New channelrhodopsin VR3.0 and application thereof

Abstract

Provided in the present application are a new channelrhodopsin VR3.0 and application thereof. The channelrhodopsin VR3.0 series provided in the present application has a relatively wide photosensitive wavelength range, a higher sensitivity to photoreaction and relatively fast photoreaction kinetics, maintains the stability of a current signal under a high-frequency response, and shows a good response amplitude and frequency under light stimulation of a plurality of wavelengths (especially white light and natural light). The channelrhodopsin VR3.0 series provided in the present application has clear treatment effects on retinal photoreceptor cell degenerative diseases, and can be used for preparing drugs for restoring a retinal photoreceptor function, restoring the vision or light perception of a subject, and treating retinal degenerative diseases.

Description

Novel photosensitive channel protein VR3.0 and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and in particular relates to a novel photosensitive channel protein VR3.0 and use thereof. BACKGROUND

[0002] Retinal photoreceptor degenerative diseases are a class of degenerative diseases characterized by progressive loss of function of photoreceptor cells and pigment epithelial cells. Such diseases are mainly caused by gene mutations or dysfunction of retinal pigment epithelial cells (RPE cells), and common typical examples are retinitis pigmentosa (RP) and age-related macular degeneration (AMD), which are two major and intractable blinding eye diseases. The prevalence of photoreceptor degenerative diseases caused by genetic causes is about 1 / 3500-1 / 4000, and 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 degenerative diseases caused by drugs and diseases and other reasons is also increasing.

[0003] Because the apoptosis of photoreceptor cells in retinal photoreceptor degenerative diseases is irreversible and most of such diseases have high genetic heterogeneity, the treatment of related diseases also becomes very difficult. Current treatment methods mainly include stem cell transplantation, gene therapy, retinal prosthesis implantation, and optogenetic therapy. Among them, optogenetic therapy is to use the integrity of the remaining cell structure of retinal degenerative diseases to target the expression of photosensitive proteins in cone cells (early stage of degenerative diseases), bipolar cells or ganglion cells (middle and late stages of degenerative diseases) using recombinant adeno-associated virus (AAV) as a carrier to restore the photosensitive ability of the retina. In addition, stem cell transplantation, virtual reality system and holographic imaging technology can also be combined to restore visual function.

[0004] Optogenetic therapy, as an eye treatment strategy that does not depend on restoring the function of specific gene sites and can respond to light stimulation at the single-cell level, has great application potential in treating retinal photoreceptor degenerative diseases. The photosensitive proteins used to exert visual recovery function by using optogenetic strategy mainly include two categories: microbial photosensitive proteins and mammalian photosensitive proteins. Microbial photosensitive proteins usually have fast kinetic characteristics, but low photosensitivity, such as the cation-permeable photosensitive protein Channelrhodopsin-2 (ChR2) first applied to visual function recovery. The mammalian endogenous photosensitive protein belongs to the G Protein-Coupled Receptors (GPCRs) family, has a 7-transmembrane alpha helix structure, and usually has high photosensitivity, but insufficient kinetics, such as Rhodopsin derived from rod photoreceptor cells (Rods). At present, the existing optogenetic tools (i.e. photosensitive proteins) applied to visual recovery cannot well meet the high photosensitivity and fast photoreaction kinetic characteristics, and there are problems such as differences in ion selectivity, side effects of easily leading to intracellular acidification. Therefore, it is necessary to develop a kind of photosensitive protein with high sensitivity to light stimulation, fast photoreaction kinetic characteristics, small side effects, and stable current signal under high-frequency light response, combined with a more efficient gene delivery carrier targeting retinal cells, so as to better apply optogenetic therapy to the treatment of retinal photoreceptor degenerative diseases. SUMMARY

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

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

[0007] 1. A photosensitive channel protein comprising a channel rhodopsin protein comprising an amino acid sequence shown in SEQ ID NO. 1 or a variant thereof, 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 channel rhodopsin protein;

