Recombinant adeno-associated viral vectors for treating 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 an expression cassette and a recombinant adeno-associated virus (rAAV) vector having a nucleic acid encoding CYP4V2, and further provides a viral particle having the rAAV vector, the composition of the viral particle, and its use.

Background Art

[0002] In the treatment of human genetic disorders, it has been found from recent progress that gene therapy is very promising [1]. More than 6,000 genetic diseases have been reported so far [2], and the number is only increasing with further disclosure of genomic sequencing technology [3]. With safer transduction vectors and gene editing techniques seen in recent years, the options for treatment methods for such genetic diseases have been greatly improved. As a result, it is possible to supplement defective genes with exogenous DNA carried by a therapeutic vector or to permanently correct them in situ by gene editing [4]. However, despite such promising prospects, the number of treatable genetic disorders has not increased so much at present [5]. This is mainly because there is no animal model convertible to humans, so the effect of gene therapy cannot be evaluated [6].

[0003] The first mention of Bietti crystalline dystrophy (BCD, MIM 210370), an autosomal recessive genetic disease, was made by the Italian ophthalmologist G. B. Bietti, who clearly confirmed the precipitation of yellowish-white crystals in the fundus of patients in 1937, so it is named after the doctor [7]. BCD accounts for up to 3% of all asymptomatic retinitis pigmentosa (RP) in Europe and up to 10% of asymptomatic autosomal recessive RP [8]. BCD is commonly seen in East Asia, especially in China, and it is said that gene mutations occur at a rate of 1 in 20,000 people [9].

[0004] Genetically, it was confirmed in 2000 that the cause of BCD is associated with chromosome 4q35 [8]. In 2004, it was revealed that CYP4V2, a member of the cytochrome P450 superfamily, is the BCD gene that causes the disease

[10] . In addition to crystal deposition in the retina and cornea, an altered fatty acid composition could also be confirmed in the serum of BCD patients, which suggested dysregulation of lipid metabolism [11&12]. Furthermore, BCD patients develop blindness or night blindness between the ages of 20 and 40 and progress to legal blindness by the ages of 50 to 60

[11] . Unfortunately, even 80 years after the discovery of BCD, no treatment has yet been found for this severe disease that leads to blindness.

[0005] AAV is currently prioritized as a suitable means for treating genetic disorders such as BCD because its long-term transgene expression has been demonstrated.

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

[0007] CN109136266A also provides a construct for expressing CYP4V2 by rAAV. In its packaging plasmid, a sequence encoding the short peptide C9 is inserted to enable specific binding to the CD59 antigen on the human RPE cell membrane. Furthermore, a sequence encoding the HRH peptide is inserted together with the CYP4V 2 coding sequence to inhibit VEGF.

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

[0009] Therefore, there is a need for improved techniques and methods for gene therapy compositions that can efficiently and safely restore the CYP4V2 gene function in BCD patients at present.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The inventor of the present invention developed a certain combination of an rAAV vector consisting of a codon-optimized coding sequence CYP4V2 and a gene expression regulatory sequence, and as a result, the expression level was significantly improved (up to 26.1-fold) compared to the previously published form

[13] , thus completing the present invention.

Means for Solving the Problems

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

[0012] In the present disclosure, first, the above isolated nucleic acid molecule further has a promoter functionally linked to the 5' of the nucleotide encoding CYP4V2. This promoter is preferably the CAG promoter of SEQ ID NO: 35.

[0013] In one embodiment, the isolated nucleic acid molecule further contains a polyadenylation sequence at the 3' of the nucleotide sequence encoding the CYP4V2 polypeptide. This polyadenylation sequence is preferably bovine growth hormone polyA, synthetic polyA (SPA), or simian virus 40 (SV40). More preferably, it is SV40 polyA.

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

[0015] In a preferred embodiment, this isolated nucleic acid molecule comprises the following.

[0016] (a) Any one nucleotide sequence 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 to 34.

[0017] In the present disclosure, second, a recombinant AAV (rAAV) vector constituting the nucleic acid molecule provided first is provided.

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

[0019] Thirdly provided in the present disclosure are the recombinant AAV vector virus particles provided second, and an AAV capsid selected from AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8, AAVAnc80L65, and variants thereof. Here, the capsid is preferably AAV8.

[0020] Fourthly provided in the present disclosure is a pharmaceutical composition comprising the virus particles provided third and a pharmaceutically acceptable excipient.

