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WO2024226667A2 - Modulation optogénétique par opsines à caractéristiques multiples pour la restauration de la vision et d'autres applications associées - Google Patents

Modulation optogénétique par opsines à caractéristiques multiples pour la restauration de la vision et d'autres applications associées Download PDF

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WO2024226667A2
WO2024226667A2 PCT/US2024/026082 US2024026082W WO2024226667A2 WO 2024226667 A2 WO2024226667 A2 WO 2024226667A2 US 2024026082 W US2024026082 W US 2024026082W WO 2024226667 A2 WO2024226667 A2 WO 2024226667A2
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emco1
light
retinal
mice
patient
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WO2024226667A3 (fr
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Samarendra Kumar Mohanty
Sulagna BHATTACHARYA
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Nanoscope Therapeutics Inc
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Nanoscope Therapeutics Inc
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    • 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
    • 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

  • This invention relates generally to compositions and methods for modulating cellular activities by synthetic opsins.
  • the invention provides enhanced light sensitivity to neurons for vision restoration and other therapeutic applications.
  • the present disclosure provides several light-sensitive ion-channel and ligand molecules, methods of their preparation and different uses, including vision restoration.
  • the invention comprises isolated nucleic acid sequences that encode light-sensitive ionchannels, ligands and constructs, including plasmids and other nucleic acid vectors that comprise such nucleic acid sequences.
  • the disclosure provides light-sensitive ion-channel and ligands (Multi-Characteristics Opsins) that are synthetic in origin, These Multi-Characteristics Opsins or MCOs: (i) have high photosensitivity at multiple visible wavelengths and (ii) can be packaged into a genomic vector such as a plasmid that can be packaged into a virus.
  • the virus can include an adeno-associated virus or a lentivirus.
  • the disclosure in some aspects provides expression of MCOs in cells in-vitro or in-vivo as well as methods for modulating cellular activities by these synthetic opsins.
  • the MultiCharacteristics Opsins are highly sensitive to visible light and ambient-light activatable.
  • expression of a specific MCO in cell produces a long-lasting inward current in response to white light similar to that found in an unmodified photoreceptor-rod signal.
  • the disclosure provides a synthetic, ambient-light activatable, fast, enhanced Multi-Characteristics Opsin (eMCO1) which has a stabilizer-biomarker that play an active role in stabilizing eMCO1 expression on the cell membrane.
  • eMCO1 has a higher percentage of beta sheets and a lower percentage of a disordered structure (i.e. less prone to cleavage).
  • the stabilized eMCO1 enhances the photo-induced current in the cells expressing eMCO1 .
  • the disclosure provides a synthetic, ambient-light activatable, fast, enhanced Multi-Characteristic Opsin (eMCO1) that contains a stabilizer-biomarker can be used to confirm the MCO gene expression in targeted cells.
  • eMCO1 enhanced Multi-Characteristic Opsin
  • the light emitted from the stabilizer-biomarker present in the enhanced Multi-Characteristics Opsin enhances the photo-induced current in the cells expressing eMCO1 by light emitted/re-emitted from the stabilizer-biomarker molecule.
  • the disclosed invention provides method for the use of synthetic opsins for vision restoration and other applications.
  • the amino acid sequence of the synthetic opsin is modified to provide enhanced light sensitivity, kinetics and ionselectivity.
  • a method of delivering MCO to degenerated retinas in order to restore light sensitivity is provided.
  • efficient and stable in-vivo expression of MCO- reporter protein in the retina occurs after intravitreal injection of Adeno-Associated Virus carrying MCO.
  • expression of MCO in the retina of an individual suffering from retinal degeneration can provide for the behavioral restoration of vision.
  • the improvement in visually guided behavior occurs even at light intensity levels that are orders of magnitude lower than that required for Channelrhodopsin-2 opsin.
  • the present disclosure provides a method of efficient restoration of vision in human.
  • the method include use of MCO that is expressed in retinal cells.
  • the MCO produces a slower depolarizing phase after initial response to a white light that is similar to a photoreceptor-rod signal.
  • an opsin can be delivered to retinal cells in-vivo by Adeno- Associated Virus (AAV) or a lentivirus administered to the eye of an individual that contains a nucleic acid vector comprising a promoter-MCO-gene, and/or a Pronase E and/or Alpha-Aminoadipic Acid (AAA).
  • AAV and/or lentivirus can be modified or enhanced to increase efficiency to a targeted retinal layer that crosses the thick inner limiting membrane in humans.
  • an individual is administered a virus mediated MCO to treat diverse retinal degenerations in individuals suffering such diseases, including retinal degeneration or dystrophy.
  • the present disclosure provides the use of opsin that produces a slower depolarizing phase after an initial response to white light, which is similar to a characteristic photoreceptorrod signal. In an aspect, this results in the restoration of vision in blind individuals.
  • the nucleic acid molecule(s) encode for any of the polypeptides described herein.
  • the nucleic acid molecule(s) is formulated to include a pharmaceutically acceptable carrier.
  • a method is provided wherein cells are contacted with or have been transformed with an isolated nucleic acid molecule that encodes for an isolated polypeptide molecule of the invention, including an MCO, and MCO1 and an eMCO1.
  • the cells are rod bipolar cells, ON-type retinal ganglion cells, or ON-type bipolar cells.
  • a method is provided to use an opsin to modulate a cell and tissue function, and for use in diagnosis and treatment of retinal disorders.
  • the present invention discloses a recombinant, ambient-light activatable, enhanced, fast Multi-Characteristics Opsin (eMCO1) chimeric protein comprising: an MCO1 protein mutated to modulate at least one of ion selectivity, light sensitivity, or kinetics of the MCO1 protein.
  • the MCO1 protein has SEQ ID NO: 1 , 3, 5, 7, or 1 1 .
  • one or more of the following single or combinations of mutations modulate ion selectivity, light sensitivity, or kinetics, wherein the mutation is selected from at least one of: S to C substitution at an amino acid residue corresponding to amino acid 132 of the MCO1 sequence; E to A substitution at an amino acid residue corresponding to amino acid 123 of the MCO1 sequence; D to A substitution at an amino acid residue corresponding to amino acid 253 of the MCO1 sequence; R to A substitution at an amino acid residue corresponding to amino acid 120 of the MCO1 sequence; Q to A, substitution at an amino acid residue corresponding to amino acid 56 of the MCO1 sequence; K to A substitution at an amino acid residue corresponding to amino acid 93 of the MCO1 sequence; E to A substitution at an amino acid residue corresponding to amino acid 90 of the MCO1 sequence; E to Q substitution at an amino acid residue corresponding to amino acid 90 of the MCO1 sequence; E to A substitution at an amino acid residue corresponding to amino acid 97 of the MCO1 sequence; E to A substitution at an
  • a protein is a recombinant, ambient-light activatable, slow Multi-Characteristics Opsin (MCO2) chimeric protein; wherein 7 amino acid residues (VNKGTGK) from 309 to 315 are deleted in the molecule of claim 1 to improve the gene expression on membrane; wherein S132L mutation is carried out in the trans-membrane domain 2 of SEQ ID NO: 1 to cause increased binding affinity towards retinal and increased light sensitivity; wherein the opsin is encoded in 658 amino acids; and wherein the MCO2- sensitized cell generates a slowly decaying inward current after initial fast current response to a pulse of white light.
  • MCO2 slow Multi-Characteristics Opsin
  • a single or a combination of mutations is selected from E473A, D603A, R469A of SEQ ID NO:1 that further modulate at least one of the ion selectivity, light sensitivity, or kinetics of the molecule.
  • a trans-membrane sequence is selected from E473A, D603A, R469A of SEQ ID NO:1 that further modulate at least one of the ion selectivity, light sensitivity, or kinetics of the molecule.
  • TPARWVWISLYYAAFYVVMTGLFALCIYVLMQTI is inserted after amino acid residue 315 in MCO1 (SEQ ID NOS:1 or 2) or 308 amino acid residues in MCO2 (SEQ ID NOS:3 or 4).
  • eMCO1 comprises MCO1 sequence (SEQ ID NO: 1) and a stabilizer-biomarker sequence.
  • the recombinant eMCO1 further comprises at least one of: the stabilizer-biomarker is 900 amino acids of SEQ ID NO: 11 ; the stabilizer-biomarker is connected downstream with the ligand non-4- transmembrane domain by a linking sequence; a light emitted from the stabilizer-biomarker stabilizes eMCO1 expression in a membrane with higher percentage of beta sheets and lower percentage of disordered structure and is less prone to cleavage that a non-modified MCO1 ; the stabilizer-biomarker molecule enhances a photo-induced current in cells expressing eMCO1 by better orientation-stabilization of eMCO1 across a membrane; the stabilizer-biomarker molecule enhances a photo-induced current in cells expressing eMCO1 by light emitted/re-emitted from the stabilizer-biomarker molecule; a promoter is used upstream to eMCO1 to target specific cells; the promoter-eMCOI gene is packaged
  • the recombinant cMOC1 further comprises a reporter-gene is downstream from the MCO1 gene to detect cellular expression/activation, wherein the promoter-MCOI -reporter gene is packaged in a viral vector; and wherein cells can be transfected by the promoter-MCOI -reporter gene using either chemical, viral or physical method.
  • MCO-sensitized cells are highly sensitive to light and can be activated at low intensity (-0.02 mW/mm 2 ) ambient light.
  • the MCO-sensitized retinal neurons e.g.
  • the opsin is sensitive to any wavelength of light in a visible and a near-infrared range.
  • the opsin is activated by a single-photon including direct, and indirect (e.g., fluorescence, phosphorescence, up/down conversion) illumination light in a visible and a near-infrared range.
  • the present invention discloses methods and uses of the MCO1 , MCO2 or eMCO1 , or mutants thereof for restoration of lost vision.
  • the vision loss is due to any degenerative retinal disease; wherein delivery of a recombinant MCO-gene to targeted cells is carried out by an intravitreal/sub-retinal injection of a virus carrying promoter-MCO-gene in an eye, in combination with Pronase E and/or alpha-aminoadipic acid (AAA) for enhancing delivery efficiency, or both; wherein delivery of the MCO-gene is carried out by intravitreal/sub-retinal injection of promotor-MCO-gene plasmids in eye, followed by either chemical, or physical transduction method or a combination thereof; wherein the MCO- gene delivery into eye does not cause either undesired expression in non-targeted cells and organs, or any adverse reaction or cytotoxicity in the treated eye; wherein significant visually guided behavioral improvement is
  • the present invention discloses methods and uses of the MCO1 , MCO2 or eMCO1 for preventing or slowing down the vision loss, wherein the MCO-gene is delivered to retinal cells during progressive photoreceptor loss through transduction, including, through the use of a viral vector, including an AAV or lentivirus; wherein light stimulation of the MCO-sensitized retinal cells is carried out to prevent or slow down the photoreceptor loss; and wherein the light stimulation dose is optimized for maximal efficacy.
