Models of AT Deficiency Using Human Hepatocytes

摘要 这项拟议的研究旨在利用患者来源的细胞开发ATD模型，并使用这些模型来 确认先前已确定的ATD修饰剂，并在人类细胞中测试治疗ATD的潜在药物疗法。我们 研究表明，干细胞来源的肝细胞样细胞(IHEPs)是由干细胞从 ATD患者的肝和肺表现在超微结构形态和动力学上有所不同。 变种ATZ的处置。基于这些发现，我们假设ATZ以外的基因变异 突变决定了ATD的临床表型。作为本计划项目上一个周期的一部分，我们 我们的同事使用siRNA文库对线虫进行了高通量分析，以确定几个 改变ATZ积累的基因。拟议的项目将揭示ATZ积累的差异 以及其在不同亚群ATD患者中的蛋白毒性 疾病，不同的肝脏表型，共存的肺部疾病或肝细胞癌， 以及ATZ等位基因的杂合性。我们还将检查a)体外特征的程度 IHEPs对应于不同的ATD临床亚群，b)遗传调节剂在ATD肝病中的作用 表型，c)iHEPs对治疗ATD的候选药物的反应，以及d)在 有严重肝病的ATD患者的iHEPS的ATZ处置是由于聚集倾向所致 ATZ的属性。最后，我们将通过重新填充人类ATD的肝脏来产生体内模型 用ATD患者的原代人肝细胞和iHEPS治疗免疫缺陷大鼠 控制。使用患者来源的细胞建立体内和体外ATD模型将大大 有利于发现和验证ATD新疗法的靶基因并评估其潜力 治疗药物。

SUMMARY The proposed study aims to develop models of ATD using patient-derived cells and use these models to confirm previously identified modifiers of ATD and test potential drug therapies for ATD in human cells. We have shown that stem cell-derived hepatocyte-like cells (iHeps) generated by differentiating stem cells from patients with liver vs. lung manifestations of ATD exhibit differences in ultrastructural morphology and kinetics of mutant ATZ disposal. Based on these findings, we hypothesize that genetic variations other than the ATZ mutation determine the clinical phenotype of ATD. As a part of the previous cycle of this program project, we and our colleagues have used high throughput analysis in C. elegans using a siRNA library to identify several genes that modify ATZ accumulation. The proposed project will reveal the differences in accumulation of ATZ and its proteotoxicity in iHeps from various subsets of ATD patients delinieated by age of onset of liver disease, disparate hepatic phenotype, presence of co-existing pulmonary disease or hepatocellular carcinoma, and heterozygosity for the ATZ allele. We will also examine a) the extent to which in vitro characteristics of iHeps correspond to various ATD clinical subsets, b) the role of genetic modulators on ATD liver disease phenotypes, c) the response of iHeps to candidate drugs for the treatment of ATD, and d) whether the delay in ATZ disposal that characterizes iHeps from ATD patients with severe liver disease is due to aggregation-prone properties of ATZ. Finally, we will generate in vivo models of human ATD by repopulating the livers of retrorsine-treated immune deficient rats with primary human hepatocytes and iHeps from ATD patients and controls. The development of in vivo and in vitro models of ATD using patient-derived cells would greatly benefit the discovery and validation of target genes for novel therapies of ATD and evaluation of potential therapeutic drugs.