Improved treatment for rare eye disease through gene replacement

Abstract

The present invention relates to recombinant adeno-associated virus (AAV) vectors carrying a nucleic acid sequence encoding the retinal transcription factor cone-rod homeobox (CRX) for use in the treatment of CRX-associated IRDs in subjects in need thereof, or for use in the treatment of inherited retinal dystrophies caused by hypomorphic mutations in CRX target genes.

Description

[Technical field]

[0001] The present invention relates to a method for treating retinal diseases and to a pharmaceutical composition comprising CRX as an active substance. [Background technology]

[0002] The progressive loss of photoreceptors (PR) in inherited retinal diseases (IRD) is a common cause of visual impairment, with a prevalence of 1 / 4,000, implying approximately 17,000 patients in France. They form a group of genetically and clinically heterogeneous diseases with more than 60 identified human genes. The efficacy and safety of gene therapy by intraocular injection of adeno-associated viruses (AAV) have been proven with preclinical successes that translate into clinical efficacy. Such techniques are most often used in patients with recessive loss of function mutations. The aim is to compensate the mutated gene causing the disease with a fully functional copy.

[0003] Dominant forms of PR degeneration represent at least 20% of cases of retinitis pigmentosa (RP). Today, a very limited number of therapeutic approaches have been initiated with these dominant forms. In fact, dominant mutations can increase the activity of gene products or provide new activities of gene products. Thus, in contrast to recessive forms, dominant forms of PR degeneration are more difficult to cure, since it may not be sufficient to restore the lost function. In fact, dominant forms may require the expression of a healthy copy, with the concomitant silencing of the mutated gene product.

[0004] Over 50 mutations in the human CRX gene are associated with autosomal dominant Leber congenital amaurosis (LCA), cone-rod dystrophy (CORD), and RP.

[0005] The transcription factor Cone-Rod Homeobox (CRX) plays a central role in controlling gene expression in rod and cone photoreceptors, the primary light-sensing cells in the retina. Cone-Rod Homeobox (CRX) proteins are "paired-like" homeodomain transcription factors that are important in regulating rod and cone photoreceptor transcription. CRX acts as a transcriptional activator by interacting with coactivators, promoting histone acetylation at target gene promoters, and mediating intrachromosomal looping interactions of enhancer / promoter genes of target photoreceptor genes.

[0006] In this context, it has surprisingly been found that expression of the transcription factor CRX can be used to treat subjects with a CRX-related disease, i.e. an inherited dystrophy associated with a dominant retinal mutation in CRX, or subjects with at least one hypomorphic mutation in at least one target gene of CRX, without any side effects.

[0007] The present invention relates generally to recombinant viral vectors and methods of using same to express a CRX protein in the retina of a subject suffering from a retinal disease. Summary of the Invention

[0008] Thus, the present invention relates to a recombinant adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding the retinal transcription factor cone-rod homeobox (CRX), for use in treating CRX-associated IRD in a subject in need thereof.

[0009] In one embodiment of the invention, the AAV vector is of the AAV2 serotype.

[0010] In more specific embodiments of the invention, the AAV vector is of the AAV2 / 5 or AAV2 / 8 serotype.

[0011] In a further aspect, the polynucleotide contained in the AAV vector of the invention is under the control of a promoter driving expression in rod and cone photoreceptors selected from the rhodopsin (Rho), β-phosphodiesterase (PDE), retinitis pigmentosa 1 (RP1), or human rhodopsin kinase 1 (GRK1 or RK1) promoters.

[0012] In a more specific embodiment, the polynucleotide is under the control of the GRK1 promoter.

[0013] In one embodiment, the vector of the present invention is for use in the treatment of a CRX-associated IRD, wherein the CRX-associated IRD is selected from a retinopathies resulting from dominant mutations in the CRX gene, or resulting in symptoms due to hypomorphic mutations that are cured by expression of CRX.

[0014] In a further embodiment, the vector of the present invention is for use in the treatment of a CRX-associated disease, wherein the CRX-associated IRD is selected from retinitis pigmentosa, Leber's congenital amaurosis, or cone-rod dystrophy, or is associated with a hypomorphic mutation in a target gene of CRX, such as the PDE6B, NMNAT1, ARL13b, AIPL1, or ABCA4 gene.

[0015] In a further aspect, the vector for use according to the invention is administered to a subject in a therapeutically effective amount.

[0016] In one embodiment, the vector for use in the present invention is 8 ~10 12 vg / eye, more preferably 1×10 9 ~1×10 12 It is administered in vg / eye.

[0017] In one embodiment, the vector of the invention is administered before the onset of the disease.

[0018] In another embodiment, a vector of the invention is administered after the initiation of photoreceptor degeneration.

[0019] In a further embodiment, the vector of the invention is administered so long as functional cone and / or rod photoreceptors are present.

[0020] In one embodiment, the vector for use according to the invention is comprised in a composition, more particularly a pharmaceutical composition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition In the present invention, the following terms have the following meanings.

[0022] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives.

[0023] "AAV-CRX or AAV-hRK1-CRX" refers to an AAV2 / 5 vector containing the human rhodopsin kinase 1 promoter driving expression of human CRX.

[0024] "AAV-GFP or AAV-hRK1-GFP" refers to an AAV2 / 5 vector containing the human rhodopsin kinase 1 promoter driving expression of GFP.

[0025] "CRX" stands for cone-rod homeobox CRX (NCBI: Nucleotide NM_000554.6; Protein NP_000545.1, or Ensembl No. ENSG00000105392), an Otd / OTX-like "paired" homeodomain transcription factor preferentially expressed in vertebrate rod and cone photoreceptor cells in the retina and pineal cells of the brain. CRX plays an important role in the development and maintenance of functional mammalian rod and cone photoreceptors. CRX encodes a 299 amino acid protein that contains a homeodomain (HD) near its N-terminus that contributes to DNA binding. One of skill in the art will understand that a CRX coding sequence can include any nucleic acid sequence that encodes a CRX gene product. The CRX coding sequence may or may not contain intervening regulatory elements (eg, introns, enhancers, or other non-coding sequences).

[0026] "Dominantly inherited retinal disease" refers to a pattern of inheritance of disease through families. Dominant means that only one allele carrying a mutated gene is sufficient to induce disease instances, which are certain forms of RP, CORD, and LCA.

[0027] "hCRX" refers to the human CRX gene.

[0028] A "heterologous gene" or "heterologous nucleotide sequence" generally refers to a gene or nucleotide sequence that is not naturally occurring in the virus. Alternatively, a heterologous gene or nucleotide sequence can refer to a viral sequence that is placed into a non-naturally occurring environment (e.g., by being linked to a promoter that is not naturally associated with the virus).

[0029] "Hypomorphic mutation" refers to a mutation that provides a reduced level of activity for an altered gene product or that results in a reduced level of expression of an altered gene product. Non-limiting examples of such mutations are certain subtypes of phosphodiesterase 6B (PDE6B) mutations, and other mutations identified, for example, in the NMNAT1, ARL13b, AIPL1, or ABCA4 genes.

