Enhancing Suicide Gene Therapy Through Mechanism-Based Approaches

描述（由申请人提供）：自杀基因治疗是一种有吸引力的癌症治疗方法，因为它比传统的癌症化疗更具选择性。由于更昔洛韦（GCV）的上级细胞毒性和其独特的作用机制，我们集中研究了单纯疱疹病毒胸苷激酶（HSV-TK），它是抗病毒药物更昔洛韦（GCV）对其细胞毒性三磷酸的初始激活剂。基因治疗的主要限制是自杀基因向肿瘤细胞的转移较低，因此所有基因治疗方法必须具有杀死非转基因表达（旁观者）细胞的机制。HSV-TK/GCV依赖于间隙连接细胞间通讯（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。在上一个资助期，我们根据GCV的作用机制评价了药理学调节与增加GJIC以增强HSV-TK/GCV治疗的对比。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，在裸鼠模型中，只有10%至50%的细胞表达HSV-TK的人肿瘤异种移植物，我们证明，无论是GCV或单独的药理学调节剂可以抑制肿瘤生长。然而，GCV和调节剂的组合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）协同旁观者杀伤的新机制，产生抗癌药物5-氟尿嘧啶的自杀基因模型。此外，我们证明了顺序给药的重要性，这种双自杀基因治疗的协同杀伤。我们将通过确定药物测序在临床试验准备中的影响，在前列腺癌的小鼠模型中扩展这些结果。此外，我们提出了机制研究，旨在阐明的类型和频率的DNA损伤诱导的HSV-TK/GCV单独和调制的影响与dFdCyd，羟基脲或CD/5-FC，以及参与修复这种损伤的途径。我们将利用人类肿瘤细胞的遗传操作以及酵母基因缺失模型来鉴定对DNA损伤和修复具有重要意义的基因。这些结果将有助于我们优化目前的基因治疗方案，并启动新的方法以获得更大的疗效。公共卫生相关性：本项目将利用从阐明自杀基因疗法杀死癌细胞的机制中获得的知识，开发新的方法来增强抗肿瘤活性，同时减轻正常组织的毒性。这些新方法在细胞培养和动物模型中的成功测试将证明这些方案特别适用于前列腺癌患者，并且可能对其他肿瘤具有更广泛的适用性。

DESCRIPTION (provided by applicant): 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 DNA 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. PUBLIC HEALTH RELEVANCE: This project will utilize knowledge gained from elucidating the mechanisms by which suicide gene therapy kills cancer cells to develop novel approaches to enhance antitumor activity while lessening normal tissue toxicity. Successful testing of these new approaches both in cell culture and in animal models as proposed will justify application of these regimens specifically in men with prostate cancer, and it may have wider applicability to other tumors.