Optogenetic regulation by multi-character opsins for vision restoration and other applications.

Abstract

In one aspect, the present invention relates to compositions and methods for regulating cellular activity using synthetic opsins. Furthermore, the present invention provides methods for using synthetic opsins in vision restoration and other applications, wherein the amino acid sequence of the synthetic opsin is modified to improve its photosensitivity, kinetics, and ion selectivity.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is a continuation of U.S. Patent Application Publication No. 20220162275, filed on 22 November 2021, which claims the interests of U.S. Patent Application No. 16 / 347,375, filed on 3 May 2019, which claims the interests of PCT Application PCT / US2017 / 059922, filed on 3 November 2017, which further claims the interests of U.S. Provisional Application 62 / 418,196, filed on 11 November 2016, the entire disclosures of all these applications are incorporated herein by reference.

[0002] (Technical field) The present invention relates, in general, to compositions and methods for regulating cell activity with synthetic opsins. The present invention provides enhanced photosensitivity to neurons for vision restoration and other therapeutic applications. [Background technology]

[0003] In retinal degenerative diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), photoreceptors (e.g., rods and cones) involved in the conversion of light into electrochemical signals degenerate. This prevents the generation of light-induced signals in the retina, disrupting the sequential visual sensory events within the visual system. The loss of photoreceptor cells and / or loss of function of photoreceptor cells is a major cause of reduced photosensitivity and blindness. [Overview of the Initiative]

[0004] In one aspect of the present invention, the disclosure provides a plurality of photosensitive ion channel and ligand molecules, methods for preparing them, and various uses including vision restoration. In another aspect, the present invention comprises isolated nucleic acid sequences encoding photosensitive ion channels, ligands, and constructs, including plasmids and other nucleic acid vectors containing such nucleic acid sequences.

[0005] In one embodiment, the disclosure provides synthetically derived photosensitive ion channels and ligands (multi-characteristic opsins). These multi-characteristic opsins or MCOs (i) are highly photosensitive at multiple visible wavelengths and (ii) can be packaged into genomic vectors such as plasmids that can be packaged into viruses. The viruses may include adeno-associated viruses or lentiviruses.

[0006] In addition, the disclosure provides, in some embodiments, intracellular MCO expression in vitro or in vivo, and methods for regulating cellular activity with these synthetic opsins. In one embodiment, multi-characteristic opsins are highly sensitive to visible light and ambient light activation. In some embodiments, intracellular expression of specific MCOs generates long-lasting inward currents in response to white light, similar to the signaling of unmodified photoreceptor rods.

[0007] In another embodiment, the disclosure provides a synthetic ambient light-activatable, rapidly enhancing, multi-characteristic opsin (eMCO1) having a stabilization biomarker that plays an active role in stabilizing eMCO1 expression on the cell membrane. In another embodiment, the stabilized eMCO1 has a structure with a high proportion of β-sheets and a low proportion of disordered structures (i.e., it is less prone to cleavage). In a further embodiment, the stabilized eMCO1 enhances photo-inducible currents in cells expressing eMCO1.

[0008] In another aspect, the disclosure provides a synthetic ambient light-activatable, rapidly enhancing, multi-characteristic opsin (eMCO1) that includes a stabilized biomarker that can be used to confirm MCO gene expression in target cells.

[0009] According to another aspect of the present invention, light emitted from a stabilizing biomarker present in an enhanced multi-characteristic opsin enhances the photo-inducible current in cells expressing eMCO1 by light emitted / re-emitted from the stabilizing biomarker molecule.

[0010] According to another aspect of the present invention, the disclosed invention provides a method of using synthetic opsin for vision restoration and other applications. In one aspect of the present invention, the amino acid sequence of the synthetic opsin is modified to provide enhanced light sensitivity, kinetics, and ion selectivity.

[0011] In one aspect of the present invention, a method of delivering MCO to a degenerated retina to restore light sensitivity is provided. In another aspect, efficient and stable in vivo expression of MCO reporter protein in the retina occurs after intravitreal injection of adeno-associated virus carrying MCO. In a further aspect, MCO expression in the retina of an individual with retinal degeneration can result in a behavioral restoration of vision. In one aspect of the present invention, improvement of visually guided behavior occurs at light intensity levels much lower than those required for channelrhodopsin-2 opsin.

[0012] According to yet another aspect, the present disclosure provides an efficient method of restoring vision in humans. This method includes the use of MCO expressed within retinal cells. In another aspect, the MCO produces a slower depolarization phase similar to photoreceptor rod signals after an initial response to white light. In a further aspect, the opsin can be delivered in vivo to retinal cells by administering an adeno-associated virus (AAV) or lentivirus having a nucleic acid vector containing a promoter-MCO gene into the human eye. It may also contain pronase E and / or α-aminoadipic acid (AAA). In one aspect, the AAV and / or lentivirus can be modified or enhanced to improve delivery efficiency to the target retinal layer across the thick inner limiting membrane in humans.

[0013] In one aspect of the present invention, virus-mediated MCO is administered to an individual suffering from such diseases, including retinal degeneration or dystrophy, for the treatment of various retinal degenerations.

[0014] In another aspect, the present disclosure provides the use of an opsin that produces a slower depolarization phase similar to photoreceptor rod signals after an initial response to white light. In one aspect, this results in restoration of vision in blind individuals.

[0015] In one aspect of the invention, the nucleic acid molecule encodes any of the polypeptides described herein. In another aspect, the nucleic acid molecule is formulated to include a pharmaceutically acceptable carrier.

[0016] In another aspect, a method is provided where a cell is contacted or transformed with an isolated nucleic acid molecule encoding an isolated polypeptide molecule of the invention. This includes MCO, MCO1, and eMCO1. In one aspect, the cell is a rod bipolar cell, an ON-type retinal ganglion cell, or an ON-type bipolar cell.

[0017] In another aspect, methods of modulating cell and tissue function using opsin, as well as methods for use in the diagnosis and treatment of retinal diseases, are provided.

[0018] In one embodiment, the present invention discloses a recombinant ambient light-activatable enhanced high-speed pluripotent opsin (eMCO1) chimeric protein comprising an MCO1 protein mutated to regulate at least one of the following: ion selectivity, photosensitivity, or kinetics of the MCO1 protein. In one embodiment, the MCO1 protein has SEQ ID NOs: 1, 3, 5, 7, or 11. In one embodiment, one or more of the following single mutations or combinations of mutations regulate ion selectivity, photosensitivity, or kinetics, the mutation being an S-to-C substitution at the amino acid residue corresponding to amino acid 132 of the MCO1 sequence, an E-to-A substitution at the amino acid residue corresponding to amino acid 123 of the MCO1 sequence, a D-to-A substitution at the amino acid residue corresponding to amino acid 253 of the MCO1 sequence, an R-to-A substitution at the amino acid residue corresponding to amino acid 120 of the MCO1 sequence, a Q-to-A substitution at the amino acid residue corresponding to amino acid 56 of the MCO1 sequence, a K-to-A substitution at the amino acid residue corresponding to amino acid 93 of the MCO1 sequence, and an A-to-A substitution at the amino acid residue corresponding to amino acid 90 of the MCO1 sequence. At least one of the following is selected: substitution of E to A at an acid residue, substitution of E to Q at the amino acid residue corresponding to amino acid 90 in the MCO1 sequence, substitution of E to A at the amino acid residue corresponding to amino acid 97 in the MCO1 sequence, substitution of E to A at the amino acid residue corresponding to amino acid 101 in the MCO1 sequence, substitution of N to D at the amino acid residue corresponding to amino acid 258 in the MCO1 sequence, substitution of E to T at the amino acid residue corresponding to amino acid 83 in the MCO1 sequence, substitution of E to T at the amino acid residue corresponding to amino acid 123 in the MCO1 sequence, or substitution of S to D at the amino acid residue corresponding to amino acid 63 in the MCO1 chimeric protein sequence.

[0019] In another embodiment, the protein is a recombinant ambient light-activatable slow pluripotency opsin (MCO2) chimeric protein, wherein seven amino acid residues (VNKGTGK) from 309 to 315 are deleted in the molecule described in claim 1 to improve gene expression on the membrane, the S132L mutation is implemented in the transmembrane domain 2 of SEQ ID NO: 1 to improve binding affinity to the retina and photosensitivity, the opsin is encoded by 658 amino acids, and the MCO2-sensitized cells contain a recombinant ambient light-activatable slow pluripotency opsin (MCO2) protein that generates a slowly decaying inward current after an initial rapid current response to a pulse of white light. In one embodiment, a single mutation or combination of mutations is selected from E473A, D603A, and R469A of SEQ ID NO: 1 to further modulate at least one of the molecule's ion selectivity, photosensitivity, or kinetics. In another embodiment, the transmembrane sequence (TPARWVWISLYYAAFYVVMTGLFALCIYVLMQTI) is inserted after amino acid residue 315 in MCO1 (SEQ ID NO: 1 or 2) or amino acid residue 308 in MCO2 (SEQ ID NO: 3 or 4).

[0020] In another embodiment, recombinant peripheral photoactivatable fast-enhancing pluripotent opsin (eMCO1) comprises an MCO1 sequence (SEQ ID NO: 1) and a stabilizing biomarker sequence. In one embodiment, recombinant eMCO1 has a stabilizing biomarker which is 900 amino acids of SEQ ID NO: 11, the stabilizing biomarker which is linked downstream of the ligand non-quadrupermembrane domain by a ligation sequence, light emitted from the stabilizing biomarker which stabilizes eMCO1 expression with a higher proportion of β-sheets and a lower proportion of disordered structures in the membrane and is less prone to cleavage than unmodified MCO1, the stabilizing biomarker molecule which enhances photo-inducible current in cells expressing eMCO1 by better orientation stabilization of eMCO1 across the membrane, the stabilizing biomarker molecule which enhances photo-inducible current in cells expressing eMCO1 by light emitted / re-emitted from the stabilizing biomarker molecule, and a promoter which targets specific cells. To that end, the recombinant cMOC1 further includes at least one of the following: it is used upstream of eMCO1; the promoter-eMCO1 gene is packaged in a viral vector; cells can be transfected with the promoter-eMCO1 gene using chemical, viral or physical transfection; testing of eMCO1 containing stabilized biomarker expression in the retina (by fundus examination) is an indicator for determining the effectiveness of gene delivery to target tissue; light emitted / re-emitted by the stabilized biomarker is monitored and used to discriminate the presence of eMCO1 expression; or loss of expression requires re-delivery of the promoter-eMCO1 gene to restore photosensitivity to target cells / make target cells functional. In another embodiment, recombinant cMOC1 further includes a reporter gene located downstream of the MCO1 gene for detecting cell expression / activation; the promoter-MCO1-reporter gene is packaged in a viral vector; and cells can be transfected with the promoter-MCO1-reporter gene using chemical, viral or physical methods. In another embodiment, MCO-sensitized cells are highly sensitive to light and low intensities (approximately 0.02 mW / mm²). 2Opsins can be activated by ambient light. In another embodiment, MCO-sensitized retinal neurons (e.g., retinal ganglion cells, bipolar cells) produce a slower depolarization phase after an initial response to white light similar to that of wild-type photoreceptor rod signals. In another embodiment, opsins are sensitive to any wavelength of light in the visible and near-infrared ranges. In yet another embodiment, opsins are activated by single photons, including direct and indirect (e.g., fluorescence, phosphorescence, up / down conversion) illumination light in the visible and near-infrared ranges.

[0021] In another embodiment, the present invention discloses a method for restoring lost vision and the use of MCO1, MCO2, or eMCO1, or mutants thereof, for the purpose of restoring lost vision. In one embodiment, the loss of vision is due to some degenerative retinal disease, and the delivery of recombinant MCO genes to target cells is carried out by intravitreal / subretinal injection of a virus carrying the promoter-MCO gene in the eye, combined with pronase E and / or α-aminoadipic acid (AAA) or both, to enhance delivery efficiency; or the delivery of MCO genes is carried out by either a chemical or physical transduction method or a combination thereof, following intravitreal / subretinal injection of a promoter-MCO gene plasmid in the eye; or the delivery of MCO genes in the eye is not due to unwanted expression in non-target cells and organs, or any adverse reaction or cytotoxicity in the treated eye; or a visually induced significant behavioral improvement is observed after delivery of MCO genes; or reinjection and transfection of MCO genes are carried out in cases of MCO gene deficiency.

[0022] In another aspect, the present invention discloses a method for preventing or delaying vision loss and the use of MCO1, MCO2, or eMCO1 therefor, wherein the MCO gene is delivered to retinal cells during progressive photoreceptor loss through transduction including the use of a viral vector comprising AAV or lentivirus, and photostimulation of MCO-sensitized retinal cells is carried out to prevent or delay photoreceptor loss, and the photostimulation dose is optimized for maximum effect.

[0023] In another aspect, the present invention discloses a method for restoring vision by regenerating damaged RGC axons and the use of MCO1, MCO2, or eMCO1 therefor, wherein the MCO gene is delivered to retinal ganglion cells during or after axonal degeneration by transduction including the use of a viral vector including AAV or lentivirus, and photostimulation of MCO-sensitized RGCs is performed to slow the rate of degeneration and / or regenerate axons, and the photostimulation dose is optimized to minimize degeneration and / or maximize axonal regeneration.

[0024] In another aspect, the present invention discloses a method for stimulating different types of excitable cells, including neurons and cardiac cells, and the use of MCO1, MCO2, or eMCO1 therefor, the use of which includes delivery of the MCO gene by any chemical, viral, or physical transduction method, the activation of MCO being achieved by light illumination, and the effect being measured electrophysiologically.

[0025] In another aspect, the present invention discloses a method for treating a disease and the use of MCO1, MCO2, or eMCO1 therefor, the use of which comprises the delivery of the MCO gene to different organs by any chemical, viral, or physical transduction method, the activation of MCO being achieved by light illumination, and the effect being measured by electrophysiological or other functional and behavioral analysis.

