Novel GPCR chimeric opsin for the treatment of retinal neurodegenerative diseases

Abstract

This application relates to a novel GPCR chimeric opsin for the treatment of retinal neurodegenerative diseases. Specifically, this application relates to a GPCR chimeric opsin comprising a recombinant opsin formed by replacing a corresponding domain of the natural opsin with at least one domain derived from mGluR4; a nucleic acid molecule and a vector encoding the chimeric opsin; a transgenic cell comprising the chimeric opsin, the nucleic acid molecule, or the vector; and uses thereof.

Description

Technical Field

[0001] This application relates to the field of treatment of eye diseases. Specifically, this application relates to a GPCR chimeric opsin comprising a recombinant opsin formed by replacing a corresponding domain of a natural opsin with at least one domain derived from mGluR4; a nucleic acid molecule and a vector encoding the chimeric opsin; a transgenic cell comprising the chimeric opsin, the nucleic acid molecule, or the vector; and uses thereof. Background Technology

[0002] Retinal neurodegenerative diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are blinding eye diseases caused by the degeneration and death of key retinal cells such as photoreceptor cells, leading to loss of visual function or even blindness. This pathogenesis of retinal neurodegenerative diseases severely impairs patients' vision, and current treatments cannot prevent irreversible visual loss. The pathogenesis of retinal neurodegenerative diseases is often due to apoptosis caused by gene mutations in photoreceptor cells or an imbalanced retinal microenvironment (such as neovascularization and death of retinal pigment epithelial cells) that exacerbates photoreceptor cell apoptosis. Based on the pathogenesis, treatment strategies mainly focus on: 1) overexpressing or gene editing to repair related mutated genes, delaying photoreceptor cell degeneration; 2) inhibiting angiogenesis to reduce damage to photoreceptor cells; and 3) enabling other retinal neurons (such as bipolar cells or ganglion cells) to express photosensitive proteins and perform photosensitivity.

[0003] The strategy of restoring vision by utilizing the expression of photosensitive proteins by other neurons in the retina is currently the most popular research topic. Photosensitive proteins include endogenous and exogenous photosensitive proteins. Among them, the use of exogenous photosensitive proteins, namely light-sensitive ion channel proteins (chr2, chrimsonR, MCO, etc.), has achieved vision restoration in mice, and related products are already in clinical trials, with preliminary clinical results being quite positive.

[0004] Endogenous photosensitive proteins mainly include opsin, rhodopsin, and melanopsin. Opsin is expressed in retinal cone cells and, based on its optimal sensing wavelength, is divided into LW-opsin (sensing long wavelengths), MW-opsin (sensing medium wavelengths), and SW-opsin (sensing short wavelengths). Rhodopsin is expressed in retinal rod cells, and melanopsin is expressed in photosensitive retinal ganglion cells. All three photosensitive proteins belong to the G protein-coupled receptor (GPCR) family. GPCRs are an important superfamily of membrane proteins, with over 800 members identified to date. They consist of a classically conserved seven-transmembrane (7TM) helix, three extracellular loops, three intracellular loops, an N-terminal region, and a C-terminal region. Extracellular chemical substances, acting as signaling molecules (ligands), bind to GPCRs to mediate downstream intracellular signal transduction, thereby triggering a series of physiological processes. Based on sequence and structural similarity, GPCRs can be divided into five subfamilies: rhodopsin (family A), secretin (family B1), adhesin (family B2), glutamate (family C), and Taste2 / Frizzled (family F). Different families have different structural characteristics and endogenous ligand binding sites. All three endogenous photosensitizing proteins belong to family A. For family A receptors, in most cases, endogenous ligands are recognized by the orthocortical site in the 7TM domain. Researchers have found that these three endogenous photosensitizing proteins can be applied to vision restoration in blind mice, and this is currently in the early stages of development.

[0005] The study also found that fusing endogenous photosensitive proteins with metabolized glutamate receptors (mGluR) could partially restore vision in blind mice. mGluR belongs to the class C G protein-coupled receptor (GPCR) family and is one of the most important neurotransmitter receptors in the human body. Based on amino acid sequence homology and differences in intracellular signal transduction mechanisms, the eight mGluRs were divided into three groups: Group I includes mGluR1 and mGluR5, which mainly work by coupling with G… q Activation of phospholipase C promotes the hydrolysis of intracellular phosphatidylinositol diphosphate to inositol triphosphate and diacylglycerol, leading to intracellular calcium... 2+ Concentration increased; Group II includes mGluR2 and mGluR3, via coupling with G i / o Inhibition of cyclic adenosine monophosphate formation and voltage-sensitive calcium 2+ Channel, activate K + Channels; Group III includes mGluR4, mGluR6, mGluR7, and mGluR8, which are also related to G. i / oCoupled with inhibition of adenylate cyclase activity. To date, researchers have fused melanopsin and MW-opsin with mGluR6 (ICL2, ICL3, CT) and expressed them in retinal bipolar cells, resulting in partial vision recovery in blind rd1 mice. This strategy of using photosensitive proteins fused with mGluR chimeras to treat retinal neurodegenerative diseases is currently in the early stages of development.

[0006] US20140171376A1 describes a chimeric GPCR protein suitable for coupling light signals with a signal transduction cascade of mGluR6 in ON bipolar cells of the inner retina, comprising domains of at least two members of the G protein-coupled receptor (GPCR) protein family, characterized in that the first of the at least two members is a photosensitive GPCR that facilitates at least some of its light-mediated activation domains, and the second of the at least two GPCR family members is a domain that facilitates at least some of its ability to couple light activation with a signal transduction cascade of mGluR6, wherein fusion of the endogenous photosensitive protein melanopsin with mGluR6 enables visual restoration in blind rd1 mice.

[0007] US20230049217A1 describes a recombinant expression vector comprising a first inverted terminal repeat (ITR) polynucleotide sequence, a promoter polynucleotide sequence operatively linked to a polynucleotide sequence encoding a mid-wavelength cone opsin (MW-opsin) transgene, a polyA polynucleotide sequence, and a second ITR polynucleotide sequence.

