WO2024084075A1 - Compositions et méthodes de traitement de troubles dégénératifs de la rétine - Google Patents

Compositions et méthodes de traitement de troubles dégénératifs de la rétine Download PDF

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WO2024084075A1
WO2024084075A1 PCT/EP2023/079355 EP2023079355W WO2024084075A1 WO 2024084075 A1 WO2024084075 A1 WO 2024084075A1 EP 2023079355 W EP2023079355 W EP 2023079355W WO 2024084075 A1 WO2024084075 A1 WO 2024084075A1
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nucleic acid
viral vector
acid encoding
pharmaceutical composition
girk1
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Deniz DALKARA
Hanen KHABOU
Florence LORGET
José-Alain Sahel
Stéphane BOISSEL
Mehdi Gasmi
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Sparingvision
Institut National de la Santé et de la Recherche Médicale
Centre National De La Recherche Scientifique
Sorbonne Universite
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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

  • compositions and methods for treating retinal degenerative disorders are provided.
  • the present invention relates to the treatment of retinal neurodegenerative disorders, and more particularly to the treatment by maintaining the integrity of the cone photoreceptors and by reactivating cones which have already lost their outer segment.
  • Retina is the light sensitive tissue of the eye composed of three layers of neurons interconnected by synapses.
  • the primary neurons of the retina are the light-sensing photoreceptors (PR), which are of two types: the rods for night vision and the cones for daylight vision.
  • PR light-sensing photoreceptors
  • Cone-mediated vision is mostly supported by the fovea and is responsible for high acuity central vision most valuable to our daily visual tasks [1].
  • the light sensitive G protein coupled receptors that link photon capture to intracellular signaling leading to membrane hyperpolarization in photoreceptors are called opsins [2]
  • rod opsin found in rods and three types of cone opsins - responsible for trichromatic vision - in the primate retina. The structural properties and phototransduction cascades are similar between these opsins.
  • Photoreceptors such as rods and cones, are light-sensitive sensory neurons found on the posterior layer of the retina. They are also called photoreceptor cells or photoreceptor neurons.
  • the phototransduction cascade is composed of several proteins that are concentrated in the photoreceptor outer segments in normal retinas ( Figure 1A).
  • the role of the photoreceptor is to sense light via this phototransduction cascade and induce an electrical signal that is then processed and transmitted towards downstream neurons [3].
  • the activated PDE hydrolyses cGMP into GMP.
  • the reduction of cGMP clones the nucleotide-gated channels (CNG) and this stops cation entry, resulting in PR hyperpolarization and reduction in glutamate release by the photoreceptor [4],
  • this phototransduction cascade is deactivated by two mechanisms: (i) the transducin inactivates itself by hydrolyzing the bound GTP and (ii) the rhodopsin kinase (GRK) phosphorylates the opsin that interacts with the regulatory protein arrestin, leading to opsin inactivation. Retinal is then recycled by the retinal pigment epithelium (RPE) and Muller glial cells.
  • RPE retinal pigment epithelium
  • Muller glial cells Each and every protein of this cascade plays an important role in converting the light signal into an electrical signal conveyed to the second and third order neurons [5].
  • Neurodegenerative disorder encompasses a range of seriously debilitating conditions that are characterized by neuron degeneration.
  • Retinal neurodegenerative disorders or retinal degenerative diseases encompass different subgroups of pathologies: Rod-cone dystrophies, Cone dystrophies, Cone-rod dystrophies, and atrophic age-related macular degeneration.
  • Rod-cone dystrophies such as retinitis pigmentosa (RP)
  • RP retinitis pigmentosa
  • Rod-cone dystrophies are genetically heterogeneous retinal neurodegenerative diseases characterized by the progressive death of rod photoreceptors followed by the consecutive loss of cones.
  • RP is one of the most common forms of inherited retinal degeneration, affecting around 1 :3,500 people worldwide [6], which represents 2 million patients worldwide. Mutations causing RP in over 63 distinct genes have been identified to date with a significant proportion of these mutations in rodspecific transcripts.
  • Cones dystrophies are characterized by the vision loss (age of onset ranging from the late teens to the sixties), sensitivity to bright lights, and poor color vision. Therefore, patients see better at dusk. Visual acuity usually deteriorates gradually, but it can deteriorate rapidly to 20/200. Later, in more severe cases, it drops to "counting fingers" vision. Color vision testing using color test plates (HRR series) reveals many errors on both red-green and blueyellow plates.
  • HRR series color test plates
  • Cone-Rod Dystrophies refer to a group of inherited retinal degenerations (1 :30 - 40,000 people) that affect the photoreceptor (light sensing) cells that are responsible for capturing images from the visual field. These cells line the back of the eye in the region known as the retina. Cone photoreceptor cells are present throughout the retina but are concentrated in the central region (the macula). They are useful for central (reading) vision. Rod photoreceptor cells are present throughout the retina except for the very center of the macula called the fovea where only cones are present. They are responsible for night vision.
  • Cone-Rod Dystrophies In contrast to typical retinitis pigmentosa (known as the Rod-Cone Dystrophies), which results from the loss of rod cells and later the cone cells, Cone-Rod Dystrophies can reflect the opposite sequence of events, where cone cells are primarily first affected with later loss of rods. The degree of vision loss becomes more severe over time. There are multiple types of Cone-Rod Dystrophies, which are determined by their genetic cause and pattern of inheritance.
  • Atrophic age-related macular degeneration or advanced dry AMD, is an advanced form of age-related macular degeneration that can result in the progressive and irreversible loss of retina (photoreceptors, retinal pigment epithelium, choriocapillaris) which can lead to a loss of visual function over time [27, 28, 29, 30]. It is estimated that atrophic AMD affects more than 5 million people worldwide and approximately 1 million patients in the US [31 , 32], which is similar to the prevalence of neovascular (wet) AMD, the other advanced form of the disease.
  • the final stage of the disease corresponds to the degeneration of the foveolar cone photoreceptors, which leads to a total loss of vision in the patient.
  • this stage some of the cones present viable cell bodies despite the degeneration of their light sensing outer segments.
  • transplanted healthy retinal tissue has been shown to support cone survival in areas distant from the grafted tissue in the rd 1 mouse [11 , 12].
