CREATING MUTANT ENZYMES FOR GENE THERAPY OF CANCER

我们建议将突变酶用于人类癌症的基因治疗，两者都 在化疗期间切除肿瘤和保护骨髓。我们 已经建立了通过偶联来创造新的突变酶的技术 酶基因选择的随机核苷酸突变 改变了底物的特性。我们将使用这项技术来创造 具有所需性质的突变酶用于癌症基因治疗，并将 在各种实验系统中测试它们的有效性。 我们将采取四种策略，分别针对DNA中的不同步骤 新陈代谢。我们已经建立了一个选择系统来鉴定突变 使用对5-氟尿嘧啶有抗药性的胸苷合成酶 用于在治疗过程中保护骨髓 探员。我们已经鉴定出突变的DNA甲基转移酶 活性高于野生型，并对抑制剂具有抵抗力 用于增加骨髓抵抗力的苄基鸟嘌呤 前体细胞到烷基化剂。我们将评估变种人是否 DNA聚合酶β可以用来增强复制旁路和 从而提高骨髓细胞对未修复的耐受性 阻碍DNA合成的损伤。我们已经建立了一个大型图书馆 优先磷酸化的疱疹病毒胸苷激酶的突变 核苷类似物。这些突变的胸苷激酶使哺乳动物变得敏感 细胞对更昔洛韦和阿昔洛韦的致死作用更大 比野生型酶的范围更大，并将在 肿瘤消融。

We propose to use mutant enzymes for gene therapy of human cancer, both to ablate tumors and to protect bone marrow during chemotherapy. We have established techniques for creating new mutant enzymes by coupling random nucleotide mutagenesis with genetic selection for enzymes with altered substrate specificities. We will use this technology to create mutant enzymes with desired properties for cancer gene therapy, and will test their efficacity in a variety of experimental systems. We will pursue four strategies that target different steps in DNA metabolism. We have established a selection system to identify mutant thymidylate synthases that are resistant to 5-fluorouracil, to be used for protection of bone marrow during therapy with this commonly employed agent. We have identified mutant DNA methyltransferases that are more active than the wild type, and are resistant to the inhibitor benzylguanine, to be used to increase the resistance of bone marrow precursor cells to alkylating agents. We will evaluate whether mutant DNA polymerase beta's can serve to enhance replication bypass and thereby increase the tolerance of bone marrow cells for unrepaired lesions that block DNA synthesis. We have established a large library of mutant herpes thymidine kinases that preferentially phosphorylate nucleoside analogs. These mutant thymidine kinases sensitize mammalian cells to the lethal effects of gancyclovir and acyclovir to a greater extent than the wild type enzyme, and will be tested for efficacity in tumor ablation.