GENE THERAPY VECTOR FOR THE TREATMENT OF PARKINSON'S DISEASE AND ITS APPLICATION

Claims

1. An adeno-associated virus (AAV) vector comprising nucleotide sequences encoding tyrosine hydroxylase (TH), GTP cyclohydrolase 1 (GCH1) and aromatic amino acid decarboxylase (AADC), wherein the nucleotide sequences encoding TH, GCH1 and / or AADC are linked in frame by a nucleic acid sequence encoding a linker, wherein any one or more of TH, GCH1 and AADC is a truncated form of the wild type or a functional variant thereof and has catalytic activity required for the dopamine synthesis pathway.

2. The AAV vector according to claim 1, characterized in that TH is a truncated form of wild-type TH or a functional variant thereof and has the activity of catalyzing the synthesis of levodopa from tyrosine; preferably, TH contains 330-390 amino acid residues.

3. The AAV vector according to claim 1 or 2, characterized in that TH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

11.

4. The AAV vector according to any one of claims 1-3, characterized in that GCH1 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

9.

5. The AAV vector of any one of claims 1-4, wherein the AADC comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

7.

6. An AAV vector according to any one of claims 1-5, wherein the linker comprises a fusion linker, a 2A peptide linker and / or an internal ribosome entry site (IRES).

7. The AAV vector of claim 6, wherein the fusion linker is a peptide linker; preferably, the peptide linker is selected from (GGS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1 to 5.

8. The AAV vector of claim 7, wherein the fusion linker is (G4S)3.

9. The AAV vector according to claim 6, wherein the peptide linker 2A comprises a peptide 2A selected from foot-and-mouth disease virus peptide 2A (F2A), porcine teschovirus peptide 2A (P2A), Thosea asigna virus peptide 2A (T2A) and / or equine rhinitis virus peptide 2A (E2A).

10. The AAV vector according to claim 9, characterized in that the N-terminus and / or C-terminus, preferably the N-terminus of the peptide linker 2A further comprises the amino acid sequence GSG.

11. The AAV vector of claim 10, wherein the peptide linker 2A comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 13 or SEQ ID NO:

15.

12. The AAV vector according to any one of claims 1-11, characterized in that the nucleotide sequences encoding TH, GCH1 and AADC are arranged in the direction from upstream to downstream in the sequence in an order selected from any of: (1) TH, GCH1, AADC; (2) AADC, GCH1, TH; (3) AADC, TH, GCH1; (4) GCH1, TH, AADC; (5) GCH1, AADC, TH; or (6) TH, AADC, GCH1.

13. The AAV vector of claim 12, wherein the nucleotide sequences encoding TH, GCH1 and AADC are linked in an upstream to downstream direction in a manner selected from any of: 1) TH-F2A-GCH1-P2A-AADC; 2) GCH1-P2A-TH-P2A-AADC; 3) GCH1-P2A-AADC-P2A-TH; 4) AADC-E2A-GCH1-(G4S)3-TH; 5) GCH1-F2A-TH-F2A-AADC; 6) AADC-(G4S)3-TH-T2A-GCH1; 7) AADC-(G4S)4-TH-T2A-GCH1; 8) AADC-P2A-GCH1-P2A-TH; 9) AADC-T2A-GCH1-(G4S)3-TH; 10) GCH1-F2A-TH-P2A-AADC; 11) GCH1-F2A-AADC-F2A-TH; 12) AADC-(G4S)3-GCH1-F2A-TH; 13) GCH1-E2A-TH-T2A-AADC; 14) GCH1-F2A-AADC-P2A-TH; 15) AADC-(G4S)3-GCH1-P2A-TH; 16) AADC-(G4S)5-GCH1-P2A-TH; 17) TH-(G4S)3-GCH1-P2A-AADC; 18) GCH1-T2A-AADC-E2A-TH; 19) AADC-(G4S)3-GCH1-T2A-TH; 20) GCH1-E2A-TH-P2A-AADC; 21) AADC-(G4S)3-GCH1-E2A-TH; 22) GCH1-E2A-AADC-P2A-TH; 23) AADC-(G4S)3-GCH1-(G4S)3-TH; 24) GCH1-T2A-TH-E2A-AADC; 25) GCH1-(G4S)3-TH-T2A-AADC; 26) GCH1-(G4S)2-TH-T2A-AADC; 27) GCH1-P2A-AADC-T2A-TH; 28) TH-P2A-GCH1-P2A-AADC; 29) TH-(G4S)3-GCH1-T2A-AADC; 30) GCH1-(G4S)3-TH-E2A-AADC; 31) GCH1-T2A-TH-(G4S)3-AADC; 32) TH-P2A-GCH1-F2A-AADC; 33) GCH1-F2A-TH-(G4S)3-AADC; 34) TH-F2A-GCH1-F2A-AADC; 35) TH-(G4S)3-GCH1-E2A-AADC; 36) TH-G4S-GCH1-E2A-AADC; 37) GCH1-P2A-TH-(G4S)3-AADC; 38) AADC-F2A-GCH1-P2A-TH; 39) TH-T2A-GCH1-(G4S)3-AADC; 40) GCH1-E2A-TH-(G4S)3-AADC; 41) TH-E2A-GCH1-(G4S)3-AADC; 42) TH-T2A-GCH1-E2A-AADC; 43) AADC-F2A-GCH1-F2A-TH; 44) TH-F2A-GCH1-(G4S)3-AADC; 45) GCH1-(G4S)3-TH-(G4S)3-AADC; 46) TH-(G4S)3-GCH1-F2A-AADC; 47) TH-E2A-GCH1-T2A-AADC; 48) AADC-P2A-TH-(G4S)3-GCH1; 49) TH-(G4S)3-GCH1-(G4S)3-AADC; 50) AADC-F2A-TH-(G4S)3-GCH1; 51) TH-F2A-AADC-F2A-GCH1; 52) TH-(G4S)3-AADC-T2A-GCH1; 53) TH-(G4S)3-AADC-E2A-GCH1; 54) AADC-P2A-GCH1-(G4S)3-TH; 55) TH-F2A-AADC-(G4S)3-GCH1; 56) AADC-F2A-GCH1-(G4S)3-TH; 57) TH-P2A-AADC-F2A-GCH1; 58) TH-P2A-AADC-(G4S)3-GCH1; 59) GCH1-(G4S)3-TH-P2A-AADC; 60) TH-T2A-AADC-(G4S)3-GCH1; 61) TH-T2A-AADC-E2A-GCH1; 62) GCH1-(G4S)3-TH-F2A-AADC; 63) TH-E2A-AADC-(G4S)3-GCH1; 64) AADC-E2A-GCH1-T2A-TH; 65) GCH1-P2A-TH-F2A-AADC; 66) TH-(G4S)3-AADC-(G4S)3-GCH1; 67) TH-E2A-AADC-T2A-GCH1; 68) GCH1-P2A-AADC-F2A-TH; 69) AADC-T2A-TH-(G4S)3-GCH1; 70) AADC-T2A-GCH1-E2A-TH; 71) TH-F2A-AADC-P2A-GCH1; 72) AADC-E2A-TH-(G4S)3-GCH1; 73) AADC-P2A-GCH1-F2A-TH; 74) TH-(G4S)3-AADC-F2A-GCH1; 75) AADC-P2A-TH-F2A-GCH1; 76) AADC-(G4S)3-TH-P2A-GCH1; 77) AADC-T2A-TH-E2A-GCH1; 78) AADC-(G4S)3-TH-E2A-GCH1; 79) AADC-E2A-TH-T2A-GCH1; or 80) AADC-(G4S)3-TH-F2A-GCH1.

