Black M E, Newcomb T G, Wilson H M, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, School of Medicine, University of Washington, Seattle, 98195-7705, USA.
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3525-9. doi: 10.1073/pnas.93.8.3525.
Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.
单纯疱疹病毒1型（HSV-1）胸苷激酶目前在癌症基因治疗中用作自杀基因。这种治疗方法基于表达HSV-1胸苷激酶的肿瘤细胞对核苷类似物的优先磷酸化作用。然而，HSV-1胸苷激酶的使用在一定程度上受到核苷类似物毒性的限制。我们利用随机序列诱变技术创造了新的HSV-1胸苷激酶，与野生型胸苷激酶相比，这些新的胸苷激酶使细胞对特定核苷类似物的敏感性大大提高。在假定的核苷结合位点处的HSV-1胸苷激酶基因片段被随机核苷酸序列所取代。从超过一百万个大肠杆菌转化子中筛选出了对核苷类似物更昔洛韦或阿昔洛韦具有优先磷酸化作用的突变酶。在我们分离出的426个活性突变体中，有26个对更昔洛韦的敏感性增强，54个对阿昔洛韦更敏感。在大肠杆菌中表达时，只有6种突变酶对更昔洛韦和阿昔洛韦均表现出敏感性。对3种药物敏感的酶进行分析表明，其中1种产生了稳定的哺乳动物细胞转染子，这些转染子对更昔洛韦的敏感性提高了43倍，对阿昔洛韦的敏感性提高了20倍。