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

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

[0011] (4) a protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to (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 set forth 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 and 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 set forth in any one of SEQ ID NOs. 8-26,

[0017] or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to 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 the variant thereof,

[0019] and / or a T polypeptide connected to the C-terminus of the channelrhodopsin protein or the variant thereof to increase the cell membrane expression efficiency and an endoplasmic reticulum export signal sequence E polypeptide connected 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 set forth in SEQ ID NO. 2, the amino acid sequence of the T polypeptide is set forth in SEQ ID NO. 4, and the amino acid sequence of the E polypeptide is set forth 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 comprised between any two of the channelrhodopsin protein or the variant thereof, 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 connecting peptide is set forth 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 comprising 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 for the manufacture of a medicament for treating a retinal photoreceptor degenerative disease.

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

[0030] 18. A method of treating a retinal photoreceptor degenerative disease, 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 degenerative disease comprises retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, Leber congenital amaurosis (LCA).

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

[0033] The photosensitive channel protein VR3.0 series provided in the present application has a wider photosensitive wavelength range, higher sensitivity to light response, faster light response kinetics, maintains stable current signals under high frequency response, and exhibits good response amplitude and frequency under light stimulation of various wavelengths (especially white light, natural light). The photosensitive channel protein VR3.0 series provided in the present application has a clear therapeutic effect on retinal photoreceptor degenerative diseases, and can be used to prepare drugs for restoring the photoreceptor function of the retina, restoring the vision or light sensitivity of the subject, and treating retinal degenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

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

[0035] Figure 2 is an amino acid structure diagram of the photosensitive protein VR3.0 series modified from the photosensitive protein NCR1.

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

[0037] Figure 4A is a current change trend diagram of part of the variants of the photosensitive protein VR3.0 series at the level of Xenopus oocytes with increasing light intensity; 4B is the photoelectric current level of part of the variants of the photosensitive protein VR3.0 series at the level of Xenopus oocytes in response to light between the wavelength range of 420nm to 600nm.

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

[0039] Figure 6 is a comparison of the light response current diagrams of part of the variants of the photosensitive protein VR3.0 series and the control group PsCatch2.0 photosensitive protein at the level of HEK293T cells, A. under the condition of wavelength 470nm, light intensity 1.7x10 15 photons / cm 2 s; B. under the condition of wavelength 530nm, light intensity 1.9x10 15 photons / cm 2 s; C. under the condition of wavelength 590nm, light intensity 2.1x10 15 photons / cm 2 s.

[0040] Figure 7 is a graph showing the light response current of different photoreceptor VR3.0 variants at different wavelengths (470 nm, 530 nm, 590 nm) and different light intensities after expressing the partial variants of photoreceptor VR3.0 series at HEK293T cell level (7A); and a graph showing the change of photocurrent of different photoreceptor VR3.0 variants at 470 nm wavelength and different light intensities (7B).

[0041] Figure 8 is a graph showing the light response current of different photoreceptor VR3.0 variants and control group PsCatch2.0 photoreceptor at 470 nm wavelength, light intensity of 1.7 x 10 15 photons / cm 2 s, stimulation frequency of 2 Hz, 4 Hz, 8 Hz, 16 Hz, 32 Hz after expressing the partial variants of photoreceptor VR3.0 series and control group PsCatch2.0 photoreceptor at HEK293T cell level.

[0042] Figure 9 shows the photophobic response of C57BL / 6J mice, rd10 mice treated with rAAV2-CMV-VR3.0-EYFP by intravitreal injection and littermates without any treatment in the light-dark box. A is a schematic diagram of the light-dark box experiment; B is a statistical column chart of the activity time of the above mice in the light box. P<0.05, significant difference, marked with *; when p value is less than 0.01, extremely significant, marked with **; when p value is less than 0.001, more significant, marked with ***; when p value is less than 0.0001, particularly significant, marked with ****; ns represents no significant difference.