  • the present invention discloses methods and uses of the MCO1 , MCO2 or eMCOI for restoration of vision by regenerating the damaged RGC axons: wherein the MCO-gene is delivered to retinal ganglion cells through transduction, including, through the use of a viral vector, including an AAV or lentivirus during or after axonal degeneration; wherein light stimulation of the MCO-sensitized RGCs is carried out to slow down the rate of degeneration and/or to regenerate the axons; and wherein the light stimulation dose is optimized for minimizing the degeneration and/or maximizing the axonal regeneration.
  • the present invention discloses methods and uses of the MCO1 , MCO2 or eMCO1 for stimulation of different types of excitable cells including neurons, cardiac cells: wherein the use comprises delivery of the MCO-gene by either chemical, viral or physical transduction transformation method; wherein activation of MCO is achieved upon illumination of light; and wherein the effect is measured by electro/opto-physiology.
  • the present invention discloses methods and uses of the MCO1 , MCO2 or eMCO1 fortreatment of disorders: wherein the use comprises delivery of the MCO-gene to different organs by either chemical, viral or physical transduction method; wherein activation of MCO is achieved upon illumination of light; and wherein an effect is measured by an electrophysiology or other functional and behavioral analysis.
  • the present invention discloses a polypeptide comprising a sequence comprising at least 75%, 85%, 95% or 100% identity to SEQ ID NO: 1 , 3, 5, 7 or 11 , wherein said polypeptide exhibits the photosensitivity characteristics of the protein of at least one of SEQ ID NO: 1 , 3, 5, 7, or 11 .
  • the present invention discloses a recombinant nucleic acid encoding a polypeptide having at least 75%, 85%, 95% or 100% identity to SEQ ID NO: 1 , 3, 5, 7 or 11 , wherein said polypeptide exhibits the photosensitivity characteristics of the protein of at least one of SEQ ID NO: 1 , 3, 5, 7 or 11 .
  • the nucleic acid has at least one of 75%, 85%, 95% or 100% identity to SEQ ID NO: 2, 4, 6, or 8.
  • the invention discloses a vector comprising the nucleic acid having 75%, 85%, 95% or 100% identity to at least one of SEQ ID NO: 1 , 3, 5, 7 or 11 .
  • the vector is selected from an adenovirus, adeno-associated virus or lentivirus vector.
  • the present invention discloses a method of treating blindness comprising administering to a patient in need thereof a vector comprising the nucleic acid having 75%, 85%, 95% or 100% identity to at least one of SEQ ID NO: 1 , 3, 5, 7 or 11 .
  • a light-sensitive ion-channel molecule (expressed in the transmembrane) is used that is capable of capturing blue light, along with a light-sensitive ligand to capture light from a green-red wavelength and a light-emitting enhancer that in an embodiment is located at the C- terminus.
  • an eMCO1 results in a fast light induced activity in a target cell at a low light threshold and over a broad spectral range.
  • a blue light activates the trans-membrane (TM) domain of an eMCO1 and the eMCO1 acts as an ion channel.
  • red light absorbed by a non-TM, middle domain (non-ion channel) leads to a conformational change of a TM domain of an eMCO1 , which results in an ion flow through the TM-domain of the eMCO1.
  • the C-terminus domain (RFP) of the eMCO1 emits a red light that results in an enhancement of eMCO1 efficacy.
  • an enhanced Multi-Characteristic Opsin is transduced into cells in-vitro or in-vivo and expressed by the transduced cells.
  • the transduced eMCO1 modulates a cellular activity following exposure to different wavelengths of visible light.
  • a method is provided for delivering (through for example transduction) an eMCO1 to a degenerated retina to restore light sensitivity.
  • an eMCO1 transduced into a retina, and in a further aspect, one or more cells that comprise the retina) is efficiently and stably expressed in vivo.
  • an eMCO1 -reporter protein is administered to a patient through an intravitreal injection of an Adeno-Associated Virus (AAV) that includes a gene that codes for an eMCO1 into the retina.
  • AAV Adeno-Associated Virus
  • the administration of an eMCO1 including an AAV containing a gene coding for an eMCO1 into the retina of a patient results in the behavioral restoration of vision.
  • administering results in the restoration of part of all of the vision of the patient.
  • administration of an eMCO1 to a patient, including an AAV containing a gene coding for an eMCO1 into the retina results in results in the arrest of retinal degeneration and disorganization in the retina of the patient.
  • administering results in the arrest of the degeneration and disorganization of an inner nuclear layer of the retina and its connectivity with ganglion cell layer.
  • methods for use in treating a patient suffering from a retinal degenerative disease in one or both eyes by administering an eMCO1 (including through administration of an AAV containing the gene coding for an eMCO1 , through an intraocular injection into one eye.
  • administering can be used to treat a retinal dystrophy or a retinal degeneration disease.
  • a retinal dystrophy or a retinal degeneration disease is characterized by the loss of part of all of the outer retina, including photoreceptor cells and/or retinal pigment epithelium.
  • administration of eMCO1 can be used to treat macular degeneration diseases such as Stargardt macular degeneration and advanced macular degeneration, wherein degeneration or dysfunction of outer retinal cells including photoreceptor cells occur.
  • administering can be used to treat macular degeneration diseases with geographic atrophy or disciform scar, wherein the central portion of the retina (macula) is targeted.
  • administration of eMCO1 can lead to an improvement in the vision function of subjects suffering from macular degenerative diseases.
  • the eMCO1 treated patients can further increase the quality of vision via virtual reality (VR) headset by enhancing the contrast and /or optical zooming of the visual field.
  • VR virtual reality
  • the eMCO1 treated macular degeneration subjects demonstrated an increase in visual acuity.
  • the eMCO1 treated macular degeneration subjects demonstrated further increase in visual acuity while using the VR headset.
  • MCOs Multi-Characteristics Opsins
  • MCO2 contains mutation (S 132 L) of MCO1 and deletion of 7 amino acid residues 6(VNKGTGK (SEQ ID NO: 13)) after 308.
  • MCO1T and MCO2T contain additional trans-membrane sequence (TPARWVWISLYYAAFYVVMTGLFALCIYVLMQTI (SEQ ID NO: 14)) after 315 and 308 amino acid residues respectively.
  • Table-05 shows the DNA sequences of promoter (mGluR6) used upstream of MCO-sequences for targeting specific cells as an example; and Table-06 shows the DNA sequences of reporter (mCherry) used downstream of MCO-sequences for confirming expression in specific cells as an example.
  • promoter mGluR6
  • reporter mCherry
  • Table-06 shows DNA sequences of reporter-stabilizer (mCherry) used downstream of MCO- sequences for confirming expression in specific cells as an example.
  • Table-07 shows Amino acid and DNA sequences of Enhanced Multi-Characteristics Opsin-1 (eMCO1). It contains MCO1 sequence (Table-01) and biomarker-stabilizer sequence (Table-06).
  • Table-08 shows the comparison of stability of the MCO1 and eMCO1 based on secondary structure and folding using theoretical modeling by RaptorX.
  • Fig. 1 A illustrates domain architecture of Multi-Characteristics Opsins (MCOs) with reporter protein, which includes eMCO1 .
  • Fig. 1 B shows typical circular map showing the insertion of MCO gene cloned at the restriction sites (BamH I and Sal I).
  • FIG. 2 shows Theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsins.
  • Fig. 2A shows the theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, MCO 1 .
  • Fig. 2B depicts the theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, MCO 2.
  • Fig. 2C shows the theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, eMCO1 .
  • Figs. 3A and 3 B show expression of eMCO1 in model HEK 293 cells.
  • Fig. 3A shows Expression of eMCO1 is localized in plasma membrane.
  • Confocal fluorescence images of HEK293 cells transfected with mGluR6-MCO1-mCherry (mGluR6-eMCO1) Fig. 3B shows Intensity of MCO1 reporter fluorescence along line across representative cells.
  • Figs. 4A and 4B illustrate functioning of enhanced Multi-Characteristics Opsin (eMCO1).
  • Fig. 4A shows inward current profiles in MCO1 -expressing cells in response to light (average intensity: 0.024 mW/mm 2 ).
  • Fig. 4B shows Activation spectrum of eMCO1 . Average ⁇ SEM.
  • Figs. 5A and 5B show the effect of eMCO1 (e.g., presence of mCherry on MCO1) function measured by cellular activity. Inward current profiles in HEK cells measured by Port-a-Patch automated Patch clamp electrophysiology.
  • Fig. 5A shows photocurrent measured at white light intensity of 0.02 mW/mm 2 in cell transfected with mGluR6-eMCO1 (mGluR6-MCO1 -mCherry).
  • Fig. 5B depicts photocurrent measured at white light intensity of 0.02 mW/mm 2 in cell transfected with mGluR6-MCO1 .
  • Figs. 6A and 6B illustrate functioning of Multi-Characteristics Opsin (MCO2).
  • Fig. 6A shows Fluorescence upon lipofection of MCO2-mCherry into HEK293 cells.
  • Fig. 6B shows Inward current in MCO2-expressing cells in response to light (average intensity: 0.024 mW/mm 2 ) measured by Patch-clamp electrophysiology.
  • Figs. 7A and 7B illustrate AAV2-carried mGluR6-eMCO1 (vMCO1) transfection of cells.
  • Fig. 7A depicts Three-dimensional reconstruction of eMCO1 expression in HEK 293 cells, scale bar: 30 pm.
  • Fig. 7B shows Three-dimensional reconstruction of eMCO1 expression in Whole retinal cup, scale bar: 0.8 mm.
  • Figs. 8A and 8B show the patch-clamp recording of MCO1 transfected retina.
  • Fig. 8A shows eMCO1 expression in the cells of mice retina explant.
  • Fig. 8B shows Inward photocurrent induced by light pulse (100 ms) train.
  • Figs.9A -9F shows dose and time dependent layer-specific expression of MCO1 in rd10 mice after vMCO1 injection.
  • Fig. 9A shows Fluorescence confocal image of rd10 mouse retina cup after 1 week of intravitreal vMCO1 injection.
  • Fig. 9B shows Fluorescence confocal image of rd10 mouse retina cup 8 weeks after intravitreal injection of vMCO1. Scale bar: 200 pm.
  • Fig. 9C shows Confocal fluorescence image of folded edge of retinal cup transfected with vMCO1 at dose of 1 .6 x 10 11 VG/ml. Scale bar: 100 pm.
  • Fig. 9A shows Fluorescence confocal image of rd10 mouse retina cup after 1 week of intravitreal vMCO1 injection.
  • Fig. 9B shows Fluorescence confocal image of rd10 mouse retina cup 8 weeks after intravitreal injection of vMCO1. Scale bar: 200 pm
  • FIG. 9D shows Cross-sectional view of vMCO1 expression in retina 16 weeks after intravitreal injection at dose of 1 .6 x 10 12 VG/ml. Scale bar: 50 pm.
  • Fig. 9E shows Kinetics of MCO1 expression in rd 10 mice retina at two different doses of vMCO1 . Average! SD.
  • Fig. 9F shows Inter-animal variation of MCO1 -mCherry (eMCO1) expression (after background subtraction) in retina of rd10 mice 16 weeks after transfection at dose of 1 .6 x 10 12 VG/ml. Average+ SD. * p ⁇ 0.01 vMCO1 injected vs. non-injected. [0056] Figs.
