Mutant enzymes of tyrosine hydroxylase for an effective gene therapy of PD

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Several studies have suggested that the direct virus vector-mediated delivery of transgenes of tyrosine hydroxylase (TH) involved in atecholamine synthesis in vivo may be available for gene therapy of Parkinson's disease (PD). However, TH expressed in mammalian cells cannot produce efficiently L-dopa, because the catalytic activity of TH is inhibited by the end-products catecholamines accumulated in the cells. The purpose of our study is to produce TH with high capability of L-dopa synthesis in the mammalian cells for an effective gene therapy of PD. We produced the mutant enzymes of human TH type1 (hTH1) and then examined the characteristic of them. 1)The replacement of N-terminal residues 30-40 of hTH1 by neutral or negatively charged residues decreased the inhibitory effect of dopamine on the catalytic activity. 2)Especially, the replacement of Arg^<37> -Arg^<38> and Ser^<40> of hTH1 by negatively charged Glu or Asp gave an efficient production of DA in vitro. 3)The efficient production of DA by the mutant enzymes was detected in AtT-20 neuroendcrine cells. 4)Moreover, the mutant enzymes of hTH1 revealed a high stability in other mammalian cell lines in addition to AtT-20 cells. Collectively, our study provides useful information for the refinement of the gene-therapy approach to obtain increased production of DA in vivo.