Novel light-sensitive channel protein VR2.0 and application thereof

Abstract

The invention provides a novel light-sensitive channel protein VR2.0 and application thereof. The photosensitive channel protein VR2.0 series provided by the invention has an obvious treatment effect in treatment of retina photoreceptor cell degenerative diseases. The novel light-sensitive channel protein has higher light sensitivity, fast dynamics and low side effect; under the same light stimulation condition, the response frequency is fast, and stable current signals can be kept under high frequency response. The application provides a better choice for developing photogenetic therapy for retina photoreceptor cell degenerative diseases, and has important application and popularization values.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically, relates to a novel light-sensitive channel protein VR2.0 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] 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, optogenetic therapy has great application potential in the treatment of retinal photoreceptor degenerative diseases. The photosensitive proteins that utilize optogenetic strategies to play a role in visual restoration 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) that was 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, few side effects, and the ability to maintain a stable current signal under high-frequency light responses. Combining with a more efficient gene delivery vector that targets retinal cells can better apply optogenetic therapy to the treatment field of retinal photoreceptor degenerative diseases. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the present application provides a novel photosensitive channel protein VR2.0 and its uses.

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

[0007] 1. A photosensitive channel protein, including any one of the following PsCatCh variants:

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

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

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

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

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

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

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

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

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

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

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

[0019] 8. The light-sensitive channel protein according to any one of items 1-7, wherein the amino acid sequence of the light-sensitive channel protein is as shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, or SEQ ID NO.36,

[0020] or has a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% with SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, or SEQ ID NO.36.

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

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

[0023] 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.

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

[0025] 11. A vector, comprising the nucleic acid molecule described in item 10.

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

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

[0028] 14. A pharmaceutical composition, comprising the light-sensitive channel protein described in any one of items 1-9, the nucleic acid molecule described in item 10, the vector described in item 11, or the recombinant virus described in item 12 or 13, and a pharmaceutically acceptable carrier.

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

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

[0031] Advantages and beneficial effects of the present application:

[0032] The light-sensitive channel protein VR2.0 series provided by the present application has a definite therapeutic effect in the treatment of retinal photoreceptor cell degenerative diseases. This novel light-sensitive channel protein takes into account high sensitivity and fast kinetics, can maintain the stability of the photocurrent signal under high-frequency response, and has a faster response frequency under the same light stimulation conditions. The present application provides a new and better option for the development of optogenetic therapy for retinal photoreceptor cell degenerative diseases, expands the scope of application of clinical optogenetic therapy for diseases, and has great application and promotion value. Description of the Drawings

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

[0034] Figure 2 is a schematic diagram of the amino acid structure of the photosensitive protein VR2.0 series obtained by modifying the photosensitive protein PsCatCh;

[0035] Figure 3 is a diagram of the photocurrent intensity signal of the mutant of the photosensitive protein VR2.0 series;

[0036] Figure 4 It is the electro-signal waveform diagram of the light response of some mutants of the photosensitive protein VR2.0 series at the level of Xenopus oocytes;

[0037] Figure 5 It is the diagram of the Na + ion and H + ion permeability of some mutants of the photosensitive protein VR2.0 series;

[0038] Figure 6 It is the diagram of the core vector pAAV-CMV-VR2.0-EYFP carrying the expression frames of some mutants of the photosensitive protein VR2.0 series;

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

[0040] Figure 8 It shows the current magnitudes of some mutants of the VR2.0 series under different light intensity conditions at a wavelength of 470 nm after patch clamp recording after expressing some mutants of the photosensitive protein VR2.0 series and the control group PsCatCh2.0 at the level of HEK293T cells. Among them, "photons / cm 2 s" is the light intensity unit.

[0041] Figure 9 It shows the visual evoked potentials of C57BL / 6J mice, rd1 mice treated with intravitreal injection of rAAV2-CMV-VR2.0-EYFP, and untreated littermate rd1 mice. Figure 9 A: The representative waveform diagram of the visual evoked potentials of the above mice; Figure 9 B: The statistical bar chart of the N1 wave amplitude of the visual evoked potentials of the above mice. Among them, P < 0.001, indicating a significant difference;

[0042] Figure 10Shows the light avoidance response of C57BL / 6J mice, rd1 mice treated with intravitreal injection of rAAV2-CMV-VR2.0-EYFP, and untreated littermate rd1 mice in a light-dark box. Figure 10 A: Schematic diagram of the light-dark box experiment; Figure 10 B: Statistical bar graph of the activity time of the above mice in the light box. Among them, P < 0.05, indicating a significant difference.

