 430 nm wavelength blue light (referred to as “blue light” hereafter) to introduce the oxidative stress (FIG.1C, left, schemed chart). Next, we investigated reactive oxygen species (ROS), a marker for oxidative stress, and 4-HNE levels in iRPEs from six BCD patients and wild-type donors to determine if ROS and the terminal product of omega-6 PUFA correlate with BCD iRPE death. F&R Ref No.: 43219-0011WO1 After exposure to blue light, the average ROS level increased in both BCD and WT iRPEs, indicating elevated levels of oxidative stress (FIG.1C, left bar graph). However, after blue light exposure, the average ROS level in BCD iRPEs increased more than in WT iRPEs. The relative change of average ROS fluorescence intensity, relative fluorescent units (RFU) before and after blue light exposure in WT iRPEs was 8584 RFU, and in BCD iRPEs it was 37119 RFU. The relative ROS change in BCD iRPEs is more than four times higher than in WT iRPEs (FIG.1C, left bar graph). To monitor the 4-HNE level in iRPE cells, we measured 4-HNE protein-adduct concentration by ELISA. In the WT group, the 4-HNE average concentration was stable after blue light exposure (46.19 µg / ml without blue light exposure and 40.96 µg / ml after exposure). In the BCD group, before blue light exposure, the average 4-HNE concentration was 49.75 µg / ml, similar to that of the WT group. However, after blue light exposure, average 4-HNE concentration more than doubled in BCD iRPE, reaching 100.18 µg / ml (FIG. 1C, middle bar graph). This trend was shown also in cell viability tests. Cell viability reagents Calcein AM and Propidium Iodide (PI) were used to label live and dead iRPE cells to quantify iRPE cell death rates. Without blue light exposure, the average iRPE cell death rates in the WT and BCD groups were 2.30% and 2.40%, respectively. After blue light exposure, the iRPE cell death rate in the WT group remained stable at 2.76%, but in the BCD group, the cell death rate increased dramatically to 21.06% (***P<0.001) (FIG.1C, right bar graph), which is about 7.6 times of the average cell death rate in WT iRPEs exposed to blue light (2.76%). The BCD patient iRPE cell-based disease model showed significant increases in 4-HNE concentrations and cell death rates after exposure to blue light. There was no significant difference in both outcomes before and after blue light exposure in WT iRPEs. The relative change of ROS, 4-HNE and cell death rates in WT iRPE and BCD iRPE, before and after blue light exposure, are summarized in FIG.1D. Our results revealed that BCD patient RPE cells are highly susceptible to blue light-induced cell death. To further validate the cellular phenotype that we found in BCD patient-specific iRPEs, we established an isogenic iPSC line from BCD-P1 with a CRISPR / Cas9 genome editing system and successfully repaired in both alleles the homozygous 17-base pair (bp) deletion mutation c.802-8_810del17insGC mutation in the CYP4V2 gene, the most common F&R Ref No.: 43219-0011WO1 mutation among BCD patients (9, 17) (FIG.8A). We then differentiated this repaired isogenic iPSC line into the RPE cell fate (FIG.8B). We found that BCD-P1 isogenic iRPE cells express higher levels of CYP4V2 compared to BCD-P1 parental iRPE cells (FIG.8B). There was no significant change in ROS level, 4-HNE concentration and cell death rate of BCD isogenic iRPE before and after blue light exposure (FIG.8C and 8D). This evidence further validated that the increase in cell death in BCD iRPEs after blue light exposure were directly linked to CYP4V2 mutations. Example 17—Cellular Phenotype Differences Can Be Observed Among BCD Individual iRPE Subjects of Divergent Mutations in CYP4V2 Gene During the BCD disease modeling process, we observed that the individual BCD cellular phenotype (ROS, 4-HNE and cell death rate) varied in BCD iRPEs from patients of different mutations in their CYP4V2 gene. According to these observations, we performed a statistical analysis of direct comparison of the ROS, 4-HNE and cell death rate among all the six BCD iRPE subjects. Before blue light exposure, there was no significant difference in ROS and cell death rates among all the six BCD iRPE subjects. For 4-HNE, before blue light, BCD-P5 had a significantly lower concentration of 4-HNE than the other 5 BCD iRPE samples. After blue light exposure, there were significant cellular phenotype differences among the six BCD iRPE subjects in all 3 biomarkers. We also performed multi-comparison with ANOVA tests and observed significant phenotype differences among various subjects. In brief, there was significant phenotype differences between BCD-P1 and BCD-P2, as well as between BCD-P1 and BCD-P3, in ROS levels and 4-HNE; significant phenotype differences between BCD-P2 and BCD-P4 in ROS, and among BCD-P2 to BCD-P6 in 4- HNE; significant phenotype differences between BCD-P3 and BCD-P4 in ROS, and between BCD-P2 and BCD-P3, as well as among BCD-P3 to BCD-P6 in 4-HNE and cell death rate. In addition, there was no significant phenotype difference among BCD-P4, BCD-P5 and BCD-P6 in all 3 tested phenotype biomarkers (FIG.2). Example 18—AAV Serotypes Screening to Discover Optimal Vectors for Clinical Trials We performed a preliminary screen to test multiple AAV vectors of different serotypes (vector details summarized in Table 1) on 5-6 iRPE cell lines from BCD-P1 and F&R Ref No.: 43219-0011WO1 BCD-P2 (2-3 iRPE lines from 2-3 individual iPSC clones of each BCD subject). We also used multiple individual WT iRPE cell lines as controls to measure the AAV treatment efficacy. The accumulated PUFA levels in these AAV treated BCD iRPEs were examined. FFA lipidomic results showed that all the tested AAV-CYP4V2 vectors reduced the average level of major PUFAs (e.g., AA and DHA) that had accumulated in BCD iRPE cells (FIG. 7B). Overall, AAV2 and AAV5 decreased abnormal PUFA levels the most in BCD iRPE. Example 19—Application of Optimal AAV Vectors with Different Dosages to BCD Patient- Specific Cell-Based Disease Model Platforms and Patient-Specific Cell Model Elucidates Individual Differences in Gene Therapy Treatment Efficacy Based on the FFA lipidomic result from AAV serotype testing described above, we decided to use AAV2 and AAV5 as the candidate vectors for further study. Human CYP4V2 cDNA driven by a CAG promoter and WPRE enhancer were packaged into AAV2 or AAV5, respectively. Both vectors were transduced into our BCD patient-cell based disease model platforms: BCD iRPE cell lines from six individual BCD subjects. A detailed AAV treatment plan is shown in FIG.3A. In brief, we applied a high dose (MOI=1x105vg / cell) and low dose (MOI=1x104vg / cell) of each AAV2 and AAV5 vector on mature iRPEs from BCD patients which had been cultured for two months (60 days). Three months after AAV transfection (150 days in total), we exposed BCD iRPE cell-based disease models from each AAV-treated group to blue light and measured the levels of ROS, 4-HNE and cell viability to assess the therapeutic effect of each of the four AAV treatment strategies. WT iRPE cell lines from 4-6 individual healthy donors were used as a control group. After blue light exposure, the high dosage of both AAV2 and AAV5 significantly reduced the ROS level in BCD iRPE cells and reduced the ROS levels back to those seen in WT samples. The low dose of AAV2 also significantly reduced ROS levels; however, not as well as the high dose treatment. The low-dose AAV5 treatment failed to show any significant decrease in ROS levels in response to blue light treatment (FIG.3B, left chart, group comparison). Next, from the 4-HNE results, all four AAV treatment strategies reduced the average 4-HNE concentration in BCD iRPE after blue light exposure, and only low-dose AAV5 treatments failed to show significant reductions. Moreover, none of the four AAV treatments reduced the 4-HNE levels to those seen in WT iRPE samples (FIG.3C, left chart, F&R Ref No.: 43219-0011WO1 group comparison). The high dose AAV2 achieved the strongest cell death rescue effect in iRPE samples from six patients. Both high dose AAV2 and AAV5 showed significant rescue effects (FIG.3D, left chart, group comparison). As negative control to AAV2-CYP4V2 and AAV5-CYP4V2 treatments, we applied high doses of AAV2 and AAV5 vectors without CYP4V2 cDNA packaged (labeled as AAV- null in FIG.9) to BCD iRPEs. After exposure to blue light, there were no significant differences in ROS levels or cell death rates in these cells compared with the ones without any AAV treatment (FIG.9). When we processed the data from each AAV treatment group, we noticed the outcome variations among all the six BCD iRPE cell lines. Then we present the ROS (FIG. 3B, right chart), 4-HNE (FIG.3C, right chart), and cell death rate (FIG.3D, right chart) of individual BCD iRPE subjects from each AAV treatment strategy. The exact value of all the three outcome measurements are summarized in Table 2. In brief, in BCD-P1, both high and low doses of AAV2 showed similar levels of average ROS reduction. In AAV5, only the high dose of AAV5 significantly reduced ROS levels in BCD-P1. In addition, the high dose of AAV5 reduced ROS levels more than that of AAV2 in BCD-P1. In BCD-P2 and BCD-P3, all four treatment strategies significantly reduced ROS levels. In BCD-P4 iRPEs, all four tested AAV treatments reduced average ROS levels slightly; only the high dose of AAV2 showed significant efficacy. All AAV treatments on BCD-P5 iRPEs significantly reduced average ROS levels. The high dose AAV2 and AAV5 treatments significantly reduced more ROS levels compared with the low dosage treatment. In BCD-P6 iRPEs, the high dose of AAV2 and AAV5 both significantly reduced average ROS levels significantly. Low-dose AAV2 also showed a significant reduction in average ROS levels, but high doses of AAV2 and AAV5 had more significant reductions in average ROS levels compared to the low doses of each respective AAV. Reduction of 4-HNE concentrations from each AAV treatment had similar trends to those observed in the tests of ROS. In BCD-P1 iRPEs, AAV5 had the best performance in 4- HNE reduction. In BCD-P2 and BCD-P3, the treatment effective in reducing 4-HNE average level from different treatment are similar. In iRPEs of BCD-P4, BCD-P5 and BCD-P6, all four tested AAV treatments showed significant efficacy, and high doses of AAV2 and AAV5 reduced 4-HNE average levels more than the low doses of each respective AAV. F&R Ref No.: 43219-0011WO1 The high doses of AAV2 and AAV5 each generated a significant reduction in cell death rate in all six patients’ iRPEs except in BCD-P2 where only the high dose of AAV5 generated a significant reduction. BCD-P1 and BCD-P5 iRPEs showed more statistically significant reduction in death rates in response to the high dose AAV5 treatment, while the high dose of AAV2 produced more statistically significant reductions in cell death rates in BCD-P4 and BCD-P6 iRPEs. In BCD-P2 and BCD-P3, the high doses of AAV5 and AAV2, respectively, provided the strongest efficacy as shown by lower levels in the cell death rates. Notably, in both BCD-P2 and BCD-P3, the best gene therapy treatment strategy reduced the cell death rates to below 10%. Among all the other 4 BCD iRPEs (BCD-P1, BCD-P4, BCD- P5 and BCD-P6), a reduction in cell death rates to levels below 10% was only observed in BCD-P1’s iRPE in response to both high dose AAV2 and high dose AAV5 treatments. AAV caused reduction of ROS, 4-HNE and cell death rate normalized against value of blue light induced ROS, 4-HNE and cell death rate, respectively, presented in FIG.10. In addition, the average cell death rate after 20-23 replicates were consistent with each other. The metabolite proportions for BCD iRPE cell lines ranged from 10% to 50%. This range may reflect regional differences in human RPE cells cultured in vitro (24). Example 20—Distinct AAV Transduction Rate from Individual BCD iRPE Subjects We used two methods to determine the AAV transduction rate in BCD iRPEs. First, we use immunoblots