  • the rod-derived cone viability factor was originally identified from a high- throughput method of screening cDNA libraries as a candidate molecule responsible for this rescue effect [9]. Rods secrete RdCVF, and therefore, as rods die, the source of this paracrine factor is lost and RdCVF levels decrease. The loss of expression of RdCVF, and secreted factors like it, may therefore contribute to the secondary wave of cone degeneration observed in rod-cone dystrophies.
  • RdCVF has been shown to mediate cone survival both in culture [13] and when injected subretinally in mouse and rat models of recessive and dominant forms of retinitis pigmentosa [9, 14], In 2010, Leveillard and Sahel [26] have shown that expression of RdCVF preserves cone-mediated vision by allowing the maintenance of cone outer segments, thereby increasing cone functional life.
  • Nxnll encodes two protein isoforms through differential splicing.
  • the isoform mediating cone survival, RdCVF is a truncated thioredoxin-fold protein of its longer counterpart, RdCVFL, which includes a C-terminal extension conferring enzymatic thiol- oxidoreductase activity [16].
  • RdCVFL which contains all the amino acids of RdCVF, is encoded by exons 1 and 2 of the Nxnll gene and is a member of the thioredoxin family [17].
  • Thioredoxins have diverse functions, including maintaining the proper reducing environment in cells and participating in apoptotic pathways. These functions are accomplished via thioloxidoreductase reactions mediated by a conserved CXXC catalytic site within a thioredoxin fold [18].
  • RdCVF retinal pigmented epithelium
  • RPE retinal pigmented epithelium
  • RdCVFL protects the cones against oxidative damage in a cell autonomous manner, due to its thioloxidoreductase function.
  • GIRK channels are composed of two subunits. There are four types of subunits: GIRK1 to 4. GIRK1 , 4 and 3 cannot form homotetramers; they have to be associated with another subunit to be functional [36].
  • GIRK2 alone can form homotetramers.
  • a single point mutant GIRK1 at position F137 was suggested to form functional homomeric channels [37], GIRK channel is predominantly closed at resting membrane potentials.
  • Gi/ 0 protein After its activation by the By subunit of a Gi/ 0 protein, potassium ions flow out of the cell, thus, hyperpolarizing the neuron (Figure 1 B).
  • G protein coupled inwardly rectifying potassium channel 2 can delay vision loss, by preserving cone light-sensitivity in rd10 and RhoP347S mice and enhance visual acuity [23] (International application W02021/204407A1 ).
  • Dormant cones which are cones that have diminished outer segments and thus that became dysfunctional, could be rendered functional again thanks to the expression of GIRK2.
  • the present invention relates to the combination of:
  • RdCVF rod-derived cone viability factor
  • GIRK1 G-protein-gated inwardly rectifying potassium channel
  • GIRK4 S143T a nucleic acid encoding a mutated form of the subunit 1 of G-protein-gated inwardly rectifying potassium channel (GIRK1 ) (GIRK1 F137S) or a mutated form of the subunit 4 of G-protein-gated inwardly rectifying potassium channel (GIRK4) (GIRK4 S143T).
  • the nucleic acid encoding RdCVF, the nucleic acid encoding RdCVFL, and the nucleic acid encoding GIRK1 F137S or GIRK4 S143T may be expressed through one, two or three viral vectors. Said vectors may be within a single pharmaceutical composition or within several different pharmaceutical compositions (two or three).
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or several viral vectors, wherein said one or several viral vectors comprise a nucleic acid encoding RdCVF, a nucleic acid encoding RdCVFL, and a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the pharmaceutical composition comprises a single viral vector.
  • a second aspect of the invention deals with a viral vector comprising three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRK1 F137S or GIRK4 S143T.
  • a third aspect of the invention relates to a kit comprising two or three pharmaceutical compositions.
  • the invention also relates to a pharmaceutical composition, a viral vector or a kit above mentioned, for the treatment of a retinal degenerative disease.
  • a pharmaceutical composition for the treatment of a retinal degenerative disease.
  • the combination of RdCVF, RdCVFL, and GIRK1 F137S or GIRK4 S143T described in the present application allows to both preserve cone cells along with keeping them light responsive. This overcomes the light sensitivity loss that occurs when only trophic factors such as RdCVF and RdCVFL, are used, by implementing an additional approach to increase light sensitivity.
  • the present invention relates to a single or several viral vectors comprising a nucleic acid encoding a short isoform of rod-derived cone viability factor (RdCVF), a nucleic acid encoding a long isoform of rod-derived cone viability factor (RdCVFL), and a nucleic acid encoding a mutated form of the subunit 1 of G-protein-gated inwardly rectifying potassium channel (GIRK1 ) (GIRK1 F137S) or a mutated form of the subunit 4 of G-protein-gated inwardly rectifying potassium channel (GIRK4) (GIRK4 S143T), and the use thereof.
  • the viral vectors may be in a single or in separate pharmaceutical compositions such as distributed among two or three pharmaceutical compositions.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or several viral vectors, said one or several viral vectors comprising:
  • RdCVF rod-derived cone viability factor
  • GIRK1 G-protein-gated inwardly rectifying potassium channel
  • GIRK4 S143T a nucleic acid encoding a mutated form of the subunit 1 of G-protein-gated inwardly rectifying potassium channel (GIRK1 ) (GIRK1 F137S) or a mutated form of the subunit 4 of G-protein-gated inwardly rectifying potassium channel (GIRK4) (GIRK4 S143T).
  • nucleic acid encoding RdCVF, said nucleic acid encoding RdCVFL, and said nucleic acid encoding GIRK1 F137S or GIRK4 S143T may be comprised in a single vector or in separate vectors such as 2 or 3 vectors.
  • the pharmaceutical composition comprises three viral vectors respectively comprising a nucleic acid encoding RdCVF, a nucleic acid encoding RdCVFL, and a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the pharmaceutical composition comprises two viral vectors wherein the first viral vector comprises a nucleic acid encoding RdCVF and a nucleic acid encoding RdCVFL, and the second viral vector comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the pharmaceutical composition comprises two viral vectors wherein the first viral vector comprises a nucleic acid encoding RdCVF and a nucleic acid encoding GIRK1 F137S or GIRK4 S143T, and the second viral vector comprises a nucleic acid encoding RdCVFL.
