WO2024110770A1

WO2024110770A1 - A new promoter for retinal pigment epithelium (rpe) targeted gene therapy

Info

Publication number: WO2024110770A1
Application number: PCT/IB2022/000679
Authority: WO
Inventors: Thierry Leveillard; Frédéric BLOND; Léa THIÉBAULT
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Sorbonne Université; Centre National De La Recherche Scientifique
Priority date: 2022-11-22
Filing date: 2022-11-22
Publication date: 2024-05-30

Abstract

Retinal pigment epithelium (RPE) structure and function are essential to normal vision, and alterations in the RPE can impair function and lead to retinopathy. Gene therapy is currently investigated for the treatment of retinal pigment epithelial diseases but there is a need for identifying promoter polynucleotides that will allow specific expression of polynucleotides of interest in said tissue. The SLC16A8 gene encodes for a RPE-specific lactate transporter MCT3. The inventors aimed at defining the sequence sufficient to drive specific expression of SLC16A8 in the RPE. To identify the promoter region of SLC16A8, different candidate fragments were then cloned into plasmids expressing a reporter gene to test the ability of each of these regions to activate the expression of this reporter. They also chose to exploit the restricted expression of SLC16A8 and to perform this experiment by a new in vivo approach using recombinant adenovirus associated virus (rAAV). By combining the methods, the inventors succeed in identifying the promoter region of SLC16A8.

Description

A NEW PROMOTER FOR RETINAL PIGMENT EPITHELIUM (RPE) TARGETED GENE THERAPY FIELD OF THE PRESENT INVENTION: The present invention is in the field of medicine, in particular ophthalmology. BACKGROUND OF THE PRESENT INVENTION: Retinal pigment epithelium (RPE) is formed from a single layer of regular polygonal cells arranged at the outermost layer of the retina. The outer side of the RPE is connected to Bruch’s membrane and the choroid, while the inner side is connected to the outer segment of photoreceptor cells. The outer side exhibits basal infolding, which increases cell surface area and facilitates substance exchange. The basement membrane is closely connected to the basal folds by half desmosomes located in the innermost layer of Bruch’s membrane. The inside of RPE cells harbors microvillous structures extending between photoreceptor outer segments (POS), which participate in the phagocytic function of the RPE. The tight junction formed between the single-layer RPE and the gap junction control the movement of substances and at the same time forms the choroid-blood-retinal barrier with Bruch’s membrane and choroid at the lateral retina. The RPE appears dark brown due to its melanin content, which reduces damage to the retina and internal nerves from ultraviolet light. The RPE also harbors a complex metabolic system that reduces excessive accumulation of reactive oxygen species (ROS) and consequent oxidative damage. Therefore, RPE structure and function are essential to normal vision, and alterations in the RPE can impair function and lead to retinopathy. For example, retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt disease (SD) are degenerative retinal diseases in which RPE dysfunction has been implicated in their pathogenesis. Gene therapy is currently investigated for the treatment of retinal pigment epithelial diseases but there is a need for identifying promoter polynucleotides that will allow specific expression of polynucleotides of interest in said tissue. The SLC16A8 gene encodes for a RPE-specific lactate transporter MCT3 and several risk alleles at the SLC16A8 locus for AMD. The promoter of said gene could represent an interesting candidate for restraining gene expression in RPE but its characterization has not been carried out. SUMMARY OF THE PRESENT INVENTION: The present invention is defined by the claims. In particular, the present invention relates to a new promoter for Retinal pigment epithelium (RPE) targeted gene therapy. DETAILED DESCRIPTION OF THE PRESENT INVENTION: Main definitions: As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. As used herein, the term “polynucleotide” or “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits. The promoter of the present invention can be prepared by any method known to one skilled in the art, including chemical synthesis, recombination, and mutagenesis. In particular, the promoter of the present invention is a DNA molecule, typically synthesized by recombinant methods well known to those skilled in the art. As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide or polynucleotide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide or polynucleotide that is different from the first one). As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleic acid sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “nucleic acid sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleic acid sequence encoding the protein may in some version contain an intron(s). As used herein, the term “transgene” refers to a polynucleotide that is introduced into the cells of a tissue or an organ and is capable of being expressed under appropriate conditions, or otherwise conferring a beneficial property to the cells. A transgene is selected based upon a desired therapeutic outcome. As used herein, the term “transgene product” refers to any molecule that is encoded by a transgene and confers a beneficial property to the cells or a desired therapeutic outcome. Typically, the transgene product is a polypeptide. As used herein, the terms "vector" refers to the vehicle by which a polynucleotide can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. As used herein, the term “viral vector” encompasses vector DNA as well as viral particles generated thereof. Viral vectors can be replication-competent, or can be genetically disabled so as to be replication-defective or replication-impaired. The term “replication-competent” as used herein encompasses replication-selective and conditionally-replicative viral vectors which are engineered to replicate better or selectively in specific host cells (e.g. tumoral cells). As used herein, the term “non-viral vector” notably refers to a vector of plasmid origin, and optionally such a vector combined with one or more substances improving the transfectional efficiency and/or the stability of said vector and/or the protection of said vector. As used herein, the term “promoter” refers to a polynucleic acid sequence (such as, for example, a DNA sequence) recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleic acid sequence, thereby allowing the expression of a gene product operably linked to the promoter/regulatory sequence. In particular, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter sequence may, for example, be one which expresses the gene product in a tissue specific manner. As used herein, the term “promoter activity” refers to the ability of a promoter to initiate transcription of a nucleic acid to which it is operably linked. Promoter activity can be measured using procedures known in the art or as described in the Examples. For example, promoter activity can be measured as an amount of mRNA transcribed by using, for example, Northern blotting or polymerase chain reaction (PCR). Alternatively, promoter activity can be measured as an amount of translated protein product, for example, by Western blotting, ELISA, colorimetric assays and various activity assays, including reporter gene assays and other procedures known in the art. As used herein, the term "operably linked" or "transcriptional control" refers to functional linkage between a regulatory sequence and a heterologous polynucleic acid sequence resulting in expression of the latter. For example, a first polynucleic acid sequence is operably linked with a second polynucleic acid sequence when the first polynucleic acid sequence is placed in a functional relationship with the second polynucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame. As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or polynucleic acid sequence are identical irrespective of any chemical and/or biological modification. According to the invention a first amino acid sequence having at least 80% of identity with a second amino acid sequence means that the first sequence has 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. As used herein, the term “retinal pigment epithelium” or “RPE” has its general meaning in the art and refers to the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells, and that is firmly attached to the underlying choroid and overlying retinal visual cells. RPE provides vital metabolic support to other retinal layers but is not directly involved in encoding visual stimuli into neurological signals, and is not responsive to light. RPE cells are darkly pigmented and absorb stray photons that would otherwise contribute to light scatter within the eye. As used herein, the term "gene therapy" refers to the introduction of a polynucleotide into a cell's genome that restores, corrects, or modifies the gene and/or expression of the gene. Thus the term “RPE gene therapy” refers to a gene therapy that is applied to the RPE, in particular for expressing a transgene product in RPE cells. As used herein, the term “therapeutic gene” refers to a gene encoding a therapeutic protein which is useful in the treatment of a pathological condition. The therapeutic gene, when expressed, confers a beneficial effect on the cell or tissue in which it is present, or on a patient in which the gene is expressed. Examples of beneficial effects include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desired characteristic. Therapeutic genes include genes that partially or wholly correct a genetic deficiency in the patient. In particular, the therapeutic gene may be, without limitation, a nucleic acid sequence encoding a protein useful in gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of said protein in a cell or tissue of a subject. The therapeutic polypeptide may, e.g., supply a polypeptide and/or enzymatic activity that is absent, defective or present at a sub-optimal level in RPE cells, supply a polypeptide and/or enzymatic activity that indirectly counteracts an imbalance in RPE cells. The therapeutic polypeptide may also be used to reduce the activity of a polypeptide by acting, e.g., as a dominant-negative polypeptide. Typically, the therapeutic polypeptide supplies a polypeptide and/or enzymatic activity that is absent, defective or present at a sub-optimal level in RPE cells, more typically a polypeptide and/or enzymatic activity that is absent or defective in RPE cells. As used herein, the term “expression cassette” refers to a nucleic acid construct comprising a coding sequence and one or more control sequences required for expression of said coding sequence. In particular, one of these control sequence is the promoter of the present invention that has a promoter activity in RPE. Generally, the expression cassette comprises a coding sequence and regulatory sequences preceding (5′ non-coding sequences) and following (3′ non- coding sequences) the coding sequence that are required for expression of the selected gene product. Thus, an expression cassette typically comprises a promoter sequence, a coding sequence and a 3′ untranslated region that usually contains a polyadenylation site and/or transcription terminator. The expression cassette may also comprise additional regulatory elements such as, for example, enhancer sequences, a polylinker sequence facilitating the insertion of a DNA fragment within a vector and/or splicing signal sequences. The expression cassette is usually included within a vector, to facilitate cloning and transformation. As used herein, the term "transformation" means the introduction of a "foreign" (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed". As used herein, the term "transduced cell" relates to a genetically modified cell i.e. a cell wherein the transgene has been introduced deliberately. The herein provided transduced cell comprises the transgene of the present invention. As used herein, the term "patient" or "patient in need thereof", is intended for a human or non-human mammal. Typically, the patient is affected or likely to be affected with a retinal pigment epithelial disease. As used herein, the term “retinal pigment epithelial disease” refers to an inherited retinal disease or an acquired retinal disease that affects the function of the RPE. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). As used herein, the term “therapeutic level” refers to the amount of a transgene product or the level of activity of a transgene product sufficient to confer its therapeutic or beneficial effect(s) in the host receiving the transgene. Expression levels of the transgene or the levels of activity of the transgene product can be measured at the protein or the mRNA level using methods known in the art. As used herein, the term “therapeutically efficient amount” is intended an amount of pharmaceutical composition of the present invention administered to a subject that is sufficient to constitute a treatment as defined above of ocular disease. Promoter of the present invention: The first object of the present invention relates to a polynucleotide having a promoter activity in retinal pigment epithelium (RPE) having a nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:1. > SEQ ID NO:1 CGCTGCCTCTGTTGGGAGGGGGCGGGGACAAGAGGGAGGGGCTGGGCCAGCAGCCTAGAG AAGGAGGGGGATGGAGACGAAGCATGGGGAAACTGAGGCACAGGGCTACCCACGTGAGGG TGGCTCAGGTCTGGGAACCAAAAGGGGAAACGGAGGTAGGACTCTCACCCCAAGTCTCCT TCTCCTGCAAAGGGCGCTGAAGGACGAGGGGAGGACGACGGGTCCCACCTGACTCTGCAC CTCTGCAGGCTCCCTCTGAAGGACAACTGCTGGCCCCTCAGGGCCTCAGGTGGAAGGCGT CGGGAAGTGGAGCAGGTATGTGCCTGGAGGTGGGCGTCCAGCACCAAAGTCGCCCCGTAC GCGTGAGGGTCTCGGGACAACAGAACAAACAGCAGAGGGGCAGATGCCGGCTGCAAAGGG CCGCTGTGCGTGATTTTCCTCTTTAGCCTTCCGCCATGTTCCTAATGTTTCTCAGTGATC ACAAATGACTATTGTCATTAGAACATTAAGGGGACAAAAGCCACTTTCAACAGATATCAG CCAGGGGAGCTGAGTTCCCATGCATCTCAGCTCAGCCATGGATTCCCTCCCTGACTTGGG CCGGTCGCCTCCCCTCTCCCAACCTCCCTTTCCTCCGATGTAAGATGGGTGATTGATCAC CCCGAGGCCAGCTGGGAATCTTGCGGGAGAGGAGTCAGGTCTCTGGCTTCTGCCAGACAG GTAGGAACCTTAGCAGCACTGAGAGAGTGGCCTTCTGCCCTCCAAACCTCAAACCCTCTA TATGAAAAGAATGGCTCCAAAGCCTGTCTCATTCCTGGGGCTCCACCCGGGGCCCTCACA GCAGTTACAAGCGCCCCCATAACAGATGAGGAGGGTGTACTCAGAGACAGGTGCACCAGG AGCCGGGGGCTGGGGATAGCCTCCTGCCCTCCTGCCTGGAGTGTCCCTCCTGGCACTGGG CAGTGCCACCACCCCCAGGGTATCCGGGACTCTGCCCCTCCTCCACTAGGACCAGGTTGT GGCTTTTCCTGCCTGTTGTCCTCTTGTCACAGGGGCAGCCACTTGAAGGTGGAGACAGGA GGGTGATCCCAGGCAGCCCTCACATCACACTCACCTGAGCCTGCCTCGTGGGTTTGGGGG TCCAGCCTCACATCTCCTGAGGCCTGCAGGGAGGGGCCGCCGCCGCAGCTCCCGGTGAAG CTTACCCGGATGGGGGCTGCCAGACACCCCAGCTTCCCACCAGCCTGGGCTGAGCCTCTG GCCGACTCCCGCCTGACGCTCCCTGCCCCAGGCCTGGAGCTTAGAGTGTGTGCCCCCACC ACACCTCACCCCTGCTAAGAGGCTTTTGTCCAGGAGGGGAGAAGAGACTGGGAGCCCCAG GCCAGGCTGGGCCTCGGCGGTGGGCTCCCTGTCCCAAGCCAGTCCAGGAGGGAGGAAGAG AATCCCTGTGCCTCCTGGAGCCTGGGGTGGTAGACACAGAGACCTTGCCCAGGGGGTCCT AGTCTGGTGCTGGGGGCACAGCCCTGCCCTGAAGAGACCCCAACGGTTCTAATCACCTCA GTGCCTCATCTGCCCCCGCCCCAACCCCCCGCCCCAGGCCACATTCCAGGAGGGGCTGGC AGCTTTCTCTAGAATGAAGCTAATCCACGCCTGTGCCTGGGAATGTGGGGGGGGGAGGGG CCAAAACAAAGGGTGCTGTGTCCAGGCCGCCTGAGGGCAGCTTGCTGGTGGTCCCAGCCC TTGGCCACTGCCACCTCTCCCCTGACCCGAGCAGCCGCTACTTCCTAGGAAACCCACAGG TCGAAATGAGAGCAGGTGGAGATGGGGGCTGCAGCGCGAGGGCTTTGGGGCAGCCCTAAA GAGTTCTCCAAACAAACAAACAAGGGGCTGGGAGTGAGATGGCTTCCCGGGGAACAGCCA GGACCTCAGTACCCCCTAGACCCCATTCTCCAATGGGGGAGTCTCTATCCCTCACCCCCT GCCCTGTCCCCCAAATAAGACACCACCAGTCCTG The nucleic acid sequence of SEQ ID NO: 1 was derived from the regulatory region of the SLC16A8 gene encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3. The promoter of the present invention thus exhibits a promoter activity in RPE, i.e. when introduced in RPE, it can initiate transcription of a nucleic acid to which it is operably linked. Typically, the promoter activity is specific of RPE cells. The term “specific of RPE cells” shall be understood to mean a promoter mainly active in RPE cells. It shall be understood that a residual expression, generally lower, in