to investigate the expression level of human CYP4V2 protein in BCD iRPEs from each AAV treatment strategy (FIG.4A). In BCD-P1, BCD-P2 and BCD-P3 iRPEs, all four treatment strategies (both high and low doses of AAV2 and AAV5) produced higher levels of CYP4V2 protein. In BCD P4 iRPEs, the high dose of AAV2 resulted in an increase of CYP4V2 expression, while the low dose of AAV2 and high dose of AAV5 also increased CYP4V2 expression but at much lower levels than that seen from the AAV2 high dose. In BCD-P5 iRPEs, AAV2 high dose and AAV5 (both high and low doses) increased CYP4V2 expression. The low dose of AAV2 also resulted in higher CYP4V2 expression, but not as obvious as other treatments. In BCD-P6 iRPEs, the AAV2 high dose showed the most obviously increase in CYP4V2 expression. The AAV2 low dose and AAV5 high and low doses also increased CYP4V2 protein expression. F&R Ref No.: 43219-0011WO1 Second, we transfected BCD iRPEs with AAV2-eGFP and AAV5-eGFP at a MOI=1x104(vg / cell), then quantified GFP-positive iRPE cells by flowcytometry. For each BCD iRPE cell line, fluorescence intensity of GFP more than 103(beyond the iRPE GFP background) was counted as a positive cell (FIG.4B). We have tested nine iRPE samples, six of them were BCD iRPE and three were WT iRPEs. Among all tested samples, BCD-P1, BCD-P2, BCD-P3, and all three WT iRPEs transduction rate of AAV5 are higher than AAV2 (FIG.4C). While in BCD-P4, the AAV transduction rate of AAV2 was higher than that of AAV5, and there was no significant difference in the AAV transduction rates between AAV2 and AAV5 in BCD-P5 and BCD-P6 (FIG.4C). The exact transduction rates of AAV2 and AAV5 for each sample is summarized in Table 3. Overall, the average rate of GFP-positive iRPE cells of the nine samples tested, in the levels were higher in AAV5 compared to AAV2, however, there was no significant difference in expression between the two vectors (FIG. 4D). To further investigate the AAV vector preference, we also transfected AAV2-eGFP and AAV5-eGFP on the same subject (BCD-P1) at two different time points, respectively. Results demonstrated that the culturing time only affected the amplitude of transduction rate, but not the vector serotype preference (FIG.11). Example 21—BCD iRPE Cells Harboring Homozygous Deletion Mutations in the CYP4V2 Gene Exhibit a More Pronounced Improvement Following AAV-Mediated Gene Augmentation Therapy than BCD iRPE Cells with Homozygous Missense Mutations in CYP4V2 Among the four homozygous BCD iRPE subjects (BCD-P1, BCD-P4, BCD-P5 and BCD-P6) enrolled in this study, BCD-P1 carries a 17-base pair deletion mutation, while BCD-P4, BCD-P5 and BCD-P6 carry three distinct homozygous missense mutations, respectively (Table 5). From the AAV transduction rate result, BCD-P1 had the lowest AAV transduction rate among all the four homozygous BCD iRPE subjects, however, when we measured the outcomes from each AAV treatment, BCD-P1 have similar rescue level with others. A regression was done to establish the correlation of AAV transduction rate and iRPE cell death rate with AAV treatments among the four homozygous BCD iRPE subjects (FIG. 5A). In AAV2, AAV transduction rates and iRPE cell death rates did not correlate when F&R Ref No.: 43219-0011WO1 linear regression analysis was done on all 4 homozygous subjects (r2=0.08799), but they highly correlated among the 3 homozygous missense subjects (r2=0.8685). In AAV5, the correlation level of AAV transduction rate and iRPE cell death rate was higher among the 3 homozygous missense BCD subjects (r2=0.9996) compared to the 4 homozygous subjects (r2=0.7351). Next, we investigated if there was any difference on AAV rescue efficacy among the four homozygous BCD iRPE subjects. We found the cell death rate of BCD-P1 was significantly lower than the other three homozygous BCD iRPE subjects under the AAV2 treatment, and there was no significant difference among the three iRPE subjects of homozygous missense mutations. In AAV5 treatment, we obtained a similar result, however, there was no significant difference among the four homozygous subjects (FIG.5B). To validate the difference of AAV rescue efficacies between BCD genotypes with deletion mutation and missense mutations, we calculated the changes of ROS, 4-HNE and cell death rates before and after AAV2 and AAV5 treatments on each BCD iRPE samples with homozygous mutations (P1, P4, P5, P6), respectively, then normalized the changes with each their own AAV transduction rates. In AAV2 treatment, BCD-P1 was different from the other three missense mutation subjects on all three outcome measurements. Due to individual variation in transduction rates of AAV5, there was no statistically significant difference in AAV5 treatments among the four subjects (FIG.5C-5E). Example 22—Sequences SEQ ID NO: 1 (human CYP4V2 protein, NP_997235.3, 525 aa): MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVARAYPLVGHALLMKPDGREFF QQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVEVILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWRSRRK MLTPTFHFTILEDFLDIMNEQANILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNIGAQSNDDSEYVRA VYRMSEMIFRRIKMPWLWLDLWYLMFKEGWEHKKSLQILHTFTNSVIAERANEMNANEDCRGDGRGSAPSKNKR RAFLDLLLSVTDDEGNRLSHEDIREEVDTFMFEGHDTTAAAINWSLYLLGSNPEVQKKVDHELDDVFGKSDRPA TVEDLKKLRYLECVIKETLRLFPSVPLFARSVSEDCEVAGYRVLKGTEAVIIPYALHRDPRYFPNPEEFQPERF FPENAQGRHPYAYVPFSAGPRNCIGQKFAVMEEKTILSCILRHFWIESNQKREELGLEGQLILRPSNGIWIKLK RRNADER SEQ ID NO: 2 (human CYP4V2 cDNA encoding the human CYP4V2 protein of SEQ ID NO: 1, 1578 bp including stop codon): ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTGCCCTTTCCCTGGC CGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACGCGCGGAAATGGCAGCAGATGCGGCCCA TCCCCACGGTGGCCCGCGCCTACCCACTGGTGGGCCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTTTT CAGCAGATCATTGAGTACACAGAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCAT GGTGGCCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAATCCTCTATGT ACAAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGAAACAAATGGCGCTCCAGGAGAAAG F&R Ref No.: 43219-0011WO1 ATGTTAACACCCACTTTCCATTTTACCATTCTGGAAGATTTCTTAGATATCATGAATGAACAAGCAAATATATT GGTTAAGAAACTTGAAAAACACATTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAG ATATCATCTGTGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCGTGCA GTTTATAGAATGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGGCTTGATCTCTGGTACCTTAT GTTTAAAGAAGGATGGGAACACAAAAAGAGCCTTCAGATCCTACATACTTTTACCAACAGTGTCATCGCTGAAC GGGCCAATGAAATGAACGCCAATGAAGACTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGC AGGGCCTTTCTTGACTTGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATTCGAGA AGAAGTTGACACCTTCATGTTTGAGGGGCACGATACAACTGCAGCTGCAATAAACTGGTCCTTATACCTGTTGG GTTCTAACCCAGAAGTCCAGAAAAAAGTGGATCATGAATTGGATGACGTGTTTGGGAAGTCTGACCGTCCCGCT ACAGTAGAAGACCTGAAGAAACTTCGGTATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGT TCCTTTATTTGCCCGTAGTGTTAGTGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAGCCG TCATCATTCCCTATGCATTGCACAGAGATCCGAGATACTTCCCCAACCCCGAGGAGTTCCAGCCTGAGCGGTTC TTCCCCGAGAATGCACAAGGGCGCCATCCATATGCCTACGTGCCCTTCTCTGCTGGCCCCAGGAACTGTATAGG TCAAAAGTTTGCTGTGATGGAAGAAAAGACCATTCTTTCGTGCATCCTGAGGCACTTTTGGATAGAATCCAACC AGAAAAGAGAAGAGCTTGGTCTAGAAGGACAGTTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAGTTGAAG AGGAGAAATGCAGATGAACGCTAA SEQ ID NO: 3 (CYP4V2 cDNA (codon-optimized) encoding the human CYP4V2 protein of SEQ ID NO: 1, 1578 bp including stop codon) ATGGCTGGACTGTGGCTGGGACTGGTGTGGCAGAAACTGCTGCTGTGGGGGGCCGCTTCCGCACTGTCACTGGC TGGGGCTTCACTGGTGCTGAGCCTGCTGCAGAGGGTGGCCTCCTACGCCAGAAAGTGGCAGCAGATGAGGCCCA TCCCTACCGTGGCCAGAGCCTATCCACTGGTGGGACACGCACTGCTGATGAAGCCTGACGGCAGAGAGTTCTTT CAGCAGATCATCGAGTACACAGAGGAGTATAGGCACATGCCACTGCTGAAGCTGTGGGTGGGACCCGTGCCTAT GGTGGCCCTGTACAACGCCGAGAATGTGGAAGTGATCCTGACCAGCAGCAAGCAGATCGATAAGTCTAGCATGT ATAAGTTCCTGGAGCCTTGGCTGGGCCTGGGCCTGCTGACCTCTACAGGCAACAAGTGGAGGAGCCGGAGAAAG ATGCTGACCCCAACATTCCACTTTACAATCCTGGAGGACTTCCTGGACATCATGAACGAGCAGGCCAATATCCT GGTGAAGAAGCTGGAGAAGCACATCAACCAGGAGGCCTTTAATTGCTTCTTTTACATCACCCTGTGCGCCCTGG ACATCATCTGTGAGACAGCTATGGGCAAGAACATCGGCGCCCAGTCTAATGACGATAGCGAGTACGTGCGGGCC GTGTATAGAATGAGCGAGATGATCTTTAGGCGCATCAAGATGCCCTGGCTGTGGCTGGATCTGTGGTATCTGAT GTTCAAGGAGGGCTGGGAGCACAAGAAGTCCCTGCAGATCCTGCACACCTTTACAAACTCTGTGATCGCCGAGA GAGCCAATGAGATGAACGCCAATGAGGACTGTAGGGGCGATGGAAGGGGCAGCGCCCCTTCCAAGAACAAGCGG AGAGCCTTCCTGGACCTGCTGCTGAGCGTGACCGACGATGAGGGCAATCGCCTGTCCCACGAGGACATCCGGGA GGAGGTGGATACATTCATGTTTGAGGGACACGACACCACAGCCGCCGCCATCAACTGGTCCCTGTACCTGCTGG GCTCTAATCCAGAGGTGCAGAAGAAGGTGGATCACGAGCTGGACGACGTGTTCGGCAAGTCCGACAGGCCAGCA ACCGTGGAGGATCTGAAGAAGCTGAGATACCTGGAGTGCGTGATCAAGGAGACACTGCGCCTGTTCCCCTCTGT GCCTCTGTTTGCCCGGTCCGTGTCTGAGGACTGTGAGGTGGCCGGCTATCGCGTGCTGAAGGGCACCGAGGCCG TGATCATCCCTTACGCCCTGCACCGGGACCCCAGGTATTTCCCTAACCCAGAGGAGTTTCAGCCAGAGAGATTC TTTCCCGAGAATGCCCAGGGCAGGCACCCTTACGCCTATGTGCCATTCTCCGCCGGACCAAGGAACTGCATCGG ACAGAAGTTTGCCGTGATGGAGGAGAAAACCATCCTGTCTTGTATCCTGAGACACTTCTGGATCGAGAGCAATC AGAAGAGGGAGGAGCTGGGCCTGGAGGGACAGCTGATCCTGCGGCCAAGCAACGGCATCTGGATCAAACTGAAA AGAAGGAACGCTGACGAGAGGTAA SEQ ID NO: 4 (functional variant of human CYP4V2 protein; 525 aa, comprising amino acid Gln259Lys (Q259K) change compared to the human CYP4V2 protein of SEQ ID NO: 1): MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVARAYPLVGHALLMKPDGREFF QQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVEVILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWRSRRK MLTPTFHFTILEDFLDIMNEQANILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNIGAQSNDDSEYVRA VYRMSEMIFRRIKMPWLWLDLWYLMFKEGWEHKKSLKILHTFTNSVIAERANEMNANEDCRGDGRGSAPSKNKR RAFLDLLLSVTDDEGNRLSHEDIREEVDTFMFEGHDTTAAAINWSLYLLGSNPEVQKKVDHELDDVFGKSDRPA TVEDLKKLRYLECVIKETLRLFPSVPLFARSVSEDCEVAGYRVLKGTEAVIIPYALHRDPRYFPNPEEFQPERF FPENAQGRHPYAYVPFSAGPRNCIGQKFAVMEEKTILSCILRHFWIESNQKREELGLEGQLILRPSNGIWIKLK RRNADER F&R Ref No.: 43219-0011WO1 SEQ ID NO: 5 (CYP4V2 cDNA encoding a functional variant (SEQ ID NO: 4) of the human CYP4V2 protein, 1578 bp including stop codon): ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTGCCCTTTCCCTGGC CGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACGCGCGGAAATGGCAGCAGATGCGGCCCA TCCCCACGGTGGCCCGCGCCTACCCACTGGTGGGCCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTTTT CAGCAGATCATTGAGTACACAGAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCAT GGTGGCCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAATCCTCTATGT ACAAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGAAACAAATGGCGCTCCAGGAGAAAG ATGTTAACACCCACTTTCCATTTTACCATTCTGGAAGATTTCTTAGATATCATGAATGAACAAGCAAATATATT GGTTAAGAAACTTGAAAAACACATTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAG ATATCATCTGTGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCGTGCA GTTTATAGAATGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGGCTTGATCTCTGGTACCTTAT GTTTAAAGAAGGATGGGAACACAAAAAGAGCCTTAAGATCCTACATACTTTTACCAACAGTGTCATCGCGGAAC GGGCCAATGAAATGAACGCCAATGAAGACTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGC AGGGCCTTTCTTGACTTGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATTCGAGA AGAAGTTGACACCTTCATGTTTGAGGGGCACGATACAACTGCAGCTGCAATAAACTGGTCCTTATACCTGTTGG GTTCTAACCCAGAAGTCCAGAAAAAAGTGGATCATGAATTGGATGACGTGTTTGGGAAGTCTGACCGTCCCGCT ACAGTAGAAGACCTGAAGAAACTTCGGTATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGT TCCTTTATTTGCCCGTAGTGTTAGTGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAGCCG TCATCATTCCCTATGCATTGCACAGAGATCCGAGATACTTCCCCAACCCCGAGGAGTTCCAGCCTGAGCGGTTC TTCCCCGAGAATGCACAAGGGCGCCATCCATATGCCTACGTGCCCTTCTCTGCTGGCCCCAGGAACTGTATAGG TCAAAAGTTTGCTGTGATGGAAGAAAAGACCATTCTTTCGTGCATCCTGAGGCACTTTTGGATAGAATCCAACC AGAAAAGAGAAGAGCTTGGTCTAGAAGGACAGTTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAGTTGAAG AGGAGAAATGCAGATGAACGCTAA SEQ ID NO: 6 (human CYP4V2 protein variant; 525 aa, comprising amino acid Leu22Val change compared to the human CYP4V2 protein of SEQ ID NO: 1): 1 MAGLWLGLVW QKLLLWGAAS AVSLAGASLV LSLLQRVASY ARKWQQMRPI PTVARAYPLV 61 GHALLMKPDG REFFQQIIEY TEEYRHMPLL KLWVGPVPMV ALYNAENVEV ILTSSKQIDK 121 SSMYKFLEPW LGLGLLTSTG NKWRSRRKML TPTFHFTILE DFLDIMNEQA NILVKKLEKH 181 INQEAFNCFF