Engineering enzymes for anti-tumor suicide gene therapy

用于抗肿瘤自杀基因治疗的工程酶

基本信息

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
项目类别：
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

项目摘要

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）：前药基因疗法（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强效细胞毒素的代谢酶。几种酶/前药组合正在积极研究中。这种策略本质上受到基因向癌细胞传递效率低下的限制（实际上，用基因传递问题取代了药物传递问题）。为了解决这个重大问题，必须优化酶的药代动力学特性（其稳定性、半衰期和动力学活性）、前药（其毒性和代谢）以及两者的组合（它们对转染细胞的独特性），以获得最大的治疗效果。功效。在该项目中，三个合作实验室正在设计和优化两种用于 PGT 的核苷挽救/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）被设计用于有效地将 5-氟胞嘧啶 (5-FC) 转化为 5-氟尿嘧啶 (5-FU)，5-氟尿嘧啶 (5-FU) 是 DNA 合成和 RNA 功能的代谢抑制剂。相比之下，dCK（一种人类酶）可催化嘧啶核苷的 β-磷酸化，并且经过改造可有效激活嘧啶类似物，例如吉西他滨和地西他滨。在这两种情况下，该项目都遵循晶体结构测定、计算蛋白质工程、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。我们来自上一个资助周期的数据表明，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会限制前药治疗的性能。此外，任何一个限制都可以通过设计和选择改进的酶构建体来克服。根据之前对该更新申请的审查的建议，我们现在描述该项目的一系列修订后的具体目标如下：（1）我们将确定 yCD 或 bCD 的优化是否通过可识别的酶增加机制带来显着的治疗效果增益肿瘤细胞中的表达和/或药物产生。 (2) 我们将创建一种新的酶/前药组合（dCK 和地西他滨，它是一种有效的细胞毒素，但不稳定且不能被 dCK 有效磷酸化）。我们将比较酶重新设计以增强抗地西他滨活性的结果与吉西他滨（相比之下，吉西他滨是 dCK 的有效底物）的平行实验。除了向 PGT 库中添加新的酶/前药组合外，这些实验还将检查底物依赖性酶在 PGT 中的性能限制。我们的假设是，由于非癌组织中缺乏这种活性，地西他滨最终应与工程酶变体结合以改善 dCK 的性能。