Models of AT Deficiency Using Human Hepatocytes

使用人肝细胞的 AT 缺陷模型

• 批准号: 10630350
• 负责人: Ira J. Fox
• 金额: $ 60.47万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10630350
• 关键词: Adolescence; Adult; Age of Onset; Alleles; Applications Grants; Biology; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Characteristics; Childhood; Chronic; Cirrhosis; Clinical; Custom; Degradation Pathway; Development; Disparate; Effectiveness; Endoplasmic Reticulum; Evaluation; Exhibits; Exons; Fibrinogen; Fibroblasts; Gene Modified; Genes; Genetic; Genetic Variation; Goals; Hepatic; Hepatocyte; Hepatotoxicity; Hereditary Disease; Heterozygote; Hong Kong; Human; Human Engineering; Immune; Impairment; In Vitro; Kinetics; Libraries; Liver; Liver Fibrosis; Liver diseases; Lung; Lung diseases; Modeling; Morphology; Mutation; Pathologic; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physical shape; Polymers; Predisposition; Primary carcinoma of the liver cells; Property; Proteins; Rattus; Rodent; Role; Signal Pathway; Skin; Small Interfering RNA; System; Testing; Therapeutic; Transforming Growth Factor beta; Trypsin; Update; Validation; Variant; alpha 1-Antitrypsin Deficiency; clinical phenotype; differentiation protocol; disease phenotype; drug candidate; endoplasmic reticulum stress; genome editing; high throughput analysis; human model; human tissue; in vitro Model; in vivo; in vivo Model; induced pluripotent stem cell; infancy; liver injury; liver transplantation; middle age; mutant; novel therapeutics; programs; proteotoxicity; response; retrorsine; stem cell differentiation; stem cells

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.

概括 拟议的研究旨在使用患者来源的细胞开发ATD模型，并使用这些模型来 确认先前确定的ATD的修饰剂，并测试人类细胞中ATD的潜在药物疗法。我们 已经表明，通过区分干细胞与 ATD的肝脏与肺部表现的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，我们假设除了ATZ以外的遗传变异 突变决定了ATD的临床表型。作为该计划项目上一个周期的一部分，我们 我们的同事使用siRNA库在秀丽隐杆线虫中使用了高吞吐量分析来识别几个 改变ATZ积累的基因。拟议的项目将揭示ATZ积累的差异 及其在肝脏发病年龄的各种ATD患者子集的IHEP中的蛋白质毒性 疾病，不同的肝表型，共存肺疾病或肝细胞癌的存在， 和ATZ等位基因的杂合性。我们还将检查a）体外特征的程度 IHEPS对应于各种ATD临床子集，b）遗传调节剂对ATD肝病的作用 表型，c）IHEPS对ATD治疗的候选药物的反应，d）是否延迟 ATZ处置的特征是来自严重肝病的ATD患者的IHEPS是由于容易发生的 ATZ的性质。最后，我们将通过重现 用ATD患者的原代人肝细胞和IHEPS重新治疗的免疫缺陷大鼠， 控件。使用患者衍生细胞的体内和ATD体外模型的发展将大大 使目标基因发现和验证ATD的新疗法和潜在评估的发现和验证 治疗药物。