[0043] Figure 10 shows the optokinetic response of C57BL / 6J mice, rd10 mice treated with rAAV2-CMV-VR3.0-EYFP by intravitreal injection and littermates without any treatment. A: experimental mode diagram of optokinetic response; B: statistical column chart of visual acuity of the above mice. P<0.05, significant difference, marked with *; when p value is less than 0.01, extremely significant, marked with **; when p value is less than 0.001, more significant, marked with ***; when p value is less than 0.0001, particularly significant, marked with ****; ns represents no significant difference. DETAILED DESCRIPTION

[0044] The present application will be further described in conjunction with the 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, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described below. However, if there is a conflict between the present specification and any document incorporated by reference, the present specification controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0046] As used herein, "light-sensitive channel protein" and "light-sensitive protein" can be used interchangeably to refer to a class of proteins on the cell membrane that can sense light stimulation to produce a specific effect (such as: change the open state of ion channels), which can be divided into two types: activation and inhibition, which can cause excitation or inhibition of neurons.

[0047] Light-sensitive channel protein

[0048] The present application provides a light-sensitive channel protein, comprising a channel rhodopsin protein (NCR1) comprising the amino acid sequence shown in SEQ ID NO. 1 or a variant thereof, or a channel rhodopsin protein (NCR1) comprising 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:

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

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

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

[0052] (4) a protein having at least 60% sequence identity to (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 99% or more sequence identity.

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

[0054] In one specific embodiment, the variant is a protein obtained by truncating 1-23 amino acids from the N-terminus of a channelrhodopsin protein having an amino acid sequence as set forth 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 one specific embodiment, the variant is a protein obtained by truncating 5-15 amino acids from the N-terminus of a channelrhodopsin protein having an amino acid sequence as set forth in SEQ ID NO. 1.

[0056] In one specific embodiment, the amino acid sequence of the variant is as set forth in any one of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, or has 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11.

[0057] In one embodiment, the variant is a protein obtained by truncating 90-189 amino acids from the C-terminal end of a channel rhodopsin protein having an amino acid sequence represented by 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 one embodiment, the variant is a protein obtained by truncating 102-179 amino acids from the C-terminal end of a channel rhodopsin protein having an amino acid sequence represented by SEQ ID NO. 1.

[0059] In a particular embodiment, the amino acid sequence of said variant is as set forth in any 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 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with any 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 one embodiment, the variant is a protein obtained by truncating 1-23 amino acids from the N-terminus and 90-189 amino acids from the C-terminus of a channel rhodopsin protein having an amino acid sequence represented by 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. 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 one embodiment, the variant is a protein obtained by truncating 5-15 amino acids from the N-terminus and 102-159 amino acids from the C-terminus of a channel rhodopsin protein having an amino acid sequence represented by SEQ ID NO. 1.

[0062] In one specific embodiment, the amino acid sequence of the variant is as set forth 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 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to 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, or SEQ ID NO. 26.

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

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

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

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

[0067] It can be understood by those skilled in the art that there can also be a connecting peptide sequence consisting of a small number of amino acids between any two of the channel rhodopsin protein or the variant thereof, the T polypeptide, and the E polypeptide, which does not affect the function of the light-sensitive channel protein.

[0068] In one specific embodiment, the amino acid sequence of the connecting peptide is as set forth 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 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 be replicated and / or expressed in a cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule can be expressed in a eukaryotic cell and can be replicated in a prokaryotic cell. Thus, the DNA molecule comprises, in addition to the nucleotide sequence encoding the light-sensitive channel protein, genetic 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 encompass a linear DNA molecule. Specifically, the term "plasmid" also encompasses molecules obtained by linearizing a circular plasmid, for example, by cleaving the circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule, as well as linear molecules that are replicable in prokaryotes. Plasmids can replicate, i.e., amplify in a cell, independently of the genomic genetic information stored in the prokaryotic cell's nucleus 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.

