Novel light-sensitive channel protein VR3.0 and application thereof

Abstract

The invention provides a novel light-sensitive channel protein VR3.0 and application thereof. The photosensitive channel protein VR3.0 series provided by the invention has a relatively wide photosensitive wavelength range, relatively high photoreaction sensitivity and relatively fast photoreaction kinetics, keeps the stability of a current signal under high frequency response, and shows good response amplitude and frequency under the photostimulation of multiple wavelengths (especially white light and natural light). The light-sensitive channel protein VR3.0 series provided by the invention has a definite treatment effect on retina photoreceptor cell degenerative diseases, and can be used for preparing medicines for recovering the photoreceptor function of retina, recovering the vision or light-sensitive ability of a subject and treating the retina degenerative diseases.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically, relates to a novel light-sensitive channel protein VR3.0 and its use. Background Art

[0002] Retinal photoreceptor cell degenerative diseases are a type of degenerative disease characterized by progressive functional loss of photoreceptor cells and pigment epithelial cells. This type of disease is mainly caused by gene mutations or dysfunction of retinal pigment epithelial cells (RPE cells). Common typical examples are retinitis pigmentosa (RP) and age-related macular degeneration (AMD), which are also two major and difficult blinding eye diseases. The prevalence of photoreceptor cell degeneration caused by genetic reasons is about 1 / 3500-1 / 4000. There are currently 400,000 patients with retinitis pigmentosa in China and more than 1.5 million patients worldwide. The incidence of secondary retinal photoreceptor cell degenerative diseases caused by acquired reasons 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 uses the integrity of the remaining cell structure of retinal degenerative lesions to target the expression of photosensitive proteins in cones (early degenerative lesions), bipolar cells or ganglion cells (mid-to-late degenerative lesions) using recombinant adeno-associated virus (AAV) as a vector to restore the photosensitivity of the retina. In addition, stem cell transplantation, virtual reality systems and holographic imaging technology can also be combined to restore visual function.

[0004] Optogenetic therapy, as an ophthalmic treatment strategy that does not rely on restoring the gene function of specific gene locus mutations and can respond to light stimulation at the single-cell level, has great application potential in the treatment of retinal photoreceptor degenerative diseases. The photosensitive proteins that play a role in visual restoration using optogenetic strategies are mainly divided into two categories: microbial photosensitive proteins and mammalian photosensitive proteins. Microbial photosensitive proteins usually have fast kinetic characteristics but low light sensitivity, such as the cation-permeable photosensitive protein Channelrhodopsin-2 (ChR2) first applied to visual function restoration. Mammalian endogenous photosensitive proteins belong to the G Protein-Coupled Receptors (GPCRs) family and have a seven-transmembrane α-helix structure. They usually have high light sensitivity but insufficient kinetics, such as Rhodopsin derived from rod photoreceptor cells (Rods). Currently, the existing optogenetic tools (i.e., photosensitive proteins) applied to visual restoration cannot well meet the requirements of high light sensitivity and fast light response kinetics, and there are problems such as differences in ion selectivity and side effects that easily lead to intracellular acidification. Therefore, it is necessary to develop a class of photosensitive proteins with high sensitivity to light stimulation, fast light response kinetics, small side effects, and the ability to maintain a stable current signal under high-frequency light response. Combining with a more efficient gene delivery vector targeting retinal cells can better apply optogenetic therapy to the treatment field of retinal photoreceptor degenerative diseases. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present application provides a novel photosensitive channel protein VR3.0 and its uses.

[0006] Specifically, the present application relates to the following aspects:

[0007] 1. A photosensitive channel protein, comprising a channelrhodopsin protein or a variant thereof containing the amino acid sequence shown in SEQ ID NO.1, wherein the variant is selected from any one of the following:

[0008] (1) A protein obtained by truncating 1-23 amino acids at the N-terminus of the channelrhodopsin protein;

[0009] (2) A protein obtained by truncating 90-189 amino acids at the C-terminus of the channelrhodopsin protein;

[0010] (3) A protein obtained by truncating 1-23 amino acids at the N-terminus of the channelrhodopsin protein and truncating 90-189 amino acids at the C-terminus; or

[0011] A protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity with (1), (2), or (3).

[0012] 2. The light-sensitive channel protein according to item 1, wherein the amino acid sequence of the channelrhodopsin protein is as shown in SEQ ID NO.1.

[0013] 3. The light-sensitive channel protein according to item 1 or 2, wherein the variant is a protein obtained by truncating 5-15 amino acids at the N-terminus of the channelrhodopsin protein.

[0014] 4. The light-sensitive channel protein according to item 1 or 2, wherein the variant is a protein obtained by truncating 102-179 amino acids at the C-terminus of the channelrhodopsin protein.

[0015] 5. The light-sensitive channel protein according to item 1 or 2, wherein the variant is a protein obtained by truncating 5-15 amino acids at the N-terminus of the channelrhodopsin protein and truncating 102-179 amino acids at the C-terminus of the channelrhodopsin protein.

[0016] 6. The light-sensitive channel protein according to any one of items 1-5, wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NOs.8-26,

[0017] or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity with any one of SEQ ID NOs.8-26.

