Engineered 3-D Human Liver Tissue for Hepatitis Studies

用于肝炎研究的工程 3D 人类肝脏组织

• 批准号: 6803105
• 负责人: Brian A Naughton
• 金额: $ 29.99万
• 依托单位: REGENEMED, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2006-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6803105
• 关键词: bioengineering /biomedical engineering; biomarker; clinical research; disease /disorder model; hepatitis; hepatitis C virus; hepatitis D virus; immunocytochemistry; liver infection; model design /development; northern blottings; organ culture; polymerase chain reaction; technology /technique development; tissue engineering; transfection; virus replication; western blottings

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).

描述（由申请人提供）：丙型肝炎病毒（HCV）是导致肝病的主要原因，目前的治疗方法对此还不够。全球范围内，有 1.7 亿人感染 HCV，并且根据未来 20 年内死亡率可能增加两倍，预计该疾病在几年内杀死的人数将超过艾滋病。肝脏的HCV感染进展为肝硬化，随后是肝细胞癌，最终导致肝功能衰竭，并且是肝移植的主要原因。此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。目前的护理标准采用干扰素-a 和利巴韦林的组合，但只有一半的治疗患者表现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疗法的一个关键先决条件是能够支持有效的真实病毒复制的便捷模型系统的可用性。不幸的是，迄今为止唯一的非人类动物模型是黑猩猩，以及具有多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、非生理性和低复制水平分别对其在快速有效的药物发现和开发中的有用性造成了巨大的实际限制。能够支持病毒感染的工程人类肝组织的可用性相对便宜、方便，并且是评估新型抗病毒疗法以及研究 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 3 维肝组织领域的突破性专利技术，将其打造为分子病毒学和抗病毒药物开发的新平台。这项技术成功的关键包括其可扩展性、可重复性以及产生肝组织的既定能力，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能够进行高水平复制的高效肝炎病毒，即丁型肝炎病毒（HDV），并且有高度敏感和特异性的检测试剂可供使用。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项核心技术的潜力。工程化肝组织将接种 HDV 或 HCV 感染性血清，或用感染性克隆转录的 RNA 转染。免疫组织化学、免疫印迹分析、通过链特异性RNA印迹进行RNA基因组复制、通过连续传代从头产生病毒释放以及对培养基上清液的定量PCR分析将确定是否发生感染和复制。该提案中详细介绍的项目旨在测试上述假设，并将结果转化为一个有价值的新模型系统，用于研究病毒性肝炎、生成用于研究的病毒和开发抗病毒药物。未来的适应症包括一种独特且宝贵的试剂，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，例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）。