[0074] The present application provides a recombinant virus comprising any of the above-mentioned nucleic acid molecule or vector.

[0075] 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 an 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 a variant thereof.

[0076] Pharmaceutical composition

[0077] The present application provides a pharmaceutical composition comprising any of the above photosensitive 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 extent of the disease, or the dosage of administration, etc.

[0079] In some embodiments, the pharmaceutical composition can be formulated to be delivered into a subject through an appropriate route, including, but not limited to, through an oral route, an injection route (such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracardiac injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc.), a mucosal route (such as intranasal administration, intraoral administration, etc.), a sublingual route, a rectal route, a transdermal route, an intraocular route, a pulmonary route. Depending on the desired route of administration, the pharmaceutical composition can be formulated as tablets, capsules, pills, dragees, powders, granules, sachets, lozenges, suppositories, suspensions, emulsions, syrups, aerosols (as solids or in liquid media), sprays, ointments, pastes, patches, creams, lotions, gels, inhalants, etc.

[0080] It is understood by those skilled in the art that the dosage of administration and the frequency of administration of the pharmaceutical composition can vary depending on the age, weight, or individual response to the vaccine of the subject, and the particular administration selected.

[0081] Therapeutic methods, therapeutic uses

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

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

[0084] Among them, the retinal photoreceptor degenerative disease 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, congenital blindness (LCA) and the like.

[0085] It should be understood that the present application includes various aspects, embodiments described herein, and combinations of the aspects and / or embodiments. The above description and the following 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 should recognize that the scope of the present application also includes the described improvements and modifications to the aspects and embodiments.

[0086] Examples

[0087] Example 1. Construction of VR3.0 series variant expression plasmid based on microbial light-sensitive protein NCR1 modification

[0088] Channelrhodopsin-2 (ChR2) is a membrane protein containing seven transmembrane helices and a covalently bound retinal chromophore. After ChR2, researchers have discovered channelrhodopsins with different ion selectivity. In the genomic data of H. catenoides, we identified another class of ChR sequences, which were found to have high Na+ ion permeability, so we named it as a Na+ ion channelrhodopsin, named HcNCR1 (abbreviated as NCR1). The full-length NCR1 protein consists of 432 amino acids. In order to improve its photosensitivity in eukaryotic cells, we truncated NCR1 (432 aa) and added several plasma membrane targeting enhancement peptides at both ends to improve the efficiency of plasma membrane transport.

[0089] Based on our previous research and understanding of the structure (Figure 1) and function of NCR1 light-sensitive protein, we performed amino acid truncation modification on NCR1 protein (amino acid sequence as shown in SEQ ID NO. 1). NCR1 protein has 7 transmembrane regions, with about 20 amino acids in the N-terminal extracellular region and about 220 amino acids in the C-terminal extracellular region. The specific truncation and mutation design scheme (Figure 2), wherein:

[0090] Four truncated forms of NCR1 with 5, 10, 15, 24 amino acid deletion at N-terminus, named NCR1 e1, NCR1 e2, NCR1 e3, and NCR1 e4, respectively;

[0091] Six truncated forms of NCR1 with 102, 126, 149, 159, 179, 190 amino acid deletion at C-terminus, named NCR1 e5, NCR 1e6, NCR 1e7, NCR1 e8, NCR 1e9, and NCR1 e10, respectively;