YITLCALDII CETAMGKNIG AQSNDDSEYV RAVYRMSEMI FRRIKMPWLW 241 LDLWYLMFKE GWEHKKSLQI LHTFTNSVIA ERANEMNANE DCRGDGRGSA PSKNKRRAFL 301 DLLLSVTDDE GNRLSHEDIR EEVDTFMFEG HDTTAAAINW SLYLLGSNPE VQKKVDHELD 361 DVFGKSDRPA TVEDLKKLRY LECVIKETLR LFPSVPLFAR SVSEDCEVAG YRVLKGTEAV 421 IIPYALHRDP RYFPNPEEFQ PERFFPENAQ GRHPYAYVPF SAGPRNCIGQ KFAVMEEKTI 481 LSCILRHFWI ESNQKREELG LEGQLILRPS NGIWIKLKRR NADER SEQ ID NO: 7 (human CYP4V2 protein variant; 525 aa, comprising amino acid Met123Val change compared to the human CYP4V2 protein of SEQ ID NO: 1): 1 MAGLWLGLVW QKLLLWGAAS ALSLAGASLV LSLLQRVASY ARKWQQMRPI PTVARAYPLV 61 GHALLMKPDG REFFQQIIEY TEEYRHMPLL KLWVGPVPMV ALYNAENVEV ILTSSKQIDK 121 SSVYKFLEPW LGLGLLTSTG NKWRSRRKML TPTFHFTILE DFLDIMNEQA NILVKKLEKH 181 INQEAFNCFF YITLCALDII CETAMGKNIG AQSNDDSEYV RAVYRMSEMI FRRIKMPWLW 241 LDLWYLMFKE GWEHKKSLQI LHTFTNSVIA ERANEMNANE DCRGDGRGSA PSKNKRRAFL 301 DLLLSVTDDE GNRLSHEDIR EEVDTFMFEG HDTTAAAINW SLYLLGSNPE VQKKVDHELD 361 DVFGKSDRPA TVEDLKKLRY LECVIKETLR LFPSVPLFAR SVSEDCEVAG YRVLKGTEAV 421 IIPYALHRDP RYFPNPEEFQ PERFFPENAQ GRHPYAYVPF SAGPRNCIGQ KFAVMEEKTI 481 LSCILRHFWI ESNQKREELG LEGQLILRPS NGIWIKLKRR NADER F&R Ref No.: 43219-0011WO1 SEQ ID NO: 8 (human CYP4V2 protein variant; 525 aa, comprising amino acid Glu275Lys change compared to the human CYP4V2 protein of SEQ ID NO: 1): 1 MAGLWLGLVW QKLLLWGAAS ALSLAGASLV LSLLQRVASY ARKWQQMRPI PTVARAYPLV 61 GHALLMKPDG REFFQQIIEY TEEYRHMPLL KLWVGPVPMV ALYNAENVEV ILTSSKQIDK 121 SSMYKFLEPW LGLGLLTSTG NKWRSRRKML TPTFHFTILE DFLDIMNEQA NILVKKLEKH 181 INQEAFNCFF YITLCALDII CETAMGKNIG AQSNDDSEYV RAVYRMSEMI FRRIKMPWLW 241 LDLWYLMFKE GWEHKKSLQI LHTFTNSVIA ERANKMNANE DCRGDGRGSA PSKNKRRAFL 301 DLLLSVTDDE GNRLSHEDIR EEVDTFMFEG HDTTAAAINW SLYLLGSNPE VQKKVDHELD 361 DVFGKSDRPA TVEDLKKLRY LECVIKETLR LFPSVPLFAR SVSEDCEVAG YRVLKGTEAV 421 IIPYALHRDP RYFPNPEEFQ PERFFPENAQ GRHPYAYVPF SAGPRNCIGQ KFAVMEEKTI 481 LSCILRHFWI ESNQKREELG LEGQLILRPS NGIWIKLKRR NADER SEQ ID NO: 9 (CAG promoter, 1715 bp) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTC CGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT GACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTG CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCC GCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC TATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATT TTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGG CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA AAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGC TGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTAC TCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTT TCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGG GTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGC GCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGG GGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGG TCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCG TGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGC CGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGA GCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGC CGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAA TGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGG GGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCC TCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCA TCATTTTGGCAAA SEQ ID NO: 10 (WPRE enhancer, 589 bp) AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATG TGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATA AATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTT GCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTG ACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTG CGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGC TCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC SEQ ID NO: 11 (bGH polyA, 225 bp) CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACT CCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG TGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA TGG F&R Ref No.: 43219-0011WO1 SEQ ID NO: 12 (EFS promoter, 235 bp) g attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acag SEQ ID NO: 13 (Small PolyA (SPA), 54 bp) GATCCAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 14 (Kozak sequence, 6 bp) GCCACC SEQ ID NO: 15 (Kozak sequence, 5 bp) CCACC SEQ ID NO: 16 (SV40 late PolyA, 120 bp) TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTC ACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAT SEQ ID NO: 17 (CMV promoter, 576 bp) TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT CATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGG AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAG SEQ ID NO: 18 (EF-1 alpha promoter, 1184 bp) cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcct ttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttg ccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtg ccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagag ttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgcc gcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttga tgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttc ggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgag cgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccg tgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcc cggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttcccca cactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttg agtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtga F&R Ref No.: 43219-0011WO1 SEQ ID NO: 19 (AAV25' Left-ITR, 141 bp) cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc t SEQ ID NO: 20 (AAV23' Right-ITR, 141 bp) ag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcagg SEQ ID NO: 21 (mutant AAV25’ ITR in scAAV construct, 117 bp) cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtgg SEQ ID NO: 22 (AAV23’ ITR in scAAV construct, 141 bp) aggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc agg SEQ ID NO: 23 (a shorter version of the CAG promoter, 936 bp): 1 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 61 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 121 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 181 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 241 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc 301 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg 361 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg 421 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 481 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg 541 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 601 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 661 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg 721 ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg gggacggctg 781 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag 841 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct 901 ggttattgtg ctgtctcatc attttggcaa agaatt SEQ ID NO: 24 – CYP4V2 expression cassette: Left-ITR: 1-141 CAG promoter: 237-1951 Human CYP4V2 cDNA (codon-optimized): 2002-3579 WPRE enhancer: 3736-4324 bGH polyA: 4350-4574 Right-ITR 4659-4799 1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 51 CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC 101 GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCAAT 151 TCAGTCGATA ACTATAACGG TCCTAAGGTA GCGATTTAAA TACGCGCTCT 201 CTTAAGGTAG CCCCGGGACG CGTCAATTGA GATCTCGACA TTGATTATTG 251 ACTAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA F&R Ref No.: 43219-0011WO1 301 TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC 351 CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA 401 GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG ACTATTTACG 451 GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC 501 CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG 551 TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT 601 CATCGCTATT ACCATGGGTC GAGGTGAGCC CCACGTTCTG CTTCACTCTC 651 CCCATCTCCC CCCCCTCCCC ACCCCCAATT TTGTATTTAT TTATTTTTTA 701 ATTATTTTGT GCAGCGATGG GGGCGGGGGG GGGGGGGGCG CGCGCCAGGC 751 GGGGCGGGGC GGGGCGAGGG GCGGGGCGGG GCGAGGCGGA GAGGTGCGGC 801 GGCAGCCAAT CAGAGCGGCG CGCTCCGAAA GTTTCCTTTT ATGGCGAGGC 851 GGCGGCGGCG GCGGCCCTAT AAAAAGCGAA GCGCGCGGCG GGCGGGAGTC 901 GCTGCGTTGC CTTCGCCCCG TGCCCCGCTC CGCGCCGCCT CGCGCCGCCC 951 GCCCCGGCTC ACAGGTGAGC GGGCGGGACG 1001 GCCCTTCTCC TGGTTTAATG ACGGCTCGTT 1051 TCTTTTCTGT GGCTGCGTGA AAGCCTTAAA GGGCTCCGGG AGGGCCCTTT 1101 GTGCGGGGGG GAGCGGCTCG GGGGGTGCGT GCGTGTGTGT GTGCGTGGGG 1151 AGCGCCGCGT GCGGCCCGCG CTGCCCGGCG GCTGTGAGCG CTGCGGGCGC 1201 GGCGCGGGGC TTTGTGCGCT CCGCGTGTGC GCGAGGGGAG CGCGGCCGGG 1251 GGCGGTGCCC CGCGGTGCGG GGGGGCTGCG AGGGGAACAA AGGCTGCGTG 1301 CGGGGTGTGT GCGTGGGGGG GTGAGCAGGG GGTGTGGGCG CGGCGGTCGG 1351 GCTGTAACCC CCCCCTGCAC CCCCCTCCCC GAGTTGCTGA GCACGGCCCG 1401 GCTTCGGGTG CGGGGCTCCG TGCGGGGCGT GGCGCGGGGC TCGCCGTGCC 1451 GGGCGGGGGG TGGCGGCAGG TGGGGGTGCC GGGCGGGGCG GGGCCGCCTC 1501 GGGCCGGGGA GGGCTCGGGG GAGGGGCGCG GCGGCCCCGG AGCGCCGGCG 1551 GCTGTCGAGG CGCGGCGAGC CGCAGCCATT GCCTTTTATG GTAATCGTGC 1601 GAGAGGGCGC AGGGACTTCC TTTGTCCCAA ATCTGGCGGA GCCGAAATCT 1651 GGGAGGCGCC GCCGCACCCC CTCTAGCGGG CGCGGGCGAA GCGGTGCGGC 1701 GCCGGCAGGA AGGAAATGGG CGGGGAGGGC CTTCGTGCGT CGCCGCGCCG 1751 CCGTCCCCTT CTCCATCTCC AGCCTCGGGG CTGCCGCAGG GGGACGGCTG 1801 CCTTCGGGGG GGACGGGGCA GGGCGGGGTT CGGCTTCTGG CGTGTGACCG 1851 GCGGCTCTAG AGCCTCTGCT AACCATGTTC ATGCCTTCTT CTTTTTCCTA 1901 CAGCTCCTGG GCAACGTGCT GGTTATTGTG CTGTCTCATC ATTTTGGCAA 1951 AGAATTCTAA TACGACTCAC TATAGGGAGA CCCAAGCTGG CTAGAGCCAC 2001 CATGGCTGGA CTGTGGCTGG GACTGGTGTG GCAGAAACTG CTGCTGTGGG 2051 GGGCCGCTTC CGCACTGTCA CTGGCTGGGG CTTCACTGGT GCTGAGCCTG 2101 CTGCAGAGGG TGGCCTCCTA CGCCAGAAAG TGGCAGCAGA TGAGGCCCAT 2151 CCCTACCGTG GCCAGAGCCT ATCCACTGGT GGGACACGCA CTGCTGATGA 2201 AGCCTGACGG CAGAGAGTTC TTTCAGCAGA TCATCGAGTA CACAGAGGAG 2251 TATAGGCACA TGCCACTGCT GAAGCTGTGG GTGGGACCCG TGCCTATGGT 2301 GGCCCTGTAC AACGCCGAGA ATGTGGAAGT GATCCTGACC AGCAGCAAGC 2351 AGATCGATAA GTCTAGCATG TATAAGTTCC TGGAGCCTTG GCTGGGCCTG 2401 GGCCTGCTGA CCTCTACAGG CAACAAGTGG AGGAGCCGGA GAAAGATGCT 2451 GACCCCAACA TTCCACTTTA CAATCCTGGA GGACTTCCTG GACATCATGA 2501 ACGAGCAGGC CAATATCCTG GTGAAGAAGC TGGAGAAGCA CATCAACCAG 2551 GAGGCCTTTA ATTGCTTCTT TTACATCACC CTGTGCGCCC TGGACATCAT 2601 CTGTGAGACA GCTATGGGCA AGAACATCGG CGCCCAGTCT AATGACGATA 2651 GCGAGTACGT GCGGGCCGTG TATAGAATGA GCGAGATGAT CTTTAGGCGC 2701 ATCAAGATGC CCTGGCTGTG GCTGGATCTG TGGTATCTGA TGTTCAAGGA 2751 GGGCTGGGAG CACAAGAAGT CCCTGCAGAT CCTGCACACC TTTACAAACT 2801 CTGTGATCGC CGAGAGAGCC AATGAGATGA ACGCCAATGA GGACTGTAGG F&R Ref No.: 43219-0011WO1 2851 GGCGATGGAA GGGGCAGCGC CCCTTCCAAG AACAAGCGGA GAGCCTTCCT 2901 GGACCTGCTG CTGAGCGTGA CCGACGATGA GGGCAATCGC CTGTCCCACG 2951 AGGACATCCG GGAGGAGGTG GATACATTCA TGTTTGAGGG ACACGACACC 3001 ACAGCCGCCG CCATCAACTG GTCCCTGTAC CTGCTGGGCT CTAATCCAGA 3051 GGTGCAGAAG AAGGTGGATC ACGAGCTGGA CGACGTGTTC GGCAAGTCCG 3101 ACAGGCCAGC AACCGTGGAG GATCTGAAGA AGCTGAGATA CCTGGAGTGC 3151 GTGATCAAGG AGACACTGCG CCTGTTCCCC TCTGTGCCTC TGTTTGCCCG 3201 GTCCGTGTCT GAGGACTGTG AGGTGGCCGG CTATCGCGTG CTGAAGGGCA 3251 CCGAGGCCGT GATCATCCCT TACGCCCTGC ACCGGGACCC CAGGTATTTC 3301 CCTAACCCAG AGGAGTTTCA GCCAGAGAGA TTCTTTCCCG AGAATGCCCA 3351 GGGCAGGCAC CCTTACGCCT ATGTGCCATT CTCCGCCGGA CCAAGGAACT 3401 GCATCGGACA GAAGTTTGCC GTGATGGAGG AGAAAACCAT CCTGTCTTGT 3451 ATCCTGAGAC ACTTCTGGAT CGAGAGCAAT CAGAAGAGGG AGGAGCTGGG 3501 CCTGGAGGGA CAGCTGATCC TGCGGCCAAG CAACGGCATC TGGATCAAAC 3551 TGAAAAGAAG GAACGCTGAC GAGAGGTAAA AGCTTGGTAC CGATATCGCG 3601 GCCGCCCTAG GGAGCTCCTC GAGGCGGCCC GCTCGAGTCT AGAGGGCCCT 3651 TCGAAGGTAA GCCTATCCCT AACCCTCTCC TCGGTCTCGA TTCTACGCGT 3701 ACCGGTCATC ATCACCATCA CCATTGAGTT TCGATAATCA ACCTCTGGAT 3751 TACAAAATTT GTGAAAGATT GACTGGTATT CTTAACTATG TTGCTCCTTT 3801 TACGCTATGT GGATACGCTG CTTTAATGCC TTTGTATCAT GCTATTGCTT 3851 CCCGTATGGC TTTCATTTTC TCCTCCTTGT ATAAATCCTG GTTGCTGTCT 3901 CTTTATGAGG AGTTGTGGCC CGTTGTCAGG CAACGTGGCG TGGTGTGCAC 3951 TGTGTTTGCT GACGCAACCC CCACTGGTTG GGGCATTGCC ACCACCTGTC 4001 AGCTCCTTTC CGGGACTTTC GCTTTCCCCC TCCCTATTGC CACGGCGGAA 4051 CTCATCGCCG CCTGCCTTGC CCGCTGCTGG ACAGGGGCTC GGCTGTTGGG 4101 CACTGACAAT