[0043] Figure 11 Shows the optokinetic response of C57BL / 6J mice, rd1 mice treated with intravitreal injection of rAAV2-CMV-VR2.0-EYFP, and untreated littermate rd1 mice. Figure 11 A: Schematic diagram of the optokinetic response experiment; Figure 11 B: Statistical bar graph of the visual acuity of the above mice. Among them, P < 0.05, indicating a significant difference. Specific 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 in this specification have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are still described below. In case of conflict, the present specification including the definitions therein shall prevail. Additionally, the materials, methods, and examples are for illustrative purposes only and are not restrictive. The following further illustrates the present application with specific embodiments, but does not 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), which can be divided into two types: activating type and inhibitory type, and can cause the excitation or inhibition of neurons.

[0047] The PsCatCh variant in the present application is obtained by modifying the light-sensitive channel protein (Platymonas subcordiformis channelrhodopsin, PsChR) derived from Platymonas subcordiformis.

[0048] Light-sensitive channel protein

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

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

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

[0052] (3) A protein obtained by mutating around the retinol-binding site of the reference protein shown in SEQ ID NO.1;

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

[0054] (5) A protein having at least 60% sequence identity with (1), (2), (3), or (4), 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.

[0055] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 1 to 33 amino acids at the N-terminus of the reference protein shown in SEQ ID NO.1. The 1 to 33 amino acids can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids.

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

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

[0058] In a specific embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, 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.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13.

[0059] In a specific embodiment, the PsCatCh variant is a protein obtained by truncating 1 - 29 amino acids at the C-terminus of the reference protein shown in SEQ ID NO.1. The 1 - 29 amino acids can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

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

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

[0062] In a specific embodiment, the amino acid sequence of the PsCatCh variant is as shown in any one of SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, 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.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19.

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

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

[0065] In a specific embodiment, as shown by any one of SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, or having 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.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36.

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

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

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

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

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

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

[0072] Those skilled in the art can understand that between any two of the PsCatCh 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.

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

[0074] Nucleic acid molecule, vector, recombinant virus

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

[0076] In some embodiments, the nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can 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 can replicate in prokaryotic cells. Therefore, in addition to the nucleotide sequence encoding the light-sensitive channel protein, the DNA molecule further comprises gene operation 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.

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

[0078] In some embodiments, the vector is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid composed of double-stranded DNA molecules. 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, so that the circular plasmid molecule is transformed 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.

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

[0080] 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 serotype or variants thereof.

[0081] Pharmaceutical composition

[0082] 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.

[0083] The form of the pharmaceutical composition depends on multiple criteria, including, for example, the route of administration, the degree of the disease, or the dose of administration, etc.

[0084] 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 required 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.

[0085] Those skilled in the art can understand that the administration dose and frequency of the pharmaceutical composition can vary depending on the age, weight of the subject, or the individual response to the vaccine and the particular administration selected.

[0086] Therapeutic method, therapeutic use

[0087] The present application provides the use of the above-mentioned light-sensitive channel protein in the treatment of retinal photoreceptor degenerative diseases.

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

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

[0090] Among them, in the above method or use, the retinal photoreceptor degenerative diseases can cover various retinal photoreceptor degenerative diseases known in the art, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), cone-rod dystrophy, congenital amaurosis (LCA), etc.

[0091] It should be understood that the present application includes various aspects, embodiments 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 the described improvements and modifications to the described aspects and embodiments.

[0092] Examples

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

[0094] In order to improve the characteristics of the photosensitive protein, mainly to improve the plasma membrane transport efficiency and light sensitivity, based on the previous research foundation and the understanding of the structure ( Figure 1 ) and function of the PsCatch 2.0 photosensitive protein, we carried out amino acid truncation and mutagenesis on the PsCatch part of PsCatch 2.0 (the amino acid sequence is shown in SEQ ID NO.1). The PsCatch 2.0 protein has 7 transmembrane regions, with approximately 30 amino acids in the extracellular region at the N-terminus and approximately 40 amino acids in the extracellular region at the C-terminus.

