Recombinant adeno-associated virus vectors for the treatment of Bietti crystalline dystrophy

Abstract

To provide recombinant adeno-associated viral vectors for treating Bietti Crystalline Dystrophy.SOLUTION: Provided are a recombinant adeno-associated vector comprising a codon-optimized sequence encoding CYP4 V2 linked to predetermine gene expression regulatory sequences, and its use in treating Bietti Crystalline Dystrophy (BCD).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure provides recombinant adeno-associated virus (rAAV) vectors containing expression cassettes and nucleic acids encoding CYP4V2, as well as viral particles containing the rAAV vectors, compositions of the viral particles, and uses thereof. [Background technology]

[0002] Recent advances have demonstrated the great promise of gene therapy for the treatment of human genetic disorders. [1] More than 6,000 genetic diseases have been described to date. [2] This number is only expected to grow with the development of new genome sequencing technologies. [3] Safer delivery vectors and recent gene editing technologies have greatly improved treatment options for these diseases. Consequently, defective genes can be supplemented with exogenous DNA delivered by therapeutic vectors or permanently corrected in situ by gene editing. [4] Despite these promising prospects, however, the number of treatable genetic disorders has not expanded significantly. [5] This is primarily due to the lack of animal models that can be translated into humans, making it impossible to evaluate the efficacy of gene therapy. [6]

[0003] The autosomal recessive disorder Bietti crystalline dystrophy (BCD, MIM 210370) was first described in 1937 by the Italian ophthalmologist GB Bietti, who clearly identified yellow-white crystalline deposits in the fundus of a patient's eye.[7] BCD accounts for up to 3% of all nonsyndromic retinitis pigmentosa (RP) cases in Europe and up to 10% of nonsyndromic autosomal recessive RP cases.[8] BCD is common in East Asia, particularly China, where the genetic mutation is thought to occur in 1 in 20,000 people.[9]

[0004] Genetic studies of BCD, linked to chromosome 4q35, were identified in 2000.[8] In 2004, CYP4V2, a member of the cytochrome P450 superfamily, was identified as the BCD gene responsible for the disease.

[10] In addition to retinal and corneal crystal deposition, altered fatty acid profiles were also identified in the serum of BCD patients, suggesting dysregulated lipid metabolism.[11&12] Furthermore, BCD patients develop blindness and night blindness between the ages of 20 and 40, progressing to legal blindness by the age of 50 or 60.

[11] Unfortunately, 80 years after the discovery of BCD, no cure has yet been found for this devastating disease.

[0005] AAV is nowadays preferred as a suitable tool for the treatment of genetic disorders such as BCD due to its demonstrated long-term transgene expression.

[0006] CN111733174B provides a construct that can be packaged into an AAV vector and also carries the coding sequences for CYP4V2 and RdCVF for the treatment of BCD.

[0007] CN109136266A also provides a rAAV-mediated CYP4V2 expression construct. The packaging plasmid contains a sequence encoding the short peptide C9, which allows specific binding to the CD59 antigen on the human RPE cell membrane. Furthermore, a sequence encoding the HRH peptide is inserted into the CYP4V2 expression construct. 2 inserted together with the coding sequence to inhibit VEGF.

[0008] CN111733174B and CN109136266A both use the wild-type coding sequence of CYP4V2.

[0009] In light of the above, there is currently a need for improved techniques and methods for gene therapy compositions that efficiently and safely restore CYP4V2 gene function in BCD patients. Summary of the Invention [Problem to be solved by the invention]

[0010] The inventors of the present invention have developed a combination of rAAV vectors consisting of codon-optimized coding sequences of CYP4V2 and gene expression regulatory sequences, which significantly improved expression levels (up to 26.1-fold) compared to previously published forms

[13] , thus completing the present invention. [Means for solving the problem]

[0011] Accordingly, the present disclosure relates primarily to an isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-17, and encoding human CYP4V2. In preferred embodiments, the nucleotide sequence is selected from SEQ ID NOs: 8, 9, 15, 16, and 17. In even more preferred embodiments, the nucleotide sequence is SEQ ID NO: 8 or SEQ ID NO: 16. Of these, SEQ ID NO: 16 is the most preferred nucleotide sequence.

[0012] In the present disclosure, first, the isolated nucleic acid molecule further comprises a promoter operably linked to the nucleotides 5' encoding CYP4V2, preferably the CAG promoter of SEQ ID NO: 35.

