Enhancing Suicide Gene Therapy Through Mechanism-Based Approaches

描述(申请人提供)：自杀基因疗法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。单纯疱疹病毒胸苷激酶(HSV-TK)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，由于其优越的细胞毒性和独特的作用机制，我们将重点放在HSV-TK上。基因治疗的主要局限性是自杀基因对肿瘤细胞的低转移率，因此所有的基因治疗方法都必须有一个杀死非转基因表达(旁观者)细胞的机制。HSV-TK/GCV依赖缝隙连接细胞间通讯(GJIC)将表达HSV-TK的细胞毒三磷酸转移到旁观者细胞。在之前的资助期间，我们根据GCV的作用机制，与增加GJIC相比，评估了药物调节以加强HSV-TK/GCV的治疗。结果表明，药物调节(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，在裸鼠移植人肿瘤模型中，只有10%到50%的细胞表达HSV-TK，我们证明了GCV和两种药物调节剂都不能单独抑制肿瘤的生长。但联合应用GCV和调节剂后，肿瘤生长明显延缓，部分肿瘤完全消退。新的结果证明了HSV-TK/GCV和胞嘧啶脱氨酶(CD)/5-氟胞嘧啶(5-FC)协同杀伤旁观者的新机制，胞嘧啶脱氨酶/5-氟胞嘧啶(5-FC)是产生抗癌药物5-氟尿嘧啶的自杀基因模型。此外，我们证明了序贯给药与这种双自杀基因疗法协同杀伤的重要性。我们将通过确定药物排序在准备临床试验中的影响，将这些结果推广到前列腺癌的小鼠模型中。此外，我们提出了一些机制研究，旨在阐明单纯HSV-TK/GCV诱导的DNA损伤的类型和频率，以及dFdCyd、羟基脲或CD/5-FC的调节作用，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识别对DNA损伤和修复至关重要的基因，并使用这些治疗方法。这一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高的疗效。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，同时降低正常组织的毒性。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患有前列腺癌的男性，并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.