Novel photosensitive channel protein VR2.0 and its use

Abstract

This application provides a novel photosensitive channel protein VR2.0 and its use. [Solution] The VR2.0 series of photosensitive channel proteins provided in this application exhibits remarkable therapeutic effects in the treatment of retinal photoreceptor cell degeneration diseases. This novel photosensitive channel protein exhibits higher photosensitivity, faster photoresponse dynamics, and reduced side effects. Under the same photostimulation conditions, it can maintain a faster response frequency and a stable current signal even in high-frequency responses. This application offers a better option for the development of optogenetic therapies for retinal photoreceptor cell degeneration diseases and has significant application and promotional value.

Description

[Technical Field]

[0001] This application belongs to the biomedical field and specifically relates to a novel photosensitive channel protein VR2.0 and its use. [Background technology]

[0002] Retinal photoreceptor cell degeneration is a type of degenerative disease characterized primarily by the progressive loss of function of photoreceptor cells and pigment epithelial cells. These diseases are mainly caused by gene mutations or retinal pigment epithelial cell (RPE) dysfunction. Representative examples include retinitis pigmentosa (RP) and age-related macular degeneration (AMD), which are two of the most serious eye diseases that can lead to blindness. The prevalence of photoreceptor cell degeneration due to genetic factors is approximately 1 in 3,500 to 1 in 4,000 people. Currently, there are 400,000 people with retinitis pigmentosa in China and more than 1.5 million people worldwide. The incidence of secondary retinal photoreceptor cell degeneration caused by drugs or other diseases is also increasing.

[0003] In retinal photoreceptor cell degeneration, apoptosis of photoreceptor cells is irreversible, and many of these diseases exhibit high genetic heterogeneity, making the treatment of related diseases extremely difficult. Current treatments mainly include stem cell transplantation, gene therapy, artificial retina transplantation, and optogenetic therapy. Among these, optogenetic therapy utilizes the soundness of the remaining cellular structure in retinal degenerative diseases, using recombinant adeno-associated virus (AAV) as a vector to express photosensitive proteins in cone cells (early degeneration), bipolar cells, or ganglion cells (mid to late degeneration), thereby restoring retinal photosensitivity. Furthermore, by combining this with stem cell transplantation, virtual reality systems, and holographic imaging technology, it is possible to restore visual function.

[0004] Optogenetic therapy holds great potential for treating retinal photoreceptor cell degeneration diseases as an ophthalmic treatment strategy that can respond to light stimulation at the single-cell level without relying on the restoration of function of mutated genes in specific gene regions. Photosensitive proteins that play a role in restoring vision through optogenetic therapy are mainly classified into two types: microbial and mammalian. Microbial photosensitive proteins generally have fast dynamic characteristics but low light sensitivity. An example is channelrhodopsin-2 (ChR2), a cation-permeable photosensitive protein that was first used for restoring visual function. Endogenous photosensitive proteins of mammalian origin belong to the G protein-coupled receptor (GPCR) family, have seven transmembrane α-helical structures, and generally exhibit high light sensitivity but limited reaction rates. An example is rhodopsin derived from rod photoreceptor cells (Rods). Currently, existing optogenetic tools for vision restoration (i.e., photosensitive proteins) do not meet the requirements of high photosensitivity and rapid photoresponse dynamics, and suffer from problems such as differences in ion selectivity and side effects of intracellular acidification. Therefore, there is a need to develop photosensitive proteins that possess high sensitivity to light stimulation, rapid photoresponse dynamics, minimal side effects, and the ability to maintain a stable current signal even under high-frequency light responses. These proteins, when combined with more efficient gene delivery vectors that target retinal cells, can be used more effectively in the treatment of retinal photoreceptor cell degeneration diseases. [Overview of the project] [Problems that the invention aims to solve]

[0005] To address the problems present in the prior art, this application provides a novel photosensitive channel protein VR2.0 and its use. [Means for solving the problem]

[0006] Specifically, this application relates to the following aspects.

[0007] 1. A photosensitive channel protein containing one of the following PsCatCh variants: (1) A protein obtained by cleaving 1 to 33 amino acids from the N-terminus of a reference protein shown in Sequence ID No. 1; (2) A protein obtained by cleaving 1 to 29 amino acids from the C-terminus of a reference protein shown in Sequence ID No. 1; (3) Proteins obtained by mutations near the retinal dehyde binding site of the reference protein shown in Sequence ID No. 1; (4) Any combination of two or three of the above (1), (2), and (3); or (5) Proteins having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with (1), (2), (3), and (4) above.

[0008] 2. The photosensitive channel protein according to claim 1, wherein the photosensitive channel protein includes a protein obtained by cleaving 12 to 26 amino acids from the N-terminus of a reference protein shown in SEQ ID NO: 1.

[0009] 3. The photosensitive channel protein according to item 2, wherein the photosensitive channel protein includes a protein obtained by cleaving 17 to 21 amino acids from the N-terminus of a reference protein shown in SEQ ID NO: 1.

[0010] 4. The photosensitive channel protein according to any one of claims 1 to 3, wherein the photosensitive channel protein includes a protein obtained by cleaving 9 to 23 amino acids from the C-terminus of a reference protein shown in SEQ ID NO: 1.

[0011] 5. The photosensitive channel protein according to claim 4, wherein the photosensitive channel protein includes a protein obtained by cleaving 9 to 18 amino acids from the C-terminus of a reference protein shown in SEQ ID NO: 1.

[0012] 6. The photosensitive channel protein according to claim 1, wherein the photosensitive channel protein includes a protein obtained by cleaving 12 to 21 amino acids from the N-terminus and 13 to 23 amino acids from the C-terminus based on the reference protein shown in SEQ ID NO: 1.