other tissues or cells cannot be entirely excluded. In some embodiments, the promoter of the present invention is not active in Muller cells or in photoreceptors. In some embodiments, the promoter of the present invention comprises, or consists of, the sequence of SEQ ID NO: 1. In some embodiments, the promoter of the present invention comprises, or consists of, a functional variant of SEQ ID NO: 1. As used herein, the term “variant” refers to a nucleic acid sequence differing from the original sequence, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the original polynucleotide. The sequence of the variant may differ by nucleotide substitutions, deletions or insertions of one or more nucleotides in the sequence, which do not impair the promoter activity. The variant may have the same length of the original sequence, or may be shorter or longer. The term “functional variant” refers to a variant of SEQ ID NO: 1 that exhibits a promoter activity of SEQ ID NO: 1, i.e. that exhibits a promoter activity in RPE cells, typically a promoter activity specific of RPE cells. In some embodiments, the promoter of the present invention comprises, or consists of, a functional variant having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 1, typically over the entire sequence of SEQ ID NO: 1. The promoter of the present invention may differ from the polynucleotide of SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, deletions and/or insertions. In some embodiments, the promoter of the present invention comprises, or consists of, a functional variant having a sequence capable of hybridizing under low, medium or high stringency conditions with the nucleic acid sequence of SEQ ID NO: 1 or its complementary strand, typically under medium stringency conditions, more typically under high stringency conditions. As used herein, the term “low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 50°C. The term “medium stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 55°C. The term “high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 65°C. Expression cassettes of the present invention: A further object of the present invention relates to an expression cassette comprising a polynucleotide of the present invention operably linked to a polynucleotide of interest. Typically, the nucleic acid operably linked to the promoter of the present invention is a transgene that encodes a polypeptide of interest or a polynucleotide of interest. Typically, the promoter of the present invention is operably linked to a heterologous polynucleotide. As used herein, the term “heterologous” means a nucleic acid other than the nucleic acid that the promoter is operably linked to in a naturally occurring genome. In some embodiments, the promoter of the present invention is not operably linked to SLC16A8 gene, and in particular to the human SLC16A8 gene. In some embodiments, the polynucleotide operably linked to the promoter of the present invention encodes a polypeptide of interest. The polypeptide of interest may be any polypeptide of which expression in RPE cells is desired. In particular, the polypeptide of interest may be a therapeutic polypeptide, or reporter protein. In some embodiments, the nucleic acid operably linked to the promoter of the present invention is a therapeutic gene. Examples of therapeutic genes include, but are not limited to, nucleic acids for replacement of a missing or mutated gene known to cause retinal disease such as ARSG, BEST1, DFNB31, IQCB1, KCNJ13, LCA5, LRAT, MERTK, RGR, RPE65, SPATA7. In some embodiments, the therapeutic gene may also encode neurotrophic factors such as GDNF (Gene ID: 2668), CNTF (Gene ID: 1270), FGF2 (Gene ID: 2247), BDNF (Gene ID: 627) and EPO (Gene ID: 2056), anti-apoptotic genes such as BCL2 (Gene ID: 596) and BCL2L1 (Gene ID: 598), anti-angiogenic factors such as endostatin, angiostatin and sFlt, anti-inflammatory factors such as IL10 (Gene ID: 3586), IL1R1 (Gene ID: 3554), TGFBI (Gene ID; 7045) and IL4 (Gene ID: 3565), or nucleoredoxin-like 1 the rod-derived cone viability factor (RdCVF) and RdCVFL (Gene ID: 115861). In some embodiments, the transgene product of interest is an endonuclease that provides for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a RPE disease. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a retinal structural protein and/or provides for normal retinal function, a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele. In addition to knocking out a defective allele, a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, e.g., the method of the invention can be used to deliver both a site-specific endonuclease that knocks out a defective allele, and can be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional retinal protein (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.). See, e.g., Li et al. (2011) Nature 475:217. In some embodiments, the DNA targeting endonuclease of the present invention is a TALEN. In some embodiments, the DNA targeting endonuclease of the present invention is a ZFN. In some embodiments, the DNA targeting endonuclease of the present invention is a CRISPR-associated endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. In some embodiments, the CRISPR-associated endonuclease is a Cpf1 nuclease. In some embodiments, the polynucleotide operably linked to the promoter of the present