[0092] Nine truncated forms of NCR1 with N6-C330 (5 amino acid deletion at N-terminus and 102 amino acid deletion at C-terminus), N6-C283 (5 amino acid deletion at N-terminus and 149 amino acid deletion at C-terminus), N6-C273 (5 amino acid deletion at N-terminus and 159 amino acid deletion at C-terminus), N11-C283 (11 amino acid deletion at N-terminus and 149 amino acid deletion at C-terminus), N11-C273 (11 amino acid deletion at N-terminus and 159 amino acid deletion at C-terminus), N16-C306 (15 amino acid deletion at N-terminus and 126 amino acid deletion at C-terminus), N16-C283 (15 amino acid deletion at N-terminus and 149 amino acid deletion at C-terminus), N16-C273 (15 amino acid deletion at N-terminus and 159 amino acid deletion at C-terminus), N16-C242 (15 amino acid deletion at N-terminus and 190 amino acid deletion at C-terminus) at N- and C-terminus, named NCR1 e11, NCR1 e12, NCR1 e13, NCR1 e14, NCR1 e15, NCR1 e16, NCR1 e17, NCR1 e18, and NCR1 e19, respectively.

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

[0094] wherein NCR 2.0 is obtained by adding the LR signal peptide sequence to the N-terminus of NCR1, and connecting the T sequence, the YFP sequence, the E sequence, and the connecting peptide sequence to the C-terminus. NCR 2.0e1-e19 is obtained by adding the LR signal peptide sequence to the N-terminus of NCR1 e1-e19, and connecting the T sequence and the E sequence to the C-terminus.

[0095] Example 2. Comparison of the performance of the VR3.0 series of variants based on the microbial light-sensitive protein NCR1 at the oocyte level of Xenopus laevis

[0096] We used the AmpliCap-MaxT7 kit to synthesize the above-mentioned RNA for expressing the light-sensitive protein, and injected 30 ng of the above-mentioned RNA into different Xenopus laevis oocytes, respectively. Two days after injection, the photoreaction of the Xenopus laevis oocytes expressing the light-sensitive protein was recorded using a two-electrode voltage clamp. Specifically, the electrophysiological measurement of the Xenopus laevis oocyte was carried out in the oocyte Ringer 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. The 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 light irradiation conditions (wavelength of 532 nm, 0.5 mW / mm 2The light response current of the above-mentioned photosensitive protein NCR 2.0 and NCR2.0 e series variants (also referred to as VR3.0 series) was detected compared with the light response current of photosensitive protein NCR1 at -40 mV, and the results are shown in Figure 3.

[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 light response current level of the NCR2.0 variant constructed by truncating the N-terminal or (and) C-terminal of the NCR1 protein as shown above is significantly improved compared with the photosensitive protein NCR1, which can achieve stronger photosensitivity than the photosensitive protein NCR1, and has great application potential.

[0099] We studied the light response current trend of the VR3.0 series photosensitive proteins with increasing light intensity. At a wavelength of 532 nm, the light intensity was 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 (as shown in Figure 4A). It can be observed that with the increase of light intensity, the light response current of the NCR2.0 series photosensitive proteins gradually increases, and slowly increases gradually to a platform at 3 mW / mm 2 . The experimental results show that within a certain wavelength (532 nm) and 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 light response current increases with the increase of light intensity.

[0100] To test the wavelength range of the VR3.0 series of light-sensitive proteins in response to light, we carried out 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 of 420 nm to 620 nm. These include: 422 nm, 439 nm, 459 nm, 481 nm, 496 nm, 516 nm, 540 nm, 562 nm, 595 nm, and 620 nm. In order to achieve similar light intensity at different wavelengths, we adjusted the output power of the light source and used gray filters to ensure that the light intensity was about 0.5 mW / mm 2 The detection results show that the NCR2.0 series of light-sensitive proteins have obvious photocurrent response between 450 nm and 600 nm wavelengths, with the highest response wavelength at about 532 nm (as shown in FIG. 4B). The experimental results show that the VR3.0 series of light-sensitive proteins have a very wide response continuous wavelength range, and are sensitive to the main visible light bands represented by blue, green, and red. Therefore, these in vitro cell level light response tests show that the light response characteristics of the VR3.0 series of light-sensitive protein variants we constructed have been significantly improved compared to the natural NCR1 light-sensitive protein, laying a solid foundation for carrying out functional research and applications at the level of living animals.

