Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present inventions relate to methods of expressing a polynucleotide of interest in the cone photoreceptors of a subject comprising the intravitreal delivery of a therapeutically effective amount of a recombinant AAV2-derived vector.
• the fovea -located at the center of the macula- is a specialized region of the retina that dominates the visual perception of primates by providing high acuity color vision (1).
• the highest density of cones is found at the center of the fovea ( ⁇ 0.3 mm from the foveal center), devoid of rod photoreceptors (2).
• Cone density decreases by up to 100 fold with distance from the fovea (3).
• Cone cells in the fovea are the primary targets of gene therapies aiming to treat inherited retinal diseases like mid-stage retinitis pigmentosa (4, 5) and achromatopsia (6).
• the present inventions relates to methods of expressing a polynucleotide of interest in the cone photoreceptors of a subject comprising the intravitreal delivery of a therapeutically effective amount of a recombinant AAV2 -variant vector.
• the present invention is defined by the claims.
• AAV adeno-associated viral
• the first object of the present invention relates to a method of expressing a polynucleotide of interest in the cone photoreceptors of a subject comprising the intravitreal delivery of a therapeutically effective amount of a recombinant AAV2-variant vector comprising the VP1 capsid protein as set forth in SEQ ID NO: l encoding a polynucleotide of interest under the control of the PR1.7 promoter as set forth in SEQ ID NO:2.
• the term "subject" is typically intended for a human. Typically the subject is affected or likely to be affected with a retinal disease affecting cone photoreceptors. Accordingly a wide variety of retinal diseases impacting retinal cone photoreceptors may thus be treated given the teachings provided herein and typically include age-related macular degeneration, Bassen-kornzweig syndrome, choroideremia, gyrate atrophy, Refsum syndrome, Usher syndrome, color blindness, blue cone monochromacy, achromatopsia, incomplete achromatopsia, oligocone trichromacy, retinitis pigmentosa (RP), macular degeneration, Stargardt's Disease, Bardet-Biedl syndrome, Bornholm eye disease, Best's Disease and Leber's congenital amaurosis.
• age-related macular degeneration Bassen-kornzweig syndrome, choroideremia, gyrate atrophy, Refsum syndrome, Usher syndrome, color blindness, blue cone monochro
• a further object of this invention is to provide a method for treating a retinal disease affecting cone photoreceptors in a subject in need thereof comprising the intravitreal delivery of a therapeutically effective amount of a recombinant AAV2 -variant vector comprising the VP1 capsid protein as set forth in SEQ ID NO: l and the polynucleotide of interest under the control of the PR1.7 promoter as set forth in SEQ ID NO:2 wherein the polynucleotide of interest which when expressed in cone photoreceptor has a beneficial effect on the retinal disease.
• treatment refers 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 subject 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.
• 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.
• 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.
• maintenance regimen 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 regular intervals, 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., disease manifestation, etc.]).
• cone photoreceptors has its general meaning in the art and are one of three types of photoreceptor cells in the retina of mammalian eyes. They are responsible for color vision and function best in relatively bright light, as opposed to rod cells, which work better in dim light.
• the combination between the specific vector and the PR1.7 promoter specially drives the expression of the polynucleotide of interest in cone photoreceptors.
• vitreous humour also called vitreous body or simply vitreous
• the clear gel fills the space between the lens and the retina of the eyeball of humans and other vertebrates.
• polynucleotide of interest designates any nucleotide sequence coding for any polypeptide, structural protein, enzyme etc., the expression of which is wanted in a target cell, for any kind of reason. It can also designate a non-coding sequence, for example an antisense sequence or the sequence of an interfering RNA aimed at decreasing the expression of a gene.
• a non-coding sequence for example an antisense sequence or the sequence of an interfering RNA aimed at decreasing the expression of a gene.
• Gene therapy can be performed either by introducing in cone photoreceptor a functional copy of a polynucleotide of interest (e.g. a gene) that is deficient therein (gene replacement therapy), or by delivering to cone photoreceptors a polynucleotide of interest which will have a beneficial effect on the eye disease to be treated (symptomatic therapy).