invention encodes a polynucleotide of interest. The polynucleotide of interest may be any nucleic acid of which expression in RPE cells is desired. In particular, the polynucleotide of interest may be a therapeutic nucleic acid. The nucleic acid may be, for example, an siRNA, an shRNA an RNAi, a miRNA, an antisense RNA, a ribozyme or a DNAzyme. In some embodiments, the nucleic acid encodes an RNA that when transcribed from the nucleic acid operably linked to the promoter of the present invention can treat or prevent a retinal pigment epithelial disease by interfering with translation or transcription of an abnormal or excess protein associated with said disorder. For example, the polynucleotide of interest may encode for an RNA, which treats the disease by highly specific elimination or reduction of mRNA encoding the abnormal and/or excess proteins. The expression cassette of the present invention may comprise one or more nucleic acids operably linked to the promoter of the present invention. For example, the promoter may be operably linked to one or more therapeutic genes and a nucleic acid encoding a reporter protein or to a therapeutic gene. Vectors of the present invention: A further object of the present invention relates to a vector comprising the promoter of the present invention or the expression cassette of the present invention. Typically, the vector of the present invention is a vector suitable for use in gene therapy, and in particular is suitable to target RPE cells. The vector of the present invention is typically a viral genome vector including any element required to establish the expression of the polypeptide of interest in a host cell such as, for example, a promoter, e.g., a polynucleotide of the present invention, an ITR, a ribosome binding element, terminator, enhancer, selection marker, intron, polyA signal, and/or origin of replication. In some embodiments, the vector is a viral vector, such as vectors derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV or SNV, lentiviral vectors (e.g. derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or equine infectious anemia virus (EIAV)), adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Ban virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors. In some embodiments, the vector is a retroviral vector, typically a lentiviral vector or a non- pathogenic parvovirus. As is known in the art, depending on the specific viral vector considered for use, suitable sequences should be introduced in the vector of the present invention for obtaining a functional viral vector, such as AAV ITRs for an AAV vector, or LTRs for lentiviral vectors. In some embodiments, the vector is an AAV vector. As used herein, the term “AAV vector” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequence (ITR), typically two ITRs. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins). An “inverted terminal repeat” or “ITR” sequence is a term well understood in the art and refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation. An “AAV inverted terminal repeat (ITR)” sequence is an approximately 145-nucleic acid sequence that is present at both termini of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (designated A, A′, B, B′, C, C and D regions), allowing intra-strand base-pairing to occur within this portion of the ITR. AAV ITRs for use in the vectors of the present invention may have a wild-type nucleic acid sequence or may be altered by the insertion, deletion or substitution. The serotype of the inverted terminal repeats (ITRs) of the AAV vector may be selected from any known human or nonhuman AAV serotype. The promoter or expression cassette of the present invention may be introduced into the vector by any method known by the skilled person. The vector of the present invention may be packaged into a virus capsid to generate a “viral particle”. Thus, in a further aspect, the present invention also relates to a viral particle comprising a vector of the present invention. In some embodiments, the vector is an AAV vector and is packaged into an AAV-derived capsid to generate an “adeno-associated viral particle” or “AAV particle”. Thus, used herein, the term “AAV particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV vector genome. The capsid serotype determines the tropism range of the AAV particle. Multiple serotypes of adeno-associated virus (AAV), including 12 human serotypes and more than 100 serotypes from nonhuman primates have now been identified (Howarth al., 2010, Cell Biol Toxicol 26: 1-10). Among these serotypes, human serotype 2 was the first AAV developed as a gene transfer vector. Other currently used AAV serotypes include, but are not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74 and AAVdj, etc. In addition, non-natural engineered variants and chimeric AAV can also be useful. In particular, the capsid proteins may be variants comprising one or more amino acid substitutions enhancing transduction efficiency. Different AAV serotypes are used to optimize transduction of particular target cells or to target specific cell types within a particular target tissue (e.g., RPE cells). An AAV particle can comprise viral proteins and viral nucleic acids of the same serotype or any natural or artificial sequence variant of AAV. For example, the AAV particle may comprise AAV2 capsid proteins and at least one, typically