Targeting AAV vectors to cell types involved in alcohol-induced liver injury

项目总结/摘要 酒精性肝病是一个主要的临床挑战，因为它与严重的并发症有关，如肝脏 肝硬化和酒精性肝炎，死亡率很高。我们对潜在机制的理解 酒精性肝病仍在发展;然而，以前的研究已经确定了参与肝脏疾病的细胞。 包括肌成纤维细胞、巨噬细胞和卵圆细胞（称为小管细胞）。 人类的反应）。由于它们独特而重要的功能，这些细胞类型中的每一种都是有希望的。 治疗靶点巨噬细胞启动和促进肝脏炎症并激活肝星状细胞， 这导致形成分泌胶原的肌成纤维细胞和肝纤维化和肝硬化。其他类型的 巨噬细胞解决纤维化和卵圆细胞可以产生新的肝细胞，虽然卵圆细胞积累， 并不能预防酒精性肝炎患者的肝功能衰竭。向这些细胞有效地递送基因 类型将有助于抑制或激活这些功能，这将在研究中有许多应用 和酒精性肝病的治疗。为了实现这一总体目标，我们的目标是针对非整合无毒 腺相关病毒（AAV）载体至（1）肌成纤维细胞，（2）巨噬细胞亚群，即，枯否细胞和前- 炎性和抗炎性浸润巨噬细胞，和（3）体内卵圆细胞。为此，我们成立了一个 合作，结合我们在AAV载体工程和细胞和分子生物学的专业知识， 肝损伤和再生。为了促进特定的实验和支持临床翻译，我们不会 AAV载体不仅靶向这些细胞类型中的每一种，而且使它们从每一种其它器官和细胞类型中去靶向 使用结合了最先进的AAV衣壳进化技术的工作流程， 酒精性肝病模型，基于下一代测序的载体生物分布分析， 载体基因表达靶向和脱靶调控。为了证明新合成的 AAV衣壳，我们将使用它们来确定哪种靶细胞类型最容易在体内感染 重编程为肝细胞。我们假设，卵圆细胞可以最有效地诱导成为 肝细胞，因为它们来源于密切相关的胆管细胞或肝细胞本身。因此在 除了为酒精性肝损伤的研究提供有效和精确的工具外， 可能为肝衰竭和其他危及生命的并发症的新疗法创造机会。 酒精性肝病

Project Summary/Abstract Alcoholic liver disease is a major clinical challenge because it is associated with severe complications like liver cirrhosis and alcoholic hepatitis, which carry a high mortality. Our understanding of the mechanisms underlying alcoholic liver disease is still evolving; however, previous research has identified cells involved in the liver's response to alcohol-induced injury, including myofibroblasts, macrophages and oval cells (called ductular reaction in humans). Because of their unique and important functions, each of these cell types is a promising therapeutic target. Macrophages initiate and promote liver inflammation and activate hepatic stellate cells, which leads to the formation of collagen-secreting myofibroblasts and liver fibrosis and cirrhosis. Other types of macrophages resolve fibrosis and oval cells may produce new hepatocytes, although oval cell accumulation does not appear to prevent liver failure in patients with alcoholic hepatitis. Efficient gene delivery to these cell types would facilitate inhibiting or activating these functions, which would have many applications in research and therapy of alcoholic liver disease. To achieve this overall goal, we aim to target nonintegrating nontoxic adenoassociated viral (AAV) vectors to (1) myofibroblasts, (2) macrophage subsets, i.e., Kupffer cells and pro- inflammatory and anti-inflammatory infiltrating macrophages, and (3) oval cells in vivo. For this, we formed a collaboration that combines our expertise in AAV vector engineering and the cellular and molecular biology of liver injury and regeneration. To facilitate specific experimentation and support clinical translation, we will not only target AAV vectors to each of these cell types but also detarget them from every other organ and cell type in the body using a workflow that combines state-of-the-art AAV capsid evolution technology, faithful mouse models of alcoholic liver disease, next-generation sequencing-based analysis of vector biodistribution and on- target and off-target regulation of vector gene expression. To demonstrate the efficacy of the new synthetic AAV capsids, we will use them to determine which of the targeted cell types is most susceptible to in vivo reprogramming into hepatocytes. We hypothesize that oval cells can be most efficiently induced to become hepatocytes because they derive from closely related cholangiocytes or hepatocytes themselves. Therefore, in addition to providing efficient and precise tools for studies of alcohol-induced liver injury, the proposed project may establish opportunities for new therapies for liver failure and other life-threatening complications of alcoholic liver disease.