[0018] 7. The light-sensitive channel protein according to any one of items 1-6, wherein the light-sensitive channel protein further comprises an LR signal peptide fused to the N-terminus of the channelrhodopsin protein or its variant,

[0019] and / or a T polypeptide linked to the C-terminus of the channelrhodopsin protein or its variant to increase the cell membrane expression efficiency and an endoplasmic reticulum export signal sequence E polypeptide linked to the T polypeptide.

[0020] 8. The light-sensitive channel protein according to item 7, wherein the amino acid sequence of the LR signal peptide is as shown in SEQ ID NO.2, the amino acid sequence of the T polypeptide is as shown in SEQ ID NO.4, and the amino acid sequence of the E polypeptide is as shown in SEQ ID NO.5.

[0021] 9. The light-sensitive channel protein according to item 7 or 8, wherein a linker peptide sequence is further included between any two of the channelrhodopsin protein or its variant, the T polypeptide, and the E polypeptide.

[0022] 10. The light-sensitive channel protein according to item 9, wherein the amino acid sequence of the linker peptide is as shown in SEQ ID NO. 28.

[0023] 11. A nucleic acid molecule comprising a nucleotide sequence encoding the light-sensitive channel protein according to any one of items 1-10.

[0024] 12. A vector comprising the nucleic acid molecule according to item 11.

[0025] 13. A recombinant virus, wherein the recombinant virus comprises the nucleic acid molecule according to item 11 or the vector according to item 9.

[0026] 14. The recombinant virus according to item 13, wherein the recombinant virus is a recombinant adeno-associated virus.

[0027] 15. A pharmaceutical composition comprising the light-sensitive channel protein according to any one of items 1-10, the nucleic acid molecule according to item 11, the vector according to item 12, or the recombinant virus according to item 13 or 14, and a pharmaceutically acceptable carrier.

[0028] 16. Use of the pharmaceutical composition according to item 15 in the preparation of a drug for treating retinal photoreceptor cell degenerative diseases.

[0029] 17. The use according to item 16, wherein the retinal photoreceptor cell degenerative diseases include retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and Leber congenital amaurosis (LCA).

[0030] 18. A method for treating retinal photoreceptor cell degenerative diseases, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to item 15.

[0031] 19. The method according to item 18, wherein the retinal photoreceptor cell degenerative diseases include retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and Leber congenital amaurosis (LCA).

[0032] Advantages and beneficial effects of the present application:

[0033] The light-sensitive channel protein VR3.0 series provided by the present application has a relatively wide light-sensitive wavelength range, higher light response sensitivity, faster light response kinetics, maintains the stability of the current signal under high-frequency response, and exhibits good response amplitude and frequency under light stimulation of multiple wavelengths (especially under white light and natural light). The light-sensitive channel protein VR3.0 series provided by the present application has a definite therapeutic effect on retinal photoreceptor degenerative diseases and can be used to prepare drugs for restoring the photoreceptor function of the retina, restoring the visual acuity or light-sensing ability of a subject, and treating retinal degenerative diseases. Description of the Drawings

[0034] Figure 1 It is a simulated three-dimensional structure diagram of the photosensitive protein NCR1 (1-432aa).

[0035] Figure 2 It is a schematic diagram of the amino acid structure of the photosensitive protein VR3.0 series obtained by modifying the photosensitive protein NCR1.

[0036] Figure 3 It is a waveform diagram of the light response electrical signal of some variants of the photosensitive protein VR3.0 series at the level of Xenopus oocytes.

[0037] Figure 4A It is a graph showing the trend of current change with increasing light intensity of some variants of the photosensitive protein VR3.0 series at the level of Xenopus oocytes; 4B is the photocurrent level generated by some variants of the photosensitive protein VR3.0 series at the level of Xenopus oocytes in the wavelength range from 420 nm to 600 nm under light illumination.

[0038] Figure 5 It is a schematic diagram of the core viral vector carrying the expression cassette of the photosensitive protein VR3.0 series.

[0039] Figure 6 It is after expressing some variants of the photosensitive protein VR3.0 series and the control group PsCatch2.0 photosensitive protein at the level of HEK293T cells. A. Comparison of the light response current diagrams under the conditions of a wavelength of 470 nm and a light intensity of 1.7×10 15 photons / cm 2 s; B. Comparison of the light response current diagrams under the conditions of a wavelength of 530 nm and a light intensity of 1.9×10 15 photons / cm 2 s; C. Comparison of the light response current diagrams under the conditions of a wavelength of 590 nm and a light intensity of 2.1×10 15 photons / cm 2 s.

[0040] Figure 7 shows the light response current graphs (7A) of different variants of the photosensitive protein VR3.0 series at different wavelengths (470 nm, 530 nm, 590 nm) and different light intensities after expression at the HEK293T cell level; and the light current change curve graphs (7B) of different variants of the photosensitive protein VR3.0 at 470 nm wavelength and different light intensities.

[0041] Figure 8 After expressing some variants of the photosensitive protein VR3.0 series and the control group PsCatch2.0 photosensitive protein at the HEK293T cell level, at a wavelength of 470 nm and a light intensity of 1.7×10 15 photons / cm 2 s, the light response current graphs generated at stimulation frequencies of 2 Hz, 4 Hz, 8 Hz, 16 Hz, and 32 Hz.

[0042] Figure 9 Shows the light avoidance responses of C57BL / 6J mice, rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0-EYFP, and littermate rd10 mice untreated in a light-dark box. A is a schematic diagram of the light-dark box experiment; B is a statistical bar graph of the activity time of the above mice in the light box. Among them, P<0.05, there is a significant difference, marked with *; when the p value is less than 0.01, it is extremely significant, marked with **; when the p value is less than 0.001, the degree of significance is even greater, marked with ***; when the p value is less than 0.0001, the degree of significance is extremely high, marked with ****; ns indicates no significant difference.