[0013] 7. The photosensitive channel protein according to any one of claims 1 to 6, wherein the photosensitive channel protein includes a protein obtained by mutating the 66th E of the reference protein shown in SEQ ID NO: 1 to D, and / or the 165th C to L, based on the reference protein shown in SEQ ID NO: 1 or the PsCatCh variant.

[0014] 8. The amino acid sequence of the photosensitive channel protein is shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or Sequence IDs 8, 9, 13, 14, 15, 16, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, or 36 have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity. A photosensitive channel protein as described in any one of items 1 to 7.

[0015] 9. The photosensitive channel protein is fused to the N-terminus of the PsCatCh variant, and / or The present invention further comprises a T polypeptide linked to the C-terminus of the PsCatCh mutant, and an E polypeptide linked to the T polypeptide. The photosensitive channel protein according to any one of items 1 to 8, wherein the amino acid sequence of the LR signal peptide is shown in SEQ ID NO: 2, the amino acid sequence of the T polypeptide is shown in SEQ ID NO: 4, and the amino acid sequence of the E polypeptide is shown in SEQ ID NO: 5.

[0016] 10. A nucleic acid molecule comprising a nucleotide sequence encoding the photosensitive channel protein according to any one of items 1 to 9.

[0017] 11. A vector comprising the nucleic acid molecule according to item 10.

[0018] 12. A recombinant virus comprising the nucleic acid molecule according to item 10 or the vector according to item 11.

[0019] 13. The recombinant virus according to item 12, which is a recombinant adeno-associated virus.

[0020] 14. A pharmaceutical composition comprising the photosensitive channel protein according to any one of items 1 to 9, the nucleic acid molecule according to item 10, the vector according to item 11, or the recombinant virus according to item 12 or 13, and a pharmaceutically acceptable carrier.

[0021] 15. Use of the photosensitive channel protein according to any one of items 1 to 9 in the preparation of a medicament for treating retinal photoreceptor cell degeneration diseases.

[0022] 16. The use according to item 15, wherein the retinal photoreceptor cell degeneration diseases include retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and Leber congenital amaurosis (LCA).

Advantages of the Invention

[0023] Advantages and beneficial effects of the present application: The VR2.0 series of photosensitive channel proteins provided in this application exhibits clear therapeutic effects in the treatment of retinal photoreceptor cell degeneration diseases. These novel photosensitive channel proteins possess higher photosensitivity, faster photoresponse dynamics, maintain stable current signals in high-frequency responses, and exhibit faster response frequencies under the same photostimulation conditions. This application offers a new and better option for the development of optogenetic therapies for retinal photoreceptor cell degeneration diseases, expanding the scope of clinical optogenetic therapy for these diseases and possessing significant application and promotional value. [Brief explanation of the drawing]

[0024] [Figure 1] These are 3D structural diagrams of the light-sensitive proteins PsCatch (1-305aa), PsCatch2.0 (1-343aa), and the representative mutant PsCatch2.0 e26, obtained through simulation. [Figure 2] This is a schematic diagram of the amino acid structure of the VR2.0 series of photosensitive proteins, which are modified versions of the photosensitive protein PsCatCh. [Figure 3] This is a signal diagram of the photoresponse current intensity of mutants in the photosensitive protein VR2.0 series. [Figure 4] This figure shows the waveforms of photoresponsive electrical signals from several mutants of the photosensitive protein VR2.0 series in African clawed frog oocytes. [Figure 5] This figure shows the Na+ and H+ ion permeability of several mutants of the photosensitive protein VR2.0 series. [Figure 6] This figure shows the pAAV-CMV-VR2.0-EYFP core vector, which carries expression cassettes of several variants of the light-sensitive protein VR2.0 series. [Figure 7]This figure shows the currents after photostimulation recorded by patch clamp in HEK293T cells after expression of several mutants of the photosensitive protein VR2.0 series and the control group PsCatch2.0. This reflects the photosensitivity and response frequency of the photosensitive protein to photostimulation. Figure 7A: Current diagram of VR2.0 generated at a light intensity of 1.66 × 10¹⁵ photons / cm²s with a wavelength of 470 nm and stimulation frequencies of 2 Hz, 4 Hz, 8 Hz, 16 Hz, and 32 Hz; Figure 7B: Current diagram of VR2.0 generated after 1 second of stimulation with a light intensity of 1.66 × 10¹⁵ photons / cm²s with a wavelength of 470 nm. [Figure 8] This figure shows patch-clamp recordings of current at a wavelength of 470 nm and various light intensities after expression of several mutants of the photosensitive protein VR2.0 series and the control group PsCatch2.0 in HEK293T cells. Here, "photons / cm2s" represents the unit of light intensity. [Figure 9] This figure shows the visual evoked potentials of C57BL / 6J mice, rd1 mice administered intravitreally with rAAV2-CMV-VR2.0-EYFP, and rd1 littermates that were not administered the drug. Figure 9A: Representative waveform of the visual evoked potentials of the above mice; Figure 9B: Statistical bar graph of the N1 amplitude of the visual evoked potentials of the above mice. Here, P<0.001, indicating a statistically significant difference. [Figure 10] This figure shows the light avoidance responses of C57BL / 6J mice, rd1 mice administered intravitreously with rAAV2-CMV-VR2.0-EYFP, and unadministered rd1 littermates in a light-dark chamber. Figure 10A: Schematic diagram of the light-dark chamber experiment; Figure 10B: Statistical bar graph of the activity time of the above mice in the light chamber. Here, P<0.05 indicates a statistically significant difference. [Figure 11] This figure shows the viscomotor responses of C57BL / 6J mice, rd1 mice administered intravitreally with rAAV2-CMV-VR2.0-EYFP, and rd1 littermates that were not administered the drug. Figure 11A: Schematic diagram of the viscomotor response; Figure 11B: Statistical bar graph of visual sensitivity for the above mice. Here, P<0.05 indicates a statistically significant difference. Detailed description of the invention

[0025] This application will be further described with reference to the following embodiments. These embodiments are intended solely to illustrate and illustrate this application and are not intended to limit it.