[0013] In one embodiment, the isolated nucleic acid molecule further comprises a polyadenylation sequence 3' to the nucleotide sequence encoding the CYP4V2 polypeptide, preferably bovine growth hormone polyA, synthetic polyA (SPA), or hybrid virus 40 (SV40), and more preferably SV40 polyA.

[0014] In another embodiment, the isolated nucleic acid further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) located between the woodchuck CYP4V2 coding sequence and the SV40 polyA sequence.

[0015] In one preferred embodiment, the isolated nucleic acid molecule comprises:

[0016] (a) a nucleotide sequence of any one of SEQ ID NOs: 18 to 34, or (b) A nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NOs: 18-34.

[0017] Second, the present disclosure provides a recombinant AAV (rAAV) comprising the nucleic acid molecule provided in the first section. A vector is provided.

[0018] In one embodiment, the rAAV vector has at least one ITR, and preferably two, and in a preferred embodiment, the two ITRs are derived from the AAV2 ITRs.

[0019] The present disclosure third provides a recombinant AAV vector virus particle as provided second, and an AAV capsid selected from AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrhlO, AAV2.7m8, AAVAnc80L65, and variants thereof, wherein the capsid is preferably AAV8.

[0020] Fourthly, the present disclosure provides a pharmaceutical composition comprising the virus particles provided thirdly and a pharmaceutically acceptable excipient.

[0021] Fifth, the present disclosure provides for the use of rAAV vectors by pharmaceutical manufacturers in drug manufacturing processes to treat or prevent Bietti crystalline dystrophy (BCD) or other diseases of the retinal pigment epitheliopathy (RPE) atrophy system. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the structures of CMV-hCyp4v2 (top) and CAG-hCyp4v2 (BCD1, bottom). [Figure 2] Figure 2 is a histogram showing the expression levels of the CYP4V2 transgene in the CMV-hCyp4v2 construct compared to the CAG-hCyp4v2 (BCD1) construct in HEK293. [Figure 3] Figure 3 shows the evaluation of the expression rate of the CYP4V2 transgene in constructs BCD1 to BCD17 in HEK293 cells. [Figure 4] Figure 4 shows the evaluation of the expression rate of the CYP4V2 transgene in constructs BCD1 to BCD17 in ARPE-19 cells. [Figure 5A-5B] 5A-5B show the expression rate of the CYP4V2 transgene in HEK293 cells at the constructs BCD8, BCD9, BCD15, BCD16, and BCD17 by Western blot (A) and histogram (B). [Figures 6A-6B] 6A-6B show the expression rate of the CYP4V2 transgene in the constituents BCD8, BCD9, BCD15, BCD16, and BCD17 in ARPE-19 cells by Western blot (A) and histogram (B). [Figures 7A-7B] Figures 7A-7B show the expression rate of CYP4V2 transgene protein in ARPE-19 cells transfected with the constructs AAV8-BCD1, AAV8-BCD8, and AAV8-BCD16 by Western blot (A) and histogram (B). DETAILED DESCRIPTION OF THE INVENTION

[0023] Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art of the present invention.

[0024] As used in this document, including the appended claims, unless the context clearly dictates otherwise, the terms "a," "an," "one," and "the" are intended to include the plural forms.

[0025] In the context of this disclosure, unless otherwise specified, variations of the terms "having", "including", "consisting of", etc. shall be understood to implicitly include the stated moiety, such as the amino acid sequence, nucleotide sequence, property, step, group, etc., and not to exclude other moieties, such as the amino acid sequence, nucleotide sequence, property, step, etc. Throughout this document, the term "having" or its variations "consisting of", "including", or "composed of" or equivalent variations can be substituted. In some instances, the term "having" also includes "consisting of".

[0026] The abbreviation "CYP4V2" refers to subfamily V of the cytochrome P450 family. CYP4V2, a member of the cytochrome P450 heme thiolate protein superfamily involved in the oxidation of various substrates in metabolic pathways. It is a genetic code for a protein. Defects in the gene CYP4V2 are known to cause BCD and fundus dystrophy. CYP4V2 as referred to herein refers to human CYP4V2 in this disclosure unless otherwise specified.

