Rad51C启动子在肿瘤基因治疗中的作用研究

Developing a method of specifically targeting malignant cells with minimal or no toxicity to normal tissues is the ultimate goal in the field of cancer therapy. Our most recent work compared the activity of Rad51C promoter in a panel of cancer cells to that in various types of normal cells. Our result reveals that on average Rad51C promoter activity in cancer cells was elevated by ~ 250 fold in comparison to normal cells. A vector bearing Rad51C promoter driving diphtheria toxin A gene specifically killed several types of cancer cells with no significant effect on normal human cells. Notably, the size of Rad51C promoter is only ~ 2 kb, which is within the insert size range that can be accommodated by most of viral vectors. The features of Rad51C promoter allow us for further study on its potential therapeutic and diagnostic applications. Therefore, in this proposal, we plan to accomplish the following aims. Aim 1: Create adeno associate virus vector, which contains Rad51C promoter driving luciferase or DTA, and test them in in vitro cell culture. Aim 2: Further examine the specificity of the Rad51C promoter in cancer diagnosis and gene therapy using xenograft model with the AAV virus.

运用特定的基因表达调控原件，使治疗基因特异性地在肿瘤细胞中表达，是实现肿瘤基因疗法可控或靶向性的有效手段。本课题组研究发现，相较于一系列正常细胞，Rad51C启动子活性在不同肿瘤细胞株中平均增强约250倍，而由Rad51C启动子介导白喉毒素A（DTA）蛋白的表达能够于体外特异性地杀伤肿瘤细胞。利用这一特性，基于Rad51C启动子具有片段长度短及在正常和肿瘤细胞中活性差异大等诸多适宜应用于基因治疗的优势，我们将在本课题的研究中将其与荧光素酶（luciferase）或DTA基因共同克隆入腺相关病毒载体中，通过建立荷瘤裸鼠模型，系统评价由Rad51C启动子介导luciferase或DTA基因表达是否具有使体内肿瘤可视化的协助诊断或治疗肿瘤的作用，以期为肿瘤的基因治疗提供全新的治疗策略。

由于在肿瘤细胞中活性增强，同源重组修复通路（HR）通常被认为是潜在有效的肿瘤治疗靶点。利用HR通路重要蛋白RAD51或RAD51C启动子在正常及肿瘤细胞中的活性差异，已有研究分别于体内外证实，由RAD51或RAD51C启动子介导的荧光素酶或白喉毒素A蛋白的表达能够特异性地靶向诊断或治疗肿瘤。然而上述启动子片段较长使其难以被克隆入病毒载体而实现高效转运，很大程度上限制了其临床应用。本研究发现，相较于其他肿瘤特异性表达基因的启动子，RAD51家族成员蛋白XRCC2启动子具有活性差异大及长毒较短等适合应用于肿瘤基因治疗的优势。本研究深入阐明了XRCC2启动子活性在肿瘤细胞中显著增强，由XRCC2启动子介导的GFP、Luciferase或DTA在肿瘤细胞中呈高表达，利用XRCC2启动子的上述特性可在裸鼠移植瘤模型中实现在体肿瘤的可视性及肿瘤治疗，该研究结果对于肿瘤诊断和治疗具有一定的指导意义。