[0043] Figure 10 Shows the optokinetic responses of C57BL / 6J mice, rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0-EYFP, and littermate rd10 mice untreated. A: Schematic diagram of the optokinetic response experiment; B: Statistical bar graph of the visual acuity of the above mice. Among them, P<0.05, there is a significant difference marked with *; when the p value is less than 0.01, it is extremely significant, marked with **; when the p value is less than 0.001, the degree of significance is even greater, marked with ***; when the p value is less than 0.0001, the degree of significance is extremely high, marked with ****; ns indicates no significant difference. Detailed implementation manners

[0044] The present application will be further described below in conjunction with embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not used to limit the present application.

[0045] Unless otherwise defined, the technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in experiments or practical applications, the materials and methods are described hereinafter. In case of conflict, the present specification, including its definitions, will prevail. In addition, the materials, methods, and examples are for illustrative purposes only and not restrictive. The present application will be further described below in conjunction with specific embodiments, but it is not intended to limit the scope of the present application.

[0046] As used herein, "light-sensitive channel protein" and "photosensitive protein" can be used interchangeably and refer 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: activation type and inhibition type, and can cause the excitation or inhibition of neurons.

[0047] Light-sensitive channel protein

[0048] The present application provides a light-sensitive channel protein, which includes channelrhodopsin protein (NCR1) containing the amino acid sequence shown in SEQ ID NO.1 or its variant, or includes channelrhodopsin protein (NCR1) containing the amino acid sequence shown in SEQ ID NO.1 or its variant, wherein the variant is selected from any one of the following:

[0049] (1) A protein obtained by truncating 1-23 amino acids at the N-terminus of the channelrhodopsin protein;

[0050] (2) A protein obtained by truncating 90-189 amino acids at the C-terminus of the channelrhodopsin protein;

[0051] (3) A protein obtained by truncating 1-23 amino acids at the N-terminus of the channelrhodopsin protein and truncating 90-189 amino acids at the C-terminus; or

[0052] (4) A protein having at least 60% sequence identity with (1), (2), or (3), such as a protein 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 more than 99% sequence identity.

[0053] In a specific embodiment, the amino acid sequence of the channelrhodopsin protein is as shown in SEQ ID NO.1.

[0054] In a specific embodiment, the variant is a protein obtained by truncating 1 - 23 amino acids at the N-terminus of the channelrhodopsin protein with the amino acid sequence as shown in SEQ ID NO.1. The 1 - 23 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, or 23 amino acids.

[0055] In a specific embodiment, the variant is a protein obtained by truncating 5 - 15 amino acids at the N-terminus of the channelrhodopsin protein with the amino acid sequence as shown in SEQ ID NO.1.

[0056] In a specific embodiment, the amino acid sequence of the variant is as shown in any one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, or has a sequence identity of 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%, or 99% or more with any one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11.

[0057] In a specific embodiment, the variant is a protein obtained by truncating 90 - 189 amino acids from the C-terminus of the channelrhodopsin protein with the amino acid sequence as shown in SEQ ID NO.1. The 90 - 189 amino acids can be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, or 189 amino acids.

[0058] In a specific embodiment, the variant is a protein obtained by truncating 102 - 179 amino acids from the C-terminus of the channelrhodopsin protein with the amino acid sequence as shown in SEQ ID NO.1.

[0059] In a specific embodiment, the amino acid sequence of the variant is as shown in any one of SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, or has a sequence identity of 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%, or more than 99% with any one of SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17.

[0060] In a specific embodiment, the variant is a protein obtained by truncating 1 - 23 amino acids at the N-terminus and 90 - 189 amino acids at the C-terminus of the channelrhodopsin protein with the amino acid sequence as shown in SEQ ID NO.1. Among them, the 1 - 23 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, or 23 amino acids; the 90 - 189 amino acids can be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, or 189 amino acids.

[0061] In a specific embodiment, the variant is a protein obtained by truncating 5 - 15 amino acids at the N-terminus and 102 - 159 amino acids at the C-terminus of the channelrhodopsin protein with the amino acid sequence as shown in SEQ ID NO.1.

[0062] In a specific embodiment, the amino acid sequence of the variant is as shown in any one of SEQ ID NO.18, SEQ ID NO.19, 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, or has a sequence identity of 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%, or more than 99% with any one of SEQ ID NO.18, SEQ ID NO.19, 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.

[0063] In a specific embodiment, the light-sensitive channel protein further comprises an LR signal peptide fused to the N-terminus of the channelrhodopsin protein or its variant.

[0064] In a specific embodiment, the amino acid sequence of the LR signal peptide is as shown in SEQ ID NO.2.

[0065] In a specific embodiment, the light-sensitive channel protein further comprises a T polypeptide linked to the C-terminus of the channelrhodopsin protein or its variant to increase the cell membrane expression efficiency and an endoplasmic reticulum export signal sequence E polypeptide linked to the T polypeptide.

[0066] In a specific embodiment, the amino acid sequence of the T polypeptide is as shown in SEQ ID NO.4, and the amino acid sequence of the E polypeptide is as shown in SEQ ID NO.5.