[0008] CN111417339A describes a method for restoring or enhancing visual function in an individual, the method comprising administering to the individual a nucleic acid containing a nucleotide sequence encoding one or more intermediate-wavelength cone opsin (MW-opsin), long-wavelength cone opsin (LW-opsin), and short-wavelength cone opsin (SW-opsin), wherein one or more of the MW-opsin, LW-opsin, and SW-opsin are expressed in the individual's retinal cells, thereby restoring or enhancing visual function.

[0009] WO2021105509A1 describes chimeric opsin mGluR6 proteins that exhibit one or more particularly advantageous properties for use in gene therapy, for example, in patients with partial or complete vision loss due to a lack or inadequacy of photosensitive signal transduction activity provided by natural photoreceptors. In this study, fusing the endogenous photosensitive protein melanopsin with mGluR6 or fusing the endogenous photosensitive protein MW-opsin with mGluR6 can both achieve vision restoration in blind rd1 mice.

[0010] Gaub, Benjamin M. et al. (Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity, 2015, 23(10), 1562-1571) described the endogenous photosensitive protein rhodopsin as being used to restore vision.

[0011] Therefore, the field has been dedicated to researching and finding GPCR chimeric opsins with low immune rejection, high sensitivity, strong efficacy and / or wide distribution to treat various retinal neurodegenerative diseases. Summary of the Invention

[0012] The inventors have unexpectedly discovered that expressing GPCR chimeric opsins in other retinal neurons (retinal ganglion cells) can restore vision in blind mice, providing a new treatment strategy for restoring sight to blind patients in clinical practice through similar means. Furthermore, this GPCR chimeric opsin exhibits low immune rejection, high sensitivity, strong efficacy, and / or wide distribution. Additionally, using the GPCR chimeric opsin (mGluR4 chimeric opsin) of this invention has the potential to achieve better results in improving vision and / or treating various retinal neurodegenerative diseases than the existing mGluR6 chimeric opsin. Moreover, the GPCR chimeric opsin (mGluR4 chimeric opsin) of this invention can be expressed in different target cells than the existing mGluR6 chimeric opsin (the mGluR4 chimeric opsin of this invention can be expressed in retinal ganglion cells, while the existing mGluR6 chimeric opsin is expressed in bipolar cells).

[0013] In one aspect, the present invention provides a GPCR chimeric opsin comprising a recombinant opsin formed by replacing a corresponding domain of a native opsin with at least one domain derived from mGluR4. In some embodiments, the native opsin is a mid-wavelength opsin (MW-opsin). In some embodiments, the at least one domain derived from mGluR4 is selected from ICL2, ICL3, a C-terminus, and combinations thereof. In some embodiments, the at least one domain derived from mGluR4 is composed of ICL2, ICL3, and a C-terminus. In some embodiments, the chimeric opsin comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:3.

[0014] MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWM

[0015] IFVVIASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISVVNQVYGYFVL

[0016] GHPMCVLEGYTVSLCGITGLWSLAIISWERIYRIFEQGKRSVSAPRFISPASQVGIAFSWIWA

[0017] AVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCITPLSIIVLCYLQ

[0018] VWLKTRGVPETFNEAKMVVVMVLAFCFCWGPYAFFACFAAANPGYPFHPLMAALPAFFA

[0019] KSATIYNPVIYVFMNRQPEQNVPKRKRSLKAVVTAATMSNKFTQKGNFRPNGEAKSELCENLEAPALATKQTYVTYTNHAI (SEQ ID NO: 3).

[0020] In some embodiments, the chimeric opsin further comprises other light-sensitive ion channel proteins, such as chr2, chrimsonR, MCO, etc., or their functional derivatives. In some embodiments, the recombinant opsin is formed by replacing the ICL2, ICL3, and C-terminus of the native opsin with ICL2, ICL3, and C-terminus derived from mGluR4, respectively. In some embodiments, the ICL2, ICL3, and C-terminus derived from mGluR4 have the sequences IYRIFEQGKRSVSAPRFISPASQ (SEQ ID NO:5), KTRGVPETFNEAK (SEQ ID NO:6), and PEQNVPKRKRSLKAVVTAATMSNKFTQKGNFRPNGEAKSELCEN LEAPALATKQTYVTYTNHAI (SEQ ID NO:7), or are composed of said sequences. In some embodiments, the GPCR chimeric opsin further comprises a signal peptide at its C-terminus, such as a Golgi output signal sequence like RSRFVKKDGHCNVQFINV (SEQ ID NO:8) and / or a rhodopsin transport sequence like TETSQVAPA (SEQ ID NO:9). In some embodiments, the chimeric opsin comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:1.

[0021]

[0022] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the GPCR chimeric opsin described herein, or comprising a nucleic acid molecule described herein. In some embodiments, the vector is an AAV vector. In some embodiments, the nucleotide sequence encoding the GPCR chimeric opsin described herein is operatively linked to a bipolar cell-specific promoter (e.g., mGlu6) or a ganglion cell-specific promoter (e.g., SNCG), preferably a ganglion cell-specific promoter (e.g., SNCG).

[0023] In another aspect, the present invention provides transgenic cells comprising the GPCR chimeric opsin, the nucleic acid molecule, or the vector described herein. In some embodiments, the transgenic cells are transgenic retinal ganglion cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are non-human cells. In some embodiments, the cells are retinal cells. In some embodiments, the cells are bipolar cells or ganglion cells. In some embodiments, the cells are in vitro. In some embodiments, the cells are in vivo.

[0024] In another aspect, the present invention provides the use of the GPCR chimeric opsin, the nucleic acid molecule, the vector, or the transgenic cell described herein in the preparation of a medicament for improving the vision of a subject in need. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the improvement includes administering the GPCR chimeric opsin, the nucleic acid molecule, or the vector to retinal ganglion cells. In some embodiments, the transgenic cell described herein is a transgenic retinal ganglion cell.

