New photosensitive channel protein VR2.0 and use thereof

Abstract

Provided in the present application are a new photosensitive channel protein VR2.0 and the use thereof. The photosensitive channel protein VR2.0 series provided in the present application has a significant therapeutic effect in the treatment of retinal photoreceptor degenerative diseases. The new photosensitive channel protein has a higher photosensitivity, fast kinetics and low side effects. Under the same light stimulation conditions, the new photosensitive channel protein has a relatively fast response frequency and can maintain a stable current signal during high-frequency responses. The present application provides a better choice for the development of optogenetic therapy for retinal photoreceptor degenerative diseases and has an important application and popularization value.

Description

Novel light-sensitive channel protein VR2.0 and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a novel light-sensitive channel protein VR2.0 and its uses. Background Art

[0002] Retinal photoreceptor degenerative diseases are a type of degenerative disease characterized by the progressive loss of function of photoreceptor cells and pigment epithelial cells. These diseases are primarily caused by gene mutations or dysfunction of the retinal pigment epithelium (RPE cells). Common examples include retinitis pigmentosa (RP) and age-related macular degeneration (AMD), two major and intractable blinding eye diseases. The prevalence of photoreceptor degeneration caused by genetic factors is approximately 1 / 3500-1 / 4000. There are currently 400,000 patients with retinitis pigmentosa in China and over 1.5 million patients worldwide. The incidence of secondary retinal photoreceptor degenerative diseases caused by acquired factors such as drugs and diseases is also increasing.

[0003] Since the apoptosis of photoreceptor cells in retinal photoreceptor degenerative diseases is irreversible and most of these diseases have a high degree of genetic heterogeneity, the treatment of related diseases has become very difficult. Current treatment methods mainly include stem cell transplantation, gene therapy, retinal prosthesis implantation and optogenetic therapy. Among them, optogenetic therapy utilizes the integrity of the remaining cell structure of retinal degenerative lesions and uses recombinant adeno-associated virus (AAV) as a vector to target the expression of photosensitive proteins in cone cells (early degenerative lesions), bipolar cells or ganglion cells (mid-to-late degenerative lesions) to restore the retina's photosensitivity. In addition, stem cell transplantation, virtual reality systems and holographic imaging technologies can also be combined to restore visual function.

[0004] Optogenetic therapy, as an ophthalmic therapeutic strategy that does not rely on restoring gene function at specific gene loci and can respond to light stimulation at the single-cell level, holds great potential for treating retinal photoreceptor degenerative diseases. Photosensitive proteins that have been exploited for visual restoration using optogenetics fall into two main categories: microbial and mammalian. Microbial photosensitive proteins typically exhibit fast kinetics but low photosensitivity, such as the cation-permeable photosensitive protein Channelrhodopsin-2 (ChR2), which was first used for visual restoration. Mammalian endogenous photosensitive proteins belong to the G protein-coupled receptor (GPCR) family and possess seven transmembrane α-helices. These proteins typically exhibit high photosensitivity but limited kinetics, such as rhodopsin from rod photoreceptors. Currently, existing optogenetic tools for visual restoration (i.e., photosensitive proteins) do not meet the requirements for both high photosensitivity and fast photoreaction kinetics, suffering from issues such as ion selectivity and the potential for side effects such as intracellular acidification. Therefore, it is necessary to develop a class of light-sensitive proteins with high sensitivity to light stimulation, faster photoreaction kinetics, fewer side effects, and the ability to maintain stable current signals under high-frequency light response, combined with more efficient gene delivery vectors targeting retinal cells, so that optogenetic therapy can be better used in the treatment of retinal photoreceptor degenerative diseases. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present application provides a novel light-sensitive channel protein VR2.0 and its use.

[0006] Specifically, this application involves the following aspects:

[0007] 1. A light-sensitive channel protein comprising any one of the following PsCatCh variants:

[0008] (1) Proteins obtained by truncating 1-33 amino acids at the N-terminus of the reference protein shown in SEQ ID NO. 1;

[0009] (2) proteins obtained by truncating 1-29 amino acids at the C-terminus of the reference protein shown in SEQ ID NO. 1;

[0010] (3) a protein obtained by mutation around the retinal binding site of the reference protein shown in SEQ ID NO. 1;

[0011] (4) a combination of two or three of (1), (2), and (3) above; or

[0012] (5) A protein having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to (1), (2), (3), or (4).

[0013] 2. The light-sensitive channelrhodopsin according to item 1, wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 12-26 amino acids at the N-terminus of the reference protein shown in SEQ ID NO. 1.

[0014] 3. The light-sensitive channelrhodopsin according to item 2, wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 17-21 amino acids at the N-terminus of the reference protein shown in SEQ ID NO. 1.

[0015] 4. The light-sensitive channelrhodopsin according to any one of items 1 to 3, wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 9 to 23 amino acids at the C-terminus of a reference protein shown in SEQ ID NO. 1.

[0016] 5. The light-sensitive channelrhodopsin according to item 4, wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 9-18 amino acids at the C-terminus of the reference protein shown in SEQ ID NO. 1.

[0017] 6. The light-sensitive channelrhodopsin according to item 1, wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 12-21 amino acids at the N-terminus and 13-23 amino acids at the C-terminus based on the reference protein shown in SEQ ID NO. 1.