[0030] "ITR" or "inverted terminal repeat" refers to a stretch of nucleic acid sequence present in an AAV vector and / or recombinant adeno-associated virus vector (rAAV) that can form a T-shaped palindrome structure, which is necessary for the completion of the lytic and latent life cycle of AAV (Muzyczka N and Berns KI 2001). The term "non-degradable ITR" refers to an ITR that has been modified to reduce degradation by Rep proteins. A non-degradable ITR can be an ITR sequence without a terminal resolution site (TRS), which results in low or no degradation of the non-degradable ITR, resulting in 90-95% of self-complementary AAV vectors (McCarty et al 2003).

[0031] "Operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Usually, the term refers to the functional relationship of a transcription control sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcription control sequences that are operably linked to a transcribed sequence are contiguous with the transcribed sequence, i.e., they are cis-acting. However, some transcription control sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0032] "Polynucleotide of interest" refers to any nucleotide sequence that encodes a CRX.

[0033] A "promoter" refers to a sequence that regulates the transcription of an operably linked gene or nucleotide sequence that codes for a protein. A promoter can provide sufficient sequences to direct transcription and recognition sites for RNA polymerase and other transcription factors necessary for efficient transcription, directing cell-specific expression. In addition to sequences sufficient to direct transcription, the promoter sequences of the present invention can also include sequences of other regulatory elements involved in regulating transcription (e.g., enhancers, Kozak sequences, and introns). Furthermore, standard techniques are known in the art for creating functional promoters by mixing and matching known regulatory elements. "Truncated promoters" can also be created from promoter fragments or by mixing and matching fragments of known regulatory elements.

[0034] "Subject" or "subject in need thereof" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who is waiting to receive medical care or is receiving medical care, or has been / is / may be the subject of past / present / future medical care, or is being monitored for the development of a disease. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is male. In another embodiment, the subject is female.

[0035] "Treating" or "treatment" of an inherited retinal dystrophy (IRD) refers to alleviating the IRD, for example by delaying or arresting or reducing its onset or at least one of its clinical symptoms. "Treating" or "treatment" may also refer to alleviating or reducing at least one physical parameter, including those that may not be discernible by the patient. "Treating" or "treatment" may also refer to modulating the IRD, either physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. More specifically, "treating" an IRD refers to any action that results in the improvement or preservation of visual function and / or local anatomy of a subject with an IRD. "Treatment" as used herein also encompasses "preventing" or "prevention," which refers to preventing or delaying the onset or development or progression of an IRD. "Prevention," in the context of an IRD, refers to any action that prevents or delays the deterioration, as described below, in patients having an IRD and in patients at risk for deterioration of visual function, retinal anatomy, and / or IRD parameters. Methods for assessing disease treatment and / or prevention are known in the art and are described herein below.

[0036] "Virus vector" or "viral vector" is intended to refer to a non-wild-type recombinant viral particle that functions as a gene delivery vehicle and contains a recombinant viral genome packaged within a viral (e.g., AAV) capsid. A particular type of viral vector may be a "recombinant adeno-associated viral vector" or an "AAV vector." The recombinant viral genome packaged in a viral vector is also referred to herein as a "vector genome."

[0037] Detailed Description of the Invention The present invention is based on the discovery of viral vectors that express a heterologous CRX gene in both cone and rod photoreceptor cells.

[0038] Thus, the present invention relates to recombinant adeno-associated virus (AAV) vectors that direct expression of the retinal transcription factor cone-rod homeobox (CRX) in cone and rod cells, compositions comprising such vectors, and methods of using the vectors or compositions.

[0039] The viral vector of the present invention can be a recombinant adeno-associated (rAAV or AAV) vector. AAV vectors are small single-stranded DNA viruses that require a helper virus to facilitate efficient replication. The viral vector comprises a vector genome and a protein capsid. The viral vector capsid can be sourced from any of the AAV serotypes known in the art, including currently identified human and non-human AAV serotypes and yet to be identified AAV serotypes. The viral capsid can be mixed and matched with other vector components to form a hybrid viral vector, for example, the ITRs and capsid of the viral vector can come from different AAV serotypes. In one embodiment, the ITRs can be from the AAV2 serotype, and the capsid is, for example, from the AAV2 or AAV5 serotype. Moreover, one of skill in the art will recognize that the capsid of the vector can be a mosaic capsid (e.g., a capsid composed of a mixture of capsid proteins from different serotypes) or even a chimeric capsid (e.g., a capsid protein containing foreign or unrelated protein sequences to generate markers and / or alter tissue tropism). It is contemplated that the viral vector of the present invention can include an AAV2 capsid. It is further contemplated that the present invention can include an AAV5 capsid.

[0040] In the present invention, the term "AAV" refers to AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8) and AAV type 9 (AAV9). The genomic sequences of various AAV serotypes and the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases, such as GenBank. See, for example, GenBank accession numbers NC_001401 (AAV-2), AF043303 (AAV2), and NC_006152 (AAV-5).

[0041] As used herein, "AAV vector" refers to an AAV vector that contains a polynucleotide of interest (i.e., a heterologous polynucleotide) for gene expression in rod and / or cone cells. A rAAV vector contains 5' and 3' adeno-associated virus inverted terminal repeats (ITRs) and a polynucleotide of interest (CRX) operably linked to a sequence that controls its expression in target cells.

[0042] In certain embodiments, the AAV vector of the invention is an AAV2 serotype.

[0043] In another embodiment, the AAV vector of the invention is an AAV2 / 5 or AAV2 / 8 serotype.

[0044] The AAV2 / 5 or AAV2 / 8 vector of the present invention is produced using methods known in the art. In summary, the method generally includes (a) introducing an AAV vector into a host cell, (b) introducing an AAV helper construct into the host cell (wherein the helper construct contains the viral functions that are missing from the AAV vector), and (c) introducing the helper virus into the host cell. All functions for the replication and packaging of AAV virions must be present to achieve the replication and packaging of AAV vectors into AAV virions. The introduction into the host cell can be performed simultaneously or sequentially using standard virology techniques. Finally, the host cell is cultured to produce AAV virions, which are purified using standard techniques such as cesium chloride (CsCl) gradients. The activity of remaining helper virus can be inactivated using known methods, such as heat inactivation. The purified AAV virions are then ready to be used in the method of the present invention.

[0045] The vector may also include control sequences that allow expression and secretion of the encoded protein, such as promoters, enhancers, polyadenylation signals, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTDs), and the like. In this regard, the vector includes a promoter region operably linked to a polynucleotide of interest to effect or enhance expression of the protein in infected cells. Such promoters may be ubiquitous, tissue-specific, strong, weak, controlled, chimeric, inducible, and the like to allow efficient and appropriate production of the protein in infected tissues. The promoter may be homologous to the encoded protein or may be heterologous, including cellular, viral, fungal, plant, or synthetic promoters. Preferred promoters for use in the present invention are selected from among promoters capable of driving expression in cone and rod cells, such as retinitis pigmentosa 1 (RP1) or human rhodopsin kinase 1 (GRK1).

[0046] In a preferred embodiment, the present invention refers to an AAV vector, in which a polynucleotide encoding CRX is under the control of the human rhodopsin kinase 1 (GRK1) promoter.