[0026] Unless otherwise defined, the technical and scientific terms used herein have the meanings generally understood by those skilled in the art. Methods and materials similar to or identical to those described herein may be used in experiments or practical applications, but these materials and methods are described below herein. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples are provided for illustrative purposes only and are not intended to limit the scope of this application. This application will be further described with reference to the following specific examples, but these examples are not intended to limit the scope of this application.

[0027] As used herein, “photosensitive channel proteins” and “photosensitive proteins” are used interchangeably and refer to a class of proteins on the cell membrane that can produce specific effects (e.g., altering the open state of ion channels) in response to light stimulation. These proteins are classified into activating and inhibitory types and can cause excitation or inhibition of neurons.

[0028] The PsCatCh variant in this application is a modified version based on the photosensitive channel protein derived from the subcordiformis alga (Platymonas subcordiformis channelrhodopsin, PsChR).

[0029] Light-sensitive channel proteins This application provides a photosensitive channel protein comprising any one of the following PsCatCh variants: (1) A protein obtained by cleaving 1 to 33 amino acids from the N-terminus of a reference protein shown in Sequence ID No. 1; (2) A protein obtained by cleaving 1 to 29 amino acids from the C-terminus of a reference protein shown in Sequence ID No. 1; (3) Proteins obtained by mutations near the retinal dehyde binding site of the reference protein shown in Sequence ID No. 1; (4) Any combination of two or three of the above (1), (2), and (3); or (5) Proteins having at least 60% sequence identity with (1), (2), (3), and (4) above, for example, proteins having 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity.

[0030] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 1 to 33 amino acids from the N-terminus of a reference protein shown in SEQ ID NO: 1. Here, 1 to 33 amino acids may be, for example, 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, or 33 amino acids.

[0031] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 12 to 26 amino acids from the N-terminus of the reference protein shown in Sequence ID No. 1.

[0032] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 17 to 21 amino acids from the N-terminus of the reference protein shown in Sequence ID No. 1.

[0033] In a particular embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, or 60%, 61%, 62%, 63%, 64%, 65%, 66%, and 67% of any one of SEQ ID NOs: 8, 9, 10, 11, 12, and 13. They have sequence identity of 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0034] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 1 to 29 amino acids from the C-terminus of a reference protein shown in Sequence ID No. 1. Here, 1 to 29 amino acids may be, for example, 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, or 29 amino acids.

[0035] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 9 to 23 amino acids from the C-terminus of the reference protein shown in Sequence ID No. 1.

[0036] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 9 to 18 amino acids from the C-terminus of the reference protein shown in Sequence ID No. 1.

[0037] In a particular embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NOs: 14, 15, 16, 17, 18, and 19, or as one of SEQ ID NOs: 14, 15, 16, 17, 18, and 19 with 60%, 61%, 62%, 63%, 64%, 65%, 66%, 6%. They have sequence identity of 7%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0038] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 1 to 33 amino acids from the N-terminus and 1 to 29 amino acids from the C-terminus of a reference protein shown in Sequence ID No. 1.

[0039] In one particular embodiment, the PsCatCh variant is a protein obtained by cleaving 12 to 21 amino acids from the N-terminus and 13 to 23 amino acids from the C-terminus of the reference protein shown in Sequence ID No. 1.

[0040] In a particular embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, and 36, or 60%, 61%, 62%, and 63% of any one of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, and 36. They have sequence identity of 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0041] In one particular embodiment, the PsCatCh mutant is a protein obtained by changing the 66th E to D and / or the 165th C to L in the reference protein shown in SEQ ID NO: 1 or the PsCatCh mutant. Here, for the point mutation in the PsCatCh mutant, the 66th or 165th site both correspond to the reference protein shown in SEQ ID NO: 1.

[0042] In one particular embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, and 28, or 60%, 61%, of any one of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, and 28. They have sequence identity of 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0043] In one particular embodiment, the photosensitive channel protein further comprises an LR signal peptide fused to the N-terminus of the PsCatCh variant.

[0044] In one particular embodiment, the amino acid sequence of the LR signal peptide is shown in SEQ ID NO: 2.

[0045] In one particular embodiment, the photosensitive channel protein further comprises a T polypeptide ligated to the C-terminus of the PsCatCh variant to enhance cell membrane expression efficiency, and an endoplasmic reticulum transport signal sequence E polypeptide ligated to the T polypeptide.

[0046] In one particular embodiment, the amino acid sequence of the T polypeptide is shown in SEQ ID NO: 4. In one particular embodiment, the amino acid sequence of the E polypeptide is shown in SEQ ID NO: 5.

[0047] Those skilled in the art will understand that a linker peptide sequence consisting of a small number of amino acids that does not affect the function of the photosensitive channel protein may exist between any two of the PsCatCh variant, T polypeptide, and E polypeptide.

[0048] In one particular embodiment, the amino acid sequence of the linker peptide is shown in SEQ ID NO: 37.

[0049] Nucleic acid molecules, vectors, and recombinant viruses This application provides a nucleic acid molecule comprising a nucleotide sequence encoding any one of the aforementioned photosensitive channel proteins.

[0050] In some embodiments, the nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule may be replicated and / or expressed in a cell. In some embodiments, the DNA molecule may be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule may be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule may be expressed in a eukaryotic cell and may be replicated in a prokaryotic cell. Thus, in addition to comprising a nucleotide sequence encoding the photosensitive channel protein, the DNA molecule also comprises a genetically engineered or regulatory element for replication and / or expression in prokaryotic and / or eukaryotic cells. In some embodiments, the eukaryotic cell is a human retinal photoreceptor cell. In some embodiments, the eukaryotic cell is a human cone cell. In some embodiments, the eukaryotic cell is a bipolar cell or ganglion cell.