[0095] Specific truncation and mutagenesis design schemes (such as Figure 2As shown in the figure, there are 6 truncated forms with 12, 17, 19, 21, 26, and 33 amino acid deletions at the N-terminus of PsCatch 2.0, named PsCatch2.0 (e1, e2, e3, e4, e5, e6) respectively; there are 6 truncated forms with 9, 13, 16, 18, 23, and 29 amino acid deletions at the C-terminus of PsCatch 2.0, named PsCatch2.0 (e7, e8, e9, e10, e11, e12) respectively; there are 9 truncated forms with amino acid deletions at the N-terminus and C-terminus of PsCatch 2.0, including N12-C13 (12 amino acids truncated at the N-terminus and 13 amino acids truncated at the C-terminus), N12-C18 (12 amino acids truncated at the N-terminus and 18 amino acids truncated at the C-terminus), N12-C29 (12 amino acids truncated at the N-terminus and 29 amino acids truncated at the C-terminus), N17-C18 (17 amino acids truncated at the N-terminus and 18 amino acids truncated at the C-terminus), N19-C16 (19 amino acids truncated at the N-terminus and 16 amino acids truncated at the C-terminus), N19-C18 (19 amino acids truncated at the N-terminus and 18 amino acids truncated at the C-terminus), N21-C16 (21 amino acids truncated at the N-terminus and 16 amino acids truncated at the C-terminus), N21-C18 (21 amino acids truncated at the N-terminus and 18 amino acids truncated at the C-terminus), N26-C22 (26 amino acids truncated at the N-terminus and 22 amino acids truncated at the C-terminus), named PsCatch2.0 (e13, e14, e15, e16, e17, e18, e19, e20, e21) respectively.

[0096] In addition to the truncation strategy, we selected single-point mutations of amino acids E66D, C165L, and H177R at the amino acid sites of the ion channel functional region on the basis of PsCatCh 2.0 and the truncated mutant PsCatCh 2.0-e18 respectively, named PsCatch2.0 (e22, e23, e24, e25, e26, e27) respectively. PsCatch2.0-e28 was obtained by changing the amino acid T at the N-terminus to G and adding 2 amino acids LE at the C-terminus on the basis of PsCatCh2.0-e26; PsCatch2.0-e29 was obtained by adding the E66D mutation on the basis of PsCatch2.0-e28, with double mutations of E66D and C165L.

[0097] We first used gene synthesis and molecular cloning methods to construct expression plasmids (using the pGEMHE plasmid as the backbone) for the above-mentioned series of mutants of the photosensitive protein PsCatch 2.0 (e1 to e29). 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 the convenience of detecting the expression product of the photosensitive protein, a fusion expression fluorescent protein YFP sequence (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 endoplasmic reticulum export signal E sequence (amino acid sequence SEQ ID NO.5) were added to the C-terminus of the photosensitive protein. Specifically, for the photosensitive protein VR2.0, the connection order is that at the N-terminus, first is the LR signal peptide sequence, then the PsCatch2.0 amino acid sequence, 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 VR2.0 is used for clinical gene therapy, it does not contain the YFP fluorescent protein.

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

[0099] Table 1

[0100]

[0101] Among them, the current of PsCatch is about 1.55 μA, the current of PsCatch 2.0 is about 4.83 μA. The current of PsCatch2.0-e1 is about 4.34 μA, the current of PsCatch 2.0-e18 is about 8.23 μA, the current of PsCatch 2.0-e26 is about 15.49 μA, the current of PsCatch 2.0-e28 is about 15.03 μA, and the current of PsCatch 2.0-e29 is about 18.59 μA. There are 10 kinds with decreased photocurrent compared to PsCatch 2.0, and 19 kinds with increased photocurrent. The research results show that appropriate N-terminal or (and) C-terminal truncation can effectively improve the photosensitivity of the photosensitive protein PsCatch 2.0.

[0102] For the point mutation strategy, the photocurrent of the point mutation E66D based on PsCatch 2.0 has an increase of about 20%; the point mutation C165L has an increase of about 100.4% in photocurrent; while the point mutation H117R results in a decrease of about 58%. The point mutations based on PsCatch2.0-e18 also have a similar trend of change. The photocurrent of PsCatch2.0-e29 with the double mutations E66D and C165L is also increased by about 3.56 μA compared to PsCatch 2.0-e28 containing the single mutation C165L. Thus, we infer that there is a superimposed effect of performance improvement in the combined use of the truncation strategy and the point mutation strategy for the modification of the photosensitive protein PsCatch. We compared the photocurrents of the representative photosensitive proteins PsCatCh2.0-e9, PsCatCh2.0-e18, PsCatCh 2.0-e26 and PsCatCh2.0, and the results are as Figure 4 shown.