[0026] In another aspect, the present invention discloses a polypeptide comprising a sequence having at least 75%, 85%, 95%, or 100% identity with SEQ ID NOs: 1, 3, 5, 7, or 11, wherein the polypeptide exhibits the photosensitive properties of at least one of the proteins SEQ ID NOs: 1, 3, 5, 7, or 11.

[0027] In another embodiment, the present invention discloses a recombinant nucleic acid encoding a polypeptide having at least 75%, 85%, 95%, or 100% identity to SEQ ID NOs: 1, 3, 5, 7, or 11, wherein the polypeptide exhibits photosensitive properties of at least one of the proteins among SEQ ID NOs: 1, 3, 5, 7, or 11. In one embodiment, the nucleic acid has at least one of 75%, 85%, 95%, or 100% identity to SEQ ID NOs: 2, 4, 6, or 8. In another embodiment, the present invention discloses a vector comprising a nucleic acid having 75%, 85%, 95%, or 100% identity to at least one of the proteins among SEQ ID NOs: 1, 3, 5, 7, or 11. In one embodiment, the vector is selected from adenovirus, adeno-associated virus, or lentiviral vectors.

[0028] In another aspect, the present invention discloses a method for treating blindness, comprising administering to a patient in need of treatment for blindness a vector containing a nucleic acid having 75%, 85%, 95%, or 100% identity with at least one of SEQ ID NOs: 1, 3, 5, 7, or 11.

[0029] In one aspect of the present invention, a photosensitive ion channel molecule (expressed transmembranely) capable of capturing blue light, a photosensitive ligand for capturing light from green-red wavelengths, and, in one embodiment, a luminescence enhancing factor located at the C-terminus are used. In a further aspect of the present invention, eMCO1 provides rapid photo-induced activity in target cells with a low light threshold and a broad spectral range. In another aspect of the present invention, blue light activates the transmembrane (TM) domain of eMCO1, and eMCO1 functions as an ion channel. In a further aspect, red light absorbed by a non-TM intermediate domain (non-ion channel) induces a structural change in the TM domain of eMCO1, resulting in ion flow through the TM domain of eMCO1. In one aspect of the present invention, after absorption of green light by eMCO1, the C-terminal domain (RFP) of eMCO1 emits red light, thereby enhancing the effectiveness of eMCO1.

[0030] In one aspect of the present invention, an enhanced multi-characteristic opsin (eMCO1) is introduced into cells in vitro or in vivo and expressed by the introduced cells. In a further aspect, the introduced eMCO1 modulates cell activity after exposure to different wavelengths of visible light.

[0031] In one aspect of the present invention, a method is provided for restoring photosensitivity by delivering eMCO1 to a degenerated retina (e.g., via transduction). In a further aspect of the present invention, eMCO1 introduced into the retina, and in a further aspect, one or more cells constituting the retina, are efficiently and stably expressed in vivo. In another aspect, the eMCO1-reporter protein is administered to a patient by intravitreal injection of an adeno-associated virus (AAV) containing the gene encoding eMCO1 into the retina. In one aspect of the present invention, behavioral recovery of vision is achieved by administering eMCO1, including an AAV containing the gene encoding eMCO1, to the patient's retina.

[0032] In one aspect of the present invention, intraretinal administration of eMCO, including an AAV containing the gene encoding eMCO1, results in partial or complete recovery of the patient's vision. In one aspect of the present invention, intraretinal administration of eMCO1, including an AAV containing the gene encoding eMCO1, suppresses the progression of retinal degeneration and structural disturbance in the patient's retina. In one aspect of the present invention, intraretinal administration of eMCO1, including an AAV containing the gene encoding eMCO1, suppresses degeneration and structural disturbance of the inner retinal layer, as well as degeneration of the connections with the retinal ganglion cell layer.

[0033] In another embodiment, a method is provided for treating a patient suffering from retinal degenerative disease in one or both eyes by intravitreal injection of eMCO1 (including administration of AAV containing the gene encoding eMCO1) into one eye.

[0034] In one aspect of the present invention, administration of eMCO1 may be used to treat retinal dystrophy or retinal degenerative disease. In a further aspect, retinal dystrophy or retinal degenerative disease is characterized by the loss of part or all of the outer layer of the retina, including photoreceptor cells and / or retinal pigment epithelium.

[0035] In another aspect of the present invention, administration of eMCO1 may be used to treat macular degenerative diseases in which degeneration or dysfunction of the outer retinal cells, including photoreceptor cells, occurs, such as Stalgardt macular degeneration and progressive macular degeneration.

[0036] In another aspect of the present invention, administration of eMCO1 may be used to treat macular degenerative diseases with geographic atrophy or discoid scarring, targeting the central part of the retina (macula).

[0037] In a further aspect of the present invention, administration of eMCO1 may lead to an improvement in the visual function of subjects suffering from macular degeneration disease.

[0038] In another aspect of the present invention, a patient treated with eMCO1 may have their visual quality further improved by enhancing the contrast and / or optical zoom of their field of view using a virtual reality (VR) headset.

[0039] In a further aspect of the present invention, it has been shown that visual acuity improved in subjects with macular degeneration treated with eMCO1.

[0040] In another aspect of the present invention, it has been shown that subjects with macular degeneration treated with eMCO1 experienced further improvement in visual acuity while using a VR headset.

[0041] The following drawings are illustrative and not limiting. For the sake of brevity and clarity, not all features of a given structure are always shown in every drawing in which that structure appears.

[0042] Tables 1 to 4 show the amino acid sequences of the multi-characteristic opsins (MCOs): MCO1, MCO2, MCO1T, and MCO2T. MCO2 contains a mutation in MCO1 after 308 amino acids (S132L) and the deletion of seven amino acid residues (VNKGTGK (SEQ ID NO: 13)). MCO1T and MCO2T contain additional transmembrane sequences (TPARWVWISLYYAAFYVVMTGLFALCIYVLMQTI (SEQ ID NO: 14)) after 315 and 308 amino acid residues, respectively.

[0043] Table 05 shows, as an example, the DNA sequence of a promoter (mGluR6) used upstream of an MCO sequence to target specific cells, and Table 06 shows, as an example, the DNA sequence of a reporter (mCherry) used downstream of an MCO sequence to confirm expression in specific cells.

[0044] Table 06 shows, as an example, the reporter stabilizer (mCherry) DNA sequence used downstream of the MCO sequence to confirm expression in specific cells.

[0045] Table 07 shows the amino acid and DNA sequences of enhanced multi-characteristic opsin-1 (eMCO1). This contains the MCO1 sequence (Table 01) and the biomarker stabilizer sequence (Table 06).

