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: 7628052
• 负责人: BARRY L. STODDARD
• 金额: $ 31.01万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628052
• 关键词: Amidohydrolases; Animal Model; Bacteria; Behavior; Belief; Biological Assay; Cancerous; Cell Culture Techniques; Cells; Chemicals; Clinical Trials; Complex; Cytidine; Cytosine; Cytosine deaminase; Cytotoxin; DNA Synthesis Inhibitors; Data; Decitabine; Deoxycytidine Kinase; Drug Delivery Systems; Drug Kinetics; Engineering; Enzymes; Flucytosine; Fluorouracil; Funding; Gene Delivery; Half-Life; Human; Investigation; Kinetics; Laboratories; Measurement; Measures; Metabolic; Metabolism; Nucleosides; Performance; Phosphorylation; Physiologic pulse; Predisposition; Prodrugs; Production; Property; Protein Engineering; Proteins; Protocols documentation; Pyrimidine Nucleosides; RNA; Research Personnel; Resolution; Specificity; Structure; Suggestion; Suicide Gene Therapy; Testing; Therapeutic; Tissues; Toxic effect; Treatment Efficacy; Tumor Cell Line; Uracil; Variant; X-Ray Crystallography; Yeasts; analog; base; cancer cell; chemotherapy; design; directed evolution; drug production; enzyme structure; gemcitabine; gene therapy; improved; microbial; neoplastic cell; novel; pre-clinical; preclinical study; programs; pyrimidine analog; research study; suicide gene; thermostability; tumor; uptake

DESCRIPTION (provided by applicant): Prodrug gene therapy (PGT) is a therapeutic strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. Several enzyme/prodrug combinations are under active investigation. This strategy is inherently limited by inefficient delivery of the gene to cancer cells (in effect, replacing the problem of drug delivery with the problem of gene delivery). To offset this significant issue, the pharmacokinetic properties of the enzyme (its stability, half-life and kinetic activity), the prodrug (its toxicity and metabolism) and combination of the two (their uniqueness to transfected cells) must be optimized for maximum therapeutic efficacy. In this project, three collaborating laboratories are engineering and optimizing two nucleoside salvage/synthesis enzymes for PGT: cytosine deaminase (CD) and deoxycytidine kinase (dCK). CD (a microbial enzyme) is being engineered to efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which is a metabolic inhibitor of DNA synthesis and RNA function. In contrast, dCK (a human enzyme) catalyzes the ?-phosphorylation of pyrimidine nucleosides, and is being engineered to efficiently activate pyrimidine analogues such as gemcitabine and decitabine. In both cases, the project follows a 'design cycle' of crystallographic structure determination, computational protein engineering, directed evolution and subsequent kinetic and structural analyses. The ability of the best enzyme variants to induce sensitivity to the prodrug is assayed in tumor cell lines, animal models and ongoing clinical trials. Our data from the previous funding cycle demonstrate that either the stability or the substrate-specific activity and specificity of a given enzyme/prodrug combination can be limiting for performance in prodrug therapy. Furthermore, either limitation can be overcome by design and selection of improved enzyme constructs. Based on suggestions from previous review of this renewal application, we now describe a set of revised specific aims for this project as follows: (1) We will determine whether optimization of yCD or bCD leads to significant therapeutic efficacy gains via recognizable mechanisms of increased enzyme expression and/or drug production in tumor cells. (2) We will create a new enzyme/prodrug combination (dCK and decitabine, which is a potent cytoxin but is both unstable and inefficiently phosphorylated by dCK). We will compare the results of enzyme redesign for enhanced activity against decitabine to parallel experiments with gemcitabine (which, in contrast, is an efficient substrate for dCK). In addition to adding a new enzyme/prodrug combination to the PGT arsenal, these experiments will examine limitations on an enzyme's performance in PGT that are substrate-dependent. Our hypothesis is that decitabine should ultimately couple with engineered enzyme variants to yield improvements in the performance of dCK, due to the lack of this activity in non-cancerous tissues.

描述（由申请人提供）：前药基因治疗（PGT）是一种治疗策略，其中肿瘤细胞被“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。几种酶/前药组合正在积极研究中。这种策略固有地受到基因向癌细胞的低效递送的限制（实际上，用基因递送的问题代替药物递送的问题）。为了解决这个重要问题，必须优化酶的药代动力学性质（其稳定性、半衰期和动力学活性）、前药（其毒性和代谢）以及两者的组合（其对转染细胞的独特性），以获得最大的治疗效果。在该项目中，三个合作实验室正在设计和优化两种用于PGT的核苷补救/合成酶：胞嘧啶脱氨酶（CD）和脱氧胞苷激酶（dCK）。CD（一种微生物酶）正在被改造以有效地将5-氟胞嘧啶（5-FC）转化为5-氟尿嘧啶（5-FU），5-FU是DNA合成和RNA功能的代谢抑制剂。相反，dCK（一种人体酶）催化？本发明涉及嘧啶核苷的磷酸化，并且被工程化以有效地活化嘧啶类似物，如吉西他滨和地西他滨。在这两种情况下，该项目遵循晶体结构测定，计算蛋白质工程，定向进化和随后的动力学和结构分析的“设计周期”。在肿瘤细胞系、动物模型和正在进行的临床试验中测定最佳酶变体诱导对前药敏感性的能力。我们之前的资助周期数据表明，给定酶/前药组合的稳定性或底物特异性活性和特异性可能限制前药治疗的性能。此外，可以通过设计和选择改进的酶构建体来克服这两种限制。基于先前对该更新申请的审查的建议，我们现在描述了一组修订后的该项目的具体目标，如下所示：（1）我们将确定yCD或bCD的优化是否通过增加肿瘤细胞中酶表达和/或药物产生的可识别机制导致显著的治疗功效增益。(2)我们将创建一种新的酶/前药组合（dCK和地西他滨，这是一种有效的细胞毒素，但不稳定且不能被dCK有效磷酸化）。我们将比较针对地西他滨的增强活性的酶重新设计的结果与使用吉西他滨的平行实验（相比之下，吉西他滨是dCK的有效底物）。除了将新的酶/前药组合添加到PGT库之外，这些实验还将检查底物依赖性的酶在PGT中的性能的限制。我们的假设是，地西他滨最终应该与工程酶变体偶联，以改善dCK的性能，因为在非癌组织中缺乏这种活性。