[0103] As Figure 5 shown, in terms of ion selectivity, compared to PsCatCh2.0, the permeability of sodium ions (Na + ) of PsCatCh2.0-e9, PsCatCh2.0-e18, PsCatCh2.0-e26 has increased to varying degrees, and the permeability of hydrogen ions (H + ) has decreased slightly, which is beneficial to reducing the side effects caused by excessive acidification due to the increase of H+ ions in the cell.

[0104] In summary, through the strategies of amino acid truncation and point mutation, we designed a variety of mutants. After testing and screening at the in vitro cell level, we obtained a variety of VR2.0 series photosensitive protein mutants with different levels of improvement in light response characteristics.

[0105] Example 2. Construction of the core plasmid vector for expressing photosensitive proteins and preparation of rAAV viruses

[0106] After initially screening out multiple mutants with better photosensitivity than PsCatCh2.0 in in vitro experiments, we selected 3 photosensitive protein mutants, PsCatCh2.0-e9, PsCatCh 2.0-e18, and PsCatCh 2.0-e26, to construct the core plasmid vectors and package them into rAAV viruses, and then carried out the next verification and functional tests on them.

[0107] To enable the efficient expression of photosensitive proteins in retinal cells, we used a broad-spectrum CMV promoter; to facilitate the detection of the expression products of photosensitive proteins, we added a fusion expression fluorescent protein YFP sequence to the C-terminus of the photosensitive protein, as well as sequences T for increasing the 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 and constructed the pAAV-CMV-VR2.0-T-EYFP-E-WPRE-HGHpA series of core plasmids through molecular cloning and gene synthesis techniques, as Figure 6 shown. The sequencing verification results showed that the constructed plasmids were correct.

[0108] We used the AAV serotype 2 with higher efficiency of infecting retinal cells by intravitreal injection, and used the method of triple plasmid transfection of HEK293 cells to co-transfect the pAAV-RC2 serotype plasmid, the pAAV-AdHelper helper plasmid, and the pAAV-CMV-VR2.0-T-EYFP-E-WPRE-HGHpA core plasmid 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, with the titer unit of vg / ml. After aliquoting, it was stored in a -80 °C refrigerator.

[0109] Example 3. Patch clamp recording of the light response of HEK293T cells expressing the VR2.0 series

[0110] We transfected adherently cultured HEK293T cells with the core plasmid carrying the preferred light-sensitive protein VR2.0 series expression cassette. After transfection, the cells were cultured for another 48 h, and then whole-cell voltage clamp mode recording was performed under the condition of a constant room temperature of 25 °C. The main experimental conditions were as follows: The extracellular solution had a composition 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 had a composition of 115 mM cesium methanesulfonate, 20 mM cesium chloride, 2.5 mM magnesium chloride, 0.6 mM ethylene glycol bis(2-aminoethyl ether)-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 using 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, and then the patch clamp experiment was performed after the cell state was stable. To verify the light sensitivity of the light-sensitive protein to be tested, the current was recorded at a light intensity of 1.66×10 15 photons / cm 2 2 s, and the open time constant and closed time constant were analyzed in Clampfit 10.6 software. In addition, to explore the light response frequency of the light-sensitive protein to be tested, pulsed light stimuli of 2, 4, 8, 16, and 32 Hz were set. The light source was a Mightex external optical fiber, the stimulation time was set by the BioLED control software, and the specific light intensity was measured by a light power meter.

[0111] The experimental results are shown in Figure 7 . As can be seen from Figure 7 A, under the conditions of a wavelength of 470 nm and a light intensity of 1.66×10 15 photons / cm 2 2 s, at a high frequency of 32 Hz, VR2.0 could still produce a good photocurrent response to light stimulation. As can also be seen from Figure 7 B, under the conditions of a wavelength of 470 nm and a light intensity of 1.66×10 15 photons / cm 2 2 s, for the current generated by stimulating for 1 s, some mutants of VR2.0 had varying degrees of improvement compared to PsCatCh2.0.

[0112] We also tested the current response of some mutants of the VR2.0 series under different light intensity conditions at a wavelength of 470 nm, and the results are as shown in Figure 8As shown. The results show that multiple photosensitive protein mutants all exhibit good photocurrent responses, showing an upward trend with the increase in light intensity. Among them, PsCatch2.0-e2, PsCatch2.0-e4, PsCatch2.0-e5; PsCatch2.0-e13, PsCatch2.0-e16, PsCatch2.0-e17, PsCatch2.0-e18, PsCatch2.0-e19, PsCatch2.0-e20, PsCatch2.0-e26 are all improved compared to PsCatCh2.0.