[0046] Table 08 shows a comparison of the stability of MCO1 and eMCO1 based on secondary structure and folding using theoretical modeling with RaptorX. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1A shows the domain structure of a multi-characteristic opsin (MCO) containing a reporter protein, including eMCO1. Figure 1B shows a typical circular map showing the insertion of the MCO gene cloned at restriction sites (BamH I and Sal I). [Figure 2]Figure 2 shows theoretical models of the three-dimensional arrangement of amino acid chains of multi-property opsins. Figure 2A shows theoretical models of the three-dimensional arrangement of amino acid chains of the multi-property opsin, MCO1. Figure 2B shows theoretical models of the three-dimensional arrangement of amino acid chains of the multi-property opsin. Figure 2C shows theoretical models of the three-dimensional arrangement of amino acid chains of the multi-property opsin. Figure 2C shows theoretical models of the three-dimensional arrangement of amino acid chains of the multi-property opsin. Figure 2C shows theoretical models of the three-dimensional arrangement of amino acid chains of the multi-property opsin, eMCO1. [Figure 3] Figures 3A and 3B show eMCO1 expression in model HEK293 cells. Figure 3A shows that eMCO1 expression is localized to the plasma membrane. Confocal fluorescence images of HEK293 cells transfected with mGluR6-MCO1-mCherry (mGluR6-eMCO1) are shown. Figure 3B shows the intensity of MCO1 reporter fluorescence along a line crossing a representative cell. [Figure 4] Figures 4A and 4B illustrate the function of enhanced multi-characteristic opsin (eMCO1). Figure 4A shows the inward current profile in MCO1-expressing cells in response to light (average intensity: 0.024 mW / mm2). Figure 4B shows the activation spectrum of eMCO1. Mean ± SEM. [Figure 5] Figures 5A and 5B show the effect of eMCO1 (e.g., the presence of mCherry on MCO1) on its function as measured by cell activity. Inward current profiles in HEK cells were measured by port-a-patch automated patch-clamp electrophysiology. Figure 5A shows the photocurrent measured at a white light intensity of 0.02 mW / mm2 in cells transfected with mGluR6-eMCO1 (mGluR6-MCO1-mCherry). Figure 5B shows the photocurrent measured at a white light intensity of 0.02 mW / mm2 in cells transfected with mGluR6-MCO1. [Figure 6]Figures 6A and 6B illustrate the function of multi-characteristic opsin (MCO2). Figure 6A shows fluorescence on lipofection of MCO2-mCherry into HEK293 cells. Figure 6B shows the inward current in MCO2-expressing cells in response to light (average intensity: 0.024 mW / mm2), as measured by patch-clamp electrophysiology. [Figure 7] Figures 7A and 7B show cell transfection with AAV2-supported mGluR6-eMCO1 (vMCO1). Figure 7A shows the three-dimensional reconstruction of eMCO1 expression in HEK293 cells, with a scale bar of 30 μm. Figure 7B shows the three-dimensional reconstruction of eMCO1 expression in the entire retinal cup, with a scale bar of 0.8 mm. [Figure 8] Figures 8A and 8B show patch-clamp recordings of retinas transfected with eMCO1. Figure 8A shows eMCO-1 expression in cells of mouse retinal explants. Figure 8B shows inward photocurrents induced by a series of light pulses (100 ms). [Figure 9] Figures 9A–9F show the dose- and time-dependent layer-specific expression of MCO1 in rd10 mice after vMCO1 injection. Figure 9A shows a fluorescence confocal image of the retinal cup of rd10 mice one week after intravitreal vMCO injection. Figure 9B shows a fluorescence confocal image of the retinal cup of rd10 mice eight weeks after intravitreal injection of vMCO1. Scale bar: 200 μm. Figure 9C shows a focal fluorescence image of the folded edge of a retinal cup transfected with vMCO1 at a dose of 1.6 × 10¹¹ VG / mL. Scale bar: 100 μm. Figure 9D shows a cross-sectional view of vMCO1 expression in the retina 16 weeks after intravitreal injection at a dose of 1.6 × 10¹² VG / mL. Scale bar: 50 μm. Figure 9E shows the dynamics of MCO1 expression in the retina of rd10 mice at two different doses of vMCO1. Mean ± SD. Figure 9F shows the inter-animal variability of MCO1-mCherry (eMCO1) expression in the retina of rd10 mice 16 weeks after transfection with a dose of 1.6 × 10¹² VG / mL (after background removal). Mean + SD. *p<0.01 vMCO1 injected vs. uninjected. [Figure 10]Figures 10A–10H show visually induced improvements in the behavior of rd10 mice in a radial water maze. Figure 10A shows time-course images of visually induced rd10 mouse behavior in a radial water maze with white LED light, before intravitreal vMCO1 injection. Figure 10B shows the behavior of rd10 mice with the LED light on, 6 weeks after vMCO1 injection. Figure 10C shows the time it took for vMCO1-treated rd10 mice to find a platform after falling into the center of the maze with and without light. Mean ± SEM. N=5 per mouse. Figure 10D shows the time it took for vMCO1-treated rd10 mice to find a platform after falling into side arms 2 and 4 of the maze with and without light. Mean ± SEM. N=5 per mouse. Figure 10E shows the time it took for vMCO1-treated rd10 mice to find the platform after falling at arm 3 on the edge of the maze, both with and without light. Mean ± SEM. N=5 per mouse. Figure 10F shows the number of error arms passed through by vMCO1-treated rd10 mice that fell at the center before finding the platform, both with and without light. Mean ± SEM. N=5 per mouse. Figure 10G shows the number of error arms passed through by vMCO1-treated rd10 mice that fell at the side arms before finding the platform, both with and without light. Mean ± SEM. N=5 per mouse. Figure 10H shows the number of error arms passed through by vMCO1-treated rd10 mice that fell at the edge before finding the platform, both with and without light. Mean ± SEM. N=5 per mouse. [Figure 11]Figures 11A and 11B show a follow-up study of visually induced improvements in the behavior of rd10 mice in a radial water maze. Figure 11A shows a schematic diagram of the radial arm water maze used to test visually induced improvements in the behavior of rd10 mice injected with vMCO1. Figure 11B shows the time it took for rd10 mice to reach the platform from the center of the maze (light intensity: 0.007 mW / mm2) before vMCO1 injection, depending on the time elapsed after injection. N=5; mean ± SD. *P<0.05. Figure 11C shows the time it took for rd10 mice to reach the platform from a nearby arm of the maze (light intensity: 0.014 mW / mm2) before vMCO1 injection, depending on the time elapsed after injection. N=5; mean ± SD. *P<0.05. Figure 11D shows the time from the side arm (light intensity: 0.004 mW / mm2) before vMCO1 injection to the time from injection to when the rd10 mouse reached the platform, depending on the post-injection period. N=5; mean ± SD. *P<0.05. [Figure 12] Figure 12 shows the light intensity dependence of behavioral improvement in rd10 mice in a radial water maze. It compares the time from the center of the maze to the platform at two different light intensities, depending on the time elapsed since injection. Mean ± SD. *P<0.01. L0=0.0005mW / mm2; L2=0.007mW / mm2. Bright ambient level is approximately 0.01mW / mm2. [Figure 13] Figures 13A and 13B show the visual performance evaluation of rd10 and MCO-sensitized rd10 mice. Figure 13A shows a quantitative comparison of the number of head movements in rd10 mice before and 8 weeks after vMCO1 injection at a vertical stripe rotation speed of 1 rpm. N=4 mice. Mean + SD. *p<0.05. The light intensity at the center of the chamber was 0.001 mW / mm2. Figure 13B shows a quantitative comparison of the number of head movements in rd10 mice before and 8 weeks after vMCO1 injection at a vertical stripe rotation speed of 2 rpm. N=4 mice. Mean + SD. *p<0.05. The light intensity at the center of the chamber was 0.001 mW / mm2. [Figure 14]Figures 14A–14D show the viability of MCO1-sensitized retinal cells after chronic light exposure. Figure 14A shows that rd10 mice treated with vMCO1 avoid bright light by staying away from and blocking it (by piling up bedding material, as indicated by the arrows), similar to wild-type (open-label) mice. Figure 14B shows a fluorescence image of the caspase-3 (green) stained retina of rd10 mice treated with vMCO1 after 4 weeks of 8 hours of white light (intensity: 0.1 mW / mm2) illumination per day. Figure 14C shows a fluorescence image of the caspase-3 (green) stained retina of wild-type mice after 4 weeks of 8 hours of white light (intensity: 0.1 mW / mm2) illumination per day. Figure 14D shows a quantitative comparison of the percentage of apoptotic retinal cells between wild-type mice and rd10 mice treated with vMCO1. Apoptotic cells in the inner nucleus layer of rd10 mice treated with vMCO1 are 0%. [Figure 15] Figures 15A and 15B show the results of the evaluation of retinal structural integrity after vMCO1 injection in rd10 mice. Figure 15A shows OCT images of the retina of rd10 mice after vMCO1 injection. Figure 15B shows a comparison of retinal thickness in four different rd10 mice before and one week after injection. N=10 B-scans / mouse. Mean + SD. [Figure 16] Figures 16A to 16C show the immunotoxicity results in rd10 mice injected with vMCO1. Figure 16A shows a quantitative comparison of plasma IL-6 (pro-inflammatory marker) between group 1 (1.6 × 10¹⁰ VG / mL) and group 2 (1.6 × 10¹¹ VG / mL) before, 7 days after, and 14 days after vMCO1 injection. N=5 mice / group. Mean ± SD. Figure 16B shows a quantitative comparison of plasma IL-10 (anti-inflammatory marker) between group 1 (1.6 × 10¹⁰ VG / mL) and group 2 (1.6 × 10¹¹ VG / mL) before, 7 days after, and 14 days after vMCO1 injection. N=5 mice / group. Mean ± SD. Figure 16C shows a quantitative comparison of plasma IFN-Y (pro-inflammatory marker) levels between Group 1 (1.6 × 10¹⁰ VG / mL) and Group 2 (1.6 × 10¹¹ VG / mL) before, 7 days after, and 14 days after vMCO1 injection. N=5 mice / group. Mean ± SD. [Figure 17]Figure 17 shows the biodistribution of the multi-characteristic opsin (vMCO1) packaged in AAV2. QPCR detection of the vector sequence in rd10 mice at different doses and post-injection time points revealed that the vector DNA in the treated outer eye tissue was present in very small or undetectable amounts. N=5 mice / dose / time point. [Figure 18] Figures 18A–18F show immunohistochemistry of the eyes of rd10 mice injected with vMCO1. Figure 18A shows that MCO-mCherry (red) is selectively targeted and expressed in the INL of rd10 mice 8 weeks after intravitreous injection of vMCO1. The absence of arrestin (green) suggests complete loss of the photoreceptor. Figure 18B shows PKCa staining (green) in rod bipolar cells expressing mCherry (red, endogenous) in rd10 mice 8 weeks after intravitreous injection of vMCO1. Figure 18C shows mGluR6 staining (green) in ON-type bipolar cells expressing mCherry (red) in rd10 mice 8 weeks after intravitreous injection of vMCO1. Figure 18D shows mCherry (green immunostaining) expression in the rd10 retina 8 weeks after intravitreous delivery of vMCO1 to rd10 mice. Figure 18E shows GFAP (green) in rd10 mice 18 weeks after intravitreal injection of vMCO1, as reported in retinas with photoreceptor degeneration. Figure 18F shows the absence of CD45 (green) expression, suggesting the lack of immune cells in rd10 mice 8 weeks after intravitreal injection of vMCO1. [Figure 19] Figure 19 shows the structure of eMCO1 and how its different domains are activated by light. Blue light activates the transmembrane (TM) domain (ion channel) of eMCO1, allowing cation flow. Green and red light activate the non-TM intermediate domain (non-ion channel). [Figure 20]Figure 20A shows the inward current profile measured at a blue (450 nm) light intensity of 0.06 mW / mm2 in HEK cells transfected with eMCO1. Figure 20B shows the inward current profile measured at a green (520 nm) light intensity of 0.06 mW / mm2 in HEK cells transfected with eMCO1. Figure 20C shows the inward current profile measured at a red (630 nm) light intensity of 0.06 mW / mm2 in HEK cells transfected with eMCO1. Figure 20D shows the inward current profile measured at a blue (450 nm) light intensity of 0.06 mW / mm2 in the presence of a Ca2+ chelating agent (BAPTA) in HEK cells transfected with eMCO1. Figure 20E shows a comparison of photocurrents generated in cells by light of different wavelengths and the effect of the presence of a Ca2+ chelating agent (BAPTA), verified by blue (450 nm) photoactivation, on eMCO1 transmembrane domain function. Mean ± SEM. Figure 20F shows a comparison of photoactivation ON times generated by blue and green light. Mean ± SEM, *p<0.05. [Figure 21]Figure 21A shows the results of SDOCT time-course monitoring of the retina of rd mice before and after different doses of AAV2-eMCO1 or AAV2-vehicle injection. Group AA: 1.0 × 10¹² VG / mL AAV2-eMCO1 (vMCO1); Group BB: 1.0 × 10¹² VG / mL AAV2 (no transgene); Group CC: 1.0 × 10¹⁰ VG / mL AAV2-eMCO1. Comparison of retinal thickness before injection, 1 day, 1 month, and 4 months before injection, across different groups and within each group by sex. Mean ± SEM. Figure 21B is a scatter plot showing a comparison of retinal thickness before injection and 4 months after injection in different groups. Group AA: 1.0 × 10¹² VG / mL AAV2-eMCO1; Group BB: 1.0 × 12 VG / mL AAV2 (no transgene); Group CC: 1.0 × 10¹⁰ VG / mL AAV2-eMCO1. Figure 21C shows the mean difference in retinal thickness between baseline and intravitreal-injected mice as a Gardner-Altman estimated plot. Group AA: 1.0 × 10¹² VG / mL AAV2-eMCO1; Group BB: 1.0 × 10¹² VG / mL AAV2 (without eMCO1); Group CC: 1.0 × 10¹⁰ VG / mL AAV2-eMCO1. The curves show the resampling distribution of the mean difference based on observed data. The 95% confidence interval for the mean difference is shown by the black vertical line. *p<0.05 between baseline and 4 months after vehicle injection in Group BB. [Figure 22]Figure 22A shows PKCα staining (green) in rod bipolar cells expressing mCherry (red, endogenous) in rd10 mice after intravitreous injection of AAV2-eMCO1. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Figure 22B shows eMCO1 (mCherry: red reporter) expression in bipolar cells (INL) of rd10 mice after intravitreous injection of AAV2-eMCO1. Figure 22C shows quantification of eMCO1 (reporter mCherry) expression in bipolar cells of three rd10 mice. Figure 22D shows immunohistochemical cross-sectional images of the retina of AAV2-eMCO1-treated rd10 mice, showing that bipolar cell terminals (green: PKCa) in close contact with retinal ganglion cells (RGCs) co-localize with CtBP (synaptic ribbon marker, red). Figure 22E shows an immunohistochemical cross-sectional image of the retina of an untreated rd10 mouse, showing that bipolar cell terminals (green: PKCa) in close contact with retinal ganglion cells (RGCs) colocalize with CtBP (synaptic ribbon marker, red). Figure 22F shows that in the retina of an AAV2-eMCO1-treated rd10 mouse, the PKCa immunohistochemical signal at the axon terminals is stronger (*p<0.05) and the CtBP signal is observed. [Figure 23]Figure 23A shows the time it took for Stargard (ABCA4- / -) mice to reach the platform from the side arm (light intensity: 0.004 mW / mm2) of a radial arm water maze before and after injection of AAV2 (no transgene, -ve control). N=5; mean ± SD, ns: no statistically significant difference. Figure 23B shows the time it took for Stargard (ABCA4- / -) mice to reach the platform from the side arm (light intensity: 0.004 mW / mm2) of a radial arm water maze before and after injection of AAV2-eMCO1 (vMCO1). N=5; mean ± SD. ****p<0.001. Figure 23C shows the time it took for Rpe65rd12 (LCA) mice to reach the platform from the side arm (light intensity: 0.004 mW / mm2) of a radial arm water maze before and after injection of AAV2 (no transgene, -ve control). N=5; mean ± SD, ns: no statistically significant difference. Figure 23D shows the time it took Rpe65rd12 (LCA) mice to reach the platform from the side arm (light intensity: 0.004 mW / mm2) of the radial arm water maze before and after AAV2-eMCO1 injection. N=5; mean ± SD. **p<0.01. [Figure 24] Figure 24A shows the results of monitoring the retina of Abca4tm1Ght / J Stalgard mice over time using SDOCT before and after AAV2-eMCO1 injection and compared with the control (non-injection group). Comparison of retinal thickness before injection, 4 weeks, 12 weeks, and 16 weeks. Mean ± SEM. Figure 24B shows the change in ERG b-wave amplitude at 1 cd.s / m2 (relative to baseline) in Abca4tm1Ght / J Stalgard mice after AAV2-eMCO1 injection compared with the control (non-injection group). Figure 24C shows the change in ERG b-wave amplitude at 10 cd.s / m2 (relative to baseline) in Abca4tm1Ght / J Stalgard mice after AAV2-eMCO injection compared with the control (non-injection group). [Figure 25]Figure 25 shows the method of visual acuity restoration by intraocular eMCO1 administration. 100: Retina; 110: Retinal ganglion cells; 120: Bipolar cells; 130: Photoreceptors; 140: Retinal pigment epithelium; 150: Disappearance of photoreceptors / retinal pigment epithelium; 160: Delivery device; 170: AAV-carrying eMCO1; 180: Optic nerve; 190: Optic tract; 200: Optic chiasm; 210: Transfer of AAV2-carrying eMCO1 to the contralateral eye; 220: Retinal cells introducing AAV2-eMCO1; 230: Projection of the outer visual field; 240: Photoactivated bipolar cells; 250: Activated retinal ganglion cells; 260: Electrochemical signals transmitted via the optic nerve; 270: Brain for visual processing of photoactivation signals received from the eMCO1-sensitized retina. [Figure 26] Figure 26A shows eMCO1 gene expression in the retina after intravitreal injection in mice. It shows reporter (mCherry) fluorescence images of retinal cups in mice (N=4) 6 months after intravitreal injection of 1 μl of 1E12vg / ml AAV2-eMCO1 (vMCO1). Figure 26B shows contralateral translocation and retinal expression of the eMCO1 gene after unilateral intravitreal injection in mice. It shows reporter (mCherry) fluorescence images of retinal cups in mice (N=4) 6 months after intravitreal injection of 1 μl of 1E12vg / ml AAV2-eMCO1 (vMCO1). Figure 26C shows reporter (mCherry) immunofluorescence images of canine retinal slices 4 months after unilateral (right eye) intravitreal injection of 75 μl of 2E11VG / ml vMCO1. GCL: ganglion cell layer; INL: inner nucleus layer; ONL: outer nucleus layer. Figure 26D shows evidence of contralateral translocation and retinal expression of the eMCO1 gene after unilateral intravitreal injection in dogs. Immunofluorescence images of the eMCO1 reporter (mCherry) in the contralateral (left eye) retina are shown. GCL: ganglion cell layer; INL: inner nucleus layer; ONL: outer nucleus layer. Figure 26E shows qPCR detection of vMCO1 vector sequences in visual system tissues of dogs 1 and 2 after unilateral intravitreal injection of 75 μl of 2E11VG / ml AAV2-eMCO into the right eye. Mean ± SD. [Figure 27]Figure 27A shows fundus images of mCherry (reporter for eMCO1) fluorescence. Figure 27B shows fundus images of mCherry (reporter) fluorescence in the contralateral eye of a retinitis pigmentosa patient 8 weeks after AAV2-eMCO1 injection. Figure 27C shows the results of monitoring retinal thickness in the injected eye and the contralateral eye over time using OCT images after AAV2-eMCO1 injection in a retinitis pigmentosa patient. N=11, mean ± SD. X31 week data is missing for patients 2-4 and 7-9 due to COVID-related lockdowns; XX52 week data is missing for patient 8 due to the COVID situation. [Figure 28] Figure 28 shows the time course of visual acuity (LogMAR) in the eye and contralateral eye treated with AAV2-eMCO1 injection (3.5E11vg) in patients with severe retinal degeneration. Mean ± SEM (N=8). [Figure 29] Figures 29A and 29B show improvement in visual induction behavior in mice in which opsin-sensitized retinal ganglion cells were stimulated with stroboscopic light at a frequency of approximately 0.5 Hz. Figure 29A shows a schematic diagram of the mouse visual induction Y-mobility assay. +light: light ON; -light: light OFF. Figure 29B shows improvement in visual induction behavior in mice in which opsin-sensitized retinal ganglion cells were stimulated with stroboscopic light at a frequency of approximately 0.5 Hz. The number of events (finding light ON vs. OFF) in mice under different conditions is shown. Baseline: no optic nerve damage; ON damage: optic nerve damage; Post-light stimulation: after 2 weeks (8 hours / day) of stroboscopic light (0.5 Hz) stimulation of opsin-sensitized retinal ganglion cells in mice with optic nerve damage. [Figure 30] Figure 30A shows the time-dependent improvement in visual acuity character scores measured using the ETDRS visual acuity chart in subjects treated with eMCO1. Figure 30B shows the time-dependent improvement in visual acuity character scores measured using the ETDRS visual acuity chart in subjects treated with eMCO1 using a VR headset (100% contrast, 3x zoom). [Modes for carrying out the invention]

[0048] The regulation of cell activity by electrical and other means has enabled the quantitative evaluation of cellular characteristics and changes associated with disease progression. Opsins (photosensitive ion channel proteins) or ligands have been used in combination with light to regulate cell activity and may be used for various therapeutic applications, including vision restoration, as well as drug discovery screening.