TCCGTGGTGT TGTCGGGGAA ATCATCGTCC TTTCCTTGGC 4151 TGCTCGCCTG TGTTGCCACC TGGATTCTGC GCGGGACGTC CTTCTGCTAC 4201 GTCCCTTCGG CCCTCAATCC AGCGGACCTT CCTTCCCGCG GCCTGCTGCC 4251 GGCTCTGCGG CCTCTTCCGC GTCTTCGCCT TCGCCCTCAG ACGAGTCGGA 4301 TCTCCCTTTG GGCCGCCTCC CCGCATCGAA ACCCGCTGAT CAGCCTCGAC 4351 TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT 4401 CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG 4451 GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG 4501 GGTGGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG 4551 CTGGGGATGC GGTGGGCTCT ATGGCTTCTG AGGCGGAAAG AACCAGATCC 4601 TCTCTTAAGG TAGCATCGAG ATTTAAATTA GGGATAACAG GGTAATGGCG 4651 CGGGCCGCAG GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG 4701 CTCGCTCGCT CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC 4751 TTTGCCCGGG CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG SEQ ID NO: 25 – CYP4V2 expression cassette: Left-ITR: 1-141 CAG promoter: 166-1880 Human CYP4V2 cDNA: 1938-3515 WPRE enhancer: 3551-4139 bGH polyA: 4163-4387 Right-ITR: 4399-4539 1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 51 CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC 101 GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCTAA F&R Ref No.: 43219-0011WO1 151 GGCAATTGAG ATCTCGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 201 ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA 251 ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT 301 TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC 351 CATTGACGTC AATGGGTGGA CTATTTACGG TAAACTGCCC ACTTGGCAGT 401 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG 451 GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT 501 CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGGTCG 551 AGGTGAGCCC CACGTTCTGC TTCACTCTCC CCATCTCCCC CCCCTCCCCA 601 CCCCCAATTT TGTATTTATT TATTTTTTAA TTATTTTGTG CAGCGATGGG 651 GGCGGGGGGG GGGGGGGCGC GCGCCAGGCG GGGCGGGGCG GGGCGAGGGG 701 CGGGGCGGGG CGAGGCGGAG AGGTGCGGCG GCAGCCAATC AGAGCGGCGC 751 GCTCCGAAAG TTTCCTTTTA TGGCGAGGCG GCGGCGGCGG CGGCCCTATA 801 AAAAGCGAAG CGCGCGGCGG GCGGGAGTCG CTGCGTTGCC TTCGCCCCGT 851 GCCCCGCTCC GCGCCGCCTC GCGCCGCCCG CCCCGGCTCT GACTGACCGC 901 GTTACTCCCA CAGGTGAGCG GGCGGGACGG CCCTTCTCCT CCGGGCTGTA 951 ATTAGCGCTT GGTTTAATGA CGGCTCGTTT CTTTTCTGTG GCTGCGTGAA 1001 AGCCTTAAAG GGCTCCGGGA GGGCCCTTTG TGCGGGGGGG AGCGGCTCGG 1051 GGGGTGCGTG CGTGTGTGTG TGCGTGGGGA GCGCCGCGTG CGGCCCGCGC 1101 TGCCCGGCGG CTGTGAGCGC TGCGGGCGCG GCGCGGGGCT TTGTGCGCTC 1151 CGCGTGTGCG CGAGGGGAGC GCGGCCGGGG GCGGTGCCCC GCGGTGCGGG 1201 GGGGCTGCGA GGGGAACAAA GGCTGCGTGC GGGGTGTGTG CGTGGGGGGG 1251 TGAGCAGGGG GTGTGGGCGC GGCGGTCGGG CTGTAACCCC CCCCTGCACC 1301 CCCCTCCCCG AGTTGCTGAG CACGGCCCGG CTTCGGGTGC GGGGCTCCGT 1351 GCGGGGCGTG GCGCGGGGCT CGCCGTGCCG GGCGGGGGGT GGCGGCAGGT 1401 GGGGGTGCCG GGCGGGGCGG GGCCGCCTCG GGCCGGGGAG GGCTCGGGGG 1451 AGGGGCGCGG CGGCCCCGGA GCGCCGGCGG CTGTCGAGGC GCGGCGAGCC 1501 GCAGCCATTG CCTTTTATGG TAATCGTGCG AGAGGGCGCA GGGACTTCCT 1551 TTGTCCCAAA TCTGGCGGAG CCGAAATCTG GGAGGCGCCG CCGCACCCCC 1601 TCTAGCGGGC GCGGGCGAAG CGGTGCGGCG CCGGCAGGAA GGAAATGGGC 1651 GGGGAGGGCC TTCGTGCGTC GCCGCGCCGC CGTCCCCTTC TCCATCTCCA 1701 GCCTCGGGGC TGCCGCAGGG GGACGGCTGC CTTCGGGGGG GACGGGGCAG 1751 GGCGGGGTTC GGCTTCTGGC GTGTGACCGG CGGCTCTAGA GCCTCTGCTA 1801 ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG CAACGTGCTG 1851 GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTCTAAT ACGACTCACT 1901 ATAGGGAGAC CCAAGCTGGC TAGCCAAAGC TTCCACCATG GCGGGGCTCT 1951 GGCTGGGGCT CGTGTGGCAG AAGCTGCTGC TGTGGGGCGC GGCGAGTGCC 2001 CTTTCCCTGG CCGGCGCCAG TCTGGTCCTG AGCCTGCTGC AGAGGGTGGC 2051 GAGCTACGCG CGGAAATGGC AGCAGATGCG GCCCATCCCC ACGGTGGCCC 2101 GCGCCTACCC ACTGGTGGGC CACGCGCTGC TGATGAAGCC GGACGGGCGA 2151 GAATTTTTTC AGCAGATCAT TGAGTACACA GAGGAATACC GCCACATGCC 2201 GCTGCTGAAG CTCTGGGTCG GGCCAGTGCC CATGGTGGCC CTTTATAATG 2251 CAGAAAATGT GGAGGTAATT TTAACTAGTT CAAAGCAAAT TGACAAATCC 2301 TCTATGTACA AGTTTTTAGA ACCATGGCTT GGCCTAGGAC TTCTTACAAG 2351 TACTGGAAAC AAATGGCGCT CCAGGAGAAA GATGTTAACA CCCACTTTCC 2401 ATTTTACCAT TCTGGAAGAT TTCTTAGATA TCATGAATGA ACAAGCAAAT 2451 ATATTGGTTA AGAAACTTGA AAAACACATT AACCAAGAAG CATTTAACTG 2501 CTTTTTTTAC ATCACTCTTT GTGCCTTAGA TATCATCTGT GAAACAGCTA 2551 TGGGGAAGAA TATTGGTGCT CAAAGTAATG ATGATTCCGA GTATGTCCGT 2601 GCAGTTTATA GAATGAGTGA GATGATATTT CGAAGAATAA AGATGCCCTG 2651 GCTTTGGCTT GATCTCTGGT ACCTTATGTT TAAAGAAGGA TGGGAACACA F&R Ref No.: 43219-0011WO1 2701 AAAAGAGCCT TCAGATCCTA CATACTTTTA CCAACAGTGT CATCGCTGAA 2751 CGGGCCAATG AAATGAACGC CAATGAAGAC TGTAGAGGTG ATGGCAGGGG 2801 CTCTGCCCCC TCCAAAAATA AACGCAGGGC CTTTCTTGAC TTGCTTTTAA 2851 GTGTGACTGA TGACGAAGGG AACAGGCTAA GTCATGAAGA TATTCGAGAA 2901 GAAGTTGACA CCTTCATGTT TGAGGGGCAC GATACAACTG CAGCTGCAAT 2951 AAACTGGTCC TTATACCTGT TGGGTTCTAA CCCAGAAGTC CAGAAAAAAG 3001 TGGATCATGA ATTGGATGAC GTGTTTGGGA AGTCTGACCG TCCCGCTACA 3051 GTAGAAGACC TGAAGAAACT TCGGTATCTG GAATGTGTTA TTAAGGAGAC 3101 CCTTCGCCTT TTTCCTTCTG TTCCTTTATT TGCCCGTAGT GTTAGTGAAG 3151 ATTGTGAAGT GGCAGGTTAC AGAGTTCTAA AAGGCACTGA AGCCGTCATC 3201 ATTCCCTATG CATTGCACAG AGATCCGAGA TACTTCCCCA ACCCCGAGGA 3251 GTTCCAGCCT GAGCGGTTCT TCCCCGAGAA TGCACAAGGG CGCCATCCAT 3301 ATGCCTACGT GCCCTTCTCT GCTGGCCCCA GGAACTGTAT AGGTCAAAAG 3351 TTTGCTGTGA TGGAAGAAAA GACCATTCTT TCGTGCATCC TGAGGCACTT 3401 TTGGATAGAA TCCAACCAGA AAAGAGAAGA GCTTGGTCTA GAAGGACAGT 3451 TGATTCTTCG TCCAAGTAAT GGCATCTGGA TCAAGTTGAA GAGGAGAAAT 3501 GCAGATGAAC GCTAAGCGGC CGCAACTCGA GACTCTAGAG GTTAATCGAT 3551 AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA 3601 CTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT 3651 ATCATGCTAT TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA 3701 TCCTGGTTGC TGTCTCTTTA TGAGGAGTTG TGGCCCGTTG TCAGGCAACG 3751 TGGCGTGGTG TGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCA 3801 TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT CCCCCTCCCT 3851 ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG 3901 GGCTCGGCTG TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAATCAT 3951 CGTCCTTTCC TTGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGG 4001 ACGTCCTTCT GCTACGTCCC TTCGGCCCTC AATCCAGCGG ACCTTCCTTC 4051 CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT CGCCTTCGCC 4101 CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCA TCGAAACCCG 4151 CTGACTAGAC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC 4201 TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC 4251 CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA 4301 TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC 4351 AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCCG CGGGCCGCAG 4401 GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG CTCGCTCGCT 4451 CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC TTTGCCCGGG 4501 CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG SEQ ID NO: 26 – CYP4V2 expression cassette: Left-ITR (truncated): 1-117 EFS promoter: 130-364 Human CYP4V2 cDNA (codon-optimized): 520-2097 SPA: 2116-2169 Right-ITR: 2263-2403 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggacg 121 cgtaggcctg attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 181 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt 241 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 301 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 361 acaggtgtcg tgacgcgacc aggtatgcat ctgcagctct aaggtaaata taaaattttt F&R Ref No.: 43219-0011WO1 421 aagtgtataa tgtgttaaac tactgattct aattgtttct ctcttttaga ttccaacctt 481 tggaactgac tgcagggatc caagctttct agagccacca tggctggact gtggctggga 541 ctggtgtggc agaaactgct gctgtggggg gccgcttccg cactgtcact ggctggggct 601 tcactggtgc tgagcctgct gcagagggtg gcctcctacg ccagaaagtg gcagcagatg 661 aggcccatcc ctaccgtggc cagagcctat ccactggtgg gacacgcact gctgatgaag 721 cctgacggca gagagttctt tcagcagatc atcgagtaca cagaggagta taggcacatg 781 ccactgctga agctgtgggt gggacccgtg cctatggtgg ccctgtacaa cgccgagaat 841 gtggaagtga tcctgaccag cagcaagcag atcgataagt ctagcatgta taagttcctg 901 gagccttggc tgggcctggg cctgctgacc tctacaggca acaagtggag gagccggaga 961 aagatgctga ccccaacatt ccactttaca atcctggagg acttcctgga catcatgaac 1021 gagcaggcca atatcctggt gaagaagctg gagaagcaca tcaaccagga ggcctttaat 1081 tgcttctttt acatcaccct gtgcgccctg gacatcatct gtgagacagc tatgggcaag 1141 aacatcggcg cccagtctaa tgacgatagc gagtacgtgc gggccgtgta tagaatgagc 1201 gagatgatct ttaggcgcat caagatgccc tggctgtggc tggatctgtg gtatctgatg 1261 ttcaaggagg gctgggagca caagaagtcc ctgcagatcc tgcacacctt tacaaactct 1321 gtgatcgccg agagagccaa tgagatgaac gccaatgagg actgtagggg cgatggaagg 1381 ggcagcgccc cttccaagaa caagcggaga gccttcctgg acctgctgct gagcgtgacc 1441 gacgatgagg gcaatcgcct gtcccacgag gacatccggg aggaggtgga tacattcatg 1501 tttgagggac acgacaccac agccgccgcc atcaactggt ccctgtacct gctgggctct 1561 aatccagagg tgcagaagaa ggtggatcac gagctggacg acgtgttcgg caagtccgac 1621 aggccagcaa ccgtggagga tctgaagaag ctgagatacc tggagtgcgt gatcaaggag 1681 acactgcgcc tgttcccctc tgtgcctctg tttgcccggt ccgtgtctga ggactgtgag 1741 gtggccggct atcgcgtgct gaagggcacc gaggccgtga tcatccctta cgccctgcac 1801 cgggacccca ggtatttccc taacccagag gagtttcagc cagagagatt ctttcccgag 1861 aatgcccagg gcaggcaccc ttacgcctat gtgccattct ccgccggacc aaggaactgc 1921 atcggacaga agtttgccgt gatggaggag aaaaccatcc tgtcttgtat cctgagacac 1981 ttctggatcg agagcaatca gaagagggag gagctgggcc tggagggaca gctgatcctg 2041 cggccaagca acggcatctg gatcaaactg aaaagaagga acgctgacga gaggtaaaag 2101 cttgaattcc tcgaggatcc aataaaagat ctttattttc attagatctg tgtgttggtt 2161 ttttgtgtgt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 2221 ctggaaggtg ccactcccag tttaaactta attaagggcc gcaggaaccc ctagtgatgg 2281 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 2341 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc 2401 agg SEQ ID NO: 27 – CYP4V2 expression cassette: Left-ITR: 1-141 CAG promoter: 166-1880 CYP4V2 cDNA encoding the Q259K variant of the human CYP4V2 protein: 1938-3515 WPRE enhancer: 3551-4139 bGH polyA: 4163-4387 Right-ITR: 4399-4539 1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 51 CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC 101 GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCTAA 151 GGCAATTGAG ATCTCGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 201 ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA 251 ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT 301 TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC 351 CATTGACGTC AATGGGTGGA CTATTTACGG TAAACTGCCC ACTTGGCAGT 401 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG 451 GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT F&R Ref No.: 43219-0011WO1 501 CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGGTCG 551 AGGTGAGCCC CACGTTCTGC TTCACTCTCC CCATCTCCCC CCCCTCCCCA 601 CCCCCAATTT TGTATTTATT TATTTTTTAA TTATTTTGTG CAGCGATGGG 651 GGCGGGGGGG GGGGGGGCGC GCGCCAGGCG GGGCGGGGCG GGGCGAGGGG 701 CGGGGCGGGG CGAGGCGGAG AGGTGCGGCG GCAGCCAATC AGAGCGGCGC 751 GCTCCGAAAG TTTCCTTTTA TGGCGAGGCG GCGGCGGCGG CGGCCCTATA 801 AAAAGCGAAG CGCGCGGCGG GCGGGAGTCG CTGCGTTGCC TTCGCCCCGT 851 GCCCCGCTCC GCGCCGCCTC GCGCCGCCCG CCCCGGCTCT GACTGACCGC 901 GTTACTCCCA CAGGTGAGCG GGCGGGACGG CCCTTCTCCT CCGGGCTGTA 951 ATTAGCGCTT GGTTTAATGA CGGCTCGTTT CTTTTCTGTG GCTGCGTGAA 1001 AGCCTTAAAG GGCTCCGGGA GGGCCCTTTG TGCGGGGGGG AGCGGCTCGG 1051 GGGGTGCGTG CGTGTGTGTG TGCGTGGGGA GCGCCGCGTG CGGCCCGCGC 1101 TGCCCGGCGG CTGTGAGCGC TGCGGGCGCG GCGCGGGGCT TTGTGCGCTC 1151 CGCGTGTGCG CGAGGGGAGC GCGGCCGGGG GCGGTGCCCC GCGGTGCGGG 1201 GGGGCTGCGA GGGGAACAAA GGCTGCGTGC GGGGTGTGTG CGTGGGGGGG 1251 TGAGCAGGGG GTGTGGGCGC GGCGGTCGGG CTGTAACCCC CCCCTGCACC 1301 CCCCTCCCCG AGTTGCTGAG CACGGCCCGG CTTCGGGTGC GGGGCTCCGT 1351 GCGGGGCGTG GCGCGGGGCT CGCCGTGCCG GGCGGGGGGT GGCGGCAGGT 1401 GGGGGTGCCG GGCGGGGCGG GGCCGCCTCG GGCCGGGGAG GGCTCGGGGG 1451 AGGGGCGCGG CGGCCCCGGA GCGCCGGCGG CTGTCGAGGC GCGGCGAGCC 1501 GCAGCCATTG CCTTTTATGG TAATCGTGCG AGAGGGCGCA GGGACTTCCT 1551 TTGTCCCAAA TCTGGCGGAG CCGAAATCTG GGAGGCGCCG CCGCACCCCC 1601 TCTAGCGGGC GCGGGCGAAG CGGTGCGGCG CCGGCAGGAA GGAAATGGGC 1651 GGGGAGGGCC TTCGTGCGTC GCCGCGCCGC CGTCCCCTTC TCCATCTCCA 1701 GCCTCGGGGC TGCCGCAGGG GGACGGCTGC CTTCGGGGGG GACGGGGCAG 1751 GGCGGGGTTC GGCTTCTGGC GTGTGACCGG CGGCTCTAGA GCCTCTGCTA 