Engineering enzymes for anti-tumor suicide gene therapy

描述（由申请人提供）：前药基因治疗是一种治疗策略，其中肿瘤细胞用“自杀”基因转染，该基因编码能够将无毒前药转化为有效细胞毒素的代谢酶。这种方法允许选择性根除肿瘤细胞，同时使正常组织免于显著的细胞杀伤。该策略的有效性取决于旁观者效应，其中未转染的肿瘤细胞通过细胞毒性酶产物的主动或被动转运而被杀死。几种酶/前药组合正在积极研究中，证明在组织培养和动物模型中的有效性。然而，低转染效率和前药底物的不良周转的组合限制了肿瘤中细胞杀伤的效率。为了改善这种疗法，必须选择和工程化酶变体以增强前药底物的周转。在该提案中，两个实验室的合作提出优化核苷补救酶胞嘧啶脱氨酶和脱氧胞苷激酶用于前药自杀基因治疗，使用结构生物学和定向进化筛选的组合，并在细胞系和动物模型中测试酶变体的功效。胞嘧啶脱氨酶（CD）催化胞嘧啶脱氨为尿嘧啶和氨。胞嘧啶脱氨酶存在于细菌和真菌中，但不存在于哺乳动物细胞中。表达CD的细胞对核苷类似物5-氟胞嘧啶敏感。由于5 FC酶促转化为5-氟尿嘧啶（5 FU）。这种化合物及其脱氧核糖核苷，氟脱氧尿苷（FUdR），是DNA合成和RNA功能的有效抑制剂，广泛用于癌症治疗。相反，脱氧胞苷激酶（dCK）从胞苷核苷产生胞苷-单磷酸，并且还激活抗肿瘤剂吉西他滨和阿糖胞苷。本项目的具体目标是：（1）确定细菌CD、酵母CD和人dCK的结构。(2)进行基于结构的诱变和遗传筛选，以分离具有增强的前药底物结合和周转的酶变体。这一目标将利用晶体学信息来指导酶的特定区域的诱变，并直接可视化增强的前药的结构基础。（3）使用细胞系和动物模型，在已建立的肿瘤模型系统中测试酶变体对肿瘤细胞的杀伤。

DESCRIPTION (provided by applicant): Prodrug gene therapy is a therapeutic strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme which is capable of converting a nontoxic prodrug into a potent cytotoxin. Such a method allows selective eradication of tumor cells while sparing normal tissue from significant cell killing. The effectiveness of this strategy is dependent on a bystander effect in which untransfected tumor cells are killed through active or passive transport of the cytotoxic enzyme product. Several enzyme/prodrug combinations are under active investigation, demonstrating effectiveness in both tissue culture and animal models. However, the combination of low transfection efficiencies and poor turnover of prodrug substrates limit the efficiency of cell killing in the tumor. In order to improve such therapies, enzyme variants must be selected and engineered for enhanced turnover of the prodrug substrate. In this proposal, a collaboration of two laboratories propose to optimize the nucleoside salvage enzymes cytosine deaminase and deoxycytidine kinase for prodrug suicide gene therapy, using a combination of structural biology and directed evolution screens, and to test the efficacy of enzyme variants in cell line and animal models. Cytosine deaminase (CD) catalyzes the deamination of cytosine to uracil and ammonia. Cytosine deaminase is found in bacteria and fungi but is not present in mammalian cells. Cells expressing CD are sensitive to the nucleoside analog, 5-fluorocytosine. Due to the enzymatic conversion of 5FC to 5-fluorouracil (5FU). This compound and its deoxyribonucleoside, fluorodeoxyuridine (FUdR), are potent inhibitors of DNA synthesis and RNA function and are widely used in cancer treatment. In contrast, deoxycytidine kinase (dCK) generates cytidine-monophosphate from cytidine nucleoside, and also activates the antineoplastic agents gemcitabine and cytarabine. The specific aims for this project are: (1) Determine the structure of bacterial CD, yeast CD and human dCK. (2) Perform structure-based mutagenesis and genetic screens to isolate enzyme variants with enhanced binding and turnover of prodrug substrates. This aim will exploit crystallographic information both to direct the mutagenesis of specific regions of the enzyme, and to directly visualize the structural basis of enhanced prodrug. activation by selected enzyme variants (3) Test enzyme variants for tumor cell killing in established tumor model systems, using cell lines and animal models.