[0049] Since higher-order neurons remain intact within the degenerated retina, some stimulation methods target higher-order neurons (e.g., bipolar cells and retinal ganglion cells), which carry visual information to the visual cortex. Direct electrical stimulation methods require mechanical contact of electrodes with retinal cells, while indirect stimulation methods (e.g., optogenetic stimulation) do not require such physical contact. Therefore, indirect methods offer the clear advantage of being non-invasive. In addition, cell specificity and high (single-cell) resolution can be achieved while using optogenetic stimulation.

[0050] To achieve optogenetic stimulation of retinal neurons, cells are generally transfected with viruses to express opsins (photosensitive molecular ion channels and ligands, as well as enhancers), and then the opsins are activated. Opsins depolarize opsin-expressing cells upon irradiation with light of specific visible wavelengths. For example, retinal cells expressing channelrhodopsin-2 (ChR2) are sensitive to blue light.

[0051] To enhance the photosensitivity of cells or to be activated by visible light of different wavelengths, various light-activated ion channels (opsins) and ligands have been developed. To be activated by broadband visible light, a complex of three opsins (blue ChR2, green C1V1, and red ReaChr photosensitive opsins) has been delivered to cells by chemical or physical means. However, such large complexes cannot be packaged into commonly used viral vectors (i.e., adeno-associated viruses). Furthermore, the use of chemical or physical delivery methods is inefficient and / or impairs cell viability, limiting their rapid usefulness.

[0052] Opsins developed and used to date for vision restoration do not generate characteristic photoreceptor rod signals when stimulated by light. That is, the voltage signal lacks a slower depolarization phase following an initial fast response. This invention provides effective optogenetic vision restoration at low light levels.

[0053] The present invention provides vision restoration by optogenetics, protein administration, or gene therapy, by administering opsin or other genes into the vitreous humor of the eye using a method that includes a viral means, such as recombinant adeno-associated virus, rAAV, etc.

[0054] In one embodiment, the present disclosure has the ability to generate characteristic photoreceptor rod signals that do not require an external active stimulator. This embodiment avoids the obstacles associated with or encountered in existing opsin-based approaches. The present invention provides vision recovery for individuals lost due to retinal degenerative diseases. Furthermore, an advantage of the present invention is that the method of delivering opsin / other therapeutic genes can transiently penetrate the internal limiting membrane of the human eye through a combination of rAAV and chemical agents.

[0055] This invention highlights that the unique structure of eMCOs enables rapid photo-induced activation of cells with a low light threshold and a broad spectral range. Blue light activates the transmembrane (TM) domain of eMCO1, which in one embodiment functions as an ion channel. In yet another embodiment, a non-TM intermediate domain (non-ion channel) absorbs red light, causing a conformational change in the eMCO1-TM domain, thereby generating ion flow through the eMCO1-TM domain. In yet another embodiment, the C-terminal domain (RFP) absorbs green light and emits red light, enhancing the effect of eMCO1.

[0056] In another embodiment, the present invention provides a method for expressing an enhanced pluripotent opsin (eMCO1) intracellularly or in vivo, and a method for regulating cell activity using different visible light wavelengths.

[0057] Currently, the use of optogenetic sensitization of retinal cells combined with activation / inhibition has shown potential as an alternative to retinal implants, eliminating the need to place electrodes near individual neurons for high resolution (4). Optogenetic stimulation offers high temporal precision (5–10) by introducing photoactivatable molecular channels (e.g., channelrhodopsin-2, ChR2; halorhodopsin, NpHR) into cells via gene targeting. In addition to high temporal and spatial resolution, optogenetics offers several advantages, including cell specificity (e.g., unused cones, ganglia, or bipolar cells) and minimal invasiveness (11). Photo-induced activation of the non-selective cation channel ChR2 depolarizes only those cells expressing ChR2. Selective activation of neurons by ms-pulsed blue light has been demonstrated in cultures (9), brain sections, and small animals (12–15). This optogenetic activation method uses medium-intensity light (approximately 0.1 mW / mm²) that can be delivered from a light-emitting diode (LED) or laser (5, 6). 2 Because it requires only ), it is very promising for controlling cell activity in vitro and in vivo.

[0058] This disclosure provides several photosensitive ion channel and ligand molecules (multi-characteristic opsins) that are produced by synthetic means and (i) have high photosensitivity at multiple visible wavelengths and (ii) have a plasmid size small enough to be packaged in a safe adeno-associated virus. The present invention also includes isolated nucleic acid sequences encoding the photosensitive ion channel and ligand of the present invention, and constructs comprising such nucleic acid sequences. In some embodiments, the MCOs found using the methods disclosed herein comprise amino acids shown in Tables 1-4, 7 and represented by SEQ ID NOs: 1, 3, 5, 7, or 11. In some embodiments, the MCOs have at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the sequences shown in SEQ ID NOs: 1, 3, 5, 7, or 11, and the MCOs have the photosensitive properties of SEQ ID NOs: 1, 3, 5, 7, or 11. In some embodiments, the MCOs are encoded by nucleic acids shown in Tables 1-4, 7 and represented by SEQ ID NOs: 2, 4, 6, 8, or 12. In some embodiments, the nucleic acid encoding the MCO has at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with the sequence shown in SEQ ID NOs. 2, 4, 6, 8, or 12, and the encoded MCO has the photosensitive properties of SEQ ID NOs. 1, 3, 5, 7, or 11.

[0059] Nucleic acids encoding MCOs are useful when incorporated into vectors for delivery to patients who need them. In some embodiments, the vector is a plasmid having a suitable promoter known in the art. In some embodiments, the vector is a viral vector. Viral vectors that may be used in the methods disclosed herein include adenovirus vectors, adeno-associated virus vectors, and the like.

[0060] In some embodiments, the present invention includes the expression of multi-characteristic opsins (MCOs) intracellularly or in vivo, and methods for regulating cell activity using these synthetic opsins.

[0061] One example of the use of MCO in treating diseases is blindness resulting from degenerative diseases of the retina. Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) refer to diseases characterized by the degeneration of photoreceptors in the eye, resulting in impaired vision due to non-functional neuronal activity and signal propagation to the visual cortex (16-20). AMD is the leading cause of new vision loss in approximately 15 million people over the age of 65 (21), and the prevalence of RP is at least 1 million worldwide (22, 23). RP is most often inherited as autosomal recessive inheritance, with the majority of cases having this form of inheritance (18, 22, 24). Furthermore, the degree of vision loss increases with age (25), which is a major concern in demographic shifts toward older populations.

[0062] Most current clinical treatments focus primarily on slowing disease progression (26), there are no treatments that can halt degeneration (27), and there are no treatments other than retinal prostheses that can restore vision loss caused by degeneration (28). Partial vision restoration involves invasive surgical procedures for retinal implants (29). Two different types of retinal implants, subretinal and supraretinal, have been developed (30). Subretinal implants are positioned between the pigment epithelium and bipolar cells in the region of the retina where photoreceptor cells are present (31). These retinal prostheses have been successful in generating vision in blind subjects (32-34). Disadvantages of using such subretinal implants include (i) chronic damage to the implanted electrodes and (ii) insufficient current generated by microphotodiodes from ambient light to stimulate adjacent neurons (35, 36). Supraretinal implants are placed within the region of retinal ganglion cells (RGCs), and the device functions by stimulating the RGCs in response to input from a camera placed outside the eye or within the intraocular lens (36, 37). Disadvantages of supraretinal implants include (i) cell proliferation due to surgical implantation and (ii) irregular stimulation patterns resulting from electrical stimulation of both the axons and cell bodies of RGCs (36). Apart from being inherently invasive, these methods for restoring vision in blind patients are based on nonspecific cell activation and have low spatial resolution due to a low number of electrodes (more electrodes or higher density electrodes require more power and result in thermal damage to nerve tissue), and therefore can improve vision with low spatial resolution.

[0063] Cytogenetic methods have been employed for vision restoration in blind mouse models, either by nonspecific stimulation of the retina (38) or by mGluR6 (44, 45) targeting ON-type bipolar cells in a promoter-specific manner including Thy1 for RGCs (39-43). Attempts have also been made to stimulate RGCs using melanopsin (46) or photogenetics (47). Furthermore, the use of activating halorhodopsin, a chloride channel expressed in long-lasting cone photoreceptors (48), represents a novel possibility for therapeutic interventions for vision restoration (49). Resensitized photoreceptors have been shown to activate retinal circuits, activate cortical circuits, and mediate vision-driven behaviors.

[0064] Early approaches to visual acuity restoration through optogenetic stimulation of retinal cells utilize opsins such as ChR2(38) and others, which require higher light intensity than ambient light conditions. Therefore, clinical success of such opsin molecules in ambient environments for visual acuity restoration has not yet been achieved. Furthermore, activation of such opsins using external light sources or devices (e.g., LED arrays(50)) can substantially damage retinal cells with prolonged use. In addition, these opsins (used for visual acuity restoration) have a fast (millisecond) ON and OFF response to light pulses. That is, when opsin-sensitized cells are stimulated with light, characteristic photoreceptor rod signals are not generated. In other words, the voltage signal does not have a slower depolarization phase after the initial fast response to the light pulse. Therefore, effective optogenetic visual restoration at ambient light levels has not been demonstrated to date.

[0065] The disclosed invention includes methods for preparing highly photosensitive ion channels and ligands and their various uses, including vision restoration. In some embodiments, the expression of a specific MCO within a cell generates a long-lasting inward current in response to white light, similar to characteristic photoreceptor rod signaling. According to another embodiment of the invention, the disclosed invention provides methods for using synthetic opsins for vision restoration and other applications, wherein the amino acid sequence of the synthetic opsin is modified to provide enhanced photosensitivity, kinetics, and ion selectivity.

[0066] The results presented in this invention demonstrate efficient and stable in vivo expression of the MCO receptor protein in the mouse retina after intravitreal injection of adeno-associated virus carrying MCO. The results also demonstrate that MCO expression in the retina of a mouse model of retinal degeneration enables behavioral recovery of visual acuity. Following delivery of MCO to mice with degenerated retinas, the number of error arms in a radial arm water maze and the time to reach the platform were significantly reduced. In particular, improvements in visually induced behavior were observed even at light intensity levels much lower than those required for channelrhodopsin-2 opsin(1).

[0067] In one embodiment, a method is provided for restoring photosensitivity by administering eMCO1 to a patient's degenerated retina. In another embodiment, efficient expression of the eMCO1 gene and production of the eMCO1 protein are obtained by administering eMCO1, including intravitreal injection of an adeno-associated virus containing the gene encoding eMCO1, to a patient suffering from retinal degeneration or retinal dystrophy. Yet another embodiment includes a method for obtaining long-term improvement in retinal function and visual behavior compared to a patient who does not receive eMCO1 by administering eMCO1 to a patient's retina-degenerated eye. Yet another embodiment shows that administration of eMCO1 to a patient's retina reverses retinal degeneration (including Stargardt disease and LCA measurements) and restores visual behavior. Yet yet another aspect of the present invention provides a method for efficiently restoring visual acuity in humans. This method involves the use of MCO, which, when expressed in retinal cells, generates a slower depolarization phase after an initial response to white light similar to characteristic photoreceptor rod signaling, and the in-vivo delivery of the opsin to retinal cells by adeno-associated virus (AAV) carrying the promoter-MCO gene in the eye, and / or in combination with pronase E or α-aminoadipic acid (AAA) to enhance the efficiency of delivery to the target retinal layer across the thick internal limiting membrane in humans.

[0068] In yet another embodiment, a method is described in which the progression of retinal degeneration or retinal dystrophy is stopped by administering eMCO1 to an eye with retinal degeneration. In yet another embodiment, a method is described in which the progression of structural disorder of the inner nucleus layer and disorder of connections with the ganglion cell layer is stopped or delayed by administering eMCO1 to an eye with retinal degeneration. In yet another embodiment, eMCO1 expression is obtained in the other eye by administering eMCO1 to one eye of a patient. In one embodiment of the present invention, by administering eMCO1 to one eye of a patient, expression is observed in the non-administered eye, the progression of retinal degeneration or retinal dystrophy in the non-administered eye is stopped, and retinal thickness is maintained.

[0069] The present invention provides an approach for treating retinal dystrophy or retinal degeneration characterized by the loss / mutation of the outer retina, including photoreceptors and / or retinal pigment epithelium.

[0070] In another embodiment, administration of eMCO1 (including administration of AAV containing the eMCO1 gene) to one eye of a patient results in eMCO1 expression in the other eye of the patient, and long-term improvement in retinal function and visual behavior in the non-administered eye of the patient compared to patients who did not receive eMCO1. In yet another embodiment, eMCO1 administered to one eye of a patient is expressed only in the other eye of the patient.

[0071] In yet another embodiment, after administration of eMCO1 to a patient, the patient's visual function and behavior improve. Administration of eMCO1 results in improvements in visual function and behavior regardless of the type of retinal dystrophy or retinal degeneration.

[0072] In one embodiment, administration of eMCO1 to a patient results in the restoration of vision and the cessation of symptoms associated with the progression of retinal dystrophy or retinal degeneration.

[0073] In yet another embodiment, an enhanced multi-characteristic opsin (eMCO1) is administered to a patient suffering from a neurodegenerative disease. In one embodiment, eMCO1 is administered to the patient to regulate retinal cell activity.

[0074] In one embodiment, a method of administering eMCO1, including an AAV containing the gene encoding eMCO1, involves using a device capable of injecting eMCO1 (protein alone or AAV) through one or more routes in the eye, vitreous, or retina.