  • the pharmaceutical composition comprises two viral vectors wherein the first viral vector comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T and a nucleic acid encoding RdCVFL, and the second viral vector comprises a nucleic acid encoding RdCVF.
  • the pharmaceutical composition comprises a single viral vector, said single viral vector comprising three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRK1 F137S or GIRK4 S143T.
  • the present invention relates to a kit comprising two or three pharmaceutical compositions.
  • each pharmaceutical composition comprises respectively one viral vector as follows:
  • a viral vector comprising a nucleic acid encoding RdCVF and a nucleic acid encoding RdCVFL, and a viral vector comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T;
  • a viral vector comprising a nucleic acid encoding RdCVF and a nucleic acid encoding GIRK1 F137S or GIRK4 S143T, and a viral vector comprising a nucleic acid encoding RdCVFL;
  • a viral vector comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T, and a nucleic acid encoding RdCVFL, and a viral vector comprising a nucleic acid encoding RdCVF.
  • the invention relates to a kit comprising two pharmaceutical compositions, wherein:
  • the first pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding RdCVF and a nucleic acid encoding RdCVFL, and
  • the second pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the invention also relates to a kit comprising two pharmaceutical compositions, wherein: - the first pharmaceutical composition comprises a viral vector comprising a nucleic acid encoding RdCVF, and a viral vector comprising a nucleic acid encoding RdCVFL, and
  • the second pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the invention also relates to a kit comprising two pharmaceutical compositions, wherein:
  • the first pharmaceutical composition comprises a viral vector comprising a nucleic acid encoding RdCVF, and a viral vector comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T, and
  • the second pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding RdCVFL.
  • the invention also relates to a kit comprising two pharmaceutical compositions, wherein:
  • the first pharmaceutical composition comprises a viral vector comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T, and a viral vector comprising a nucleic acid encoding RdCVFL, and
  • the second pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding RdCVF.
  • each pharmaceutical composition comprises a single viral vector respectively comprising a nucleic acid encoding RdCVF, a nucleic acid encoding RdCVFL, and a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the invention relates to kit comprising three pharmaceutical compositions, wherein:
  • the first pharmaceutical composition comprises a viral vector, said viral vector comprising a nucleic acid encoding RdCVF,
  • the second pharmaceutical composition comprises a viral vector, said viral vector comprising a nucleic acid encoding RdCVFL, and
  • the third pharmaceutical composition comprises a viral vector, said viral vector comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the pharmaceutical composition according to the present disclosure may further comprise an excipient pharmaceutically acceptable.
  • excipients pharmaceutically acceptable means that said excipient is generally safe and well tolerated for human or animal use following ocular administration, and should not interfere with the efficacy of the active ingredient (i.e. a viral vector as described in the present disclosure).
  • excipients pharmaceutically acceptable may be isotonic, sterile, saline solutions such as monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts. These solutions may further comprise nonionic surfactants such as, e.g., Tween, Pluronic.
  • an excipient pharmaceutically acceptable is a phosphate-buffered saline (PBS) solution or a balanced-salt solution (BSS), even more particularly supplemented with 0.001 % Pluronic.
  • PBS phosphate-buffered saline
  • BSS balanced-salt solution
  • the present invention relates to a viral vector comprising three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRK1 F137S or GIRK4 S143T.
  • Rod-derived Cone Viability Factor refers to the short isoform encoded by the thioredoxin-like 6 (TXNL6) or Nucleoredoxin-like 1 (NXNL1 ) gene. It encompasses the RdCVF proteins of any animal species.
  • the RdCVF proteins according to the present invention can be mammalian RdCVF proteins, including, but not limited to human, mice, rats, non-human primates, cats and dogs.
  • the short isoform (RdCVF) is a 109 amino-acid long protein references under Uniprot accession number Q91 W38.
  • the short isoform RdCVF is in particular the human short isoform (hRdCVF) as set forth in SEQ ID NO:1 .
  • said short isoform hRdCVF may be encoded by the nucleic acid as set forth in SEQ ID NO:3.
  • the nucleic acid encoding the short isoform hRdCVF can be a nucleic acid which differs from SEQ ID NO: 3 but encodes the same amino acid sequence SEQ ID NO:1.
  • Suitable nucleic acid sequences include but are not limited to: polymorphisms of the cDNA encoding human RdCVF;
  • rare haplotypes polymorphisms (rare haplotypes) of the cDNA encoding human RdCVF.
  • An example of rare haplotype cDNA is set forth as SEQ ID NO: 6; - “optimized” sequences in which certain codons are replaced by codons that code for the same amino-acid.
  • Suitable codon-optimized sequences encoding human RdCVF include, but are not limited to, the sequence as set forth in SEQ ID NO: 7;
  • chimpanzee cDNA sequence encoding the short isoform of chimpanzee RdCVF can be used, since it encodes the same amino acid sequence as the human cDNA.
  • the chimpanzee cDNA has the sequence as set forth in SEQ ID NO: 4.
  • the short isoform hRdCVF is encoded by a nucleic acid corresponding to a codon-optimized cDNA as set forth in SEQ ID NO:7.
  • RdCVFL refers to the long isoform encoded by the thioredoxin-like 6 (TXNL6) or Nucleoredoxin-like 1 (NXNL1 ) gene. It encompasses the RdCVFL proteins of any animal species. Typically, the RdCVFL proteins according to the present invention can be mammalian RdCVFL proteins, including, but not limited to human, mice, rats, non-human primates, cats and dogs.
  • the murine long isoform (RdCVFL) is a 217 amino-acid long protein referenced under Q8VC33.
  • the long isoform RdCVFL is in particular the human long isoform (hRdCVFL) having the sequence referenced under accession number Q96CM4 and as set forth in SEQ ID NO:2.
  • said short isoform hRdCVFL may be encoded by the nucleic acid as set forth in SEQ ID NO:5.
  • the nucleic acid encoding the long isoform hRdCVFL can be a nucleic acid which differs from SEQ ID NO:5 but encodes the same amino acid sequence SEQ ID NO:2.