[0021] Fifthly provided in the present disclosure is the use of rAAV vectors in the drug manufacturing process by pharmaceutical manufacturers for treating or preventing Best vitelliform macular dystrophy (BCD) or other diseases of the retinal pigment epithelium (RPE) atrophy system.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

Figures 5A-5B

Figures 6A-6B

Figures 7A-7B

Best Mode for Carrying Out the Invention

[0023] Unless otherwise specified in this document, the technical and scientific terms used in this document have meanings that are normally understood by those skilled in the art in the field of the present invention.

[0024] As used in this document, including the appended claims, unless otherwise clearly specified from the context, expressions such as "one", "a", and "the" shall include their plural forms.

[0025] In the context of the present disclosure, unless otherwise specified, variations of expressions such as "having", "comprising", and "consisting of" are implicitly understood to include the described portions such as the amino acid sequence, nucleotide sequence, characteristics, steps, groups, etc., and are not understood to exclude other portions such as amino acid sequences, nucleotide sequences, characteristics, and steps. In this document, the expression "having" or variations thereof such as "consisting of", "comprising", or "composed of" or corresponding variations can be replaced. There are also examples where the expression "having" includes "consisting of".

[0026] The abbreviation "CYP4V2" means member 2 of subfamily V of the cytochrome P450 family which is the gene code of the CYP4V2 protein of the cytochrome P450 heme thiolate protein superfamily involved in the oxidation of various substrates within the metabolic pathway. It is known that defects within the gene CYP4V2 cause BCD and retinal dystrophy. Unless otherwise specified, CYP4V2 as referred to in this document means human CYP4V2 in the present disclosure.

[0027] An isolated nucleic acid encoding the CYP4V2 protein. The present disclosure provides an isolated nucleic acid sequence consisting of the nucleotide sequence encoding the CYP4V2 protein, particularly the human CYP4V2 protein. "Isolated nucleic acid" means DNA or RNA isolated from part or all of a polynucleotide, in which the isolated polynucleotide is naturally occurring within this nucleotide or is associated with nucleotides that do not naturally bind. An isolated nucleic acid molecule "having" a certain nucleotide sequence can include, in addition to the certain sequence, regulatory sequences functionally associated with controlling the expression of the coding region of the recited nucleic acid sequence. Due to codon degeneracy, those skilled in the art can understand that a certain amino acid sequence can be coded by different nucleotide sequences.

[0028] The nucleotide sequence coding for CYP4V2 of the present disclosure is subject to codon optimization and screening, and as a result, the expression rate is improved compared to the wild-type coding sequence without codon optimization.

[0029] For example, in the CYP4V2 coding sequence with codon optimization, Western blot analysis of the expression rate of the CYP4V2 protein in HEK293 cells and ARPE-19 cells can achieve an expression rate about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold compared to the wild-type coding sequence.

[0030] The coding sequence of codon-optimized CYP4V2 preferably contains a CpG number and / or a reduced number of CpG islands. For example, the coding sequence after codon optimization contains 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 coding sequence after codon optimization does not contain a CpG island. "CpG island" means a DNA region with a length of at least 200 bp, a GC ratio of 50% or more, and an observed / expected CpG ratio of 60% or more.

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

[0032] The coding sequence after codon optimization can be selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 2 to 17. It is desirable that the coding sequence after codon optimization is any one of SEQ ID NOs: 8, 9, 15, 16, and 17. Among the coding sequences after codon optimization, more preferably, it is SEQ ID NO: 16.

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

[0034] The promoter in the present disclosure may be any of a constitutive promoter, a tissue-specific promoter, or a cell type-specific promoter. For example, this promoter may be an RPE cell-specific promoter or the native promoter of CYP4V2. In a more preferred embodiment, this promoter is the CAG promoter having the sequence of SEQ ID NO: 35. The CAG promoter is a strong constitutive promoter that exhibits a high gene expression rate in mammalian expression vectors. The CAG promoter contains a promoter selected from the early enhancer element of cytomegalovirus (CMV), the first exon and the first intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin 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 functions naturally as a protein translation start site

[14] . The Kozak sequence enables correct translation of the protein and improves protein expression.

[0036] Furthermore, the nucleic acid in the present disclosure also includes an intron inserted between the promoter and the coding sequence. As is well known to those skilled in the art, there are also introns that improve the expression of eukaryotic genes. This intron in the present disclosure can also form part of the naturally occurring intron of the CYP4V2 gene.