[0018] 7. The light-sensitive channelrhodopsin according to any one of items 1 to 6, wherein the light-sensitive channelrhodopsin comprises a protein obtained by mutating E at position 66 to D and / or C at position 165 to L of the reference protein shown in SEQ ID NO.1 or the PsCatCh variant.

[0019] 8. The light-sensitive channelrhodopsin according to any one of items 1 to 7, wherein the amino acid sequence of the light-sensitive channelrhodopsin is as 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,

[0020] or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to 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.

[0021] 9. The light-sensitive channelrhodopsin according to any one of items 1 to 8, wherein the light-sensitive channelrhodopsin further comprises an LR signal peptide fused to the N-terminus of the PsCatCh variant,

[0022] and / or a T polypeptide at the C-terminus of the PsCatCh variant and an E polypeptide connected to the T polypeptide,

[0023] 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.

[0024] 10. A nucleic acid molecule comprising a nucleotide sequence encoding the light-sensitive channelrhodopsin according to any one of items 1 to 9.

[0025] 11. A vector comprising the nucleic acid molecule according to item 10.

[0026] 12. A recombinant virus, comprising the nucleic acid molecule described in item 10 or the vector described in item 11.

[0027] 13. The recombinant virus according to item 12, wherein the recombinant virus is a recombinant adeno-associated virus.

[0028] 14. A pharmaceutical composition comprising the light-sensitive channelrhodopsin 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.

[0029] 15. Use of the light-sensitive channel protein according to any one of items 1 to 9 in the preparation of a drug for treating retinal photoreceptor cell degenerative diseases.

[0030] 16. The method according to claim 15, wherein the retinal photoreceptor cell degenerative diseases include retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and congenital amaurosis (LCA).

[0031] Advantages and benefits of this application:

[0032] The VR2.0 series of channelrhodopsin proteins provided in this application demonstrates clear therapeutic efficacy in treating retinal photoreceptor degenerative diseases. This novel channelrhodopsin combines high sensitivity with fast kinetics, maintaining stable photocurrent signals at high frequencies and exhibiting a faster response frequency under the same light stimulation conditions. This application provides a new and improved option for developing optogenetic therapies for retinal photoreceptor degenerative diseases, expanding the scope of clinical optogenetic therapy and possessing significant application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a simulated three-dimensional structure diagram of the light-sensitive protein PsCatch (1-305aa), PsCatch2.0 (1-343aa) and the representative mutant PsCatch2.0e26;

[0034] FIG2 is a schematic diagram of the amino acid structure of the photosensitive protein VR2.0 series obtained by modifying the photosensitive protein PsCatCh;

[0035] FIG3 is a diagram of light-responsive current intensity signals of mutants of the photosensitive protein VR2.0 series;

[0036] FIG4 is a diagram showing the light-responsive electrical signal waveforms of some mutants of the photosensitive protein VR2.0 series at the Xenopus laevis oocyte level;

[0037] Figure 5 is the Na of some mutants of the photosensitive protein VR2.0 series + ions, H + a graph of ion permeability;

[0038] FIG6 is a diagram of the pAAV-CMV-VR2.0-EYFP core vector carrying expression cassettes for some mutants of the photosensitive protein VR2.0 series;

[0039] Figure 7 shows the currents recorded by patch clamp after expressing some mutants of the photosensitive protein VR2.0 series and the control group PsCatCh2.0 in HEK293T cells, which reflects the photosensitivity and response frequency of the photosensitive protein to light stimulation. Figure 7A: VR2.0 under 470nm wavelength and 1.66×10 15 photons / cm 2 s, the current generated by stimulation frequencies of 2 Hz, 4 Hz, 8 Hz, 16 Hz, and 32 Hz; Figure 7B: VR2.0 at a wavelength of 470 nm and a light intensity of 1.66 × 10 15 photons / cm 2 s, the current diagram of stimulating 1s;

[0040] Figure 8 shows the current size of some mutants of the VR2.0 series of light-sensitive proteins and the control group PsCatCh2.0 after expressing them in HEK293T cells, recorded by patch clamp at 470nm wavelength and different light intensities, where "photons / cm 2 s" is the unit of light intensity.

[0041] Figure 9 shows the visual evoked potentials (VEPs) of C57BL / 6J mice, rd1 mice treated with intravitreal rAAV2-CMV-VR2.0-EYFP, and untreated littermates of rd1 mice. Figure 9A: Representative waveforms of the VEPs of the aforementioned mice; Figure 9B: Statistical histogram of the N1 amplitude of the VEPs of the aforementioned mice. P < 0.001 indicates a significant difference.

[0042] Figure 10 shows the light-avoidance responses of C57BL / 6J mice, rd1 mice treated with intravitreal rAAV2-CMV-VR2.0-EYFP, and untreated littermates of rd1 mice in a light-dark chamber. Figure 10A: Schematic diagram of the light-dark chamber experiment; Figure 10B: Statistical bar graph of the time the mice spent in the light chamber. P < 0.05 indicates significant differences.

