Mitochondrially Targeted p53 DBD for Treatment of Ovarian Cancer

 描述（由申请人提供）：卵巢癌仍然是最致命的妇科恶性肿瘤，69%的患者死于这种疾病。卵巢癌的异质性是一个巨大的挑战，这表明靶向多种生物学途径几乎肯定是疾病治疗所必需的。然而，p53肿瘤抑制基因的突变/功能丧失发生在>96%的所有高级别浆液性癌（HGSC）病例中，HGSC是卵巢癌的最常见形式。虽然使用p53的许多工作都集中在其作为细胞核中的转录因子的作用上，但这项提议将利用p53在线粒体中的直接细胞杀伤作用。p53对线粒体的快速作用代表了执行p53死亡信号传导的最短途径，其触发细胞凋亡。该建议的目标是利用线粒体中的p53内在的直接凋亡途径用于卵巢癌治疗。与野生型p53不同，p53的线粒体靶向结构域从未尝试用于卵巢癌治疗。我们建议：A）用高度有效的线粒体靶向信号（MTS）重新工程化p53的小的单体结构域，其杀死任何癌细胞而不管p53状态或遗传学，并且绕过癌细胞中的显性负效应，和B）使用聚合物WSLP递送系统（目前在临床试验中）有效递送至细胞。我们的核心假设是，重新设计， 采用经过验证的递送方法腹腔内给予p53将有效治疗卵巢癌。使用由基因编码的线粒体靶向蛋白而不是细胞毒性剂的优点是能够掺入用于该蛋白的癌症特异性表达的启动子。最终，p53通路的校正和细胞凋亡的激活可能是一种通用的方法：功能性的、靶向的单体p53重新引入癌细胞将作为一个“大锤”，在任何情况下都有效（无论遗传学或癌症发展的途径如何）。提出了三个目标：目标1：基于p53的DBD设计一种新的凋亡基因治疗构建体（称为DBD-MTS），其与由癌症特异性启动子驱动的最佳线粒体靶向信号（MTS）偶联，能够激活内在凋亡。目标二：确定DBD-MTS在具有不同p53状态的卵巢癌细胞系中单独以及与标准护理、卡铂和紫杉醇（CPTX）组合的凋亡潜力。目标3：使用水溶性脂质聚合物（WSLP）通过腹膜内注射将DBD-MTS递送到我们新的同基因原位转移性小鼠卵巢癌模型（由我们的合作者开发）中，单独使用以及与卡铂和紫杉醇（CPTX）联合使用。如果成功，我们预计这项工作将推进到一项针对诊断为晚期（>III）或复发性高级别浆液性卵巢癌的女性的新临床试验，并推进大部分未探索的卵巢靶向p53基因治疗领域。

 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.