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治疗癌症的方法，因为它比传统的癌症化疗更具选择性。单纯疱疹病毒胸苷激酶（HSV-TK）是抗病毒药物更昔洛韦（GCV）细胞毒性三磷酸的初始激活剂，由于GCV具有优异的细胞毒性及其独特的作用机制。基因治疗的主要限制是自杀基因向肿瘤细胞的转移率低，因此所有的基因治疗方法都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的细胞转移到旁细胞。在之前的资助期内，我们根据GCV的作用机制评估了药物调节与增加GJIC以增强HSV-TK/GCV治疗的对比。结果表明，药物调节（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，在人类肿瘤异种移植的裸鼠模型中，只有10%至50%的细胞表达HSV-TK，我们证明GCV或单独的药物调节剂都不能抑制肿瘤的生长。然而，GCV与调节剂联合使用可产生较强的肿瘤生长延迟，并有部分完全回归。新的研究结果证明了HSV-TK/GCV和胞嘧啶脱氨酶(CD)/5-氟胞嘧啶（5- FC）协同杀死旁观者的新机制，胞嘧啶脱氨酶是一种产生抗癌药物5-氟尿嘧啶的自杀基因模型。此外，我们证明了序贯药物管理的重要性与这种双重自杀基因治疗协同杀伤。我们将通过确定药物测序在准备临床试验中的影响，将这些结果扩展到前列腺癌的小鼠模型中。此外，我们提出了机制研究，旨在阐明HSV-TK/GCV单独诱导的DNA损伤的类型和频率，以及dFdCyd，羟基脲或CD/5-FC调制的影响，以及参与这种损伤修复的途径。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识别对这些治疗方法的DNA损伤和修复重要的基因。这一结果将有助于我们优化当前的基因治疗方案，并开创更有效的新方法。公共卫生相关性：该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，同时减少正常组织的毒性。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在前列腺癌患者中的应用是合理的，并且它可能对其他肿瘤有更广泛的适用性。

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.