Dual-function vectors for in vivo gene therapy of AAT Liver disease

描述(申请人提供)：α-1抗胰蛋白酶缺乏症是一种常见的单基因疾病，其中最常见的突变“Z”等位基因(Glu342Lys)在北欧和北美出现的频率非常高。纯合子Z患者患有肺部疾病，这是由于野生型AAT缺乏正常的抗蛋白酶功能，还可能患有肝脏疾病，似乎是由于聚合和/或聚集的Z-AAT蛋白滞留在肝细胞内而引发的。表达人类Z等位基因的小鼠模型在检查分子终点方面很有用，但不能忠实地复制在严重感染患者中观察到的炎症和肝硬变。在这一应用中，我们建议利用环境挑战和人类干细胞来源的肝脏嵌合体来开发更好的AAT相关肝病模型，并进一步测试联合基因治疗/RNA抑制策略作为潜在的未来治疗方法。我们的第一个目标将使用乙醇诱导具有人类Z等位基因的小鼠的肝毒性来评估增加肝细胞癌风险的风险，并更好地了解这些肝脏中的促炎和促纤维化途径。然后，这一概念将扩展到使用人源化小鼠肝脏的研究，这些小鼠肝脏来自iPS细胞，具有正常或Z等位基因阳性的人肝干细胞。最后，这两个模型都将被用来评估基于rAAV的RNAi介导的Z-AAT mRNA下调的效果。

DESCRIPTION (provided by applicant): Alpha-1 antitrypsin deficiency is a common single-gene disorder, in which the most common mutant "Z" allele (Glu342Lys) occurs with remarkably high frequency in Northern Europeans and North Americans. Homozygous Z patients suffer from a lung disease that is due to the lack of normal antiprotease function of the wild-type AAT, and may also suffer from a liver disease, that appears to be triggered by retention of polymeric and/or aggregated Z-AAT protein within hepatocytes. A mouse model expressing the human Z-allele has utility in examining molecular endpoints but does not faithfully reproduce the inflammation and cirrhosis observed in severely affected patients. In this application, we propose to develop better models of AAT-related liver disease using environmental challenges and human stem cell derived liver chimeras, and further to test a combined gene therapy/RNA inhibition strategy as a potential future therapy. Our first aim will use an ethanol induce hepatotoxicity in mice with the human Z allele to assess the risk of increased risk hepatocellualr carcinoma and to better understand the pro-inflammatory and pro-fibrotic pathways in these livers. This concept will then be extended to studies using humanized mouse livers with either normal or Z-allele positive human liver stem cells derived from iPS cells. Finally both of these models will be utilized to assess the effect of rAAV-based RNAi mediated knockdown of the Z-AAT mRNA.