[0047] An exemplary amino acid sequence of CRX is NCBI Reference Sequence: NP_000545.1, and an exemplary nucleic acid sequence of CRX is NCBI Reference Sequence: NM_000554.6.

[0048] The present invention also refers to compositions and pharmaceutical compositions comprising the viral vector of the present invention. The pharmaceutical compositions are formulated with a pharmaceutically acceptable carrier. The compositions may further comprise one or more other therapeutic agents suitable for treating or preventing, for example, CRX-associated IRD. The pharmaceutically acceptable carrier may be used to enhance or stabilize the composition or to facilitate the preparation of the composition. A pharmaceutically acceptable excipient refers to any kind of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. Pharmaceutically acceptable excipients that may be used in the compositions of the invention include, but are not limited to, physiologically compatible solvents, surfactants, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0049] Pharmaceutical compositions of the present invention may further comprise an antioxidant, including, but not limited to, ascorbic acid, ascorbyl palmitate, butylated hydroxytoluene, potassium sorbate, or rosemary (Rosmarinus officinalis) extract.

[0050] Pharmaceutical compositions according to the present invention may further include pharma- ceutically acceptable salts, including, but not limited to, acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with the free carboxyl groups may also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0051] The excipient can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils such as oleic acid. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin (i.e., soybean lecithin or defatted soybean lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0052] The vector or pharmaceutical composition of the present invention can be administered by various methods known in the art. The route and / or form of administration varies according to the desired result. Preferably, administration is subretinal. The pharmaceutically acceptable carrier is suitable for subretinal, intravitreal, intravenous, subcutaneous or topical administration.

[0053] The composition should be sterile and fluid. The proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0054] Those skilled in the art can prepare suitable solutions well using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary. For delayed release, the vector can be included in pharmaceutical compositions that are formulated for sustained release, such as microcapsules formed from biocompatible polymers or liposomal carrier systems by methods known in the art.

[0055] The pharmaceutical compositions of the invention may be prepared by methods well known and routinely performed in the art.

[0056] The pharmaceutical composition is preferably manufactured under GMP conditions. Usually, a therapeutically effective dose or effective dose of the viral vector is used in the pharmaceutical composition of the present invention. The viral vector can be formulated into a pharma- ceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased, such as indicated by the exigencies of the therapeutic situation. It is particularly suitable to formulate parenteral compositions into dosage unit forms for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dose for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier.

[0057] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dosage level will vary depending on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health, previous medical history, and other factors of the patient being treated.

[0058] The present invention provides a method for treating a CRX-associated IRD in a subject by delivering to the eye of a subject in need thereof an AAV vector comprising a polynucleotide encoding a CRX that has a favorable effect on retinal diseases when expressed in cone and rod cells.

[0059] More specifically, the present invention provides methods for treating CRX-associated IRD in a subject in need thereof, which relates to a retinopathy resulting from a dominant mutation in the CRX gene or which results in symptoms due to a hypomorphic mutation that is cured by expression of CRX, and even more specifically, provides methods for treating dominant forms of retinal degenerative diseases such as RP, CORD, or LCA, or for treating hypomorphic mutations in PDE6B.

[0060] In a further aspect, the present invention refers to an AAV vector expressing CRX for use in the treatment of a CRX-associated IRD in a subject in need thereof.

[0061] In the present invention, CRX-associated IRD is associated with a retinopathy resulting from dominant mutations in the CRX gene or resulting in a condition due to hypomorphic mutations that are cured by expression of CRX.

[0062] Typically, the subject is afflicted with or likely to be afflicted with a CRX-associated IRD resulting from a dominant mutation in the CRX gene, which is associated with a dominant form of a retinal degenerative disease, such as RP, CORD, or LCA.

[0063] In another embodiment, the present invention also refers to a vector or a composition or a pharmaceutical composition for use in the treatment of a CRX-associated IRD resulting from a dominant mutation in the CRX gene.

[0064] In another aspect, the present invention also refers to a vector or a composition or a pharmaceutical composition for use in the treatment of CRX-associated IRD caused by hypomorphic mutations in target genes of CRX.

[0065] In a more specific embodiment, the present invention also refers to a vector or a composition or a pharmaceutical composition for use in the treatment of hypomorphic mutations of PDE6B.

[0066] In the present invention, treatment is either therapeutic treatment or preventative or prophylactic treatment.

[0067] Thus, the subjects to be treated are either those already suffering from an IRD as well as those prone to have the disease or those in whom the disease is to be prevented.

[0068] In one embodiment, a subject is successfully "treated" if, after being administered a therapeutically effective amount of a compound according to the invention, the subject exhibits an observable and / or measurable reduction in one or more symptoms associated with a particular disease; and / or an improvement in quality of life.

[0069] Parameters for assessing successful treatment and improvement of the disease are readily measurable by routine means familiar to physicians.

[0070] In another aspect, the vector, composition, or pharmaceutical composition of the invention is administered in a therapeutically effective amount.

[0071] The doctor can start administering the viral vector of the present invention used in the pharmaceutical composition at a level lower than the dose required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Generally, the effective dose of the composition of the present invention varies depending on many different factors, including the administration means, the target site, the physiological condition of the patient, whether the patient is human or animal, other drug therapies being administered, and whether the treatment is preventative or therapeutic, in the treatment of CRX-associated retinal dystrophy described herein.

[0072] As used herein, a "therapeutically effective amount" refers to an amount of an AAV vector that provides a therapeutic benefit to a subject. In one embodiment, the term "effective amount" refers to a level or amount of vector that is intended to (1) delay or prevent the onset of CRX-associated IRD, (2) delay or stop the progression, progression, or worsening of one or more symptoms of CRX-associated IRD, (3) provide relief from symptoms of CRX-associated IRD, (4) reduce the severity or frequency of occurrence of CRX-associated IRD, or (5) cure CRX-associated IRD, without causing significant negative or adverse side effects to the target.

[0073] The dose of the vector may be adapted depending on the disease state, the subject (e.g. according to the patient's weight, metabolism, etc.), the treatment schedule, etc. A preferred effective dose within the context of the present invention is one that allows optimal expression in PR cells.

[0074] Treatment doses should be titrated to optimize safety and efficacy. For subretinal administration with viral vectors, doses range from 1×10 8 Vector genome (vg) / eye ~1×10 12 For example, the dose can range from 1×10 8 vg / eye, 1.5×10 8 vg / eye, 2×10 8 vg / eye, 2.5×10 8 vg / eye, 3×10 8 vg / eye, 4×10 8vg / eye, 5×10 8 vg / eye, 6×10 8 vg / eye, 7.5×10 8 vg / eye, 8×10 8 vg / eye, 9×10 8 vg / eye, 0.5×10 9 vg / eye, 1×10 9 vg / eye, 1.5×10 9 vg / eye, 2.5×10 9 vg / eye, 3×10 9 vg / eye, 4×10 9 vg / eye, 5×10 9 vg / eye, 6×10 9 vg / eye, 7×10 9 vg / eye, 7.5×10 9 vg / eye, 8×10 9 vg / eye, 9×10 9 vg / eye, 0.5×10 10 vg / eye, 1×10 10 vg / eye, 1.5×10 10 vg / eye, 2.5×10 10 vg / eye, 3×10 10 vg / eye, 4×10 10 vg / eye, 5×10 10 vg / eye, 6×10 10 vg / eye, 7×10 10 vg / eye, 7.5×10 10 vg / eye, 8×10 10 vg / eye, 9×10 10 vg / eye, 0.5×10 11 vg / eye, 1×10 11 vg / eye, 1.5×10 11 vg / eye, 2.5×10 11 vg / eye, 3×10 11 vg / eye, 4×10 11 vg / eye, 5×10 11 vg / eye, 6×10 11 vg / eye, 7×10 11 vg / eye, 7.5×10 11 vg / eye, 8×10 11 vg / eye, 9×10 11 vg / eye, 0.5×10 12 vg / eye, 1×10 12 vg / eyes であり got る.