[0025] In another aspect, the present invention provides the use of the GPCR chimeric opsin described herein, the nucleic acid molecule described herein, the vector described herein, or the transgenic cells described herein in the preparation of a medicament for treating retinal neurodegenerative diseases in a subject of need. In some embodiments, the retinal neurodegenerative disease is retinitis pigmentosa (RP) or age-related macular degeneration (AMD). In some embodiments, the subject is a mammal, such as a human. In some embodiments, the treatment comprises administering the GPCR chimeric opsin described herein, the nucleic acid molecule described herein, or the vector described herein to retinal ganglion cells. In some embodiments, the transgenic cells described herein are transgenic retinal ganglion cells.

[0026] In another aspect, the present invention provides a composition comprising the GPCR chimeric opsin described herein, the nucleic acid molecule described herein, the vector described herein, or the transgenic cell described herein.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising the GPCR chimeric opsin described herein, the nucleic acid molecule described herein, the vector or transgenic cell described herein, and a pharmaceutically acceptable carrier.

[0028] In another aspect, the present invention provides a cell transduced by or containing the viral vector described herein. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a non-human cell. In one embodiment, the cell is a retinal cell. In one embodiment, the cell is a bipolar cell or ganglion cell. In one embodiment, the cell is in vitro. In one embodiment, the cell is in vivo.

[0029] In another aspect, the present invention provides a method for treating retinal neurodegenerative diseases, the method comprising administering a therapeutically effective amount of the GPCR chimeric opsin described herein, the nucleic acid molecule described herein, the vector described herein, the transgenic cells described herein, or the composition or pharmaceutical composition described herein to the eye of a subject in need. In some embodiments, the retinal neurodegenerative disease is retinitis pigmentosa (RP) or age-related macular degeneration (AMD). In some embodiments, the subject is a mammal, such as a human. In some embodiments, the method comprises administering the GPCR chimeric opsin described herein, the nucleic acid molecule described herein, or the vector described herein to retinal ganglion cells. In some embodiments, the transgenic cells described herein are transgenic retinal ganglion cells. Attached Figure Description

[0030] The invention will be better understood in conjunction with the accompanying drawings.

[0031] Figure 1 The construction and in vitro validation of the GPCR chimeric opsin AAV expression vector are shown. A, Schematic diagram of the GPCR chimeric opsin AAV expression vector. B, The GPCR chimeric opsin EGR0 C-terminus co-expresses green fluorescent protein EGFP via P2A. Transfection of 293T cells in vitro revealed EGFP expression, indirectly indicating that EGR0 can fold and express normally. C, Flow cytometry sorting using a FLAG tag showed that EGR0 can be located normally and precisely on the cell membrane surface.

[0032] Figure 2This study demonstrates that the RGC promoter is specifically expressed in retinal ganglion cells. Adult normal C57 mice (12 weeks old) were injected subvitreal with AAV. Two weeks later, confocal imaging of frozen retinal sections showed that EGFP was specifically expressed in retinal ganglion cells. The left image shows the DAPI image (blue), the middle image shows the EGFP image (green), and the right image shows the fusion of DAPI and EGFP (blue + green).

[0033] Figure 3 The results show that EGR0 is efficiently expressed in retinal ganglion cells. In C3H mice (12 weeks old) with photoreceptor degeneration and blindness, AAV was injected subvitrectally. Retinal retinal slices 7 weeks later showed that EGR0 was efficiently expressed in retinal ganglion cells (A), with their axons converging at the optic nerve (C). Image B shows a local image of the optic mastoid process.

[0034] Figure 4 The placement response scores of C3H mice at different time points after drug administration are shown. Detailed Implementation

[0035] Several aspects of the invention are described below with reference to illustrative examples. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. However, those skilled in the art will readily recognize that the invention may be practiced without one or more of these specific details or may be practiced in other ways.

[0036] Retinitis pigmentosa, age-related macular degeneration, and other retinal neurodegenerative diseases ultimately end in the degeneration and apoptosis of retinal photoreceptor cells, causing irreversible vision loss. This invention utilizes gene therapy to express photosensitive GPCR chimeric opsins in retinal ganglion cells using adeno-associated virus (AAV) as a delivery vector. This allows these cells to retain their original physiological functions while also acquiring the ability to sense external light.

[0037] The GPCR chimeric opsin developed in this invention is formed by replacing the ICL2, ICL3, and CT domains of MW-opsin with the ICL2, ICL3, and CT domains of mGluR4, respectively. mGluR4 is widely distributed in retinal ganglion cells. Therefore, without being bound by theoretical constraints, if the GPCR chimeric opsin is specifically expressed in retinal ganglion cells and localized on the cell membrane surface, the photosensitive portion of MW-opsin is responsible for sensing external light. The chimeric opsin is activated, and mGluR4 utilizes the retinal ganglion cell's own endogenous downstream signaling pathway (GPCR-G protein pathway) to further integrate the information, ultimately transmitting it to the brain's visual center via the optic nerve in the form of nerve impulses, i.e., visual recovery.

[0038] This invention utilizes this treatment strategy to express GPCR chimeric opsin in adult C3H mice with photoreceptor degeneration blindness (>7 weeks). The placement response indicates that their behavior is partially restored, meaning that blind mice can achieve repair of the entire visual pathway from eye to brain after expressing GPCR chimeric opsin.

[0039] In this invention, unless otherwise specifically stated, the use of the singular includes the plural, the term "a / a" means "at least one / a," and the use of "or" means "and / or." Furthermore, the open-ended expressions "comprising" and "including" are interpreted as potentially containing structural components or method steps not mentioned, but it should be noted that this open-ended expression also covers situations consisting solely of the stated components and method steps (i.e., it covers the closed-ended expression "consisting of").

[0040] As used in this article, the term "about" when used with a percentage or other quantity means that percentage or other quantity plus or minus 10%. For example, "about 80%" includes 80% plus or minus 8%.