[0043] Figure 11 shows the optokinetic responses of C57BL / 6J mice, rd1 mice treated with intravitreal rAAV2-CMV-VR2.0-EYFP, and untreated littermates of rd1 mice. Figure 11A: Schematic diagram of the optokinetic response experiment; Figure 11B: Statistical bar graph of the visual acuity of the aforementioned mice. P < 0.05 indicates significant differences. DETAILED DESCRIPTION

[0044] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0045] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0046] As used in this article, "photosensitive channel protein" and "photosensitive protein" can be used interchangeably, referring to a class of proteins on the cell membrane that can sense light stimulation and produce specific effects (such as changing the open state of ion channels). They can be divided into two types: activating and inhibitory types, which can cause neuronal excitation or inhibition.

[0047] The PsCatCh variant in the present application is modified based on the light-sensitive channel protein (Platymonas subcordiformis channelrhodopsin, PsChR) derived from subcordiform algae.

[0048] channelrhodopsin

[0049] The present application provides a light-sensitive channel protein, including any one of the following PsCatCh variants:

[0050] (1) Proteins obtained by truncating 1-33 amino acids at the N-terminus of the reference protein shown in SEQ ID NO. 1;

[0051] (2) proteins obtained by truncating 1-29 amino acids at the C-terminus of the reference protein shown in SEQ ID NO. 1;

[0052] (3) a protein obtained by mutation around the retinal binding site of the reference protein shown in SEQ ID NO. 1;

[0053] (4) a combination of two or three of (1), (2), and (3) above; or

[0054] (5) A protein having at least 60% sequence identity to (1), (2), (3), or (4), such as 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 more than 99% sequence identity.

[0055] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 1-33 amino acids at the N-terminus of the reference protein shown in SEQ ID NO. 1. The 1-33 amino acids can 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.

[0056] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 12-26 amino acids at the N-terminus of the reference protein shown in SEQ ID NO.1.

[0057] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 17-21 amino acids at the N-terminus of the reference protein shown in SEQ ID NO.1.

[0058] In a specific embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13, or a sequence of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13. %, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity to any of NO. 13.

[0059] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 1-29 amino acids at the C-terminus of the reference protein shown in SEQ ID NO. 1. The 1-29 amino acids can 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.

[0060] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 9-23 amino acids at the C-terminus of the reference protein shown in SEQ ID NO.1.

[0061] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 9-18 amino acids at the C-terminus of the reference protein shown in SEQ ID NO.1.

[0062] In a specific embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19, or a sequence thereof. 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity to any one of the peptides of the invention.

[0063] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 1-33 amino acids at the N-terminus and 1-29 amino acids at the C-terminus based on the reference protein shown in SEQ ID NO.1.

[0064] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 12-21 amino acids at the N-terminus and 13-23 amino acids at the C-terminus based on the reference protein shown in SEQ ID NO.1.

[0065] In a specific embodiment, as shown in any one of SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, or a combination of SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO. %, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity to any of NO. 36.

[0066] In a specific embodiment, the PsCatCh variant is a protein obtained by mutating E at position 66 to D and / or C at position 165 to L based on the reference protein shown in SEQ ID NO. 1 or the PsCatCh variant. For the point mutations in the PsCatCh variant, positions 66 and 165 correspond to the reference protein shown in SEQ ID NO. 1.

[0067] In a specific embodiment, as shown in any one of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, or a combination of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID %, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity to any of NO. 28.

[0068] In a specific embodiment, the light-sensitive channelrhodopsin further comprises a LR signal peptide fused to the N-terminus of the PsCatCh variant.

[0069] In a specific embodiment, the amino acid sequence of the LR signal peptide is shown as SEQ ID NO.2.

[0070] In a specific embodiment, the light-sensitive channelrhodopsin further comprises a T polypeptide connected to the C-terminus of the PsCatCh variant to increase the efficiency of cell membrane expression, and an endoplasmic reticulum export signal sequence E polypeptide connected to the T polypeptide.

[0071] In a specific embodiment, the amino acid sequence of the T polypeptide is shown as SEQ ID NO. 4. In a specific embodiment, the amino acid sequence of the E polypeptide is shown as SEQ ID NO. 5.

[0072] Those skilled in the art will appreciate that a connecting peptide sequence consisting of a small number of amino acids that does not affect the function of the light-sensitive channel protein may be present between any two of the PsCatCh variant, T polypeptide, and E polypeptide.

[0073] In a specific embodiment, the amino acid sequence of the connecting peptide is shown as SEQ ID NO.37.

[0074] Nucleic acid molecules, vectors, and recombinant viruses

[0075] The present application provides a nucleic acid molecule comprising a nucleotide sequence encoding any one of the above-mentioned light-sensitive channel proteins.

[0076] In some embodiments, the nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can be replicated and / or expressed in cells. In some embodiments, the DNA molecule can be replicated and / or expressed in eukaryotic cells. In some embodiments, the DNA molecule can be replicated and / or expressed in prokaryotic cells. In some embodiments, the DNA molecule can be expressed in eukaryotic cells and can be replicated in prokaryotic cells. Therefore, in addition to comprising the nucleotide sequence encoding the channelrhodopsin protein, the DNA molecule also comprises genetic manipulation or regulatory elements for replication and / or expression in prokaryotic and / or eukaryotic cells. In some embodiments, the eukaryotic cells are human retinal photoreceptor cells. In some embodiments, the eukaryotic cells are human cone cells. In some embodiments, the eukaryotic cells are bipolar cells or ganglion cells.

