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

项目摘要 粘膜纤毛清除是预防慢性气道疾病的基本功能。在健康的肺中， 活动纤毛同步跳动，将吸入的颗粒和粘液输送出气道。黏膜纤毛不良 当运动纤毛功能受损时，清除就会出现，并且是许多遗传性和 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）。由于运动纤毛是复杂和高度专业化的细胞器，大量的基因， 许多尚未被发现的，可能有助于各种形式的PCD，其中纤毛可能缺失，减少， 数字，或失踪的关键结构，使一个有效的，协调的动力冲程。这片广阔的 病理学使得诊断困难，需要高度专业化的专业知识来解释电子 显微照片和纤毛跳动频率，治疗主要是对症。理解复杂性 纤毛疾病驱动的肺部疾病和这些疾病的靶向治疗的发展受到缺乏 可重复的患者特异性体外模型，以研究控制人类多纤毛的分子机制 电池（MCC）规格和功能。在本申请中，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传导机制 潜在的遗传性和获得性形式的睫状体功能障碍。我们凭借我们的专业知识， 纤毛发生、基因编辑（CRISPR/Cas9）和人iPSC以完成以下具体目标： 评估来自iPSC的MCC分化并生成完整的人MCC转录组;（目标2） 评估和纠正源自DNAH 5突变PCD患者iPSC的肺上皮细胞中的纤毛功能障碍; (Aim 3）鉴定和评估PCD患者中的新的缺陷纤毛基因型， 致病基因突变这一创新提案的预期总体影响是获得机械性 使用稳健的体外模型了解人MCC的规格和功能，其中直接 对照和PCD患者细胞之间的比较将导致更好地理解已知的人类细胞， 导致纤毛功能障碍的基因此外，这种实验性的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 以纤毛功能障碍为特征的潜在遗传和获得性疾病。拟议的研究是 创新，因为我们将利用我们的人类iPSC方法来确定MCC分化的关键调节因子。 来自PCD患者的人iPSC衍生的MCC的系统性比较将导致以下的功能验证： 已知的和新的致病突变，同时解决了一个可重复的和定义的人的关键需要， 模型系统，在其中进行这些实验。这些研究应该会导致 新的治疗方法和更好的诊断/遗传测试PCD的临床。

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.