[0041] All documents referenced in this application, including but not limited to patents, patent applications, articles, books, and treaties, are expressly incorporated herein by reference for any purpose. If a definition of a term in one or more included documents and similar materials contradicts the definition of that term in this application, the definition in this application shall prevail.

[0042] The terms “protein,” “polypeptide,” and “peptide” used in this invention are interchangeable unless otherwise specified.

[0043] When used in this invention, unless otherwise stated, the terms "treatment" and "therapy" refer to an action that occurs when a subject has a disease (e.g., retinal neurodegenerative disease) to reduce the severity of one or more symptoms or the impact of the disease. When used in this invention, unless otherwise stated, the term "prevention" refers to an action that occurs before a subject develops a disease (e.g., retinal neurodegenerative disease), delaying the onset of the disease and / or inhibiting or reducing the severity of the disease. It should be understood that treatment can be preventative or administered after a disease or condition has been diagnosed. The treatments of this invention can reduce or eliminate symptoms or features of an impairment, disease, or condition, or can eliminate the impairment, disease, or condition itself. It should be understood that the treatments of this invention can slow or eliminate the progression of a disease or impairment, and in some cases may lead to regression of the disease, impairment, or condition. In some specific embodiments of this invention, the GPCR chimeric opsin of this invention is expressed in a population of cells and used to treat retinal neurodegenerative diseases.

[0044] In this invention, unless otherwise stated, a "therapeuticly effective amount" of a compound is an amount sufficient to provide any therapeutic benefit in the treatment of a disease, or an amount sufficient to delay or alleviate one or more symptoms associated with the disease. A therapeutically effective amount of a compound refers to the amount of a compound used alone or in combination with one or more other therapies and therapeutic agents that provides any therapeutic benefit in the treatment of a disease. The term "therapeuticly effective amount" can include amounts that alleviate retinal neurodegenerative diseases, improve or reduce visual impairment, improve overall treatment, or enhance the efficacy of another therapeutic agent.

[0045] When used in this invention, "patient" or "subject" includes mammals that are suffering from or susceptible to the diseases described in this invention, such as humans and non-human mammals. Non-limiting examples of non-human mammals include rodents, mice, rats, non-human primates, companion animals (such as dogs and cats), and livestock (such as sheep, cattle, horses, etc.).

[0046] As used herein, “vision” or “visual acuity” is used interchangeably and is defined as the ability of an organism to effectively detect light as a stimulus. “Vision” or “visual acuity” is intended to encompass: (i) light detection or perception, i.e., the ability to discern the presence of light; (ii) light projection, i.e., the ability to discern the origin of a light stimulus; (iii) resolution, i.e., the ability to detect different levels of brightness (i.e., contrast) within a grid or letter target; and (iv) recognition, i.e., the ability to recognize the shape of a visual target by referring to different contrasts within the target. Therefore, “vision” or “visual acuity” includes the simple ability to detect the presence of light, preferably visible light, more preferably light with wavelengths between about 365 nm and about 700 nm, between about 480 nm and about 590 nm, and in some specific embodiments, peak activation may occur upon contact with light of about 530 nm wavelength.

[0047] When used in this invention, "functional derivative" includes "mutant," "variant," and "fragment," whether these terms are used together or alternately. While conserved substitutions of, for example, 2, 3, 4, or 5 residues are also in accordance with the spirit of this invention, preferred variants are single-amino acid conserved substitution variants. In some specific embodiments, the functional derivative is at least 70% homologous to the full-length amino acid sequence of the parent polypeptide, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, and more preferably 100%. The percentage of homology is determined based on the length of the relevant amino acid sequence. Thus, if the polypeptide according to the invention is contained within a larger polypeptide, the percentage of homology is determined only relative to the polypeptide portion corresponding to the polypeptide according to the invention, rather than determining the percentage of homology for the entire larger polypeptide. The "percentage of homology" associated with a polypeptide sequence refers to the percentage of identical amino acids between at least two polypeptide sequences aligned using a base local alignment search tool (BLAST) engine. The BLAST engine is made publicly available by the National Center for Biotechnology Information (NCBI) in Bethesda, Maryland. According to a specific embodiment, the functional derivative is a polypeptide comprising an amino acid sequence that has at least 70% homology to the full-length sequence of a parent polypeptide, wherein it differs from the parent polypeptide only in substitutions at one or more positions. These substitutions are preferably “conserved substitutions.” Furthermore, the functional derivative has at least 70% homology to the full-length amino acid sequence of the parent polypeptide, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, and more preferably 100%. Methods for determining sequence homology or similarity are known in the art.

[0048] Functional derivatives may contain amino acid residues that are not present in the corresponding natural protein or are missing relative to the corresponding natural protein. Functional derivatives may also be truncated "fragments" compared to the corresponding natural protein, i.e., only a portion of the full-length protein. Functional derivatives also include peptides having at least one D-amino acid.

[0049] When used in this invention, the term "conservative substitution" generally refers to an amino acid substitution that maintains the structural and functional properties of a protein or polypeptide. Such functionally equivalent (conservative substitution) peptide amino acid sequences include, but are not limited to, the addition or substitution of amino acid residues in an amino acid sequence encoded by a nucleotide sequence that results in a silencing change, thereby producing a functionally equivalent gene product. Conservative amino acid substitutions can be based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the relevant residues. For example: nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0050] Conserved amino acid substitutions can also be based on the hydrophilicity index of the amino acids. Each amino acid is assigned a hydrophilicity index according to its hydrophobicity and charge properties. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The use of hydrophilic amino acid indices in conferring biological functions of protein interactions is known in the art (Kyte and Doolittle, J. Mol. Biol., 157:105-132, 1982). It is known that in certain cases, certain amino acids can be substituted with other amino acids having similar hydrophilic indices or fractions while still retaining similar biological activity. When making changes based on similar hydrophilic indices, some embodiments include amino acid substitutions with hydrophilic indices in the range of ±2, others include substitutions with hydrophilic indices in the range of ±1, and still others include substitutions with hydrophilic indices in the range of ±0.5.

