Engineered 3-D Human Liver Tissue for Hepatitis Studies

描述（由申请人提供）：丙型肝炎病毒（HCV）是目前治疗不足的肝脏疾病的主要原因。全世界有1.7亿人感染HCV，根据未来20年死亡率可能增加两倍的情况，预计在几年内这种疾病造成的死亡人数将超过艾滋病。肝的HCV感染进展为肝硬化，随后是肝细胞癌，最终是肝衰竭，并且是肝移植的主要原因。这类感染已成为一个日益受到重视的公共卫生问题，特别是因为目前的治疗方式不足。目前的标准治疗采用干扰素和利巴韦林的组合，但只有一半的患者治疗显示出足够的抗病毒反应。因此，显然存在对更好疗法的需求和市场。 识别和开发更好的病毒性肝炎治疗方法的一个关键前提是能够支持有效真实病毒复制的方便模型系统的可用性。不幸的是，到目前为止，唯一的非人类动物模型是黑猩猩和具有各种严重局限性的免疫缺陷异种移植小鼠模型。与这些动物模型相关的巨大费用以及非生理性和低复制水平分别对它们用于快速和有效的药物发现和开发的有用性施加了很大的实际限制。能够支持病毒感染的工程化人肝组织的可用性相对便宜，方便，并且是评价新型抗病毒疗法和研究HCV和其他病毒相关病理学的理想选择。 该提案旨在利用工程化三维肝脏组织领域的专利突破技术，为分子病毒学和抗病毒开发提供一个新的平台。这项技术成功的关键包括其可扩展性，可重复性和产生肝脏组织的能力远远超过以前的器官工程尝试。我们试图确定工程组织是否可以感染一种有效的肝炎病毒，能够高水平复制，并具有高度敏感性和特异性的检测试剂，即丁型肝炎病毒（HDV）。然后，我们将通过确定这些组织支持的丙型肝炎病毒（HCV）感染和复制水平来扩展这一核心技术的潜力。工程化肝组织将用HDV或HCV感染性血清接种，或用从感染性克隆转录的RNA转染。免疫组织化学、免疫印迹分析、通过链特异性北方印迹进行的RNA基因组复制、通过连续传代重新产生的病毒释放以及培养基上清液的定量PCR分析将确定是否发生感染和复制。本提案中详细介绍的项目旨在测试上述假设，并将结果转化为研究病毒性肝炎的有价值的新模型系统，生成用于研究用途的病毒和开发抗病毒药物。未来的适应症包括一种独特而宝贵的试剂，用于研究这些病毒生命周期的许多关键方面，其相关的发病机制，以及抗病毒治疗的新方法，包括确定肝细胞亚群的感染潜力，以及其他肝脏疾病，如非酒精性脂肪性肝炎（NASH），酒精性肝炎，肝纤维化和肝细胞癌（HCC）。

DESCRIPTION (provided by applicant): The Hepatitis C virus (HCV) is a leading cause of liver disease for which current treatments are inadequate. Worldwide, 170 million are infected with HCV and the disease is projected to kill more people than AIDS within a few years based on a death rate that is likely to triple in the next 20 years. HCV infection of the liver progresses to cirrhosis followed by hepatocellular carcinoma and ultimately liver failure, and is the leading reason for liver transplantation. Such infections have become an increasingly appreciated public health problem, especially because current treatment modalities are inadequate. The current standard of care employs a combination of interferon-a and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus, clearly there is a need and market for better therapies. A critical prerequisite for identifying and developing better therapies for viral hepatitis is the availability of convenient model systems capable of supporting efficient authentic viral replication. Unfortunately, to date the only non-human animal models are the chimpanzee and, with a variety of severe limitations, an immunodeficient xenotransplant mouse model. The great expense, and non-physiologic and low replication levels, respectively, associated with these animal models place great practical limits on their usefulness for rapid and efficient drug discovery and development. The availability of engineered human liver tissue capable of supporting viral infection would be relatively inexpensive, convenient, and ideal for the evaluation of novel antiviral therapies and the study of HCV and other virus-related pathology. This proposal seeks to leverage patented breakthrough technology in the field of engineered, 3-dimensional liver tissue into a novel platform for molecular virology and antiviral development. Keys to the success of this technology include its scalability, reproducibility, and an established ability to yield liver tissues far exceeding previous attempts at organ engineering. We seek to determine whether the engineered tissues can be infected with an efficient hepatitis virus capable of high-level replication and for which highly sensitive and specific detection reagents are available, namely hepatitis delta virus (HDV). We will then extend the potential of this core technology by determining the level of hepatitis C virus (HCV) infection and replication supported by these tissues. Engineered liver tissues will be inoculated with HDV or HCV infectious serum, or transfected with RNA transcribed from an infectious clone. Immunohistochemistry, immunoblot analysis, RNA genome replication by strand-specific northern blots, and de novo produced virus release by serial passaging and quantitative PCR analyses of the media supernatant will determine if infection and replication have occurred. The project detailed in this proposal is designed to test the above hypotheses and translate the results into a valuable new model system for studying viral hepatitis, generating virus for research use and the development of antivirals. Future indications include a unique and invaluable reagent for studying many key aspects of these viral life cycles, their associated pathogenesis, and novel approaches to antiviral therapy, including determination of the infection potential of subsets of hepatic cells, as well as other liver diseases such as non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, hepatic fibrosis, and hepatocellular cancer (HCC).