  • FIG. 10A - 10H show visually guided improvement in rd1O mice behavior in radial water maze.
  • Fig. 10A shows Time-lapse images of visually guided rd10 mice behavior in radial water maze with white LED light before intravitreal vMCO1 injection.
  • Fig. 10B shows Behavior of rd10 mouse with LED light ON six weeks after vMCO1 injection.
  • Fig. 10D depicts Latency to find the platform by the vMCO1 treated rd10 mouse, with and without light, dropped at side arms-2 & 4 of the maze.
  • ⁇ SEM. N 5 for each mouse.
  • FIGs. 11 A and 11 B show longitudinal study of visually guided improvement in rd10 mice behavior in radial water maze.
  • FIG. 11A depicts Schematic of the radial-arm water maze used to test improvement in visually guided behavior of vMCO1 injected rd10 mice.
  • Fig. 11 B shows the Time to reach platform by the rd10 mice from center of the maze (light intensity: 0.007 mW/mm2) before vMCO1 injection and as a function of post-injection period.
  • N 5; Average ⁇ S.D. *P ⁇ 0.05.
  • Fig. 11A depicts Schematic of the radial-arm water maze used to test improvement in visually guided behavior of vMCO1 injected rd10 mice.
  • Fig. 11 B shows the Time to reach platform by the rd10 mice from center of the maze (light intensity: 0.007 mW/mm2) before vMCO1 injection and as a function of post-injection period.
  • N 5
  • 1 1 C shows the Time to reach platform by the rd10 mice from near arm of the maze (light intensity: 0.014 mW/mm 2 ) before vMCO1 injection and as a function of post-injection period.
  • N 5; Average ⁇ S.D. *P ⁇ 0.05.
  • Fig. 11 D plots the Time to reach platform by the rd10 mice from side arm (light intensity: 0.004 mW/mm 2 ) before vMCO1 injection and as a function of post-injection period.
  • N 5; Average ⁇ S.D. *P ⁇ 0.05.
  • Fig. 12 shows the Light-intensity dependence of improvement in rd10 mice behavior in radial water maze. Comparison of time to reach platform from center of the maze between two different light intensities as a function of post-injection period. Average ⁇ S.D. *P ⁇ 0.01 . LOO.0005 mW/mm 2 ; L2 .007 mW/mm 2 . Bright ambient level is ⁇ 0.01 mW/mm 2 .
  • Figs. 13A and 13B show optokinetic assessment of rd 10 and MCO-sensitized rd10 mice.
  • Figs. 14A - 14D show viability of MCO1 sensitized retinal cells after chronic light exposure.
  • Fig. 14A shows that Similar to the wild-type (non-blind) mice, vMCO1 treated rd10 mice avoid bright light by staying away and blocking light (via creating a heap out of bedding material, as shown in the arrow).
  • Fig. 14B shows Fluorescence image of retina stained with Caspase-3 (green) for vMCO1 -treated rd10 mouse 4 weeks after 8-hr/day illumination of white light (intensity: 0.1 mW/mm 2 ).
  • Fig. 14A shows that Similar to the wild-type (non-blind) mice, vMCO1 treated rd10 mice avoid bright light by staying away and blocking light (via creating a heap out of bedding material, as shown in the arrow).
  • Fig. 14B shows Fluorescence image of retina stained with Caspase-3 (green) for vMCO1 -treated r
  • FIG. 14C shows Fluorescence image of retina stained with Caspase-3 (green) for wild-type mouse 4 weeks after 8-hr/day illumination of white light (intensity: 0.1 mW/mm 2 ).
  • Fig. 14D shows Quantitative comparison of % apoptotic retinal cells between wild type and vMCO1 treated rd10 mice. 0 % apoptotic cells in inner nuclear layer of vMCO1 treated rd 10 mice.
  • Figs. 15A and 15B show results of evaluation of structural integrity of retina after vMCO1 injection in rd10 mice.
  • Fig. 15A shows an OCT image of rd10 mice retina after vMCO1 injection.
  • Fig. 15B shows the Comparison of retinal thickness of 4 different rd10 mice before and 1 week after injection.
  • N 10 B-scans/ mice. Average + SD.
  • Figs. 16A -16C show results of immune-toxicity in vMCO1 injected rd10 mice.
  • Fig. 17 shows biodistribution of AAV2 packaged Multi-Characteristics Opsin (vMCO1).
  • vMCO1 Multi-Characteristics Opsin
  • Figs. 18A-18F show immunohistochemistry of vMCOI injected rd10 mice eye.
  • Fig. 18A shows that MCO-mCherry (red) is selectively targeted and expressed in INL of rd10 mice 8 wks after intravitreal injection of vMCOI .
  • the absence of arrestin (green) suggests a complete loss of photoreceptors.
  • Fig. 18B shows PKCa stain (green) in rod bipolar cells expressing mCherry (red, intrinsic) in rd10 mice 8 wks after intravitreal injection of vMCO1.
  • Fig. 18A shows that MCO-mCherry (red) is selectively targeted and expressed in INL of rd10 mice 8 wks after intravitreal injection of vMCOI .
  • the absence of arrestin (green) suggests a complete loss of photoreceptors.
  • Fig. 18B shows PKCa stain (green) in rod bipolar cells expressing
  • FIG. 18C shows mGluR6 stain (green) in ON bipolar cells expressing mCherry (red) in rd10 mice 8 wks after intravitreal injection of vMCOI .
  • Fig. 18D shows mCherry (green- immunostained) expression in rd10 retina 8 wks following intravitreal delivery of vMCOI to rd10 mice.
  • Fig. 18E shows that GFAP (green) in rd10 mice 18 wks after intravitreal injection of vMCO1 as reported in photoreceptor degenerated retina.
  • Fig. 18F shows no CD45 (green) expression suggesting no immune cells in rd10 mice 8 wks after intravitreal injection of vMCOI .
  • Fig. 19 illustrates the structure of eMCO1 and activation of its different domains by light.
  • Blue light activates the trans-membrane (TM) domain (ion-channel) of eMCO1 allowing a flow of cations.
  • Green & Red light activates the non-TM, middle domain (non-ion channel).
  • Fig. 20A shows the Inward current profile measured at blue (450 nm) light intensity of 0.06 mW/mm 2 in HEK cell transfected with eMCO1 .
  • Fig. 20B shows the inward current profile measured at green (520 nm) light intensity of 0.06 mW/mm 2 in HEK cell transfected with eMCO1.
  • Fig. 20C shows the inward current profile measured at red (630 nm) light intensity of 0.06 mW/mm 2 in HEK cell transfected with eMCO1 .
  • Fig. 20D shows Inward current profile measured at blue (450 nm) light intensity of 0.06 mW/mm 2 in HEK cell transfected with eMCO1 , in presence of Ca 2+ chelator (BAPTA).
  • Fig. 20E shows the comparison of photocurrent in cells generated by light of different wavelengths and the effect of the presence of Ca 2+ chelator (BAPTA) on eMCO1 transmembrane-domain function that has been probed by blue (450 nm) light activation. Average ⁇ SEM.
  • Fig. 20F shows the comparison of light activation ON-time generated by blue and green light. Average ⁇ SEM, *p ⁇ 0.05.
  • Fig 21 A shows results of longitudinal monitoring of retina of rd mice with SDOCT before and after different doses of AAV2-eMCO1 or AAV2-vehicle injection.
  • Group AA 1.0 x 10 12 VG/ml AAV2-eMCO1 (vMCO1); group BB: 1 ,0 x 10 12 VG/ml AAV2 (no transgene); and group CC: 1.0 x 10 10 VG/ml AAV2-eMCO1.
  • Fig. 21 B shows the scattered plot showing comparison of retinal thickness before, and 4 months after injection in different groups.
  • Group AA 1.0 x 10 12 VG/ml AAV2-eMCO1
  • group BB 1 .0 x 10 12 VG/ml AAV2 (no transgene)
  • group CC 1 .0 x 10 10 VG/ml AAV2-eMCO1 .
  • Fig. 21 C shows the mean difference of retina-thickness between baseline and intravitreally-injected mice shown as Gardner-Altman estimation plot.
  • Group AA 1 .0 x 10 12 VG/ml AAV2-eMCO1
  • group BB 1 .0 x 10 12 VG/ml AAV2 (no eMCO1)
  • group CC 1 .0 x 10 10 VG/ml AAV2-eMCO1 .
  • the curve indicates the resampled distribution of the mean difference, given the observed data.
  • the 95% confidence level of the mean difference is illustrated by the black vertical line.
  • Fig. 22A shows the PKCa stain (green) showing rod bipolar cells expressing mCherry (red, intrinsic) in rd10 mouse after intravitreal injection of AAV2-eMCO1 .
  • ONL Outer Nuclear layer
  • INL Inner Nuclear Layer
  • GCL Ganglion Cell Layer.
  • Fig. 22B shows eMCO1 (mCherry: red reporter) expression in bipolar cells (INL) of rd10 mouse after intravitreal injection of AAV2-eMCO1.
  • Fig. 22C shows the quantification of eMCO1 (reporter mCherry) expression in bipolar cells of three rd10 mice.
  • Fig. 22D shows the immunostained cross-sections of the retina showing Bipolar cell terminals (green: PKCa) costoring CtBP (a synaptic ribbon marker, red) in close contact with retinal ganglion cells (RGCs) in the AAV2-eMCO1 treated rd10 mouse retina.
  • Bipolar cell terminals green: PKCa
  • CtBP a synaptic ribbon marker, red
  • RRCs retinal ganglion cells
  • Fig. 22E shows the images of immunostained cross-sections of the retina showing Bipolar cell terminals (green: PKCa) costoring CtBP (a synaptic ribbon marker, red) in close contact with retinal ganglion cells (RGCs) in the untreated rd10 mouse retina.
  • Fig. 22F illustrates stronger PKCa immunostained signal in axonal terminals (*p ⁇ 0.05) and CtBP signal is found in the AAV2-eMCO1 treated rd10 mice retina.
  • Fig. 23A illustrates the time to reach platform by the Stargardt (ABCA4-/-) mice from side-arms of the radial-arm water maze (light intensity: 0.004 mW/mm 2 ) before and after AAV2 (no transgene, -ve control) injection.
  • N 5; Average ⁇ S.D, ns: Not statistically significant.
  • Fig. 23B shows the time to reach platform by the Stargardt (ABCA4-/-) mice from side-arms of the radialarm water maze (light intensity: 0.004 mW/mm 2 ) before and after AAV2-eMCO1 (vMCO1) injection.
  • N 5; Average ⁇ S.D. ****p ⁇ 0.001 .
  • Fig. 23D shows the time to reach platform by the Rpe65rd12 (LCA) mice from side-arms of the radial-arm water maze (light intensity: 0.004 mW/mm 2 ) before and after AAV2-eMCO1 injection.
  • N 5; Average ⁇ S.D. **p ⁇ 0.01 .
  • FIG. 24A shows results of longitudinal monitoring of retina of Abca4 tm1Ght/J Stargardt mice using SDOCT before and after AAV2-eMCO1 injection as compared to control (non-injected group). Comparison of retinal thickness before, Week 4, 12 and 16 after injection. Average ⁇ SEM.