[0075] In a preferred embodiment of the invention, the vector or a composition comprising said vector is administered in a concentration of 1×10 9 ~1×10 12 It is administered in vg / eye.

[0076] Administration of the vectors of the invention to a subject can be by direct subretinal injection for expression of CRX in cone and rod cells.

[0077] In another embodiment, the vector or composition for use according to the invention is administered prior to the onset of the disease and when necessary to prevent this onset.

[0078] In another aspect, the vector or composition for use according to the invention is administered after the onset of photoreceptor degeneration, more specifically after the onset of photoreceptor degeneration for as long as functional cones and / or photoreceptors are present and there is a need for them.

[0079] The viral vector described herein is mainly used as a single dose per eye, with the possibility of repeated administration to treat areas of the retina that are not covered by previous administration.The dose of administration can vary depending on whether the treatment is preventive or therapeutic.

[0080] Various features and embodiments of the invention referred to in individual sections and embodiments above apply mutatis mutandis to other sections and embodiments as appropriate. As a result, features described in one section or embodiment may be combined with features described in other sections or embodiments as appropriate.

[0081] The invention is further illustrated by the following figures and examples, which should not, however, be construed in any way as limiting the scope of the invention. [Brief description of the drawings]

[0082] [Figure 1]Plasmid map of the final vector, 822-pKL.AAV.GRK1.hCRX, containing the human rhodopsin kinase 1 promoter driving expression of human CRX. The promoter was subcloned using restriction sites SpeI and EcoRI. The cDNA encoding human CRX was subcloned and blunt-ended using Kpn1 digestion, which results in a cohesive site, followed by Xba1 digestion. [Diagram 2] The selected AAV vectors combined with the selected promoters allow efficient transduction of WT photoreceptors and CrxRip / + immature cone-like photoreceptors. WT or CrxRip / + mice received a single subretinal injection of 2.5x1010 AAV-GFP at P30. Eyes were harvested 14 days later for sectioning and immunolabeling with anti-GFP antibody. Nuclei were stained with DAPI. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Scale bar, 20 μm. [Diagram 3] Overexpression of CRX after subretinal injection of AAV-CRX in CrxRip / + CrxRip / + mice were given or not given a single subretinal injection of three different doses of AAV-CRX (0.5×1010, 1×1010 and 2.5×1010 vg / eye) at P30. After 14 days, CRX expression was assessed by immunoblotting. [Figure 4] Quantification of CRX expression after subretinal injection of AAV-GFP or AAV-CRX. CrxRip / + mice received a single subretinal injection of three different doses of AAV-GFP or AAV-CRX at P30–P40: 0.5×1010, 1×1010, or 2.5×1010 vg / eye. 14 days later, CRX expression was quantified by immunoblotting. Expression was normalized using tubulin. All quantifications reported relative to endogenous CRX expression in animals injected with 0.5.1010 vg of AAV-GFP. Black bars: endogenous CRX expression in CrxRip / + injected with AAV-GFP; white bars: CRX expression in CrxRip / + mice injected with AAV-CRX. [Diagram 5]Differentiated PRs can be observed in CrxRip / + retinas using AAV-CRX vectors. CrxRip / + mice received a single subretinal injection of 2.5x1010 AAV-CRX at P30. The other eye was not injected and served as a control. One month later, eyes were harvested for sectioning and labeling with anti-rhodopsin and anti-cone arrestin antibodies. Nuclei were stained with DAPI. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 20 μm. [Figure 6] Differentiated PRs in the outer segment can be observed in CrxRip / + retinas using a low dose of AAV-CRX vector (0.5.1010vg). (A) Both eyes of CrxRip / + mice received subretinal injections of 0.5×1010vg of AAV-CRX or AAV-GFP at P30. Eyes were harvested 2 months later and flat-mounted after labeling with anti-rhodopsin and anti-cone arrestin antibodies. GFP expression was directly observed after subretinal injection of AAV-GFP. Scale bar, 20 μm. (B) A single subretinal injection of 0.5×1010 AAV-CRX and a low dose (<1.109vg) of AAV-GFP was performed in CrxRip / + mice at P30 to low-density label transduced photoreceptors with GFP and visualize their morphology. Confocal acquisition of multiple z-sections allowed 3D reconstruction. 1, outer segment; 2: nucleus; 3, synaptic terminal, scale bar, 5 μm. [Figure 7]Subretinal injection of AAV-CRX allowed the recovery of photoreceptor light sensitivity. Both eyes of CrxRip / + mice received a single subretinal injection of 0.5x1010 AAV-CRX or AAV-GFP at P30. Light responses were assessed 4 months after injection. (A) The increase in the dark-adapted b-wave response was statistically significant in animals treated with AAV-CRX compared to AAV-GFP controls at a stimulus intensity of 2.2 log cd.sec / m2. (B) The increase in the light-adapted b-wave response was statistically significant in animals treated with AAV-CRX compared to AAV-GFP controls at a stimulus intensity of 1.6 log cd.sec / m2. (C) The light / dark choice test showed that the number of times that CrxRip / + mice injected with AAV-CRX crossed the line toward the light compartment was comparable to that of uninjected WT mice. In contrast, CrxRip / + mice injected with AAV-GFP crossed this line more frequently, indicating their characteristic failure to detect light.Grey bars, uninjected WT; black bars, CrxRip / + mice injected with AAV-GFP; white bars, CrxRip / + mice injected with AAV-CRX, *p<0.05, **p<0.01. [Figure 8] Photoreceptor degeneration was reduced in rd10 mice after AAV-CRX vector injection at a dose of 2.5.1010vg. (A) AAV-CRX vector was injected or not injected into P14 Rd10 mice. Retinas were harvested 2 months after injection. Retinal sections were immunolabeled with anti-rhodopsin (Rho) and anti-cone arrestin (CA) antibodies. Nuclei were stained with DAPI. (B) Histograms represent measurements of the thickness of rd10 ONL injected (white bars) or not (NI, black bars) with AAV-CRX. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 20 μm. [Figure 9]Photoreceptor degeneration was reduced in rd10 mice after AAV-CRX vector injection at doses of 0.5.1010 and 1.1010 vg. The histogram represents measurements of the thickness of the rd10 ONL 2 months after injection of AAV-CRX at P14. Specific detection of human CRX on paraffin sections allowed differentiation of transduced or non-transduced areas. Black bars, areas not transduced with AAV-CRX; white bars, areas transduced with AAV-CRX. [Figure 10] Expression of CRXR41W in the cone-only retina results in cone degeneration that can be prevented using AAV-CRX. (A) Histogram showing reduction in photopic b-wave amplitude in 2-month-old Nrl- / -;Tg(CRXR41W) compared to Nrl- / - at a light stimulation of 2.8 log cd.sec / m2. (B) Histogram showing preservation of photopic b-wave amplitude 2 months after injection in Nrl- / -;Tg(CRXR41W) treated at 2 months with AAV-CRX compared to AAV-GFP. *p<0.05. EXAMPLES

[0083] The invention is further illustrated by the following examples.