[0067] Those skilled in the art can understand that between any two of the channelrhodopsin protein or its variant, the T polypeptide, and the E polypeptide, there may also be a linker peptide sequence composed of a small number of amino acids that does not affect the function of the light-sensitive channel protein.

[0068] In a specific embodiment, the amino acid sequence of the linker peptide is as shown in SEQ ID NO.28.

[0069] Nucleic acid molecule, vector, recombinant virus

[0070] The present application provides a nucleic acid molecule comprising a nucleotide sequence encoding any one of the above-mentioned light-sensitive channel proteins.

[0071] In some embodiments, the nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can replicate and / or express in cells. In some embodiments, the DNA molecule can replicate and / or express in eukaryotic cells. In some embodiments, the DNA molecule can replicate and / or express in prokaryotic cells. In some embodiments, the DNA molecule can express in eukaryotic cells and replicate in prokaryotic cells. Therefore, in addition to the nucleotide sequence encoding the light-sensitive channel protein, the DNA molecule further comprises gene manipulation or regulatory elements 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 a ganglion cell.

[0072] The present application provides a vector comprising the above-mentioned nucleic acid molecule.

[0073] In some embodiments, the vector is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid composed of a double-stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also cover linear DNA molecules. Specifically, the term "plasmid" also covers molecules obtained by linearizing a circular plasmid, for example, by cutting the circular plasmid with a restriction endonuclease to transform the circular plasmid molecule into a linear molecule, and linear molecules that can replicate in prokaryotes. Plasmids can replicate, that is, they can be amplified independently of the genomic genetic information stored in the prokaryotic cell nucleoid or nucleoid in cells, and can be used for cloning, that is, 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.

[0074] The present application provides a recombinant virus, and the recombinant virus comprises any one of the above-mentioned nucleic acid molecules or vectors.

[0075] In some embodiments, the recombinant virus is an adeno-associated virus (AAV), chimeric AAV, adenovirus, retrovirus, lentivirus, herpes simplex virus, 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 serotypes or variants thereof.

[0076] Pharmaceutical composition

[0077] The present application provides a pharmaceutical composition comprising any one of the above-mentioned light-sensitive channel proteins, nucleic acid molecules, vectors or recombinant viruses, and a pharmaceutically acceptable carrier.

[0078] The form of the pharmaceutical composition depends on multiple criteria, including for example the route of administration, the degree of the disease, or the dosage of administration, etc.

[0079] In some embodiments, the pharmaceutical composition can be formulated to be delivered to a subject by a suitable route, including but not limited to, by the oral route, injection routes (such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracardiac injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc.), mucosal routes (such as intranasal administration, intraoral 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 solids or in liquid media), sprays, ointments, pastes, patches, creams, lotions, gels, inhalants, etc.

[0080] Those skilled in the art can understand that the dosage and frequency of administration of the pharmaceutical composition can vary depending on the age, weight of the subject, or the individual response to the vaccine and the specific administration selected.

[0081] Therapeutic method, therapeutic use

[0082] The present application provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating retinal photoreceptor degenerative diseases.

[0083] The present application provides a method for treating retinal photoreceptor degenerative diseases, comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to a subject.

[0084] Among them, the retinal photoreceptor degenerative diseases can cover various retinal photoreceptor degenerative diseases known in the art, for example, can include retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, Leber congenital amaurosis (LCA), etc.

[0085] It should be understood that the present application includes various aspects, embodiments described herein, and combinations of the described aspects and / or embodiments. 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 obvious to those skilled in the art to which the present application pertains. Therefore, those of ordinary skill in the art should recognize that the scope of the present application also includes such improvements and modifications to the described aspects and embodiments.

[0086] Examples

[0087] Example 1. Construction of VR3.0 series variant expression plasmids based on the modification of the microbial photosensitive protein NCR1

[0088] Channelrhodopsin-2 (ChR2) is a membrane protein containing seven transmembrane helices and a covalently bound retinal chromophore. After ChR2, researchers discovered channelrhodopsin proteins with different ion selectivities. In the genomic data of the alga H. catenoides, we identified another class of ChR sequences and found that it has a high Na+ ion permeability. Therefore, we named this Na+ ion channel rhodopsin HcNCR1 (abbreviated as NCR1). The full-length NCR1 protein consists of 432 amino acids. To improve its photosensitive properties in eukaryotic cells, we truncated NCR1 (432aa) and added several plasma membrane targeting enhancing peptides at both ends to improve the plasma membrane transport efficiency.

[0089] Based on our previous research foundation and the structure of the NCR1 photosensitive protein ( Figure 1) and understanding of its functions, we performed amino acid truncation modification on the NCR1 protein (amino acid sequence shown in SEQ ID NO.1). The NCR1 protein has 7 transmembrane regions, with approximately more than 20 amino acids in the extracellular region at the N-terminus and approximately more than 220 amino acids in the extracellular region at the C-terminus. The specific truncation and mutation design schemes ( Figure 2 ) are as follows:

[0090] There are 4 truncated forms of the N-terminus of NCR1 with deletions of 5, 10, 15, and 24 amino acids, named NCR1 e1, NCR1 e2, NCR1 e3, and NCR1 e4, respectively;

[0091] There are 6 truncated forms of the C-terminus of NCR1 with deletions of 102, 126, 149, 159, 179, and 190 amino acids, named NCR1 e5, NCR 1e6, NCR 1e7, NCR1 e8, NCR 1e9, and NCR1 e10, respectively;