[0051] This application provides a vector containing the aforementioned nucleic acid molecule.

[0052] In some embodiments, the vector is a DNA plasmid. In this specification, the term “DNA plasmid” refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the term “plasmid” refers to a circular DNA molecule. In some embodiments, the term “plasmid” also encompasses linear DNA molecules. Specifically, the term “plasmid” includes molecules obtained by linearizing a circular plasmid, such as by cleaving the circular plasmid with restriction enzymes to convert the circular plasmid molecule into a linear molecule, as well as linear molecules that can replicate within prokaryotes. Plasmids can replicate, i.e., amplify, within a cell independently of the nucleoid or genomic genetic information stored in the nucleoid of a prokaryotic cell, and can also be used for cloning, i.e., for amplifying genetic information within a bacterial cell. For example, the DNA plasmid relating to this application is a plasmid constructed on a pGEMHE plasmid.

[0053] This application provides a recombinant virus comprising any one of the nucleic acid molecules or vectors described above.

[0054] In some embodiments, the recombinant virus is an adeno-associated virus (AAV), a chimeric AAV, an adenovirus, a retrovirus, a lentivirus, a herpes simplex virus, a baculovirus, or any variant or derivative thereof. Preferably, the recombinant virus is an AAV. In some embodiments, the AAV includes one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh36, AAVrh37, AAVrh74, AAVrh79, AAV-DJ, AAV-DJ / 8, AAV.Anc80, AAV.Anc80L65, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, AAV-PHP.S, AAV2i8, MyoAAV, AAVMYO, AAV.CPP.16 capsid serotypes, or their variants.

[0055] Pharmaceutical composition This application provides a pharmaceutical composition comprising any one of the aforementioned photosensitive channel proteins, nucleic acid molecules, vectors, or recombinant viruses, and a pharmaceutically acceptable carrier.

[0056] The form of a pharmaceutical composition depends on several criteria, including, for example, the route of administration, the severity of the disease, or the dosage.

[0057] In some embodiments, the pharmaceutical composition can be formulated to be delivered to the target via any suitable route, including but not limited to oral, injectable (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal, etc.), mucosal (e.g., intranasal, oral, etc.), sublingual, rectal, transdermal, intraocular, or pulmonary routes. Depending on the desired route of administration, the pharmaceutical composition can be formulated as tablets, capsules, pills, sugar-coated tablets, powders, granules, cachets, lozenges, suppositories, suspensions, emulsions, syrups, aerosols (solid or in a liquid medium), sprays, ointments, pastes, patches, creams, lotions, gels, inhalants, etc.

[0058] Those skilled in the art will understand that the dosage and frequency of administration of a pharmaceutical composition may vary depending on the subject's age, weight, or individual response to the vaccine, and the specific method of administration chosen.

[0059] Treatment methods and therapeutic uses This application provides the use of the aforementioned photosensitive channel protein in the treatment of retinal photoreceptor cell degeneration disease.

[0060] This application provides the use of the aforementioned photosensitive channel protein in the preparation of drugs for treating retinal photoreceptor cell degeneration diseases.

[0061] This application provides a method for treating retinal photoreceptor cell degeneration disease, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition to a subject.

[0062] In the above method or use, the retinal photoreceptor cell degeneration disease may include various retinal photoreceptor cell degeneration diseases known in the art, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and congenital amaurosis (LCA).

[0063] It should be understood that this application encompasses various aspects, embodiments, and combinations thereof as described herein. The above description and the following examples are intended to be illustrative and not to limit the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be obvious to those skilled in the art. Therefore, those skilled in the art should recognize that the scope of this application also includes such improvements and modifications to the aforementioned aspects and embodiments. [Examples]

[0064] Example 1. Construction of VR2.0 series mutant expression plasmids modified based on the microbial photosensitive protein PsCatCh and performance comparison in Xenopus laevis oocytes. To improve the properties of the photosensitive protein, particularly its cell membrane transport efficiency and photosensitivity, we introduced amino acid cleavage and mutations into the PsCatch portion of PsCatch 2.0 (the amino acid sequence is shown in SEQ ID NO: 1), based on previous research and knowledge of the structure (Figure 1) and function of the PsCatch 2.0 photosensitive protein. The PsCatch 2.0 protein has seven transmembrane regions, with approximately 30 amino acids in the N-terminal extramembrane region and approximately 40 amino acids in the C-terminal extramembrane region.

[0065] The specific cleavage and mutation design scheme (Figure 2) is as follows. The N-terminus of PsCatch 2.0 has six shortened forms with 12, 17, 19, 21, 26, and 33 amino acids deleted, named PsCatch 2.0 (e1, e2, e3, e4, e5, e6) respectively. The C-terminus of PsCatch 2.0 also has six shortened forms with 9, 13, 16, 18, 23, and 29 amino acids deleted, named PsCatch 2.0 (e7, e8, e9, e10, e11, e12) respectively. 2.0 has the following N-terminuses and C-terminuses: N12-C13 (12 amino acids cleaved at the N-terminus and 13 amino acids cleaved at the C-terminus), N12-C18 (12 amino acids cleaved at the N-terminus and 18 amino acids cleaved at the C-terminus), N12-C29 (12 amino acids cleaved at the N-terminus and 29 amino acids cleaved at the C-terminus), N17-C18 (17 amino acids cleaved at the N-terminus and 18 amino acids cleaved at the C-terminus), and N19-C16 (19 amino acids cleaved at the N-terminus and 16 amino acids cleaved at the C-terminus). There are nine shortened forms of PsCatch 2.0 (e13, e14, e15, e16, e17, e18, e19, e20, e21), each with a deletion of an amino acid: N19-C18 (19 amino acids cleaved at the N-terminus and 18 amino acids cleaved at the C-terminus), N21-C16 (21 amino acids cleaved at the N-terminus and 16 amino acids cleaved at the C-terminus), N21-C18 (21 amino acids cleaved at the N-terminus and 18 amino acids cleaved at the C-terminus), and N26-C22 (26 amino acids cleaved at the N-terminus and 22 amino acids cleaved at the C-terminus).