[0084] Example 1 material and method animal Male and female adult (3-8 weeks old) C57BL / 6J mice, Crx Rip / + and Tg(CRX R41W ) mice, as well as male and female juvenile (2 weeks old) rd10 mice, were produced in the research animal facility (Animalerie central - campus CNRS Gif sur Yvette).

[0085] All animal experiments were performed in accordance with European guidelines for the care and use of laboratory animals and were approved by the local ethical committee (CEEA59).

[0086] vector rAAV2 / 5 vectors were generated expressing green fluorescent protein (GFP) or human cone-rod homeobox protein (CRX) under the control of the human rhodopsin kinase (GRK1) promoter.

[0087] 1. Vector plasmid cloning (822-pKL.AAV.GRK1.hCRX vector #6556 batch) The vector plasmid was constructed in two steps as described below. First, the promoter GRK1 was extracted from 754-pAAV.GRK1.eGFP.wpre.bGH by double digestion with Spe1 (position 185) and EcoR1 (position 427), and the 778-pKL.AAV.CAG.bGH vector plasmid was pre-digested with Spe1 (position 188)-EcoR1 (position 1947) to extract the CAG promoter, obtaining the intermediate plasmid pKL.AAV.bGH, and then the promoter GRK1 was inserted. The GRK1 promoter was inserted into pKL.AAV.bGH to generate the 821-pKL.AAV.GRK1.bGH backbone plasmid. Second step: 821-pKL.AAV.GRK1.bGH backbone plasmid was linearized by EcoR1 (position 531) / Xba1 (position 569) double digestion. The hCRX gene was extracted from pcDNA4c.hCRX plasmid by Kpn1 digestion (position 1171), creating a cohesive site, blunting the ends, followed by Xba1 digestion (position 2120). The extracted hCRX gene was inserted into the linearized pKL.AAV.hCRX.bGH at position 531 to generate the final vector plasmid 822-pKL.AAV.GRK1.hCRX (Figure 1).

[0088] The final vector plasmid 822-pKL.AAV.GRK1.hCRX has been sequenced from position 106 to 2745 bp, which corresponds to the expression cassette.

[0089] 2. AAV2 / 5 Production and Purification Procedures Cell amplification, transfection, harvesting, and PEG precipitation of supernatants HEK293 cells seeded in a CellStack Cells 5 chamber (CS5) with a vented cap were cultured in DMEM supplemented with 10% FBS and 1% Pen / Strep. At approximately 80% confluence, cells were cultured with CaPO 4 Using the precipitation technique, vector plasmid and helper plasmid (containing helper genes from adenovirus (E2A, E4, and VA RNA) and the rep cap gene corresponding to rep serotype-2 and capsid serotype-5) are co-transfected. Culture medium is removed from the CS5 and replaced with transfection medium, and the cells are then incubated at 37±1°C and 5±1% CO. 2 Then, the transfection medium is removed from the CS5 and replaced with fresh medium (DMEM, 1% Pen / Strep), and then incubated at 37 ± 1 °C and 5 ± 1% CO. 2 The cells are then incubated at 4°C for 3 days. The transfected CS5 cells are then harvested. The supernatant alone is precipitated with PEG overnight at 5±3°C. The precipitated supernatant is then centrifuged. The supernatant is discarded and the PEG-pellet is resuspended in TBS and then digested with benzonase.

[0090] Vector purification by ultracentrifugation on dual CsCl gradients The virus suspension is centrifuged and the vector-containing supernatant is loaded in a step density CsCl gradient in an Ultra-Clear tube for a SW28 rotor. The gradient is centrifuged at 28000 rpm for 24 hours at 15°C. The intact particle band is collected and transferred to a new Ultra-Clear tube for a SW41 rotor. The second gradient is centrifuged at 38000 rpm for 48 hours. The concentrated intact particle band is then collected. The virus suspension is then subjected to four successive dialyses in a Slide-a Lyzer cassette against a buffer solution for ophthalmic formulations. The final purified vector is collected, sampled for vg titer and purity assays, and stored in polypropylene low-binding cryovials at less than -70°C.

[0091] 3. Quality control: quantification of vector genomes The basic unit for determining vector dosage is the vector genome (vg), which corresponds to the concentration of particles containing the gene of interest and is quantified by quantitative PCR using primers specific for ITR-2 as described in D'Costa et al. (2016).

[0092] Subretinal AAV injection in vivo Adult / juvenile mice were anesthetized with a solution consisting of xylazine (1 mg / mL) and ketamine (10 mg / mL) in phosphate-buffered saline (PBS) at 50 μL / 10 g per mouse via intraperitoneal injection. The iris was dilated using specific eye drops (Mydriaticum (Tropicamide 5 mg 0.5%) and Neosynephrine (Phenylephrine hydrochloride 2.5%)). Eyes were anesthetized with Cebesine 0.4%.

[0093] After anesthetization, C57BL / 6J, rd10 and Crx mice were Rip / + For the mouse, 5×10 9 vg / eye (n = 20 Crx Rip / + Mice; n = 3 rd10 mice; n = 2 Nrl mice- / - ;Tg(CRX R41W ) 1×10 10 vg / eye (n = 8 Crx Rip / + mice, n = 2 rd10 mice) 2.5×10 10 vg / eye (n = 3 C57BL / 6J mice; n = 10 Crx Rip / + Mice; n = 2 rd10 mice) AAV2 / 5.hGRK1.GFP (AAV-GFP), or 5×10 9 vg / eye (n = 3 C57BL / 6J mice; n = 21 Crx Rip / + Mice; n = 3 rd10 mice; n = 2 Nrl mice - / - ) 1×10 10 vg / eye (n=9 Crx Rip / + Mice; n = 4 rd10 mice) 2.5×10 10 vg / eye (n = 6 C57BL / 6J mice; n = 10 Crx Rip / + Mice; n = 14 rd10 mice) AAV2 / 5.hGRK1.hCRX (AAV-CRX) was injected into the subretinal space.

[0094] A 33G beveled needle attached to a 5 μL Hamilton syringe was inserted through the RPE / choroid near the optic nerve into the ventral subretinal space. 2 μL was slowly injected (over a period of approximately 30 seconds) to detach the retina from the RPE cells.