[0077] The present application provides a vector comprising the above-mentioned nucleic acid molecule.

[0078] In some embodiments, the vector is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of double-stranded DNA molecules. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, and linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify in a cell independently of the genomic genetic information stored in the prokaryotic nucleoid or nucleoid, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. For example, the DNA plasmid according to the present application is a plasmid constructed based on the pGEMHE plasmid.

[0079] The present application provides a recombinant virus, which comprises any one of the above-mentioned nucleic acid molecules or vectors.

[0080] 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 mutant or derivative thereof. Preferably, the recombinant virus is AAV. In some embodiments, the AAV comprises 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 serotype or variants thereof.

[0081] Pharmaceutical composition

[0082] The present application provides a pharmaceutical composition comprising any one of the above-mentioned channelrhodopsin proteins, nucleic acid molecules, vectors or recombinant viruses, and a pharmaceutically acceptable carrier.

[0083] The form of the pharmaceutical composition depends on a number of criteria, including, for example, the route of administration, the extent of the disease, or the dose to be administered.

[0084] In some embodiments, the pharmaceutical composition can be formulated for delivery to a subject by an appropriate route, including but not limited to, oral route, injection route (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracardiac injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc.), mucosal route (e.g., intranasal administration, oral administration, etc.), sublingual route, rectal route, transdermal route, intraocular route, pulmonary route. Depending on the desired route of administration, the pharmaceutical composition can be formulated as tablets, capsules, pills, dragees, powders, granules, cachets, lozenges, suppositories, suspensions, emulsions, syrups, aerosols (as solid or in liquid media), sprays, ointments, pastes, patches, creams, lotions, gels, inhalants, etc.

[0085] It will be appreciated by those skilled in the art that the dosage and frequency of administration of the pharmaceutical composition may vary depending on the age, weight, or individual response of the subject to the vaccine and the specific administration selected.

[0086] Treatment methods, therapeutic uses

[0087] The present application provides the use of the above-mentioned light-sensitive channelrhodopsin in treating retinal photoreceptor cell degenerative diseases.

[0088] The present application provides the use of the above-mentioned light-sensitive channel protein in the preparation of a drug for treating retinal photoreceptor cell degenerative diseases.

[0089] The present application provides a method for treating retinal photoreceptor cell degenerative diseases, comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to a subject.

[0090] Among them, in the above methods or uses, the retinal photoreceptor cell degenerative diseases can cover various retinal photoreceptor cell degenerative diseases known in the art, for example, can include retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, congenital amaurosis (LCA), etc.

[0091] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments.

[0092] Example

[0093] Example 1. Construction of VR2.0 series mutant expression plasmids based on the microbial photosensitive protein PsCatch and comparison of their performance in Xenopus oocytes

[0094] To enhance the properties of the light-sensitive protein, primarily its plasma membrane transport efficiency and light sensitivity, we conducted amino acid truncation and mutation modifications on the PsCatch portion of PsCatch 2.0 (amino acid sequence shown in SEQ ID NO. 1), building on previous research and our understanding of the structure and function of the PsCatch 2.0 light-sensitive protein (Figure 1). The PsCatch 2.0 protein has seven transmembrane regions, with approximately 30 amino acids in the N-terminal extramembrane region and 40 amino acids in the C-terminal extramembrane region.

[0095] The specific truncation and mutation design scheme (as shown in Figure 2) includes six truncations of PsCatch 2.0 with 12, 17, 19, 21, 26, and 33 amino acids missing at the N-terminus, respectively named PsCatch2.0 (e1, e2, e3, e4, e5, and e6); six truncations of PsCatch 2.0 with 9, 13, 16, 18, 23, and 29 amino acids missing at the C-terminus, respectively named PsCatch2.0 (e7, e8, e9, e10, e11, and e12); The N-terminus and C-terminus of 2.0 have N12-C13 (12 amino acids are truncated at the N-terminus and 13 amino acids are truncated at the C-terminus), N12-C18 (12 amino acids are truncated at the N-terminus and 18 amino acids are truncated at the C-terminus), N12-C29 (12 amino acids are truncated at the N-terminus and 29 amino acids are truncated at the C-terminus), N17-C18 (17 amino acids are truncated at the N-terminus and 18 amino acids are truncated at the C-terminus), N19-C16 (19 amino acids are truncated at the N-terminus and 16 amino acids are truncated at the C-terminus), N19-C18 (12 amino acids are truncated at the N-terminus and 29 amino acids are truncated at the C-terminus), Nine truncated forms of the PsCatch2.0 protein with amino acid deletions were identified, including N21-C16 (21 amino acids deleted from the N-terminus and 16 amino acids deleted from the C-terminus), N21-C18 (21 amino acids deleted from the N-terminus and 18 amino acids deleted from the C-terminus), and N26-C22 (26 amino acids deleted from the N-terminus and 22 amino acids deleted from the C-terminus). They were named PsCatch2.0 (e13, e14, e15, e16, e17, e18, e19, e20, and e21), respectively.