• a polynucleotide of interest e.g. a gene
• gene replacement therapy e.g. a functional copy of a polynucleotide of interest
• RPGRORF15 retinitis pigmentosa GTPase regulator
• GNAT2 cone specific alpha subunit of transducin
• CNGA3 alpha subunit of the cone cyclic nucleotide-gated cation channel
• the polynucleotide of interest may encode for a neurotrophic factor.
• the "neurotrophic factor” is a generic term of proteins having a physiological action such as survival and maintenance of nerve cells, promotion of neuronal differentiation.
• the neurotrophic factor is RdCVF.
• RdCVF has its general meaning in the art and refers to rod-derived cone viability factor (Leveillard T, Mohand-Sa ' id S, Lorentz O, Hicks D, Fintz A C, Clerin E et al. Identification and characterization of rod-derived cone viability factor. Nat Genet 2004; 36: 755-759.).
• the polynucleotide of interest encodes both a long form (RdCVF-L, 217 aa, Q8VC33) having a putative thiol-oxydoreductase activity (JEFFERY, Trends Biochem. Sci., vol.24(l):8-l 1, 1999; JEFFERY, Trends Genet, vol.19(8) :415-417, 2003) and a short form (RdCVF-S, 109 aa, Q91W38) with trophic activity for cones but no redox activity.
• the neurotrophic factor is RdCVF2, which shares many similarities with RdCVF in terms of gene structure, expression in a rod-dependent manner and protein 3D structure (see e.g. WO2008148860 and Chalmel F, Leveillard T, Jaillard C, Lardenois A, Berdugo N, Morel E, Koehl P, Lambrou G, Holmgren A, Sahel JA, Poch O.
• Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential. BMC Mol Biol. 2007 Aug 31;8:74.).
• the RdCVF2 short isoform exhibits cone rescue activity that is independent of its putative thiol-oxydoreductase activity.
• the polynucleotide of interest encodes for RdCVFL2.
• the polynucleotide product of interest is an opsin.
• the opsin sequence can be derived from any suitable single- or multicellular- organism, including human, algae and bacteria.
• the opsin is rhodopsin, photopsin, L/M wavelength (red/green) cone-opsin, or short wavelength (S) cone-opsin (blue).
• the opsin is channelrhodopsin or halorhodopsin or cruxhalorhodopsin.
• the opsin is a light-responsive opsin as described in U.S. Patent Publication No. 2007/0261127 (e.g., ChR2; Chop2); U.S.
• opsins include NpHR, eNpHR 1.0, eNpHR 2.0, eNpHR 3.0, SwiChR, SwiChR 2.0, SwiChR 3.0, Mac, Mac 3.0, Arch, ArchT, Arch 3.0, ArchT 3.0, iChR, ChR2, C1V1-T, C1V1-TT, Chronos, Chrimson, ChrimsonR, CatCh, VChRl-SFO, ChR2-SFO, ChR2-SSFO, ChEF, ChlEF, Jaws, ChloC, Slow ChloC, iClC2, iClC2 2.0, and iClC2 3.0.
• the opsin consists of the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO:
• the polynucleotide product of interest is a site-specific endonuclease that provides for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a retinal disease.
• a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.
• the vector thus comprises a polynucleotide that encodes a site-specific endonuclease; and a polynucleotide that encodes a functional copy of a defective allele, where the functional copy encodes a functional retinal protein.
• Site-specific endonucleases that are suitable for use include, e.g., zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs), and CRISPR-associated endonuclease.
• CRISPR-associated endonuclease has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
• the CRISPR-associated endonuclease is Cas9 or a derivative thereof.
• the Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence. Alternatively, the wild type Streptococcus pyrogenes Cas9 sequence can be modified.
• the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, MA).
• the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, MA).
• the Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations).
• the Cas9 nuclease can be mutated in the conserved FiNH and RuvC domains, which are involved in strand specific cleavage.
• the vector comprises one or more guide RNA.
• the term "one or more guide RNA” refers to the RNAs that guide the insertion or deletion of residues. In the context of the invention, the guide RNA is used for recruiting Cas9 to specific genomic loci.