  • Suitable nucleic acid sequences include but are not limited to:
  • Suitable codon-optimized sequences encoding human RdCVFL include, but are not limited to, the sequence as set forth in SEQ ID NO:8;
  • GIRK1 F137S means a nucleotide sequence encoding a mutated form of the wild-type subunit 1 of G-protein-gated inwardly rectifying potassium channel (GIRK1 ) comprising a substitution of Phe137 by Ser and which retain the ability to respond to light when co-expressed with an opsin.
  • GIRK1 G-protein-gated inwardly rectifying potassium channel
  • it is a mutated form of the human wild type GIRK1 (SEQ ID NO: 9) or a mutated form of the mouse wild type GIRK1 (SEQ ID NO: 12).
  • the GIRK1 F137S may differ from wild-type GIRK1 by the substitution of Phe137 by Ser, only (e.g. as in SEQ ID NO: 11 ), or by a limited number of mutation(s), e.g. substitution and/or deletion and/or insertion of at most 1 , 2, 3, 4, or 5 of an amino acid(s), in addition to the substitution of Phe137 by Ser.
  • a nucleotide sequence encoding GIRK1 F137S comprises or consists of a nucleotide sequence encoding the polypeptide of sequence SEQ ID NO:11 , or comprises or consists of the nucleotide sequence SEQ ID NO: 10, or comprises or consists of a nucleotide sequence encoding the polypeptide of sequence SEQ ID NO: 13.
  • GIRK4 S143T means a nucleotide sequence encoding a mutated form of the wild-type subunit 4 of G-protein-gated inwardly rectifying potassium channel (GIRK4) comprising a substitution of Ser143 by Thr and which retain the ability to respond to light when co-expressed with an opsin.
  • GIRK4 G-protein-gated inwardly rectifying potassium channel
  • it is a mutated form of the human wild type GIRK4 (SEQ ID NO: 38).
  • the GIRK4 S143T may differ from wild-type GIRK4 by the substitution of Ser143 by Thr, only (e.g. as in SEQ ID NO: 38), or by a limited number of mutation(s), e.g. substitution and/or deletion and/or insertion of at most 1 , 2, 3, 4, or 5 of an amino acid(s), in addition to the substitution of Ser143 by Thr.
  • a nucleotide sequence encoding GIRK4 S143T comprises or consists of a nucleotide sequence encoding the polypeptide of sequence SEQ ID NQ:40 or comprises or consists of the nucleotide sequence SEQ ID NO: 39.
  • nucleic acids encoding respectively RdCVF, RdCVFL, and GIRK1 F137S or GIRK4 S143T are under the control of a promoter that allows the expression of said proteins in the target cells.
  • Suitable promoters can be ubiquitous promoters, such as the Chicken beta actin (CBA) promoter, the chicken beta hybrid (CBh) promoter, the cytomegalovirus (CMV) promoter, the CMV/CBA promoter, CAG promoter.
  • CBA Chicken beta actin
  • CBh chicken beta hybrid
  • CMV cytomegalovirus
  • CAG CAG promoter
  • Suitable promoters can be promoters that enable the expression in the retina, preferably in retinal pigmented epithelial cells and photoreceptor cells such as cones and rods.
  • the promoter allows nucleic acids expression in retinal pigmented epithelial cells and/or photoreceptor cells.
  • Non-limiting examples are the rhodopsin kinase
  • GRK promoters which target the expression in cones and rods, such as GRK1 promoter, GRK1-93 promoter, IRBP promoter and mCAR promoter.
  • the GRK1 promoter is as set forth in SEQ ID NO: 15.
  • the GRK1 -93 promoter is as set forth in SEQ ID NO: 16.
  • the promoter allows nucleic acids expression in cone photoreceptors.
  • Non-limiting examples are the cone-opsin PR1 .7 promoter and the ProA7 promoter.
  • the promoter allows nucleic acids expression in cone photoreceptors.
  • Non-limiting examples are cone-opsin PR1 .7 promoter or ProA7 promoter.
  • the PR1.7 promoter is as set forth in SEQ ID NO: 17.
  • the ProA7 promoter is as set forth in SEQ ID NO: 18.
  • Table 5 provides particular nucleic acids sequences of the promoters.
  • the short isoform of the NXNL1 gene is expressed at least by retinal pigmented epithelial cells
  • the long isoform of the NXNL1 gene, and GIRK1 F137S or GIRK4 S143T are expressed at least by cone photoreceptor cells.
  • the expression of the nucleic acid encoding RdCVF is driven by a CBh promoter, in particular as set forth in SEQ ID NO:14.
  • the expression of the nucleic acid encoding RdCVFL is driven by the ProA7 promoter as set forth in SEQ ID NO:18 or the GRK1 promoter as set forth in SEQ ID NO:15.
  • the expression of the nucleic acid encoding and GIRK1 F137S or GIRK4 S143T is driven by the GRK1 -93 promoter as set forth in SEQ ID NO:16.
  • each nucleic acid is under the control of a different promoter.
  • the viral vector comprises three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRK1 F137S or GIRK4 S143T
  • the nucleic acid encoding RdCVF is under the control of a CBh promoter
  • the nucleic acid encoding RdCVFL is under the control of a ProA7 promotor or a GRK1 promoter
  • the nucleic acid encoding GIRK1 F137S or GIRK4 S143T is under the control of a GRK1 -93 promoter.
  • a viral vector above described comprises two or three nucleic acids encoding RdCVF, RdCVFL, and/or GIRK1 F137S or GIRK4 S143T
  • at least two nucleic acids may be under the control of a same promoter and linked by a nucleic acid sequence encoding a 2A self-cleaving peptide.
  • 2A self-cleaving peptides is a class of 18-22 amino acids- long peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP (SEQ ID NO:19).
  • the 2A self-cleaving peptide is the P2A peptide as set forth in SEQ ID NO: 20.
  • this P2A peptide is encoded by the nucleic acid sequence as set forth in SEQ ID NO:24.
  • the viral vector above described comprises three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRKI F137S or GIRK4 S143T, wherein the nucleic acid encoding RdCVF is under control of a promoter, in particular a CBh promoter, the nucleic acid encoding RdCVFL is under control of a promoter, in particular a GRK1 promoter, and it is linked to the nucleic acid encoding GIRK1 F137S or GIRK4 S143T by a nucleic acid sequence encoding a 2A self-cleaving peptide, in particular the P2A peptide.