[0101] Example 3. Construction of core plasmid vector expressing light-sensitive protein and preparation of rAAV virus

[0102] We constructed a core plasmid viral vector containing the gene of the control PsCath2.0 light-sensitive protein (amino acid sequence as shown in SEQ ID NO. 27) or the gene of the VR3.0 series of light-sensitive protein variants and packaged it into rAAV virus, which was then subjected to further verification and functional testing.

[0103] In order to enable the light-sensitive protein to be expressed efficiently in retinal cells, we used a broad-spectrum CMV promoter; in order to facilitate detection of the light-sensitive protein expression product, we added a fusion expression of the YFP sequence at the C-terminus of the light-sensitive protein, as well as the sequence T to increase cell membrane expression efficiency and the endoplasmic reticulum export signal sequence E, added the WPRE element (nucleotide sequence as shown in SEQ ID NO. 6), and the HGHpA sequence (nucleotide sequence as 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 shown in FIG. 5. Sequencing verification results show that the constructed plasmids are correct.

[0104] We used the commonly used AAV2 serotype, using the method of three plasmid transfection of HEK293 cells, the serotype plasmid pAAV-RC2, packaging helper plasmid pAAV-Helper and pAAV-CMV-VR3.0-T-EYFP-E-WPRE-HGHpA core plasmid were co-transfected into HEK293 cells to package the virus, and the cell precipitate and medium supernatant were collected after 72 hours. Then the rAAV virus was obtained by using the method of idox ultracentrifugation, the titer unit was vg / ml, and it was stored in the -80 degree refrigerator after being divided.

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

[0106] We transfected the core plasmid carrying the photoreceptor protein VR3.0 series expression frame prepared in Example 3 into adherent cultured HEK293T cells, and continued to culture for 48 hours after transfection, and then performed whole-cell voltage clamp mode recording at a constant room temperature of 25°C. The main experimental conditions are as follows: the extracellular fluid composition is 140 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 16 mM glucose, and the pH is adjusted to 7.4 with sodium hydroxide, and it is placed at room temperature; the intracellular fluid composition is 115 mM cesium methanesulfonate, 20 mM cesium chloride, 2.5 mM magnesium chloride, 0.6 mM ethylene glycol bis(2-aminoethylether) tetraacetic acid, 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 4 mM adenosine 5'-triphosphate magnesium salt, 0.4 mM guanosine 5'-triphosphate sodium salt, 10 mM phosphocreatine, and the pH is adjusted to 7.2 with cesium hydroxide, and it is placed on ice. The extracellular fluid was pre-oxygenated with 100% O2 30 min before the experiment. The glass microelectrode was drawn with a horizontal puller P-1000 (Sutter Instrument Company). The tested HEK293T cells were dark adapted in the extracellular fluid for 30 min, and then the patch clamp experiment was performed after the cell state was stable.

[0107] To verify the photosensitivity of the tested photoreceptor protein, the current was recorded under 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] In FIG. 6 and Table 1, we can see the light response current levels of the VR3.0 series variants at different wavelengths and different light intensities, respectively. The results show that the control group PsCatCh2.0 only has a light response to 470 nm wavelength, and has almost no light response to 530 nm and 590 nm. From Table 1, we can see that compared with the control group PsCatCh2.0, the experimental group VR3.0 series variants also have better photosensitivity to 530 nm and 590 nm light stimulation, i.e., a wider light response wavelength range. Thus, the light response sensitivity of the VR3.0 series variants in the present application has greater advantages under natural light conditions.

