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.

肝脏胆汁淤积性疾病的特征在于胆汁流动受损和胆汁酸在肝脏中的有害积累。遗传学是胆汁淤积性疾病风险的一个关键因素，转运蛋白基因的罕见突变是胆汁淤积的公认原因。最近的研究强调，肝脏转录增强子内的常见遗传变异也有助于胆汁淤积。然而，与编码序列不同的是，增强子在其他物种中保守性较差，使得动物模型用于胆汁淤积遗传因素研究的有效性受到限制。用人类模型系统取代动物模型用于胆汁淤积风险变体研究的主要障碍是缺乏适当表征的人类肝脏体外模型。人诱导多能干细胞（iPSC）衍生的肝细胞（iHep）是研究人类遗传变异对肝脏转录程序和最终疾病相关细胞输出的影响的有吸引力的模型。特别地，在极化诱导条件下培养的iHep显示增强的功能性。我们设想，更好地表征iPSC衍生肝脏体外系统的分子特征将有助于其用于研究肝脏疾病遗传学，并在本研究和未来的研究中取代动物模型。在该项目中，一个多学科团队将人类胆汁淤积，调控基因组学，基于干细胞的建模和脂质组学，将研究两种培养系统（经典的2D与优化胆汁淤积建模的新三明治模型），生成全局表达和染色质活性图谱。这些系统将根据人类肝脏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，以及（2）更适合在功能上研究与人类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比较，突出显示啮齿动物不适合模型的基因座。然后，我们将对胆汁淤积位点进行CRISPR激活筛选，以识别非编码变体的靶基因，因为增强子可以与不同距离的基因相互作用。最后，我们将专注于一个基因座，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方案相结合来建模。然后将使用编辑的iHep研究胆汁淤积相关的细胞输出，包括小管的形成和脂质组学表征。我们希望证明iHep在研究胆汁淤积相关非编码变异方面的上级适用性，为iHep中风险位点的变异-功能研究提供框架。更广泛地说，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脏疾病的风险位点。