[0075] In another aspect of the present invention, administration of eMCO1 treats macular degenerative diseases such as Stargardt macular degeneration and progressive macular degeneration, in which degeneration or dysfunction of extraretinal cells, including photoreceptor cells, occurs.

[0076] In another embodiment, administration of eMCO1 targets the central part of the retina (macula), enabling the expression of eMCO1 in higher-order neurons of the retina, thereby treating macular degeneration disease.

[0077] In yet another embodiment of the present invention, administration of eMCO1 improves the visual function of patients suffering from macular degeneration disease.

[0078] In another embodiment, subjects treated with eMCO1 experience improved visual quality through the use of a virtual reality (VR) headset. The VR headset enhances the contrast and / or optical zoom of the field of view, facilitating improvement in patients after eMCO1 treatment.

[0079] In another embodiment of the present invention, subjects with macular degeneration treated with eMCO1 show improved visual acuity (visual resolution).

[0080] In yet another embodiment, subjects with macular degeneration treated with eMCO1 show further improvement in vision when using a VR headset.

[0081] In yet another embodiment, the VR headset has variable magnification of 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, and 18x. In yet another embodiment, the VR headset has magnification of 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, and 18.5x. In another embodiment, the VR headset has a magnification of approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, and 18x. In another embodiment, the VR headset has a magnification of 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, and at least 18x. In another embodiment, the VR headset has magnifications of 1x or less, 1.5x or less, 2x or less, 2.5x or less, 3x or less, 3.5x or less, 4x or less, 4.5x or less, 5x or less, 5.5x or less, 6x or less, 6.5x or less, 7x or less, 7.5x or less, 8x or less, 8.5x or less, 9x or less, 9.5x or less, 10x or less, 10.5x or less, 11x or less, 11.5x or less, 12x or less, 12.5x or less, 13x or less, 13.5x or less, 14x or less, 14.5x or less, 15x or less, 15.5x or less, 16x or less, 16.5x or less, 17x or less, 17.5x or less, and 18x or less.

[0082] In another embodiment, the VR headset has a contrast of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%. In yet another embodiment, the VR headset has a contrast of approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In another embodiment, the VR headset has a contrast of 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%. In another embodiment, the VR headset has a contrast of 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, or 95% or less.

[0083] In yet another embodiment, the VR headset has different contrast options, namely normal view, black on white, or white on black.

[0084] Visual acuity letter scores were measured by presenting the ETDRS visual acuity chart to patients at distances of 10cm, 20cm, 30cm, 40cm, 50cm, 60cm, 70cm, 80cm, 90cm, and 100cm or more. Visual acuity letter scores were measured by presenting the ETDRS visual acuity chart to patients at distances of at least 10cm, at least 20cm, at least 30cm, at least 40cm, at least 50cm, at least 60cm, at least 70cm, at least 80cm, at least 90cm, and 100cm or more. Visual acuity letter scores were measured by presenting the ETDRS visual acuity chart to patients at distances of 10cm or less, 20cm or less, 30cm or less, 40cm or less, 50cm or less, 60cm or less, 70cm or less, 80cm or less, 90cm or less, and 100cm or less. Visual acuity test scores were measured by presenting the ETDRS visual acuity chart to patients at distances of approximately 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, and 100 cm or more.

[0085] When using a VR headset, the increase in character score after eMCO1 injection is 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, Numbers of 47, 48, 49, and 50 characters or more increased in weeks 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, and 50.

[0086] When using a VR headset with 3x zoom (and 100% contrast), the increase in letter score after eMCO1 injection was further enhanced to approximately 30 letters at 24 weeks. [Examples]

[0087] Example 1 - Figure 1A shows the domain structure of multi-characteristic opsins (MCOs) containing a reporter protein. These MCOs were synthesized using a typical circular map with insertions of cloned MCO genes at restriction sites, as shown in Figure 1B. The MCO genes were synthesized using a DNA synthesizer and their sequences were verified. Gel electrophoresis was performed on the amplified MCO1 genes (classified by restriction enzymes BamH I and Sal I containing restriction fragments) using 0.8% agarose. Western blotting was performed to confirm that MCOs were expressed in retinal cells. Mouse retinas were transfected with lipofectamine, and the expressed proteins were extracted for Western blotting. Western blots were developed using a primary (anti-mCherry polyclonal) antibody and a secondary (goat anti-rabbit IgG) antibody with a one-step NBT / BCIP substrate.

[0088] Example 2 - Figure 2 shows theoretical modeling of the three-dimensional arrangement of amino acid chains of multi-characteristic opsins using a web-based protocol (RaptorX). RaptorX uses multiple template processing procedures to develop the following predicted structures of MCOs, including conditional neural field (CNF) and conditional random field variants. Figure 2A shows theoretical modeling of the three-dimensional arrangement of amino acid chains of multi-characteristic opsin MCO1. Figure 2B shows theoretical modeling of the three-dimensional arrangement of amino acid chains of multi-characteristic opsin MCO2. Figure 2C shows theoretical modeling of the three-dimensional arrangement of amino acid chains of multi-characteristic opsin eMCO1. Gene expression and the functions of MCO1 and eMCO1 were investigated. eMCO1 was found to fold / express better within the membrane and therefore function more effectively compared to MCO1. In the design of eMCO1, a special element is placed between MCO1 and mCherry, resulting in increased interaction between the MCO1 gene and mCherry, allowing mCherry to play an active role in stabilizing the entire intramembrane therapeutic molecule (eMCO1). Table 08 shows that eMCO1 exhibits a higher proportion of β-sheets and a lower proportion of irregular structures (i.e., less prone to cleavage) compared to MCO1. Furthermore, the presence of mCherry in eMCO1 serves as an indicator for determining the effectiveness of gene delivery to target tissue and helps determine the presence of opsin at different time points. Therefore, in the case of opsin expression loss, reinjection of the opsin gene can be performed to re-sensitize target cells. For example, if visual acuity declines or is lost over time after initial improvement (by injection of vMCO-1), examination of mCherry expression in the retina (by fundus examination) serves as a biomarker to determine whether vMCO-1 expression has been lost (requiring reinjection). If mCherry expression is not impaired (but visual improvement is lost / decreased), it suggests that targeted retinal cells have lost their connection to retinal ganglion cells that transmit visual information to the visual cortex.

[0089] Example 3 - To evaluate the membrane transport of MCO, the expression of MCO in the cell membrane (vs. cytoplasm) of transfected HEK293 cells was quantified using the fluorescence intensity of the reporter protein (mCherry). HEK293 cells were transfected with an MCO construct using lipofectamine 3000 (Life Technologies). After transfection, HEK293 cells were maintained in petri dishes in DMEM / F-12 containing 10% fetal bovine serum and 0.2 mg / mL gentamicin. The culture was maintained at 37°C in 5% CO2 humidified air. Cells were incubated for 48 hours after transfection to enable MCO expression. Reporter (mCherry) fluorescence was visualized under epifluorescence microscopy. Fluorescence images of HEK293 cells expressing eMCO1 (MCO1-mCherry) and MCO2-mCherry are shown in Figures 3A and 6A, respectively. Furthermore, intensity profiles are plotted to quantify the relative expression of eMCO1 in the cell membrane and intracellular components. Figure 3B shows the intensity of eMCO1 reporter fluorescence along a line crossing representative HEK293 cells transfected with mGluR6-MO1-eMCO1 (mGluR6-mCherry). No significant intracellular (cytoplasmic) aggregation, implicitly suggesting effective trafficking of MCO to the plasma membrane, was observed.

[0090] Example 4 - To determine the light-dependent inward photocurrent, MCO-expressing cells were subjected to 0.024 mW / mm². 2 The samples were exposed to pulses of light with a specified intensity. A single-mode optical fiber coupled to a supercontinuous laser source (NKT Photonics) delivered broadband light to the samples for optogenetic stimulation. A power meter (818-SL, Newport) was used to quantify the light intensity on the sample plane. The light pulse width was synchronized with an electrophysiological recording system controlled by an Axon Instruments Digidata system (Molecular Devices). Cells transfected with MCO were incubated with all-trans retinal (ATR, 1 μM) for 4 hours, followed by patch-clamp experiments.

[0091] The patch-clamp recording setup included an inverted Nikon fluorescence microscope (TS 100) platform with an amplifier system (Axon Multiclamp 700B, Molecular Devices). A micropipette was pulled using a two-stage pipette puller (Narshinghe) to achieve a resistance of 3–5 MΩ when filled with a solution containing 130K-gluconic acid, 7KC1, 2NaCl, 1MgCl2, 0.4 EGTA, 10 HEPES, 2 ATP-Mg, 0.3GTP-Tris, and 20 sucrose (in mM). The micropipette electrode was mounted on a micromanipulator. An extracellular solution containing 150NaCl, 10 glucose, 5KC1, 2CaCl2, and 1MgCl2 (in mM) was buffered with 10 mM HEPES (pH 7.3). Photocurrent was measured while holding cells within a voltage clamp at -70 mV. The electrophysiological signals from the amplifier were digitized using a Digidata 1440 (Molecular Devices) interfaced with patch-clamp software (Clampex, Molecular Devices). A light-stimulating beam was delivered via optical fiber to activate MCO-expressing cells. pClamp 10 software was used for data analysis. Figure 4A shows the light intensity (average intensity: 0.024 mW / mm²) measured by patch-clamp electrophysiology. 2 This shows a typical inward photocurrent in MCO1-expressing cells in response to ambient light level (0.02 mW / mm²). The inward photocurrent was found to be significantly higher in eMCO1-sensitized cells than in ChR2-expressing cells. 2) The inward photocurrent (195 ± 32 pA) in eMCO1-sensitized cells is above the threshold of action potential (AP) (51), different from that in cells sensitized with ChR2 and white opsin. Note that a faster response time is required for the good fidelity of light-induced spikes in opsin-sensitized cells. The ON response time of peripherally light-activatable MCO1 (Figure 4A) was measured to be 2.94 ± 0.70 ms, which is similar to that measured for other fast opsins (52). However, the ON response time is known to depend on the intensity of the activating light and increases as the light intensity decreases (53).

[0092] To obtain the activation spectrum of eMCO1, the inward photocurrent was measured using stimulating light of different wavelengths (bandwidth: 30 mm). Figure 4B shows the normalized activation spectrum of eMCO1. In addition to acting as a stabilizing biomarker, mCherry enhances the light-induced current in cells expressing eMCO1 by (i) better orientation stabilization of eMCO1 across the membrane and (ii) enhancing the activation of eMCO1 by the light emitted / re-emitted from the stabilizing biomarker molecule. Figure 5 shows the inward current profiles in HEK cells measured by Nanion Port-a-Patch automated patch-clamp electrophysiology. Figure 5A shows the photocurrent measured at a white light intensity of 0.02 mW / mm 2 in cells transfected with mGluR6-eMCO1 (mGluR6-MCO1-mCherry). Figure 5B shows the photocurrent measured at a white light intensity of 0.02 mW / mm 2 in cells transfected with mGluR6-MCO1. The effect of the presence of mCherry on the enhanced MCO1 function is clearly demonstrated here. MCO1 was found to have a broad activation spectrum that coincides with white peripheral light.

[0093] The inward photocurrent within MCO2-expressing cells in response to light of the same average intensity (0.024 mW / mm 2 ) is shown in Figure 6B. 0.024 mW / mm 2At the light intensity, the peak photocurrent generated in MCO1 cells was approximately 160 pA, compared to approximately 320 pA in MCO2-expressing cells. While the on-rate of inducible photocurrent in light-responsive MCO1 and MCO2-expressing cells did not differ significantly, the off-response (attenuation of current in the absence of light) of MCO2 was found to be significantly slower than that of MCO1 (Figure 6B, Figure 4A). In MCO2 expressing HEK293 cells, the threshold peak current (54) for generating an action potential was the ambient light level of 0.02 mW / mm². 2 This can be achieved at a light intensity of [value]. Therefore, ambient light is expected to generate sufficient photocurrent (in terms of action potential) in MCOs expressing retinal cells. Figure 8 shows a patch-clamp recording of a mouse retina transfected with MCO1. Figure 8A shows eMCO-1 expression in cells of a mouse retinal explant. Figure 8B shows the inward photocurrent induced by a series of light pulses (100 ms). The rapid dynamics and small size of eMCO1, combined with its spectral and intensity sensitivity (allowing for AAV packaging), make it unparalleled suitable for conferring photosensitivity to higher retinal neurons in subjects with retinal degeneration, enabling visual acuity recovery in ambient light environments.

[0094] Example 5 - MCO1 and MCO2 plasmids were packaged with the mGluR6 promoter and mCherry reporter in adeno-associated virus (serotype 2). The synthetic plasmids were cloned into a pAAV MCS vector via the BamHl and Sal1 sites. AAV physical titer was obtained by quantitative PCR using a primer designed to selectively bind the AAV inverted terminal sequence. The TCID50 assay was performed according to the ATCC protocol. The purity of the purified virus was verified by SDS / PAGE. Figure 7A shows a fluorescence image of HEK293 cells expressing mCherry 2 days after transfection with AAV2-mGluR6-MCO1-mCherry. Robust expression was observed without any detectable morphological changes, confirming that the transfected cells were healthy. For in vivo transfection of rd10 mice, intravitreous injection of 1 μL of AAV2-mGluR6-MCO1-mCherry (vMCO1) was performed for targeted expression in ON-type bipolar cells. The uniformity of MCO expression was confirmed by 3D reconstruction from confocal mCherry expression in z-slice of the entire retinal cup of rd10 mice injected intravitreously with vMCO1 (Figure 7B).