[0110] 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 of the corresponding wavelength. The safety threshold of blue light with a wavelength of 470 nm should not exceed 7.6 x 10 14 photons / cm 2 s, the safety threshold of green light with a wavelength of 530 nm should not exceed 2.5 x 10 16 photons / cm 2 s, and the safety threshold of red light with a wavelength of 590 nm should not exceed 5.9 x 10 17 photons / cm 2 s. As shown in FIG. 7A, the experimental results show that most of the photoreceptor proteins VR3.0 series variants can generate photocurrents under the conditions of 6.9 x 10 12 photons / cm 2 s (wavelength 470 nm), 6.7 x 10 13 photons / cm 2 s (wavelength 530 nm), and 7.4 x 10 13 photons / cm 2 s (wavelength 590 nm), all of which can generate photocurrents under conditions lower than the safe light intensity threshold of the retina, and do not have phototoxic effects on the retina.

[0111] As shown in FIGS. 7A and 7B, the light-induced current of most of the photoreceptor proteins VR3.0 series variants also shows an increasing trend with the increase of light intensity, which is significantly improved compared with the control photoreceptor protein PsCatCh2.0. At a wavelength of 470 nm and a light intensity of 1.7 x 10 15 photons / cm 2NCR2.0 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 all have improved compared with PsCatCh2.0 under the condition of 1 s.

[0112] Visual recovery requires high spatiotemporal resolution, which requires the photoreceptor protein to have high spatiotemporal resolution and fast kinetics. Therefore, we also detected the light response frequency of the photoreceptor protein, and set the pulse light stimulation at 2 Hz, 4 Hz, 8 Hz, 16 Hz and 32 Hz. The light source is an external optical fiber of Mightex, and the stimulation time is set by BioLED control software, and the specific light intensity is measured by a light power meter. At a wavelength of 470 nm, the light intensity is 1.7 x 10 15 photons / cm 2 Under the condition of 1 s, the current map generated by the stimulation is shown in Figure 8. The results show that the visual signal processing requires 24 Hz in response frequency, and the VR3.0 series of photoreceptor protein variants can respond to 32 Hz light stimulation, meeting the demand of visual signal.

[0113] Example 5. Intravitreal injection of rAAV virus in rd10 mice

[0114] We selected a model mouse of retinal pigmentosa disease (rd10 mouse) at 4 weeks after birth and wild type C57BL / 6J mouse for animal experiments. We anesthetized the mice by intraperitoneal injection of a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine. After sufficient anesthesia, the eye surface and periorbital skin were disinfected with 0.5% iodine. To reduce the discomfort of the mice caused by intravitreal injection, we used proparacaine hydrochloride eye drops (Alcaine) to perform topical anesthesia on the eyeball of the mice. After fixing the mice and exposing the eyeball, we used a Nanoject III high-precision microsyringe with a glass microelectrode to aspirate 1.5 μL of rAAV2-CMV-VR3.0 virus (titer about 5.0 x 10 12 vg / mL) from the lower 0.5 mm of the corneoscleral limbus on the nasal side of the mouse to complete the intravitreal injection. The other side of the eye was injected in the same way, and a control group was set up with only the injection preparation. After the injection was completed, we applied levofloxacin hydrochloride eye gel (Ji Qi) to the mouse eyeball to prevent infection. One month after the virus injection, we detected the therapeutic effect of the rd10 mouse and observed the behavior.

[0115] Example 6. Light-induced light / dark box behavior experiment in mice

[0116] The light / dark box consisted of two identical compartments (18 cm x 20 cm x 18 cm) connected by an arched door (7 cm x 5 cm). The light compartment was equipped with LED light source (Mightex, Canada) and the dark compartment was covered with a black cloth. All mice were between 10-12 weeks old and 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, rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0 and normal rd10 mice were individually placed in the light compartment with white light intensity of 190 lux and allowed to freely explore. The head position of the mice in the light / dark box was analyzed. The collected data were then imported into GraphPad Prism 7 software and one-way ANOVA was used to evaluate the significance, with P < 0.05 as the significant level.

