CREATING MUTANT ENZYMES FOR GENE THERAPY OF CANCER

我们建议使用突变酶进行人类癌症的基因治疗， 以切除肿瘤并在化疗期间保护骨髓。 我们 已经建立了通过偶联产生新的突变酶的技术， 随机核苷酸诱变和酶的遗传选择， 改变底物特异性。 我们将利用这项技术来创造 具有癌症基因治疗所需特性的突变酶， 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，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， 对5-氟尿嘧啶具有抗性的胸苷酸脱氢酶， 为了在治疗过程中保护骨髓， 剂 我们已经确定了突变的DNA甲基转移酶， 活性高于野生型，并且对抑制剂具有抗性 苄基鸟嘌呤，用于增加骨髓抵抗力 烷化剂的前体细胞。 我们将评估突变体是否 DNA聚合酶β可以用于增强复制旁路， 从而增加骨髓细胞对未修复的 阻碍DNA合成的病变 我们建立了一个大图书馆 突变的疱疹胸苷激酶， 核苷类似物。 这些突变的胸苷激酶使哺乳动物 细胞对更昔洛韦和阿昔洛韦的致死作用更大， 程度比野生型酶，并将测试的效力， 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.