Improving Suicide Genes for Cancer Gene Therapy

描述（申请人提供）：基因治疗提供了选择性地将基因导入癌细胞的前景，使其对特定的抗肿瘤药物敏感。目前引起肿瘤缩小的方案利用单纯疱疹病毒1型（HSV）胸苷激酶（TK）与前药更昔洛韦（GCV）或E.大肠杆菌或酵母胞嘧啶脱氨酶（CD）与前体药物5-氟胞嘧啶（5 FC）。虽然具有自杀基因的功能，但一些警告限制了它们的全部效力。这些包括对前药的不良Km或结合亲和力以及与引发肿瘤反应所必需的高前药剂量相关的毒副作用。我们寻求确定最佳的自杀基因和前药组合的最安全和最有效的癌症基因治疗。该项目的具体目标是使用诱变策略优化三个独立的自杀基因系统[胞嘧啶脱氨酶、鸟苷酸激酶/TK（途径工程）和CD/TK（会聚途径工程）]，并测试酶变体在肿瘤细胞系和动物模型中的功效。本研究致力于克服目前自杀基因治疗策略中发现的动力学限制，并将解决和比较：1）增加活化前药的产生和对肿瘤细胞杀伤的影响; 2）增强旁观者效应，因为它与增加的细胞毒素产生有关; 3）减少前药剂量以抵消毒副作用; 4）增强双自杀基因方法的协同作用（会聚途径工程）。该项目的结果不仅将影响用于癌症治疗的基因的选择，而且它们在其他地方也有广泛的应用，包括移植物抗宿主病、再狭窄、艾滋病、非侵入性肿瘤成像、细胞谱系消融研究、阴性选择系统和针对非同源重组的选择以产生转基因小鼠。

DESCRIPTION (provided by applicant): Gene therapy offers the prospect of selectively introducing genes into cancer cells, leaving them susceptible to specific antitumor drugs. Current protocols to elicit tumor reduction utilize Herpes Simplex Virus type 1 (HSV) thymidine kinase (TK) with the prodrug ganciclovir (GCV), or the E. coli or yeast cytosine deaminase (CD) with the prodrug 5-fluorocytosine (5FC). While functional as suicide genes, a number of caveats restrict their full effectiveness. These include a poor Km or binding affinity for prodrugs and toxic side effects associated with the high prodrug doses necessary to elicit tumor response. We seek to identify the optimal suicide gene and prodrug combination for the safest and most effective cancer gene therapy. The specific aims of this project are to optimize three separate suicide gene systems [cytosine deaminase, guanylate kinase/TK (pathway engineering) and CD/TK (converging pathway engineering)] using mutagenesis strategies and to test the efficacy of enzyme variants in tumor cell lines and animal models. This research endeavors to overcome the kinetic limitations found in current suicide gene therapy strategies and will address and compare: 1) increasing production of activated prodrugs and the impact on tumor cell killing; 2) enhancing the bystander effect as it relates to increased cytotoxin production; 3) reducing prodrug doses for therapeutic efficacy to offset toxic side effects and; 4) augmenting synergy of the dual suicide gene approach (converging pathway engineering). Not only will the results from this project impact the choice of gene(s) used for cancer treatment but they also have broad application elsewhere including for graft versus host disease, restenosis, AIDS, in noninvasive tumor imaging, cell lineage ablation studies, in negative selection systems and selection against non-homologous recombination for the generation of transgenic mice.