IMPROVING SUICIDE GENES FOR CANCER GENE THERAPY

基因治疗提供了有选择地将基因导入癌细胞的前景，使它们对特定的抗肿瘤药物敏感。目前诱导肿瘤减少的方案使用单纯疱疹病毒1型(HSV)胸苷激酶(TK)与前药更昔洛韦(GCV)或大肠杆菌胞嘧啶脱氨酶(5FC)与前药5-氟胞嘧啶(5FC)。虽然野生型TK具有自杀基因的功能，但一些警告限制了其充分的有效性。这些问题包括对GCV的Km或结合亲和力较差(约47微米)，以及与高剂量GCV相关的毒性。另一种前药阿昔洛韦(ACV)，主要用作抗疱疹药物，即使在非常高的剂量下也不能表现出GCV的免疫抑制属性。然而，HSV TK对阿昔洛韦的KM非常高(约320微米)，排除了它作为前药用于消融性基因治疗的可能性。同样，胞嘧啶脱氨酶和5FC也有一些警告，排除了它作为安全有效的自杀基因的潜力。我们寻求确定最安全和最有效的癌症基因治疗的最佳自杀基因和前药组合。为此，我们试图1)了解核苷代谢酶对自杀基因治疗重要的结构-功能关系，以及2)操纵HSV-1胸苷激酶和胞嘧啶脱氨酶基因，以在消融性基因治疗环境中获得更好的表现。这项工作的目标是1)通过突变创造新的基因，2)评估突变基因提高肿瘤对各种前体药物的敏感性，3)构建微通道，4)创造与其他基因的融合蛋白，以增强前体药物的激活和肿瘤的消融。该项目的结果不仅将影响用于癌症治疗的基因(S)的选择，而且它还具有广泛的应用，包括移植物抗宿主病、再狭窄、艾滋病、肿瘤成像、细胞谱系消融研究、阴性选择系统以及针对非同源重组的转基因小鼠的选择。此外，了解核苷代谢酶功能的分子基础以及与现有药物的相互作用将对新型抗病毒、抗真菌和抗菌药物的设计、开发和使用具有深远的影响。

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 cytosine deaminase with the prodrug 5-fluorocytosine (5FC). While the wild-type TK is functional as a suicide gene, a number of caveats restrict its full effectiveness. These include a poor Km or binding affinity for GCV (approximately 47muM) and the toxicity associated with high doses of GCV. Another prodrug, acyclovir (ACV), predominantly used as an anti-herpetic drug, does not demonstrate the immunosuppressive attributes of GCV, even at very high doses. However, the very high Km that HSV TK displays towards ACV (approximately 320 muM), precludes its use as a prodrug in ablative gene therapy. Similarly, there are caveats with cytosine deaminase and 5FC that preclude its potential as a safe and effective suicide gene. We seek to identify the optimal suicide gene and prodrug combination for the safest and most effective cancer gene therapy. Towards this end we seek 1) to understand the structure-function relationship of nucleoside metabolizing enzymes important to suicide gene therapy and 2) to manipulate HSV-1 thymidine kinase and cytosine deaminase genes for superior performance in ablative gene therapy settings. The goal of this work is to 1) create novel genes by mutagenesis, 2) evaluate mutant genes for improved tumor sensitivity to various prodrugs, 3) construct mini-pathways and 4) create fusion proteins with other genes to enhance prodrug activation and tumor ablation. Not only will the results from the project impact the choice of gene(s) used for cancer treatment, but it also has wide-reaching applications including graft versus host disease, restenosis, AIDS, tumor imaging, cell lineage ablation studies, in negative selection systems and selection against non-homologous recombination for the generation of transgenic mice. Furthermore, understanding the molecular basis of nucleoside metabolizing enzyme function and interaction with current drugs will have far-reaching ramifications in the design, development and use of novel antiviral, antifungal and antibacterial drugs.