Novel photosensitive channel protein VR3.0 and its use

Abstract

This application provides a novel photosensitive channel protein VR3.0 and its use. [Solution] The VR3.0 series of photosensitive channel proteins provided in this application exhibits a broad photosensitive wavelength range, higher photosensitivity, and faster photoresponse dynamics, maintains a stable current signal at high frequencies, and shows good response amplitude and response frequency under light stimulation of multiple wavelengths (particularly white light and natural light). The VR3.0 series of photosensitive channel proteins provided in this application has a clear therapeutic effect on retinal photoreceptor cell degeneration diseases and can be used to restore retinal photoreceptor function, restore the visual acuity or photosensitivity of subjects, and prepare drugs for the treatment of retinal degenerative diseases.

Claims

1. A photosensitive channel protein comprising a channelrhodopsin or a variant thereof containing the amino acid sequence shown in Sequence ID No. 1, wherein the variant is selected from any one of the following: (1) Proteins obtained by cleaving 1 to 23 amino acids from the N-terminus of the channelrhodopsin; (2) Proteins obtained by cleaving 90 to 189 amino acids from the C-terminus of the channelrhodopsin; (3) A protein obtained by cleaving 1 to 23 amino acids from the N-terminus and 90 to 189 amino acids from the C-terminus of the channelrhodopsin; or (4) A protein having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with (1), (2), or (3) above.

2. The photosensitive channel protein according to claim 1, wherein the amino acid sequence of the channelrhodopsin is shown in SEQ ID NO:

1.

3. The photosensitive channel protein according to claim 1 or 2, wherein the mutant is a protein obtained by cleaving 5 to 15 amino acids from the N-terminus of the channelrhodopsin.

4. The photosensitive channel protein according to claim 1 or 2, wherein the mutant is a protein obtained by cleaving 102 to 179 amino acids from the C-terminus of the channelrhodopsin.

5. The photosensitive channel protein according to claim 1 or 2, wherein the mutant is a protein obtained by cleaving 5 to 15 amino acids from the N-terminus and 102 to 179 amino acids from the C-terminus of the channelrhodopsin.

6. The amino acid sequence of the aforementioned mutant is 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 more sequence identity with any one of sequence numbers 8-26, A photosensitive channel protein according to any one of claims 1 to 5.

7. The aforementioned photosensitive channel protein is fused to the N-terminus of the channelrhodopsin or its variant, and is an LR signal peptide. The photosensitive channel protein according to any one of claims 1 to 6, further comprising a T polypeptide for enhancing cell membrane expression efficiency ligated to the C-terminus of the channelrhodopsin or a variant thereof, and an endoplasmic reticulum transport signal sequence E polypeptide ligated to the T polypeptide.

8. The photosensitive channel protein 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 photosensitive channel protein according to claim 7 or 8, further comprising a linker peptide sequence between any two of the channelrhodopsin or its variant, the T polypeptide, and the E polypeptide.

10. The photosensitive channel protein according to claim 9, wherein the amino acid sequence of the linker peptide is shown in SEQ ID NO:

28.

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

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

13. A recombinant virus comprising the nucleic acid molecule described in claim 11 or the vector described in claim 9.

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

15. A pharmaceutical composition comprising a photosensitive channel protein according to any one of claims 1 to 10, a nucleic acid molecule according to claim 11, a vector according to claim 12, or a 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 drug for treating retinal photoreceptor cell degeneration disease.

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

18. A method for treating retinal photoreceptor cell degeneration disease, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 15 to a subject.

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

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