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

多项研究表明，病毒载体介导的酪氨酸羟化酶(TH)转基因参与体内阿儿茶酚胺合成，可用于帕金森病(PD)的基因治疗。然而，在哺乳动物细胞中表达的TH不能高效地产生L-多巴，因为TH的催化活性受到细胞中积累的最终产物儿茶酚胺的抑制。本研究的目的是在哺乳动物细胞中产生具有高合成L多巴能力的TH，为帕金森病的有效基因治疗奠定基础。我们生产了人TH1型突变酶(HTH1)，并对其特性进行了检测。1)用中性或带负电荷的残基取代hTH1的30-40个N端残基，可减弱多巴胺对催化活性的抑制作用。2)用带负电荷的Glu或Asp取代hTH1的Arg^&lt；37&gt；-Arg^&lt；38&gt；和Ser^&lt；40&gt；3)突变酶在AtT-20神经内分泌细胞中能有效地产生DA。4)此外，hTH1的突变酶在除AtT-20细胞外的其他哺乳动物细胞系中表现出很高的稳定性。总之，我们的研究为改进基因治疗方法以提高体内DA的产量提供了有用的信息。

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.