[0027] An isolated nucleic acid encoding a CYP4V2 protein. The present disclosure provides isolated nucleic acid sequences comprising the nucleotide sequence encoding a CYP4V2 protein, particularly a human CYP4V2 protein. "" refers to DNA or RNA isolated from part or all of a polynucleotide in which the isolated polynucleotide is naturally occurring or associated with nucleotides with which it is not naturally associated. An isolated nucleic acid molecule "having" a given nucleotide sequence can include, in addition to the given sequence, operably associated regulatory sequences that control expression of the coding region of the recited nucleic acid sequence. Due to codon degeneracy, one of skill in the art will understand that a given amino acid sequence can be coded for by different nucleotide sequences.

[0028] The nucleotide sequence coding for CYP4V2 of the present disclosure has been subjected to codon optimization and screening, resulting in improved expression rates compared to the wild-type coding sequence without codon optimization.

[0029] For example, Western blot analysis of CYP4V2 protein expression in HEK293 and ARPE-19 cells shows that a codon-optimized CYP4V2 coding sequence can achieve approximately 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or 20-fold higher expression than the wild-type coding sequence.

[0030] It is desirable for the codon-optimized CYP4V2 coding sequence to contain CpG numbers and / or a reduced number of CpG islands. For example, the codon-optimized coding sequence may contain 100 CpGs or less, 70 CpGs or less, 20 CpGs or less, 15 CpGs or less, 10 CpGs or less, 5 CpGs or less, or 0 CpGs. For example, the codon-optimized coding sequence does not contain any CpG islands. A "CpG island" refers to a DNA region at least 200 bp in length, with a GC ratio of 50% or greater, and an observed-to-expected CpG ratio of 60% or greater.

[0031] For example, the codon-optimized coding sequence, as shown in SEQ ID NO: 1, has a sequence identity of less than 80% compared to the wild-type coding sequence of CYP4V2. The homology ratio between two sequences can be calculated using any of the programs already used in the art. Due to differences in orientation parameters and homology definitions, the calculation of the homology ratio between two sequences may vary depending on the program used. In the present disclosure, the homology ratio between two sequences was calculated using blastn, and the "highly similar sequences (MegaBLAST)" mode was specifically selected.

[0032] The codon-optimized coding sequence can be selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOS: 2-17. Preferably, the codon-optimized coding sequence is any one of SEQ ID NOS: 8, 9, 15, 16, and 17. More preferably, the codon-optimized coding sequence is the sequence The number is 16.

[0033] The control sequences present in the nucleic acid molecule can be selected from one or more of a promoter, enhancer, polyadenylation sequence, and translation termination signal. Some combinations of control sequences in the present disclosure produce unexpected effects on the efficiency of expression of the coding sequence.

[0034] The promoter in the present disclosure may be a constitutive promoter, a tissue-specific promoter, or a cell type-specific promoter. For example, the promoter may be an RPE cell-specific promoter or the native promoter of CYP4V2. In a more preferred embodiment, the promoter is a CAG promoter having the sequence of SEQ ID NO: 35. The CAG promoter is a robust constitutive promoter that drives high gene expression in mammalian expression vectors. The CAG promoter includes a promoter selected from the cytomegalovirus (CMV) early enhancer element, the first exon and first intron of the chicken beta actin gene, and the splice acceptor of the rabbit betaglobin gene.

[0035] The Kozak consensus sequence (Kozak sequence), named after the scientist who discovered it, is a nucleic acid motif present in most eukaryotic mRNA transcripts that naturally functions as a protein translation initiation site.

[14] The Kozak sequence ensures proper protein translation and improves protein expression.

[0036] Additionally, the nucleic acids of the present disclosure may also contain an intron inserted between the promoter and the coding sequence. As those skilled in the art will appreciate, some introns enhance expression of eukaryotic genes. The introns of the present disclosure may be part of a naturally occurring intron in the CYP4V2 gene.

[0037] The polyadenylation sequence of the present disclosure may be bGH polyA, SPA, or SV40 polyA.

[15] SV40 polyA is preferred. Either polyA can be combined with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[16] This enhances expression by forming a three-dimensional structure during transcription, resulting in WPRE-bGH polyA, WPRE-SPA, or WPRE-SV40 polyA sequences, respectively.

[0038] In preferred embodiments of the present disclosure, the nucleic acid sequence comprises a nucleotide sequence encoding a CAG promoter, a Kozak sequence, a codon-optimized coding sequence of the CYP4V2 gene, and a WPRE-SV40 polyA. For example, the isolated nucleic acid sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 18-34. In more preferred embodiments, the isolated nucleic acid sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 25, 26, 32, 33, and 34. In a most preferred embodiment, the isolated nucleic acid sequence comprises SEQ ID NO: 33.