[0066] In addition to the cleavage strategy, PsCatCh 2.0 and its cleavage mutant PsCatCh 2.0-e18 were modified by introducing single point mutations in amino acids at the amino acid sites E66D, C165L, and H177R of the ion channel functional domain, respectively. These were named PsCatch2.0 (e22, e23, e24, e25, e26, and e27). PsCatch2.0-e28 was obtained by converting the N-terminal amino acid T to G and adding two amino acids LE to the C-terminus, based on PsCatCh 2.0-e26. PsCatch2.0-e29 is obtained by adding the E66D mutation to PsCatCh2.0-e28, and has a double mutation in E66D and C165L.

[0067] First, expression plasmids (using the pGEMHE plasmid as a backbone) of the aforementioned photosensitive protein PsCatCh2.0 (e1~e29) series mutants were constructed using gene synthesis and molecular cloning methods. The LR signal peptide sequence (amino acid sequence shown in SEQ ID NO: 2) was added to the N-terminus of the photosensitive protein. A fluorescent protein YFP fusion expression sequence (amino acid sequence shown in SEQ ID NO: 3) to facilitate detection of the photosensitive protein expression product, as well as a T sequence (amino acid sequence shown in SEQ ID NO: 4) and an endoplasmic reticulum transport signal E sequence (amino acid sequence shown in SEQ ID NO: 5) to enhance cell membrane expression efficiency, were added to the C-terminus of the photosensitive protein. Specifically, the linking order of the photosensitive protein VR2.0 is as follows: first the LR signal peptide sequence at the N-terminus, followed by the PsCatCh2.0 amino acid sequence, then the T sequence, and finally the E sequence. If fusion of the YFP fluorescent protein tag is required, YFP is inserted between the T sequence and the E sequence. When the photosensitive protein VR2.0 is used in clinical gene therapy, the YFP fluorescent protein is not included.

[0068] All plasmids constructed in this example were validated by sequencing, and the plasmid sequences were confirmed to be correct. Next, RNA expressing the above-mentioned photosensitive protein was synthesized using the AmpliCap-Max T7 kit, and the RNA was injected into Xenopus laevis oocytes. The photoresponse of Xenopus laevis oocytes expressing the photosensitive protein was recorded using a two-electrode voltage clamp method. Specifically, Xenopus laevis oocytes were placed in ND96 solution, 1 μM all-trans retinal was added, and cultured at 16°C. 2+ Because it inhibits the opening of activated intracellular intrinsic chloride channels, Ca 2+ 50 nL (200 mM concentration) of the chelating agent BAPTA was injected into African clawed frog oocytes. The BAPTA-injected clawed frog oocytes were incubated at 16°C for 90 minutes. Subsequently, recording was performed using a two-electrode voltage clamp method at 25°C. As shown in Figure 3 and Table 1, the light irradiation conditions (wavelength 450 nm, 5 mW / mm²) were used. 2 Under -60mV conditions, stable photocurrent results were detected for the photosensitive response of the above-mentioned photosensitive protein PsCatch 2.0 e-series mutant.

[0069] [Table 1]

[0070] The current of PsCatch was approximately 1.55 μA, while the current of PsCatch 2.0 was approximately 4.83 μA. The current of PsCatch 2.0-e1 was approximately 4.34 μA, PsCatch 2.0-e18 was approximately 8.23 ​​μA, PsCatch 2.0-e26 was approximately 15.49 μA, PsCatch 2.0-e28 was approximately 15.03 μA, and PsCatch 2.0-e29 was approximately 18.59 μA. Compared to PsCatch 2.0, the photocurrents decreased in 10 species and increased in 19 species. The experimental results indicate that appropriate N-terminal and / or C-terminal cleavage can effectively enhance the photosensitivity of the photosensitive protein PsCatch 2.0.

[0071] Regarding point mutation strategies, the photocurrent of the E66D point mutation based on PsCatch 2.0 increased by approximately 20%, the photocurrent of the C165L point mutation increased by approximately 100.4%, and the photocurrent of the H117R point mutation decreased by approximately 58%. Similar trends were observed with point mutations based on PsCatch2.0-e18. PsCatch2.0-e29, which has a double mutation of E66D and C165L, also showed an increase in photocurrent of approximately 3.56 μA compared to PsCatch 2.0-e28, which has a single mutation of C165L. Therefore, it is presumed that combining cleavage and point mutation strategies in the modification of the photosensitive protein PsCatch has an additive effect on performance improvement. The photocurrents of representative photosensitive proteins, PsCatCh2.0-e9, PsCatCh2.0-e18, and PsCatCh2.0-e26, were compared with those of PsCatCh2.0. The results are shown in Figure 4.

[0072] As shown in Figure 5, in terms of ion selectivity, PsCatCh2.0-e9, PsCatCh2.0-e18, and PsCatCh2.0-e26 are more selective than PsCatCh2.0 in terms of sodium ion (Na + ) Permeability is improved to various degrees, and hydrogen ions (H + ) Permeability was slightly reduced. This is because H inside the cell + It helps to mitigate the side effects of excessive acidification caused by increased ions.

[0073] In summary, various mutants were designed using amino acid cleavage and point mutation strategies. In vitro cell studies and screening yielded multiple VR2.0 series photosensitive protein mutants with varying degrees of improved photoresponsiveness.

[0074] Example 2. Construction of a core plasmid vector expressing a photosensitive protein and preparation of rAAV virus. After a preliminary in vitro screening of several variants with improved photosensitivity compared to PsCatCh 2.0, three photosensitive protein variants, namely PsCatCh 2.0-e9, PsCatCh 2.0-e18, and PsCatCh 2.0-e26, were selected. Core plasmid vectors were constructed and packaged in rAAV virus for further validation and functional testing.