  • Fig. 24B shows the measured change in ERG b-wave amplitude (with respect to baseline) at 1 cd. s/m 2 of Abca4 tm1Ght/J Stargardt mice after AAV2-eMCO1 injection as compared to control (non-injected group).
  • Fig. 24C shows the change in ERG b-wave amplitude (with respect to baseline) at 10 cd. s/m 2 of Abca4 tm1Ght/J Stargardt mice after AAV2-eMCO injection as compared to control (non-injected group).
  • Fig. 25 illustrates the method of vison restoration via intraocular eMCO1 administration.
  • 100 Retina; 110: Retinal Ganglion cells; 120: Bipolar cells; 130: photoreceptors; 140: Retinal pigment epithelium; 150: Loss of photoreceptors/ retinal pigment epithelium; 160: Delivery device; 170: AAV-carried eMCO1 ,; 180: Optic nerve; 190: Optic tract; 200: Optic Chiasm; 210: Transfer of AAV2-carried eMCO1 to contralateral eye; 220: AAV2-eMCO1 Transduced retinal cells; 230: Projected external visual field; 240: Light-activated bipolar cells; 250: Activated retinal ganglion cells; 260: Electro-chemical signal transmitted via optic nerve; 270: Brain for visual processing of light-activated signal received from eMCO1 -sensitized retina.
  • FIG. 26A shows eMCO1 gene expression in retina subsequent to intravitreal injection in mice.
  • Reporter (mCherry) fluorescence images of retina cups from mice (N 4) eyes, 6 months after intravitreal injection with 1 pl of 1 E12 vg/ml AAV2-eMCO1 (vMCO1).
  • Fig. 26B shows contralateral transfer of eMCO1 gene and expression in retina subsequent to uniocular intravitreal injection in mice.
  • Reporter (mCherry) fluorescence images of retina cups from mice (N 4) eyes, 6 months after intravitreal injection of 1 pl of 1 E12 vg/ml AAV2-eMCO1 (vMCO1).
  • Fig. 26C shows reporter (mCherry) immuno-fluorescence image of retina slice from a dog eye 4 months after uniocular (right eye) intravitreal injection with 75g I of 2E11 VG/ml vMCO1 .
  • GCL Ganglion Cell Layer
  • INL Inner Nuclear layer
  • ONL Outer Nuclear layer.
  • Fig. 26D shows evidence of contralateral transfer of eMCO1 gene and expression in retina subsequent to uniocular intravitreal injection in a dog.
  • GCL Ganglion Cell Layer
  • INL Inner Nuclear layer
  • ONL Outer Nuclear layer.
  • Fig. 26E shows qPCR detection of vMCO1 vector sequences in tissues of visual system of dogs 1 and 2, after uniocular intravitreal injection of right eye with 75 pl of 2E11 VG/ml AAV2-eMCO. Av ⁇ SD.
  • Fig. 27A shows the fundus imaging of mCherry (reporter for eMCO1) fluorescence.
  • Fig. 27B shows the fundus imaging of mCherry(reporter) fluorescence showing enhancement in contralateral eye, 8 week after AAV2-eMCO1 injection in Retinitis Pigmentosa patient.
  • Fig. 27C shows results of longitudinal monitoring of retinal thickness in injected and contralateral eyes by OCT imaging after injection of AAV2- eMCO1 in in Retinitis Pigmentosa patient.
  • N 1 1 , Av ⁇ SD.
  • X 31 weeks data is missing patients 2-4, and 7- 9 due to COVID-related lockdown; xx 52 weeks data does not include patient 8, who did not travel due to COVID-situation.
  • Fig. 29A and 29B show improved visually-guided behavior in mice with opsin-sensitized retinal ganglion cells being stimulated by a strobe light of frequency ⁇ 0.5Hz.
  • Fig. 29A shows the Schematic of Visually-guided Y-mobility assay to evaluate vision in mice. +Light: Light ON; -Light: Light OFF.
  • Fig. 29B shows the Number of events (finding Light ON vs. OFF) by mice for different conditions. Baseline: No optic nerve crush; ON crush: Optic nerve crush; After Light Stimulation: 2 weeks after (8 hours/day) 0.5 Hz strobe light stimulation of opsin-sensitized retinal ganglion cells in optic nerve crush mice.
  • Fig 30A shows longitudinal improvement in an eMCO1 treated subject for visual acuity letter score measured using ETDRS eye chart.
  • Fig 30B shows longitudinal improvement in an eMCO1 treated subject with VR headset (with 100% contrast and 3X zoom) for visual acuity letter score measured using ETDRS eye chart.
  • the cells are generally transfected by a virus to express opsin (light-sensitive molecular ion-channel and ligand as well as enhancer), which is then activated.
  • opsin light-sensitive molecular ion-channel and ligand as well as enhancer
  • the opsin depolarizes the opsin-expressing cells following illumination by light of a specific visible wavelength.
  • retinal cells expressing Channelrhodopsin-2 (ChR2) are sensitive to blue light.
  • opsins light-activated ion channels
  • ligands have been developed to either enhance photosensitivity of cells, or to be activated by different wavelengths of visible light.
  • complex of three opsins ChoR2 for blue, C1V1 for green, and ReaChR for red photosensitivity
  • complex of three opsins has been delivered to cells by chemical or physical method.
  • such large complexes cannot be packaged into commonly used viral vectors (i.e. Adeno-Associated Virus).
  • use of a chemical or physical method for delivery is less efficient and/or compromises cell viability, thus limiting their ready usefulness.
  • the opsins developed and utilized to date for vision restoration when stimulated by light, do not produce a characteristic photoreceptor-rod signal, i.e., the voltage signal does not have a slower depolarizing phase after an initial fast response.
  • the present invention provides for an effective optogenetic vision restoration at a low light level.
  • the present invention provides for vision restoration by optogenetics, protein administration or gene therapy methods by delivering an opsin or other genes, through methods that include viral means (e.g. recombinant adeno-associated virus, rAAV), and where the administration is into the vitreous of the eye.
  • viral means e.g. recombinant adeno-associated virus, rAAV
  • the present disclosure comprises an ability to produce characteristic photoreceptor-rod signal that do not require an external active stimulation device. This embodiment avoids obstacles that are associated with or encountered by existing opsin-based approaches.
  • the present invention provides for the restoration of an individual’s vision that is lost due to a retinal degenerative disease. Further advantage of the present invention is that the method of delivering opsin/other therapeutic gene include a combination of rAAV and chemical agent that can transiently permeabilize the inner limiting membrane of the human eye.
  • the present invention highlights the unique structure of eMCO leading to fast light induced activity of cells at low light threshold and over a broad spectral range.
  • the blue light activates the trans-membrane (TM) domain of eMCO1 that in an embodiment acts as an ion channel.
  • TM trans-membrane
  • a red light absorbed by the non-TM, middle domain (non-ion channel) leads to a conformational change of an eMCO1 - TM domain, which in turn leads to an ion flow via the eMCO1 TM-domain.
  • absorption of a green light by a C-terminus domain (RFP) results in the emission of a red light that enhances the eMCO1 efficacy.
  • the present invention provides for the expression of an enhanced MultiCharacteristic Opsin (eMCO1) in cells in-vitro or in-vivo, as well as methods for modulating cellular activities by different wavelengths of visible light.
  • eMCO1
  • Optogenetic stimulation provides high temporal precision (5-10) by introducing light-activatable molecular channels (e.g. channelrhodopsin-2, ChR2; halorhodopsin, NpHR) into cells by genetic targeting.
  • light-activatable molecular channels e.g. channelrhodopsin-2, ChR2; halorhodopsin, NpHR
  • optogenetics has several advantages over electrical stimulation such as cellular specificity (e.g. spared cones, ganglion or bipolar cells) and minimal invasiveness (11).
  • ChR2 Light-induced activation of ChR2, a non-selective cation channel, results in depolarization of only those cells that express ChR2.
  • Selective activation of neurons by ms-pulsed blue light has been demonstrated in culture (9), brain slices, as well as in small animals (12-15).
  • This optogenetic activation method is very promising for controlling cellular activities in-vitro as well as in- vivo as it only requires light of moderate intensity ( ⁇ 0.1 mW/mm 2 ) that can be delivered from a light emitting diode (LED) or laser (5, 6).
  • the present disclosure provides several light-sensitive ion-channel and ligand molecules (MultiCharacteristics Opsins) made by synthetic means: (i) having high photosensitivity at multiple visible wavelengths, (ii) with plasmid size small enough to be packaged into safe Adeno Associated Virus.
  • the invention also includes isolated nucleic acid sequences that encode light-sensitive ion-channels and ligands of the invention, and constructs that comprise such nucleic acid sequences.
  • MCOs that find use the methods disclosed herein comprise amino acids as shown in Tables 1-4, 7 and as represented by SEQ ID NOS: 1 , 3, 5, 7, or 11 .
  • the MCO has at least around 70, or 75, or 80, or 85 or 90 or 95, or 96 or 97, or 98 or 99% identity with a sequence as shown in SEQ ID NOS:
  • MCO has the photosensitivity characteristics of SEQ ID NOS: 1 , 3, 5, 7, or 11 , wherein said MCO has the photosensitivity characteristics of SEQ ID NOS: 1 , 3, 5, 7, or 11 , wherein said MCO has the photosensitivity characteristics of SEQ ID NOS: 1 , 3, 5, 7, or
  • the MCO is encoded by a nucleic acid as shown in Tables 1-4, 7 and as represented by SEQ ID NOS: 2, 4, 6, 8, or 12.
  • the nucleic acids encoding the MCO have at least around 70, or 75, or 80, or 85, or 90, or 95, or 96, or 97, or 98 or 99% identity with a sequence as shown in SEQ ID NOS: 2, 4, 6, 8, or 12, wherein said encoded MCO has the photosensitivity characteristics of SEQ ID NOS: 1 , 3, 5, 7, or 11 .
  • the nucleic acids encoding the MCO find use when incorporated into vectors for delivery to a patient in need thereof.
  • the vectors are plasmids with appropriate promoters as is known in the art.
  • the vectors are viral vectors.
  • Viral vectors that find use in the methods disclosed herein include adenovirus vectors, adeno-associated virus vectors, and the like.
  • the invention in some aspects includes expression of Multi-Characteristics Opsins (MCOs) in cells in-vitro or in-vivo as well as methods for modulating cellular activities by these synthetic opsins.
  • MCOs Multi-Characteristics Opsins
  • RP Retinitis Pigmentosa
  • AMD age-related macular degeneration
  • RP Retinitis Pigmentosa
  • AMD age-related macular degeneration
  • retinal implants Two different types of retinal implants are being developed: subretinal and epiretinal implants (30). The subretinal implants are positioned in the area of the retina where the photoreceptor cells reside, between the pigmented epithelium and the bipolar cells (31). These retinal prostheses have been successful in generating visual perception in blind subjects (32- 34).
  • the disadvantages of using such subretinal implants include (i) chronic damage of the implanted electrodes, and (ii) insufficient current produced by microphotodiode from the ambient light to stimulate adjacent neurons (35, 36).