[0095] Example 6 - rd10 mice (retinal degeneration 10, spontaneous missense point mutation in Pde6b) have a late-onset progressive retinal degeneration, which closely resembles the phenotype of human retinal photodegeneration. After anesthesia, rd10 mice were treated with AAV2-mGluR6-MCO1-mCherry (1 μL) solution (1.6 × 10⁻⁶). 12AAV2-mGluR6-MCO1-mCherry solution was injected into the vitreous cavity through the sclera using a sterile needle from a Hamilton syringe. The AAV2-mGluR6-MCO1-mCherry solution was injected into both eyes. The cornea was kept moist with buffered salt solution throughout the procedure. In vivo transfection of vMCO1 with four different final doses of vMCO1 was performed in the rd10 mouse retina. Mice in each group were euthanized and retinal tissue was collected at different time points after vMCO1 injection. Confocal fluorescence microscopy was performed to analyze eMCO1 expression in the retina. Reporter fluorescence expression levels (fluorescence intensity) of the transfected retina were assessed using confocal microscopy to evaluate the retention of MCO. Mice were sacrificed, the retina extracted, and imaged by confocal microscopy at different time points after vMCO1 injection. The rd10 mouse retina transfected with MCO showed clear expression of the reporter (mCherry) on the cell membrane in the target cell layer. In contrast to significant expression in eyes injected with vMCO1, no characteristic mCherry expression (background autofluorescence only) was observed in eyes injected with PBS, monitored for up to 16 weeks. Furthermore, no significant increase in mCherry expression (background autofluorescence only) was observed one week after injection of three different vMCO1 doses. eMCO1 expression was significantly elevated 4–8 weeks after intravitreal injection of vMCO1 (Figure 9B). In vivo viral transfection was performed to deliver eMCO1 to bipolar cells in the retina of the rd10 mouse model. eMCO1 expression was found to be localized to targeted retinal cells (Figure 9C). Figure 9D shows 1.6 × 10⁶ 12 Figure 9E shows a cross-sectional view of eMCO1 expression in the retina 16 weeks after intravitreal injection at a dose of VG / mL. Furthermore, the expression level was significant even 4 months after injection. Figure 9E shows the dynamics of MCO1 expression in the rd10 mouse retina at two different doses of vMCO1. Figure 9F shows 1.6 × 10⁻⁶ 12 This shows the inter-animal variability in MCO1-mCherry (eMCO1) expression in the retina of rd10 mice 16 weeks after transfection with vMCO1 at a dose of VG / mL (after background removal).

[0096] Example 7 – A visible radial-arm water maze was used to test the spatial memory and learning abilities of light-oriented, vMCO-treated rd10 mice (55). Briefly, the mouse was placed in the center of the maze, and the platform was positioned just below the water surface at one end of the arm. The mouse quickly learned to determine the location of the platform by utilizing a visual cue (an LED emitting light having the visible spectrum). The platform (one of the arms) provided the mouse with the reward of being able to rest instead of having to swim. The time to reach the platform and the number of errors made before finding the platform were quantified under both on and off light conditions. Data (video) recording was stopped when the mouse found the platform or before 60 seconds had elapsed after the mouse was dropped into the water to prevent the mouse from becoming exhausted from swimming. The selection of the drop location (center, side, edge) was randomized per mouse and per test. Exclusion criteria consisted of mice that did not swim (floated). Visual acuity in this study was determined by measuring the time it took to reach the platform and the number of errors the mouse made before reaching the platform as the quality of the visual stimuli (cues) decreased. Approximately 10 weeks postnatally, rd10 mice were intravitreal-injected with vMCO targeting bipolar cells. The platform provided the mouse with the reward of being able to rest instead of having to swim. Intravitreal injection of the vMCO-carrying virus resulted in a significant improvement in the visually induced behavior of rd10 mice when assessed by the radial arm water maze assay. Approximately 8 weeks postnatally, rd10 mice were intravitreal-injected with MCO targeting bipolar cells in the retina. Figure 10 shows the visually induced improvement in the behavior of rd10 mice in the radial water maze. Figure 10A shows time-course images of the visually induced behavior of rd10 mice in a radial water maze with white LED light before intravitreal vMCO1 injection. Figure 10B shows the behavior of rd10 mice with LED lights on, 6 weeks after vMCO1 injection.The distance and time traveled by rd10 mice transfected with MCO before reaching the platform was significantly shorter than that of rd10 mice. Figure 10C shows the time it took for rd10 mice treated with vMCO1 to find the platform after being dropped at the center of the maze with and without light. Mean ± SEM. N=5 per mouse. Figure 10D shows the time it took for rd10 mice treated with vMCO1 to find the platform after being dropped at side arms 2 and 4 of the maze with and without light. Figure 10E shows the time it took for rd10 mice treated with vMCO1 to find the platform after being dropped at edge arm 3 of the maze with and without light. Consistently with the time it took to find the platform, the number of errors made by rd10 mice transfected with MCO before reaching the platform was significantly smaller (<1) than that of untransfected mice (>2) (56). Figure 10F shows the number of error arms passed through by vMCO1-treated rd10 mice that fell in the center before finding the platform, both in the presence and absence of light. Figure 10G shows the number of error arms passed through by vMCO1-treated rd10 mice that fell on the side arms before finding the platform, both in the presence and absence of light. Mean ± SEM. N=5 per mouse. Figure 10H shows the number of error arms passed through by vMCO1-treated rd10 mice that fell on the edge before finding the platform, both in the presence and absence of light. Mean ± SEM. N=5 per mouse.

[0097] Figure 11 shows a follow-up study of visually induced improvements in the behavior of rd10 mice in a radial water maze. Data were collected to determine baseline (pre-viral transfection) and longitudinal (every 4 weeks for 4 months) visual acuity. Figure 11A shows a schematic diagram of the radial arm water maze used to test visually induced behavioral improvements in rd10 mice injected with vMCO1. Four weeks after injection, all mice significantly recovered visually induced behavior, which persisted throughout the 16-week trial. The number of errors made by rd10 mice transfected with MCO before arriving at the platform was significantly smaller (<1) than that of untransfected mice (>2) (56). Consistently with the number of error arms, the distance and time traveled by MCO-transfected mice before arriving at the platform was much shorter than that of rd10 mice (n=5 for both groups). Figure 11B shows the center of the maze (light intensity: 0.007 mW / mm²) before vMCO1 injection. 2 The time from injection to arrival at the platform is shown, depending on the post-injection period. N=5; mean ± SD. * P<0.05. Figure 11C shows the vicinity arm of the labyrinth before vMCO1 injection (light intensity: 0.014 mW / mm²). 2 The time from injection to the arrival of the rd10 mouse is shown, depending on the post-injection period. N=5; mean ± SD. * P<0.05. Figure 11D shows the side arm before vMCO1 injection (light intensity: 0.004 mW / mm²). 2 The time from injection to the arrival of the rd10 mouse is shown, depending on the post-injection period. N=5; mean ± SD. * P<0.05.

[0098] Most importantly, when rd10 mice treated with MCO were randomly placed in a series on five different arms of a radial water maze, the mice were able to find the platform (the sixth arm) from all the other arms without a single error. Furthermore, rd10 mice treated with MCO were able to find the platform in low light intensities (0.005~0.01 mW / mm²) comparable to ambient light levels. 2Even with this, the mice performed better in visually guided tasks. To determine the light intensity dependence of behavioral improvement in mice treated with vMCO1, the intensity of diverging LED light was set to 0.0005~0.03 mW / mm². 2 The following was changed: The average time it took for rd10 mice treated with vMCO1 to reach the platform was at an ambient light intensity level of 0.007 mW / mm². 2 The response time was <20 seconds. The behavioral score correlated with the light intensity and threshold for improving visually induced behavior, at 0.004 mW / mm². 2 It was determined that this was the case. Figure 12 shows the light intensity dependence of behavioral improvement in rd10 mice in a radial water maze. This is a comparison of the time from the center of the maze to the platform, depending on the time elapsed since injection, between two different light intensities. For the first time, opsin-treated mice were able to perform significantly better at such low light levels. Early behavioral studies using ChR2-treated mice used much higher light intensities, which is not suitable for the practical application of optogenetics in visual acuity recovery without the use of active light sources. Example 8 - Since the measurement of optokinetic response is commonly used to measure the threshold of the visual system in humans and animals (57, 58), this tool was used to evaluate the improvement of visual characteristics in rd10 mice with MCO-sensitized retinas. The advantage of this method is that it does not require prior training of the animals. Briefly, rd10 mice were placed on a platform (at the center of a drum) surrounded by rotating stripes (Figure 10). Optokinetic stimuli of varying speeds were applied, and the mean optokinetic response and the mouse scores were measured. Figure 13 shows the optokinetic evaluation of rd10 mice and rd10 mice sensitized with MCO. Figure 13A shows a quantitative comparison of the number of head movements in rd10 mice before and 8 weeks after vMCO1 injection at a vertical stripe rotation speed of 1 rpm (0.07 cpd). The light intensity at the center of the chamber was 0.001 mW / mm². 2 Figure 13B shows a quantitative comparison of head motility in rd10 mice before and 8 weeks after vMCO1 injection at a vertical stripe rotation speed of 2 rpm. The light intensity at the center of the chamber was 0.001 mW / mm². 2Even at this low light intensity, mice treated with MCO rotated their heads in response to rotating stripes, suggesting improved spatial vision.

[0099] Example 9 - Similar to wild-type (open-label) mice, rd10 mice treated with vMCO were observed to avoid bright light by moving away from and blocking it (Figure 14).

[0100] Example 10 - Chronic exposure of opsin-transfected retinal cells to light may raise concerns regarding their viability. Therefore, to evaluate any adverse effects of light exposure on retinal cell viability, wild-type mice and rd10 mice injected with MCO were exposed to ambient light intensity (i.e., approximately 0.01 mW / mm²). 2 ) has an intensity approximately 10 times higher (i.e., (0.1 mW / mm²) 2 The mice were exposed to white light (intensity: 0.1 mW / mm²) for 4 weeks (8 hours per day). After 4 weeks of light exposure, rd10 and wild-type (control) mice transfected with MCO were sacrificed, and retinal tissue was collected for immunohistochemical analysis. The retinas were immunostained with apoptosis markers and imaged using confocal microscopy. Figure 14 shows the viability of eMCO1-sensitized retinal cells after chronic light exposure. Figure 14A shows that rd10 mice treated with vMCO1, as well as wild-type (open-label) mice, avoid bright light by moving away from and blocking it (by piling up bedding material as indicated by the arrows). Figure 14B shows the results of exposure to 8 hours of white light (intensity: 0.1 mW / mm²) per day. 2 Figure 14C shows a fluorescence image of the caspase-3 (green) stained retina of vMCO1-treated rd10 mice four weeks after exposure to white light (intensity: 0.1 mW / mm²). 2The image shows a fluorescence image of the retina stained with caspase-3 (green) in wild-type mice four weeks after illumination. Quantitative comparison (Figure 14D) shows no significant cell death in either wild-type mice or rd10 mice injected with MCO, indicating that cell viability under chronic light exposure is not impaired. Apoptotic cells in the inner nucleus layer of rd10 mice treated with vMCO1 are 0%. Furthermore, the photosensitivity of MCO-expressing cells significantly reduces the light intensity required to generate an action potential, thus minimizing photo-induced chronic damage to retinal cells.

[0101] Example 11 - Optical incision / imaging using SDOCT was performed to monitor any changes in ocular structure following intravitreal injection of vMCO1. SDOCT images of the cornea, lens, and retina lwk after intravitreal vMCO injection in rd10 mice were compared with images before injection. Figure 15 shows the results of the evaluation of the structural integrity of the retina after vMCO1 injection in rd10 mice. Figure 15A shows the OCT image of the retina of rd10 mice after vMCO1 injection. Figure 15B shows a comparison of retinal thickness in four different rd10 mice before and one week after injection. No detectable changes (e.g., detachment) in the cornea, lens, or retina were observed after intravitreal injection of vMCO1. SDOCT images were analyzed using ImageJ. A quantitative comparison of retinal thickness before and one week after vMCO1 injection (Figure 15D) did not show any change in retinal thickness.

[0102] Example 12 - Gene therapy has been controversial over the past decade due to undesirable side effects (59, 60), but opsins (e.g., ChR2) have been reported to be non-toxic, not induce an immune response, and maintain stable cell membrane properties. Therefore, the safety of this approach was confirmed by monitoring the health of mice. For immunotoxicity studies, blood was collected in two different doses (Group 1: 1.66 × 10⁶). 10 , group 2: 1.66×10 11Blood was collected from mice before, 7 days after, and 14 days after intravitreal injection of vMCO (GC / mL) (N=5 / dose). After anesthesia, blood (approximately 0.2 mL) was collected from the facial vein (using a germ-free animal lancet) one week before intravitreal injection. After vMCO injection, blood was collected for analysis (Table 6.1). After completion of the study period, the mice were euthanized. To collect blood from the facial vein of the mice, a sparse freckle on the side of the jaw was selected and punctured with a lancet. Pro-inflammatory (IL-6 and IFN-γ) and anti-inflammatory (IL-10) cytokines in plasma were quantified using an ELISA kit. Figure 16 summarizes the ELISA quantification results of inflammatory cytokines, indicating that the intravitreal dose of vMCO was within safe limits. Figure 16A shows a quantitative comparison of plasma IL-6 (a pro-inflammatory marker) between Group 1 and Group 2 before, 7 days after, and 14 days after vMCO1 injection. Figure 16B shows a quantitative comparison of plasma IL-10 (an anti-inflammatory marker) between the two groups. Figure 16C shows a quantitative comparison of plasma IFN-γ (a pro-inflammatory marker) between the two groups before, 7 days after, and 14 days after vMCO1 injection.