[0075] To ensure efficient expression of photosensitive proteins in retinal cells, a broad-spectrum CMV promoter was used. To facilitate detection of the photosensitive protein expression product, a T sequence to enhance the cell membrane expression efficiency of the fluorescent protein YFP fusion expression sequence and an endoplasmic reticulum transport signal sequence E were added to the C-terminus of the photosensitive protein. A WPRE element (nucleotide sequence shown in SEQ ID NO: 6) and an HGHpA sequence (nucleotide sequence shown in SEQ ID NO: 7) were also added. Using the pAAV-MCS plasmid backbone, the core plasmids of the pAAV-CMV-VR2.0-T-EYFP-E-WPRE-HGHpA series were constructed by molecular cloning and gene synthesis, as shown in Figure 6. The accuracy of the constructed plasmids was confirmed by sequencing.

[0076] Using AAV type 2 serotype, which has high infection efficiency in retinal cells, HEK293 cells were co-transfected with the serotype plasmid pAAV-RC2, the helper plasmid pAAV-Helper, and the pAAV-CMV-VR2.0-T-EYFP-E-WPRE-HGHpA core plasmid via intravitreous injection using a three-plasmid transfection method, and the virus was packaged. After 72 hours, the cell pellet and culture supernatant were collected. The target rAAV virus was purified by iodixanol (idox) ultracentrifugation, and the titer was vg / ml. The virus was aliquoted and stored in a refrigerator at -80°C.

[0077] Example 3. Patch-clamp recording of photoresponse in HEK293T cells expressing the VR2.0 series. A preferred core plasmid carrying the expression cassette of the photosensitive protein VR2.0 series was transfected into adherently cultured HEK293T cells. After transfection, the cells were cultured for 48 hours, and whole-cell potential clamp recordings were performed at a constant temperature of 25°C. The main experimental conditions were as follows. The components of the extracellular solution were 140 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 16 mM glucose, adjusted to pH 7.4 with sodium hydroxide and placed at room temperature. The components of the intracellular solution were 115 mM cesium methanesulfonate, 20 mM cesium chloride, 2.5 mM magnesium chloride, 0.6 mM ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 4 mM magnesium adenosine 5'-triphosphate, 0.4 mM sodium guanosine 5'-triphosphate, and 10 mM creatine phosphate, adjusted to pH 7.2 with cesium hydroxide and kept on ice. The extracellular solution was pre-oxygenated with 100% O2 30 minutes before the experiment. Glass microelectrodes were pulled using a micropipette puller P-1000 (Sutter Instrument) with a resistance of 6 - 8 MΩ. The test HEK293T cells were placed in the extracellular solution and dark-adapted for 30 minutes. After the cells were stabilized, patch-clamp experiments were carried out. To verify the photosensitivity of the test photosensitive protein, current was recorded at a light intensity of 1.66×10 15 photons / cm 2 2 s, and the opening and closing time constants were analyzed using Clampfit 10.6 software. Furthermore, to examine the light response frequency of the test photosensitive protein, pulsed light stimulation was performed at 2, 4, 8, 16, and 32 Hz. A Mightex external optical fiber was used as the light source. The stimulation time was set using BioLED control software, and the light intensity was measured using an optical power meter.

[0078] The experimental results are shown in Figure 7. Figure 7A shows that VR2.0, at a wavelength of 470 nm and a light intensity of 1.66×10 15 photons / cm 2Under the conditions of s, it is still possible to generate a good photocurrent response to optical stimulation at a high frequency of 32 Hz. Figure 7B also shows the results at a wavelength of 470 nm and an optical intensity of 1.66 × 10⁻¹⁶. 15 photon / cm 2 The graph shows the current generated after s stimulation for 1 second, and it can be seen that the current generated by some variants of VR2.0 is improved to varying degrees compared to PsCatCh2.0.

[0079] Furthermore, the current response of several VR2.0 series mutants was tested at a wavelength of 470 nm and various light intensities. The results are shown in Figure 8. These results indicate that many photosensitive protein mutants showed good photocurrent responses, and that the response tended to increase with increasing light intensity. Among these mutants, PsCatch2.0-e2, PsCatch2.0-e4, PsCatch2.0-e5; PsCatch2.0-e13, PsCatch2.0-e16, PsCatch2.0-e17, PsCatch2.0-e18, PsCatch2.0-e19, PsCatch2.0-e20, and PsCatch2.0-e26 all showed improvement compared to PsCatCh2.0.

[0080] Figure 8 shows that PsCatch2.0-e18 and PsCatch2.0-e26 are 7.9 × 10⁻¹⁰ 13 photon / cm 2 This indicates that at light intensity s, photocurrent signals of approximately 50.00 pA and 130.00 pA were generated, respectively. This represents an increase of 2.22 times and 5.78 times, respectively, compared to 22.50 pA for PsCatCh2.0.

[0081] According to the International Commission on Non-Ionizing Radiation Protection (ICIRP) guidelines, the light intensity irradiated to the retina must not exceed the safety threshold for the corresponding wavelength, and high spatiotemporal resolution is required for vision recovery. To achieve these two characteristics, photosensitive proteins must possess both high photosensitivity that is safe for the retina and fast dynamics characteristics with high spatiotemporal resolution. Therefore, the safety threshold for blue light intensity at a wavelength of 470 nm for the retina is 7.62 × 10⁻¹⁰. 14 photon / cm 2 It must be less than or equal to s. In this example, some of the photosensitive protein VR2.0 mutants used were 5.92 × 10⁻⁶. 13 photon / cm 2 A clear photocurrent can be generated at s. This is far below the retinal safe light intensity threshold and will not cause phototoxic effects on retinal cells. Visual signal processing requires a response frequency of 24 Hz, and since the photosensitive protein VR2.0 can respond to light stimuli of at least 32 Hz, it can meet the visual signal requirements.