[0092] There are 9 truncated forms of NCR1 with amino acid deletions at both the N-terminus and C-terminus, namely N6-C330 (5 amino acids truncated at the N-terminus and 102 amino acids truncated at the C-terminus), N6-C283 (5 amino acids truncated at the N-terminus and 149 amino acids truncated at the C-terminus), N6-C273 (5 amino acids truncated at the N-terminus and 159 amino acids truncated at the C-terminus), N11-C283 (11 amino acids truncated at the N-terminus and 149 amino acids truncated at the C-terminus) at the N-terminus and C-terminus of NCR10, N11-C273 (11 amino acids truncated at the N-terminus and 159 amino acids truncated at the C-terminus) at the N-terminus and C-terminus of NCR1, N16-C306 (15 amino acids truncated at the N-terminus and 126 amino acids truncated at the C-terminus) at the N-terminus and C-terminus of NCR1, N16-C283 (15 amino acids truncated at the N-terminus and 149 amino acids truncated at the C-terminus) at the N-terminus and C-terminus of NCR1, N16-C273 (15 amino acids truncated at the N-terminus and 159 amino acids truncated at the C-terminus) at the N-terminus and C-terminus of NCR1, N16-C242 (15 amino acids truncated at the N-terminus and 190 amino acids truncated at the C-terminus) at the N-terminus and C-terminus of NCR 1, named NCR1 e11, NCR1 e12, NCR1 e13, NCR1 e14, NCR1 e15, NCR1 e16, NCR1 e17, NCR1 e18, and NCR1 e19, respectively.

[0093] We constructed expression plasmids (with the pGEMHE plasmid as the backbone) for the above-mentioned series of variants of the photosensitive protein NCR1 (NCR 1e1 to e19). An LR signal peptide sequence (shown by the amino acid sequence SEQ ID NO.2) was added to the N-terminus of the photosensitive protein; for facilitating the detection of the expression product of the photosensitive protein, a sequence for fusing and expressing the fluorescent protein YFP (shown by the amino acid sequence SEQ ID NO.3), as well as a T sequence (amino acid sequence as shown by SEQ ID NO.4) for increasing the cell membrane expression efficiency and an E sequence (amino acid sequence SEQ ID NO.5) for endoplasmic reticulum export signal were added to the C-terminus of the photosensitive protein. Specifically, for the photosensitive protein VR3.0, the connection order is that at the N-terminus, first is the LR signal peptide sequence, then the amino acid sequence of NCR1 or its variant, then the T sequence, and finally the E sequence. When the YFP fluorescent protein tag needs to be fused, YFP is inserted between the T sequence and the E sequence. When the photosensitive protein VR3.0 is used for clinical gene therapy, it does not contain the YFP fluorescent protein.

[0094] Among them, NCR 2.0 was obtained by adding an LR signal peptide sequence to the N-terminus based on NCR1 and connecting a T sequence, a YFP sequence, an E sequence, and a linker peptide sequence to the C-terminus. NCR 2.0e1-e19 was obtained by adding an LR signal peptide sequence to the N-terminus based on NCR1 e1-e19 and connecting a T sequence and an E sequence to the C-terminus.

[0095] Example 2. Performance comparison of the VR3.0 series of variants modified based on the microbial photosensitive protein NCR1 at the level of Xenopus laevis oocytes

[0096] We synthesized the above-mentioned RNA for expressing the photosensitive protein using the AmpliCap-MaxT7 kit, and injected 30 ng of the above-mentioned RNA into different Xenopus laevis oocytes respectively. Two days after injection, the light response of the Xenopus laevis oocytes expressing the photosensitive protein was recorded using a two-electrode voltage clamp. Specifically, the electrophysiological measurement of Xenopus laevis oocytes was carried out in the oocyte Ringer's solution (Ori, 110 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES and pH 7.6). The electrode capillary (Φ = 1.5 mm, wall thickness 0.178 mm) was filled with 3M KCl, and the tip opening produced a resistance of 0.4–1 MΩ. Stimulation and data acquisition were controlled using an AD-DA converter and WinWCP software (v4.1.7). The light power was measured using a PLUS2 power and energy meter.

[0097] Under the light irradiation condition (wavelength 532 nm, 0.5 mW / mm 2, when (-40 mV), the photocurrent responses of the above-mentioned photosensitive protein NCR 2.0 and the NCR2.0e series variants (also known as the VR3.0 series) were detected and compared with those of the photosensitive protein NCR1. The results are as Figure 3 shown.

[0098] Among them, the current of NCR1 is about 0.41 μA; the current of NCR2.0 is about 6.75 μA, which is about 16.46 times higher than that of NCR1; the current of NCR2.0 e5 is about 13.82 μA, which is about 33.71 times higher than that of NCR1; the current of NCR2.0 e7 is about 17.79 μA, which is about 43.39 times higher than that of NCR1; the current of NCR2.0 e14 is about 27.01 μA, which is about 65.88 times higher than that of NCR1. The experimental results show that the photocurrent response levels of the NCR2.0 variants constructed by truncating the N-terminal or (and) C-terminal of the NCR1 protein as shown above are significantly higher than those of the photosensitive protein NCR1, and can achieve stronger photosensitivity than the photosensitive protein NCR1, with great application potential.

