Developing preclinical human iPSC-based HTS assays to identify therapeutic agents for biliary atresia

项目摘要 该研究的目的是开发基于患者iPSC的HTS测定， 用于治疗胆道闭锁（BA）纤维化的治疗发现。BA是儿科最常见的原因 终末期肝病在美国，如果不治疗，在生命的头两年内不可避免地致命。 值得注意的是，BA是人类中最快速的纤维化肝脏疾病，并且与显著的肝纤维化相关。 儿童发病率和死亡率。与其他肝脏疾病相比， 肝硬化数十年，BA婴儿特征性地在数周至数月内发展纤维化/肝硬化 出生后尽管有一种姑息性手术，但目前还没有已知的治疗方法可以阻止这种情况的发生。 进行性纤维化;大多数患有这种疾病的婴儿需要进行肝移植， 生存在开发有效的抗纤维化药物中的主要挑战是缺乏纤维化的模型。 人类疾病 人诱导多能干细胞（iPSC）技术提供了用于产生多能干细胞的替代方案。 用于使用患者组织进行疾病建模的功能性、可再生和相关的细胞来源。基于 我们在体外疾病建模方面的专业知识，我们最近成功地开发了BA患者- 我们已经证明，这些细胞产生的胶原蛋白和其他胶原蛋白 纤维化标志物沿着胆道分化缺陷（BA的关键疾病特征），与 健康儿童的iPSCs。对BA患者iPSC的这一新的研究路线使其可行， 在更人性化的环境中评估潜在药物的有效性和安全性。因此我们相信 这种BA的人细胞模型可以作为鉴定有效抗纤维化的理想系统， 化合物治疗BA中的肝纤维化。 在本研究中，我们提出：1）开发一种新的高通量检测方法来评估抗纤维化 使用COL 1A 1报告基因BA-iPSC系观察化合物对BA纤维化的影响。康贝特人将以 用于使测定稳定化和用于稳健的测定自动化的条件。2)执行试点屏幕， 使用临床药物库验证和优化测定，以确保自动化可靠性， 测定重现性。3)开发独立的二次检测，通过进一步优化命中选择 验证命中的抗纤维化作用，并评估其对 来源于患者iPSC的肝胆组织。在这项研究结束时，我们将有 开发了一种强大的，基于患者细胞的HTS检测方法，能够识别新的疾病靶点和线索 为BA患者开发新的治疗方法。此外，该项目的成功将是一个进步 在将基本iPSC发现转化为治疗应用方面， 发展再生医学

Project Summary The objective of the research is to develop patient iPSC-based HTS assays that can facilitate therapeutic discovery for treating biliary atresia (BA) fibrosis. BA is the most common cause of pediatric end-stage liver disease in the U.S. and is inevitably fatal within the first two years of life if untreated. Notably, BA is the most rapidly fibrosing liver disease in humans and is associated with significant morbidity and mortality in children. Compared to other liver diseases that gradually progress into cirrhosis over decades, BA infants characteristically develop fibrosis/cirrhosis within weeks to months after birth. Although there is a palliative surgical procedure, there is no known treatment to halt the progressive fibrosis; most infants born with the disease will need liver transplantation in order to survive. A main challenge in developing effective anti-fibrotic drugs has been the lack of a model of the human disease. The human induced pluripotent stem cell (iPSC) technology provides an alternative for generating functional, renewable and relevant cell sources for disease modeling using patient tissues. Based on our expertise on in vitro disease modeling, we have recently succeeded in developing BA patient- specific iPSCs and have demonstrated that these cells produce significantly more collagen and other fibrosis markers along with deficiency in biliary differentiation (key disease features of BA), compared to the iPSCs of healthy children. This new line of research on BA patient iPSCs makes it feasible to evaluate both efficacy and safety of potential drugs in a more human-relevant setting. Thus we believe this human cellular model of BA can serve as an ideal system to identify effective anti-fibrotic compounds in treating liver fibrosis in BA. In the current study, we propose to: 1) Develop a novel high throughput assay to assess anti-fibrotic effects of compounds on BA fibrosis using COL1A1 reporter BA-iPSC lines. We will determine the conditions for miniaturizing the assay and for robust assay automation. 2) Perform pilot screens to validate and optimize the assay using a clinical drug library in order to ensure automation reliability and assay reproducibility. 3) Develop independent secondary assays to prioritize hit selection by further verifying the anti-fibrotic effects of the hits and evaluating their protective/adverse effects on hepatobiliary tissues derived from patient iPSCs. At the conclusion of this study, we will have developed a robust, patient cell-based HTS assay capable of identifying new disease targets and leads for developing novel therapies for BA patients. Moreover, success of this project will be a step forward in translating basic iPSC discoveries to therapeutic applications, helping to fulfill their promise in developing regenerative medicine.