[0096] In addition to the truncation strategy, we selected PsCatCh 2.0 and its truncated mutant PsCatCh 2.0-e18, respectively, and performed single-point amino acid mutations at amino acid sites E66D, C165L, and H177R in the ion channel functional region. These mutations were named PsCatch2.0 (e22, e23, e24, e25, e26, and e27). PsCatch2.0-e28 was derived from PsCatCh 2.0-e26 by replacing the N-terminal amino acid T with a G and adding two amino acids, LE, to the C-terminus. PsCatch2.0-e29 was derived from PsCatch2.0-e28 by adding the E66D mutation, resulting in a double mutation of E66D and C165L.

[0097] We first constructed expression plasmids for the aforementioned photosensitive protein PsCatch 2.0 (e1 to e29) series of mutants (using the pGEMHE plasmid as a backbone) using gene synthesis and molecular cloning. An LR signal peptide sequence (amino acid sequence shown in SEQ ID NO. 2) was added to the N-terminus of the photosensitive protein. To facilitate detection of the photosensitive protein expression product, a fusion expression fluorescent protein YFP sequence (amino acid sequence shown in SEQ ID NO. 3) was added to the C-terminus of the photosensitive protein, along with a T sequence (amino acid sequence shown in SEQ ID NO. 4) to increase cell membrane expression efficiency and an endoplasmic reticulum export signal E sequence (amino acid sequence shown in SEQ ID NO. 5). Specifically, the order of attachment of the photosensitive protein VR2.0 is as follows: the LR signal peptide sequence is first at the N-terminus, followed by the PsCatch 2.0 amino acid sequence, then the T sequence, and finally the E sequence. When a YFP fluorescent protein tag is required, YFP is inserted between the T and E sequences. When the photosensitive protein VR2.0 is used for clinical gene therapy, it does not contain the YFP fluorescent protein.

[0098] The plasmids constructed in this example have been verified by gene sequencing, and the results show that the plasmid sequences are correct. Then, the RNA expressing the light-sensitive protein was synthesized using the AmpliCap-MaxT7 kit, and the RNA was injected into African clawed oocytes. The light response of African clawed oocytes expressing the light-sensitive protein was recorded using a two-electrode voltage clamp. Specifically, the African clawed oocytes were cultured in ND96 solution, and 1 μM all-trans-retinal was added. The culture temperature was 16 ° C. In order to block Ca 2+ To activate the opening of endogenous chloride channels, 50 nl (200 mM) Ca2+ was injected into Xenopus oocytes. 2+ The oocytes injected with BAPTA were incubated at 16°C for 90 min. Then, two-electrode voltage clamp recording was performed at room temperature (25°C). Under light irradiation conditions (450 nm wavelength, 5 mW / mm 2 , -60 mV), the stable photocurrent results of the photosensitivity reaction of the above-mentioned photosensitive protein PsCatch 2.0e series mutants were detected, as shown in Figure 3 and Table 1.

[0099] Table 1

[0100] Among them, the current of PsCatch was approximately 1.55μA, and the current of PsCatch 2.0 was approximately 4.83μA. The current of PsCatch2.0-e1 was approximately 4.34μA, the current of PsCatch 2.0-e18 was approximately 8.23μA, the current of PsCatch 2.0-e26 was approximately 15.49μA, the current of PsCatch 2.0-e28 was approximately 15.03μA, and the current of PsCatch 2.0-e29 was approximately 18.59μA. Compared with PsCatch 2.0, the photocurrent of 10 types was reduced, and the photocurrent of 19 types was improved. The results show that appropriate N-terminal or (and) C-terminal truncation can effectively improve the photosensitivity of the light-sensitive protein PsCatch 2.0.

[0101] Regarding the point mutation strategy, the E66D point mutation based on PsCatch 2.0 increased the photocurrent by approximately 20%, the C165L point mutation increased the photocurrent by approximately 100.4%, and the H117R point mutation resulted in an approximately 58% decrease. Similar trends were observed for point mutations based on PsCatch2.0-e18. PsCatch2.0-e29, with the E66D and C165L double mutations, also showed a photocurrent increase of approximately 3.56 μA compared to PsCatch 2.0-e28, with the C165L single mutation. Therefore, we infer that the combined use of truncation and point mutation strategies for photosensitive protein PsCatch modification has an additive effect in improving performance. We compared the photocurrents of the representative photosensitive proteins PsCatCh2.0-e9, PsCatCh2.0-e18, PsCatCh 2.0-e26 and PsCatCh2.0, and the results are shown in Figure 4.

[0102] As shown in Figure 5, in terms of ion selectivity, PsCatCh2.0-e9, PsCatCh2.0-e18, and PsCatCh2.0-e26 have a higher selectivity for sodium ions (Na + ) permeability has been improved to varying degrees, hydrogen ions (H + ) The permeability is slightly reduced, which helps to reduce the side effects caused by excessive acidification due to the increase of H+ ions in the cells.

[0103] In summary, we designed a variety of mutants through the strategy of amino acid truncation and point mutation. After testing and screening at the in vitro cell level, we obtained a variety of VR2.0 series photosensitive protein mutants with different levels of improvement in light response characteristics.