1801 ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG CAACGTGCTG 1851 GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTCTAAT ACGACTCACT 1901 ATAGGGAGAC CCAAGCTGGC TAGCCAAAGC TTCCACCATG GCGGGGCTCT 1951 GGCTGGGGCT CGTGTGGCAG AAGCTGCTGC TGTGGGGCGC GGCGAGTGCC 2001 CTTTCCCTGG CCGGCGCCAG TCTGGTCCTG AGCCTGCTGC AGAGGGTGGC 2051 GAGCTACGCG CGGAAATGGC AGCAGATGCG GCCCATCCCC ACGGTGGCCC 2101 GCGCCTACCC ACTGGTGGGC CACGCGCTGC TGATGAAGCC GGACGGGCGA 2151 GAATTTTTTC AGCAGATCAT TGAGTACACA GAGGAATACC GCCACATGCC 2201 GCTGCTGAAG CTCTGGGTCG GGCCAGTGCC CATGGTGGCC CTTTATAATG 2251 CAGAAAATGT GGAGGTAATT TTAACTAGTT CAAAGCAAAT TGACAAATCC 2301 TCTATGTACA AGTTTTTAGA ACCATGGCTT GGCCTAGGAC TTCTTACAAG 2351 TACTGGAAAC AAATGGCGCT CCAGGAGAAA GATGTTAACA CCCACTTTCC 2401 ATTTTACCAT TCTGGAAGAT TTCTTAGATA TCATGAATGA ACAAGCAAAT 2451 ATATTGGTTA AGAAACTTGA AAAACACATT AACCAAGAAG CATTTAACTG 2501 CTTTTTTTAC ATCACTCTTT GTGCCTTAGA TATCATCTGT GAAACAGCTA 2551 TGGGGAAGAA TATTGGTGCT CAAAGTAATG ATGATTCCGA GTATGTCCGT 2601 GCAGTTTATA GAATGAGTGA GATGATATTT CGAAGAATAA AGATGCCCTG 2651 GCTTTGGCTT GATCTCTGGT ACCTTATGTT TAAAGAAGGA TGGGAACACA 2701 AAAAGAGCCT TAAGATCCTA CATACTTTTA CCAACAGTGT CATCGCGGAA 2751 CGGGCCAATG AAATGAACGC CAATGAAGAC TGTAGAGGTG ATGGCAGGGG 2801 CTCTGCCCCC TCCAAAAATA AACGCAGGGC CTTTCTTGAC TTGCTTTTAA 2851 GTGTGACTGA TGACGAAGGG AACAGGCTAA GTCATGAAGA TATTCGAGAA 2901 GAAGTTGACA CCTTCATGTT TGAGGGGCAC GATACAACTG CAGCTGCAAT 2951 AAACTGGTCC TTATACCTGT TGGGTTCTAA CCCAGAAGTC CAGAAAAAAG 3001 TGGATCATGA ATTGGATGAC GTGTTTGGGA AGTCTGACCG TCCCGCTACA F&R Ref No.: 43219-0011WO1 3051 GTAGAAGACC TGAAGAAACT TCGGTATCTG GAATGTGTTA TTAAGGAGAC 3101 CCTTCGCCTT TTTCCTTCTG TTCCTTTATT TGCCCGTAGT GTTAGTGAAG 3151 ATTGTGAAGT GGCAGGTTAC AGAGTTCTAA AAGGCACTGA AGCCGTCATC 3201 ATTCCCTATG CATTGCACAG AGATCCGAGA TACTTCCCCA ACCCCGAGGA 3251 GTTCCAGCCT GAGCGGTTCT TCCCCGAGAA TGCACAAGGG CGCCATCCAT 3301 ATGCCTACGT GCCCTTCTCT GCTGGCCCCA GGAACTGTAT AGGTCAAAAG 3351 TTTGCTGTGA TGGAAGAAAA GACCATTCTT TCGTGCATCC TGAGGCACTT 3401 TTGGATAGAA TCCAACCAGA AAAGAGAAGA GCTTGGTCTA GAAGGACAGT 3451 TGATTCTTCG TCCAAGTAAT GGCATCTGGA TCAAGTTGAA GAGGAGAAAT 3501 GCAGATGAAC GCTAAGCGGC CGCAACTCGA GACTCTAGAG GTTAATCGAT 3551 AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA 3601 CTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT 3651 ATCATGCTAT TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA 3701 TCCTGGTTGC TGTCTCTTTA TGAGGAGTTG TGGCCCGTTG TCAGGCAACG 3751 TGGCGTGGTG TGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCA 3801 TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT CCCCCTCCCT 3851 ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG 3901 GGCTCGGCTG TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAATCAT 3951 CGTCCTTTCC TTGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGG 4001 ACGTCCTTCT GCTACGTCCC TTCGGCCCTC AATCCAGCGG ACCTTCCTTC 4051 CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT CGCCTTCGCC 4101 CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCA TCGAAACCCG 4151 CTGACTAGAC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC 4201 TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC 4251 CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA 4301 TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC 4351 AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCCG CGGGCCGCAG 4401 GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG CTCGCTCGCT 4451 CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC TTTGCCCGGG 4501 CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG SEQ ID NO: 28 – CYP4V2 expression cassette: Left-ITR: 1-141 CAG promoter: 166-1880 Human CYP4V2 cDNA: 1938-3515 bGH polyA: 3574-3798 Right-ITR: 3810-3950 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggcctaa ggcaattgag atctcgacat tgattattga 181 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 241 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 301 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 361 aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 421 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 481 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 541 ccatgggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 601 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 661 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 721 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 781 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgttgcc 841 ttcgccccgt gccccgctcc gcgccgcctc gcgccgcccg ccccggctct gactgaccgc F&R Ref No.: 43219-0011WO1 901 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt 961 ggtttaatga cggctcgttt cttttctgtg gctgcgtgaa agccttaaag ggctccggga 1021 gggccctttg tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga 1081 gcgccgcgtg cggcccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct 1141 ttgtgcgctc cgcgtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg 1201 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg 1261 gtgtgggcgc ggcggtcggg ctgtaacccc cccctgcacc cccctccccg agttgctgag 1321 cacggcccgg cttcgggtgc ggggctccgt gcggggcgtg gcgcggggct cgccgtgccg 1381 ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggag 1441 ggctcggggg aggggcgcgg cggccccgga gcgccggcgg ctgtcgaggc gcggcgagcc 1501 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa 1561 tctggcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcgggcgaag 1621 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc 1681 cgtccccttc tccatctcca gcctcggggc tgccgcaggg ggacggctgc cttcgggggg 1741 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta 1801 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc 1861 tgtctcatca ttttggcaaa gaattctaat acgactcact atagggagac ccaagctggc 1921 tagccaaagc tgccaccatg gcggggctct ggctggggct cgtgtggcag aagctgctgc 1981 tgtggggcgc ggcgagtgcc ctttccctgg ccggcgccag tctggtcctg agcctgctgc 2041 agagggtggc gagctacgcg cggaaatggc agcagatgcg gcccatcccc acggtggccc 2101 gcgcctaccc actggtgggc cacgcgctgc tgatgaagcc ggacgggcga gaattttttc 2161 agcagatcat tgagtacaca gaggaatacc gccacatgcc gctgctgaag ctctgggtcg 2221 ggccagtgcc catggtggcc ctttataatg cagaaaatgt ggaggtaatt ttaactagtt 2281 caaagcaaat tgacaaatcc tctatgtaca agtttttaga accatggctt ggcctaggac 2341 ttcttacaag tactggaaac aaatggcgct ccaggagaaa gatgttaaca cccactttcc 2401 attttaccat tctggaagat ttcttagata tcatgaatga acaagcaaat atattggtta 2461 agaaacttga aaaacacatt aaccaagaag catttaactg ctttttttac atcactcttt 2521 gtgccttaga tatcatctgt gaaacagcta tggggaagaa tattggtgct caaagtaatg 2581 atgattccga gtatgtccgt gcagtttata gaatgagtga gatgatattt cgaagaataa 2641 agatgccctg gctttggctt gatctctggt accttatgtt taaagaagga tgggaacaca 2701 aaaagagcct tcagatccta catactttta ccaacagtgt catcgctgaa cgggccaatg 2761 aaatgaacgc caatgaagac tgtagaggtg atggcagggg ctctgccccc tccaaaaata 2821 aacgcagggc ctttcttgac ttgcttttaa gtgtgactga tgacgaaggg aacaggctaa 2881 gtcatgaaga tattcgagaa gaagttgaca ccttcatgtt tgaggggcac gatacaactg 2941 cagctgcaat aaactggtcc ttatacctgt tgggttctaa cccagaagtc cagaaaaaag 3001 tggatcatga attggatgac gtgtttggga agtctgaccg tcccgctaca gtagaagacc 3061 tgaagaaact tcggtatctg gaatgtgtta ttaaggagac ccttcgcctt tttccttctg 3121 ttcctttatt tgcccgtagt gttagtgaag attgtgaagt ggcaggttac agagttctaa 3181 aaggcactga agccgtcatc attccctatg cattgcacag agatccgaga tacttcccca 3241 accccgagga gttccagcct gagcggttct tccccgagaa tgcacaaggg cgccatccat 3301 atgcctacgt gcccttctct gctggcccca ggaactgtat aggtcaaaag tttgctgtga 3361 tggaagaaaa gaccattctt tcgtgcatcc tgaggcactt ttggatagaa tccaaccaga 3421 aaagagaaga gcttggtcta gaaggacagt tgattcttcg tccaagtaat ggcatctgga 3481 tcaagttgaa gaggagaaat gcagatgaac gctaagcggc cgcaactcga gactctagag 3541 gttaatcgat atcgaaaccc gctgactaga cgactgtgcc ttctagttgc cagccatctg 3601 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 3661 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 3721 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 3781 atgcggtggg ctctatggcc gcgggccgca ggaaccccta gtgatggagt tggccactcc 3841 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 3901 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg SEQ ID NO: 29 – CYP4V2 expression cassette: Left-ITR: 1-141 CAG promoter (a shorter version): 166-1101 F&R Ref No.: 43219-0011WO1 Human CYP4V2 cDNA: 1159-2736 bGH polyA: 2795-3019 Right-ITR: 3031-3171 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggcctaa ggcaattgag atctcccatt gacgtcaata 181 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 241 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 301 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 361 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag 421 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg 481 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc 541 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg 601 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg 661 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc 721 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 781 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa 841 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct 901 ttgtgcgggg ggagcggctc ggggctgtcc gcggggggac ggctgccttc gggggggacg 961 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca 1021 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc 1081 tcatcatttt ggcaaagaat tgaattctaa tacgactcac tatagggaga cccaagctgg 1141 ctagccaaag ctgccaccat ggcggggctc tggctggggc tcgtgtggca gaagctgctg 1201 ctgtggggcg cggcgagtgc cctttccctg gccggcgcca gtctggtcct gagcctgctg 1261 cagagggtgg cgagctacgc gcggaaatgg cagcagatgc ggcccatccc cacggtggcc 1321 cgcgcctacc cactggtggg ccacgcgctg ctgatgaagc cggacgggcg agaatttttt 1381 cagcagatca ttgagtacac agaggaatac cgccacatgc cgctgctgaa gctctgggtc 1441 gggccagtgc ccatggtggc cctttataat gcagaaaatg tggaggtaat tttaactagt 1501 tcaaagcaaa ttgacaaatc ctctatgtac aagtttttag aaccatggct tggcctagga 1561 cttcttacaa gtactggaaa caaatggcgc tccaggagaa agatgttaac acccactttc 1621 cattttacca ttctggaaga tttcttagat atcatgaatg aacaagcaaa tatattggtt 1681 aagaaacttg aaaaacacat taaccaagaa gcatttaact gcttttttta catcactctt 1741 tgtgccttag atatcatctg tgaaacagct atggggaaga atattggtgc tcaaagtaat 1801 gatgattccg agtatgtccg tgcagtttat agaatgagtg agatgatatt tcgaagaata 1861 aagatgccct ggctttggct tgatctctgg taccttatgt ttaaagaagg atgggaacac 1921 aaaaagagcc ttcagatcct acatactttt accaacagtg tcatcgctga acgggccaat 1981 gaaatgaacg ccaatgaaga ctgtagaggt gatggcaggg gctctgcccc ctccaaaaat 2041 aaacgcaggg cctttcttga cttgctttta agtgtgactg atgacgaagg gaacaggcta 2101 agtcatgaag atattcgaga agaagttgac accttcatgt ttgaggggca cgatacaact 2161 gcagctgcaa taaactggtc cttatacctg ttgggttcta acccagaagt ccagaaaaaa 2221 gtggatcatg aattggatga cgtgtttggg aagtctgacc gtcccgctac agtagaagac 2281 ctgaagaaac ttcggtatct ggaatgtgtt attaaggaga cccttcgcct ttttccttct 2341 gttcctttat ttgcccgtag tgttagtgaa gattgtgaag tggcaggtta cagagttcta 2401 aaaggcactg aagccgtcat cattccctat gcattgcaca gagatccgag atacttcccc 2461 aaccccgagg agttccagcc tgagcggttc ttccccgaga atgcacaagg gcgccatcca 2521 tatgcctacg tgcccttctc tgctggcccc aggaactgta taggtcaaaa gtttgctgtg 2581 atggaagaaa agaccattct ttcgtgcatc ctgaggcact tttggataga atccaaccag 2641 aaaagagaag agcttggtct agaaggacag ttgattcttc gtccaagtaa tggcatctgg 2701 atcaagttga agaggagaaa tgcagatgaa cgctaagcgg ccgcaactcg agactctaga 2761 ggttaatcga tatcgaaacc cgctgactag acgactgtgc cttctagttg ccagccatct 2821 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 2881 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 2941 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 3001 gatgcggtgg gctctatggc cgcgggccgc aggaacccct agtgatggag ttggccactc 3061 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 3121 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag g F&R Ref No.: 43219-0011WO1 SEQ ID NO: 30 – AAV2 capsid protein (735 aa) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKG EPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKR VLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWA LPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLM NPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNN NSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAK IPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEI EWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 31 – AAV5 capsid protein (724 aa) MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGE PVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRV LEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPAS SLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYR EIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQ VKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLN RDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQY LYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRME LEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPV NRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHP SPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKR WNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL SEQ ID NO: 32 – AAV204 capsid protein (736 aa) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGA KTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMA SGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNH YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTST VQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN NFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPK NWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFG KESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQNSSTDPATGDVHVMGALPGMVWQDRDV YLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVS VEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTVPRPIGTRYLTRPL SEQ ID NO: 33 – Nucleic acid sequence encoding AAV204 capsid protein (2211 bp) 1 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc F&R Ref No.: 43219-0011WO1 61 gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 121 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 181 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 241 cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 301 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 361 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 421 ggaaagaaac gtccggtaga gcagtcacca caagagccag actcctcctc gggcatcggc 481 aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 541 tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 601 actacaatgg cttcaggcgg tggcgcacca atggcggaca ataacgaagg cgccgacgga 661 gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 721 accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 781 tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 841 gggtattttg atttcaacag attccactgc catttctcac cacgtgactg gcagcgactc 901 atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 961 gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1021 gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1081 ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcagtacgg ctacctaacg 1141 ctcaacaatg gcagccaggc agtgggacgg tcatcctttt actgcctgga atatttccca 1201 tcgcagatgc tgagaacggg caataacttt accttcagct acaccttcga ggacgtgcct 1261 ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1321 cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1381 ttgctgttta gccgggggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1441 ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1501 tttacctgga caggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1561 ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1621 atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1681 acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1741 gcagtcaatc tccagaacag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1801 gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1861 aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1921 aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1981 gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2041 gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2101 tatacatcta actatgcaaa atctgccaac gttgatttca ctgtagacaa caatggactt 2161 tatactgtgc ctcgccccat tggcacccgt tacctcaccc gtcccctgta a SEQ ID NO: 34 (region of human CYP4V2 gene containing c.802- 8_810del17insGC mutation) CAAACAGAAGCATGTGATTATCATTCAAAGCGAACGGGCCAATGAAATGAACGCCAATGA SEQ ID NO: 35 (region of wild-type human CYP4V2 gene without the c.802-8_810del17insGC mutation) CAAACAGAAGCATGTGATTATCATTCAAATCATACAGGTCATCGCTGAACGGGCCAATGAAATGAACG CCAATGA SEQ ID NO: 36 – gRNA for CRISPR-mediated gene editing of the c.802- 8_810del17insGC mutation of the CYP4V2 gene (20 nt): UUCAUUGGCGUUCAUUUCAU F&R Ref No.: 43219-0011WO1 SEQ ID NO: 37 - Donor template sequence for genetic correction / repair of the c.802-8_810del17insGC mutation: TAGCATATTTTATAAGAAAATGTGTTAACTAGGGTGCATCCAAGTCCAAACAGAAGCATGTGATTATC ATTCAAATCATACAGGTCATCGCTGAACGGGCtAATGAAATGAACGCtAATGAAGACTGTAGAGGTGA TGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTTCTTGACTTGCTTTTAAGTGT SEQ ID NO: 38 - Primer of amplifying the CYP4V2 HDR (Forward): AGAGCCTATGTTGTCGAAATGTTG SEQ ID NO: 39 - Primer of amplifying the CYP4V2 HDR (Reverse): GCCTGTTCCCTTCGTCATCA References 1. Xue K, and MacLaren RE. Ocular gene therapy for choroideremia: clinical trials and future perspectives. Expert Rev Ophthalmol.2018;13(3):129-38. 2. Cowan CS, Renner M, De Gennaro M, Gross-Scherf B, Goldblum D, Hou Y, et al. Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution. Cell. 2020;182(6):1623-40 e34. 3. Ji C, Li Y, Kittredge A, Hopiavuori A, Ward N, Yao P, et al. Investigation and Restoration of BEST1 Activity in Patient-derived RPEs with Dominant Mutations. Sci Rep. 2019;9(1):19026. 4. Tsai YT, Li Y, Ryu J, Su PY, Cheng CH, Wu WH, et al. Impaired cholesterol efflux in retinal pigment epithelium of individuals with juvenile macular degeneration. Am J Hum Genet.2021;108(5):903-18. 5. Vasireddy V, Mills JA, Gaddameedi R, Basner-Tschakarjan E, Kohnke M, Black AD, et al. AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models. PLoS One.2013;8(5):e61396. 6. Li A, Jiao X, Munier FL, Schorderet DF, Yao W, Iwata F, et al. Bietti crystalline corneoretinal dystrophy is caused by mutations in the novel gene CYP4V2. Am J Hum Genet.2004;74(5):817-26. 7. Halford S, Liew G, Mackay DS, Sergouniotis PI, Holt R, Broadgate S, et al. Detailed phenotypic and genotypic characterization of bietti crystalline dystrophy. Ophthalmology.2014;121(6):1174-84. F&R Ref No.: 43219-0011WO1 8. Garcia-Garcia GP, Martinez-Rubio M, Moya-Moya MA, Perez-Santonja JJ, and Escribano J. Current perspectives in Bietti crystalline dystrophy. Clin Ophthalmol. 2019;13:1379-99. 9. Hanany M, Yang RR, Lam CM, Beryozkin A, Sundaresan Y, and Sharon D. An In-Depth Single-Gene Worldwide Carrier Frequency and Genetic Prevalence Analysis of CYP4V2 as the Cause of Bietti Crystalline Dystrophy. Transl Vis Sci Technol. 2023;12(2):27. 10. Zhang Z, Yan B, Gao F, Li Q, Meng X, Chen P, et al. PSCs Reveal PUFA- Provoked Mitochondrial Stress as a Central Node Potentiating RPE Degeneration in Bietti's Crystalline Dystrophy. Mol Ther.2020;28(12):2642-61. 11. Hata M, Ikeda HO, Iwai S, Iida Y, Gotoh N, Asaka I, et al. Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes. Proc Natl Acad Sci U S A. 2018;115(15):3936-41. 12. Wang JH, Lidgerwood GE, Daniszewski M, Hu ML, Roberts GE, Wong RCB, et al. AAV2-mediated gene therapy for Bietti crystalline dystrophy provides functional CYP4V2 in multiple relevant cell models. Sci Rep.2022;12(1):9525. 13. Ma Z, Jiao X, Agbaga MP, Anderson RE, Qian H, Li Q, et al. A Bietti Crystalline Dystrophy Mouse Model Shows Increased Sensitivity to Light-Induced Injury. Int J Mol Sci.2022;23(21). 14. Lockhart CM, Nakano M, Rettie AE, and Kelly EJ. Generation and characterization of a murine model of Bietti crystalline dystrophy. Invest Ophthalmol Vis Sci. 2014;55(9):5572-81. 15. Jia R, Meng X, Chen S, Zhang F, Du J, Liu X, et al. AAV-mediated gene- replacement therapy restores viability of BCD patient iPSC derived RPE cells and vision of Cyp4v3 knockout mice. Hum Mol Genet.2023;32(1):122-38. 16. Yang RR. A patient advocating for transparent science in rare disease research. Orphanet J Rare Dis.2023;18(1):14. 17. Jiao X, Li A, Jin ZB, Wang X, Iannaccone A, Traboulsi EI, et al. Identification and population history of CYP4V2 mutations in patients with Bietti crystalline corneoretinal dystrophy. Eur J Hum Genet.2017;25(4):461-71. F&R Ref No.: 43219-0011WO1 18. Xiao X, Mai G, Li S, Guo X, and Zhang Q. Identification of CYP4V2 mutation in 21 families and overview of mutation spectrum in Bietti crystalline corneoretinal dystrophy. Biochem Biophys Res Commun.2011;409(2):181-6. 19. Lockhart CM, Smith TB, Yang P, Naidu M, Rettie AE, Nath A, et al. Longitudinal characterisation of function and structure of Bietti crystalline dystrophy: report on a novel homozygous mutation in CYP4V2. Br J Ophthalmol.2018;102(2):187-94. 20. Kelly EJ, Nakano M, Rohatgi P, Yarov-Yarovoy V, and Rettie AE. Finding homes for orphan cytochrome P450s: CYP4V2 and CYP4F22 in disease states. Mol Interv. 2011;11(2):124-32. 21. Nakano M, Kelly EJ, Wiek C, Hanenberg H, and Rettie AE. CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant. Mol Pharmacol.2012;82(4):679- 86. 22. Tanito M, Brush RS, Elliott MH, Wicker LD, Henry KR, and Anderson RE. High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress- induced degeneration. J Lipid Res.2009;50(5):807-19. 23. Dalleau S, Baradat M, Gueraud F, and Huc L. Cell death and diseases related to oxidative stress: 4-hydroxynonenal (HNE) in the balance. Cell Death Differ. 2013;20(12):1615-30. 24. Ortolan D, Sharma R, Volkov A, Maminishkis A, Hotaling NA, Huryn LA, et al. Single-cell-resolution map of human retinal pigment epithelium helps discover subpopulations with differential disease sensitivity. Proc Natl Acad Sci U S A. 2022;119(19):e2117553119. 25. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005;85(3):845-81. 26. Garcia-Garcia GP, Martinez-Rubio M, Moya-Moya MA, Perez-Santonja JJ, and Escribano J. Identification of novel CYP4V2 genotypes associated with Bietti crystalline dystrophy and atypical anterior segment phenotypes in Spanish patients. Acta Ophthalmol. 2018;96(7):e865-e73. F&R Ref No.: 43219-0011WO1 27. Bright SR, Brown TE, and Varnum MD. Disease-associated mutations in CNGB3 produce gain of function alterations in cone cyclic nucleotide-gated channels. Mol Vis.2005;11:1141-50. 28. Childs B. Sir Archibald Garrod's conception of chemical individuality: a modern appreciation. N Engl J Med.1970;282(2):71-7. 29. Garrod A. The incidence of alkaptonuria, a study in chemical individuality. Lancet.1902;2:1616–20. 30. Rosenberg LE. Legacies of Garrod's brilliance. One hundred years--and counting. J Inherit Metab Dis.2008;31(5):574-9. 31. Scriver CR. Garrod's foresight; our hindsight. J Inherit Metab Dis. 2001;24(2):93-116. 32. Li Y, Nguyen HV, and Tsang SH. Skin Biopsy and Patient-Specific Stem Cell Lines. Methods Mol Biol.2016;1353:77-88. 33. Li Y, Wu WH, Hsu CW, Nguyen HV, Tsai YT, Chan L, et al. Gene therapy in patient-specific stem cell lines and a preclinical model of retinitis pigmentosa with membrane frizzled-related protein defects. Mol Ther.2014;22(9):1688-97. 34. Li Y, Zhang Y, Xu Y, Kittredge A, Ward N, Chen S, et al. Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca(2+)-dependent Cl(-) currents in human RPE. Elife.2017;6. 35. Idelson M, Alper R, Obolensky A, Ben-Shushan E, Hemo I, Yachimovich- Cohen N, et al. Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell.2009;5(4):396-408. 36. Sonoda S, Spee C, Barron E, Ryan SJ, Kannan R, and Hinton DR. A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nat Protoc.2009;4(5):662-73. 37. Heller KN, Montgomery CL, Janssen PM, Clark KR, Mendell JR, and Rodino-Klapac LR. AAV-mediated overexpression of human alpha7 integrin leads to histological and functional improvement in dystrophic mice. Mol Ther.2013;21(3):520-5. 38. Frederick A, Sullivan J, Liu L, et al. Engineered Capsids for Efficient Gene Delivery to the Retina and Cornea. Hum Gene Ther.2020;31(13-14):756-774. doi:10.1089 / hum.2020.070 F&R Ref No.: 43219-0011WO1 It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims. Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