[0039] rAAV vectors and viral particles The nucleic acid molecules of the present disclosure can be incorporated into recombinant AAV vectors to obtain rAAV particles and provide them to a subject.

[0040] In addition to the inserted nucleotide sequence, the rAAV vector is in single-stranded form. rAAV vectors typically contain two inverted terminal end sequences on either side of the inserted nucleotide sequence. The ITRs of the present disclosure may be any ITRs derived from an AAV serotype. When referring to an AAV ITR serotype, the term "derived from" is used. The phrase "ITR" indicates that the ITR may be that of a particular serotype or a variant thereof with modifications derived therefrom. In a preferred embodiment of the present disclosure, the rAAV vector comprises two ITRs derived from AAV2

[17] . For example, the rAAV vector may have two AAV2 ITRs, or may have a wild-type AAV2 ITR and a variant of the AAV2 ITR lacking the C region or the C' region. The wild-type AAV2 ITR is located 5' of the inserted nucleotide sequence. while the AAV2 ITR variants are positioned at the It may be placed at the 3' position, or vice versa.

[0041] The rAAV genome is packaged into an AAV capsid. The capsid may be derived from any AAV serotype known to those of skill in the art or that will be characterized in the future. The capsid and ITS may be derived from the same AAV serotype or different AAV serotypes. The capsid may be delivered to the eye. Preferably, the AAV vector is suitable for delivery to a target organ (e.g., subretinal, intravitreal, or intraocular site). In some embodiments, the AAV vector comprises a capsid of the AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrhlO, AAV2.7m8, or AAVAnc80L65 serotype, or a variant thereof.

[0042] In a preferred embodiment of the present disclosure, the rAAV contains eight AAV serotype capsids.

[18] Transduction of photoreceptors and retinal pigment epithelial cells (RPE) with ssAAV8 has been demonstrated to be more efficient than transduction with ssAAV2 and ssAAV5. Guided by the capsid protein, the virus transduces the target cells, and the transduced genome containing the CYP4V2 gene and regulatory elements is subsequently released into the target RPE. The released rAAV genome remains stable and independent of the host genome, enabling stable production of functional CYP4V2 protein in the treated subject.

[0043] Pharmaceutical Composition The term "pharmaceutical composition" refers to a composition suitable for administration to a subject. A pharmaceutical composition of the present disclosure comprises an isolated nucleic acid, a rAAV vector or viral particle of the present disclosure, and a pharmaceutically acceptable excipient. Conventional pharmaceutically acceptable excipients are well known to those skilled in the art and can be solid or liquid.

[0044] therapeutic use The rAAV, viral particles, or compositions of the present disclosure can be used to treat or prevent BCD associated with CYP4V2 mutations. Additionally, the rAAV, viral particles, or compositions of the present disclosure may also be effective in treating or preventing other conditions or diseases associated with retinal pigment epithelium (RPE) atrophy, such as fundus dystrophies.

[0045] The terms "treat," "therapeutic," or "treatment" include curing or at least alleviating the symptoms of BCD or RPE atrophy-related conditions or diseases.

[0046] Administration As used herein, the terms "administration" and "administered," when applied to a subject such as an animal or human, or to a cell, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, or diagnostic agent or composition with the subject, cell, tissue, organ, or biological fluid. The term "administration" also includes the treatment of cells, etc., with reagents, diagnostic or binding compounds, or other cells, both in vitro and ex vivo.

[0047] In the present disclosure, the viral particles or pharmaceutical compositions of the present invention are administered intraocularly, such as by subretinal injection. It is desirable to apply

[0048] example Example 1. Promoter selection We first prepared a CYP4V2 expression AAV vector consisting of two ITRs, one CMV promoter, one wild-type CYP4V2 CDS, and SV40 polyA

[13] . Since it was reported that the CMV promoter suppresses expression

[19] , we subsequently replaced the CMV promoter with a CAG promoter, resulting in a new vector called "CAG-hCyp4v2 (BCD1)." As a result, the expression rate of CYP4V2 protein was significantly improved (Figure 1, bottom).

