Establishing patient-derived iPSCs as a platform for discovery research in NAFLD

建立源自患者的 iPSC 作为 NAFLD 发现研究的平台

• 批准号: 10647450
• 负责人: JACQUELYN J. MAHER
• 金额: $ 161.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647450
• 关键词: Address; Affect; Biological; Biological Assay; Biological Models; CRISPR interference; CRISPR screen; Cataloging; Catalogs; Cell Culture Techniques; Cell Line; Cell Lineage; Cells; Cirrhosis; Clinic; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Collection; Communities; Data; Data Set; Development; Disease; Disease Outcome; Disease Progression; Disease model; Ensure; Environmental Risk Factor; Evaluation; Exhibits; Fibrosis; Foundations; Future; Gene Expression Profile; Genes; Genetic; Genetic Predisposition to Disease; Goals; Health Care Costs; Hepatic Stellate Cell; Hepatocyte; Hepatology; Human; Image; Impairment; In Vitro; Individual; Inflammation; Inflammatory; Information Dissemination; Insulin Resistance; Libraries; Lipids; Liver; Liver diseases; Macrophage; Malignant neoplasm of liver; Measures; Mediating; Metabolic; Mitochondria; Modeling; Morphology; Multiomic Data; Obesity; Outcome; Parents; Pathogenesis; Patients; Persons; Pharmaceutical Preparations; Phenotype; Process; Proteomics; Publishing; RNA Interference; Reporter; Research; Research Project Grants; Resources; Risk; Risk Factors; Services; Stains; Standardization; System; Testing; Therapeutic Intervention; Translations; Validation; Variant; biobank; cell type; cohort; combinatorial; comparative; data library; defined contribution; disease phenotype; experimental study; fibrogenesis; gene correction; genetic risk factor; genetic variant; genome wide association study; genome-wide; individual patient; induced pluripotent stem cell; knock-down; miniaturize; non-alcoholic fatty liver disease; novel; population based; protective allele; risk variant; screening; stem cell model; stem cells; targeted treatment; theories; transcriptomics; web platform; whole genome

PROJECT SUMMARY/ABSTRACT Our research group studies human NAFLD using patient-derived induced pluripotent stem cells (iPSCs) for in vitro disease modeling. We recently showed that iPSCs from a cohort of NAFLD patients, when differentiated to hepatocytes (iPSC-Heps), display a spontaneous disease signature in cell culture. This underscores the importance of genetic background to NAFLD disease modeling and offers a unique opportunity to study the impact of NAFLD risk genes on disease phenotype. We theorize that the disease phenotype in NAFLD iPSC- Heps is due in part to established genetic risk factors identified through GWAS and in part to others that are either poorly characterized or unknown. To address the impact of established and emerging genetic risk factors on the NAFLD phenotype in iPSC-derived liver cells, we will leverage our unparalleled collection of 61 disease-specific iPSC lines (41 NAFLD, 19 control) and our ability to differentiate iPSCs along multiple liver cell lineages to create mono- and co-cultures. In the course of three aims we will systematically study these cells and catalogue the resulting resources and information for dissemination to the hepatology community. In Aim 1 we will develop a scorecard comprising the results of 15 transcriptomic, proteomic and functional assays for all 61 iPSC lines. The data will be used to develop individual and aggregate measures distinguishing normal from diseased cellular phenotypes and correlate phenotypic profiles with individual and polygenic risk factors. This aim will generate a large body of multi-omic data in the NAFLD iPSC model system that will be used as the foundation for subsequent gene editing. Aim 2 will constitute a systematic effort to correct 113 variant genes in 33 NAFLD iPSC lines and repeat the full scorecard analysis after each edit. Comparisons will be made between scorecards from individual gene-edited vs. parent lines, as well as in groups of iPSCs with similar edits. Many iPSC lines will be subjected to sequential gene corrections and may revert to normal; iPSC lines whose scorecard does not normalize will be scrutinized for the presence of novel variants with a plausible disease association, followed by direct testing with further gene correction. Aim 3 will employ a complementary but independent strategy involving whole-genome CRISPR screening in a NAFLD iPSC line to identify genes whose inhibition suppresses a NAFLD signature. This aim will make use of fluorescent reporter iPSC lines and high-content imaging to assess NAFLD-related outcomes. The CRISPR screen will enable us to discover novel genes that have a direct impact on cellular phenotype and may be suitable for translation to the clinic. This RC2 project will yield several deliverables: (a) rich, multi-omic datasets from a large cohort of parent iPSC lines and isogenic gene-edited derivatives following multicellular differentiation and NAFLD modeling; (b) > 100 iPSC lines from which the data were generated; and (c) an iPSC line from a NAFLD subject transduced with an arrayed whole-genome CRISPRi library suitable for future screening studies. Each of these resources will be made freely available on open web-based platforms or biorepositories.

项目总结/摘要 我们的研究小组使用患者来源的诱导多能干细胞（iPSC）研究人类NAFLD， 体外疾病建模。我们最近发现，来自NAFLD患者队列的iPSC在分化时， 转化为肝细胞（iPSC-Heps），在细胞培养物中显示自发性疾病特征。这突出表明 遗传背景对NAFLD疾病建模的重要性，并提供了一个独特的机会来研究 NAFLD风险基因对疾病表型影响。我们的理论是NAFLD iPSC中的疾病表型- 肝炎部分是由于通过GWAS确定的遗传风险因素，部分是由于其他因素， 要么特征不明显要么不为人知解决已确定和新出现的遗传风险的影响 在iPSC衍生的肝细胞中NAFLD表型的因素，我们将利用我们无与伦比的61 疾病特异性iPSC系（41个NAFLD，19个对照）和我们使iPSC沿着多个肝细胞分化能力 创造单一文化和共同文化。在三个目标的过程中，我们将系统地研究这些细胞， 并对所得到的资源和信息进行编目，以传播给肝病学界。目标1 我们将开发一个记分卡，包括15个转录组学，蛋白质组学和功能测定的结果， 61个iPSC细胞系。这些数据将用于制定个体和总体措施， 患病细胞表型和相关的表型谱与个人和多基因的危险因素。这 aim将在NAFLD iPSC模型系统中生成大量的多组学数据，这些数据将被用作 为后续基因编辑奠定基础。目标2将构成一个系统的努力，以纠正113个变异基因， 33个NAFLD iPSC系，并在每次编辑后重复完整记分卡分析。将进行比较 在来自个体基因编辑与亲本系的记分卡之间，以及在具有类似基因编辑的iPSC组中， 编辑。许多iPSC系将经历顺序基因校正，并可能恢复正常; 其记分卡未正常化的患者将被仔细检查是否存在新的变体， 疾病关联，然后进行直接检测，进一步进行基因校正。目标3将采用一种互补的 但涉及NAFLD iPSC系中全基因组CRISPR筛选以识别基因的独立策略 其抑制抑制NAFLD信号。这一目标将利用荧光报告基因iPSC系， 高内涵成像评估NAFLD相关结果。CRISPR屏幕将使我们能够发现新的 对细胞表型有直接影响并可能适合于临床翻译的基因。这 RC2项目将产生几个可交付成果：（a）来自大量亲本iPSC系的丰富的多组学数据集 和多细胞分化和NAFLD建模后的同基因基因编辑的衍生物;（B）> 100 产生数据的iPSC系;和（c）来自用CD34/CD38转导的NAFLD受试者的iPSC系。 阵列式全基因组CRISPRi文库适合未来的筛选研究。这些资源中的每一个都将 在开放的网络平台或生物储存库上免费提供。