• the guide RNA can be a sequence complementary to a coding or a non-coding sequence.
• the subject is administered with a combination of at least one vectors comprising one polynucleotide encoding for a Cas9 endonuclease and at least one vector comprising the guide RNA.
• the polynucleotide product is an interfering RNA (RNAi), in particular a siRNA.
• RNAi interfering RNA
• a "small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest (a "target gene”).
• An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule.
• siRNA is "targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene.
• the length of the duplex of siRNAs is less than 30 nucleotides.
• the polynucleotide product is an antisense oligonucleotide.
• antisense oligonucleotide is understood to refer to a nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre- mRNA molecule, hnRNA (heterogenous nuclear RNA) or mRNA molecule.
• the degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions.
• AAV refers to more than 30 naturally occurring and available adeno-associated viruses, as well as artificial AAVs.
• AAV capsid, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rhlO, AAVrh64Rl, AAVrh64R2, rh8, variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. See, e.g., WO 2005/033321.
• GenBank and PDB Accession Numbers NC_002077 and 3NG9 (AAV-1), AF043303 and 1LP3 (AAV-2), NC_001729 (AAV-3), U89790 and 2G8G (AAV- 4), NC_006152 and 3NTT (AAV-5), 30AH (AAV6), AF513851 (AAV-7), NC_006261 and 2QA0 (AAV-8), AY530579 and 3UX1 (AAV-9 (isolate hu.14)); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J.
• the term "recombinant AAV2-derived vector” refers to an AAV2 -based vector comprising the VPl capsid protein as set forth in SEQ ID NO: l and the polynucleotide sequence of interest (i.e., a polynucleotide heterologous to AAV).
• the native VPl capsid protein of AAV2 is substituted by the VPl capsid protein as set forth in SEQ ID NO: l .
• the recombinant AAV2- derived vector of the present invention typically comprises 5' and 3' adeno-associated virus inverted terminal repeats (ITRs), the polynucleotide of interest (i.e a heterologous polynucleotide) operatively linked to the promoter PR1.7.
• ITRs adeno-associated virus inverted terminal repeats
• the vectors of the invention are produced using methods known in the art.
• the methods generally involve (a) the introduction of the DNA necessary for AAV replication and synthesis of the capsid, (b) the introduction of a helper construct into the producer cell line, wherein the helper construct comprises the viral functions missing from the AAV vector (c) introducing a helper virus into the producer cell line, (d) the plasmid construct containing the genome of the AAV vector, e.g. ITRs, promoter and transgene sequences, etc.... All functions for AAV virion replication and packaging need to be present, to achieve replication and packaging of the AAV vector into AAV virions.
• the introduction into the producer host cell can be carried out using standard virology techniques simultaneously or sequentially.
• the host cells are cultured to produce AAV virions and are purified using standard techniques such as iodixanol or CsCl gradients or other purification methods.
• the purified AAV virion is then ready for use in the methods.
• promoter has its general meaning in the art and refers to a nucleic acid fragment that controls the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent R A polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
• PR1.7 promoter refers to the 1.7-kb L-opsin promoter described in Hum Gene Ther. 2016 Jan;27(l):72-82 and characterized by the nucleic acid sequence as set forth in SEQ ID NO:2.
• the promoter and the polynucleotide of interest are operatively linked.
• operably linked refers to two or more nucleic acid or amino acid sequence elements that are physically linked in such a way that they are in a functional relationship with each other.
• a promoter is operably linked to a coding sequence if the promoter is able to initiate or otherwise control/regulate the transcription and/or expression of a coding sequence, in which case the coding sequence should be understood as being "under the control of the promoter.
• the coding sequence should be understood as being "under the control of the promoter.
• two nucleic acid sequences when operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may not be required.
• a “therapeutically effective amount” is meant a sufficient amount of the vector to treat the retinal disease at a reasonable benefit/risk ratio. It will be understood that the total daily usage of the vector will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
• the doses of vectors may be adapted depending on the disease condition, the subject (for example, according to his weight, metabolism, etc.), the treatment schedule, etc.
