Catecholamine enzyme fusions
Provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding TH is linked to the nucleotide sequence encoding CH1 such that they encode a fusion protein TH-CH1. Also provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding AADC is linked to the nucleotide sequence encoding TH such that they encode a fusion protein AADC-TH or TH-AADC. Further provided is a viral vector comprising such nucleotide sequences and its use in the treatment and/or prevention of Parkinson's disease.
1. A nucleic acid construct comprising:
(i) a nucleotide sequence which encodes tyrosine hydroxylase (TH),
(ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and
(iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC);
wherein the nucleotide sequence encoding TH is linked to the nucleotide sequence encoding CH1 such that they encode a fusion protein TH-CH1,
wherein the construct comprises TH- L -CH1- IRES -AADC or TH- L -CH1- P -AADC, wherein L is a linker-encoding sequence, IRES is an Internal Ribosome Entry Site, and P is a promoter.
2. The nucleic acid construct according to claim 1 , comprising TH- L -CH1- IRES -AADC.
3. The nucleic acid construct according to claim 1 , wherein the linker is not codon optimized for human usage.
4. The nucleic acid construct according to claim 1 , wherein the linker comprises the nucleic acid sequence as set forth in SEQ ID No. 1.
5. A viral vector genome comprising a nucleic acid construct according to claim 1 .
6. The viral vector genome according to claim 5 , which is a lentiviral vector genome or an adeno-associated viral vector genome.
7. An adeno-associated viral vector system comprising: (i) the adeno-associated viral vector genome according to claim 6 ; (ii) a nucleotide sequence or sequences encoding Rep and Cap proteins; and (iii) nucleotide sequences encoding other essential viral packaging components not encoded by the nucleotide sequence as set forth in (ii).
8. A lentiviral vector system comprising (i) the lentiviral vector genome according to claim 6 ; (ii) a nucleotide sequence or sequences encoding gag and pol proteins; and (iii) a nucleotide sequences encoding other essential viral packaging components not encoded by the nucleotide sequence as set forth in (ii).
9. A method for producing a lentiviral vector particle, which method comprises introducing into a producer cell:
i) the lentiviral vector genome according to claim 6 ,
ii) a nucleotide sequence or sequences coding for gag and pol proteins; and
iii) nucleotide sequences encoding other essential viral packaging components not encoded by one or more of the nucleotide sequences of ii.
10. A viral particle comprising the viral vector genome according to claim 5 .
11. The viral vector particle according to claim 10 , which is an EIAV vector particle, and which is pseudotyped with VSV-G.
12. A pharmaceutical composition comprising the viral particle according to claim 10 and a pharmaceutically acceptable carrier or diluent.