[0049] Example 2. Improving Cyp4v2 expression by codon optimization To improve CYP4V2 protein expression through transgene construction, the CYP4V2 coding sequence was codon-optimized to increase codon usage in human cells. Furthermore, to minimize the possibility of TLR9-mediated immune responses in vitro, the modified coding sequence was modified to reduce GC content and CpG island number. Based on the above requirements, 10 different coding sequences (SEQ ID NOS: 2-17) were designed, synthesized, and cloned into the CAG promoter, resulting in 16 additional transgene constructs (BCD2-BCD17). Figure 1 below shows the CpG numbers and CpG island numbers of SEQ ID NOS: 2-17, as well as their sequence homology to the wild-type sequence.

[0050] Table 1. GC content, CpG island number, and homology comparison of the 16 codon-optimized sequences with the wild-type coding sequence. TIFF0007811415000001.tif129170

[0051] Example 3. Gene transfection of HEK293 cells and ARPE-19 cells and detection of expression rate HEK293 and ARPE-19 cells were maintained in DMEM + 10% FBS and passaged every 3 days using TrypLE. The day before transfection, 1 × 10 5 cells / cm 2 HEK293 cells were seeded into 24-well plates at a density of 7 × 10 4 cells / cm 2 ARPE-19 cells were seeded in 24-well plates at a density of 1000 kJ / well. Plasmids were transfected using Lipofectamine according to the manufacturer's instructions. Transfection was performed using 3000 transfection reagent (Invitrogen, L3000008). 72 hours after transfection, the cells were transfected with a protease inhibitor cocktail (Roche, 04693159001) and SDS-PAGE additive (Cowin). The cells were lysed in RIPA lysis buffer (Biyuntian Bio, CW0027) at 12,000 rpm for 10 minutes, denatured at 95°C for 15 minutes, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was separated on a 4%-10% SDS-PAGE gel (Cowin Bio, CW0022M) and transferred onto a 0.45 μm NC membrane (Merck, HATF00010).

[0052] The expression rates of CYP4V2 and the housekeeping gene GAPDH were detected using antibodies against human CYP4V2 (Sigma, HPA029122) and GAPDH (Abcam, ab8245), respectively. Gray values ​​within the bands were calculated and normalized to BCD1.

[0053] Repeated experiments confirmed that of the 16 codon-optimized vectors tested, transfection constructs BCD8, BCD9, BCD15, BCD16, and BCD17 induced high levels of CYP4V2 protein expression, as confirmed by evaluation in HEK293 cells (Figure 3) and ARPE-19 (Figure 4). Accordingly, transfection constructs BCD8, BCD9, BCD15, BCD16, and BCD17 were selected for further evaluation, using BCD1 as a control. Western blot analysis The results of (WB) are shown in the top of Figures 5 and 6. The gray values ​​across the whole band were calculated, analyzed, and normalized to BCD1 (bottom of Figures 5 and 6).

[0054] Western blot results showed that the highest expression of CYP4V2 protein was observed in BCD8 in HEK293 cells (Figure 5), and the highest expression of CYP4V2 protein was observed in BCD16 in ARPE-19 cells (Figure 6).

[0055] BCD1, BCD8, and BCD16 were then packaged into replication-deficient AAV8, respectively, and AAV8-BCD1, AAV8-BCD8, and AAV8-BCD16 viral particles were used to transduce ARPE-19 at an MOI of 5 × 10 in the presence of 2 mM hydroxyurea.

[0056] AAV transduction experiments confirmed that AAV8-BCD16 showed the highest expression rate (Figure 7).