• a preferred effective dose within the context of this invention is a dose allowing an optimal transduction of the cone photoreceptors.
• from 10 8 to 10 10 viral genomes (vg) are administered per dose in mice.
• the doses of AAV vectors to be administered in humans may range from 10 9 to 10 12 vg.
• compositions may comprise, in addition to the vector, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient (i.e. the vector of the invention).
• a pharmaceutically acceptable excipient such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
• Physiological saline solution magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
• the active ingredient will be in the form of an aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.
• isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
• Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
• the vector may be included in a pharmaceutical composition, which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.
• a pharmaceutical composition of the present invention is supplied in a prefilled syringe.
• a "ready- to-use syringe” or “prefilled syringe” is a syringe which is supplied in a filled state, i.e. the pharmaceutical composition to be administered is already present in the syringe and ready for administration.
• Prefilled syringes have many benefits compared to separately provided syringe and vial, such as improved convenience, affordability, accuracy, sterility, and safety.
• the pH of the liquid pharmaceutical composition of the present invention is in the range of 5.0 to 7.0, 5.1 to 6.9, 5.2 to 6.8, 5.3 to 6.7 or 5.4 to 6.6.
• Figure 1 Expression in all layers of the mouse retina is obtained with the combination of AAV2-7m8-GFP and the mouse cone arrestin (mCAR) promoter (A), PR2.1 promoter (B) while most cone-specific expression pattern is achieved with PR1.7 promoter (C) -after intravitreal injections.
• mCAR mouse cone arrestin
• FIG. 2 Cone-specific expression in the retinal degeneration 10 (rdlO) mouse model for Retinitis Pigmentosa.
• Figure 3 Reporter gene expression pattern using ubiquitous versus cone-specific promoter in vivo in monkeys after intravitreal injections.
• Retinal flatmount images showing that AAV2-7m8-CMV leads to pan-retinal GFP expression (A) while it is foveal cone- specific with AAV2-7m8-PR1.7 (B). Close up to the fovea with AAV2-7m8-CMV (C, left) and AAV2-7m8-PR1.7 (C, right).
• Figure 4 Foveal cone-specific expression of Jaws-GFP under the control of PR1.7 promoter in combination with AAV2-7m8 capsid in the non-human primate, after intravitreal injections.
• A In vivo eye fundus image showing foveal expression
• B flatmount image of a half fovea (arrow: foveola)
• C retinal cryosections at the level of the fovea.
• Figure 5 Cone-specific expression of GFP under the control of PR1.7 promoter and with AAV2-7m8 capsid in human tissue.
• A Retinal organoids derived from human induced pluripotent stem cells expres GFP;
• B GFP-expressing cells in the organoids are exclusively cones (cryosections),
• C expression in cones of a human post-mortem retinal explant (cryosections).
• AAV vectors were produced as previously described using the co-transfection method and purified by iodixanol gradient ultracentrifugation (49). AAV vector stocks were titered by quantitative PCR (50) using SYBR Green (Thermo Fischer Scientific). Animals and intraocular injections
• mice Wild-type C57BL6/j or rdlO mice, and cynomolgus macaques were used for this study.
• mice were anesthetized by isofluorane inhalation. Pupils were dilated and a 33 -gauge needle was inserted into the eye to deliver 2 ⁇ , of AAV vector solution intravitreally or ⁇ ⁇ subretinally.
• macaques were first selected based on absence neutralizing antibody titers against AAV. Primates were anesthetized with 10: 1 mg/kg ketamine/xylazine. After subretinal or intravitreal vector administration, opthtalmic steroid and antibiotic ointment were applied to the corneas post injections.
• Spectralis HRA+OCT system Heidelberg Engineering, Germany
• fluorescent images of GFP using the "Fundus Autofluoresence mode" which consists of and excitation wavelength of 488 nm and barrier filter of 500 nm.
• a two-photon microscope equipped with a 40x water immersion objective (LUMPLFLN40x/W/0.80, Olympus) with a pulsed femto-second laser (InSightTM DeepSeeTM - Newport Corporation) was used for imaging GFP-positive retinal cells from whole-mount retinas (with photoreceptor-cell-side up) or retina slices (vertical sections).