[0103] Example 13 - After monitoring behavioral recovery of vision following intravitreal injection of MCO, mice were sacrificed and various organs were collected to analyze the diffusion of MCO expression in non-target tissue samples (eyes, heart, liver, muscle, skin, etc.). Organs were stored in 1.8 mL cryovials and kept at -80°C. Each vial was appropriately labeled with study number, animal identification number, extraction date, and organ name. qPCR detection of vector sequences in rd10 mice at different time points after injection showed minute amounts of MCO-1 DNA in the treated outer eye tissue, confirming the safety of our molecule and therapeutic method. Intravitreal administration of vMCO1 in the eye resulted in a locally limited distribution, minimizing off-target effects. Figure 17 shows the biodistribution of enhanced pluripotent opsin (vMCO1) packaged in AAV2. At the fixation point (1 week after injection), the vector copy number measured in the eye was found to decrease with decreasing injection volume. Furthermore, very small or undetectable amounts of vector DNA were found in the injected eye 4–8 weeks after injection. Biodistribution studies showed minimal or undetectable levels of vector in non-target organs of rd10 mice injected intravitreously. Vector DNA in the outer tissue of the treated eye, as determined by qPCR detection of vector sequences in rd10 mice at different doses and post-injection periods, was very small or undetectable. The biodistribution profiles and dynamics of transgene expression after administration of vMCO1 via intravitreous administration at multiple time points correspond to the onset of detection, peak vector / transgene levels, and decline / flattening regions of these levels.

[0104] Example 14 - To further evaluate the safety, specificity, and efficacy of our opsin, immunohistochemistry was performed on rd10 retina injected with vMCO1. Figure 18 shows the immunohistochemistry of retinal sections of eyes of rd10 mice injected with vMCO1. Figure 18A shows that MCO-mCherry (red) is selectively targeted and expressed in the inner nucleus layer (INL) of rd10 mice 8 weeks after intravitreous injection of vMCO1. The absence of arrestin (green) suggests complete loss of photoreceptors. Figure 18B shows PKCα staining (green) in rod bipolar cells expressing mCherry (red, endogenous) in rd10 mice 8 weeks after intravitreous injection of vMCO1. Figure 18B shows mGluR6 staining (green) in ON-type bipolar cells expressing mCherry (red) in rd10 mice 8 weeks after intravitreous injection of vMCO1. Figure 18D shows mCherry (green immunostaining) expression in the rd10 retina 8 weeks after intravitreous delivery of vMCO1 to rd10 mice. Figure 18E shows GFAP (green) in rd10 mice 18 weeks after intravitreous injection of vMCO1, as reported in photoreceptor-degenerated retinas. Figure 18F shows the absence of CD45 (green) expression, suggesting the lack of immune cells in rd10 mice 8 weeks after intravitreous injection of vMCO1.

[0105] The present invention provides a method for improving or restoring vision, comprising administering one of the compositions described herein to a subject. The compositions of the Enhanced MCO (eMCO) Method of the present invention can be delivered and packaged in plasmids or viral vectors comprising (i) MCO plasmid, (ii) rAAV-MCO, (iii) pAAV-MCO, and (iii) Lenti Virus-MCO. Delivery of the present invention is carried out in situ by transiently permeating the internal limiting membrane of the retina using an optimized formulation of AAA together with the molecule-MCO of the present invention (naked plasmid or virus).

[0106] Table 01: Amino acid and DNA sequence of multi-characteristic opsin-1 (MCO1)

[0107] Amino acid sequence: (Sequence ID 1)

[0108] DNA sequence:

[0109] Table 02: Amino acid and DNA sequence of multi-characteristic opsin-2 (MCO2). It contains a mutation 308 units later (S 142 L) and deletions of 7 amino acid residues (VNKGTGK) from the MCO1 sequence (Table 01).

[0110] Amino acid sequence: (Sequence ID 3)

[0111] Nucleotide sequence:

[0112] Table 03: Amino acid and DNA sequence of multi-characteristic opsin-IT (MCOIT). This contains an additional transmembrane sequence (TPARWVWISLYYAAFYVVMTGLFALCIYVLMQTI) after the 315 amino acid residues of MCO1 (Table 01).

[0113] Amino acid sequence: (Sequence ID 5)

[0114] Nucleotide sequence:

[0115] Table 04: Amino acid and DNA sequence of multi-characteristic opsin-2T (MCO2T). This contains an additional transmembrane sequence (TPARWVWISLYYAAFYVVMTGLFALCIYVLMQTI) after the 308 amino acid residues of MCO2 (Table 02).

[0116] Amino acid sequence: (Sequence ID 7)

[0117] Nucleotide sequence:

[0118] Table 05: DNA sequences of promoters (mGluR6m) used upstream of MCO sequences to target specific cells, as an example. CAGGGNNGATTGATTATTGACTAGTGATCTCCAGATGGCTAAACTTTTAAATCATGAATGAAGTAGATATTACCAAATTGCTTTTTCAGCATCCATTTAGATAATCATGTTTTTTGCCTTTAATCTGTTAATGTAGTGAATTACAGAAATACATTTCCTAAATCATTACATCCCCCAAATCGTTAATCTGCTAAAGTACA (SEQ ID NO: 9)

[0119] Table 06: DNA sequences of reporter stabilizers (mCherry) used downstream of MCO sequences to confirm expression in specific cells, as an example. ATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGGAACGGCCCGAGTTCGAGATCGAGGGCGAGGGCGAGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTG TCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTT CATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACACG CTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG TAA (SEQ ID NO: 10)

[0120] Table 07: Amino acid and DNA sequences of enhanced multi-characteristic opsin-1 (e-MCO1). This contains an MCO1 sequence with a ligation sequence (Table 01) and a biomarker stabilizer sequence (Table 06).

[0121] Amino acid sequence: (Sequence ID 11)

[0122] Nucleotide sequence:

[0123] [Table A]

[0124] Figure 19 shows the structure of Example 15-eMCO1 and the photocatalytic activation of its different domains. Blue light activates the transmembrane (TM) domain (ion channel) of eMCO1, enabling cation influx. Green and red light activate the non-TM intermediate domain (non-ion channel), causing a structural change in it, which in turn causes a structural change in the TM domain, promoting cation influx through the TM domain. Upon absorption of green light, the C-terminal domain (enhancer) emits red light, enhancing the overall effect of eMCO1. These unique structures of eMCO1 allow for rapid and efficient activation of MCO1 even with low levels of light spanning the blue to red spectrum. HEK cells transfected with eMCO1 (transfected with JetPrime) were subjected to patch-clamp experiments (voltage clamping) under different stimulation wavelengths. Figure 20A shows HEK cells transfected with eMCO1 under blue light intensity of 0.06 mW / mm² at 450 nm. 2 The inward current profile measured is shown. Figure 20B shows the inward current profile measured at a green (520 nm) light intensity of 0.06 mW / mm² in HEK cells transfected with eMCO1. 2 The inward current profile measured is shown. Figure 20C shows the inward current profile measured at a red (630 nm) light intensity of 0.06 mW / mm in HEK cells transfected with eMCO1. 2 The inward current profile measured is shown. BAPTA (a known calcium chelating agent) was added to the extracellular solution (maximum final concentration 100 mM). Figure 20D shows Ca 2+ In HEK cells transfected with eMCO1 in the presence of the chelating agent BAPTA, a blue (450 nm) light intensity of 0.06 mW / mm² was observed. 2 Figure 20E shows a decrease in the inward current profile measured by Ca. Figure 20E compares the photocurrents generated in cells by light of different wavelengths and Ca 2+This shows the effect of the presence of a chelating agent (BAPTA) on the main function of the eMCO transmembrane (activated by blue (450nm) light). The use of BAPTA reduced the current by more than half at 450nm, indicating that Ca 2+ This indicates that the ratio of permeability to other cation permeability is greater than 1.

[0125] eMCOs possess a broad activation spectrum, specific ionic conductivity, and photosensitivity, in addition to a fast on-off response rate. Figure 20F shows a quantitative comparison of the on-time (time from baseline to the -ve peak) of photocurrents generated by blue and green light. The on-time of photocurrents measured in cells was approximately four times longer with 520 nm light stimulation compared to 450 nm light stimulation.

[0126] Example 16 - To determine whether sensitization of retinal bipolar cells with eMCO1 can contribute to the maintenance of their tissue structure, retinal thickness was structurally compared by OCT measurement before and after intravitreal injection of different doses of AAV2-carrying eMCO1 protein (vMCO1 vehicle) and AAV vehicle (without eMCO1) in rd mice. 1.0 × 10⁶ mice in group AA received 12 1 μL of VG / mL AAV2-eMCO was intravitrealized, and 1.0 × 10⁶ mice in group BB received a 1.0 × 10⁶ dose. 12 A VG / mL AAV2 (without transgene, -ve control) was injected at a dose of 1 μL. Another group of CC mice received a low dose (1.0 × 10⁶). 10 1 μL of AAV2-eMCO (VG / mL) was injected. Figure 21A shows the time-course measurements of rd mouse retinal thickness before and after AAV2-eMCO or AAV2-vehicle injection. For each group, retinal thickness was measured before injection, 1 day after injection, 1 month after injection, and 4 months after injection. Figure 21C shows the mean difference in retinal thickness between baseline and intravitreal-injected mice using Gardner-Altman estimated plots. In the eMCO1 injection groups (AA and CC), retinal thickness was maintained over time compared to the vehicle injection group (BB). The stable retinal thickness observed in the eMCO1 injection group (compared to the vehicle injection group) is thought to be due to the stabilization of retinal thickness by suppressing further disturbance of the retinal layer.

[0127] Example 17 - To determine whether synaptic extension of bipolar cells and / or connections between bipolar cells and retinal ganglion cells are enhanced by eMCO1-mediated bipolar cell sensitization, rd10 mice were administered vMCO1 (1 μL intravitreous injection), and retinal sections were immunostained. Figure 22B shows the expression of reporter mCherry in bipolar cells (INL) 16 weeks after AAV2-eMCO1 injection. Figure 22C shows the quantitative results of eMCO1 (reporter mCherry) expression in the bipolar cells of three rd10 mice. Figure 22D shows a co-stained cross-sectional image of the retina of eMCO-injected rd10 mice, showing bipolar cell terminals (green: PKCa) and synaptic ribbon markers (red: CtBP) closely attached to retinal ganglion cells (RGCs). Figure 22E shows a similar immunostained cross-sectional image of the retina of untreated rd10 mice. In the retinas of AAV2-eMCO1-treated rd10 mice, PKCa immunostaining signals (*p<0.05) and CtBP signals at axon terminals were more strongly observed than in untreated mice (Figure 22F). These results indicate that although synaptic connections between bipolar cells and ganglion cells deteriorate in photoreceptor-degenerated retinas, eMCO1 sensitization (and ambient light activation) strengthens synaptic terminals, enabling the transmission of photoactivation signals from eMCO1-expressing bipolar cells to the brain via retinal ganglion cell axons (optic nerves).

[0128] Example 18 - The gene-nonspecific therapeutic effect of eMCO1 was demonstrated by visual acuity improvement in retinal degeneration mouse models of retinitis pigmentosa (rd1, rd10), Stargard (ABCA4- / -), and Lieber congenital amaurosis (LCA). Intravitreal injection of vMCO1 (1 μL, 3.5E12 vg / mL) resulted in improved visual behavior in a radial water maze. Figure 23A shows the time it took for a Stargard (ABCA4- / -) mouse to reach the platform from the side arm of the radial arm water maze (light intensity: 0.004 mW / mm²). 2Figure 23B shows the latency before and after injection of AAV2 (without transgene, -control). In the absence of transgene (eMCO), there was no statistically significant difference before and after injection of the AAV2 vehicle. On the other hand, after injection of AAV2-eMCO, the latency to find the light platform was significantly shortened. Figure 23B shows the time it took for Stargard (ABCA4- / -) mice to reach the platform from the side arm before and after injection of vMCO1 (light intensity: 0.004 mW / mm²). 2 This shows the effect. Similar behavioral effects were also obtained after vMCO1 injection in LCA mouse models (Figures 23C and 23D). No improvement in the water maze was observed in vehicle (AAV2) injected LCA mice (Figure 23C). On the other hand, the time to reach the platform (latency) was significantly reduced in the vMCO1-treated LCA mouse group compared to baseline (Figure 23D).

[0129] In Example 19, to determine whether bipolar cell sensitization with eMCO1 leads to electrophysiological recovery in addition to suppression of degeneration and conferring photosensitivity to the retina, Stargard mice were injected with AAV2-eMCO. Suppression of retinal degeneration progression in the Stargard mouse model was demonstrated by time-course monitoring of retinal thickness by SDOCT after vMCO1 injection. For comparison, an untreated mouse group was also followed over time. Comparing retinal thickness before vMCO1 injection, and at 4, 12, and 16 weeks, the vMCO1 injection group did not show a significant decrease in retinal thickness (degeneration progression was suppressed), while the untreated group showed a significant decrease in retinal thickness over time (Figure 24A). Electroretinography (ERG) performed on vMCO1-treated Stargard mice showed a 1 cds / m² decrease compared to the vehicle injection group. 2 and 10 cds / m 2 The ERG response was enhanced (Figures 24B and 24C). Both SD-OCT and ERG measurements demonstrated the therapeutic effect of eMCO treatment for visual acuity restoration (inhibition of retinal degeneration progression and improvement of photosensitivity).

[0130] Example 20 - Figure 25 shows a method for restoring visual acuity by intraocular AAV2-eMCO administration. When the retina (100) degenerates, retinal ganglion cells (110) and bipolar cells (120) survive. On the other hand, photoreceptors (130) and / or retinal pigment epithelium (140) mutate or disappear (150). In subjects with such retinal degeneration, AAV-carrying eMCO (vMCO1) (170) is administered intraocularly using a delivery device (160). Our animal and human studies have shown that vMCO1 propagates to the contralateral eye (210) via the optic nerve (180), optic tract (190), and optic chiasm (200). This contralaterally propagated AAV-eMCO transduces retinal cells (220). Bipolar cells (240) are activated by natural scenes within the visual field or projection patterns onto the retina (230), which in turn activate retinal ganglion cells (250). Electrochemical signals (260) are transmitted to the brain (270) via the optic nerve, where the photoactivation signals from the eMCO1-sensitized retina are visually processed.