  • the epiretinal implants are placed in the area of the retinal ganglion cells (RGCs) and the device functions by stimulating the RGCs in response to input obtained from a camera that is placed outside of the eye or within an intraocular lens (36, 37).
  • the disadvantages of epiretinal implants include (i) cellular outgrowth due to surgical implantation, and (ii) disordered stimulation pattern resulting from the electrical stimulation of both the axons and cell bodies of the RGCs (36).
  • these methods for restoration of vision in blind patients are based on non-specific cellular activation and have low spatial resolution due to low number of electrodes (higher number or density of electrodes requires more power, leading to damage of neural tissue by heat), and hence able to improve vision with low spatial resolution.
  • Optogenetic method has been employed for vision restoration in blind mice model either by nonspecific stimulation of retina (38) or in a promoter-specific manner including Thy1 for RGCs (39-43), mGluR6 targeting ON bipolar cells (44, 45). Attempts have also been made for stimulation of RGCs by use of melanopsin (46) or photochemical genetics (47). Further, use of active light stimulation of chloride- channel opsin (Halorhodopsin) expressing in longer-persisting cone photoreceptors (48) has shown new promise for therapeutic intervention for restoration of vision (49). The re-sensitized photoreceptors have shown to drive retinal circuitry functions, activate cortical circuits, and mediate visually guided behaviors.
  • opsins such as ChR2 (38) and others, which requires light intensities order of magnitude higher than ambient lighting conditions. Therefore, clinical success of such opsin molecules in ambient environment for vision restoration is not yet achieved. Further, use of external light source or device (e.g. LED array (50)) to activate such opsins can substantially damage the retinal cells in long-term usage.
  • external light source or device e.g. LED array (50)
  • opsins used for vision restoration
  • the disclosed invention includes methods of preparation of extremely- light sensitive ion-channels and ligands as well as different uses including vision restoration.
  • expression of a specific MCO in cell produces a long-lasting inward current in response to white light similar to characteristic photoreceptor-rod signal.
  • the disclosed invention provides method for the use of synthetic opsins for vision restoration and other applications, wherein the amino acid sequence of the synthetic opsin is modified to provide enhanced light sensitivity, kinetics and ion-selectivity.
  • the results presented in this invention show efficient and stable in-vivo expression of MCO-reporter protein in mice retina after intravitreal injection of Adeno-Associated Virus carrying MCO.
  • the results also demonstrated that the expression of MCO in retina of mouse model of retinal degeneration enables behavioral restoration of vision.
  • the number of error arms and time to reach platform in a radial-arm water maze significantly reduced after delivery of MCO to the mice having degenerated retina.
  • method of efficient restoration of vision in human includes use of MCO which when expressed in retinal cells produces a slower depolarizing phase after initial response to white light similar to characteristic photoreceptor-rod signal, and delivery of the opsin to retinal cells in-vivo by Adeno- Associated Virus (AAV) carrying promoter-MCO-gene in eye, and /or in combination with pronaseE or Alpha-Aminoadipic Acid (AAA) for enhancing delivery efficiency to targeted retinal layer crossing the thick inner limiting membrane in humans.
  • AAV Adeno- Associated Virus
  • AAAA Alpha-Aminoadipic Acid
  • a method is provided to administer an eMCO1 to a degenerated retina to restore light sensitivity in a patient.
  • administration of an eMCO1 to a patient suffering from a retinal degeneration or a retinal dystrophy, including through an intravitreal injection an Adeno-Associated Virus that includes a gene that codes for an eMCO1 results in the efficient expression of the eMCO1 gene and the production of the eMCO1 protein.
  • the invention comprises a method wherein the administration of an eMCO1 to a retinal degenerated eye of a patient results in a long-term improvement in a retinal function and a visual behavior compared to a patient who is not administered an eMCO1 into a retinal degenerated eye.
  • administration of an eMCO1 into the retina of a patient reverses the retinal degeneration (including as measured by Stargardt and LCA) and results in the behavioral restoration of vision.
  • the invention describes a method wherein the administration of eMCO1 to a retinal degenerated eye results in the arrest of a retinal degeneration or retinal dystrophy.
  • the invention describes a method wherein the administration of eMCO1 to a retinal degenerated eye results in the arrest or slowing down of a disorganization of an inner nuclear layer and its connectivity with ganglion cell layer.
  • administration of an eMCO1 to a patient to one eye results in the expression of the eMCO1 in the other eye of the patient.
  • administration of an eMCO1 to a patient to one eye results in the expression in noninjected eye and the arrest of a retinal degeneration or a retinal dystrophy and the maintenance of retinal thickness in the non-injected eye.
  • the present invention provides an approach to treat a retinal dystrophy or a retinal degeneration that is characterized by loss/mutation of outer retina including photoreceptors and/or retinal pigment epithelium.
  • administering results in expression of the eMCO1 in the other eye of the patient, which results in in long term improved retinal function and visual behavior in the non-injected eye of the patient as compared to a patient who did not receive an administration of an eMCO1.
  • an eMCO1 administered into one eye of a patient is only expressed in the other eye of the patient.
  • the patient following administration of an eMCO1 into a patient, the patient has improved visual function and behavior.
  • the administration of an eMCO1 results in improved visual function and behavior regardless of the retinal dystrophy or retinal degeneration.
  • an eMCO1 administration of an eMCO1 to a patient results in a restoration of vision and arresting of the symptoms associated with the progression of a retinal dystrophy or a retinal degeneration.
  • an enhanced multi-characteristic opsin eMCO1
  • eMCO1 is administered to a patient suffering from a neurodegeneration.
  • an eMCO1 is administered to a patient to modulate retinal cellular activities.
  • a method to administer an eMCO1 including an AAV containing a gene coding for an eMCO1 is through the use of a device that is capable of injecting the eMCO1 (protein alone or an AAV) through one or more of intraocularly, intravitreally or intraretinally.
  • administering treats macular degenerative diseases such as Stargardt macular degeneration and advanced macular degeneration, wherein degeneration or dysfunction of outer retinal cells including photoreceptor cells occur.
  • administering treats macular degeneration diseases wherein the central portion of the retina (macula) is targeted, enabling expression of the eMCO1 in higher order neurons of retina.
  • administering leads to an improvement in the vision function of patients suffering from macular degenerative diseases.
  • an eMCO1 treated subject experiences increase in the quality of vision through the use of a virtual reality (VR) headset.
  • VR virtual reality
  • the VR headset enhances the contrast and/or optical zooming of the visual field to enhance a patient’s improvement following treatment with the eMCO1 .
  • an eMCO1 treated macular degeneration subject demonstrates an increase in visual acuity.
  • the eMCO1 treated macular degeneration subject demonstrates further increase in visual acuity while using a VR headset.
  • the VR headset has a variable magnification of 1X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 11X, 12X, 13X, 14X, 15X, 16X, 17X, 18X zoom.
  • the VR headset has a 1 ,5X, 2.5X, 3.5X, 4.5X, 5.5X, 6.5X, 7.5X, 8.5X, 9.5X, 10.5X, 11.5X, 12.5X, 13.5X, 14.5X, 15.5X, 16.5X, 17.5X, 18.5X zoom.
  • the VR headset has an about 1X, about 2X, about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 11X, about 12X, about 13X, about 14X, about 15X, about 16X, about 17X, about 18X zoom.
  • the VR headset has an at least 1X, at least 1 .5X, at least 2X, at least 2.5X, at least 3X, at least 3.5X, at least 4X, at least 4.5X, at least 5X, at least 5.5X, at least 6X, at least 6.5X, at least 7X, at least 7.5X, at least 8X, at least 8.5X, at least 9X, at least 9.5X, at least 10X, at least 10.5X, at least 11X, at least 11.5X, at least 12X, at least 12.5X, at least 13X, at least 13.5X, at least 14X, at least 14.5X, at least 15X, at least 15.5X, at least 16X, at least 16.5X, at least 17X, at least 17.5X, at least 18X zoom.
  • the VR headset has no more than 1X, no more than 1 ,5X, no more than 2X, no more than 2.5X, no more than 3X, no more than 3.5X, no more than 4X, no more than 4.5X, no more than 5X, no more than 5.5X, no more than 6X, no more than 6.5X, no more than 7X, no more than 7.5X, no more than 8X, no more than 8.5X, no more than 9X, no more than 9.5X, no more than 10X, no more than 10.5X, no more than 11X, no more than 11 .5X, no more than 12X, no more than 12.5X, no more than 13X, no more than 13.5X, no more than 14X, no more than 14.5X, no more than 15X, no more than 15.5X, no more than 16X, no more than 16.5X, no more than 17X, no more than 17.5X, no more than 18X zoom.
  • the VR headset has a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% contrast.
  • the VR headset has an about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% contrast.
  • the VR headset has an at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% contrast.
  • the VR headset has a no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, no more than 75%, no more than 80%, no more than 85%, no more than 90% or no more than 95% contrast.
  • the VR headset has different contrast options: Normal View, Black on White, or White on Black.
  • the visual acuity letter score was measured using an ETDRS eye chart presented to the patient at a distance of 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 100 cm or more.
  • the visual acuity letter score was measured using an ETDRS eye chart presented to the patient at a distance of at least 10 cm, at least 20 cm, at least 30 cm, at least 40 cm, at least 50 cm, at least 60 cm, at least 70 cm, at least 80 cm, at least 90 cm, at least 100 cm or more.
  • the visual acuity letter score was measured using an ETDRS eye chart presented to the patient at a distance of no more than 10 cm, no more than 20 cm, no more than 30 cm, no more than 40 cm, no more than 50 cm, no more than 60 cm, no more than 70 cm, no more than 80 cm, no more than 90 cm, no more than 100 cm or more.
  • the visual acuity letter score was measured using an ETDRS eye chart presented to the patient at a distance of about 10 cm, about 20 cm, about 30 cm, about 40 cm, about 50 cm, about 60 cm, about 70 cm, about 80 cm, about 90 cm, about 100 cm or more.
  • the gain in the letter score after eMCO1 injection was enhanced by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 or more letters at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 weeks.
  • Example 1 -Fig. 1A illustrates domain architecture of Multi-Characteristics Opsins (MCOs) with reporter protein.
  • MCOs Multi-Characteristics Opsins
  • These MCOs were synthesized using A typical circular map with insertion of MCO gene cloned at the restriction sites is shown in Fig. 1 B.
  • the MCO genes were synthesized using DNA synthesizer and sequence was verified.
  • Gel electrophoresis was carried out on amplified MCO1 gene (digested by restriction enzymes BamH I and Sal I with restriction fragments) using 0.8% agarose.
  • Western blot was performed to confirm that the MCO is expressed in retinal cells. Retinas of mice were transfected using lipofectamine and expressed protein was extracted for western blot.
  • Western blot was developed using primary (anti-mCherry polyclonal) antibody and secondary (Goat anti-Rabbit IgG) antibody with 1-step NBT/BCIP substrate.
  • Example 2 -Fig. 2 shows Theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsins using web-based protocol (RaptorX).
  • the RaptorX uses a conditional neural fields (CNF), a variant of conditional random fields and multiple template treating procedure to develop the following predicted structure of MCO.
