Engineering enzymes for anti-tumor suicide gene therapy

描述（由申请人提供）：前药基因治疗（PGT）是一种治疗策略，其中肿瘤细胞转染“自杀”基因，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。目前正在积极研究几种酶/前药组合。这种策略本身就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）。为了弥补这一重大问题，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）必须优化，以获得最大的治疗效果。在这个项目中，三个合作实验室正在为PGT设计和优化两种核苷回收/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）被设计成有效地将5-氟胞嘧啶（5-FC）转化为5-氟尿嘧啶（5-FU）， 5-氟尿嘧啶是DNA合成和RNA功能的代谢抑制剂。相反，dCK（一种人体酶）能催化？-嘧啶核苷的磷酸化，并被设计为有效地激活嘧啶类似物，如吉西他滨和地西他滨。在这两种情况下，该项目都遵循了晶体结构确定、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，检测了最佳酶变体诱导对前药敏感性的能力。我们从上一个资助周期获得的数据表明，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表现。此外，任何限制都可以通过设计和选择改进的酶结构来克服。根据先前对该更新申请的审查建议，我们现在描述了该项目的一组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着的治疗效果提高。(2)我们将创造一种新的酶/前药组合（dCK和地西他滨，后者是一种强效细胞毒素，但不稳定且不能被dCK有效磷酸化）。我们将对酶重新设计的结果进行比较，以增强对地西他滨的活性，并与吉西他滨平行实验（相反，吉西他滨是dCK的有效底物）。除了在PGT库中添加新的酶/前药组合外，这些实验将检查酶在PGT中依赖底物的性能的局限性。我们的假设是，由于在非癌组织中缺乏这种活性，地西他滨最终应该与工程酶变体偶联以提高dCK的性能。

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.