[0095] Protein extraction and immunoblot analysis Mice were euthanized using cervical dislocation. Retinas were dissected and frozen on dry ice and then stored at -80°C. Proteins were lysed in lysis buffer (20 mM NaCl, 0.1% ... 2 HPO 4, 250 mM NaCl, 5% DTT, 30 mM NaPPi, 0.1% NP-40, 5 mM EDTA). Mechanical disruption was achieved by sonication and proteins were isolated after centrifugation (10 min, 13200 rpm, 4 °C).

[0096] 20 μg of protein from each eye to be tested was loaded onto a 7.5%-12% gradient acrylamide gel (Biorad). The run was performed at constant voltage (100 V). Upon completion, Western blot transfer was performed onto nitrocellulose membranes (ThermoFisher Scientific) using the iBlot 2 Dry Blotting System.

[0097] After transfer, the nitrocellulose membrane was incubated with blocking buffer (PBST [PBS, 0.05% Triton X-100], 5% milk) for 1 h at room temperature. Primary antibodies were then added in fresh blocking buffer either overnight at 4°C (mouse anti-Crx(A-9), ref.:sc-377138 [Santa Cruz Biotechnology, Inc.], batches J1116 and H2918, dilution 1 / 5000) or for 1 h at room temperature (mouse anti-tubulin clone DM1A, ref.:T9026 [Sigma-Aldrich], batch 052M4837, dilution 1 / 10000). Excess primary antibodies were rinsed three times for 10 min with PBST.

[0098] Secondary antibodies were added in fresh blocking buffer for 2 h at room temperature (goat anti-mouse IgG-peroxidase, ref.: A4416 [Sigma-Aldrich], batch SLBH3692, dilution 1 / 5000) followed by 3 rinses for 10 min in PBST.

[0099] The ECL revelation kit (Supersignal™ West Dura Extended Duration Substrate: 34076 [ThermoFisher Scientific]; batch SK256986) was added to the nitrocellulose membrane (Carestream® Kodak® BioMax® light film, ref.: Z373508 [Sigma-Aldrich]) 5 min before revelation in the dark.

[0100] The membrane can be stripped with stripping buffer (1.5 g glycine, 1 mL SDS 10%, 1 mL Tween-20, milliQ water qs. 100 mL, pH 2.2). The membrane was then incubated twice for 10 min at room temperature, followed by two PBS washes for 10 min and finally two washes for 5 min with PBST. The membrane was incubated with primary and secondary antibodies as described above. After development, the films were scanned and analyzed with Image J.

[0101] Histology analysis Mice were euthanized using cervical dislocation. Eyes were harvested and fixed in 4% paraformaldehyde (PFA) for 1 h, rinsed in PBS, followed by dehydratation and paraffin inclusion. 7 μm thick sections were cut with a microtome (rotary microtome, ref.: HM 340E [ThermoFisher Scientific]), mounted overnight at 37°C, and then stored at room temperature. Before immunohistochemistry (IHC), slides were deparaffinized and then incubated in hot citrate buffer (pH 6, 0.1 M) at 500 W for 20 min. After cooling, slides were mounted in PBS. For immunostaining (IHC) on flat-mount retinas, retinas were dissected, fixed in 4% PFA for 1 h, rinsed in PBS, and directly incubated with primary antibodies.

[0102] Primary antibodies in Dako REAL(tm) antibody diluent (ref S2022 lot 20060091) supplemented with 0.3% Triton X-100 were added to the slides overnight at 4°C in a drying chamber (mouse anti-rhodopsin clone 4D2, ref.:MABN15 [Merck], batch 2935495, dilution 1 / 1000; rabbit anti-cone arrestin, ref.:AB15282 [Merck], batch 2802590, dilution 1 / 1000; goat anti-GFP, ref.:ab6673 [Abcam], dilution 1 / 1000). Flat-mounted retinas were incubated with primary antibodies for 2 days.

[0103] They were rinsed three times in PBST 5 min before addition of secondary antibodies (donkey anti-mouse IgG Alexa Fluor 555, ref.:A31570 [ThermoFisher Scientific], dilution 1 / 1000; donkey anti-rabbit IgG Alexa Fluor 488, ref.:A21206 [ThermoFisher Scientific], dilution 1 / 1000; donkey anti-goat Alexa Fluor 488, ref.:A11055 [ThermoFisher Scientific], dilution 1 / 1000) in Dako REAL(tm) antibody diluent (ref S2022 lot 20060091) supplemented with 0.3% Triton X-100. After 2 h incubation at room temperature in the dark, slides were rinsed 3 times for 5 min with PBST and DAPI (ref.:62248 [ThermoFisher Scientific], batch RJ2279362, dilution 1 / 1000) was added for 20 min in PBST. Finally, slides were rinsed 3 times with PBST. Flat-mount retinas were incubated overnight with secondary antibodies.

[0104] For flat mount retinas, retinas were placed photoreceptor side up on slides. FluorSave™ Reagent (ref.:345789, Millipore, batch 3034632) was used to add a coverslip. Slides were stored at 4°C. Photographs were obtained using an Imager M2 microscope (Zeiss) or an LSM710 confocal microscope (Zeiss) and analyzed using Zen software and Image J.

[0105] Electroretinogram Electroretinogram (ERG) recordings were performed using a focal ERG module attached to a Micron IV (Phoenix Research Laboratory). Briefly, mice were dark-adapted overnight and prepared for the experiment under dim red light. Mice were anesthetized using ketamine (100 mg / kg) and xylazine (10 mg / kg) and administered topical proparacaine hydrochloride (0.5%, Alcon) via eye drops. Pupils were dilated using tropicamide (1%, Alcon) and phenylephrine (2.5%, Alcon) and lightly coated with GONAK hypromellose ophthalmic demulcent solution (2.5%, Akorn). A Micron IV lens was placed directly on the cornea and a reference electrode was placed on the mouse's head. Dark-adapted responses were elicited using a series of flashes increasing in light intensity from -1.7 to 2.2 cd.s / m2. Light-adapted responses were elicited under background light to desensitize rods using a series of flashes increasing in light intensity from -0.5 to 2.8 cd.s / m2. A- and b-wave values ​​were extracted and plotted for comparison between groups of interest.

[0106] Light-dark selection The apparatus consisted of a brightly lit white box (40 x 15 cm) with 20 cm high Plexiglas walls and connected to a dark box (15 x 15 cm) by a trap door (6 x 6 cm). Illumination in the dark box was less than 10 lux. In the light box, lights were positioned at the ends of the 40 cm compartment, thus providing an illumination gradient that increased from 600 lux at the entrance to 1500 lux at the end of the compartment.

[0107] Testing took place between 9:00 and 12:00 AM. Each mouse was placed in the dark compartment for 10 seconds, then the trap door was opened and the mouse was allowed free access to the entire apparatus for 5 minutes. Step-through latency, number of entries, and total time spent in the light compartment were scored.

[0108] Example 2 Selection of promoter and serotype of AAV vector used to efficiently transduce photoreceptors in a CRX-associated retinopathy mouse model Several AAV serotypes and promoters have been tested and demonstrated transduction of mature photoreceptors in adults. Rip / + In the case of the retina, the outer nuclear layer contains only immature cone-like photoreceptors, and therefore AAV vectors commonly used to treat differentiated photoreceptors may not be efficient. Therefore, we tested several serotypes expressing GFP driven by photoreceptor-specific promoters that direct gene expression in rod and cone photoreceptors. We selected the human rhodopsin kinase 1 (GRK1) promoter because i) it has already been well characterized to express genes in rod and cone photoreceptors, ii) it has already been used in clinical trials (PDE6B), and iii) it is active in our mouse model based on our published transcriptome data. For serotypes, we selected the AAV2 / 5 vector, which has been described to specifically transduce mouse photoreceptors.