  • the viral vector above described comprises three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRKI F137S or GIRK4 S143T, wherein the nucleic acid encoding RdCVF is under control of a promoter, in particular a CBh promoter, and it is linked to the nucleic acid encoding RdCVFL by a nucleic acid sequence encoding a 2A self-cleaving peptide, this latter being linked to the nucleic acid encoding GIRK1 F137S or GIRK4 S143T by a nucleic acid sequence encoding a 2A self-cleaving peptide, in particular the P2A peptide.
  • a promoter in particular a CBh promoter
  • the viral vector above described comprises three nucleic acids respectively encoding RdCVF, RdCVFL, and GIRKI F137S or GIRK4 S143T, wherein the nucleic acid encoding RdCVF is under control of a promoter, in particular a CBh promoter, and it is linked to the nucleic acid encoding GIRK1 F137S or GIRK4 S143T by a nucleic acid sequence encoding a 2A self-cleaving peptide, in particular the P2A peptide, this latter being linked to the nucleic acid encoding RdCVFL by a nucleic acid sequence encoding a 2A self-cleaving peptide, in particular the P2A peptide.
  • the viral vector according to the invention comprises an ITR sequence in 5’ and an ITR sequence in 3’.
  • the viral vector comprises two or three nucleic acids encoding RdCVF, RdCVFL, and/or GIRK1 F137S or GIRK4 S143T and wherein at least two nucleic acids are linked by a nucleic acid sequence encoding a 2A self-cleaving peptide
  • the nucleic acids are in any order following the 5’ITR. More particularly, the first nucleic acid following the 5’ITR is the nucleic acid encoding RdCVF.
  • the viral vector comprises three nucleic acids encoding RdCVF, RdCVFL, and/or GIRK1 F137S or GIRK4 S143T and wherein the nucleic acids are all linked by a nucleic acid sequence encoding a 2A self-cleaving peptide
  • the first nucleic acid following the 5’ITR is the nucleic acid encoding RdCVF and the second is the nucleic acid encoding RdCVFL.
  • the viral vector comprises three nucleic acids encoding RdCVF, RdCVFL, and/or GIRK1 F137S or GIRK4 S143T and wherein the nucleic acids are all linked by a nucleic acid sequence encoding a 2A self-cleaving peptide
  • the first nucleic acid following the 5’ITR is the nucleic acid encoding RdCVF and the second is the nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the viral vector described in the present disclosure further comprises a posttranscriptional regulatory element (PRE).
  • PRE posttranscriptional regulatory element
  • WPRE Woodchuck Hepatitis Virus PRE
  • viral vector has its general meaning in the art. In particular, it encompasses a vector derived from an adeno-associated virus (AAV), a herpesvirus (e.g., herpes simplex virus (HSV)), an adenovirus, a retrovirus, a lentivirus, or a vaccinia/poxvirus.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • adenovirus e.g., a herpes simplex virus (HSV)
  • HSV herpes simplex virus
  • adenovirus e.g., a retrovirus, a lentivirus, or a vaccinia/poxvirus.
  • AAV vector has its general meaning in the art.
  • AAV and AAV vectors have been extensively described in the art as suitable vectors for gene delivery.
  • AAV are non-pathogenic and display a broad range of tissue specificity, depending on their serotype.
  • AAV according to the present invention are AAV able to target retinal cells. More particularly, AAV according to the present invention are AAV which efficiently transduce retinal cells through intravitreal injection.
  • a modified version may be for example an AAV comprising an insertion peptide in the capsid protein such as AAV2-7M8 capsid variant as described in the international patent application WO 2012/145601 and in Dalkara et al. (2013) [38] which is an AAV2 comprising an insertion peptide called 7m8 in the capsid protein.
  • Other modified versions may be NHP26 [39], NHP9, R100 [40], or other similar variants engineered through directed evolution, rational design and / or machine learning approaches that are commonly known in the art.
  • Said AAV may also be AAV serotypes or variants which efficiently transduce retinal cells through subretinal injection such as AAV8 (also referred to as AAV2/8), AAV5 or AAV9- 7M8 capsid variant as described in the international patent application WO 2012/145601 , which is an AAV9 comprising an insertion peptide called 7m8 in the capsid protein.
  • AAV8 also referred to as AAV2/8
  • AAV5 or AAV9- 7M8 capsid variant as described in the international patent application WO 2012/145601 , which is an AAV9 comprising an insertion peptide called 7m8 in the capsid protein.
  • AAVs may be modified by an insertion peptide in the capsid protein.
  • they may comprise a variant VP1 capsid protein, wherein the variant AAV capsid protein comprises an insertion peptide of from 7 amino acids to 1 1 amino acids in the GH loop of said capsid protein relative to a corresponding parental AAV capsid protein.
  • Said insertion peptide may be as set forth in SEQ ID NO:26 (nicknamed 7m8), SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NQ:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 as described in WQ2019/077159 and at Table 7 below.
  • the insertion peptide may also be as set forth in SEQ ID NO:35 [33] or SEQ ID NO:41.
  • the insertion peptide may also be as set forth in SEQ ID NO:36 (LAISDQTKHA).
  • AAV serotypes as above mentioned may comprise an insertion peptide as set forth in SEQ ID NO:36.
  • Further examples of AAV are thus AAV serotypes as above mentioned , i. e. AAV2, AAV5, AAV8 or AAV9, comprising such an insertion peptide as above mentioned.
  • the above cited AAVs may comprise a variant AAV capsid protein as set forth in SEQ ID NO:37.
  • the above cited AAVs may comprise a variant AAV capsid protein as described in SEQ ID NO:42 of WO2019104279A1.
  • the AAV and the AAV vector according to the present invention is obtained according to the method described in international patent application WO2012/158757.
  • the AAV capsid is obtained according to the method described in patent application US9193956B2.
  • the present invention deals with a pharmaceutical composition above described, a kit above described, or a viral vector above described for use in the treatment of a retinal degenerative disease.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition (e.g., retinal degenerative diseases).
  • the term “retinal degenerative disease” encompasses all diseases associated with rods and cones degeneration. It encompasses different subgroups of pathologies: Rod-cone dystrophies, Cone dystrophies, Cone-rod dystrophies, and atrophic age-related macular degeneration.
