Human Hepatocyte and Discovery Core

项目总结/摘要 血友病A的基因治疗有望实现持久治愈。腺相关病毒（AAV）基因 将凝血因子FVIII转移到男性的肝脏可以实现治疗校正，但这并不持久。 临床试验中肝毒性和FVIII表达下降的原因尚不清楚。AAV疗法 目前正在研究的靶向肝细胞。然而，人们对这一问题的理解有限。 载体和肝细胞之间的相互作用以及FVIII表达和免疫应答的影响。 核心B将提供原代人肝细胞模型，以解决这些基本和机制问题 AAV和FVIII的生物学。这个提议的核心假设是， AAV和FVIII生物学特性的改变限制了治疗性表达的持久性并造成严重的安全性问题。的 Core B（人肝细胞和Discovery Core）的目标是提供三种原代人肝细胞 模型来研究AAV载体和FVIII生物学。第一个模型是原代肝细胞培养物， 肝细胞以二维形式发挥功能。这些将用于体外研究。第二种模式是 嵌合小鼠，其肝脏用原代人肝细胞高度再填充。这些将用于在 体内AAV研究。第三种模型是将造血干细胞和肝脏双重移植到小鼠体内， 肝脏中人体免疫细胞的体内研究。核心B将向研究者提供这些服务，以解决 FVIII生物学、血友病基因治疗、肝脏定向基因转移和 AAV载体的分子和免疫生物学。

PROJECT SUMMARY/ABSTRACT Gene therapy for hemophilia A holds promise to accomplish a lasting cure. Adeno-associated viral (AAV) gene transfer of clotting factor FVIII to the livers of males can achieve therapeutic correction but this is not sustained. The reasons for hepatotoxicity and the decline in FVIII expression in clinical trials are unclear. The AAV therapies currently under investigation target hepatocytes in the liver. There is, however, a limited understanding of the interactions between the vector and hepatocytes as well as effect of FVIII expression and immune responses. Core B will provide primary human hepatocyte models to address these basic and mechanistic questions related to the biology of AAV and FVIII. The central hypothesis of this proposal is that multiple interconnected features of AAV and FVIII biology limit durability of therapeutic expression and pose serious safety concerns. The objectives of Core B, the Human Hepatocyte and Discovery Core, is to provide three primary human hepatocytes models to study AAV vector and FVIII biology. The first model is primary hepatocyte cultures that retain stable hepatocyte functions in 2-dimensional format. These will be used for in vitro studies. The second model is chimeric mice whose livers are highly repopulated with primary human hepatocytes. These will be used for in vivo AAV studies. And the third model is mice doubly engrafted with hematopoietic stem cells and livers for in vivo studies of human immune cells in the liver. Core B will provide these services to investigators to address major unanswered questions in FVIII biology, gene therapy for hemophilia, liver-directed gene transfer, and molecular and immunobiology of AAV vectors.