New Hepatocyte Model Systems to study Hepatitis B Virus Infection

项目总结/摘要 在世界范围内，B型肝炎病毒（HBV）感染是最常见的病毒性肝炎， 超过20亿人，慢性感染超过4亿人， 肝硬化和肝癌的风险。在美国， 慢性B型肝炎患者。临床治疗的目标是抑制病毒性 但病毒能够以非复制的共价闭合环状形式存在 称为cccDNA，具有在免疫抑制或衰老时重新激活的潜力。作为 因此，慢性HBV感染中的B型肝炎病毒难以根除， 治愈是罕见的。开发新的HBV治疗方法的困难是由于缺乏良好的 模型系统目前研究B型肝炎的模型系统是肝癌细胞系， 所述HBV瞬时或稳定过表达以产生所有病毒基因产物， 保持复制。然而，这些模型以非生理方式过度表达HBV 并且不能如实地概括成体肝细胞的表型或功能， 经历了各种遗传和代谢变化。因此，病毒进入 因子以及病毒复制的调节过程一直知之甚少。 这阻碍了实现病毒控制的治疗策略的发展， 根除该提案建立在新的工具和技术基础之上， 代谢功能的原代人肝细胞和建立多能 干细胞衍生肝细胞样细胞。这两项创新都使人们能够探索 HBV进入、病毒复制和cccDNA状态的决定因素， 更好地了解HBV生物学和开发新的靶向治疗的机会。的 建立允许HBV感染的培养模型系统， 具有代谢功能的原代人肝细胞的长期培养代表了一种新的 探索HBV进入、病毒复制和cccDNA形成的决定因素的机会 和持久性（目标1-2）。与目前的肝癌细胞系相反， 筛选平台将使研究的作用，生物途径发挥在HBV病毒 生命周期对cccDNA生产与活性HBV病毒体生产的选择（目的2）。 然后可以在诱导的多能衍生物中详细探索和剖析这些途径。 干细胞衍生变异体，以确定其对肝细胞容许性、B型肝炎的影响 病毒发病机制和cccDNA形成（目的3）。更好地了解HBV发病机制， cccDNA形成、病毒逃逸和肝细胞自主反应的决定因素 也可能转化为慢性感染HBV患者的治疗选择， 可用但不能消除cccDNA，导致终身感染， 终身治疗。

PROJECT SUMMARY/ABSTRACT Worldwide, hepatitis B virus (HBV) infection is the most common viral hepatitis having infected over two billion people and chronically infecting more than 400 million, putting them at increased risk to develop cirrhosis and hepatocellular carcinoma. In the United States over one million people have chronic hepatitis B infection. Clinical therapy is targeted to the suppression of viral replication but the virus is able to persist in a nonreplicative covalently closed circular form called cccDNA, with the potential to reactivate upon immune suppression or with aging. As a consequence, the hepatitis B virus in chronic HBV infections is challenging to eradicate and cure is rare. The difficulty in developing new HBV therapies has been due to the lack of good model systems. The current model system to study hepatitis B has been hepatoma cell lines in which HBV is over expressed transiently or stably to produce all viral gene products and maintain replication. However such models over express HBV in a non-physiological manner and do not faithfully recapitulate adult hepatocyte phenotype or function as they have undergone a variety of genetic and metabolic changes. As a consequence, the viral entry factors as well as the regulatory processes of viral replication have been poorly understood. This has prevented development of therapeutic strategies that achieve viral control and eradication. This proposal builds on new tools and techniques that enable the long-term culture of metabolically functional primary human hepatocytes and the establishment of pluripotent stem cell derived hepatocyte-like cells. Both innovations enables the exploration of the determinants of HBV entry, viral replication, and the cccDNA state which will open new opportunities to better understand HBV biology and develop new targeted therapies. The establishment of a culture model system that is permissive for HBV infection and also enables the long-term culture of metabolically functional primary human hepatocytes represents a new opportunity to explore the determinants of HBV entry, viral replication, and cccDNA formation and persistence (Aims 1-2). In contrast to current hepatoma cell lines, a functional hepatocyte screening platform will enable the study of the role that biologic pathways play in the HBV viral life cycle's choice towards cccDNA production versus active HBV virion production (Aim 2). These pathways can then be explored and dissected in detail in induced pluripotent derived stem cell derived variants to determine their impact on hepatocyte permissiveness, hepatitis B virus pathogenesis and cccDNA formation (Aim 3). Better insights in HBV pathogenesis, cccDNA formation, virus evasion and the determinants of hepatocyte autonomous responses may also be translational as treatment options for chronically infected HBV patients are available but are unable to eliminate cccDNA leading to lifelong infection and requirements for lifelong treatment.