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

[15] , with SV40 polyA being preferred. Any of the polyAs can be combined with the post-transcriptional regulatory element (WPRE) of the woodchuck hepatitis virus, which is a DNA sequence

[16] . This creates a three-dimensional structure during transcription and improves the expression rate, resulting in WPRE-bGH polyA, WPRE-SPA, or WPRE-SV40 polyA being set respectively.

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

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

[0040] In addition to the inserted nucleotide sequence, the rAAV vector is in single-stranded form. This rAAV vector usually has two inverted terminal repeats (ITRs) at both ends of the inserted nucleotide sequence. The ITRs of the present disclosure can be any ITRs derived from AAV serotypes. When referring to AAV ITR serotypes, the expression "derived from" indicates that the ITR can be an ITR of a specific serotype or a variant derived therefrom with modifications. In a preferred embodiment of the present disclosure, the rAAV vector consists of two ITRs derived from AAV2

[17] . For example, the rAAV vector has two AAV2 ITRs, or has a variant of the wild-type AAV2 ITR and an AAV2 ITR without the C region or C' region. The wild-type AAV2 ITR is placed at the 5' of the inserted nucleotide sequence, while the AAV2 ITR variant may be placed at the 3' of the inserted nucleotide sequence. The reverse is also possible.

[0041] The rAAV genome was packaged into an AAV capsid. This capsid may be derived from any of the AAV serotypes well known to those skilled in the art or whose future characteristics will become apparent. This capsid and the ITS may be derived from the same AAV serotype or different AAV serotypes. The said capsid is suitable for delivery to the eye (such as subretinal, intravitreal, or intraocular delivery, etc.). In certain embodiments, this AAV vector consists of a capsid of the AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8, or AAVAnc80L65 serotype or a variant thereof.

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

[18] . It has been demonstrated that the conversion of photoreceptor cells and retinal pigment epithelial cells (RPE) by ssAAV8 is more efficient than that by ssAAV2 or ssAAV5. Induced by the capsid protein, the virus converts the target cells, and subsequently, the introduced genome having the CYP4V2 gene and regulatory elements is released into the target RPE. The released rAAV genome maintains its stability and is independent of the host genome, enabling the stable production of the CYP4V2 functional protein in the subject to be treated.

[0043] Pharmaceutical composition The term "pharmaceutical composition" means a composition suitable for delivery to a subject. The pharmaceutical composition of the present disclosure is an isolated nucleic acid, the rAAV vector or viral particle of the present disclosure, and a pharmaceutically acceptable excipient. Conventionally pharmaceutically acceptable excipients are well known to those skilled in the art and may be either solid or liquid.

[0044] Therapeutic use The rAAV, viral particles, or the compositions of the present disclosure can be used for the treatment and prevention of BCD associated with CYP4V2 mutations. Furthermore, the rAAV, viral particles, or the compositions of the present disclosure are also effective for the treatment and prevention of other conditions and diseases associated with retinal pigment epithelium (RPE) atrophy, such as fundus dystrophy.

[0045] The expressions "to treat", "treatment of", or "treatment" include the cure or at least the alleviation of the symptoms of BCD or conditions and diseases associated with RPE atrophy.

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

[0047] In the present disclosure, the above viral particles or pharmaceutical compositions of the present invention are preferably applied intravitreally, such as by subretinal injection. Desirably, it is applied by subretinal injection or the like intravitreally.

[0048] Example Example 1. Selection of Promoter The inventors first prepared a CYP4V2-expressing AAV vector consisting of two ITRs, one CMV promoter, one wild-type CYP4V2 CDS, and SV40 polyA

[13] . Since there are reports that the expression is suppressed by the CMV promoter

[19] , subsequently, the CMV promoter was replaced with the CAG promoter, and thus a new vector named "CAG-hCyp4v2 (BCD1)" was used. As a result, the expression rate of CYP4V2 protein was significantly improved (Figure 1, lower part).

