Autophagic and Epigenetic Control of Liver-Directed Gene Therapy with Adeno-Associated Viral Vectors

我们的总体目标是详细描述独特的AAV/肝脏相互作用，并利用这一知识来提高使用rAAV载体进行肝脏导向基因治疗的效率和安全性。在这里，我们致力于两个具有重要意义的基本新方面：1)rAAV基因组转录的肝脏特异性自噬和表观遗传调控我们证明了rAAV在肝细胞中诱导自噬，这一反应是肝细胞在体外和体内有效转导所必需的。此外，我们还表明，增加肝细胞自噬的基础(生理)水平显著提高rAAV的转导效率，根据我们的初步结果，该机制可能涉及肝细胞特异性的表观遗传调节rAAV mRNA转录。在这里，我们旨在研究自噬是否激活肝细胞特异性转录因子hnf1α，导致组蛋白修饰复合体的募集，从而有助于建立转录活性的rAAV染色质状态。2)Toll样受体(Toll-like-Receptor，TLR)2介导的肝NPC对rAAVs的感知，以及在肝细胞中诱导IL-6/自噬信号，从而促进rAAV的转导。我们的初步结果表明，这种反应导致IL-6的分泌，而IL-6又对rAAV介导的肝细胞转基因表达产生积极影响。我们推测，这种促进作用涉及依赖STAT3和AMPK的肝细胞自噬的激活，也可以通过直接的HNF1α-PSTAT3相互作用来介导。在这里，我们的目标是研究这一假说，并破译各自的途径和机制。

Our overall goal is to characterize in detail the unique AAV/liver interaction and to use this knowledge to improve the efficiency and the safety of liver-directed gene therapy with rAAV vectors. Here, we aim to focus on two fundamental new aspects with major implication: I) Liver-specific autophagic and epigenetic regulation of rAAV genome transcriptionWe demonstrated that rAAV induce autophagy in hepatocytes and that this response is required for efficient transduction of hepatocytes in vitro and in vivo. Furthermore, we showed that increasing the basal (physiological) level of autophagy in hepatocytes markedly improves rAAV transduction efficiency by a mechanism which - based on our preliminary results – likely involves hepatocyte-specific epigenetic regulation of rAAV mRNA transcription. Here, we aim to investigate whether autophagy activates hepatocyte-specific transcription factor HNF1α leading to recruitment of histone modifying complexes which in turn contribute to the establishment of a transcriptionally active rAAV chromatin state. II) Toll-like-receptor (TLR) 2-mediated sensing of rAAVs by liver NPC and induction of IL-6/autophagy signaling in hepatocytes which promotes rAAV transductionWe identified TLR2 as sensor of AAV capsids in human non-parenchymal liver cells. As shown in our preliminary results, this response leads to secretion of IL-6 which in turn positively influences rAAV-mediated transgene expression in hepatocytes. We hypothesize that this promoting effect involves STAT3- and AMPK-dependent activation of hepatocellular autophagy and can also be mediated through a direct HNF1α-pSTAT3 interaction. Here, we aim to investigate this hypothesis and decipher the respective pathways and mechanisms.