[0051] Conserved amino acid substitutions can also be performed on a hydrophilic basis, particularly in implementations where the resulting biologically functional proteins or peptides involve immunological aspects. In certain specific implementations, the protein's maximum local average hydrophilicity (determined by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigenicity, i.e., to the protein's biological characteristics. These amino acid residues were assigned the following hydrophilic values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, some embodiments include amino acid substitutions with hydrophilicity values ​​in the range of ±2, other embodiments include amino acid substitutions with hydrophilicity values ​​in the range of ±1, and still other embodiments include amino acid substitutions with hydrophilicity values ​​in the range of ±0.5.

[0052] Amino acid changes are achieved by altering the codons of the corresponding nucleic acid sequences. It is known that such peptides can be obtained by substituting certain amino acids in the peptide structure with other amino acids to modify or improve biological activity. For example, by substituting alternative amino acids, small conformational changes can be imparted to peptides that result in increased activity. Alternatively, amino acid substitutions in certain peptides can be used to provide residues that can then be linked to other molecules to provide peptide-molecule conjugates that retain sufficient initial peptide properties for other purposes.

[0053] In some embodiments, the GPCR chimeric opsin of the present invention includes a functional derivative thereof. In some embodiments, the mGluR4 of the present invention or the native opsin includes a functional derivative thereof.

[0054] Metabolic glutamate receptor 4 (mGluR4) belongs to the group III metabolic glutamate receptor (mGluR) subfamily and is significantly present in presynaptic locations in the central nervous system (Benitez et al., 2000; Bradley et al., 1996; Bradley et al., 1999; Mateos et al., 1998; Phillips et al., 1997). It can act as both an autoreceptor and a heteroreceptor to regulate the release of GABA and glutamate. Metabolic glutamate receptor 4 (mGluR4) has also been shown to be expressed at low levels in some postsynaptic locations (Benitez et al., 2000). Numerous reports indicate that metabolite glutamate receptor 4 (mGluR4) is most commonly expressed in brain regions, particularly in neurons known to play a key role in the function of the basal ganglia (Bradley et al., 1999; Corti et al., 2002; Kuramoto et al., 2007; Marino et al., 2003a), in neurons known to play a key role in learning and memory (Bradley et al., 1996), in neurons known to play a key role in vision (Akazawa et al., 1994; Koulen et al., 1996; Quraishi et al., 2007), in neurons known to play an important role in cerebellar function (Makoff et al., 1996), in neurons known to play an important role in the regulation of diet and hypothalamic hormones (Flor et al., 1995), in neurons known to play an important role in sleep and wakefulness (Noriega et al., 2007), and in other neurons.

[0055] In this document, the term "domain" refers to the intracellular and extracellular loops, N-terminus and C-terminus, and transmembrane region of a member of the GPCR protein family, such as mGluR4 or opsins. The term "at least one domain derived from mGluR4" includes any domain, such as a transmembrane domain, three extracellular loops (ECL1, ECL2, ECL3), three intracellular loops (ICL1, ICL2, ICL3), an N-terminal domain (NT), a C-terminal domain (CT), or a physiologically relevant counterpart thereof, wherein the physiologically relevant counterpart has an amino acid sequence identical to or similar to that of such a domain in a physiological counterpart of mGluR4. Generally, similar amino acid sequences or similar domains show at least 60% homology, preferably at least 80% homology, and most preferably at least 90% homology. Similar domains also particularly include domains containing conserved amino acid substitutions. In some specific embodiments of the invention, the at least one domain derived from mGluR4 constitutes a binding site for a downstream G protein required for mGluR to function. In some specific embodiments of the present invention, at least one domain derived from mGluR4 includes an intracellular domain of mGluR4 capable of enabling photoactivation to be coupled to a signal transduction cascade of mGluR4.

[0056] Rhodopsin is a membrane protein with a molecular weight of approximately 30–50 kDa. It comprises an extracellular N-terminus, seven transmembrane domains (transmembrane domains, three extracellular loops (ECL1, ECL2, ECL3), and three intracellular loops (ICL1, ICL2, ICL3)), and an intracellular C-terminus, belonging to the G protein-coupled receptor (GPCR) superfamily. Rhodopsin is widely distributed in animals and microorganisms. While non-animal rhodopsin from archaea, bacteria, and fungi shares a similar three-dimensional structure with animal rhodopsin, they differ significantly in their amino acid sequences. Studies have confirmed that animal rhodopsin is diverse, widely distributed, and possesses non-visual functions such as visual perception, regulation of circadian rhythms, and participation in pupillary light reflex. Generally, the vertebrate retina contains two types of photoreceptor cells: rod cells and cone cells. The rhodopsin in rod cells is rhodopsin (Rh), associated with night vision; while cone cells contain cone proteins, associated with photovision. Based on their absorption spectral range, cone proteins can be further classified into long-wavelength sensitive opsins (LW), medium-wavelength sensitive opsins (MW), and short-wavelength sensitive opsins (SW), with the latter further subdivided into SW1 and SW2. Most vertebrate retinas contain these four types of cone proteins, but SW2 and MW opsins have not been found in most mammals. Vertebrate visual opsins include rhodopsin and cone proteins. In some embodiments of the present invention, the opsins are those of vertebrates, such as mammals like humans. In some embodiments of the present invention, the opsins are cone opsins, particularly MW-opsins.

[0057] In this invention, the phrase "replacing a corresponding domain of a native opsin with at least one domain derived from mGluR4" refers to altering one or more domains of an opsin to a corresponding domain of mGluR4 using techniques known in the art, such as recombination techniques. For example, in some embodiments, one or more of the intracellular loop, extracellular loop, N-terminus, C-terminus, and transmembrane region derived from mGluR4 may respectively replace one or more of the intracellular loop, extracellular loop, N-terminus, C-terminus, and transmembrane region of the opsin. For example, in some embodiments, the intracellular loop of the opsin may be replaced with the intracellular loop derived from mGluR4, and the C-terminus of the opsin may be replaced with the C-terminus derived from mGluR4. For example, in some embodiments, ICL2, ICL3, and the C-terminus of the opsin may be replaced with ICL2, ICL3, and the C-terminus derived from mGluR4, respectively. In some embodiments, the replacement couples photoactivation to the mGluR4 signal transduction cascade.

