Engineering enzymes for anti-tumor suicide gene therapy

用于抗肿瘤自杀基因治疗的工程酶

• 批准号: 6735680
• 负责人: BARRY L. STODDARD
• 金额: $ 31.99万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6735680
• 关键词: X ray crystallography; aminohydrolases; antineoplastics; athymic mouse; bacterial proteins; cell line; cytotoxicity; dCMP deaminase; deoxycytidine; disease /disorder model; drug metabolism; drug screening /evaluation; enzyme activity; enzyme substrate; fluorouracil; fungal proteins; gemcitabine; gene complementation; gene therapy; genetic screening; neoplastic cell; prodrugs; protein purification; site directed mutagenesis; structural biology

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.

描述(申请人提供)：前药基因疗法是一种治疗策略，在这种疗法中，肿瘤细胞被导入一种‘自杀’基因，这种基因编码一种代谢酶，能够将无毒的前药转化为一种有效的细胞毒素。这种方法可以选择性地根除肿瘤细胞，同时使正常组织免于显著的细胞杀伤。这一策略的有效性依赖于旁观者效应，在旁观者效应中，未转基因的肿瘤细胞通过细胞毒酶产物的主动或被动运输而被杀死。几种酶/前药组合正在积极研究中，在组织培养和动物模型中都证明了有效性。然而，低的转染率和前药物底物的低周转限制了肿瘤中的细胞杀伤效率。为了改进这种疗法，必须选择和设计酶变异体，以提高前药底物的周转。在这项建议中，两个实验室的合作建议优化核苷回收酶胞嘧啶脱氨酶和脱氧胞苷激酶用于前药物自杀基因治疗，使用结构生物学和定向进化筛选相结合的方法，并在细胞系和动物模型中测试酶变体的有效性。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5FC被酶转化为5-氟尿嘧啶(5FU)。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)从胞苷核苷生成胞苷一磷酸，并激活抗肿瘤药物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，以分离具有增强前药底物结合和转化的酶变异体。这一目标将利用结晶学信息来指导酶的特定区域的突变，并直接显示增强型前药的结构基础。通过选定的酶变异体激活(3)在已建立的肿瘤模型系统中，使用细胞系和动物模型测试酶变异体对肿瘤细胞的杀伤作用。