Enhancing Suicide Gene Therapy Through Mechanism-Based Approaches

自杀基因治疗是一种有吸引力的癌症治疗方法，因为它比传统的治疗方法更具选择性。 传统的癌症化疗。我们关注单纯疱疹病毒胸苷激酶（HSV-TK）， 抗病毒药物更昔洛韦（GCV）的初始激活剂转化为其细胞毒性三磷酸，因为 GCV的上级细胞毒性及其独特的作用机制。基因治疗的主要局限性是低 因此，所有的基因治疗方法都必须有一种机制， 杀死非转基因表达（旁观者）细胞。HSV-TK/GCV依赖于细胞间隙连接， 在一些实施方案中，可以使用GJIC将细胞毒性三磷酸从表达HSV-TK的细胞转移到旁观者细胞。在 在前一个资助期，我们根据以下作用机制评估了药理学调节： GCV与增加GJIC以增强HSV-TK/GCV治疗。结果表明 药理学调节（用核糖核苷酸还原酶抑制剂dFdCyd或羟基脲） 比增强GJIC更有效。此外，在人肿瘤异种移植的裸鼠模型中， 只有10%到50%的细胞表达HSV-TK，我们证明GCV和 单独的药理学调节剂可以抑制肿瘤生长。然而，GCV和 调节剂产生强烈的肿瘤生长延迟，具有一些完全消退。新的结果表明， HSV-TK/GCV和胞嘧啶脱氨酶（CD）/5- 氟胞嘧啶（5-FC），一种产生抗癌药物5-氟尿嘧啶的自杀基因模型。另外我们 证明了这种双自杀基因疗法的顺序给药的重要性， 协同杀伤我们将通过测定小鼠前列腺癌模型中的 药物测序对临床试验准备的影响。此外，我们建议进行机理研究 旨在阐明HSV-TK/GCV单独诱导的DMA损伤的类型和频率及其影响 与dFdCyd，hydroxyurea或CD/5-FC的调节，以及参与修复的途径， 损害我们将利用人类肿瘤细胞的基因操作以及酵母基因缺失模型 来鉴定对DNA损伤和修复有重要意义的基因。结果将帮助我们 优化目前的基因治疗方案，以及启动新的方法，以获得更大的疗效。

Suicide gene therapy is an attractive approach to treatment of cancer because it is more selective than traditional cancer chemotherapy. We have focused on the herpes simplex virus thymidine kinase (HSV-TK), the initial activator of the antiviral drug ganciclovir (GCV) to its cytotoxic triphosphate, because of the superior cytotoxicity of GCV and its unique mechanism of action. The major limitation of gene therapy is low transfer of the suicide gene to tumor cells, and thus all gene therapy approaches must have a mechanism for killing non-transgene-expressing (bystander) cells. HSV-TK/GCV relies on gap junctional intercellular communication (GJIC) to transfer the cytotoxic triphosphate from HSV-TK-expressing to bystander cells. In the previous funding period, we evaluated pharmacologic modulation, based on the mechanism of action for GCV, vs. increased GJIC to enhance therapy with HSV-TK/GCV. The results demonstrated that pharmacologic modulation (with ribonucleotide reductase inhibitors dFdCyd or hydroxyurea) was more efficacious than enhancing GJIC. Furthermore, in a nude mouse model with human tumor xenografts in which only 10% to 50% of the cells expressed HSV-TK, we demonstrated that neither GCV nor either pharmacologic modulator alone could inhibit tumor growth. However, the combination of GCV and modulator produced strong tumor growth delay with some complete regressions. New results demonstrate a novel mechanism for the synergistic bystander killing with HSV-TK/GCV and cytosine deaminase (CD)/5- flucytosine (5-FC), a suicide gene model that produces the anticancer drug 5-fluorouracil. In addition, we demonstrate the importance of sequential drug administration with this double suicide gene therapy for synergistic killing. We will extend these results in murine models of prostate cancer through determining the impact of drug sequencing in preparation for clinical trials. In addition, we propose mechanistic studies designed to elucidate the type and frquency of DMA damage induced by HSV-TK/GCV alone and the impact of modulation with dFdCyd, hydroxyurea or CD/5-FC, as well as the pathways involved in repair of this damage. We will utilize genetic manipulation of human tumor cells as well as a yeast genetic deletion model to identify genes important for DNA damage and repair with these therapeutics. The results will aid us in optimizing current gene therapy protocols as well as initiate novel approaches for greater efficacy.