New Hepatocyte Model Systems to study Hepatitis B Virus Infection

描述(申请人提供)：在世界范围内，乙肝病毒感染是最常见的病毒性肝炎，已感染超过20亿人，慢性感染超过4亿人，使他们患上肝硬变和肝细胞癌的风险增加。在美国，有100多万人患有慢性乙肝。临床治疗的目标是抑制病毒复制，但病毒能够以一种称为ccccDNA的非复制共价闭合环状形式存在，并有可能重新激活。 免疫抑制或衰老。因此，乙肝病毒在慢性乙肝感染中的根除和治愈是具有挑战性的，也是罕见的。开发新的乙肝治疗方法的困难是由于缺乏良好的模型系统。目前研究乙肝的模型系统一直是肝癌细胞系，在该细胞系中，乙肝病毒瞬时或稳定地过度表达，以产生所有病毒基因产物并保持复制。然而，这些模型以非生理学的方式过度表达HBV病毒，并且不能真实地概括成人肝细胞的表型或功能，因为它们经历了各种遗传和代谢变化。因此，病毒进入因素以及病毒复制的调控过程一直知之甚少。这阻碍了实现病毒控制和根除的治疗策略的发展。这项建议建立在新的工具和技术的基础上，能够长期培养具有代谢功能的原代人类肝细胞，并建立多能干细胞来源的肝细胞样细胞。这两项创新都使探索 乙肝病毒进入、病毒复制和cccDNA状态的决定因素，将为更好地了解乙肝病毒生物学和开发新的靶向治疗提供新的机会。建立一个允许乙肝病毒感染的培养模型系统，并使具有代谢功能的原代人肝细胞能够长期培养，为探索乙肝病毒进入、病毒复制、ccDNA形成和持续的决定因素提供了新的机会(目标1-2)。与目前的肝癌细胞株不同，一个功能性的肝细胞筛选平台将能够研究生物途径在乙肝病毒生命周期选择中所起的作用，以选择产生cccDNA还是产生活跃的乙肝病毒粒子(目标2)。然后，可以在诱导多能干细胞来源的变体中详细探索和剖析这些途径，以确定它们对肝细胞通透性、乙肝病毒发病机制和cccDNA形成的影响(目标3)。更好地了解乙肝病毒的发病机制、cccDNA的形成、病毒逃避和肝细胞自主反应的决定因素也可能具有翻译意义，因为针对慢性感染的乙肝患者有治疗选择，但无法消除cccDNA，从而导致终身感染和终身治疗的需要。

DESCRIPTION (provided by applicant): 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 persit 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.