[0104] Example 2. Construction of core plasmid vector expressing light-sensitive protein and preparation of rAAV virus

[0105] After preliminarily screening out multiple mutants with better photosensitivity than PsCatCh2.0 in in vitro experiments, we selected three photosensitive protein mutants, PsCatCh2.0-e9, PsCatCh 2.0-e18, and PsCatCh 2.0-e26, to construct core plasmid vectors and package them into rAAV viruses, which were then further verified and functionally tested.

[0106] To enable efficient expression of the photosensitive protein in retinal cells, we employed the broad-spectrum CMV promoter. To facilitate detection of the photosensitive protein expression product, we added a fusion expression fluorescent protein YFP sequence to the C-terminus of the photosensitive protein, as well as sequence T and endoplasmic reticulum export signal sequence E to increase cell membrane expression efficiency. We also added the WPRE element (nucleotide sequence shown in SEQ ID NO. 6) and the HGHpA sequence (nucleotide sequence shown in SEQ ID NO. 7). We used the pAAV-MCS plasmid backbone and constructed the pAAV-CMV-VR2.0-T-EYFP-E-WPRE-HGHpA series of core plasmids through molecular cloning and gene synthesis, as shown in Figure 6. Sequencing verification results confirmed that the constructed plasmids were correct.

[0107] We used intravitreal injection of AAV serotype 2, which has a high efficiency of infecting retinal cells. Using a three-plasmid transfection method, HEK293 cells were co-transfected with the pAAV-RC2 serotype plasmid, the pAAV-AdHelper helper plasmid, and the pAAV-CMV-VR2.0-T-EYFP-E-WPRE-HGHpA core plasmid for virus packaging. After 72 hours, the cell pellet and culture supernatant were collected. The desired rAAV virus was then purified by iodixanol (idox) ultracentrifugation to obtain titers of vg / ml and stored in aliquots at -80°C.

[0108] Example 3. Patch clamp recording of light response of HEK293T cells expressing VR2.0 series

[0109] Adherent HEK293T cells were transfected with a core plasmid carrying the expression cassette for the VR2.0 series of light-sensitive proteins. Following transfection, cells were cultured for 48 h, and then whole-cell voltage-clamp recordings were performed at 25°C. The following experimental conditions were used: the extracellular solution consisted of 140 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM 4-hydroxyethylpiperazineethanesulfonic acid, and 16 mM glucose, adjusted to pH 7.4 with sodium hydroxide, and maintained at room temperature; the intracellular solution consisted of 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-hydroxyethylpiperazineethanesulfonic acid, 4 mM adenosine 5'-triphosphate magnesium salt, 0.4 mM guanosine 5'-triphosphate sodium salt, and 10 mM creatine phosphate, adjusted to pH 7.2 with cesium hydroxide, and maintained on ice. The extracellular fluid was pre-oxygenated with 100% O2 30 minutes before the experiment. Glass microelectrodes were drawn using a horizontal drawer P-1000 (Sutter Instruments) with a resistance of 6-8 MΩ. HEK293T cells to be tested were placed in the extracellular fluid and dark-adapted for 30 minutes. After the cells stabilized, patch clamp experiments were performed. To verify the photosensitivity of the photosensitive protein to be tested, a 1.66×10 15 photons / cm 2 Currents were recorded at a light intensity of 10 s, and the opening and closing time constants were analyzed using Clampfit 10.6 software. Furthermore, to investigate the light response frequency of the photosensitive protein under test, pulsed light stimulation was set at 2, 4, 8, 16, and 32 Hz. The light source was a Mightex external fiber optic. The stimulation time was set using the BioLED control software, and the specific light intensity was measured using an optical power meter.

[0110] The experimental results are shown in Figure 7. From Figure 7A, we can see that VR2.0 has a wavelength of 470nm and a light intensity of 1.66×10 15 photons / cm 2 s, VR2.0 can still produce a good photocurrent response to light stimulation at a high frequency of 32 Hz. As can be seen from Figure 7B, at a wavelength of 470 nm and a light intensity of 1.66 × 10 15 photons / cm 2 Under the condition of 5% s, the current generated by stimulation for 1 s was improved to varying degrees by some mutants of VR2.0 compared with PsCatCh2.0.

[0111] We also tested the current responses of some VR2.0 mutants at 470 nm wavelength and varying light intensities. The results are shown in Figure 8. The results show that various photosensitive protein mutants exhibited robust photocurrent responses, with an increasing trend as light intensity increased. Among them, 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 improvements compared to PsCatCh2.0.

[0112] From Figure 8, we can also observe that PsCatch2.0-e18 and PsCatch2.0-e26 have a 13 photons / cm 2 s, respectively, generated photocurrent signals of approximately 50.00pA and 130.00pA, which were 2.22 times and 5.78 times higher than 22.50pA of PsCatCh2.0.

[0113] According to the guidelines of the International Commission on Non-Ionizing Radiation Protection (ICIRP), the intensity of light applied to the retina must not exceed the safety threshold of the corresponding wavelength, and visual restoration requires high temporal and spatial resolution. The above two characteristics require that the photosensitive protein has both high light sensitivity for retinal safety and fast kinetic characteristics that meet high temporal and spatial resolution. Therefore, the intensity of 470nm blue light for retinal safety should not exceed 7.62×10 14 photons / cm 2 s. In this example, we used some photosensitive protein VR2.0 mutants that can be expressed at 5.92×10 13 photons / cm 2 s generates a significant photocurrent, far below the safe light intensity threshold for the retina, and does not cause phototoxicity to retinal cells. In terms of response frequency, visual signal processing requires 24Hz, and the photosensitive protein VR2.0 can respond to light stimulation of at least 32Hz, which can meet the needs of visual signal processing.