F&R Ref No.: 43219-0011WO1 WHAT IS CLAIMED IS:

1. A method of treating, arresting, rescuing, ameliorating, preventing or slowing progression of a disease associated with oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS) or (ii) an aldehyde in a human subject, the method comprising: delivering to the human subject’s organ, tissue or cell affected by the disease, a therapeutically effective amount of a vector comprising a nucleic acid molecule comprising a sequence encoding a CYP4V2 protein, wherein said organ, tissue or cell of the human subject is transduced with the vector, whereby said disease is treated, arrested or prevented in the human subject.

2. A method of reducing, rescuing, preventing, treating, ameliorating, or slowing progression of, the death, dysfunction, degeneration, atrophy or dystrophy of a cell associated with oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS) or (ii) an aldehyde, the method comprising: delivering to the cell, a therapeutically effective amount of a vector comprising a nucleic acid molecule comprising a sequence encoding a CYP4V2 protein, wherein the cell is transduced with the vector.

3. The method of claim 1 or 2, wherein the oxidative stress, lipid peroxidation and / or abnormally high level of (i) reactive oxygen species (ROS), or (ii) an aldehyde is induced or aggravated by light.

4. The method of claim 2 or 3, wherein the cell is a retinal cell, a retinal pigment epithelium (RPE) cell, or a photoreceptor.