[0117] We analyzed the ratio of the time spent in the light compartment to the total time in the light / dark box for the mice. The results are shown in Figure 9. The results showed that the solvent-injected wild-type C57BL / 6J mice (positive control) were 13.42%, n = 9; the solvent-injected rd10 mice (negative control) were 68.78%, n = 9; and the rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0 were 36.14%, n = 9. Thus, the rd10 mice treated with intravitreal injection of rAAV2-CMV-VR3.0 recovered the light-avoidance response, demonstrating that the VR3.0 series of light-sensitive proteins (such as NCR2.0 e14) can restore the visually guided behavior of the retinal degeneration rd10 mice and are effective in treating retinal degeneration.

[0118] Example 7. Mouse optokinetic response behavior experiment

[0119] 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, rd10 mice treated with intravitreal rAAV2-CMV-VR3.0, and normal rd10 mice were individually 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.

[0120] The experimental results are shown in Figure 10. The results show that the average maximum visual acuity of the wild-type C57BL / 6J mice injected with the solvent (positive control) was 0.49 c / d (n = 7); the average maximum visual acuity of the rd10 mice injected with the solvent (negative control) was 0.08 c / d (n = 7); and the maximum visual acuity of the rd10 mice treated with rAAV2-CMV-NCR2.0 e14 virus was 0.28 c / d (n = 8). These results indicate that intravitreal injection of rAAV2-CMV-VR3.0 significantly restored light sensitivity in rd10 mice, demonstrating the effectiveness of VR3.0-series photosensitive proteins (such as NCR2.0 e14) in restoring visually guided behavior in rd10 mice with retinal degeneration.

[0121] The sequences involved in the above examples are shown in Table 2 below.

[0122] Table 2 List of related sequences

[0123] Among them, the amino acid sequence of the above-mentioned NCR2.0 e1-e19 only includes the NCR1 variant part, namely (NCR1 e1-e19), and does 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 channelrhodopsin protein comprising a channelrhodopsin protein comprising 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 from 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 and 90-189 amino acids at the C-terminus of the channelrhodopsin protein; or (4) A protein having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to (1), (2), or (3). 2 . The light-sensitive channelrhodopsin according to claim 1 , wherein the amino acid sequence of the channelrhodopsin protein is as shown in SEQ ID NO.

1. 3 . The channelrhodopsin according to claim 1 , wherein the variant is a protein obtained by truncating 5 to 15 amino acids at the N-terminus of the channelrhodopsin protein. 4 . The channelrhodopsin according to claim 1 , wherein the variant is a protein obtained by truncating 102-179 amino acids at the C-terminus of the channelrhodopsin protein.

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

6. The channelrhodopsin according to any one of claims 1 to 5, wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NOs. 8 to 26. or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to any one of SEQ ID NOs. 8-26.

7. The light-sensitive channelrhodopsin according to any one of claims 1 to 6, wherein the light-sensitive channelrhodopsin further comprises an LR signal peptide fused to the N-terminus of the channelrhodopsin protein or its variant, And / or a T polypeptide connected 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 connected to the T polypeptide.

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

5. 9 . The light-sensitive channelrhodopsin according to claim 7 , further comprising a connecting peptide sequence between any two of the channelrhodopsin protein or its variant, the T polypeptide, and the E polypeptide. 10 . The channelrhodopsin according to claim 9 , wherein the amino acid sequence of the connecting peptide is shown as SEQ ID NO.

28.

11. A nucleic acid molecule comprising a nucleotide sequence encoding the channelrhodopsin according to any one of claims 1 to 10. A vector comprising the nucleic acid molecule according to claim 11 . 13 . A recombinant virus comprising the nucleic acid molecule of claim 11 or the vector of claim 9 . The recombinant virus according to claim 13 , wherein the recombinant virus is a recombinant adeno-associated virus.

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

16. Use of the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating retinal photoreceptor cell degenerative diseases.

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

18. A method for treating retinal photoreceptor degenerative diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 15 to a subject.

19. The method according to claim 18, wherein the retinal photoreceptor cell degenerative disease comprises retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, and congenital amaurosis (LCA).

Images