Developing a renewable and dissectible human liver for the study of HBV/HCV infection

项目摘要 HBV和HCV感染是全球慢性肝病的主要原因之一。在美国， 据估计，目前有超过85万人感染HBV，超过240万人感染HCV。 尽管有高效的HBV疫苗和HCV治疗， 与HBV和HCV相关的感染率随着现有感染的个体进入 更晚期的阶段，包括纤维化、肝硬化和HCC。此外，HCV感染者，即使 治愈病毒血症，仍然处于晚期肝病的显著升高的风险中。由于它们的共同模式 根据传播途径和流行病学特点，HBV和HCV在流行区患者中经常共存， 感染风险高的受试者。合并感染通常比单一感染更复杂， 加速疾病进展和复杂的病毒相互作用。 尽管HBV和HCV感染的严重性，但它们导致肝脏疾病的机制， 合并感染如何导致并发症的严重程度和风险增加，以及HBV和HCV如何相互作用， 在HCV治愈后可能导致HBV再活化的合并感染患者的肝脏仍然很不清楚。 这部分源于缺乏相关的人类模型来概括关键的疾病表型， 详细的机制研究可以回答这些问题。 本提案的主要研究目标是建立一种新型的、高保真的多细胞体外肝脏 以弥补上述差距。在这个系统中，我们计划共培养人类多能干细胞 （hPSC）衍生的肝细胞、肝星状细胞、巨噬细胞和内皮细胞。不同于现有 传统的细胞培养模型，我们建议创建一个创新的三维平台，模仿 肝脏的多细胞结构和微环境，通过以类似的配置培养这四种细胞类型 肝脏的体内空间组织、血液动力学和细胞间相互作用。为了区分细胞类型- 针对特定的反应，我们将开发纯化单个细胞类型的方法用于下游分析。 通过这个多细胞培养平台，我们将追求我们的长期目标， 在这个新颖的平台的独特功能，以解决问题，不能充分回答任何 现有的体外人类相关系统。这些包括疾病持续进展的机制 HCV感染治愈后，HBV/HCV合并感染加速疾病进展， HBV/HCV之间的相互作用，导致HCV感染治愈后HBV再活化（目的1）。在Aim中 2、我们将联合收割机与基因编辑相结合，了解遗传变异如何影响疾病进程，并 HBV和HCV感染后的治疗。这些研究不仅将提供新的见解， 发展有效的诊断和治疗HBV/HCV感染，但也导致技术进步 这为未来专注于其他肝脏疾病的研究开辟了新的途径。

Project Summary HBV and HCV infections are among the leading causes of chronic liver disease worldwide. In the United States, it is estimated that over 850 thousand people are currently infected with HBV and more than 2.4 million for HCV. Despite the availability of highly effective HBV vaccine and HCV treatment, the mortality and the burden associated with HBV and HCV are nevertheless increasing as individuals with existing infections advance into more advanced stages including fibrosis, cirrhosis, and HCC. Moreover, HCV-infected patients, even though cured of viremia, remain at a significantly elevated risk for advanced liver diseases. Due to their shared modes of transmission and epidemiological features, HBV and HCV frequently coexist in patients in endemic areas or among subjects at high risk of infection. Coinfection is usually more complex than monoinfection, leading to an accelerated disease progression and complicated viral interaction for their treatment. Despite the severity of HBV and HCV infections, the mechanisms by which they lead to liver disease, of how coinfection causes an increased severity and risk for complications, and of how HBV and HCV interact in the liver of coinfected patients that may lead to reactivation of HBV upon the cure of HCV remains largely unclear. This stems, in part, from the lack of relevant human models that recapitulate key disease phenotypes and permit detailed mechanistic studies that could answer these questions. The primary research goal of this proposal is to establish a novel, high fidelity multicellular in vitro liver model to address the aforementioned gaps. In this system, we plan to coculture human pluripotent stem cells (hPSCs)-derived hepatocytes, hepatic stellate cells, macrophages, and endothelial cells. Unlike existing traditional cell culture models, we propose to create an innovative three dimensional platform that mimics the liver’s multicellularity and microenvironment, by culturing these four cell types in a configuration that resembles the liver’s in vivo spatial organization, its hemodynamics, and its cell-cell interaction. To distinguish cell type- specific responses, we will develop methods to purify individual cell types for downstream analysis. With this multicellular culture platform, we will pursue our long-range objectives, across which we capitalize on the unique features of this novel platform to address questions that cannot be adequately answered with any existing in vitro human-relevant system. These include the mechanisms of continued disease progression following the cure of HCV infection, an accelerated disease progression with HBV/HCV coinfection, and the interaction between HBV/HCV that leads to HBV reactivation following the cure of HCV infection (Aim 1). In Aim 2, we will combine hPSCs with gene editing to understand how genetic variants affect the course of disease and treatment following HBV and HCV infection. These studies will not only provide new insights that could inform development of effective diagnostics and treatments for HBV/HCV infection, but also lead to technology advance that open up new avenues for future studies focusing on other liver diseases.