5. A method of treating, arresting, rescuing, ameliorating, preventing or slowing progression of a disease associated with light-induced or light-aggravated retinal damage, atrophy, dystrophy or degeneration in a human subject, the method comprising:F&R Ref No.: 43219-0011WO1 delivering to the human subject’s retina, a therapeutically effective amount of a vector comprising a nucleic acid molecule comprising a sequence encoding a CYP4V2 protein, whereby said disease is treated, arrested or prevented in the human subject, wherein the disease associated with light-induced or light-aggravated retinal damage, atrophy, dystrophy or degeneration in a human subject is attributed to oxidative stress, lipid peroxidation and / or abnormally high level of (i) a polyunsaturated fatty acid (PUFA), (ii) reactive oxygen species (ROS) and / or (iii) an aldehyde.

6. The method of claim 5, wherein the PUFA is an omega-3 PUFA or an omega- 6 PUFA.

7. The method of any one of claim 5 or 6, wherein the PUFA is arachidonic acid (AA) or docosahexaenoic acid (DHA).

8. The method of any one of claim 1, 2 or 5, wherein the aldehyde is 4- hydroxynonenal (4-HNE) or 4-hydroxy-2-hexenal (4-HHE).

9. The method of any one of claims 1 to 8, wherein the CYP4V2 protein is the human CYP4V2 protein (SEQ ID NO: 1) or its non-pathological polymorphism or variant thereof.

10. The method of any one of claims 1 to 9, wherein the CYP4V2 protein comprises up to two (2) amino acid differences to the human CYP4V2 protein of SEQ ID NO:1 or comprises the following amino acid difference as compared to the human CYP4V2 protein of SEQ ID NO:1: Gln259Lys, Leu22Val, Met123Val, or Glu275Lys, optionally, wherein the CYP4V2 protein is selected from any one of SEQ ID NO: 4, 6, 7 or 8.

11. The method of any one of claim 1 to 10, wherein the vector is a viral vector or a non-viral vector.F&R Ref No.: 43219-0011WO1 12. The method of any one of claims 1 to 11, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

13. The method of any one of claims 1 to 12, wherein the vector comprises a promoter and at least one additional regulatory element operably linked to the sequence encoding the CYP4V2 protein.

14. The method of claim 13, wherein the promoter is a CAG promoter, a CBA promoter, a CB promoter, a CMV promoter, an EF-1 alpha promoter, or a EFS promoter, optionally, wherein the CAG promoter shares at least 60% sequence identity with SEQ ID NO: 9 or 23.

15. The method of claim 13, wherein the regulatory element is a bGH PolyA, a SV40 PolyA, SV40 late PolyA, or small PolyA (SPA).

16. The method of claim 13, wherein the regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

17. The method of any one of claim 1 to 16, wherein any part of the nucleic acid molecule comprised within the vector shares at least 43% sequence identity with the entirety of any one of the CYP4V2 expression cassette sequences in SEQ ID NOs: 24 to 29 or at least 43% sequence identity with the entirety of any one of the following sequences: nucleotide (nt) 237 – nt 3579 of SEQ ID NO 24; nt 166 – nt 3515 of SEQ ID NO 25; nt 130 – nt 2097 of SEQ ID NO 26; nt 166 – nt 3515 of SEQ ID NO 27; nt 166 – nt 3515 of SEQ ID NO 28; or nt 166 – nt 2736 of SEQ ID NO 29.

18. The method of any one of claims 1 to 17, wherein the rAAV vector comprises: a VP1, VP2, or VP3 capsid protein selected from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or clade of AAV, or hybrids, variants or derivatives thereof, orF&R Ref No.: 43219-0011WO1 a 5′ AAV inverted terminal repeat (ITR) or a 3′ AAV ITR selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another naturally derived serotype or isolate or clade of AAV, or mutations, chimeras, variants or fusions thereof.

19. The method of any one of claims 1 to 18, wherein the vector is a chimeric AAV, a shuffled AAV, or a capsid-modified AAV, wherein the rAAV vector is a pseudotyped AAV, or wherein the rAAV vector is a hybrid AAV.

20. The method of any one of claims 1 to 19, wherein the vector is selected from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 2, AAV2 (Y444F+Y500F+Y730F), AAV2 / 1, AAV2 / 9, AAV2 / 8(Y733F), AAV2 / 6, AAV2 / 4, AAV2 / 7, AAV5, AAV2, AAV8, AAV1, AAV9, AAV6, AAV10, AAV3, AAV4, AAV7, AAV11, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV204, AAV-PHP.B, rh10, and a hybrid, a derivative or variant thereof, or wherein the rAAV vector is a single-stranded AAV vector or a self-complementary AAV (scAAV) vector.

21. The method of any one of claims 1 to 20, wherein the sequence encoding the CYP4V2 protein shares at least 60%, at least 70%, at least 75%, at least 77%, at least 78%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any one of SEQ ID NO: 2, 3 or 5.

22. The method of any one of claim 1 to 21, wherein the sequence encoding the CYP4V2 protein is a codon-optimized sequence.

23. The method of any one of claims 1 to 22, wherein the disease is an ocular disease, a retinal disease or a disease associated with the retinal pigment epithelium (RPE) degeneration, or Bietti Crystalline Dystrophy (BCD).F&R Ref No.: 43219-0011WO1 24. A method of determining the optimal AAV vector and / or the optimal dose of the AAV vector in AAV-mediated gene therapy treatment for a human subject, the method comprising: generating iPS-derived cells from the human subject, wherein the iPS-derived cell is the cell type targeted for gene therapy treatment; contacting the iPS-derived cells with a plurality of different AAV vectors and / or a plurality of different doses of the AAV vectors; determining the treatment effect on the iPS-derived cells of the plurality of different AAV vectors and / or the plurality of different doses of the AAV vectors, thereby determining the optimal AAV vector and / or the optimal dose of the AAV vector for the human subject.

25. The method of claim 24, wherein the iPS-derived cells are iPS-RPE (iRPE) cells or iPS-photoreceptor cells.

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