[0099] We studied the trend of the photocurrent response change of the VR3.0 series photosensitive proteins with the increase of light intensity. At a wavelength of 532 nm, the light intensity increased from 0.03 mW / mm 2 to 4 mW / mm 2 (0.031, 0.062, 0.125, 0.25, 0.5, 1, 2, 3, 4 mW / mm 2 ). We detected the photocurrent of the representative photosensitive proteins among them (as Figure 4A shown). It can be observed that with the increase of light intensity, the photocurrent responses of the photosensitive proteins in the NCR2.0 series all show a gradually increasing trend, and increase slowly and gradually reach a plateau at 3 mW / mm 2 . The experimental results show that at a certain wavelength (532 nm), within a certain light intensity range (0.03 mW / mm 2 to 4 mW / mm 2 ), the tested VR3.0 series photosensitive proteins all have the photosensitive characteristics that the photocurrent increases with the increase of light intensity.

[0100] To test the wavelength range of light response of the VR3.0 series of photosensitive proteins, we conducted the following experiments. Action spectrum measurements were carried out by combining narrow-band interference filters (Edmund Optics) of different wavelengths and a white light generator PhotoFluor II to obtain a light wavelength range from 420 nm to 620 nm. This includes: 422 nm, 439 nm, 459 nm, 481 nm, 496 nm, 516 nm, 540 nm, 562 nm, 595 nm and 620 nm. To achieve similar light intensities at different wavelengths, we adjusted the output power of the light source and used gray filters to ensure that the light intensity was approximately 0.5 mW / mm 2 . The detection results showed that the NCR2.0 series of photosensitive proteins had obvious photocurrent responses to wavelengths between 450 nm and 600 nm, and the highest response wavelength was around 532 nm (as Figure 4B shown). The experimental results showed that the VR3.0 series of photosensitive proteins had a very wide continuous response wavelength range and were sensitive to the main visible light bands represented by blue, green, and red. Therefore, these tests of light responses at the in vitro cell level showed that the VR3.0 series of photosensitive protein variants we constructed had significantly improved light response characteristics compared to the natural NCR1 photosensitive protein, laying a solid foundation for carrying out functional research and applications at the in vivo animal level.

[0101] Example 3. Construction of the core plasmid vector expressing photosensitive proteins and preparation of rAAV viruses

[0102] We constructed core plasmid viral vectors containing the gene of the control PsCath2.0 photosensitive protein (amino acid sequence shown in SEQ ID NO.27) or the VR3.0 series of photosensitive protein variants, packaged them into rAAV viruses, and then carried out the next verification and functional tests.

[0103] To enable the efficient expression of photosensitive proteins in retinal cells, we used a broad-spectrum CMV promoter; to facilitate the detection of photosensitive protein expression products, we added a fusion expression fluorescent protein YFP sequence to the C-terminus of the photosensitive protein, as well as sequences T for increasing cell membrane expression efficiency and endoplasmic reticulum export signal sequence E, 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 to construct the pAAV-CMV-VR3.0-T-EYFP-E-WPRE-HGHpA series of core plasmids, as Figure 5 shown. The sequencing verification results showed that the constructed plasmids were correct.

[0104] We used the commonly used AAV2 serotype and transfected HEK293 cells with three plasmids. The serotype plasmid pAAV-RC2, the packaging helper plasmids pAAV-Helper and the core plasmid pAAV-CMV-VR3.0-T-EYFP-E-WPRE-HGHpA were co-transfected into HEK293 cells to package the virus. After 72 h, the cell pellet and the culture medium supernatant were collected. Then, the required rAAV virus was obtained by the method of iodixanol (idox) ultracentrifugation purification. The titer unit was vg / ml, and after aliquoting, it was stored in a -80 °C refrigerator.

[0105] Example 4. Patch clamp recording of the light response of HEK293T cells expressing the VR3.0 series

[0106] We transfected the core plasmid carrying the photosensitive protein VR3.0 series expression cassette prepared in Example 3 into adherently cultured HEK293T cells. After transfection, the cells were cultured for another 48 h, and then whole-cell voltage clamp mode recording was performed at a constant room temperature of 25 °C. The main experimental conditions were as follows: The extracellular solution consisted of 140 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 16 mM glucose, and the pH was adjusted to 7.4 with sodium hydroxide and placed 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-aminoethylether)-N,N,N',N'-tetraacetic acid, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 4 mM adenosine 5'-triphosphate magnesium salt, 0.4 mM guanosine 5'-triphosphate sodium salt, 10 mM creatine phosphate, and the pH was adjusted to 7.2 with cesium hydroxide and placed on ice. The extracellular solution was pre-oxygenated with 100% O2 30 min before the experiment. Glass microelectrodes were pulled with a horizontal puller P-1000 (Sutter Instrument Company), and the resistance was 6 - 8 MΩ. The HEK293T cells to be tested were placed in the extracellular solution and dark adapted for 30 min. After the cell state was stable, the patch clamp experiment was carried out.

[0107] To verify the light sensitivity of the photosensitive protein to be tested, the current was recorded at different wavelengths and different light intensities, and the open time constant and the closed time constant were analyzed in Clampfit 10.6 software. The results are shown in Figure 6 Table 1 and Figure 7.

