Mitochondrially Targeted p53 DBD for Treatment of Ovarian Cancer

 描述(申请人提供)：卵巢癌仍然是最致命的妇科恶性肿瘤，69%的患者死于他们的疾病。卵巢癌的异质性是一项艰巨的挑战，这表明几乎可以肯定的是，疾病治疗需要靶向多种生物学途径。然而，高级别浆液性癌(HGSC)是最常见的卵巢癌，在所有病例中，高级别浆液性癌(HGSC)中有96%发生P53肿瘤抑制基因的突变/功能丧失。虽然使用P53的许多工作都集中在它作为细胞核转录因子的作用上，但这一提议将利用P53对线粒体具有的直接细胞抑制作用。P53‘S在线粒体上的快速作用代表了执行P53死亡信号的最短路径，而P53死亡信号触发了细胞凋亡。这项提议的目标是利用线粒体上P53内在的、直接的凋亡途径，用于卵巢癌的治疗。与野生型P53不同，线粒体靶向的P53结构域从未被尝试用于卵巢癌的治疗。我们建议：A)用高度有效的线粒体靶向信号(MTS)重新设计P53的一个小的单体结构域，这种信号可以杀死任何癌细胞，无论P53状态或基因如何，并绕过癌细胞中的主要负面影响，以及B)使用聚合WSLP递送系统(目前正在临床试验中)有效地递送到细胞。我们的中心假设是，重组后的线粒体靶向 具有成熟的给药方法的P53经腹膜腔给药将有效对抗卵巢癌。使用由基因编码的线粒体靶向蛋白而不是细胞毒剂的优点是能够整合该蛋白的癌症特异性表达的启动子。归根结底，纠正P53途径和激活细胞凋亡可能是一种通用的方法：将功能性的、线粒体靶向的单体P53重新引入癌细胞将起到“大锤”的作用，在任何情况下都是有效的(与遗传学或癌症发展的途径无关)。目的1：设计一种新型的细胞凋亡基因治疗方法(DBD-MTS)，它以p53的DBD为基础，辅以最佳的线粒体靶向信号(MTS)，由肿瘤特异性启动子驱动，能够激活固有的细胞凋亡。目的：分别检测DBD-MTS对不同P53基因表达的卵巢癌细胞株的凋亡潜能，并与标准的卡铂和紫杉醇(CPTX)联合应用。目的：将目前用于临床试验的水溶性脂聚合物(WSLP)单独和与卡铂和紫杉醇(CPTX)联合应用于我们的新的同种原位转移小鼠卵巢癌模型(由我们的合作者开发)中，通过腹腔注射的方式传递DBD-MTS。如果成功，我们预计这项工作将推进到为被诊断为晚期(&gt；III)或复发的高级别浆液性卵巢癌的妇女启动的新的临床试验，并推进基本上未被探索的线粒体靶向p53基因治疗。

 DESCRIPTION (provided by applicant): Ovarian cancer continues to be the most lethal gynecological malignancy with 69% of patients succumbing to their disease. Heterogeneity of ovarian cancer is a formidable challenge, suggesting that targeting multiple biological pathways will almost certainly be required for disease treatment. However, mutations/loss of function in p53 tumor suppressor gene occurs in >96% of all cases of high-grade serous carcinomas (HGSC), the most common form of ovarian cancer. While much work using p53 has focused on its effects as a transcription factor in the nucleus, this proposal will exploit the direct, cell-klling effect that p53 has at the mitochondria. p53's rapid effects at the mitochondria represent the shortest pathway for executing p53 death signaling, which triggers apoptosis. The goal of this proposal is to exploit the p53 intrinsic, direct apoptotic pathway at the mitochondria for use in ovarian cancer therapy. Mitochondrially targeted domains of p53 have never been attempted for ovarian cancer therapy, unlike wild-type p53. We propose to: A) re-engineer a small, monomeric domain of p53 with a mitochondrial targeting signal (MTS) that is highly potent, that kills any cancer cell regardless of p53 status or genetics, and bypasses the dominant negative effect in cancer cells, and B) use a polymeric WSLP delivery system (currently in clinical trials) for effective delivery to cells. Our central hypothesis is that re-engineered, mitochondrially targeted p53 with a proven delivery method administered intraperitoneally will be effective against ovarian cancer. The advantage of using a mitochondrial targeted protein encoded by a gene, rather than a cytotoxic agent [21] is the ability to incorporate a promoter for cancer specific expression of that protein. Ultimately, correction of the p53 pathway and activation of apoptosis may be a universal approach: functional, mitochondrially targeted monomeric p53 re-introduced into cancer cells would act as a "sledgehammer," effective under any circumstances (regardless of genetics or the pathway upon which the cancer develops). Three aims are proposed: Aim 1: Design a novel apoptotic gene therapy construct (called DBD-MTS) based on the DBD of p53, coupled to an optimal mitochondrial targeting signal (MTS), driven by a cancer-specific promoter, capable of activating intrinsic apoptosis. Aim 2: Determine the apoptotic potential of DBD-MTS in ovarian cancer cell lines with varying p53 status, individually, and in combo with standard of care, carboplatin and paclitaxel (CPTX). Aim 3: Deliver DBD-MTS using water soluble lipopolymer (WSLP) currently being used in clinical trials by intraperitoneal injection, ino our new syngeneic orthotopic metastatic mouse ovarian cancer model (developed by our collaborator), alone and in combination with carboplatin and paclitaxel (CPTX). If successful, we anticipate this work to advance to a new clinical trial initiated for women diagnosed with late stage (>III) or recurrent high grade serous ovarian cancer and advance the largely unexplored area of mitochondrially targeted p53 gene therapy.