Murine Model of HCV-Associated Human Liver Cancer

描述（由申请人提供）：慢性丙型肝炎病毒（HCV）感染目前是肝细胞癌（HCC）的主要原因，HCC是一种在美国发病率迅速增加的高致死性癌症。由于缺乏HCV发病机制的小动物模型，因此HCV如何导致肝癌尚未得到很好的理解。这个跨学科，多PI项目的总体目标是完善最近开发的丙型肝炎人源化小鼠模型（AFC 8-hu小鼠），该模型同时具有嵌合的人类肝脏和功能正常的人类免疫系统，并使用该模型来阐明HCV相关HCC致癌的病毒机制。用CD 34+人造血干细胞和EpCAM+人成肝细胞和肝干细胞移植的AFC 8- hu小鼠实现约18%的肝嵌合，允许HCV感染，并且当感染HCV时发展出具有肝脏炎症的人HCV特异性T细胞应答。值得注意的是，这些小鼠的HCV感染导致显著的肝纤维化和氧化DNA损伤的证据-人类患者慢性丙型肝炎的标志。提出了三个相互关联的具体目标，以完善这一模型，并将其应用于研究的致癌机制负责HCV相关的肝癌。目的1：优化方案，以提高肝嵌合体在AFC 8-hu小鼠，并感染AFC 8-hu小鼠基因型1a HCVRNA在细胞培养中产生的重组DNA，从而提供一个系统支持HCV介导的致癌作用的反向分子病毒遗传学研究。在目标2中，在移植到AFC 8小鼠中之前，p53将在人成肝细胞中失活，以加速HCV感染小鼠中人肝癌的发展，并促进发现HCV介导的肝癌发生的新决定因素。目标3将利用反向分子遗传学策略来检验以下假设：在免疫介导的肝脏炎症和氧化应激的遗传毒性环境中，HCV对关键肿瘤抑制因子功能的破坏促进癌症，并具体评估HCV对视网膜母细胞瘤蛋白的靶向破坏和HCV核心蛋白对肿瘤抑制因子DDX 3的隔离所发挥的作用。AFC 8-hu小鼠模型的进一步发展，再加上使用感染性基因型1a H77 S病毒产生的重组cDNA，将提供一个强大的和完全独特的实验系统，其中复杂的相互作用的直接和间接的致癌机制可以阐明。这些研究的结果将显著增加目前对HCV感染如何导致HCC发展的理解，并推进新的预防和治疗策略的开发和评估，以减轻慢性丙型肝炎患者肝癌带来的相当大的发病率和死亡率负担。

DESCRIPTION (provided by applicant): Chronic hepatitis C virus (HCV) infection is now the leading cause of hepatocellular carcinoma (HCC), a highly lethal cancer with rapidly increasing incidence in the U.S. How HCV causes liver cancer is not well understood because of the lack of a small animal model for HCV pathogenesis. The overarching goal of this interdisciplinary, multi-PI project is to refine a recently developed humanized mouse model of hepatitis C (the AFC8-hu mouse) that possesses both a chimeric human liver and a functioning human immune system, and to use this model to elucidate viral mechanisms of carcinogenesis responsible for HCV-associated HCC. AFC8- hu mice engrafted with CD34+ human hematopoietic stem cells and EpCAM+ human hepatoblasts and hepatic stem cells achieve ~18% hepatic chimerism, are permissive for HCV infection, and develop a human HCV- specific T cell response with hepatic inflammation when infected with HCV. Remarkably, HCV infection of these mice results in significant hepatic fibrosis and evidence of oxidative DNA damage - hallmarks of chronic hepatitis C in human patients. Three interrelated specific aims are proposed to refine this model and to apply it to the study of the mechanisms of carcinogenesis responsible for HCV-associated HCC. Aim 1 focuses on optimizing protocols to enhance hepatic chimerism in AFC8-hu mice, and to infect AFC8-hu mice with genotype 1a HCVcc produced in cell culture from recombinant DNA, thereby providing a system supporting reverse molecular viral genetics studies of HCV-mediated carcinogenesis. In Aim 2, p53 will be inactivated in human hepatoblasts prior to transplantation into AFC8 mice to provide for accelerated development of human liver cancer in HCV-infected mice, and to facilitate discovery of novel determinants of HCV-mediated hepatocarcinogenesis. Aim 3 will utilize a reverse molecular genetics strategy to test the hypothesis that the disruption of critical tumor suppressor function by HCV promotes cancer in the genotoxic environment of immune-mediated hepatic inflammation and oxidative stress, and to specifically assess the role played by the targeted destruction of the retinoblastoma protein by HCV and sequestration of the tumor suppressor DDX3 by HCV core protein. Further development of the AFC8-hu mouse model, coupled with the use of infectious genotype 1a H77S virus produced from recombinant cDNA, will provide a robust and completely unique experimental system in which the complex interplay of direct and indirect mechanisms of carcinogenesis can be elucidated. The results of these studies will add significantly to current understanding of how HCV infection leads to the development of HCC, and advance the development and evaluation of new preventative and therapeutic strategies to lessen the considerable burden of morbidity and mortality imposed by liver cancer in patients with chronic hepatitis C.