Dissecting AAV silencing in humanized mice

项目总结 肝脏已成为表达治疗性转基因的一个很有希望的靶点。 使用腺相关病毒(AAV)载体介导的递送。尽管进行了无数的临床试验 持续的进展已经确定了AAV基因治疗的几个挑战。在这 建议我们将剖析一个主要的临床障碍，例如人类肝脏中的AAV转基因沉默 以及这种现象背后的分子机制。为达致这个目标，我们首先建议 利用并表征一种新的人源化肝脏模型，它将允许精确和选择性 甲型肝炎病毒转基因沉默机制的探讨 活体内病变的人肝细胞。第二个目标将建立在最近从 我们的实验室显示AAV转基因表达是由人类沉默中心(HUSH)介导的 很复杂。我们将使用人类进一步剖析和收集这一过程中的机械性信息 甲基丙二酸血症(MMA)患者体内肝细胞。因此，MMA将作为一个 肝脏导向AAV基因治疗的概念障碍的证据。最后，我们将探索不同的 拯救AAV基因沉默的载体设计和药理学策略。

PROJECT SUMMARY The liver has emerged as a promising target for expressing therapeutic transgenes utilizing Adeno-Associated Viral (AAV) vector-mediated delivery. Despite numerous clinical trials and continued progress several challenges have been identified for AAV gene therapy. In this proposal we will dissect a major clinical hurdle, e.g. AAV transgene silencing in the human liver and molecular mechanisms underlying this phenomenon. To achieve such, we first propose to utilize and characterize a novel humanized liver model, which will allow precise and selective interrogation of the mechanisms underlying AAV transgene silencing in normal as well as diseased human hepatocytes in vivo. The second aim will build on a recent observation from our lab showing AAV transgene expression mediated by the Human Silencing Hub (HUSH) complex. We will further dissect and gather mechanistic inside on this process using human hepatocytes from methylmalonic acidemia (MMA) patients in vivo. Hence MMA will serve as a proof of concept disorder for liver directed AAV gene therapy. Finally, we will explore different vector design and pharmacological strategies to rescue AAV gene silencing.