[0108] Table 1

[0109]

[0110] In Figure 6In Table 1, we can see the photocurrent levels of the VR3.0 series variants at different wavelengths and different light intensities. The results show that the control group, PsCatCh2.0, only has a photocurrent response to the 470 nm wavelength and hardly any response to 530 nm and 590 nm; from Table 1, we can see that compared with the control group PsCatCh2.0, the variants of the VR3.0 series in the experimental group also have better photosensitivity to light stimuli at 530 nm and 590 nm, that is, they have a wider range of light response wavelengths. Therefore, the VR3.0 series variants in this application have a greater advantage in photocurrent response sensitivity under natural light conditions.

[0111] According to the guidelines of the International Commission on Non-Ionizing Radiation Protection (ICIRP), the light intensity applied to the retina should not exceed the safety threshold corresponding to the wavelength. The safety threshold of the blue light intensity at 470 nm wavelength should not exceed 7.6×10 14 photons / cm 2 s, the safety threshold of the green light intensity at 530 nm wavelength should not exceed 2.5×10 16 photons / cm 2 s, and the safety threshold of the red light intensity at 590 nm wavelength should not exceed 5.9×10 17 photons / cm 2 s. As Figure 7A shown, the experimental results show that most of the variants of the photosensitive protein VR3.0 series can generate photocurrents under the light intensity conditions of 6.9×10 12 photons / cm 2 s (wavelength 470 nm), 6.7×10 13 photons / cm 2 s (wavelength 530 nm), and 7.4×10 13 photons / cm 2 s (wavelength 590 nm), and can generate photocurrents under the light intensity threshold conditions lower than the retinal safety, without causing phototoxic effects on the retina.

[0112] As Figure 7A and 7B shown, the photoinduced currents of most of the variants of the photosensitive protein VR3.0 series also show an increasing trend with the increase of light intensity, which is significantly higher than that of the control photosensitive protein PsCatCh2.0. At a wavelength of 470 nm and a light intensity of 1.7×10 15 photons / cm 2Under the condition of s, NCR2.0 e2, NCR2.0 e3, NCR2.0 e6, NCR2.0 e7, NCR2.0 e12, NCR2.0 e13, NCR2.0 e14, NCR2.0 e16, NCR2.0 e17, NCR2.0 e18 are all improved compared with PsCatCh2.0.

[0113] Visual restoration requires high spatio-temporal resolution, and this characteristic requires photosensitive proteins to have fast kinetic characteristics with high spatio-temporal resolution. Therefore, we also detected the light response frequency of photosensitive proteins, and set pulsed light stimuli of 2Hz, 4Hz, 8Hz, 16Hz and 32Hz. The light source is an external fiber optic of Mightex, the stimulation time is set by the BioLED control software, and the specific light intensity is measured by a light power meter. At a wavelength of 470nm and a light intensity of 1.7×10 15 photons / cm 2 s, the current graph generated by stimulating for 1s, and the results are as Figure 8 shown. The results show that in terms of the response frequency, visual signal processing requires 24Hz, and some variants of the photosensitive protein VR3.0 series can respond to light stimuli of 32Hz, meeting the visual signal requirements.

[0114] Example 5. Intravitreal injection of rAAV virus into rd10 mice

[0115] We selected 4-week-old mice with retinitis pigmentosa disease model (rd10 mice) and wild-type C57BL / 6J mice for animal experiments. We anesthetized the mice intraperitoneally with a mixture of 100mg / kg ketamine and 12mg / kg xylazine according to body weight. After sufficient anesthesia, the ocular surface and the skin around the orbit were disinfected with 0.5% povidone iodine. To reduce the discomfort caused by intravitreal injection to mice, the eyes of the mice were topically anesthetized with proparacaine hydrochloride eye drops (Alcaine). The mice were fixed and the eyeballs were exposed. A Nanoject III high-precision microinjector was used with a glass microelectrode to aspirate 1.5 μL of rAAV2-CMV-VR3.0 virus (titer approximately 5.0×10 12 vg / mL), and the intravitreal injection was completed by inserting the needle 0.5mm below the limbus corneae on the nasal side of the mouse. The intravitreal injection of the other eye was completed in the same way, and a control group only adding the injection preparation was set at the same time. After the injection was completed, levofloxacin hydrochloride ophthalmic gel (Jieqi) was applied to the mouse eyeballs to prevent infection. One month after the virus injection, the efficacy of rd10 mice was detected and behavioral observation was carried out.

[0116] Example 6. Light-induced light / dark box behavior experiment in mice

[0117] The light / dark box consists of two compartments of the same size (18 cm × 20 cm × 18 cm) on the left and right. The two compartments are connected by an arched door (7 cm × 5 cm). The light box is equipped with an LED light source (Mightex, Canada), and the dark box is wrapped with a black cloth cover. All experimental mice are between 10 and 12 weeks old and are dark adapted for 2 hours before the experiment. All behavioral experiments are carried out between 18:00 and 21:00. At the start of the experiment, C57BL / 6J mice, rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0, and normal rd10 mice are individually placed in the light box with a white light intensity of 190 Lux in the center of the light box, and they are allowed to explore freely. The movement of the mice in the light / dark box is analyzed based on the position of their heads. Subsequently, the collected data are imported into GraphPad Prism 7 software, and one-way ANOVA is used to evaluate its significance. P < 0.05 is considered the significant level.

