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，其中肿瘤细胞用“自杀”基因转染，该基因编码能够将无毒药物转化为有效的细胞毒素的代谢酶。几种酶/前药组合正在积极研究中。该策略固有地受到基因向癌细胞的效率低下的限制（实际上，用基因递送问题代替了药物递送问题）。为了抵消这一重要问题，必须优化酶的药代动力学特性（其稳定性，半衰期和动力活性），前药（其毒性和代谢）以及两者（其对转染细胞的独特性）的组合，以获得最大的治疗功效。在这个项目中，三个合作实验室正在工程和优化PGT的两种核苷拯救/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）正在设计，以有效地将5-氟环肽（5-FC）转换为5-氟尿嘧啶（5-FU），这是DNA合成和RNA功能的代谢抑制剂。相反，DCK（人类酶）催化了嘧啶核苷的磷酸化，并且正在设计以有效激活嘧啶类似物（例如吉西他滨和刀位）。在这两种情况下，该项目均遵循晶体学结构确定，计算蛋白工程，定向进化以及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了最佳酶变体诱导对前药敏感性的能力。我们从上一个资金周期的数据表明，给定酶/前药的稳定性或底物特异性活性和特异性可能限制在前药治疗中的性能。此外，可以通过设计和选择改进的酶构建体来克服任何一种限制。基于对此续签应用的先前综述的建议，我们现在描述了一组对该项目的修订特定目的，如下所示：（1）我们将通过可识别的酶表达和/或药物产生的肿瘤细胞中的可识别机制来确定YCD或BCD的优化是否导致显着的治疗效率提高。 （2）我们将创建一种新的酶/前药组合（DCK和Decitabine，它是一种有效的细胞毒素，但既不稳定又被DCK磷酸化）。我们将比较酶重新设计的结果，以增强针对Decitabine的活性与吉西他滨的平行实验（相比之下，这是DCK的有效底物）。除了将新的酶/前药组合添加到PGT阿森纳外，这些实验还将检查酶在PGT中依赖于底物的酶性能的局限性。我们的假设是，由于非癌组织缺乏这种活性，德替他应最终与工程酶变体相结合，以改善DCK的性能。