[0114] Example 4. Intravitreal injection of rAAV virus into rd1 mice

[0115] We performed animal experiments using 4-week-old retinitis pigmentosa model mice (rd1 mice) and wild-type C57BL / 6J mice. Mice were anesthetized with a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine, administered intraperitoneally according to body weight. After adequate anesthesia, the ocular surface and periorbital skin were disinfected with 0.5% active iodine. To minimize discomfort caused by intravitreal injection, the eyeball was topically anesthetized with proparacaine hydrochloride eye drops (Alcaine). After securing the mouse and exposing the eyeball, 1.5 μL of rAAV2-CMV-VR2.0 virus (titer approximately 2.5E+12 vg / mL) was aspirated using a Nanoject III high-precision microinjector with a glass microelectrode. The injection was performed intravitreally 0.5 mm below the nasal corneoscleral limbus. The other eye was injected in the same manner, and a control group receiving only the injection agent was also established. After the injection, levofloxacin hydrochloride ophthalmic gel (Jeqi) was applied to the mouse eyeball to prevent infection. One month after the virus injection, the efficacy of the rd1 mouse was tested and behavioral observations were performed.

[0116] Example 5. Recording Flash Visual Evoked Potentials from RGCs Expressing VR2.0 Series in Mouse Retinas

[0117] Wild-type C57BL / 6J mice (positive control), rd1 mice treated with intravitreal rAAV2-CMV-VR2.0 (experimental group), and untreated littermate rd1 mice (negative control group) were anesthetized with a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine injected intraperitoneally. Hair was trimmed from the eyes to the ears to fully expose the bregma and lambdoid sutures. The heads of the mice were fixed using a stereotaxic apparatus (RWD, Shenzhen, China). Forty-eight hours before the FVEP experiment, a 0.25 mm diameter silver wire electrode was implanted in the right primary visual cortex (recording electrode, 3.6 mm lateral to the bregma and 2.3 mm lateral to the bregma). Mice were dark-adapted for 8 hours before the experiment. Afterwards, the mice were anesthetized intraperitoneally and the pupils were dilated with 0.5% tropicamide eye drops (0.5% tropicamide + 0.5% phenylephrine hydrochloride) for 5 minutes. The reference electrode was inserted under the skin between the eyes, and the ground electrode was clipped to the rat's tail. A flash stimulator (IRC, Chongqing, China) was used to deliver 64 repetitive light stimulations (2800 μs, blue light, 5.0 cds / m 2 When the bandpass filter was set between 3.0 and 70.0 Hz, the experimental results were recorded at a sampling rate of 2000 Hz. Flash visual evoked potential (FVEP) data were generated and recorded using RetiMINER 4.0 software to obtain the N1 amplitude data table.

[0118] The experimental results are shown in Figure 9, which show that the N1 amplitude of FVEP of wild-type C57BL / 6J mice injected with PBS solvent was 53.03 μV, n=3; the N1 amplitude of FVEP of rd1 mice injected with PBS solvent was 3.14 μV, n=4; the N1 amplitude of FVEP of rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e18 virus was 12.37 μV, n=4; the N1 amplitude of FVEP of rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e26 virus was 14.81 μV, n=4.

[0119] The above experimental results show that after intravitreal injection of rAAV2-CMV-VR2.0, the visual signals generated on the retina of rd1 mice were successfully transmitted to the visual cortex V1 area.

[0120] Example 6. Light-induced light / dark box behavior experiment in mice

[0121] The light / dark box consists of two compartments of equal size (18cm x 20cm x 18cm) on the left and right, connected by an arched door (7cm x 5cm). The light box is equipped with a 470nm LED light source (Mightex, Canada), and the dark box is wrapped with a black cloth. All experimental mice were between 10 and 12 weeks old, and the mice were dark-adapted for 2 hours before the experiment. All behavioral experiments were performed between 18:00 and 21:00. At the beginning of the experiment, C57BL / 6J mice, rd1 mice treated with intravitreal injection of rAAV2-CMV-VR2.0, and ordinary rd1 mice were placed alone in a blue light (470nm wavelength, light intensity of 4.7×10 14 photons / cm 2 The mice were placed in a light box (s) and allowed to explore freely. Their movements in the light and dark box were analyzed by head position. The collected data were then imported into GraphPad Prism 7 software and analyzed using one-way ANOVA, with P < 0.05 considered significant.

[0122] We analyzed and compared the proportion of mice's total time spent active in the light box. The results are shown in Figure 10. The results showed that the proportion of active time in the wild-type C57BL / 6J mice injected with the solvent (positive control) was 17.29% (n = 7); the proportion of active time in the rd1 mice injected with the solvent (negative control) was 51.20% (n = 5); the proportion of active time in the rd1 mice treated with intravitreal rAAV2-CMV-PsCatCh2.0-e18 was 27.63% (n = 8); and the proportion of active time in the rd1 mice treated with intravitreal rAAV2-CMV-PsCatCh2.0-e26 was 26.75% (n = 6). These results indicate that intravitreal rAAV2-CMV-VR2.0 treatment restored the avoidance response to light in rd1 mice, demonstrating that VR2.0 can restore visually guided behavior in rd1 mice with retinal degeneration and demonstrates its effectiveness in treating retinal degeneration.