[0058] The chimeric opsin according to the invention can be expressed in specific cells, tissues, and / or organisms and used to control cellular responses to light pulses of suitable wavelengths in vivo, in vitro, and in vitro. The GPCR chimeric opsin is constructed according to techniques known in the art by replacing the corresponding domain of the opsin with at least one domain derived from mGluR4 and coupling photoactivation with the mGluR4 signal transduction cascade. The identification of the desired domains and the determination of suitable cleavage and connection sites at the N- and C-termini of any particular domain are primarily based on: 1) alignment of gene sequences / conserved residues, and 2) computer modeling of the secondary and tertiary structures of the photoreceptor and mGluR4 using standard software available in the art. This method has inherent variability in the precise definition of the lengths of the individual domains, and such variability is included within the scope of the invention when referring to domains. Furthermore, at the various fusion sites between domains, there are generally multiple possibilities for splicing the domains together to produce a functional protein. Furthermore, it is evident that deletions of portions of the non-functionally required amino acid sequences, conserved amino acid substitutions (e.g., exchanges of hydrophobic and hydrophobic or hydrophilic amino acids), and nucleotide substitutions are also within the scope of this invention. Therefore, a large number of sequence variants (particularly in the region of the fusion site between the GPCR and adjacent opsin domains) fall within the scope of this invention, provided they produce a functional GPCR chimeric opsin. In some embodiments, the GPCR chimeric opsin of this invention may also include, at its C-terminus, a signal peptide that promotes its expression and / or transport, such as sequences of Golgi output signals like RSRFVKKDGHCNVQFINV (SEQ ID NO:8) and / or, for example, rhodopsin transport sequences like TETSQVAPA (SEQ ID NO:9).

[0059] In this invention, the term "ion channel" refers to a transmembrane polypeptide that forms a pore, opens when activated, and allows ionic conduction to pass through the pore across the membrane. According to this invention, photosensitive ion channel proteins include chr2, chrimsonR, MCO, or functional derivatives thereof.

[0060] The chimeric opsin or photosensitive ion channel protein according to the invention is strongly activated by contact with visible light, preferably with light with wavelengths between about 365 nm and about 700 nm, and with light with wavelengths between about 480 nm and about 590 nm, and in some specific embodiments, peak activation occurs upon contact with light with a wavelength of about 530 nm.

[0061] While the peptide amino acid sequences described in this invention can be chemically synthesized, large polypeptide sequences can preferably be generated using recombinant DNA technology, utilizing techniques well-known in the art for expressing nucleic acids containing nucleotide sequences encoding the desired peptide. Such methods can be used to construct expression vectors containing nucleotide sequences encoding peptides, as well as appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination, which are well-known in the art.

[0062] Some specific embodiments are isolated nucleic acids comprising a nucleotide sequence encoding a chimeric opsin or a functional derivative thereof according to the invention. In some specific embodiments, the nucleic acid comprises, or is composed of, the nucleotide sequence shown in SEQ ID NO:2 or 4. In other specific embodiments, the invention provides isolated nucleic acids comprising a nucleotide sequence encoding a chimeric opsin or a functional derivative thereof according to the invention, the nucleotide sequence having at least 70% identity with SEQ ID NO:2 or 4, preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99% identity, and more preferably 100% identity.

[0063] The nucleic acids according to the invention may include additional sequences, including but not limited to one or more signal sequences (e.g., enhancers, polyadenylated nucleotide signals, additional restriction enzyme sites, multiple cloning sites) and / or promoter sequences, or other coding segments, or combinations thereof. The promoter may be an inducible or constitutive general or cell-specific promoter. An example of a cell-specific promoter is a bipolar cell-specific mGluR6 promoter or a ganglion cell-specific SNCG promoter, preferably a ganglion cell-specific SNCG promoter. Some specific embodiments are any of the methods disclosed, wherein the promoter is a constitutive promoter. Some specific embodiments are any of the methods disclosed, where the constitutive promoter includes, but is not limited to, the CMV promoter or the CAG promoter (composed of a cytomegalovirus (CMV) early enhancer and a chicken β-actin promoter). Promoters, vectors, enhancers, and polyadenylated nucleotide sites are conventionally chosen by those skilled in the art. These elements are well described in the literature and are commercially available.

[0064] In this invention, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid between different genetic environments, wherein the nucleic acid molecule is operatively linked to the other nucleic acid.

[0065] When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably ligated." Generally, "operably ligated" means that the ligated DNA sequences are continuous. However, enhancers do not need to be continuous. Ligation is achieved by joining at conventional restriction sites. If such sites are not present, then synthetic oligonucleotide adaptors or linkers are used according to standard practice.

[0066] The term "vector" also refers to a virus or organism capable of transporting nucleic acid molecules. One type of vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication. Some useful vectors are those capable of autonomous replication and / or expression of the nucleic acid associated with them. Vectors capable of directing the expression of genes operatively associated with them are referred to herein as "expression vectors." Expression vectors and methods of using them are well known in the art. This invention provides non-limiting examples of suitable expression vectors and methods of using them. In a preferred embodiment, the vector is suitable for gene therapy, particularly for virus-mediated gene transfer. Viral vectors suitable for gene therapy include retroviruses, adenoviruses, adeno-associated viruses (AAVs), lentiviruses, poxviruses (e.g., MVAs), alphaviruses, herpesviruses, etc. Examples of AAVs can be found in Davidson et al., PNAS (2000) 97:3428-3432. AAVs and lentiviruses can confer persistent expression, while adenoviruses can provide transient expression.

[0067] However, gene therapy further includes non-viral methods, such as using naked DNA or nucleic acids bound to liposomes. Vectors suitable for use in some methods according to the invention can genetically insert chimeric opsins into dividing and non-dividing cells, and can insert chimeric opsins into in vivo, in vitro, or ex vivo cells.

