Engineering enzymes for anti-tumor suicide gene therapy

描述(申请人提供)：前药基因疗法(PGT)是一种治疗策略，将自杀基因导入肿瘤细胞，这种基因编码一种代谢酶，能够将无毒的前药转化为有效的细胞毒素。几种酶/前药组合正在积极研究中。这一策略在本质上受到基因向癌细胞的低效传递的限制(实际上，用基因传递问题代替药物传递问题)。为了消除这一重大问题，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(其毒性和新陈代谢)以及两者的组合(它们对转基因细胞的独特性)，以实现最大的治疗效果。在这个项目中，三个合作的实验室正在设计和优化两种用于PGT的核苷回收/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)被设计成能有效地将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。相比之下，DCK(一种人类酶)催化嘧啶核苷的β-磷酸化，并被设计成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，对最好的酶变异体诱导对前药的敏感性的能力进行了测试。我们来自前一个资助周期的数据表明，给定酶/前药物组合的稳定性或底物特定的活性和特异性可能会限制前药物治疗的效果。此外，任何一种限制都可以通过设计和选择改进的酶结构来克服。根据先前对这一更新申请的审查提出的建议，我们现在描述该项目修订后的一系列具体目标如下：(1)我们将确定yCD或BCD的优化是否通过增加肿瘤细胞中的酶表达和/或药物产生的可识别机制而导致显著的治疗效果获得。(2)我们将创造一种新的酶/前药组合(DCK和地西他滨，这是一种强有力的细胞毒素，但DCK既不稳定，又不能有效地磷酸化)。我们将比较酶重新设计以增强对地西他滨的活性的结果与与吉西他滨(相比之下，吉西他滨是DCK的有效底物)的平行实验。除了在PGT库中添加一种新的酶/前药物组合外，这些实验还将检查PGT中酶性能的限制，这些限制取决于底物。我们的假设是，由于非癌组织中缺乏这种活性，地西他滨最终应该与工程酶变体结合起来，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.