14. The AAV vector of claim 13, wherein the AAV vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 19-21.

15. The AAV vector of any one of claims 1-14, further comprising a promoter operably linked to nucleotide sequences encoding TH, GCH1 and AADC; preferably, wherein the promoter is selected from the CBH promoter, the chimeric Synapsin I promoter, the CMV promoter, the CAG promoter, the CAGG promoter or the CASI promoter.

16. The AAV vector of claim 15, wherein the promoter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 1-6; preferably, the promoter comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3; more preferably, the promoter comprises the nucleic acid sequence of SEQ ID NO:

1.

17. An AAV vector according to any one of claims 1-16, wherein the AAV vector further comprises one or more elements selected from the COSAQ sequence, the SV40 virus polyadenylation signal (SV40 poly A), and an inverted terminal repeat (ITR).

18. An AAV viral particle comprising an AAV vector according to any one of claims 1-17 and a capsid protein.

19. The AAV viral particle of claim 18, wherein the capsid protein is obtained from an AAV selected from the following serotypes: AAV5, AAV9, AAVPHP.eB, AAVPHP.S or AAVPHP.B.

20. The AAV viral particle of claim 19, wherein the capsid protein comprises an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NOs: 33-37; preferably, the capsid protein comprises an amino acid sequence selected from SEQ ID NO: 34 or 37; more preferably, the capsid protein comprises the amino acid sequence of SEQ ID NO:

34.

21. A composition comprising an AAV vector according to any one of claims 1-17 and a packaging plasmid encoding a capsid protein, and optionally a helper plasmid.

22. The composition according to claim 21, characterized in that the packaging plasmid further comprises a nucleic acid sequence encoding a Rep protein, wherein the Rep protein is obtained from AAV serotype AAV2 or serotype AAVPHP.B.

23. The composition according to claim 22, characterized in that the Rep protein contains an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 45 or SEQ ID NO:

46.

24. The composition of any one of claims 21-23, wherein the packaging plasmid comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any of SEQ ID NOs: 24-28 and 48; preferably, the packaging plasmid comprises a nucleotide sequence selected from SEQ ID NO: 25 or 28; more preferably, the packaging plasmid comprises the nucleotide sequence of SEQ ID NO:

25.

25. The composition according to any one of claims 21-24, characterized in that the helper plasmid contains a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

29.

26. The composition according to any one of claims 21-25, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

27. A composition comprising an AAV viral particle according to any one of claims 18-20, optionally the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

28. The composition according to claim 26 or 27, comprising a phosphate buffer, sodium chloride and a surfactant, and preferably the surfactant is poloxamer 188.

29. A cell comprising an AAV vector according to any one of claims 1-17, an AAV viral particle according to any one of claims 18-20, or a composition according to any one of claims 21-28.

30. The cell according to claim 29, characterized in that the cell is a nerve cell, depending on the function, the nerve cell is selected from: an inhibitory neuron (preferably, said cell is a medium spiny neuron, an interneuron), an excitatory neuron (preferably, said cell is a dopaminergic neuron), a microglia, an astrocyte, an oligodendrocyte, or a Schwann cell; depending on the region of the brain, this cell is a neuron of the striatum of the midbrain and a glial cell.

31. The use of an AAV vector according to any one of claims 1-17, an AAV viral particle according to any one of claims 18-20, a composition according to any one of claims 21-28, or a cell according to claim 29 or 30 in the manufacture of a medicament for the prevention and / or treatment of a disease caused by dopamine deficiency in the body of a subject.

32. The use according to paragraph 31, characterized in that the disease is Parkinson's disease.