Re-engineered Mitochondrially Targeted p53 Gene Therapy in Liver Cancer

翻译后摘要：肝细胞癌（HCC），最常见的肝癌，是第三大原因， 全球癌症死亡人数在美国，肝癌的5年生存率仅为17%，远处转移的5年生存率仅为3 转移性肝癌手术切除和肝移植是目前治疗HCC的标准， 只有约5%的肝硬化患者适合切除。有限的器官供应也严重限制了 移植包括激酶抑制剂和免疫疗法在内的新的HCC靶向药物显示出希望，但 使用靶向药物的患者将不可避免地产生耐药性。HCC的有效新疗法是 迫切需要。我们提出了一种新的，重组的p53肿瘤抑制蛋白-凋亡蛋白杂合基因， p53-Bad* 的治疗方法。野生型p53被批准用于基因治疗，作为Gendicine®用于癌症， 中国有限的成功有限的疗效可能与突变型p53的显性负效应有关， 超过野生型p53。由于核p53活性依赖于同源四聚化，癌细胞中的突变型p53 也可以与野生型p53四聚化以使其失活。突变型p53也可以具有功能获得性和其他功能。 可以对抗WT p53的作用的活性。为了克服这一限制，一种有效的p53融合蛋白， 本文提出了一种新的作用机制，它克服了显性负性抑制并引起细胞凋亡 而不管癌细胞的p53状态如何。通过将p53融合到凋亡的BH 3蛋白， 线粒体靶向信号，如Bad，这通过结合凋亡效应进一步增加凋亡 线粒体p53蛋白与促凋亡蛋白BH 3蛋白的差异。p53-Bad*（Bad的突变形式， 线粒体定位）是我们的顶级癌细胞杀伤构建体。我们假设p53基因的结合 和BH 3蛋白在线粒体，肝癌特异性驱动的一种修饰的甲胎蛋白（AFP） 启动子，将在体外（无论p53状态如何）和体内（无论p53状态如何）都有效地诱导肝癌细胞凋亡。 斑马鱼和小鼠模型。斑马鱼是研究人类癌症的理想模型系统，具有“组织学 以及斑马鱼和人类肿瘤之间的遗传相似性”，而原位异种移植小鼠模型是一种 迈向翻译的重要一步。目标1：a. p53-BH 3融合（p53-Bad*）将 定位于线粒体并在相关肝细胞癌（HCC）细胞系中引起有效的凋亡 不管细胞p53状态如何; B.使用修饰的/组合的AFP启动子将赋予肝癌细胞 特异性和杀死HCC细胞，而不是正常的人类细胞。目的2：肝特异性肝癌细胞系中p53-Bad* 的表达 启动子fabp 10a在斑马鱼中，与肝癌斑马鱼模型杂交将产生后代， 肝癌特异性表型的表达。目的3：p53-Bad*（在腺病毒中表达，具有优化的表达条件） HCC特异性启动子）将缩小HCC的原位异种移植小鼠模型中的肝肿瘤（使用AFP- 表达人HCC肝细胞，Hep 3B）。本研究的最终目的是开发一种新的、有效的 治疗全球HCC患者。

Abstract: Hepatocellular carcinoma (HCC), the most common cancer of the liver, is the third leading cause of cancer death worldwide. In the U.S., the overall 5-year survival rate for HCC is only 17% and only 3% for distantly metastasized HCC. Surgical resection and liver transplantation are the current standard of care for HCC, but only ~5% qualify for resection due to cirrhosis. Limited organ availability also severely restricts the number of transplants. New targeted agents for HCC including kinase inhibitors and immunotherapies show promise, but patients using targeted agents will inevitably develop drug resistance. Effective new therapies for HCC are urgently needed. We propose a new, re-engineered p53 tumor suppressor-apoptotic protein hybrid for gene therapy of HCC called p53-Bad*. Wild-type p53 was approved for gene therapy as Gendicine® for cancers in China with limited success. Limited efficacy may be related to the dominant negative effect that mutant p53 exerts over wt p53. As nuclear p53 activity is dependent on homo-tetramerization, mutant p53 in cancer cells can also tetramerize with wt p53 to cause its inactivation. Mutant p53 can also have gain-of-function and other activities that can counter the effects of wt p53. To overcome this limitation, a potent p53 fusion protein with novel mechanisms of action is proposed here that overcomes dominant negative inhibition and causes apoptosis regardless of the p53 status of cancer cells. By fusing p53 to an apoptotic BH3 protein with an inherent mitochondrial targeting signal, such as Bad, this increases apoptosis further by combining the apoptotic effects of mitochondrial p53 with those of pro-apoptotic BH3 proteins. p53-Bad* (a mutant version of Bad with increased mitochondrial localization) is our top cancer cell-killing construct. We hypothesize that the combination of p53 and BH3 proteins at the mitochondria, with liver cancer specificity driven by a modified alpha fetoprotein (AFP) promoter, will potently induce apoptosis in liver cancer both in vitro (regardless of p53 status), and in vivo in zebrafish and mouse models. Zebrafish are an ideal model system for studying human cancer, with “histologic and genetic similarities between zebrafish and human tumors” while an orthotopic xenograft mouse model is an important step toward translation. The following aims are proposed: Aim 1: a. p53-BH3 fusion (p53-Bad*) will localize to the mitochondria and cause potent apoptosis in relevant hepatocellular carcinoma (HCC) cell lines regardless of cellular p53 status; b. the use of modified/combination AFP promoters will confer liver cancer cell specificity and kill HCC cells and not normal human cells. Aim 2: Expression of p53-Bad* under the liver specific promoter fabp10a in zebrafish, crossed with a liver cancer zebrafish model will produce offspring with reduced expression of liver cancer specific phenotypes. Aim 3: p53-Bad* (expressed in adenovirus with the optimized HCC specific promoter) will shrink liver tumors in an orthotopic xenograft mouse model of HCC (using AFP- expressing human HCC liver cells, Hep3B). The ultimate goal of this study is the development of a new, effective therapy for treatment of HCC patients worldwide.