  • Fig. 2A shows the theoretical modeling of the three- dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, MCO 1.
  • Fig. 2B depicts the theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, MCO 2.
  • Fig. 1 shows the theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, MCO 1.
  • 2C shows the theoretical modeling of the three-dimensional arrangement of amino acid chains of Multi-Characteristics Opsin, eMCO1.
  • the expression of the gene and functioning of the MCO1 and eMCO1 was investigated.
  • the eMCO1 was found to fold/express in membrane better, and therefore, function effectively as compared to MCO1.
  • a special element between MCO1 and mCherry was placed, thus increasing the interaction between the MCO1 gene and mCherry, which makes mCherry play an active role in stabilizing the whole therapeutic molecule (eMCO1) in the membrane.
  • Table- 08 shows higher percentage of beta sheets and lower percentage of disordered structure (i.e.
  • mCherry in eMCO1 serves as an indicator for determining efficacy of gene delivery to targeted tissue(s), and to determine presence of the opsin at different time points.
  • re-injection of the opsin-gene for rephotosensitization of targeted cells can thus be carried out. For example, if visual ability is reduced or lost with time after initial improvement (by vMCO-1 injection), examination of mCherry expression in retina (by fundoscopy) will serve as a biomarker to determine if the vMCO-1 expression is lost (requiring reinjection). If the mCherry expression is intact (but the improvement in vision is lost/degraded), it will imply that the targeted retinal cells have lost connection with retinal ganglion cells, which carry visual information to visual cortex.
  • Example 3- For evaluating membrane trafficking of MCOs, the expression of MCOs in cell membrane (vs. cytoplasm) of transfected HEK293 cells was quantified using fluorescence intensity of reporter protein (mCherry).
  • HEK293 cells were transfected with MCO constructs using lipofectamine 3000 (Life Technologies). After transfection, the HEK293 cells were maintained in DMEM/F-12 with 10% fetal bovine serum, 0.2 mg/mL Gentamycin in Petri dishes. The cultures were maintained at 37°C in a 5% CO2 humidified atmosphere. Cells were incubated for 48 hours after transfection to allow MCO expression. Visualization of the reporter (mCherry) fluorescence was carried out under epifluorescence microscope.
  • Fig. 3A and Fig.6A The fluorescence images of HEK293 cells expressing eMCO1 (MCO1 -mCherry) and MCO2-mCherry are shown in Fig. 3A and Fig.6A respectively. Further, to quantify the relative expression of the eMCO1 in cell membrane and intracellular components, intensity profiles are plotted.
  • Fig. 3B shows the Intensity of eMCO1 reporter fluorescence along line across representative HEK293 cells transfected with mGluR6- MCO1- eMCO1 (mGluR6-mCherry). No significant intracellular (cytoplasmic) aggregation was observed implying effective trafficking of MCOs to the plasma membrane.
  • Example 4- To determine the light dependent inward photocurrent, the MCOs-expressing cells were exposed to pulses of light with intensity of 0.024 mW/mm 2 .
  • a single mode optical fiber coupled to a supercontinuum laser source (NKT Photonics) delivered the broadband light to the sample for optogenetic stimulation.
  • a power meter (818-SL, Newport) was used to quantify the light intensity at the sample plane.
  • the light pulse width was synchronized with the electrophysiology recording system, controlled by Axon Instruments Digidata system (Molecular Devices). Cells, transfected with MCOs were incubated with all- trans retinal (ATR, 1 pM) for 4 hours before conducting the patch clamp experiments.
  • the patch-clamp recording setup includes an inverted Nikon fluorescence microscope (TS 100) platform using an amplifier system (Axon Multiclamp 700B, Molecular Devices). Micropipettes were pulled using a two-stage pipette puller (Narshinghe) to attain resistance of 3 to 5 MQ when filled with a solution containing (in mM) 130 K-Gluoconate, 7 KCI, 2 NaCI, 1 MgCb, 0.4 EGTA, 10 HEPES, 2 ATP-Mg, 0.3 GTP- Tris and 20 sucrose. The micropipette-electrode was mounted on a micromanipulator.
  • the electrophysiological signals from the amplifier were digitized using Digidata 1440 (Molecular devices), interfaced with patch-clamp software (Clampex, Molecular Devices). For activation of MCO expressing cells, the light stimulation beam was delivered by the optical fiber. pCIamp 10 software was used for data analysis.
  • Fig.4A shows representative inward current in MCO1 -expressing cells in response to light (average intensity: 0.024 mW/mm 2 ) measured by Patch-clamp electrophysiology.
  • the inward photocurrent was found to be order of magnitude higher in eMCO1 sensitized cells than that in the ChR2 expressing cells.
  • Inward photocurrent (195 ⁇ 32 pA) in eMCO1 -sensitized cells at ambient light level (0.02 mW/mm 2 ) is above threshold for action potential (AP) unlike that in cells sensitized with ChR2 and White-Opsin(51). It may be noted that for a good fidelity of the light-evoked spiking of opsin-sensitized cells, faster response time is required.
  • the ON response time of ambient-light activatable MCO1 (Fig. 4A) is measured to be 2.94 ⁇ 0.70 ms, which is similar to that measured for other fast-opsins (52). However, the on response time depends on the intensity of activation light and is known to increase as the light intensity decreases (53). [0135] To obtain the activation spectrum of eMCO1 , the inward photocurrent was measured using stimulation light at different wavelengths (with bandwidth: 30 nm). In Fig. 4B, we show the normalized activation spectrum of eMCO1.
  • mCherry enhances the photoinduced current in the cells expressing eMCO1 by (i) better orientation-stabilization of eMCO1 across the membrane; and (ii) light emitted/re-emitted from the stabilizer-biomarker molecule enhance the activation of eMCO1.
  • Fig.5 shows Inward current profiles in HEK cells measured by Nanion Port-a-Patch automated Patch clamp electrophysiology.
  • Fig. 5A shows photocurrent measured at white light intensity of 0.02 mW/mm 2 in cell transfected with mGluR6-eMCO1 (mGluR6-MCO1 -mCherry).
  • Fig. 5 shows Inward current profiles in HEK cells measured by Nanion Port-a-Patch automated Patch clamp electrophysiology.
  • Fig. 5A shows photocurrent measured at white light intensity of 0.02 mW/mm 2 in cell transfected with mGluR6-eMCO1 (mGluR6-MCO1 -mCherry).
  • 5B depicts photocurrent measured at white light intensity of 0.02 mW/mm 2 in cell transfected with mGluR6-MCO1 .
  • the effect of presence of mCherry on enhanced MCO1 function is clearly demonstrated here.
  • MCO1 was found to have broad activation spectrum matching to the white ambient light.
  • FIG.6B The inward photocurrent in MCO2-expressing cells in response to light at the same average intensity (0.024 mW/mm 2 ) is shown in Fig.6B.
  • the peak photocurrent generated in eMCO1 -cells at light intensity of 0.024 mW/mm 2 was -160 pA as compared to -320 pA in MCO2 expressing cells. While the on- rate of induced photocurrent in eMCO1 and MCO2 expressing cells in response to light did not differ significantly, the off-response (decay of current in absence of light) of MCO2 was found to be significantly slower than eMCO1 (Fig. 6B vs. Fig. 4A).
  • Fig. 8 shows the patch-clamp recording of MCO1 transfected rd mouse retina.
  • Fig. 8A shows eMCO-1 expression in the cells of mice retina explant.
  • Fig. 8B shows Inward photocurrent induced by light pulse (100 ms) train.
  • the spectral and intensity sensitivity combined with the fast kinetics and small size (allowing packaging by AAV) of eMCO1 makes it uniquely suitable for photosensitizing higher-order retinal neurons in subjects with retinal degeneration to enable vision restoration in ambient light environment.
  • Example 5 - MCO1 and MCO2 plasmids were packaged in Adeno-associated virus (serotype 2) with mGluR6 promoter and mCherry reporter.
  • the synthesized plasmids were cloned into pAAV MCS vector via its BamH1 and Sall sites.
  • AAV physical titers were obtained by quantitative PCR using primers designed to selectively bind AAV inverted terminal repeats.
  • TCID50 assay was conducted according to ATCC protocol. Verification of purity of purified virus was confirmed by SDS/PAGE. Fig.
  • FIG. 7A illustrates fluorescence image of HEK293 cells expressing mCherry, 2 days after transfection with AAV2-mGluR6- MCO1 -mCherry. Robust expression was observed with no detectable change in morphology, confirm that transfected cells are healthy.
  • intravitreal injection of 1 l of AAV2- mGluR6-MCO1 -mCherry (vMCO1) was carried out for targeted expression in ON bipolar cells. Uniformity of MCO expression was confirmed by the 3D reconstruction from the confocal mCherry-expression in z- slices of the whole retinal cup oirdlO mice injected with vMCO1 intravitreally (Fig. 7B).
  • Example 6- The rd10 mice (retinal degeneration 10, spontaneous missense point mutation in Pde6b) have a later onset and progressive retinal degeneration, closer to the human retinal degeneration phenotype.
  • AAV2-mGluR6-MCO1 -mCherry (1 pl) solution (1.6 x 10 12 GC/ml) was injected by a sterilized needle of a Hamilton syringe inserted through the sclera into the vitreous cavity.
  • the AAV2-mGluR6-MCO1 -mCherry solution was injected to both the eyes.
  • the cornea was kept moist with a balanced salt solution during the entire surgical procedure.
  • In-vivo transfection of vMCO1 in rd10 mouse retina was carried out for four different final doses of vMCO1.
  • the mice in each group were euthanized and retina tissues harvested. Confocal fluorescence microscopy was carried out for analysis of eMCOI expression in retina.
  • the reporter fluorescence expression level (fluorescence intensity) of transfected retina was evaluated using confocal microscope.
  • the mice were sacrificed and retina was extracted and imaged by confocal microscopy.
  • the vMCO1 -transfected rd10 mice retina showed distinct expression of reporter (mCherry) on cell membrane in targeted cell layer.
  • FIG. 9D shows cross-sectional view of eMCOI expression in retina 16 weeks after intravitreal injection at dose of 1 .6 x 10 12 VG/ml. Furthermore, expression level was significant even after 4 months of injection.
  • Fig. 9E shows kinetics of MCO1 expression in rd10 mice retina at two different doses of vMCO1.
  • Fig. 9F shows the inter-animal variation of MCO1 -mCherry (eMCO1) expression (after background subtraction) in retina of rd10 mice 16 weeks after transfection of vMCO1 at dose of 1.6 x 10 12 VG/ml.
  • Fig. 10 shows visually guided improvement in rd10 mice behavior in radial water maze.
  • Fig. 10A shows Time-lapse images of visually guided rd10 mice behavior in radial water maze with white LED light before intravitreal vMCO1 injection.
  • Fig. 10B shows Behavior of rd10 mouse with LED light ON six weeks after vMCO1 injection.
  • Fig. 10D depicts Latency to find the platform by the vMCO1 treated rd10 mouse, with and without light, dropped at side arms-2 & 4 of the maze.
  • Fig. 10E depicts the latency to find the platform by the vMCO1 treated rd10 mouse, with and without light, dropped at edge arm-3 of the maze.
