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。在世界范围内，有1.7亿人感染了丙型肝炎病毒，根据未来20年可能增加三倍的死亡率，预计在几年内，丙型肝炎导致的死亡人数将超过艾滋病。丙型肝炎病毒感染肝脏发展为肝硬化，继而发展为肝细胞癌，最终导致肝功能衰竭，是肝移植的主要原因。这种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-a和利巴韦林的联合治疗，但只有一半的治疗患者显示出足够的抗病毒反应。因此，显然有更好的治疗方法的需求和市场。

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).