[0118] We analyzed and compared the ratio of the activity time of mice in the light box to the total time, and the experimental results are as Figure 9 shown. The results show that the wild-type C57BL / 6J mouse injection solvent group (positive control) is: 13.42%, n = 9; the rd10 mouse injection solvent group (negative control) is: 68.78%, n = 9; the group of rd10 mice treated with rAAV2-CMV-NCR2.0 e14 virus injection is: 36.14%, n = 9. Thus, it can be seen that the rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0 restored the light avoidance response, demonstrating that the VR3.0 series of photosensitive proteins (such as NCR2.0 e14) can restore the visually guided behavior of retinal degenerative rd10 mice and its effectiveness in the treatment of retinal degeneration.

[0119] Example 7. Optokinetic response behavioral experiment in mice

[0120] Four Lenovo monitors (L1900pA) were used to display a moving grating. There was a mouse activity platform with a height of 17.5 cm in the middle, and the bottom surface was composed of a mirror. Matlab was used to set the parameters of the grating program. Each experiment lasted for 12 minutes. In each stage, the grating rotated clockwise for 30 seconds, counterclockwise for 30 seconds, and paused for 10 seconds. The angular velocity of the grating rotation was 12° / s. The grating density in each stage (i.e., the number of grating lines within 1°, unit: 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 sequence. Before the experiment, the mice were dark adapted for 12 hours. C57BL / 6J mice, rd10 mice after intravitreal injection of rAAV2-CMV-VR3.0 for treatment, and ordinary rd10 mice were placed separately on the activity platform, and then this optokinetic system was used to evaluate the visual acuity of the mice. Subsequently, the collected data were imported into GraphPad Prism7 software to draw a bar chart. The Student's t-test was used to evaluate its significance, and P < 0.05 was the significant level.

[0121] The experimental results are as Figure 10 shown. The results showed that the average maximum visual acuity of the wild-type C57BL / 6J mice in the solvent injection group (positive control) was: 0.49 c / d, n = 7; the maximum average visual acuity of the rd10 mice in the solvent injection group (negative control) was: 0.08 c / d, n = 7; the maximum visual acuity of the rd10 mice after treatment with rAAV2-CMV-NCR2.0 e14 virus injection was: 0.28 c / d, n = 8. Thus, it can be seen that the rd10 mice significantly restored their light sensitivity after intravitreal injection of rAAV2-CMV-VR3.0, proving that the VR3.0 series of photosensitive proteins (such as NCR2.0 e14) can restore the visually guided behavior of retinal degenerative rd10 mice and its effectiveness in the treatment of retinal degeneration.

[0122] The sequences involved in the above embodiments are shown in Table 2 below.

[0123] Table 2 Related Sequence List

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Among them, the amino acid sequences of the above NCR2.0 e1-e19 only include the NCR1 variant part, namely (NCR1 e1-e19), and do not include the LR signal peptide, T polypeptide, and E polypeptide parts; the amino acid sequence of PsCatCh2.0 also does not include the LR signal peptide, T polypeptide, and E polypeptide parts.

Claims

1. A light-sensitive channel protein, comprising a channelrhodopsin protein having the amino acid sequence shown in SEQ ID NO.1 or a variant thereof, wherein the variant is selected from any one of the following: (1) A protein obtained by truncating 1-23 amino acids at the N-terminus of the channelrhodopsin protein; (2) A protein obtained by truncating 90-189 amino acids at the C-terminus of the channelrhodopsin protein; (3) A protein obtained by truncating 1-23 amino acids at the N-terminus of the channelrhodopsin protein and truncating 90-189 amino acids at the C-terminus; or (4) A protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity with (1), (2), or (3).

2. The light-sensitive channel protein according to claim 1, wherein the amino acid sequence of the channelrhodopsin protein is as shown in SEQ ID NO.

1.

3. The light-sensitive channel protein according to claim 1 or 2, wherein the variant is a protein obtained by truncating 5-15 amino acids at the N-terminus of the channelrhodopsin protein.

4. The light-sensitive channel protein according to claim 1 or 2, wherein the variant is a protein obtained by truncating 102-179 amino acids at the C-terminus of the channelrhodopsin protein.

5. The light-sensitive channel protein according to claim 1 or 2, wherein the variant is a protein obtained by truncating 5-15 amino acids at the N-terminus of the channelrhodopsin protein and truncating 102-179 amino acids at the C-terminus.

6. The light-sensitive channel protein according to any one of claims 1-5, wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NOs.8-26, or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity with any one of SEQ ID NOs.8-26.

7. The light-sensitive channel protein according to any one of claims 1-6, wherein the light-sensitive channel protein further comprises an LR signal peptide fused to the N-terminus of the channelrhodopsin protein or its variant, and / or a T polypeptide linked to the C-terminus of the channelrhodopsin protein or its variant to increase the cell membrane expression efficiency and an endoplasmic reticulum export signal sequence E polypeptide linked to the T polypeptide.

8. The light-sensitive channel protein according to claim 7, wherein the amino acid sequence of the LR signal peptide is as shown in SEQ ID NO.2, the amino acid sequence of the T polypeptide is as shown in SEQ ID NO.4, and the amino acid sequence of the E polypeptide is as shown in SEQ ID NO.

5.

9. The light-sensitive channel protein according to claim 7 or 8, wherein a linker peptide sequence is further included between any two of the channelrhodopsin protein or its variant, the T polypeptide, and the E polypeptide.

10. The light-sensitive channel protein according to claim 9, wherein the amino acid sequence of the linker peptide is as shown in SEQ ID NO.28.

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