[0082] Example 4. Intravitreal injection of rAAV virus in rd1 mice Animal experiments were conducted using 4-week-old retinitis pigmentosa model mice (rd1 mice) and wild-type C57BL / 6J mice. Mice were intraperitoneally anesthetized with a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine based on body weight. After sufficient anesthesia, the eye surface and periorbital skin were disinfected with 0.5% activated iodine. To minimize discomfort to the mice from intravitreous injection, the mouse eyeballs were surface-anesthetized with propalacaine hydrochloride eye drops (Alkaine). The mice were fixed and their eyeballs were exposed. Using a Nanoject III high-precision microsyringe and a glass microelectrode, 1.5 μL of rAAV2-CMV-VR2.0 virus (potency approximately 2.5E+12vg / mL) was injected into the vitreous cavity 0.5 mm below the nasal corneal limbus of the mouse. The virus was similarly injected into the vitreous cavity of the other eye, and a control group receiving only the injectable preparation was also established. After injection, levofloxacin hydrochloride eye drop gel (Jieqi) was applied to the mouse eyes to prevent infection. One month after virus injection, the therapeutic effect and behavioral observations were performed in rd1 mice.

[0083] Example 5. Recording of flash visual evoked potentials of RGCs expressing the VR2.0 series in mouse retina. Wild-type C57BL / 6J mice (positive control group), rd1 mice administered intravitreal with rAAV2-CMV-VR2.0 (experimental group), and untreated rd1 littermates (negative control group) were anesthetized by intraperitoneal administration of a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine. Hair from the eyes to the ears was trimmed with clippers to completely expose the bregma and lambdoid sutures. The mice's heads were fixed using a stereotactic device (RWD, Shenzhen, China). 0.25 mm diameter silver wire electrodes were implanted in the right primary visual cortex 48 hours before the FVEP experiment (recording electrodes were positioned 3.6 mm from the bregma point and 2.3 mm on each side). Mice were dark-adapted for 8 hours before the start of the experiment. Subsequently, the animal was anesthetized by intraperitoneal injection, and its pupils were dilated for 5 minutes using tropicamide combination eye drops (0.5% tropicamide + 0.5% phenylephrine hydrochloride). A reference electrode was clipped to the subcutaneous tissue between the eyes, and a ground electrode was clipped to the tail. A flash stimulator (IRC, Chongqing, China) was used to administer 64 repetitive flash stimuli (2800 μs, blue light, 5.0 cds / m²). 2 The experimental results were recorded using a 3.0–70.0 Hz bandpass filter at a sampling rate of 2000 Hz. Flash Visual Evoked Potential (FVEP) data were generated and recorded using RetiMINER 4.0 software, and an N1 amplitude data table was created.

[0084] The experimental results are shown in Figure 9. According to these results, the N1 amplitude of FVEP in wild-type C57BL / 6J mice injected with PBS solvent was 53.03 μV (n=3), the N1 amplitude of FVEP in rd1 mice injected with PBS solvent was 3.14 μV (n=4), the N1 amplitude of FVEP in rd1 mice treated with intravitreal administration of rAAV2-CMV-PsCatCh2.0-e18 virus was 12.37 μV (n=4), and the N1 amplitude of FVEP in rd1 mice treated with intravitreal administration of rAAV2-CMV-PsCatCh2.0-e26 virus was 14.81 μV (n=4).

[0085] These experimental results demonstrate that visual signals generated in the retina of rd1 mice treated with intravitreal administration of rAAV2-CMV-VR2.0 were successfully transmitted to the visual cortex V1.

[0086] Example 6. Light-induced light-dark box behavior assay in mice The light / dark box consisted of two identical compartments (18cm x 20cm x 18cm) connected by an arched door (7cm x 5cm). The light box was equipped with a 470nm LED light source (Mightex, Canada), and the dark box was covered with black cloth. All mice used in the experiment were 10-12 weeks old and were dark-adapted for 2 hours before the experiment. All behavioral experiments were conducted between 6pm and 9pm. At the start of the experiment, C57BL / 6J mice, rd1 mice that had received intravitreal administration of rAAV2-CMV-VR2.0, and normal rd1 mice were exposed to blue light (wavelength 470nm, light intensity 4.7 x 10⁻¹⁰). 14 photon / cm 2 Each mouse was individually placed in a light-dark chamber irradiated with light (s). The mice were allowed to freely explore within the light-dark chamber, and their movement within the chamber was analyzed based on the position of their heads. The collected data was imported into GraphPad Prism 7 software, and one-way ANOVA was used to evaluate statistical significance, with P<0.05 considered statistically significant.

[0087] The proportion of time spent by mice in an open chamber relative to their total time was analyzed and compared. The results are shown in Figure 10. According to the results, the proportion of active time for the wild-type C57BL / 6J mouse group injected with the solvent (positive control) was 17.29% (n=7), the proportion of active time for the rd1 mouse group injected with the solvent (negative control) was 51.20% (n=5), the proportion of active time for the rd1 mouse group intravitreal-administered with rAAV2-CMV-PsCatCh2.0-e18 virus was 27.63% (n=8), and the proportion of active time for the rd1 mouse group intravitreal-administered with rAAV2-CMV-PsCatCh2.0-e26 virus was 26.75% (n=6). These results indicate that intravitreal injection of rAAV2-CMV-VR2.0 restored the light avoidance response in rd1 mice, suggesting that VR2.0 can restore visually induced behavior in rd1 mice with retinal degenerative disease and is effective in treating retinal degenerative disease.