[0123] Example 7. Mouse optokinetic behavior experiment

[0124] Four Lenovo monitors (L1900pA) displayed a moving grating, with a 17.5-cm-high mouse platform in the center and a mirrored bottom. Matlab was used to program the grating program. Each trial lasted 12 minutes, with the grating rotating for 30 seconds clockwise, 30 seconds counterclockwise, and a 10-second pause. The grating's angular velocity was 12° / s. The grating density (i.e., the number of gratings per degree, units: cycles / degree (c / d)) 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 in each stage. Mice were dark-adapted for 12 hours before the experiment. C57BL / 6J mice, rd1 mice treated with intravitreal rAAV2-CMV-VR2.0, and individual rd1 mice were placed on an activity platform. Visual acuity was assessed using this optokinetic system. The collected data were then imported into GraphPad Prism 7 software for plotting bar graphs. Student's t-test was used to assess significance, with P < 0.05 considered significant.

[0125] The experimental results are shown in Figure 11, which show that the average maximum visual acuity of the wild-type C57BL / 6J mouse solvent injection group (positive control) is 0.48c / d, n=7; the maximum average visual acuity of the rd1 mouse solvent injection group (negative control) is 0.09c / d, n=8; the maximum visual acuity of the rd1 mouse after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e18 virus treatment is 0.25c / d, n=6; the maximum visual acuity of the rd1 mouse after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e26 virus treatment is 0.29c / d, n=6.

[0126] After intravitreal injection of rAAV2-CMV-VR2.0, rd1 mice significantly recovered their sensitivity to light, demonstrating that VR2.0 can restore visually guided behavior in rd1 mice with retinal degeneration and is effective in treating retinal degeneration.

[0127] The sequences involved in the above examples are shown in Table 2 below.

[0128] Table 2 List of related sequences

[0129] Among them, the amino acid sequences of the above-mentioned PsCatCh2.0-e1 to PsCatCh2.0-e29 only include the PsCatch variant part, and do not include the LR signal peptide, T polypeptide, and E polypeptide parts.

Claims

1. A light-sensitive channel protein comprising any one of the following PsCatCh variants: (1) Proteins obtained by truncating 1-33 amino acids at the N-terminus of the reference protein shown in SEQ ID NO. 1; (2) proteins obtained by truncating 1-29 amino acids at the C-terminus of the reference protein shown in SEQ ID NO. 1; (3) a protein obtained by mutation around the retinal binding site of the reference protein shown in SEQ ID NO. 1; (4) a combination of two or three of (1), (2), and (3) above; or (5) A protein having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to (1), (2), (3), or (4). 2 . The light-sensitive channelrhodopsin according to claim 1 , wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 12-26 amino acids at the N-terminus of a reference protein shown in SEQ ID NO.

1. 3 . The light-sensitive channelrhodopsin according to claim 2 , wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 17-21 amino acids at the N-terminus of a reference protein shown in SEQ ID NO.

1. 4 . The light-sensitive channelrhodopsin according to claim 1 , wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 9 to 23 amino acids at the C-terminus of a reference protein shown in SEQ ID NO.

1. 5 . The light-sensitive channelrhodopsin according to claim 4 , wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 9-18 amino acids at the C-terminus of a reference protein shown in SEQ ID NO.

1. 6 . The light-sensitive channelrhodopsin according to claim 1 , wherein the light-sensitive channelrhodopsin comprises a protein obtained by truncating 12-21 amino acids at the N-terminus and 13-23 amino acids at the C-terminus of a reference protein shown in SEQ ID NO.

1.

7. The light-sensitive channelrhodopsin according to any one of claims 1 to 6, wherein the light-sensitive channelrhodopsin comprises a protein obtained by mutating E at position 66 to D and / or C at position 165 to L of the reference protein shown in SEQ ID NO. 1 or the PsCatCh variant.

8. The light-sensitive channelrhodopsin according to any one of claims 1 to 7, wherein the amino acid sequence of the light-sensitive channelrhodopsin is as 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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to 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.

9. The light-sensitive channelrhodopsin according to any one of claims 1 to 8, wherein the light-sensitive channelrhodopsin further comprises an LR signal peptide fused to the N-terminus of the PsCatCh variant, and / or a T polypeptide at the C-terminus of the PsCatCh variant and an E polypeptide connected to the T polypeptide, 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 the channelrhodopsin according to any one of claims 1 to 9. A vector comprising the nucleic acid molecule according to claim 10 . 12 . A recombinant virus comprising the nucleic acid molecule of claim 10 or the vector of claim 11 . The recombinant virus according to claim 12 , wherein the recombinant virus is a recombinant adeno-associated virus.

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

15. Use of the light-sensitive channelrhodopsin according to any one of claims 1 to 9 in the preparation of a medicament for treating retinal photoreceptor cell degenerative diseases.

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

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