Models of AT Deficiency Using Human Hepatocytes

总结 这项研究的目的是使用患者来源的细胞开发ATD模型，并使用这些模型来 确认先前鉴定的ATD修饰剂，并在人类细胞中测试ATD的潜在药物疗法。我们 已经表明，干细胞衍生的肝细胞样细胞（iHep）是通过将干细胞从 ATD肝与肺表现患者在超微结构形态和动力学方面表现出差异 突变ATZ处置。基于这些发现，我们假设ATZ以外的遗传变异 突变决定ATD的临床表型。作为本项目上一个周期的一部分，我们 我们的同事在C. elegans使用siRNA文库鉴定几种 改变ATZ积累的基因。该项目将揭示ATZ积累的差异 根据肝损害发生的年龄对来自不同ATD患者亚群的iHep及其蛋白毒性进行了研究 疾病，不同的肝脏表型，存在共存的肺部疾病或肝细胞癌， 和ATZ等位基因的杂合性。我们还将检查a）在何种程度上， iHep对应于各种ATD临床亚群，B）遗传调节剂对ATD肝病的作用 表型，c）iHep对用于治疗ATD的候选药物的反应，和d）iHep的延迟是否与ATD的治疗有关。 ATZ处置是来自患有严重肝病的ATD患者的iHep的特征， ATZ的特性最后，我们将通过重新填充人的肝脏来产生人ATD的体内模型。 用原代人肝细胞和来自ATD患者的iHep处理的免疫缺陷大鼠， 对照使用患者来源的细胞开发ATD的体内和体外模型将极大地提高ATD的治疗效果。 有助于发现和验证ATD新疗法的靶基因，并评估潜在的 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.