  • the present invention relates to a pharmaceutical composition above described, a kit above described, or a viral vector above described for treatment of a retinal degenerative disease, wherein said retinal degenerative disease is a rod-cone dystrophy (RCD), a cone dystrophy (CD), a cone-rod dystrophy (CRD) or an atrophic age-related macular degeneration (AMD).
  • RCD rod-cone dystrophy
  • CD cone dystrophy
  • CCD cone-rod dystrophy
  • AMD atrophic age-related macular degeneration
  • Retinal degenerative diseases include but are not limited to retinitis pigmentosa, age- related macular degeneration, Bardet-Biedel syndrome, Bassen-Kornzweig syndrome, Best disease, choroideremia, gyrate atrophy, Leber congenital amaurosis, Refsum disease, Stargardt disease or Usher syndrome.
  • the retinal degenerative disease is a rod-cone dystrophy, more particularly retinitis pigmentosa, in particular non-syndromic X-linked Retinitis Pigmentosa (XLRP), autosomal recessive RP or autosomal dominant RP.
  • XLRP non-syndromic X-linked Retinitis Pigmentosa
  • the retinal degenerative disease is a cone-rod dystrophy, more particularly Stargardt disease, X Linked cone dystrophy, and Bardet-Biedl syndrome.
  • the pharmaceutical composition, the kit or the viral vector is administered to a patient in need by subretinal injection, intravitreal and suprachoroidal injection.
  • the delivery of the vector may be submacular or subfoveal or a distal bleb from the fovea without detaching said region.
  • kits When the kit is administered to the patient for the treatment of a retinal degenerative disease, the pharmaceutical compositions of the kit may be administered simultaneously or separately over time.
  • compositions of the kit are administered at the same time, or one after the other within a time limit of one hour, more preferably within the time limit of fifteen minutes.
  • “Separately over time” may mean that the time between the administration of two compositions is administered at two time points, in which the time between the two time points is greater than one day. In one embodiment, this may be within a timespan of one to six months. In another embodiment, this may be between a timespan of six months to one year. In a further embodiment, this may be within a timespan of one year to ten years.
  • nucleic acid encoding RdCVF and RdCVFL is administered at an earlier stage of disease progression than the nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • nucleic acid encoding RdCVF and RdCVFL is administered at a point of disease progression with moderate to severe loss of rod cells while the nucleic acid encoding GIRK1 F137S or GIRK4 S143T is administered at a point of further disease progression wherein the outer segment of cone cells exhibits moderate to serve degeneration.
  • compositions of the kit may vary to obtain optimal protection and functional restoration.
  • composition(s) of the kit comprising a nucleic acid encoding RdCVF and RdCVFL are administered at a point of disease progression with moderate to severe loss of rod cells and the composition(s) of the kit comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T are administered at a point of further disease progression wherein the outer segment of cone cells exhibit moderate to serve degeneration.
  • kit comprises two pharmaceutical compositions, wherein:
  • the first pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding RdCVF and a nucleic acid encoding RdCVFL, and
  • the second pharmaceutical composition comprises a viral vector which comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T, or when the kit comprises three pharmaceutical compositions, wherein:
  • the first pharmaceutical composition comprises a viral vector, said viral vector comprising a nucleic acid encoding RdCVF,
  • the second pharmaceutical composition comprises a viral vector, said viral vector comprising a nucleic acid encoding RdCVFL, and
  • the third pharmaceutical composition comprises a viral vector, said viral vector comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T
  • the composition(s) of the kit comprising vector(s) which comprise a nucleic acid encoding RdCVF and/or RdCVFL are administered at a point of disease progression with moderate to severe loss of rod cells
  • the composition of the kit comprising a vector which comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T is administered at a point of further disease progression wherein the outer segment of cone cells exhibits moderate to serve degeneration.
  • composition(s) of the kit comprising vector(s) which comprise a nucleic acid encoding RdCVF and/or RdCVFL is therefore administered at an earlier stage of disease progression than the composition of the kit comprising a vector which comprises a nucleic acid encoding GIRK1 F137S or GIRK4 S143T.
  • the time between the administration of the composition(s) of the kit comprising vector(s) which comprise a nucleic acid encoding RdCVF and/or RdCVFL and the administration of the composition(s) of the kit comprising a nucleic acid encoding GIRK1 F137S or GIRK4 S143T is one day, one to six months, six months to one year or one year to ten years.
  • the present invention also relates to a method for the treatment of a retinal degenerative disease comprising a step of administering to a patient in need of a therapeutically effective amount of a pharmaceutical composition above described, of a viral vector above described, or of pharmaceutical compositions comprised in kits above described.
  • a therapeutically effective amount means an amount sufficient to achieve a desired biological effect, in this case increasing the neuron viability, and thus to reduce symptoms or progression of the disease in a patient in need. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. However, the preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.
  • the present invention also relates to the use of a pharmaceutical composition above described, a kit above described, or a viral vector above described, in the treatment of a retinal degenerative disease.
  • FIG. 1 represents phototransduction cascade (A) normal phototransduction cascade (B) short phototransduction cascade with an animal opsin and GIRK2 channel.
  • PDE phosphodiesterase.
  • CNG cyclic-nucleotic gated channels.
  • cGMP cyclic guanosine monophosphate.
  • FIG. 2 represents plasmids (A) CMV-GIRK2-GFP and (B) CMV-SWO-mCherry
  • FIG. 3 represents what remained in the phototransduction cascade in rd10 mice using immunohistochemistry
  • A-D retinal cross-section of a control WT mouse stained with (A) opsin, (B) transducin, (C) PDE and (D) cone arrestin.
  • E-H retinal cross-section of a rd10 mouse at P14 stained with (E) opsin, (F) transducin, (G) PDE and (H) cone arrestin.
  • I-L Retinal cross-section of a rd10 mouse at P150 stained with (I) opsin, (J) transducin, (K) PDE and (L) cone arrestin.
  • ONL outer nuclear layer.
  • INL inner nuclear layer.
  • GC ganglion cells. Scale bar is 50pm. Inset scale bar is 25pm.
  • FIG. 4 represents preliminary data.
  • A Eye fundus of GIRK2-GFP expression in rd 10 mouse one week post-injection (*site of injection)
  • C Representative flickers ERG at P33.