[0049] Example 2. Improvement of Cyp4v2 Expression Rate by Codon Optimization Since the expression rate of CYP4V2 protein can be improved by the composition of the introduced gene, the CYP4V2 coding sequence was codon-optimized to increase the codon usage frequency in human cells. Furthermore, in order to minimize the possibility of in vitro immune responses mediated by TLR9, the GC content and the number of CpG islands in the modified coding sequence were reduced. Based on the above requirements, ten different coding sequences (SEQ ID NOs: 2 to 17) were designed, synthesized, and cloned into the CAG promoter, and sixteen more constructs of the introduced gene were obtained (BCD2 to BCD17). Figure 1 below shows the CpG numbers, CpG island numbers, and sequence homology of these sequences to the wild-type sequence for SEQ ID NOs: 2 to 17.

[0050] Table 1. Comparison of GC content, CpG island numbers, and homology between the sixteen codon-optimized sequences and the wild-type coding sequence TIFF2025090820000001.tif129170

[0051] Example 3. Gene transfection of HEK293 cells and ARPE-19 cells and detection of expression rate HEK293 cells and ARPE-19 cells were maintained in DMEM + 10% FBS and passaged every 3 days with TrypLE. One day before gene transfection, HEK293 cells were seeded in 24-well plates at a density of 1 × 10 5 cells / cm 2 while ARPE-19 cells were seeded in 24-well plates at a density of 7 × 10 4 cells / cm 2 . The plasmid was transfected using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008) according to the instruction manual. 72 hours after gene transfection, these cells were lysed with a reaction mixture containing protease inhibitors (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Using Bio, CW0027), it was stirred at 12,000 rpm for 10 minutes, collected in RIPA lysis buffer (Beyotime Biotechnology, P0013C), 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 transfer membrane (Merck, HATF00010).

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

[0053] As a result of repeated experiments, as confirmed in the HEK293 cell (Figure 3) and ARPE-19 (Figure 4) evaluations, among the sixteen codon-optimized vectors tested, it was confirmed that CYP4V2 protein was highly expressed by the gene introduction constructs BCD8, BCD9, BCD15, BCD16, and BCD17. Accordingly, the gene introduction constructs BCD8, BCD9, BCD15, BCD16, and BCD17 were selected as further evaluation targets using BCD1 as a control. Western blot (WB) results are shown at the top of Figures 5 and 6. The gray values in the whole bands were calculated, analyzed, and normalized against BCD1 (at the bottom of Figures 5 and 6).

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

[0055] After that, BCD1, BCD8, and BCD16 were respectively packaged into replication-deficient AAV8. AAV8-BCD1, AAV8-BCD8, and AAV8-BCD16 virus particles were used to convert ARPE-19 at an MOI = 5 × 105 under the condition of 2 mM hydroxyurea.

[0056] In the AAV conversion experiment, it was confirmed that AAV8-BCD16 showed the highest expression rate (Figure 7).

[0057] References 1. Friedmann T, Roblin R. “Gene therapy for human genetic disease” (“Gene therapy for human genetic disease”); Science. 1972; 175: 949-955. 2. Mckusick VA. “Mendelian inheritance in man and its online version” (“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, 306 genomes identifies individuals resilient to severe mendelian childhood diseases” (“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” (“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” ("Vietti crystalline corneal and retinal dystrophy is caused by mutations in a 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 Vietti crystalline dystrophy" "Correlation of Pathology to Ophthalmology." 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 Vietti 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. "Gene Therapy for Bietti Crystalline Dystrophy in a High-Fat Diet-Exacerbated Murine Model". 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 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 having a nucleotide sequence of SEQ ID NO: 16, 8, 9, 15 or 17, encoding a human CYP4V2 polypeptide.

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

8.

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

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

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

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

7. The isolated nucleic acid molecule according to claim 1, further comprising a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

8. The isolated nucleic acid molecule according to claim 1, having a nucleotide sequence of SEQ ID NO: 33 having SEQ ID NO: 16, SEQ ID NO: 25 having SEQ ID NO: 8, SEQ ID NO: 26 having SEQ ID NO: 9, SEQ ID NO: 32 having SEQ ID NO: 15, or SEQ ID NO: 34 having SEQ ID NO:

17.

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

10. The recombinant AAV vector according to claim 9, having two inverted terminal repeats (ITRs).

11. The recombinant AAV vector according to claim 10, having two AAV2 inverted terminal repeats (ITRs).

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

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

14. A pharmaceutical composition comprising the viral particle according to claim 12 and a pharmaceutically acceptable excipient.

15. Use of the recombinant AAV vector according to claim 9 in the manufacture of a medicament for the treatment or prevention of Best vitelliform macular dystrophy (BCD).

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