Characterization of mechanisms regulating multiciliated cell specification using patient-specific induced pluripotent stem cells.

项目总结 粘液纤毛清除是预防慢性呼吸道疾病的重要功能。在健康的肺中，多个 活动的纤毛同步跳动，将吸入的颗粒和粘液输送出呼吸道。粘液纤毛差 当运动纤毛功能受损时，就会产生清除，这是许多遗传性和 获得性呼吸道疾病，包括原发性纤毛运动障碍(PCD)、哮喘、慢性支气管炎和囊性 纤维化(CF)。由于活动纤毛是复杂和高度专业化的细胞器，有大量的基因， 许多尚未被发现的可能是各种形式的PCD的致病因素，其中可能没有纤毛，减少了 数字，或缺少关键结构，以实现有效、协调的动力冲程。这片广阔的土地 病理学使诊断变得困难，需要高度专业的专业知识来解释电子 显微镜下观察纤毛搏动频率，治疗以对症为主。了解以下问题的复杂性 纤毛疾病引起的肺部疾病和针对这些疾病的靶向治疗的发展因缺乏而受阻 用于研究控制人类多纤毛的分子机制的可重复性的患者特异性体外模型 信元(MCC)规格和功能。在本应用程序中，通过利用 我们的新的，体外人类系统，系统地识别致病突变和信号机制 潜在的遗传性和获得性睫状体功能障碍。我们独一无二地凭借我们的专业知识在 纤毛发生、基因编辑(CRISPR/CAS9)和人类IPSC以完成以下具体目标：(目标1) 评估MCC与IPSCs的分化，并生成完整的人类MCC转录组；(目标2) 评估和纠正DNAH5突变的PCD患者IPSC来源的肺上皮细胞的纤毛功能障碍； (目的3)鉴定和评估目前尚未发现的PCD患者中新的纤毛基因缺陷类型 致病基因突变。这一创新提议的预期总体影响是获得机械化的 使用健壮的体外模型理解人类MCC的规格和功能 对照细胞和PCD患者细胞的比较将有助于更好地理解已知的人类 导致睫毛功能障碍的基因。此外，这种试验性方法将创建一个强大的管道 鉴定导致PCD的新突变，从而为机制提供重要的新见解 以睫毛功能障碍为特征的遗传性和获得性疾病。拟议的研究是 我们将利用人类IPSC方法来确定MCC差异化的关键调控因素，这是一种创新。 PCD患者来源的人IPSC来源的MCC的系统比较将导致对 已知和新的致病突变，同时满足可复制和定义的人类的关键需求 进行这些实验的模型系统。这些研究应该会导致快速的进展 PCD的新疗法和更好的诊断/基因测试将推向临床。

PROJECT SUMMARY Mucociliary clearance is an essential function to prevent chronic airway disease. In the healthy lung, multiple motile cilia beat synchronously to transport inhaled particles and mucus out of the airways. Poor mucociliary clearance arises when motile cilia function is impaired, and is a fundamental feature of many inherited and acquired respiratory diseases, including primary ciliary dyskinesia (PCD), asthma, chronic bronchitis and cystic fibrosis (CF). Since motile cilia are complex and highly specialized organelles, a large spectrum of genes, many yet to be discovered, likely contribute to the various forms of PCD, where cilia may be absent, reduced in number, or missing key structures that enable an effective, coordinated power stroke. This wide breadth of pathologies makes diagnosis difficult, requiring highly specialized expertise for interpretation of electron micrographs and ciliary beat frequency, and treatment is mainly symptomatic. Understanding the complexity of ciliopathy-driven lung disease and development of targeted therapies for these disorders is hindered by a lack of reproducible patient-specific in vitro models to study molecular mechanisms that govern human multiciliated cell (MCC) specification and function. This experimental barrier is addressed in this application by exploiting our novel, in vitro human system to systematically identify causative mutations and signaling mechanisms underlying inherited and acquired forms of ciliary dysfunction. We are uniquely poised with our expertise in ciliogenesis, gene editing (CRISPR/Cas9) and human iPSC to complete the following specific aims: (Aim 1) Evaluate MCC differentiation from iPSCs and generate a complete human MCC transcriptome; (Aim 2) Evaluate and correct ciliary dysfunction in lung epithelial cells derived from DNAH5 mutant PCD patient iPSC; (Aim 3) Identify and evaluate novel defective cilia genotypes in PCD patients with no currently identified causative genetic mutation. The expected overall impact of this innovative proposal is to gain mechanistic understanding of human MCC specification and function using a robust in vitro model where a direct comparison between control and PCD patient cells will lead to a better understanding of the known human genes that lead to ciliary dysfunction. Moreover, this experimental approach will create a robust pipeline for identification of novel mutations causative of PCD, thus providing significant new insights into mechanisms underlying inherited and acquired diseases characterized by ciliary dysfunction. The proposed research is innovative as we will exploit our human iPSC approach to determine key regulators of MCC differentiation. Systematic comparison of human iPSC-derived MCC from PCD patients will lead to the functional validation of known and novel causative mutations while addressing a critical need of a reproducible and defined human model system in which to carry out these experiments. These studies should lead to the rapid progression of novel therapeutics and better diagnostic/genetic tests for PCD to the clinic.