[0088] Example 7. Experiment on visual-motor response behavior in mice A moving grating was displayed on four Lenovo monitors (L1900pA). A mouse activity platform with a mirrored base and a height of 17.5 cm was placed in the center. Matlab was used to set the parameters of the grating program. Each trial lasted 12 minutes. In each stage, the grating rotated clockwise for 30 seconds, counterclockwise for 30 seconds, and then stopped for 10 seconds. The angular velocity of the grating was 12° / second. The grating density (number of gratings per degree, in units of cycles / degree (c / d)) for each stage was set to 0.20 (for software and system testing), 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, and 0.60 c / d, respectively. Mice were dark-adapted for 12 hours before the experiment. C57BL / 6J mice, rd1 mice that received intravitreal administration of rAAV2-CMV-VR2.0, and rd1 mice were individually placed on an activity platform, and the visual sensitivity of the mice was evaluated using this viscomotor system. The collected data were then imported into GraphPad Prism 7 software, and bar graphs were created. Significance was assessed by Student's t-test, and a P<0.05 value was considered statistically significant.

[0089] The experimental results are shown in Figure 11. The results showed that the mean maximum visual sensitivity for the wild-type C57BL / 6J mouse solvent injection group (positive control) was 0.48 c / d (n=7), the mean maximum visual sensitivity for the rd1 mouse solvent injection group (negative control) was 0.09 c / d (n=8), the mean maximum visual sensitivity for rd1 mice treated with rAAV2-CMV-PsCatCh2.0-e18 virus was 0.25 c / d (n=6), and the mean maximum visual sensitivity for rd1 mice treated with rAAV2-CMV-PsCatCh2.0-e26 virus was 0.29 c / d (n=6).

[0090] After intravitreous injection of rAAV2-CMV-VR2.0, rd1 mice showed a significant recovery in light sensitivity. This indicates that VR2.0 can restore visual guidance behavior in rd1 mice with retinal degenerative disease and is effective in treating retinal degenerative disease.

[0091] The sequences for the above-described examples are shown in Table 2 below.

[0092] [Table 2A]

[0093] [Table 2B]

[0094] [Table 2C]

[0095] [Table 2D]

[0096] Here, the amino acid sequences of PsCatCh2.0-e1 to PsCatCh2.0-e29 above contain only the PsCatCh variant portion and do not include the LR signal peptide, T polypeptide, and E polypeptide portions.

Claims

1. A light-sensitive channel protein containing any one of the following PsCatCh variants: (1) A protein obtained by cleaving 1 to 33 amino acids from the N-terminus of a reference protein shown in Sequence ID No. 1; (2) A protein obtained by cleaving 1 to 29 amino acids from the C-terminus of a reference protein shown in Sequence ID No. 1; (3) Proteins obtained by mutations near the retinal dehyde binding site of the reference protein shown in Sequence ID No. 1; (4) Any combination of two or three of (1), (2), and (3) above; or (5) Proteins having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with (1), (2), (3), and (4) above.

2. The photosensitive channel protein according to claim 1, wherein the photosensitive channel protein includes a protein obtained by cleaving 12 to 26 amino acids from the N-terminus of a reference protein shown in SEQ ID NO:

1.

3. The photosensitive channel protein according to claim 2, wherein the photosensitive channel protein includes a protein obtained by cleaving 17 to 21 amino acids from the N-terminus of a reference protein shown in SEQ ID NO:

1.

4. The photosensitive channel protein according to any one of claims 1 to 3, wherein the photosensitive channel protein includes a protein obtained by cleaving 9 to 23 amino acids from the C-terminus of a reference protein shown in SEQ ID NO:

1.

5. The photosensitive channel protein according to claim 4, wherein the photosensitive channel protein includes a protein obtained by cleaving 9 to 18 amino acids from the C-terminus of a reference protein shown in SEQ ID NO:

1.

6. The photosensitive channel protein according to claim 1, wherein the photosensitive channel protein includes a protein obtained by cleaving 12 to 21 amino acids from the N-terminus and 13 to 23 amino acids from the C-terminus of a reference protein shown in SEQ ID NO:

1.

7. The photosensitive channel protein according to any one of claims 1 to 6, wherein the photosensitive channel protein includes a protein obtained by mutating the 66th E of the reference protein shown in SEQ ID NO: 1 to D, and / or the 165th C to L, based on the reference protein shown in SEQ ID NO: 1 or the PsCatCh variant.

8. The amino acid sequence of the light-sensitive channel protein is shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or Sequence IDs 8, 9, 13, 14, 15, 16, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, or 36 have sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. A photosensitive channel protein according to any one of claims 1 to 7.

9. The photosensitive channel protein is fused to the N-terminus of the PsCatCh mutant, and / or an LR signal peptide. The present invention further comprises a T polypeptide linked to the C-terminus of the PsCatCh mutant, and an E polypeptide linked to the T polypeptide. The photosensitive channel protein according to any one of claims 1 to 8, wherein the amino acid sequence of the LR signal peptide is shown in SEQ ID NO: 2, the amino acid sequence of the T polypeptide is shown in SEQ ID NO: 4, and the amino acid sequence of the E polypeptide is shown in SEQ ID NO:

5.

10. A nucleic acid molecule comprising a nucleotide sequence encoding a photosensitive channel protein according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid molecule described in claim 10.

12. A recombinant virus comprising the nucleic acid molecule described in claim 10 or the vector described in claim 11.

13. The recombinant virus according to claim 12, which is a recombinant adeno-associated virus.

14. A pharmaceutical composition comprising a photosensitive channel protein according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, a vector according to claim 11, or a recombinant virus according to claim 12 or 13, and a pharmaceutically acceptable carrier.

15. Use of a photosensitive channel protein according to any one of claims 1 to 9 in the preparation of a drug for treating retinal photoreceptor cell degeneration disease.

16. The use according to claim 15, wherein the retinal photoreceptor cell degenerative disease includes retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and congenital amaurosis (LCA).

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