[0109] Different doses of AAV vectors expressing GFP were administered at 1 × 109 ~5×10 10 vg / Crx Rip / + The subretinal space was injected at postnatal day 30 (P30) in the area of ​​​​the eye. Immediately after, the quality of the subretinal injection was confirmed by the presence of retinal detachment observed by OCT (Optical Coherence Tomography). After 2 weeks, the retinal detachment had disappeared and the efficiency of photoreceptor transduction was evaluated by fluorescent fundus imaging followed by histological analysis. GFP immunofluorescence analysis performed on sections from the injected retinas revealed strong GFP expression in the injection area, restricted to the outer nuclear layer where the photoreceptors are localized (Figure 2).

[0110] In conclusion, the AAV vector serotype selected for further treatment is the AAV2 / 5 vector containing the GRK1 promoter.

[0111] Example 3 Quantification of CRX expression driven by the GRK1 promoter using AAV2 / 5 vectors To verify that the product (AAV vector expressing CRX or AAV-CRX) can produce CRX protein, a series of subretinal injections were performed in mice with Crx Rip / + Three different doses (0.5, 10 10 , 1.10 10 , and 2.5.10 10 vg) and CRX expression was evaluated after 14 days. An increase in CRX expression was observed with all three doses tested. The amount produced was more dependent on the injection than on the dose used (Figure 3). This experiment was repeated when the technique of subretinal injection was fully mastered, but this time with the addition of a vector expressing GFP (AAV-GFP) at three different doses (0.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 360, 380, 400, 420, 460, 480, 500, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5000, 6000, 7000, 8 10 , 1.10 10 and 2.5.10 10 vg) (Figure 4). The relative expression of CRX was quantified after normalization to tubulin and was expressed at 0.5.10 10Endogenous CRX expression was scored after AAV-GFP injection in the vg. CRX expression was clearly significantly increased after AAV-CRX injection in proportion to the dose used.

[0112] Example 4 Crx by AAV-CRX injection Rip / + Restoration of photoreceptor differentiation in the retina To evaluate the properties of the products (AAV vector expressing CRX or AAV-CRX) for restoring photoreceptor differentiation, AAV-CRX was injected at P30 with 2.5 × 10 10 vg / eye. Retinas were harvested 2 months later for histological analysis. Immunohistochemical analysis was performed using anti-rhodopsin and anti-cone arrestin to label rod and cone photoreceptors, respectively. The results clearly revealed areas of large rhodopsin- and cone arrestin-positive cells around the injected area (Figure 5). Crx Rip / + Imaging of cells positive for rhodopsin and cone arrestin on flat mount retinas 4 months after subretinal injection of AAV-CRX in 0.5.10. 10 At a dose of 1.10 vg, eyes injected with AAV-GFP showed fully differentiated rod and cone photoreceptors (Figure 6A). In contrast, eyes injected with AAV-GFP showed no cells positive for these markers. To better visualize the morphology of differentiated photoreceptors 4 months after AAV-CRX treatment, low doses of AAV-GFP (<1.10 9 ) were co-injected to obtain a low density of GFP-positive cells that were easy to image. GFP-positive photoreceptors on flat-mount retinas transduced with AAV-CRX at high magnification showed well-formed outer segments, and the presence of synaptic terminals (Figure 6B).

[0113] Example 5 Crx by AAV-CRX injection Rip / + Partial recovery of retinal function Crx Rip / +To assess the functionality of newly differentiated rod and cone photoreceptors by AAV-CRX treatment in mice, dark- and light-adapted ERG recordings were performed. 2.2 Log cd sec / m 2 A statistically significant increase in the amplitude of the dark-adapted b-wave at maximal stimulation of 1.6 Log cd sec / m was recorded (Figure 7A). The light-adapted response showed a slight overall improvement, but was 1.6 Log cd sec / m compared to AAV-GFP-treated animals. 2 The effect was statistically significant only with stimulation of Crx (Fig. 7B). Rip / + To estimate the properties of newly differentiated photoreceptors in WT mice, we used a behavior termed “light / dark choice.” WT mice spent most of their time in the dark side of the box, whereas untreated Crx mice Rip / + Mice move independently to either side due to complete blindness. In contrast, Crx mice treated with AAV-CRX Rip / + Mice spent more time in the dark side of the box by crossing significantly fewer lines leading to the light side of the box (Fig. 7C). Collectively, these results suggest that Crx Rip / + We have demonstrated the properties of AAV-CRX treatment resulting in photoreceptor differentiation and partial functional recovery, particularly with regard to light detection properties, in AAV-CRX treated mice.

[0114] Example 6 AAV-CRX protects rd10 photoreceptors from cell death. Overexpressed CRX WT The potential interest of CRX is to stimulate the expression of its target genes. Of these, many encode enzymes that are associated with IRD due to hypomorphic mutations, leading to low expression or activity, such as certain subtypes of PDE6B mutations. Overexpression of CRX WTEnhancing the expression of CRX target genes by α-aminobutyric acid allows a certain threshold of expression and / or activity to be reached, allowing proper phototransduction and maintenance of PR for a long period of time. This was done using rd10 mice carrying a hypomorphic recessive mutation of Pde6b. Notably, retinal degeneration begins at approximately P15 in these mice, and by P30, most of the rods are lost. A sufficient increase in expression levels allows a certain threshold of sufficient enzyme activity to be reached to reduce cGMP levels, thus restoring the signaling cascade. Thus, the therapeutic product of the present invention has a broad spectrum of applications that allows more patients to potentially be treated.

[0115] To evaluate the potential therapeutic effect of AAV-CRX vectors on hypomorphic mutations, rd10 mice were injected subretinal (2.5 × 10 10 vg) and retinas were harvested one month later. IHC analysis revealed good ONL preservation in treated eyes compared to controls. In contrast to uninjected eyes, which had a marked reduction in outer segments, rod and cone PRs displayed well-preserved outer segments, where rhodopsin and cone arrestin are localized as in healthy retina (Figure 8(A)). Measurement of ONL thickness confirmed preservation (Figure 8(B)). Furthermore, efficacy one month after injection at P14 was evaluated with two additional doses of AAV-CRX:0.5.10 10 vg and 1.10 10 Using paraffin sections, the transduced area was determined using anti-CRX, which specifically recognizes human CRX produced by AAV, but not endogenous mouse Crx. Measurements of ONL thickness showed that 1.10 10 It was shown that a dose of 10000 vg resulted in better preservation of the ONL compared to non-transduced areas (Figure 9). These results clearly demonstrate the therapeutic value of the use of the products of the invention for certain subtypes of IRD.

[0116] However, due to the kinetics of degeneration starting at P14 and the time of injection, AAV-CRX injection must be performed early in the life of the rd10 to improve evaluation of the efficacy of treatment by providing more time for CRX production by AAV before the onset of degeneration.