two, AAV2 ITR. Any combination of AAV serotypes for production of an AAV particle is provided herein as if each combination had been expressly stated herein. In preferred embodiment, the AAV particle comprises an AAV-derived capsid selected from the group consisting of AAV2, AAV-5, AAV-7m8 (AAV2-7m8, Dalkara et al. Sci Transl Med (2013), 5, 189ra76), AAV9 or AAV8 capsid. AAV viruses may be engineered using conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus. Alternatively, to using AAV natural serotypes, artificial AAV serotypes may be used in the context of the present invention, including, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a VP1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non- contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non- viral source. An artificial AAV serotype may be, without limitation, a chimeric AAV capsid or a mutated AAV capsid. Numerous methods are known in the art for production of viral particles, and in particular AAV particles, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J E et al., (1997) Virology 71(11):8780-8789) and baculovirus-AAV hybrids). AAV production cultures for the production of AAV virus particles all require; 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect- derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild-type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a polynucleotide of interest flanked by at least one AAV ITR sequences, e.g., a vector of the present invention; and 5) suitable media and media components to support AAV production that are well-known in the art. In practicing the invention, host cells for producing AAV particles include mammalian cells, insect cells, plant cells, microorganisms and yeast. Host cells can also be packaging cells in which the AAV rep and cap genes are stably maintained in the host cell or producer cells in which the AAV vector genome is stably maintained. Exemplary packaging and producer cells are derived from 293, A549 or HeLa cells. AAV particles are then purified and formulated using standard techniques known in the art. Host cells of the present invention: A further object of the present invention to an isolated host cell transformed or transfected with an expression cassette, vector or viral particle of the present invention. The host cell may be any animal cell, plant cell, bacterium cell or yeast. Typically, the host cell is a mammalian cell or an insect cell. More typically, the host cell is a human cell. In some embodiments, the host cell is a RPE cell, in particular a human RPE cell. The expression cassette or vector of the present invention may be transferred into host cells using any known technique including, but being not limited to, calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, microinjection, biolistic, lipofection, or viral infection, and may be maintained in the host cell in an ectopic form or may be integrated into the genome. In some embodiments, the expression cassette or vector of the present invention is transferred into the host cell by viral infection, typically using a viral particle of the present invention, more typically using an AAV particle of the present invention. Pharmaceutical compositions of the present invention: The present invention also relates to a pharmaceutical composition comprising an expression cassette, vector, viral particle or cell of the present invention. Such compositions comprise a therapeutically effective amount of the therapeutic agent (an expression cassette, vector, viral particle or cell of the present invention), and a pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and/or humans. The term “excipient” refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, pH buffering substances, and buffers. Such excipients include any pharmaceutical agent suitable for direct delivery to the eye which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various tween compounds, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences, 15th Edition. Typically, the composition is formulated to be administered to the eye, in particular by intraocular injection, e.g., by subretinal and/or intravitreal administration. Accordingly, the composition can be combined with pharmaceutically acceptable excipient such as saline, Ringer's balanced salt solution (pH 7.4), and the like. The pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms. In some embodiments, the pharmaceutical composition comprises a vector or viral particle of the present invention, more typically an AAV vector or particle. In some embodiments, the pharmaceutical composition comprises host cells of the present invention, typically human host cell of the present invention, i.e. transformed or transfected with an expression cassette, vector or viral particle of the present invention, typically with an AAV particle. Optionally, the composition comprising host cells may be frozen for storage at any temperature appropriate for storage of the cells. For example, the cells may be frozen at about −20° C., −80°C. or any other appropriate temperature. Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce risk of cell damage and maximize the likelihood that the cells will survive thawing. Alternatively, the cells may also be maintained at room temperature of refrigerated, e.g. at about 4°C. The amount of pharmaceutical composition to be administered may be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight) and type and severity of the disease being treated have to be taken into account to determine the appropriate dosage. The pharmaceutical composition of the present invention may be administered as a single dose or in multiple doses. In some embodiments, the composition comprises viral particles of the present invention and each unit dosage comprises from 108 to 1013 viral particles, typically from 109 to 1012 particles. The pharmaceutical composition may further comprise one or several additional active compounds such as corticosteroids, antibiotics, analgesics, immunosuppressants, trophic factors, or any combinations thereof. Methods of therapy of the present invention: A further object of the present invention relates to a method of therapy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an expression cassette, a vector, a viral particle, a host cell or a pharmaceutical composition of the present invention. The method of the present invention is particularly suitable for the treatment of an ocular disease. The method of the present invention is particularly suitable for the treatment of a retinal pigment epithelial disease. Examples of retinal pigment epithelial diseases include, but are not limited to age-related macular degeneration, Leber's hereditary optic neuropathy, cone-rod dystrophy, Leber congenital amaurosis, Stargardt's disease, diabetic retinopathy, retinal detachment, Best's disease, retinitis pigmentosa, choroideremia and a tapetoretinal degeneration. Alternatively, the ocular disease is selected from disease which are not necessarily or specifically associated with RPE, but which can be treated or prevented by expressing specifically nucleic acid encoding a polypeptide or polynucleotide of interest in RPE cell. In the method for treating ocular disease of the present invention, the pharmaceutical composition of the present invention is typically administered intraocularly, more typically by subretinal or intravitreal administration. The method of the present invention may also further comprise administering at least one additional therapeutic agent to the subject. In particular, said therapeutic agent may be selected from the group consisting of a corticosteroid, an antibiotic, an analgesic, an immunosuppressant, or a trophic factor, or any combinations thereof. The composition of the present invention may be administered before or after the disease becomes symptomatic, e.g., before or after partial or complete RPE degeneration and/or before or after partial or complete loss of vision. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. EXAMPLE: To study the promoter of the SLC16A8 gene, we first needed to identify the promoter region of the gene and hypothesized that the genomic sequence between the transcription start site (TSS) of SLC16A8 and the end of the BAIAP2L2 gene located immediately on 5' end (Chr22: 38,083,143-38,084,902) might contain the promoter region. To test the ability of this promoter region, we combined it with a reporter gene encoding enhanced green fluorescent protein (eGFP) in a plasmid carrying the inverted terminal repeat (ITR) sequences of AAV serotype 2. We therefore performed the cloning of the region measuring 1.7 kb (-1759 to -1 bp) in the plasmid pAAV2-CMV/CBA-eGFP which contains the transgenic cassette of the eGFP reporter gene, under the control of the chimeric promoter associating that of cytomegalovirus and chicken beta-actin or chicken β-actin (CMV/CBA). The digestion/ligation process was used to remove the CMV/CBA promoter from the recipient plasmid to introduce our candidate promoter region instead. We chose to take advantage of the restricted expression of SLC16A8 in the RPE and perform this experiment using a novel in vivo approach through the use of recombinant adeno-associated viruses (rAAV). BALB/cJ strain mice were selected as the experimental model. Knowing that the efficiency of transduction in the retina by an adeno- associated vector serotype 9 (AAV9) following systemic injection has been demonstrated previously, we generated an AAV9 vector to assess the ability of the SLC16A81.8 region(AAV2.9-SLC16A81.8-eGFP) to direct GFP expression in vivo in RPE cells. Therefore, a RPE-restricted fluorescent signal after systemic injection of AAV2.9-SLC16A81.8-eGFP (“SLC16A81.8”) would imply that the 1.8 kb fragment contains the entire promoter with its regulatory elements. Viruses were administered systemically by intracardiac injection in 3- to 4-day-old wt/wt BALB/cJ mice (D3-J4 ) to study in vivo GFP expression. Intracardiac injection of CMV/CBA- led to very strong GFP expression, confirming the correct performance of the technical procedure. In contrast, SLC16A81.8 did not show a green fluorescent signal at the macroscopic scale. To see on a microscopic scale whether the RPE of mice injected with SLC16A81.8 expressed GFP, we performed a western blot of RPE/choroid protein extracts obtained by sonication and centrifugation of the sclera/choroid/RPE cup and neural retina of injected mice following their sacrifice. As observed in fundus images, GFP expression is very high in both the RPE/choroid and retinas of CMV/CBV-injected mice. However, mice injected with SLC16A81.8, express low levels of GFP in the RPE/choroid. The absence or low expression of GFP