• AAV-treated macaque retinas were isolated and later imaged in oxygenized (95% 02, 5% C02) Ames medium (Sigma-Aldrich).
• retinas were placed in the recording chamber of the microscope, and z-stacks were acquired using the excitation laser at a wavelength of 930 nm. Images were processed offline using Image J.
• Electrodes were made from borosilicate glass (BF100- 50-10, Sutter Instruments) and pulled to 6-9 ⁇ . Pipettes were filled with 115 mM K Gluconate, 10 mM KC1, 1 mM MgC12, 0.5 mM CaC12, 1.5 mM EGTA, 10 mM HEPES, and 4 mM ATP-Na2 (pH 7.2). Cells were clamped at a potential of -40 mV in voltage-clamp configuration, or recorded in current-clamp (current zero) configuration. Retinas were dark-adapted at least half an hour in the recording chamber prior to recordings.
• the retinal organoids were infected at day 28 of differentiation at a dose of 5xl0 10 vg/organoid with AAV2-7m8 vectors carrying the GFP gene under the control of pR1.7 promoter. 10 ⁇ DAPT (Selleck) was added to the medium for a week from day 28 on to promote cell cycle arrest of the existent cell populations. Fluorescence intensity was observed for the first time 5 days after infection and continued to increase up to day 43.
• Human retinal explants were prepared using a previously described protocol (38). Briefly, eyes were dissected in C02-independent-medium (Thermo Fischer Scientific). The anterior parts were removed, retina was isolated and cut into small pieces. These explants were placed photoreceptor side-up on a Transwell cell culture insert (Corning), and 2mL of Neurobasal medium (Thermo Fischer Scientific) supplemented with B27 (Thermo Fischer Scientific) were added to each well below each explant. The following day, each explant was infected with a single 0,5 drop of AAV-pRl .7-GFP containing 10 10 viral particles. Vector- infected explants were incubated for 10-15 days to allow GFP expression, which was checked using an epifiuorescence macroscope.
• mice were enucleated and immediately fixed in 10% formalin - 4% formaldehyde for 2 hours for cryosections.
• Macaque retinas were fixed after dissection in 4% formaldehyde for 3 hours.
• Retinal organoids and human retinal explants were rinsed in PBS at the end of their culture periods and fixed in 4% paraformaldehyde for 10 minutes.
• mouse and macaque retinas, retinal organoids and human retinal explants were immersed in PBS-30% sucrose overnight at 4°C.
• TF binding site analysis was performed on red opsin gene promoter sequence -pR2.1 and pR1.7 sequences- and the cone arrestin 3 genomic region.
• the TRANSFAC database 8.3 http://alggen.lsi.upc.es/) was used for TF binding site prediction.
• Each TF from the predicted list was analyzed using the Knowledge Base for Sensory System (KBASS, http://kbass.institut-vision.org/KBaSS/transcriptomics/index.php) to select those expressed in human retina using the transcriptomic experiment RNG209 (51).
• a filter was used to retain TFs with a signal intensity value superior to 40 units in the sample prepared from the experiment RNG209 after normalization by Robust Multi-array Average (RMA) as previously described (52).
• RMA Robust Multi-array Average
• human retinal specimens used as controls were post-mortem specimens collected within 12 hours following death of patients with no past medical history of eye disease or diabetes.
• Nineteen samples were collected from 19 eyes representing 17 patients. Sex ratio was 12 men / 7 women with a mean age of 61 years (range 25-78 years).
• mCAR promoter lead to GFP expression in some cones, but was leaky towards rods as well as cells of the inner nuclear layer (INL) and ganglion cell layer (GCL) ( Figure 1). Both PR2.1 and PR 1.7 promoters lead to more cone labeling than mCAR promoter ( Figure 1). PR2.1 transduced more cones than PR1.7 but, it also produced non-specific GFP expression in the INL and GCL. Only the PR1.7 promoter showed GFP expression in cones with minimal expression in rods and was not leaky towards the inner retina (data not shown).