[0117] We demonstrated correct expression of human wild-type CRX cDNA specific to photoreceptors. Rip / + Preliminary subretinal injections of AAV-CRX in mice were performed using i) 0.5.10 10 vg dose, ii) increased CRX expression levels, and iii) resumption of PR differentiation 4 months after injection, with larger areas of rhodopsin and cone arrestin positive cells compared to untreated mice. In particular, the gene therapy product of the present invention results in greater differentiation of cones compared to rod photoreceptors. Finally, a slight recovery of dark-adapted and light-adapted electroretinogram (ERG) responses 4 months after subretinal injection was observed. Taken together, these very encouraging results support the conclusion that adult Crx mice are more likely to develop PRs in mice with 100% CRX expression levels compared to 100% CRX expression levels. Rip / + This clearly demonstrates the efficacy of the products of the invention in restoring PR differentiation in the retina.

[0118] Example 7 The second mouse model is CRX R41W It was established based on the identification of two French families, each with a proband with CORD carrying a mutation. This dominant mutation does not alter the nuclear localization of CRX, but reduces its DNA-binding properties, resulting in reduced transcriptional activity of its target genes. This mutation acts as a dominant-negative or CRX R41W This can result in a loss of function that prevents the gene from binding to the target promoter. R41W ) has been generated using a lentiviral approach (collaboration P. Charneau and L. Vives, Institut Pasteur, Paris). This system contains a repeat human CRX fused to a myc tag under the control of the Crx promoter. R41W Carrying the mutation. Tg(CRX) with multiple copies of the transgeneR41W ) The first group of founders showed loss of dark- and light-adapted ERG responses due to photoreceptor degeneration. Successive breeding with wild-type (WT) mice to reduce copy number led to partial restoration of ERG responses, implicating CRX in disease development. WT and CRX R41W These findings suggest the importance of the ratio between the amounts of CRX and CRX to compensate for the negative effects of mutated CRX. WT There is clear therapeutic interest in increasing the amount of transgene CRX. R41W Three lines carrying one copy of the gene have been established, in which normal differentiation of photoreceptors with a slight reduction in ERG responses has been found. Transgene expression in photoreceptors was verified by IHC.

[0119] Tg(CRX R41W Mice with two copies of Tg(CRX) have been derived (from a line with one copy). R41W While a line with one copy of Nrl) had no defects in ERG responses, mice with two copies showed reduced light-adapted responses at 6 months of age. Thus, this line is a good model of cone-rod dystrophy for testing the therapeutic effects of the present invention, but the degeneration is less intense and slower. To circumvent this problem, we used Nrl, a line in which all photoreceptors are cones. - / - We established a line expressing this mutation on a 36-kDa background. In this line, a reduction in the light-adapted response to high light stimuli could be observed at 2 months of age (Figure 10(A)). Pre-injection of AAV-CRX or AAV-GFP in 2-month-old animals significantly reduced the number of Nrl mice treated with AAV-CRX 2 months after injection. - / - ;Tg(CRX R41W ) was shown to have a preserved light-adapted response compared to AAV-GFP-treated animals (Figure 10(B)).

[0120] Phenotypic characterization of photoreceptor cells differentiated from patient CRX mutant iPSC lines and evaluation of the efficacy of gene replacement As iPSCs can be reprogrammed from fibroblasts of patients with IRD, it is possible to generate disease-specific retinal cell models that present an excellent model for proof-of-concept gene therapy trials. iPSC-derived photoreceptor models were generated from three patients carrying CRX mutations resulting in CORD (p.Arg41Trp);LCA (p.Pro232Argfs*139) and RP (p.Asp65His). iPSCs from patients with CORD and LCA were generated and their pluripotency and genetic stability were confirmed. Furthermore, iPSCs from control and CORD individuals were differentiated into retinal organoids containing photoreceptors. Using qPCR analysis at different times of differentiation, we observed similar expression patterns of early photoreceptor markers (SIX3, VXVX2, RAX, OTX2, NEUROD1, NRL) up to 160 days between the two lines. It was detected that CRX expression in the CORD line appeared similar before day 160 compared to the control, but decreased thereafter. In contrast, a decrease in NR2E3 expression was detected at all time points tested in the CORD line. This is particularly interesting because NR2E3 interacts with CRX for rod development. Furthermore, CORD lines were detected to have decreased expression of more mature cone (OPN1SW, CAR, OPSN1MW) and rod (RK, RCVRN) markers compared to controls. Furthermore, rhodopsin expression could not be detected at the mRNA or protein level in the CORD line. These results indicate a delay or alteration of photoreceptor differentiation in the case of the p.Arg41Trp / R41W mutation.

[0121] Patient-specific iPSC-derived photoreceptors in retinal organoids were transduced with AAV-CRX to increase wild-type CRX expression, and their differentiation profile was monitored over time and compared to untreated and control cells.

[0122] The results were: i) to obtain genotype-phenotype correlations, thus elucidating the basis for the differential clinical profiles associated with each mutation, and ii) to determine whether all morphologies could benefit from a gene replacement strategy and whether there are toxic effects associated with CRX overexpression. These studies allow the true effect of exogenous CRX expression to be assessed in the absence of an in vivo administration challenge.

Claims

1. 1. A pharmaceutical composition for use in treating a CRX-associated inherited retinal disease in a subject in need thereof, comprising: The pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising a polynucleotide comprising a nucleic acid sequence encoding a retinal transcription factor, cone-rod homeobox (CRX); The CRX-related inherited retinal disease is selected from retinopathies resulting from dominant mutations in the CRX gene, or resulting in symptoms due to hypomorphic mutations that are cured by expression of CRX, the hypomorphic mutation being present in the CRX target gene PDE6B; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the AAV vector is an AAV2 serotype.

3. The pharmaceutical composition of claim 2 , wherein the AAV vector is of the AAV2 / 5 or AAV2 / 8 serotype.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the polynucleotide is under the control of a promoter that drives expression in rod and cone photoreceptors selected from the rhodopsin (Rho), β-phosphodiesterase (PDE), retinitis pigmentosa 1 (RP1), or human rhodopsin kinase 1 (GRK1) promoters.

5. The pharmaceutical composition of claim 4, wherein the polynucleotide is under the control of a GRK1 promoter.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the CRX-related inherited retinal disease is selected from retinitis pigmentosa, Leber's congenital amaurosis, or cone-rod dystrophy.

7. The pharmaceutical composition according to any one of claims 1 to 6, for administration to said subject in a therapeutically effective amount.

8. The amount of the vector is 10 ８ ~10 １２ 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is vg / eye.

9. The amount of the vector is 1×10 ９ ~1×10 １２ 9. The pharmaceutical composition of claim 8, wherein the .alpha.-vg / eye.

10. The pharmaceutical composition according to any one of claims 1 to 9, which is intended to be administered before the onset of a disease.

11. The pharmaceutical composition according to any one of claims 1 to 10, for administration after the onset of photoreceptor degeneration.

12. The pharmaceutical composition of any one of claims 1 to 11, for administration as long as functional cone and / or rod photoreceptors are present.

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