Establishment of a variant-to-function framework for cholestatic diseases using human liver in vitro models

利用人肝脏体外模型建立胆汁淤积性疾病的变异功能框架

• 批准号: NC/Y500628/1
• 金额: $ 17.2万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FY500628%2F1
• 关键词: Establishment; variant; function; framework; cholestatic

Hepatic cholestatic diseases are characterised by impaired bile flow and harmful accumulation of bile acids in the liver. Genetics is a key contributing factor to cholestatic disease risk, with rare mutations in transporter genes being well-established causes of cholestasis. Recent studies have highlighted that common genetic variants within liver transcriptional enhancers also contribute to cholestasis. However, unlike coding sequences, enhancers are poorly conserved in other species, making the usefulness of animal models for the study of cholestasis genetic factors limited at best.A major hurdle to replace animal models for the study of cholestasis risk variants with human model systems is the lack of appropriately characterised human liver in vitro models. Human induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) are an attractive model to study the effects of human genetic variants on hepatic transcriptional programs and ultimately disease-relevant cellular outputs. In particular, iHeps cultured under polarisation-inducing conditions show enhanced functionality. We envision that a better characterisation of the molecular features of iPSC-derived liver in vitro systems will facilitate their use to investigate liver disease genetics and enable the replacement of animal models in this and future studies.In this project, a multidisciplinary team joining expertise in human cholestasis, regulatory genomics, stem cell-based modelling and lipidomics, will investigate two culture systems (classic 2D vs. new sandwich model optimised for cholestasis modelling) generating global expression and chromatin activity maps. These systems will be benchmarked against human liver datasets to identify which system (1) better recapitulates relevant features of human hepatocytes, and (2) is better suited to functionally investigate genetic loci associated with human cholestasis. These data will also be systematically compared against data obtained from rodents, highlighting loci for which rodents are not suitable models. We will then carry out a CRISPR-activation screen of cholestasis loci to identify the target genes of noncoding variants, as enhancers can interact with genes at varying distances. Finally, we will focus on one locus, which will be modelled by combining genome editing in iPSC with the cholestasis-optimised iHeps protocol. Edited iHeps will then be used to investigate cholestasis-relevant cellular outputs, including formation of canaliculi and lipidomic characterisation. We expect to demonstrate the superior suitability of iHeps to study noncoding variants associated with cholestasis, providing a framework for variant-to-function investigation of risk loci in iHeps. More broadly, these datasets and analysis framework will be applicable to investigate risk loci for other liver diseases in future studies.

肝胆固醇疾病的特征是胆汁流量受损和胆汁酸在肝脏中的有害积累。遗传学是导致胆固性疾病风险的关键因素，转运蛋白基因的罕见突变是胆汁淤积的良好原因。最近的研究强调，肝转录增强剂中的常见遗传变异也有助于胆汁淤积。但是，与编码序列不同，增强子在其他物种中的保守性很差，这使动物模型在研究胆汁淤积的遗传因子的研究中有用。最多有限。替代动物模型的主要障碍，用于研究人类模型系统的胆汁淤积风险变体的研究是缺乏适当的人体肝脏在体外模型中表征适当的人肝模型。人类诱导的多能干细胞（IPSC）衍生的肝细胞（IHEPS）是研究人类遗传变异对肝转录程序的影响以及最终与疾病相关的细胞输出的有吸引力的模型。特别是，在极化诱导条件下培养的IHEPS显示出增强的功能。我们设想，更好地表征了IPSC衍生的肝脏在体外系统中的分子特征，将促进其用于研究肝病遗传学的用途，并在这项和未来的研究中替换动物模型。在这项项目中，多学科团队，一支加入人类胆汁疾病的专业知识，在人类的胆小性基因组中，需要调查两种型号的模型，并将调查两种型号的模型。胆汁淤积建模）生成全局表达和染色质活性图。这些系统将针对人肝数据集进行基准测试，以确定哪种系统（1）更好地概括了人类肝细胞的相关特征，并且（2）更适合于功能地研究与人胆汁淤积相关的遗传基因座。这些数据还将与从啮齿动物获得的数据进行系统地进行比较，这突出了啮齿动物不是合适模型的基因座。然后，我们将进行胆汁淤积基因座的CRISPR激活筛选，以识别非编码变体的靶基因，因为增强剂可以在不同距离时与基因相互作用。最后，我们将重点关注一个基因座，该基因座将通过将IPSC中的基因组编辑与胆汁淤积优化的IHEPS协议相结合来建模。然后，编辑的IHEP将用于研究与胆汁淤积相关的细胞输出，包括结肠菌和脂肪组表征的形成。我们希望展示IHEPS研究与胆汁淤积相关的非编码变体的卓越适用性，从而为IHEPS中风险基因座的变体到功能研究提供了一个框架。从更广泛的角度来看，这些数据集和分析框架将适用于在未来的研究中调查其他肝病的风险基因座。