[0068] In some preferred embodiments, the nucleic acid expression vector including the gene for the chimeric opsin according to the invention is selected from AAV viral vectors. The term "serotype" refers to an AAV identified by a defined antiserum and distinguished from other AAVs based on reactivity with the capsid protein of said defined antiserum. For example, many known serotypes exist for primate AAVs (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, and AAVRh10, and modified capsids of these serotypes). "AAV virus" or "AAV virus particle" refers to a viral particle composed of at least one AAV capsid protein (preferably all capsid proteins of wild-type AAV) and a capsid encapsulated polynucleotide. According to preferred embodiments, the AAV viral vector used in this invention is serotype AAV2, AAV9, or AAV2 / 7m8, more preferably serotype AAV2 / 7m8.

[0069] Some specific embodiments are methods for treating or preventing retinal neurodegenerative diseases, the methods comprising: (a) delivering to target cells a nucleic acid expression vector encoding a chimeric opsin or a functional derivative thereof according to the invention, the chimeric opsin or a functional derivative thereof being expressible in the target cells, the vector comprising an open reading frame encoding a chimeric opsin or a functional derivative thereof according to the invention, operatively linked to a promoter sequence and optionally operatively linked to a transcriptional regulatory sequence; and (b) expressing the vector in the target cells, wherein the expressed chimeric opsin or a functional derivative thereof activates the target cells upon exposure to light.

[0070] The chimeric opsin according to the invention has been found to be suitable for expression and use in mammalian cells under normal cellular environmental conditions and ion concentrations, without the need for any kind of chemical supplementation. The chimeric opsin according to the invention has been found to be activated in the wavelength range of 365 nm to 700 nm, with a preferred activation wavelength of 480 nm to 590 nm and a peak activation wavelength of 530 nm.

[0071] An effective amount of chimeric opsin or its functional derivatives or their nucleic acid expression vectors is the amount that raises the level of chimeric opsin in cells, tissues, or a subject to a level beneficial to the subject. An effective amount can also be determined by assessing the physiological effects of administration to cells or a subject, such as a reduction in symptoms after administration. Other assays are known to those skilled in the art and can be used to determine the level of response to treatment. The amount of treatment can be varied, for example, by increasing or decreasing the amount of chimeric opsin or nucleic acid expression vector administered, changing the therapeutic composition containing the administered chimeric opsin or nucleic acid expression vector, changing the route of administration, changing the timing of administration, etc. The effective amount will vary with the specific condition being treated, the age and physical condition of the treated subject, the severity of the condition, the duration of treatment, the nature of concurrent therapies (if any), the specific route of administration, and similar factors within the scope of the physician's knowledge and expertise. These factors are well known to those skilled in the art and can be resolved simply through routine experiments. Those skilled in the art will understand that a subject may adhere to a lower or tolerated dose for medical, psychological, or virtually any other reason.

[0072] The chimeric opsin or its functional derivatives, or their nucleic acid expression vectors, according to the invention can be administered using methods known in the art. In some specific embodiments, a nucleic acid encoding the chimeric opsin or its functional derivative according to the invention is administered to the subject. The manner and dosage of administration can be adjusted individually by a physician, particularly in the event of any complications. The absolute amount administered will depend on various factors, including the material selected for administration, whether a single or multiple dose is administered, and the subject's personal parameters, including age, weight, physical condition, and stage of disease. These factors are well known to those skilled in the art and can be resolved through routine experimental procedures.

[0073] Pharmaceutical compositions comprising the chimeric opsin or its functional derivatives or their nucleic acid expression vectors according to the invention can be administered alone and / or in combination with other drug therapies or other treatment regimens administered to the subject. Various routes of administration are known to those skilled in the art. Suitable routes of administration may include local administration, intravenous administration, oral administration, intracavitary administration, intrathecal administration, intrasynovial administration, buccal administration, sublingual administration, intranasal administration, percutaneous administration, subvitreal administration, subretinal administration, subcutaneous administration, intramuscular administration, and intradermal administration. The invention is not limited to the specific routes of administration disclosed herein.

[0074] This invention discloses, in some aspects, the preparation of nucleic acids comprising nucleotide sequences; expression in cells and / or membranes of polypeptides encoded by the prepared nucleic acids and nucleotide sequences; irradiation of the cells and / or membranes with appropriate light; and verification of rapid depolarization and / or changes in transmembrane conductivity in response to light, as well as rapid recovery from depolarization after light expiration. The ability to controllably alter transmembrane voltage and cell depolarization by light has been verified. This invention enables light-controlled cellular function in vivo, in vitro, and in vitro, and the chimeric opsins according to this invention have broad applications in drug screening, treatment, and research, some of which are described herein.

[0075] Example

[0076] The following examples provide further detailed information on various specific embodiments. Those skilled in the art will understand that the techniques disclosed in the examples below represent technologies and / or compositions that the inventors have discovered that work well. However, according to the present invention, those skilled in the art will understand that many modifications can be made to the disclosed specific embodiments without departing from the spirit and scope of the invention, and these modifications will still yield similar or approximate results. These examples are merely illustrative and not intended to limit the scope of the invention.

[0077] Unless otherwise stated, all materials used in the embodiments herein are commercially available, and all specific experimental methods used in the experiments are conventional experimental methods in the art or are performed according to the steps and conditions recommended by the manufacturer, and can be routinely determined by those skilled in the art as needed. Some materials and methods are described in detail below.