[0131] Example 21 - This example demonstrates that contralateral transmission is possible when an AAV containing the gene encoding eMCO1 is injected intravitreously. After intravitreous injection into mice, the eMCO1 gene expressed in the retina was observed in both the injected eye and the contralateral eye (Figure 26A: injected eye, 26B: contralateral eye). In rd10 mice, eMCO1 (reporter mCherry) expression was observed in both the injected and non-injected eyes 6 months after injection, confirming contralateral transmission of vMCO1. In dogs, transmission to the contralateral eye was also observed 4 months after injection into one eye (75 μL of 2E11VG / mL AAV2-eMCO). The transgene (eMCO + mCherry enhancer / reporter) was measured by immunofluorescence staining (Figure 26C: injected eye, Figure 26D: contralateral eye). Furthermore, qPCR was performed to quantify the vMCO1 vector copy number. Figure 26E shows the number of vector copies measured in the left and right optic nerves, optic chiasm, and left and right lateral geniculate nuclei (LGN) in dogs that received intravitreal injection of vMCO1 into the right eye (only). This data indicates that vMCO1 is transported contralaterally via the optic nerve and optic chiasm.

[0132] Example 22 - To verify whether contralateral propagation of vMCO1 (as seen in animals) occurs in humans after unilateral injection, a single intravitreal injection of vMCO1 was administered to the eye with greater visual acuity loss (the eye with lower visual acuity) in patients with progressive retinitis pigmentosa. To evaluate eMCO1 expression after vMCO1 injection, the Topcon Triton Plus imaging system was used, and reporter (mCherry) fluorescence was observed in fundus autofluorescence images with a red filter. As shown in Figure 27A, reporter fluorescence increased in the injected eye. Similarly, reporter fluorescence intensity also increased in the contralateral eye 8 weeks after vMCO1 injection. In addition to increased diffuse fluorescence in the fundus, patchy and spotty fluorescence was also observed (Figures 27A, 27B). To verify whether eMCO1 expression in the retina contributes to the suppression of retinal degeneration progression in patients with retinitis pigmentosa, retinal thickness in the injected eye and the contralateral eye was monitored and quantified over time using OCT images before and after injection (Figure 27C). No change in mean retinal thickness was observed after vMCO1 injection. Compared to baseline, there was no significant change in retinal thickness at 52 weeks, suggesting suppression or delay of degenerative progression.

[0133] Example 23 - To evaluate visual acuity recovery with vMCO1 in humans, 11 patients with progressive retinitis pigmentosa received a single intravitreal injection of 1.75E11vg / eye (low-dose group n=3) and 3.5E11vg / eye (high-dose group n=8) into the eye with the lowest visual acuity. In the high-dose group, the mean visual acuity in the injected eye showed an improvement of more than 0.68 logMAR compared to 0.1 logMAR at 16 weeks after injection. Notably, at 16 weeks after injection, 7 out of 8 patients in the high-dose group showed an improvement of more than 0.3 logMAR compared to baseline, and 6 out of 8 patients showed an improvement of more than 0.6 logMAR. Figure 28 shows the time course of visual acuity (LogMAR) in the injected eye and the contralateral eye of patients with severe retinal degeneration, using vMCO1 (3.5E11vg). Visual acuity improvement in the contralateral eye was also observed with intravitreal injection of AAV2-eMCO in humans. The mean visual acuity improvement in the contralateral eye of the high-dose group was 0.34 logMAR at 16 weeks, compared to 0.68 logMAR in the injected eye (Figure 28).

[0134] Example 24 - To determine whether opsin sensitization and optogenetic stimulation of retinal ganglion cells enable axonal regeneration and lead to visual acuity recovery, visual guidance behavior in mice at baseline and after optic nerve injury was examined in two groups: (i) a group without optogenetic stimulation and (ii) a group with optogenetic stimulation. Figure 29A shows a schematic diagram of the visual guidance Y-maze assay used to evaluate mouse visual acuity. Figure 29B shows the number of events (detection of light ON vs. OFF) by mice under different conditions. Six trials were performed for each condition. In baseline measurements (no optic nerve injury), the number of mice that detected the light ON (+light) panel in the Y-maze was significantly higher than the number of events that detected the light OFF (-light) panel. After optic nerve injury (ON crash), the number of events detecting light ON and OFF became equal. After optic nerve injury, mice were exposed to strobe (0.5 Hz) light (Philips Hue) stimulation (8 hours per day). In animals with optic nerve damage, improvement in the visual guidance behavior of mice towards a light-on panel was observed two weeks after strobe light stimulation, and this behavior was similar to that of baseline (before the ON crash). This suggests that optogenetic stimulation of opsin-sensitized retinal ganglion cells led to the regeneration of axons degenerated by the injury (ON crash), resulting in improved vision.

[0135] Example 25 - To determine whether administration of eMCO1 results in any visual acuity improvement in patients with Stargardt macular degeneration, these patients were administered 1.2E11gc / eye via a single intravitreal injection. Figure 30A shows the time-course improvement in visual acuity letter scores measured using the ETDRS visual acuity chart in eMCO1-treated patients. As shown in Figure 30A, an increase of approximately 11 letters in the score was observed at 24 weeks after eMCO1 treatment compared to baseline. Figure 30B shows the time-course improvement in visual acuity in eMCO1-treated subjects using a VR headset (100% contrast, 3x zoom). Visual acuity letter scores were measured by presenting the ETDRS visual acuity chart to the patients at a distance of 50 cm. When the VR headset was used at 3x zoom (100% contrast), the increase in letter scores after eMCO1 injection was further exaggerated, with an increase of approximately 30 letters at 24 weeks (Figure 30B).

[0136] The terms “one (a)” and “one (an)” are defined as one or more unless the Disclosure expressly requires otherwise. The term “substantially” is defined as largely, though not necessarily, specified (and including, for example, substantially 90 degrees includes 90 degrees, substantially parallel includes parallel) as understood by those skilled in the art. In any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be replaced with “within ~[percent]” as specified, where the percentage includes 0.1%, 1%, 5%, and 10%.

[0137] Furthermore, a molecule or method configured in a particular manner may be configured in at least that manner, but in a manner other than those specifically disclosed.

[0138] The terms “comprise” (and any form of “comprise,” e.g., “comprises” and “comprising”), “have” (and any form of “have,” e.g., “has” and “having”), “include” (and any form of “include,” e.g., “includes” and “including”), and “contain” (and any form of “contain,” e.g., “contains” and “containing”) are inclusive conjunctions. As a result, a device that “comprises,” “has,” “includes,” or “contains” one or more elements includes one or more elements, but is not limited to having only one or more elements. Similarly, a method that “comprises,” “has,” “includes,” or “contains” one or more steps includes one or more steps, but is not limited to having only one or more steps.

[0139] Any embodiment of an apparatus, system, or method may consist of, or essentially consist of, any of the described steps, components, and / or features, rather than comprise, include, contain, or have. Therefore, in any of the claims, “consist of” or “essentially consist of” may be replaced with any of the above-mentioned comprehensive conjunctions to alter a given claim using a comprehensive conjunction in any other form.

[0140] Even if not described or shown, one or more features of one embodiment may be applied to other embodiments unless expressly prohibited by this disclosure or expressly prohibited by the nature of the embodiment.

[0141] The inventions disclosed herein will be further described below as embodiments provided for illustrative purposes only, but should not be construed as limiting the scope of the invention.

[0142] Some references, including publications, patents, and patent specifications, are cited and mentioned in the description of the present invention. Such citations and / or descriptions of references are provided solely to clarify the description of the present invention and do not constitute an endorsement that any such reference is “prior art” of the present invention as described herein. All references cited and mentioned herein are incorporated in whole as they are incorporated individually as they are incorporated individually.

[0143] This specification and examples provide a complete description of the structure and use of exemplary embodiments. While specific embodiments have been described with particular specificity or by reference to one or more individual embodiments, those skilled in the art can make many modifications to the disclosed embodiments without departing from the scope of the invention. Thus, it is not intended to limit the various exemplary embodiments of the apparatus to the specific forms disclosed. Rather, embodiments other than those shown may include some or all of the features of the shown embodiments, including all modifications and substitutions that fall within the claims. For example, components may be omitted or combined into a single structure, and / or connections may be substituted. Furthermore, where appropriate, aspects of any embodiment described above may be combined with aspects of any other embodiment described to create further embodiments having comparable or different characteristics and addressing the same or different problems. Similarly, it will be understood that the advantages and merits described above may relate to one embodiment or to several embodiments.

[0144] Furthermore, unless such a limitation is explicitly referenced in the given claims using the phrases "means for..." or "steps for...", the claims are not intended to include, and should not be interpreted as including, a means-plus-function or step-plus-function limitation.

[0145] It should be understood that the disclosures of the present invention are included, subject to the condition that any such disclosures already made are waived, to the extent that the specific disclosures of references or other documents can be considered to predict any general aspects of the present invention. Aspects of the present invention are not predicted by the disclosures of such documents, but are not obvious from the disclosures of those publications either, by at least some of the unexpected superior results disclosed herein.

Claims

1. A method for restoring vision in a patient suffering from retinal dystrophy or retinal degeneration, wherein a vector containing nucleic acids having 75%, 85%, 95%, or 100% identity with at least one of Sequence ID No. 1, 3, 5, 7, or 11 is administered to the patient suffering from retinal degeneration or retinal dystrophy.

2. The method according to claim 1, wherein the nucleic acid comprises a sequence of a broadband eMCO protein including a blue light-sensitive transmembrane ion channel domain to which a ligand, a non-transmembrane domain, and an enhancer having a fluorescent reporter and stabilizing activity are attached.

3. The method according to claim 2, wherein the eMCO1 protein responds to blue, green, and red light.

4. The method according to claim 2, wherein the eMCO1 protein has enhanced calcium ion permeability compared to the permeability of the eMCO1 protein to other cations.

5. The method according to claim 2, wherein the ligand attached to the transmembrane domain of the eMCO1 protein is sensitive to green light and red light.

6. The method according to claim 5, wherein the higher photosensitivity of the eMCO1 protein is due to the added ligand domain.

7. The method according to claim 2, wherein the non-transmembrane ligand domain of the eMCO1 protein enhances the ON dynamics of green and red light-induced currents via the transmembrane domain of eMCO1 compared to the ON dynamics of blue light-induced currents via the transmembrane domain of eMCO1.

8. The method according to claim 2, wherein the reporter domain of the eMCO1 protein is activated by light, and the re-emission of light from the reporter domain is captured by a red light-sensitive ligand, thereby enhancing the complex photosensitivity.

9. The method according to claim 1, comprising administering a nucleic acid having a coding region having 75%, 85%, 95%, or 100% identity with at least one of sequence numbers 1, 3, 5, 7, or 11 to a patient in need, to halt retinal degeneration or retinal dystrophy.

10. A method for treating a degenerative retinal disease causing vision loss in a patient, comprising administering recombinant eMCO1 gene intraocularly to the patient by intravitreous, subretinal, or choroidal injection using a vector containing nucleic acid including the promoter-eMCO1 gene, and further administering the vector containing the promoter-eMCO1 gene to the patient in combination with one or more pronase E or α-aminoadipic acid (AAA).

11. The method according to claim 10, wherein after injecting a vector containing the promoter-eMCO1 gene into the eye, one or both of the chemical and / or physical transduction methods are administered to the patient's eye.

12. The method according to claim 10, wherein the internal limiting membrane is peeled off before the injection of the vector containing the promoter-eMCO1 gene into the eye.

13. The method according to claim 10, wherein administration of a vector containing the promoter-eMCO1 gene results in local expression of the eMCO1 gene in a retinal degenerative area of ​​the targeted retina.

14. The method according to claim 10, wherein after injection of a vector containing the promoter-eMCO1 gene, the patient exhibits improved visual guidance behavior.

15. The method according to claim 10, wherein a vector containing the promoter-eMCO1 gene is administered to a patient two or more times.

16. A method for maintaining retinal thickness, comprising administering to a patient in need a vector containing nucleic acids having 75%, 85%, 95%, or 100% identity with at least one of SEQ ID NOs: 1, 3, 5, 7, or 11.

17. The method according to claim 15, wherein administration of the vector containing the nucleic acid to a retinal degenerated eye results in transgene expression in a non-injected eye.

18. The method according to claim 15, wherein improvements in retinal function and visual behavior are observed in both the injected eye and the uninjected eye.

19. The method according to claim 1, wherein the expression and photoactivation of eMCO1 in cells improves structural order and intercellular connectivity in a target tissue.

20. The method according to claim 1, wherein the method of vision restoration is carried out in a patient in need thereof through the regeneration of damaged retinal ganglion cell (RGC) axons, the patient is administered a vector containing nucleic acids having 75%, 85%, 95%, or 100% identity to at least one of SEQ ID NOs: 1, 3, 5, 7, or 11, and further, the eMCO1 gene is delivered to the RGCs before or after axonal degeneration, and photostimulation of the eMCO1-sensitized RGCs results in a reduction of the rate of degeneration and / or axonal regeneration, and the photostimulation dose is optimized to minimize degeneration and / or maximize axonal regeneration.

21. The method according to claim 10, wherein the eMCO1 gene is administered to a patient having a macular degeneration disorder, and the macular degeneration disorder includes either or both of Stargardt macular degeneration and progressive age-related macular degeneration.

22. The method according to claim 21, wherein administration of nucleic acid containing the MCO1 gene to a patient suffering from macular degeneration of the eye results in improvement of the patient's visual acuity and other visual functions.

23. The method according to claim 22, wherein the improvement of visual acuity and other visual functions is enhanced by the use of a contrast enhancement and zoom device.

24. The method according to claim 23, wherein the contrast enhancement and zoom device is a VR headset.

25. The method according to claim 10, wherein the optogenetic therapy is administered to a patient having a macular degeneration disorder, the macular degeneration disorder includes either or both of Stargardt macular degeneration and progressive age-related macular degeneration.

26. The method according to claim 25, wherein the administration of nucleic acids, including optogenetic therapy, to a patient suffering from macular degeneration of the eye results in an improvement in the patient's visual acuity and other visual functions.

27. The method according to claim 26, wherein the improvement of visual acuity and other visual functions is enhanced by the use of a contrast enhancement and zoom function device.

28. The method according to claim 27, wherein the contrast enhancement and zoom device is a VR headset.

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