  • Fig. 10F shows the number of error arms traversed by the vMCO1 treated rd10 mouse dropped at center before finding the platform in presence and absence of light.
  • FIG. 11 shows longitudinal study of visually guided improvement in rd10 mice behavior in radial water maze. We collected data to determine visual acuity at baseline (previral transfection) and over time (every 4 wks for 4 months).
  • Fig. 11A depicts Schematic of the radial-arm water maze used to test improvement in visually guided behavior of vMCO1 injected rd1 O mice. 4 wks after injection, all mice significantly restored their visually guided behavior that lasted through the 16 wks trial. The number of errors made by the MCO-transfected rd10 mice before they reached the platform is significantly smaller ( ⁇ 1) than that of the mice without transfection (>2) (56).
  • Fig. 11 C shows the Time to reach platform by the rd10 mice from near arm of the maze (light intensity: 0.014 mW/mm 2 ) before vMCO1 injection and as a function of postinjection period.
  • N 5; Average ⁇ S.D. *P ⁇ 0.05.
  • the MCO-treated rd10 mice when randomly placed in five different arms of the radial water maze in a single sequence, they could find the platform (in 6 th arm) from all the other arms without a single error. Furthermore, the MCO treated rd10 mice performed better in visually guided tasks even at low light intensities (0.005-0.01 mW/mm 2 ), comparable to ambient light levels. To determine the light intensity-dependence of improvement of behavior for the vMCO1-treated mice, the intensity of the diverging LED light was varied from 0.0005 to 0.03 mW/mm 2 .
  • Fig. 12 shows the Light-intensity dependence of improvement in rd10 mice behavior in radial water maze. Comparison of time to reach platform from center of the maze between two different light intensities as a function of post-injection period. This is the first-time opsin- treated mice could perform significantly better at such low light levels.
  • FIG. 13 shows optomotor assessment of rd10 and MCO-sensitized rd10 mice.
  • Fig. 13A shows Quantitative comparison of number of head movement of rd10 mice before and 8 weeks after vMCO1 injection at speed of rotation of the vertical stripes (0.07 cpd) at 1 rpm. The light intensity at the center of the chamber is 0.001 mW/mm 2 .
  • Fig. 13B shows Quantitative comparison of number of head movement of rd10 mice before and 8 weeks after vMCO1 injection at speed of rotation of the vertical stripes at 2 rpm. The light intensity at the center of the chamber is 0.001 mW/mm 2 . Even at this low light intensity, the MCO-treated mice rotated its head in response to rotating stripes implying improved spatial visual acuity.
  • Example 10- Chronic exposure of opsin transfected retinal cells to light may raise concern about their viability. Therefore, to evaluate any detrimental effect of light exposure on retinal cell viability, wild type and MCO-injected rd10 mice were exposed to white light with intensity (i.e. 0.1 mW/mm 2 ) ⁇ 10 times higher than that of ambient light level ( ⁇ 0.01 mW/mm 2 ) for 4 weeks (8 hr/day). 4 weeks after light exposure, the MCO transfected rd10 as well as wild-type (control) mice were sacrificed, and the retina tissue was harvested for immuno-histochemical analysis. The retina was immunostained with apoptotic markers and imaged using confocal microscopy. Fig.
  • FIG. 14 shows viability of eMCO1 sensitized retinal cells after chronic light exposure.
  • Fig. 14A shows that Similar to the wild-type (non-blind) mice, vMCO1 treated rd10 mice avoid bright light by staying away and blocking light (via creating a heap out of bedding material, as shown in the arrow).
  • Fig. 14B shows Fluorescence image of retina stained with Caspase-3 (green) for vMCO1- treated rd10 mouse 4 weeks after 8-hr/day illumination of white light (intensity: 0.1 mW/mm 2 ).
  • Fig. 14A shows that Similar to the wild-type (non-blind) mice, vMCO1 treated rd10 mice avoid bright light by staying away and blocking light (via creating a heap out of bedding material, as shown in the arrow).
  • Fig. 14B shows Fluorescence image of retina stained with Caspase-3 (green) for vMCO1- treated rd10 mouse 4 weeks after 8
  • FIG. 14C shows Fluorescence image of retina stained with Caspase-3 (green) for wild-type mouse 4 weeks after 8- hr/day illumination of white light (intensity: 0.1 mW/mm 2 ). Quantitative comparison (Fig. 14D) shows that there is no significant cell death in either of the wild type or MCO-injected rd10 mice, indicating no compromise of cell viability under chronic light exposure. 0 % apoptotic cells in inner nuclear layer of vMCO1 treated rd10 mice. Furthermore, since light-sensitivity of MCO expressing cells significantly reduces the required light intensity for generating action potential, use of MCO will minimize light-induced chronic damage to the retinal cells.
  • Example 11- Optical sectioning/imaging using SDOCT was carried out to monitor any changes in ocular structure due to intravitreal injection of vMCO1 .
  • SDOCT images of cornea, lens, and retina 1wk after intravitreal vMCO injection in rd10 mice were compared to the images before injection.
  • Fig. 15 shows results of evaluation of structural integrity of retina after vMCO1 injection in rd10 mice.
  • Fig. 15A shows an OCT image of rd10 mice retina after vMCO1 injection.
  • Fig. 15B shows the Comparison of retinal thickness of 4 different rd10 mice before and 1 week after injection. No detectable alteration to cornea, lens or retina (e.g.
  • Example 12-Though gene therapy has been controversial for the last decade due to undesired side effects (59, 60), opsins (e.g. ChR2) are reported to be non-toxic, not generate immune response, and maintain stable cell membrane properties. Therefore, the health of the mice was monitored to confirm the safety of our approach.
  • opsins e.g. ChR2
  • intravitreal injection of two different doses (Group 1 : 1.66 x 10 1 °
  • Group 2 1 .66 x 10 11 GC/ml) of vMCO at 7 and 14 days.
  • blood ⁇ 0.2 ml is drawn from facial vein (using sterile animal lancet) 1 week before intravitreal injection.
  • IL-6 and IFN-y pro-inflammatory cytokines
  • IL-10 anti-inflammatory cytokines
  • Fig. 16 summarizes the results of the ELISA quantification of inflammatory cytokines showing that the intravitreal dose of vMCO is within safe limit.
  • Fig. 16A shows quantitative comparison of IL-6 (pro- inflammatory marker) in plasma between group-1 and group-2 before and after 7 and 14 days of vMCO1 injection.
  • Fig. 16B shows the quantitative comparison of IL-10 (anti-inflammatory marker) in plasma between the two groups.
  • Fig. 16C shows the quantitative comparison of IFN-Y (pro-inflammatory marker) in plasma between the two groups before and after 7 and 14 days of vMCO1 injection.
  • Fig. 17 shows biodistribution of AAV2 packaged enhanced Multi-Characteristics Opsin (vMCO1).
  • vMCO1 enhanced Multi-Characteristics Opsin
  • Example 14- To further evaluate the safety, specificity and efficacy of our opsins, immunohistochemistry of vMCO1 injected rd10 retina was conducted.
  • Fig. 18 shows immunohistochemistry of retinal sections ofvMCOI injected rd10 mice eye.
  • Fig. 18A shows that MCO-mCherry (red) is selectively targeted and expressed in inner nuclear layer (INL) of rd10 mice 8 wks after intravitreal injection of vMCOI .
  • INL inner nuclear layer
  • FIG. 18B shows PKCa stain (green) in rod bipolar cells expressing mCherry (red, intrinsic) in rd10 mice 8 wks after intravitreal injection of vMCO1 .
  • Fig. 18C shows mGluR6 stain (green) in ON bipolar cells expressing mCherry (red) in rd10 mice 8 wks after intravitreal injection of vMCO1 .
  • Fig. 18D shows mCherry (green-immunostained) expression in rd10 retina 8 wks following intravitreal delivery of vMCOI to rd10 mice.
  • FIG. 18E shows that GFAP (green) in rd10 mice 18 wks after intravitreal injection of vMCO1 as reported in photoreceptor degenerated retina.
  • Fig. 18F shows no CD45 (green) expression suggesting no immune cells in rd 10 mice 8 wks after intravitreal injection of vMCO1 .
  • compositions of methods of the invented enhanced MCO may be delivered and packaged in the plasmid or viral vectors that includes: (i) MCO Plasmid, (ii) rAAV-MCO, (iii) pAAV-MCO and (iii) Lenti Virus-MCO.
  • Invention delivery is improvised by use of optimized formulation of AAA together with this invention molecule-MCO (naked plasmid or virus) to transiently permeabilize inner limiting membrane of retina.
  • Table-02 Amino acid and DNA sequences of Multi-Characteristics Opsin-2 (MCO2). It contains mutation (S 142 L) and deletion of 7 amino acid residues (VNKGTGK) after 308 of MCO1 sequence (TABLE-01).
  • TGAGGACACGGTT (SEQ ID NO:6) [0157] Table-04: Amino acid and DNA sequences of Multi-Characteristics Opsin-2T (MCO2T). It contains additional trans-membrane sequence (TPARVWWISLYYAAFYVVMTGLFALCIYVLMQTI) after 308 amino acid residues of MCO2 (TABLE-02).
  • Table-05 DNA sequences of promoter (mGluR6) used upstream of MCO-sequences for targeting specific cells as an example.
  • Table-06 DNA sequences of reporter-stabilizer (mCherry) used downstream of MCO-sequences for confirming expression in specific cells as an example.
  • MCO1 sequence (Table-01) and biomarker-stabilizer sequence (Table-06) with a linking sequence.
  • Table-08 Comparison of stability of the MCO1 and eMCO1 based on secondary structure and folding using theoretical modeling by RaptorX.
  • Example 15- Structure of eMCO1 and activation of its different domains by light is shown in Fig. 19.
  • Blue light activates the trans-membrane (TM) domain (ion-channel) of eMCO1 allowing for the flow of cations.
  • Green & Red light activates the non-TM, middle domain (non-ion channel) leading to its conformational change that result in the conformational change of the TM domain and thus, facilitate flow of cations via the TM-domain.
  • the C-terminus domain Upon green light absorption, the C-terminus domain (enhancer) emits red light that enhances overall eMCO1 efficacy.
  • eMCO1 allows for the activation of the eMCO1 even at a low level of light with a wavelength ranging from the blue to red spectrum in a fast and efficient manner.
  • Patch clamp experiment (voltage clamp) on eMCO1 transfected HEK cells (transfected by JetPrime) was conducted under different stimulation wavelengths.
  • Fig. 20A shows an inward current profile measured at a blue (450 nm) light intensity of 0.06 mW/mm 2 in HEK cells transfected with eMCO1 .
  • Fig. 20B shows an inward current profile measured at a green (520 nm) light intensity of 0.06 mW/mm 2 in HEK cell transfected with eMCO1 .
  • Fig. 20A shows an inward current profile measured at a blue (450 nm) light intensity of 0.06 mW/mm 2 in HEK cells transfected with eMCO1 .
  • Fig. 20B shows an inward current profile measured at
  • FIG. 20C shows an inward current profile measured at a red (630 nm) light intensity of 0.06 mW/mm 2 in HEK cell transfected with eMCO1 .