[0057] References 1. Friedmann T, Roblin R. “Gene therapy "Gene therapy for human genetic disease"; Science. 1972; 175: 949-955. 2. Mckusick VA. “Mendelian inheritance in man and its online version”, Omim. Am J Hum Genet. 2007;80:588-604. 3. Chen R, Shi LS, Hakenberg J, Naughton B, Sklar P, Zhang JG et al. “Analysis of 589 and 306 genomes identifies individuals resilient to severe Mendelian childhood diseases.” Nat Biotechnol. 2016; 34: 531-538. 4. Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. “Gene therapy comes of age.” Science. 2018; 359. 5. Ginn SL, Amaya AK, Alexander IE, Edelstein M, Abedi MR. “Gene therapy clinical trials worldwide to 2017: An update.” J Gene Med. 2018; 20. 6. Casal M, Haskins M. “Large animal models and gene therapy.” Eur J Hum Genet. 2006; 14: 266-272. 7. Bietti GB. “Retinitis punctata albescens” (verbunden mit “dystrophia marginalis cristallinea corneae”): 737-756. 8. Jiao XD, Munier FL, Iwata F, Hayakawa M, Kanai A, Lee J et al. “Genetic linkage of Bietti crystallin corneoretinal dystrophy to chromosome 4q35.” Am J Hum Genet. 2000;67:1309-1313. 9. Hu DN. “Ophthalmic genetics in China”. Ophthalmic Paed Gen. 1983;2:39-45. 10. 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” ("Bietti crystalline corneal and retinal dystrophy is caused by mutations in the novel gene cyp4v2."). Am J Hum Genet. 2004; 74: 817-826. 11. Kaiser-Kupfer MI, Chan CC, Markello TC, Crawford MA, Caruso RC, Csaky KG et al. “Clinical biochemical and pathologic correlations in bietti's crystalline Clinical biochemistry in Bietti crystalline dystrophy "Pathological Correlations." American Journal of Ophthalmology. 1994; 118: 569-582. 12. Lai TYY, Chu KO, Chan KP, Ng TK, Yam GHF, Lam DSC et al. “Alterations in serum fatty acid concentrations and desaturase activities in bietti crystalline dystrophy unaffected by cyp4v2 genotypes” ("Serum fatty acid concentrations and desaturase activities in Bietti crystalline dystrophy are unaffected by the cyp4v2 genotype."). Invest Ophth Vis Sci. 2010; 51: 1092-1097. 13. Qu B, Wu S, Jiao G, Zou X, Li Z, Guo L et al. "Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy." Gene Therapy. 2020. 14. Kozak M. “An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.” Nucleic Acids Research. 1987; 15: 8125-8148. 15. Connelly S, Manley JL. “A functional mRNA polyadenylation signal is required for transcription termination by RNA polymerase II. Genes & development.” 1988; 2: 440-452. 16. Zufferey R, Donello JE, Trono D, Hope TJ. “Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by "Posttranscriptional regulatory elements of woodchuck hepatitis virus enhance transgene expression delivered by retroviral vectors." Journal of Virology. 1999; 73: 2886-2892. 17. Grimm D, Kern A, Rittner K, Kleinschmidt JA. “Novel tools for production and purification of recombinant adenoassociated virus vectors.” Hum Gene Ther. 1998; 9: 2745-2760. 18. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.” Proceedings of the National Academy of Sciences of the United States of America. 2002; 99: 11854-11859. 19. Tenenbaum L, Chtarto A, Lehtonen E , Velu T, Brotchi J, Levivier M. “Recombinant AAV-mediated gene delivery to the central nervous system.” The Journal of Gene Medicine: A cross-disciplinary journal for research on the science of gene transfer and its clinical applications. 2004; 6: S212-S222.

Claims

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 16, 8, 9, 15 or 17, encoding a human CYP4V2 polypeptide.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence is SEQ ID NO: 16 or SEQ ID NO:

8.

3. 2. The isolated nucleic acid molecule of claim 1, further comprising a promoter operably linked to the 5' end of the nucleotide sequence encoding CYP4V2.

4. The isolated nucleic acid molecule of claim 3 , wherein the promoter is a CAG promoter.

5. 2. The isolated nucleic acid molecule of claim 1, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding CYP4V2.

6. 6. The isolated nucleic acid molecule of claim 5, wherein the polyadenylation sequence is bovine growth hormone (bGH) polyA, synthetic polyA (SPA), or hybrid virus (SV40) polyA.

7. 2. The isolated nucleic acid molecule of claim 1, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

8. 2. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence of SEQ ID NO:33 comprising SEQ ID NO:16, SEQ ID NO:25 comprising SEQ ID NO:8, SEQ ID NO:26 comprising SEQ ID NO:9, SEQ ID NO:32 comprising SEQ ID NO:15, or SEQ ID NO:34 comprising SEQ ID NO:

17.

9. A recombinant AAV vector comprising the nucleic acid molecule of any one of claims 1 to 8.

10. The recombinant AAV vector of claim 9, which has two inverted terminal repeats (ITRs).

11. The recombinant AAV vector of claim 10, which has two AAV2 inverted terminal repeats (ITRs).

12. A viral particle having the recombinant AAV vector of claim 9 packaged in an AAV capsid.

13. The viral particle of claim 12, wherein the AAV capsid is an AAV8 capsid.

14. A pharmaceutical composition comprising the virus particle of claim 12 and a pharmaceutically acceptable excipient.

15. Use of the recombinant AAV vector of claim 9 in the manufacture of a medicament for the treatment or prevention of Bietti crystalline dystrophy (BCD).

Images