[0113] From Figure 8 among them, we can also observe that PsCatch2.0-e18 and PsCatch2.0-e26 generated photocurrent signals of approximately 50.00 pA and 130.00 pA respectively under the light intensity of 7.9×10 13 photons / cm 2 s, which are 2.22 times and 5.78 times higher than the 22.50 pA of PsCatCh2.0 respectively.

[0114] 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, and visual recovery requires a high spatio-temporal resolution. These two major characteristics require that the photosensitive protein simultaneously has high light sensitivity that is safe for the retina and fast kinetic characteristics that meet high spatio-temporal resolution. Therefore, the light intensity of 470 nm blue light that is safe for the retina should not exceed 7.62×10 14 photons / cm 2 s. In this embodiment, some photosensitive protein VR2.0 mutants used by us can generate obvious photocurrent at 5.92×10 13 photons / cm 2 s, which is much lower than the light intensity threshold for generating photocurrent under the condition of retinal safety and will not produce phototoxic effects on retinal cells. In terms of response frequency, visual signal processing requires 24 Hz, and the photosensitive protein VR2.0 can respond to light stimuli of at least 32 Hz, which can meet the requirements of visual signal processing.

[0115] Example 4. Intravitreal injection of rAAV virus into rd1 mice

[0116] We selected 4-week-old postnatal mouse models of retinitis pigmentosa (rd1 mice) and wild-type C57BL / 6J mice for animal experiments. We anesthetized the mice by intraperitoneal injection with a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine. 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 the 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 equipped with a glass microelectrode was used to aspirate 1.5 μL of rAAV2-CMV-VR2.0 virus (titer approximately 2.5E+12 vg / mL), and the intravitreal injection was completed by inserting the needle 0.5 mm below the limbus corneoscleral on the nasal side of the mouse. The intravitreal injection of the other eye was completed in the same manner, and a control group that only received the injection preparation was set up at the same time. After the injection, levofloxacin hydrochloride ophthalmic gel (Jieqi) was applied to the eyeballs of the mice to prevent infection. One month after the virus injection, the efficacy of the rd1 mice was detected and behavioral observations were made.

[0117] Example 5. Recording the flash visual evoked potential of RGCs expressing the VR2.0 series in the mouse retina

[0118] Wild-type C57BL / 6J mice (positive control group), rd1 mice (experimental group) treated with intravitreal injection of rAAV2-CMV-VR2.0, and littermate rd1 mice without any treatment (negative control group) were anesthetized by intraperitoneal injection with a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine. The hair between the two eyes and the tip of the lambdoid suture of the mice was shaved off with a clipper to fully expose the anterior fontanelle. The mouse head was fixed using a brain stereotaxic apparatus (RWD, Shenzhen, China). Forty-eight hours before the FVEP experiment, a silver wire electrode with a diameter of 0.25 mm was implanted into the right primary visual cortex (recording electrode, 3.6 mm from the bregma point, 2.3 mm on both sides). Before the experiment, the mice were dark adapted for 8 h, and then the mice were anesthetized by intraperitoneal injection, and the pupils were dilated with compound tropicamide eye drops (0.5% tropicamide + 0.5% phenylephrine hydrochloride) for 5 min. The reference electrode was inserted under the skin between the eyes, and the ground electrode was clamped on the mouse tail. A flash stimulator (IRC, Chongqing, China) was used for 64 repeated light stimulations (2800 μs, blue light, 5.0 cds / m 2 ). When the band-pass filtering was between 3.0 and 70.0 Hz, the experimental results were recorded at a sampling rate of 2000 Hz, and the flash visual evoked potential (FVEP) data were generated and recorded using RetiMINER 4.0 software to obtain the N1 amplitude data table.

[0119] The experimental results are shown inFigure 9 The results showed that the N1 amplitude of FVEP in wild-type C57BL / 6J mice injected with PBS solvent was 53.03 μV, n = 3; the N1 amplitude of FVEP in rd1 mice injected with PBS solvent was 3.14 μV, n = 4; the N1 amplitude of FVEP in rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e18 virus treatment was 12.37 μV, n = 4; the N1 amplitude of rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e26 virus treatment was 14.81 μV, n = 4.

