Using Induced Pluripotent Stem Cells to Characterize Cystic Fibrosis

项目摘要/摘要 这份提案详细说明了一项为期5年的计划，为候选人王若冰的独立职业生涯做好准备 内科医生-科学家定位于影响我们对儿童肺部疾病，特别是囊性疾病的理解 纤维化(CF)。作为一名照顾CF患者的临床医生，她发现了罕见的纯合子个体 符合临床纳入标准的F508del CFTR突变为：1)CF长期无进展(LTNP)， 2)肺功能快速进展型(RP)，肺功能下降较快。完整的外显子 测序发现I)SCNN1(编码上皮性钠通道)罕见的错义多态 2)RPS上皮选择性氯通道(ANO1和SCL26A9)中的两个基因。这个 该提议的中心假设是，CF的极端表型是由于 由这些基因缺陷驱动的上皮离子和液体运输，以及极端表型疾病 可以利用重新编程的基于细胞的平台在体外模拟和研究严重程度和机制。vbl.使用 一种新的方案，王博士从诱导的多能干细胞(IPSCs)中产生呼吸道基底样细胞 然后在气液界面上分化为基础的、多纤毛的和分泌的谱系，形成功能 具有完整屏障功能和异常跨上皮氯离子转运的呼吸道上皮细胞。的正规化 通过CFTR基因编辑证实了她的模型在这些细胞中的氯运输的可靠性 表型。该项目的中心目标是建立用于CF疾病建模的新的IPSC-平台 并对极端表型患者的上皮功能进行研究。她现在独一无二地准备完成 目的：1)测试IPSC来源的呼吸道上皮细胞是否可以作为建立呼吸道模型的平台 CF上皮离子和液体转运及粘液纤毛转运，2)特异表型CF模型的建立 并询问候选修饰基因的作用及其对离子和流体运输的影响，以及3) 建立个性化药物反应IPSC平台，检验药物的治疗作用 以备选离子通道候选为目标。王医生有80%的保护时间来自波士顿儿童 医院(BCH)呼吸科和医学部。她的联合赞助商是1)博士。 Darrell Kotton在波士顿大学(BU)再生医学中心(CReM)工作，她与 2)本杰明·拉比博士，BCH呼吸疾病科主任。 此外，王博士还组建了一支由非凡的科学顾问组成的团队，每个人都带来了 他们的特定专长，帮助她的事业发展和科学研究。详细的培训计划如下 报告内容包括有指导的研究、授课课程、在会议上的陈述以及 完成研究目标，准备稿件，以及未来R01的应用。建议数 BCH和BU的研究、培训计划、指导委员会和科学-临床环境将 候选人需要在颁奖结束前过渡到独立。

Project Summary/Abstract This proposal details a 5-yr plan to prepare the candidate, Ruobing Wang, MD, for a career as an independent physician-scientist positioned to impact our understanding of pediatric lung diseases, particularly Cystic Fibrosis (CF). As a clinician taking care of CF patients, she has identified rare individuals with homozygous F508del CFTR mutations who met clinical criteria for inclusion as: 1) CF Long-term non-progressors (LTNP), with preserved lung function 2) CF Rapid progressors (RP), whose lung function declines rapidly. Whole exome sequencing uncovered rare missense polymorphisms in i) SCNN1 (which encodes epithelial sodium channel ENaC) in LTNPs, and ii) two genes in epithelial alternative chloride channels (ANO1 and SCL26A9) in RPs. The central hypothesis of the proposal is that the extreme phenotypes of CF are due to the alterations in epithelial ion and fluid transport driven by these gene defects, and that the extreme-phenotype disease severity and mechanism can be modeled and studied in vitro with a reprogrammed cell-based platform. Using a novel protocol, Dr. Wang generated airway basal-like cells from induced pluripotent stem cells (iPSCs) which can then differentiate into basal, multi-ciliated, and secretory lineages on air-liquid interface, forming a functioning airway epithelium with intact barrier function and aberrant trans-epithelial chloride transport. Normalization of chloride transport in these cells by CFTR gene editing confirms the reliability of her model to recapitulate CF phenotype. The central goals of the project are to establish this novel iPSC-platform for CF disease modeling and study the epithelial function of the extreme-phenotype patients. She is now uniquely poised to complete the aims to 1) To test whether iPSC-derived airway epithelia can serve as a platform to model airway epithelial ion and fluid transport and muco-ciliary transport in CF, 2) to model extreme-phenotype CF patient and interrogate the role of candidate modifier genes and their impact on ion and fluid transport, and 3) To establish the iPSC-platform for personalized drug response, and test the therapeutic role of pharmacologic targeting of alternative ion channel candidates. Dr. Wang has 80% protected time from Boston Children's Hospital (BCH) Division of Respiratory Diseases and the Department of Medicine. Her co-sponsors are 1) Dr. Darrell Kotton at the Center for Regenerative Medicine (CReM) at Boston University (BU) with whom she has trained for the past 1.5 years, and 2) Dr. Benjamin Raby, the chief of BCH Division of Respiratory Diseases. Furthermore, Dr. Wang has assembled a team of extraordinary scientific advisory members, each bringing their specific expertise, to assist her career development and scientific research. A detailed training plan is presented that includes mentored research, didactic coursework, presentations at meetings, and a timeline for completion of the research aims, preparation of manuscripts, and future R01 application. The proposed research, training plan, mentorship committee, and scientific-clinical environment at BCH and BU will position the candidate to transition to independence by the end of the award.