in the RPE of mice injected with SLC16A81.8 led us to question whether the TSS reported in the UCSC database corresponded to a minor TSS. Indeed, SLC16A8 expression is restricted to the RPE, a tissue that is not among those analysed by ENCODE. Results from the study of the NXNL1 and NXNL2 genes show that there are several TSSs. Thus, for SLC16A8, we used the TSS of EST AF132610 (38078137-38083142) that was deposited by Nancy Philp, the researcher who identified the gene from the RPE (Yoon, Heeyong, et al. "Identification of a unique monocarboxylate transporter (MCT3) in retinal pigment epithelium." Biochemical and biophysical research communications 234.1 (1997): 90-94.). To circumvent this problem of the position of the TSS in our construct, we constructed a new reporter whose 3' end corresponds to the transduction initiation site, located in exon 3 of the SLC16A8 gene. This construct, which includes the 3' UTR of the SLC16A8 messenger, necessarily includes the TSS(s) regardless of its position in the genome. Therefore, the sequence upstream of the translation initiation codon (ATG) of the SLC16A8 gene (-1724 to +290 bp) was cloned, designated the “SLC16A82.1 promoter”, and was packaged into an AAV9 vector (AAV2.9-SLC16A82.1-eGFP). In addition, we generated an AAV9 vector with the BEST1 gene promoter (AAV2.9-5'585BEST1-eGFP – “5'BEST1”), for use as a specific control for expression by RPE. We again performed intracardiac injections according to the previously described protocol. AAV2.9-5'585BEST1- eGFP shows a GFP signal as green fluorescent dots. The specific expression of 5'BEST1 by the RPE suggests that these dots represent expression of the GFP reporter protein under the control of the active promoter in the RPE and not in the retina. While we did not observe a fluorescent signal in the eye fundus of mice injected with the SLC16A81.8 vector, as before, mice injected with the SLC16A82.1vector show intense fluorescent dots associated with more diffuse labeling confirming the presence of regulatory elements in the SLC16A82.1 vector. These observations were confirmed by quantitative western blot analysis as quantification of specific GFP expression in the RPE/choroid and in the retina (revealed significantly higher GFP expression by the SLC16A82.1 vector compared with that expressed by the 5'BEST1 vector. Taken together, these results demonstrate that the SLC16A82.1 fragment comprises the regulatory elements controlling SLC16A8 promoter activity by RPE cells. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS: 1. A polynucleotide having a promoter activity in retinal pigment epithelium (RPE) having a nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:1. 2. The polynucleotide of claim 1 that comprises, or consists of, the sequence of SEQ ID NO: 1. 3. The polynucleotide of claim 1 that comprises, or consists of, a functional variant having at least 80% identity with the nucleic acid sequence as set forth in SEQ ID NO:1 and may differ from the polynucleotide of SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, deletions and/or insertions. 4. The polynucleotide of claim 1 that comprises, or consists of, a functional variant having a sequence capable of hybridizing under low, medium or high stringency conditions with the nucleic acid sequence of SEQ ID NO: 1 or its complementary strand, typically under medium stringency conditions, more typically under high stringency conditions. 5. An expression cassette comprising the polynucleotide according to any one of claims 1 to 4 operably linked to a polynucleotide of interest. 6. The expression cassette of claim 5 wherein the polynucleotide of interest is a transgene that encodes a polypeptide of interest or a polynucleotide of interest. 7. The expression cassette of claim 6 wherein the transgene is a therapeutic gene. 8. The expression cassette of claim 6 wherein the transgene encodes for an endonuclease that provides for site-specific knock-down of gene function. 9. The expression cassette of claim 6 wherein the polynucleotide of interest is a therapeutic nucleic acid that is an siRNA, an shRNA an RNAi, a miRNA, an antisense RNA, a ribozyme or a DNAzyme. 10. A vector comprising the polynucleotide according to any of claims 1 to 4 or the expression cassette according to any one of claims 5 to 9. 11. The vector of claim 10 that is a viral vector, in particular an AAV vector. 12. An isolated host cell transformed or transfected with the expression cassette according to any one of claims 5 to 9 or the vector according to claim 10 or 11. 13. A pharmaceutical composition comprising the expression cassette according to any one of claims 5 to 9 or the vector according to claim 10 or 11 or the cell according to claim 12. 14. A method of therapy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the expression cassette according to any one of claims 5 to 9 or the vector according to claim 10 or 11, the cell according to claim 12 or the pharmaceutical composition according to claim 13. 15. The method of claim 14 wherein the patient suffers from a retinal pigment epithelial disease such as age-related macular degeneration, Leber's hereditary optic neuropathy, cone-rod dystrophy, Leber congenital amaurosis, Stargardt's disease, diabetic retinopathy, retinal detachment, Best's disease, retinitis pigmentosa, choroideremia or a tapetoretinal degeneration.