  • FIG. 5 represents GIRK2-mediated vision.
  • C Representative flickers ERG at P41.
  • FIG. 6 represents long term efficiency.
  • Pvalue AAV-GIRK2-GFP 0,0007.
  • Pvalue PBS 0,0104.
  • FIG. 7 represents what remained in the phototransduction cascade in huP347S+/- mice using immunohistochemistry.
  • A-D Retinal cross-section of a control WT mouse stained with (A) opsin, (B) transducin, (C) PDE and (D) cone arrestin.
  • E-H retinal crosssection of a huP347S+/- mouse at P14 stained with (E) opsin, (F) transducin, (G) PDE and (H) cone arrestin.
  • ONL outer nuclear layer.
  • INL inner nuclear layer.
  • GC ganglion cells. Scale bar is 50pm. Inset scale bar is 25pm.
  • FIG. 8 [154] [Fig. 8] represents universality of the approach.
  • FIG. 9 represents the efficiency of the mouse GIRK2 in HEK cells transfected with two plasmids: CMV-SWO-mCherry and CMV-GIRK2-GFP.
  • FIG. 10 represents cone opsin and arrestin expression in normal and RCD human retinal tissue.
  • A retinal cross-section of a 91 -year-old individual with no visual impairment (40x).
  • 5B-E Retinal cross-sections of human RCD maculae from 4 different donors (40x).
  • FIG. 1 1 shows characterization of GIRK1 F137S currents elicited by activation of mOpn4L in HEK293 cells.
  • FIG. 1 shows characterization of GIRK1 or GIRK2 currents elicited by activation of mOpn4L in HEK293 cells when activated with blue light (471 nm, exposure from 10 to 20 s on the axis of abscissae) and terminated upon stimulation with lime light (560 nm, exposure from 20 to 60 s on the axis of abscissae).
  • FIG. 13 shows the current density per capacitance of HEK293 cells expressing A1) hGIRKI F137S, B1) hGIRK2 or C1) truncated rGIRK2.
  • the current response elicited by the activation of mOpn4, is shown in comparison to constructs with c-terminal eGFP fusion and untransfected cells.
  • the current density of individual cells is illustrated by individual data points. Significant differences between conditions are marked with * (Kruskal-Wallis One Way Analysis of Variance on Ranks with all pairwise multiple comparison procedures (Dunn's Method) P ⁇ 0.05).
  • D1) Cells expressing hGIRK4 S143T are shown in comparison to untransfected cells.
  • FIG. 14 A) Stimulation of HEK293 cells expressing mOpn4 and either hGIRKI F137S, hGIRKI F137S - eGFP, hGIRK4 S143T or hGIRK2 produces a reliably observable current in whole-cell patch clamp recordings, while currents of hGIRK2-eGFP, rGIRK2 or rGIRK2-eGFP after mOpn4 activation are less likely to be observable. Results of “no induced current observable” are provided in the upper part of each graph bar. Results of “Induced current observable” are provided in the lower part of each graph bar.
  • FIG. 15 represents the visual acuity in rd10 mice after bilateral subretinal injection at P15 of AAV8-pR1 ,7-hGIRK1 F137S at 5E6 or 5E5 vg/eye compared to vehicle-injected rd 10 mice or naive (not injected) rd10 mice.
  • C57BL/6jrd10/rd10 (rd10) mice were used in these experiments. They have a mutation on the rod PDE gene leading to a dysfunctional phototransduction cascade and a rod-cone dystrophy.
  • the second model used is the huRhoP347S+/- mouse.
  • the homozygous strand of this mouse present a KO of mouse rhodopsin (mRho) gene and a KI of human rhodopsin (huRho) with a mutation (P347S) (Millington-Ward et al., 201 1 ) [30].
  • the homozygous males were crossed C57BL/6] (wild-type) females to obtain heterozygous mice. These mice have a similar phenotype as the rd10 mice but the degeneration rate is lower.
  • mice were first anesthetised with intraperitoneal injections of 0.2 ml/20g ketamine (Ketamine 500, Vibrac France) and xylazine (Xylazine 2%, Rompun) diluted in 0.9% NaCI. Eyes were dilated with 8% Neosynephrine (Neosynephrine Faure 10%, Europhta) and 42% Mydriaticum (Mydriaticum 0.5%, Thea) diluted in 0.9% NaCI.
  • Neosynephrine Neosynephrine Faure 10%, Europhta
  • Mydriaticum Mydriaticum 0.5%, Thea
  • mice were anesthetised by isofluorane inhalation. Eyes were dilated and then protected with Lubrithal eye gel (VetXX). Fundus imaging was performed with a fundus camera (Micron III; Phoenix research Lab) equipped with specific filters to monitor GFP or tdTomato expression in live anesthetised mice.
  • ERG electroretinography recordings
  • Eyes were dilated with Neosyhephrine (Neosynephrine Faure 10%, Europhta) and Mydriaticum (Mydriaticum 0.5%, Thea) diluted in 0.9% NaCI. Eyes were protected with Lubrithal eye gel before putting electrodes on the corneal surface of each eye. The reference electrode was inserted under the skin into the forehead and a ground electrode under the skin in the back.
  • ERG recordings were done under two conditions: (i) photopic condition, which reflects con-driven light responses - 6ms light flashes were applied every second during 60 seconds at increasing light intensities (0.1/1/10/50cd s/m) after an adaptation of 5 minutes at 20cd s/m - and (ii) flicker condition, which are rapid frequency light stimuli that reflect cone function (70 flashes at 10Hz et 1cd s/m).
  • HEK cells were transfected with two plasmids: CMV-SWO-mCherry and CMV-GIRK2- GFP ( Figure 2) according to a well-known procedure in the art.
  • HEK293 cells were cultured and recorded in dark room conditions after transfection. Cells were placed in the recording chamber of a microscope equipped with a 25x water immersion objective (XLPIanN-25 x - W-MP/NA1 .05, Olympus) at 36 °C in oxygenated (95% 02/5% CO2) Ames medium (Sigma- Aldrich) enriched with an addition of 1 mM9-cis-retinal. KGIuconate was added to the external solution in order to get a high extracellular potassium concentration leading to a cell potassium reversal potential of -40mV.