• AAV2-7m8-PR1.7 vector-promoter combination in a mouse model of retinal degeneration.
• AAV2- 7m8-PR1.7-GFP intravitreally in the rdlO mouse model of retinitis pigmentosa.
• GFP expression was restricted to cones ( Figure 2) supporting the suitability of this vector for cone-directed gene therapy via both intravitreal and subretinal injections.
• COUP-TFI has been shown to suppress green opsin gene (Opnlmw) expression in the mouse retina (22) and might thus be accountable for lower expression with PR2.1 promoter in macaque cones when AAV is delivered subretinally as previously shown (18).
• COUP-TFI has been shown to suppress green opsin gene (Opnlmw) expression in the mouse retina (22) and might thus be accountable for lower expression with PR2.1 promoter in macaque cones when AAV is delivered subretinally as previously shown (18).
• COUP-TFI has been shown to suppress green opsin gene (Opnlmw) expression in the mouse retina (22) and might thus be accountable for lower expression with PR2.1 promoter in macaque cones when AAV is delivered subretinally as previously shown (18).
• OFPB green opsin gene
• GTF2I ubiquitous activator TFs
• the short sequence consists of a 521bp portion of the genomic proximal CAR promoter (data not shown) presenting a TATA-box, a TATA-like box, as well as binding sites for CRX (Cone-rod homeobox protein) and SP (Specificity Protein) TFs (23) (data not shown).
• CRX Cone-rod homeobox protein
• SP Specificity Protein
• CRX and SP TFs interact with each other and with RARA (Retinoic Acid Receptor Alpha), RXRA (Retinoid X Receptor Alpha) and THRB (Thyroid Hormone Receptor Beta) TFs (data not shown).
• RARA Retinoic Acid Receptor Alpha
• RXRA Retinoid X Receptor Alpha
• THRB thyroid Hormone Receptor Beta
• PRl.7 promoter drives strong and highly specific gene expression in human cones
• the fovea accounts for less than 1% of the retinal surface area in primates yet it provides the input to about 50% of the cells in the primary visual cortex (1).
• the high concentration of cones in the fovea, the thinnest and most delicate part of the retina allows for high acuity vision and it is of utmost importance to preserve the unique functions (39) and architecture (40) of the cones in this area during therapeutic interventions.
• Foveal cones can be targeted via different administration routes, using either subretinal or intravitreal injections (35, 41, 42) but detaching the fovea might lead to mechanical damage, especially in the degenerating retina (43). For all of these reasons, ways to deliver therapeutics to the fovea, without detaching this region are needed.
• Intravitreal injections are a surgically simple way to deliver therapeutics without retinal detachment.
• Gene therapy vectors can target the outer retina via intravitreal injections in rodents without damage to the photoreceptors (17, 35).
• safe and efficient gene delivery to primate cones via intravitreal injection had not been achieved so far, likely due to the substantial dilution of the vector in the vitreous and resulting loss of efficacy.
• the use of cell-type specific promoters that provide high-level gene expression with a lower local concentration is critical to overcome this challenge (29, 44).
• mCAR and PR2.1 gave rise to non-specific expression in inner retinal cells, making them unsuitable for optogenetic applications where any expression in downstream neurons would cancel out the response from the photoreceptors.
• Subsequent in silico analysis of TF binding sites within each promoter sequence proposed basis for more specific transduction with PR1.7 and the observed the lack of specificity with mCAR promoter.
• AAV2-7m8 equipped with PR1.7 promoter to transduce foveal cones.
• Table 1 AAV vector administration strategies for cone-directed gene therapy.
• High acuity vision that includes the fovea expression pattern
• SEQ ID NO: 1 VP1 capsid of the recombinant AAV2-derived vector
• Wikler KC Williams RW, Rakic P. Photoreceptor mosaic: Number and distribution of rods and cones in the rhesus monkey retina. J. Comp. Neurol. 1990;297(4):499-508.
• Vandenberghe LH et al. AAV9 targets cone photoreceptors in the nonhuman primate retina.

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