  • BARTA known calcium chelator
  • Fig. 20D shows a reduced inward current profile measured at blue (450 nm) light intensity of 0.06 mW/mm 2 in HEK cells transfected with eMCO1 , in presence of Ca 2+ chelator BAPTA.
  • eMCO has fast ON and OFF-kinetics.
  • Fig. 20F shows a quantitative comparison of photocurrent ON-time (time to the -ve peak from baseline) generated by the blue and green light. The ON-time of photocurrent measured in cell was ⁇ 4 times higher at 520 nm light stimulation as compared to that at 450 nm.
  • Example 16- To determine if eMCO1 sensitization of retinal bipolar cells could preserve their organization, a structural comparison of the retinal thickness before, and after intravitreal injection in rd mice was conducted using different doses of an AAV2 carried eMCO1 protein (vMCO1 vehicle), including AAV-vehicle (without eMCO1) using OCT based measurement.
  • vMCO1 vehicle an AAV2 carried eMCO1 protein
  • group AA mice eyes were intravitreally injected with 1 pl of 1 .0 x 10 12 VG/ml AAV2-eMCO
  • group BB mice eyes were injected by 1 pl of 1 .0 x 10 12 VG/ml AAV2 (no transgene, -ve control).
  • FIG. 21A shows longitudinally measured retinal thickness of rd mice measured by SDOCT before and after different doses of AAV2-eMCO or AAV2-vehicle injection. The measurement of the retinal thickness from priorto injection, one day after injection and one and four months after injection for each group was carried out. A scattered plot showing the comparison of retinal thickness prior to injection, and 4 months after injection in the different groups is shown in Fig. 21 B.
  • Fig. 21 C shows the mean difference of retinal-thickness between baseline and intravitreally-injected mice shown as a Gardner-Altman estimation plot.
  • Example 17- To determine if the synaptic spread of bipolar cells and/or connectivity between bipolar cells and retinal ganglion cells is enhanced due to eMCO1 sensitization of bipolar cells, immunostaining of the retinal section of rd10 mice after vMCO injection (intravitreally injected of 1 pl of vMCO1) was conducted. The expression of reporter mCherry in bipolar cells (INL) 16 weeks after AAV2-eMCO1 injection is shown in Fig. 22B. Quantification of eMCOI (reporter mCherry) expression in bipolar cells of three rd10 mice is shown in Fig. 22C. Fig.
  • FIG. 22D shows immunostained cross-sections of the retina showing Bipolar cell terminals (green: PKCa) co-stained with CtBP (a synaptic ribbon marker, red) in close contact with retinal ganglion cells (RGCs) in the eMCO injected rd 10 mouse retina.
  • Fig. 22E shows immunostained cross-sections of the retina showing Bipolar cell terminals (green: PKCa) co-stained with CtBP (a synaptic ribbon marker, red) in close contact with retinal ganglion cells (RGCs) in the untreated rd1 O mouse retina.
  • Example 18- The gene agnostic therapeutic benefit of an eMCO1 was established by demonstrating improvement of the vision in retinal degenerated mice models for Retinitis pigmentosa (rd 1 , rd10), Stargardt (ABCA4-/-) and Lieber Congenital Amaurosis (LCA). Intravitreal injection of vMCO1 (1 pL of 3.5E12 vg/mL) lead to behavioral improvement of vision assayed by a radial water maze. Fig.
  • FIG. 23A shows the time to reach the platform by the Stargardt (ABCA4-/-) mice from side-arms of the radial-arm water maze (light intensity: 0.004 mW/mm 2 ) before and after AAV2 (no transgene, -ve control) injection.
  • eMCO transgene, -ve control
  • Fig. 23B shows the time to reach the platform by the Stargardt (ABCA4-/-) mice from sidearms of the radial-arm water maze (light intensity: 0.004 mW/mm 2 ) before and after vMCO1 injection.
  • Example 19- To determine if eMCO1 sensitization of bipolar cells could lead to electrophysiological recovery in addition to arresting degeneration and imparting light-sensitivity to the retina, the Stargardt mice were injected with AAV2-eMCO. Arresting of further retinal degeneration in Stargardt mice model is demonstrated by longitudinal monitoring of retina thickness in Stargardt (ABCA4-/-) mice after vMCO1 injection using SDOCT. For comparison, a separate mouse group that was not treated was followed up longitudinally. Comparison of retinal thickness before, Week 4, 12 and 16 after vMCO1 injection found that the retinal thickness does not decrease significantly overtime (further degeneration is arrested).
  • Example 20- Fig. 25 illustrates a method of vison restoration via intraocular AAV2-eMCO administration.
  • the retina 100
  • the retinal ganglion cells (1 10) and bipolar cells (120) survive.
  • the photoreceptors (130) and /or retinal pigment epithelium (140) are mutated or lost (150).
  • AAV-carried eMCO vMCO1 (170) is delivered intraocularly.
  • the vMCO1 propagates via the Optic nerve (180), Optic tract (190) and Optic Chiasm (200) to get transferred to the contralateral eye (210).
  • AAV-eMCO transduces retinal cells (220). With a natural scene in the visual field or projected patterns to retina (230) activates bipolar cells (240), which in turn activates the retinal ganglion cells (250). The electro-chemical signal (260) is transmitted via the optic nerve to the brain (270) for visual processing of light-activated signal received from eMCO1 -sensitized retina.
  • Example 21- This example demonstrates that an AAV that includes a gene that codes for an eMCO1 that is injected intravitreal is capable of contralateral transfer.
  • An eMCO1 gene expressed in the retina subsequent to an intravitreal injection in a mouse was observed in the injected as well as the contralateral eye (Fig. 26 A: Injected eye and 26B: Contralateral eye).
  • the transfer of vMCO1 from the injected eye to the non -injected eye was also observed in rd10 mice wherein the expression of eMCO1 (reporter mCherry) expression was observed in both the injected and the non-injected eye after 6 month of injection.
  • Fig. 26C injected eye
  • Fig. 26D Contralateral eye
  • qPCR was performed to quantify the presence ofvMCOI vector copies.
  • Fig. 26E shows the measured vector copy numbers in left/ right optic nerves, optic chiasm and left/right LGN in treated dogs that were intravitreally injected with vMCO1 in the right eye (only). The data indicates contralateral transport of vMCOI via optic nerve and optic chiasm.
  • Example 22- To determine if uniocular injection of vMCO1 will lead to contralateral transfer (as seen in animals) in humans, subjects with advanced retinal pigmentosa were injected with vMCO1 through a single intravitreal injection in the eye that had the greater impaired vision (the eye with the greater reduction in sight). For the evaluation of eMCO1 expression in the subjects after vMCO1 injection, visualization of the reporter (mCherry) fluorescence was carried out by Fundus Autofluorescence imaging through a red filter using a Topcon Triton Plus imaging system. As shown in Fig. 27A, the reporter fluorescence increased in the injected eye.
  • Fig. 27A, Fig. 27B To examine if eMCO1 expression in a retina leads to arrest of further retinal degeneration in retinitis pigmentosa patients, longitudinal monitoring of retinal thickness in injected and contralateral eyes by OCT imaging was conducted before and after injection of vMCOI was carried out. Longitudinally monitoring of retinal thickness in injected and contralateral eyes was carried out by OCT imaging and quantified (Fig. 27C). Measured average retinal thickness did not change after vMCO1 injection.
  • the mean visual acuity value of injected eyes in the high-dose group showed >0.68 logMAR improvement as compared to 0.1 logMAR improvement in the low-dose group 16 weeks after injection.
  • Fig. 28 shows longitudinally monitored Visual Acuity (LogMAR) in vMCO1 injected (3.5E11 vg) and contralateral eyes of patients with severe retinal degeneration.
  • LogMAR Visual Acuity
  • AAV2-eMCO intravitreal injection of AAV2-eMCO in humans also led to improvement of vision in contralateral eyes.
  • the mean improvement in the logMAR acuity value in high-dose contralateral eyes was found to be 0.34 logMAR at 16 weeks as compared to 0.68 logMAR in the injected eyes (Fig. 28).
  • Example 24- To determine if opsin sensitization and optogenetic stimulation of retinal ganglion cells would enable axonal regeneration leading to vision restoration, visually guided behavior in mice during baseline and after optic nerve crush was examined in two different groups: (i) without optogenetic stimulation and (ii) with optogenetic stimulation.
  • Fig. 29A shows the Schematic of Visually-guided Y-mobility assay to evaluate vision in mice.
  • Fig. 29B shows the Number of events (finding Light ON vs. OFF) by mice for different conditions. 6 trials per conditions were conducted. In Baseline (No optic nerve crush) measurements, the number of mice finding the light ON (+Light) panel of the Y-maze is significantly higher than the event of finding light OFF (-Light) panel.
  • Example 25- To determine if administration of eMCO1 resulted in some level of vision restoration in patients suffering from m Stargardt macular degeneration, these patients were injected with 1.2E11gc/eye through a single intravitreal injection.
  • Fig 30A shows longitudinal improvement for visual acuity letter scores measured in an eMCO1 treated patients using an ETDRS eye chart. As shown in Fig 30A, at week 24 following eMCO1 treatment of the patient, an about an 11 letter gain was observed as compared to the baseline.
  • Fig 30B shows longitudinal improvement in visual acuity of the eMCO1 treated subject with a VR headset (with 100% contrast and 3X zoom).
  • the visual acuity letter score was measured using an ETDRS eye chart presented to the patient at a distance of 50 cm.
  • the gain in the letter score after eMCO1 injection was further enhanced to about 30 letter gain at 24 weeks (Fig. 30B).
  • the terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise.
  • the term “substantially” is defined as largely but not necessarily wholly what is specified (and includes what is specified; e.g., substantially 90 degrees includes 90 degrees and substantially parallel includes parallel), as understood by a person of ordinary skill in the art.
  • the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1 , 1 , 5, and 10 percent.
  • a molecule or method that is configured in a certain way is configured in at least that way, but it can also be configured in other ways than those specifically described.
  • any embodiment of any of the apparatuses, systems, and methods can consist of or consist essentially of - rather than comprise/include/contain/have - any of the described steps, elements, and/or features.
  • the term “consisting of’ or “consisting essentially of’ can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.

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Abstract

Selon un aspect, la présente invention concerne de manière générale des compositions et des procédés de modulation d'activités cellulaires par opsines synthétiques. En outre, l'invention concerne un procédé d'utilisation d'opsines synthétiques pour la restauration de la vision et d'autres applications, la séquence d'acides aminés de l'opsine synthétique étant modifiée pour fournir une sensibilité à la lumière, une cinétique et une sélectivité ionique améliorées.
PCT/US2024/026082 2023-04-24 2024-04-24 Modulation optogénétique par opsines à caractéristiques multiples pour la restauration de la vision et d'autres applications associées Pending WO2024226667A2 (fr)

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US11180537B2 (en) * 2016-11-06 2021-11-23 Nancoscope Technologies LLC Optogenetic modulation by Multi-Characteristic Opsins for vision restoration and other applications
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