  • a CCD camera (Hamamatsu Corp.) was used to visualize cells using a trans-illuminated infrared-light.
  • a monochromatic light source (Polychrome V, TILL photonics) was used to stimulate cells during electrophysiological experiments with light flashes at 400 nm.
  • AOSLO Adaptive optics scanning laser ophthalmoscopy
  • the phototransduction cascade was first analysed in the rd10 mouse model by studying its components using immunohistochemistry, at different time points during retinal degeneration. Immunofluorescence staining was performed against cone opsin, transducing, phosphodiesterase and cone arrestin proteins of the phototransduction cascade that interact directly with cone opsin.
  • Figure 3 shows that only the cone opsin and arrestin were still expressed and localized around the cone cell body at late stage of the disease.
  • Photopic ERG recordings were performed to monitor the cone response to light stimuli at different time points after treatment with GIRK2 and in absence of treatment. These ERGs were done under two conditions: (i) photopic with light flashes applied every second during 60 seconds at increasing light intensities and (ii) flicker stimulation with repetitive flashes during 60 seconds. Data were collected on a weekly basis until p50 and then every 10 to 13 days until 11 weeks of age and showed a gradual decline in ERG amplitudes for both controls and treated eyes (Figure 6A). Moreover, these results are consistent with the optokinetic test, both controls and treated eyes with GIRK2 show a decreased optokinetic reflex over time (Figure 6B).
  • HEK293 Human embryonic kidney 293 (HEK293) stably expressing mouse Opn4L-mCherry are maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM), 4.5 g/l D-glucose, supplemented with 10 % fetal bovine serum (Gibco) and penicillin/streptomycin in a humidified incubator under 5% CO2.
  • HEK293 cells are transfected with FuGENE® HD (Promega) according to the manufacturer’s protocol and incubated for 18-24 h before recordings. Retinaldehyde are added to a final medium concentration of 1 pM.
  • GIRK constructs are expressed in HEK293 cells stably expressing Opn4L-mCherry. Cells are cultured and recorded in dark room conditions after transfection. GIRK-mediated K -currents are measured and analyzed as described below. The external solution is as follows: 20 mM NaCI, 120 mM KCI, 2 mM CaCI2, 1 mM MgCI2, 10 mM HEPES-KOH, pH 7.3 (KOH).
  • Patch pipettes (2-5 MQs) are filled with internal solution: 100 mM potassium aspartate, 40 mM KCI, 5 mM MgATP, 10 mM HEPES-KOH, 5 mM NaCI, 2 mM EGTA, 2 mM MgCI2, 0.01 mM GTP, pH 7.3 (KOH).
  • Cells are recorded in external solution containing 1 pM 9-cis retinal (Sigma). Cells are visualized using a transilluminated red light (590 nm) or green light filter (480 nm) during experimental manipulations.
  • Whole-cell patch clamp recordings of HEK293 cells are performed with an EPC10 amplifier (HEKA). Currents are digitized and filtered with the internal 10-kHz three- pole Bessel filter (filter 1 ) in series with a 2.9-kHz 4-pole Bessel filter (filter 2) of the EPC10 amplifier. Series resistances are partially compensated between 70 and 90%.
  • HEK293 cells are voltage clamped at - 60 mV.
  • a 500 ms long voltage ramp from -100 to +50 mV is applied before light application.
  • a 10 sec light pulse of 471 nm is applied at -60 mV.
  • the size of the GIRK currents is related to the conductance of the cell before and after light activation.
  • GIRK1 F137S induces significantly more ion efflux than truncated rat GIRK2 (about 17-fold higher) in the context of a short GIRK/opsin phototransduction cascade in HEK cells, while wild-type GIRK1 is ineffective at inducing ion efflux.
  • GIRK1 F137S incorporation of GIRK1 F137S in cones will provide for an improved gene therapy over GIRK2 gene therapy of RCD.
  • Figures 13 and 14 also confirm that GIRK1 F137S (with or without GFP tag) induces significant ion efflux, as compared to either human or rat GIRK2 (with or without GFP tag).
  • EXAMPLE 4 GIRK1 F137S vision restoration in an RCD model caused by mutant rhodopsin
  • P15 rd10/rd10 mice received a subretinal injection of AAV8-PR1 ,7-hGIRK1 F137S at a dose of 5e8vg/eye or 5e7vg/eye.
  • OKT measurements were performed to assess visual function. Significant visual improvements were observed 3 weeks after administration with 5e8vg/eye, at P37.
  • EXAMPLE 5 GIRK4 S143T induces significant ion efflux, as compared to either human or rat GIRK2
  • FIGS. 13 and 14 show that GIRK4 S143T (with or without GFP tag) induces significant ion efflux, as compared to either human or rat GIRK2 (with or without GFP tag).
  • TXNL6 is a novel oxidative stress-induced reducing system for methionine sulfoxide reductase a repair of a-crystallin and cytochrome C in the eye lens.
  • PLoS ONE published online ahead of print: 2010].

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Abstract

La présente invention concerne la combinaison d'un acide nucléique codant pour une isoforme courte du facteur de viabilité des cônes émis par les bâtonnets (RdCVF), d'un acide nucléique codant pour une isoforme longue du facteur de viabilité des cônes dérivés de bâtonnets (RdCVFL) et d'un acide nucléique codant pour une forme mutée de la sous-unité 1 du canal potassique de redressement vers l'intérieur géré par la protéine G (GIRK1) (GIRK1 F137S) ou pour une forme mutée de la sous-unité 4 du canal potassique de redressement vers l'intérieur géré par la protéine G (GIRK4) (GIRK4 S143T), les trois acides nucléiques étant exprimés par l'intermédiaire d'un, de deux ou de trois vecteurs viraux, lesdits vecteurs pouvant se trouver dans une seule composition pharmaceutique ou dans plusieurs compositions pharmaceutiques différentes (deux ou trois). La présente invention porte également sur le traitement d'une maladie dégénérative rétinienne, plus particulièrement la rétinite pigmentaire, à l'aide desdits vecteurs viraux ou compositions pharmaceutiques.
PCT/EP2023/079355 2022-10-20 2023-10-20 Compositions et méthodes de traitement de troubles dégénératifs de la rétine WO2024084075A1 (fr)

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