[0078] Example 1: Construction and in vitro cell validation of GPCR chimeric opsin AAV expression vector

[0079] The full-length human GPCR chimeric opsin coding gene sequence (SEQ ID NO) containing a signal peptide (Golgi output signal + rhodopsin transport sequence) at the C-terminus was synthesized by Nanjing Genscript Biotech Co., Ltd. NO:2) Using the traditional gene cloning method of enzyme digestion and ligation cloning, the above-mentioned coding gene was cloned into an expression vector driven by the retinal ganglion cell-specific promoter RGC (SNCG). Simultaneously, using the Gibson assembly seamless ligation cloning technology, the C-terminus of this gene was linked to the reporter gene EGFP via P2A, forming the AAV-RGC promoter-EGR0-EGFP plasmid. AAV was later packaged for in vivo efficacy verification experiments, and a control viral plasmid, AAV-RGC promoter-EGFP plasmid, was constructed. Simultaneously, a Flag tag (HA tag) was added to the N-terminus of this gene, and the C-terminus was linked to the reporter gene EGFP via P2A, cloning it into a broad-spectrum promoter CMV plasmid, forming the CMV promoter-Flag-EGR0-EGFP plasmid. This plasmid was later used for in vitro expression verification experiments. Transfecting this plasmid into HEK293T cells in vitro, imaging showed that the reporter gene EGFP was expressed normally. Figure 1 b), and flow cytometry analysis using a Flag tag-specific antibody revealed that Flag tag signals could be detected on the membrane of HEK293 cells transfected with this plasmid. Figure 1 c) Since EGR0 is expressed in fusion with the Flag tag and the Flag tag is located outside the cell membrane, it proves that EGR0 can be expressed normally and located on the cell membrane surface, thus providing a structural basis for the treatment of retinal neurodegenerative diseases.

[0080] Example 2: AAV administration in mice

[0081] In this study, AAV2 / 7m8 was produced by Heyuan Biotechnology (Shanghai) Co., Ltd. Normal wild-type C57 mice and photoreceptor degeneration type C3H mice (12 weeks old, male) were administered the drug via subvitreal injection at a dose of 1.5 × 10⁻⁶. 9 VG / eye (1.5ul, 1×10) 12 VG / ml).

[0082] 1) RGC promoter-specific labeling of retinal ganglion cells

[0083] Confocal imaging of frozen retinal sections from normal wild-type C57 mice two weeks after injection of the AAV-RGC promoter-EGFP revealed that EGFP was specifically expressed in retinal ganglion cells. Figure 2 ).

[0084] 2) Highly efficient expression of GPCR chimeric opsin in the retinas of blind C3H mice after drug administration

[0085] Seven weeks after drug administration, retinal patch and optic nerve imaging in C3H mice revealed that EGR0 was highly expressed in retinal ganglion cells. Figure 3 a), and the axons converge at the optic nerve ( Figure 3 c).

[0086] 3) The behavior of blind C3H mice was partially improved after drug administration.

[0087] Seven weeks after drug administration, C3H mice underwent a visual placement response test. The mouse's tail was lifted to a height of 15 cm above a metal cage lid, and the mouse was slowly brought closer to the lid, with its head approximately 2-4 cm (1-2 seconds) near the lid. Normal mice in this position would raise their heads and attempt to reach their forelimbs towards the lid; this response is called the placement response. However, visually impaired mice, unable to see the lid, did not exhibit a similar response. We scored the placement response of the administered C3H mice, with 0 indicating no response at all; 1 indicating a weak response; and 2 indicating a significant response. We found that the placement response in blind C3H mice expressing GPCR chimeric opsin was significantly better than in the control group, indicating that GPCR chimeric opsin can partially restore the behavior of blind mice, suggesting that this chimeric opsin could be used to treat retinal neurodegenerative diseases.

[0088] The following shows the sequence information of the GPCR chimeric opsin (EGR0) containing a signal peptide (Golgi output signal + rhodopsin transport sequence) at its C-terminus that promotes expression:

[0089] amino acid sequence:

[0090] MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWMIFVVIASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISVVNQVYGYFVLGHPMCVLEGYTVSLCGITGLWSLAIISWERIYRIFEQGKRSVSAPRFISPASQVGIAFSWIWAAVWTAPPI FGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCITPLSIIVLCYLQVWLKTRGVPETFNEAKMVVVMVLAFCFCWGPYAFFACFAAANPGYPFHPLMAALPAFFAKSATIYNPVIYVFMNRQPEQNVPKRKRSLKAVVTAATMSNKFTQKGNFRPNGEAKSELCENLEAPALATKQTYVTYTNHAI RSRFVKKDGHCNVQFINVTETSQVAPA(SEQ ID NO:1, where the underlined amino acid residues are sequences of Golgi output signals, and the bolded and double-underlined sequences are rhodopsin transport sequences)

[0091] Nucleotide sequence:

[0092] AGAAGCCGCTTTGTGAAGAAAGATGGTCATTGCAATGTGCAGTTTATTAATGT GACCGAGACCTCACAGGTTGCCCCAGCC (SEQ ID NO:2, wherein the underlined nucleotide sequence encodes the Golgi output signal, while the bolded nucleotide sequence with double underscores encodes the rhodopsin transport sequence).

Claims

1. A GPCR chimeric opsin, characterized in that... The chimeric opsin consists of the amino acid sequence shown in SEQ ID NO:

3.

2. A nucleic acid molecule, characterized in that... It contains a nucleotide sequence encoding the GPCR chimeric opsin according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that... The nucleic acid molecule contains, or is composed of, the nucleotide sequence shown in SEQ ID NO:

4.

4. A carrier, characterized in that... The vector contains a nucleotide sequence encoding the GPCR chimeric opsin according to claim 1, or contains a nucleic acid molecule according to claim 2 or 3.

5. The carrier according to claim 4, characterized in that... The carrier is an AAV carrier.

6. A transgenic cell, characterized in that... It contains the GPCR chimeric opsin according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the vector according to claim 4.

7. The transgenic cell according to claim 6, characterized in that... The transgenic cells are transgenic retinal ganglion cells.

8. Use of the GPCR chimeric opsin according to claim 1, the nucleic acid molecule according to claim 2 or 3, the vector according to claim 4 or 5, or the transgenic cell according to claim 6 or 7 in the preparation of a medicament for treating retinal neurodegenerative diseases in subjects of need.

9. The use according to claim 8, characterized in that... The aforementioned retinal neurodegenerative diseases are retinitis pigmentosa (RP) or age-related macular degeneration (AMD).

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