[0120] The above experimental results indicated that the visual signals generated in the retina of rd1 mice after intravitreal injection of rAAV2-CMV-VR2.0 treatment were successfully transmitted to the visual cortex V1 area.

[0121] Example 6. Light-induced light / dark box behavior experiment in mice

[0122] The light / dark box consisted of two compartments of the same size (18 cm × 20 cm × 18 cm) on the left and right, and the two compartments were connected by an arched door (7 cm × 5 cm). The light box was equipped with an LED light source (Mightex, Canada) with a wavelength of 470 nm, and the dark box was wrapped with a black cloth cover. All experimental mice were between 10 and 12 weeks old, and the mice were dark adapted for 2 hours before the experiment. All behavioral experiments were carried out between 18:00 and 21:00. At the start of the experiment, C57BL / 6J mice, rd1 mice after intravitreal injection of rAAV2-CMV-VR2.0 treatment, and normal rd1 mice were placed alone in the light box with blue light (wavelength 470 nm, light intensity 4.7 × 10 14 photons / cm 2 s) to allow them to freely explore, and the movement of the mice in the light / dark box was analyzed based on the position of the mouse's head. Subsequently, the collected data was imported into GraphPad Prism 7 software, and one-way ANOVA was used to evaluate its significance, with P < 0.05 as the significant level.

[0123] We analyzed and compared the ratio of the activity time of the mice in the light box to the total time. The experimental results are shown in Figure 10The results showed that for the wild-type C57BL / 6J mice injected with the solvent group (positive control), it was 17.29%, n = 7; for the rd1 mice injected with the solvent group (negative control), it was 51.20%, n = 5; for the group of rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e18 virus treatment, it was 27.63%, n = 8; for the group of rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e26 virus treatment, it was 26.75%, n = 6. Thus, it can be seen that the rd1 mice recovered the light avoidance response after intravitreal injection of rAAV2-CMV-VR2.0, demonstrating that VR2.0 can restore the visually guided behavior of retinal degenerative rd1 mice and its effectiveness in treating retinal degeneration.

[0124] Example 7. Optokinetic response behavior experiment in mice

[0125] Four Lenovo monitors (L1900pA) were used to display a moving grating, with a mouse activity platform 17.5 cm high in the middle, and the bottom was composed of a mirror. Matlab was used to set the parameters of the grating program. Each trial was 12 min, and in each stage, the grating rotated clockwise for 30 s, counterclockwise for 30 s, and paused for 10 s. 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 sequentially 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. Before the experiment, the mice were dark adapted for 12 h. The C57BL / 6J mice, the rd1 mice after intravitreal injection of rAAV2-CMV-VR2.0 treatment, and the rd1 mice were separately placed on the activity platform, and then this optokinetic system was used to evaluate the visual acuity of the mice. Subsequently, the collected data was imported into GraphPad Prism 7 software to draw a bar graph. The Student's t-test was used to evaluate its significance, and P < 0.05 was the significant level.

[0126] The experimental results are shown in Figure 11, The results showed that the average maximum visual acuity of the wild-type C57BL / 6J mice injected with the solvent group (positive control) was: 0.48 c / d, n = 7; the maximum average visual acuity of the rd1 mice injected with the solvent group (negative control) was: 0.09 c / d, n = 8; the maximum visual acuity of the rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e18 virus treatment was: 0.25 c / d, n = 6; the maximum visual acuity of the rd1 mice after intravitreal injection of rAAV2-CMV-PsCatCh2.0-e26 virus treatment was: 0.29 c / d, n = 6.

[0127] After intravitreal injection of rAAV2-CMV-VR2.0, the rd1 mice significantly recovered their light sensitivity, demonstrating the effectiveness of VR2.0 in restoring visually guided behaviors in retinal degenerative rd1 mice and in the treatment of retinal degeneration.

[0128] The sequences involved in the above embodiments are shown in Table 2 below.

[0129] Table 2 Related Sequence List

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] Among them, the amino acid sequences of the above PsCatCh2.0-e1 to PsCatCh2.0-e29 only include the PsCatch variant part and do not include the LR signal peptide, T polypeptide, and E polypeptide parts.

Claims

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

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

1.

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

1.

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

1.

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

1.

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

1.

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

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

36.

9. The light-sensitive channel protein according to any one of claims 1-8, wherein the light-sensitive channel protein further comprises an LR signal peptide fused to the N-terminus of the PsCatCh variant, and / or a T polypeptide at the C-terminus of the PsCatCh variant and an E polypeptide linked to the T polypeptide, 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.

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

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