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

使用患者特异性诱导多能干细胞来表征调节多纤毛细胞规范的机制。

基本信息

批准号：
9889170
负责人：
Amy Leanne Ryan
金额：
$ 48.47万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-15 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9889170
关键词：
Address Airway Disease Asthma Attention Automobile Driving Biological Models CRISPR/Cas technology Cell Differentiation process Cells Chronic Chronic Bronchitis Cilia Clinic Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Coiled-Coil Domain Complex Cystic Fibrosis DNA Sequence Alteration Dependence Development Diagnosis Diagnostic Disease Electrons Embryo Epithelial Epithelial Cells Epithelium Etiology Evaluation Family Frequencies Functional disorder GMNN gene Gene Expression Profiling Generations Genes Genetic Genetic Transcription Genotype Goals Health Human Impairment In Vitro Inhalation Inherited Knock-in Knock-out Lead Lung Lung diseases Methods Modeling Molecular Mucociliary Clearance Mucous body substance Mutate Mutation Organelles Pathology Pathway interactions Patients Pb clearance Phenotype Power stroke Primary Ciliary Dyskinesias Proteins Protocols documentation Recurrence Reporter Reproducibility Research Respiratory Tract Infections Respiratory distress Signal Pathway Signal Transduction Skin Structural Protein Structure System Validation Xenopus airway epithelium blastomere structure causal variant ciliopathy cilium biogenesis cilium motility comparative experimental study fluid flow gene function genetic testing human model improved in vitro Model induced pluripotent stem cell innovation insight knock-down lung development lung injury mutant new therapeutic target notch protein novel novel therapeutics overexpression particle pathogen postnatal prevent progenitor programs stem cells targeted treatment tool transcriptome transcriptome sequencing

项目摘要

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.

项目概要粘膜纤毛清除是预防慢性气道疾病的重要功能。在健康的肺部，多种活动的纤毛同步跳动，将吸入的颗粒和粘液输送出气道。粘膜纤毛较差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遗传性的基本特征。获得性呼吸系统疾病，包括原发性纤毛运动障碍 (PCD)、哮喘、慢性支气管炎和囊性支气管炎纤维化（CF）。由于运动纤毛是复杂且高度专业化的细胞器，大量的基因，许多尚未被发现，可能导致各种形式的 PCD，其中纤毛可能缺失，纤毛减少数量，或缺少能够实现有效、协调的动力冲程的关键结构。这种宽广的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照片和纤毛跳动频率，治疗主要是对症治疗。了解复杂性纤毛病驱动的肺部疾病和针对这些疾病的靶向治疗的开发因缺乏可重复的患者特异性体外模型来研究控制人类多纤毛的分子机制细胞（MCC）规格和功能。本申请通过利用我们新颖的体外人体系统可以系统地识别致病突变和信号机制潜在的遗传性和后天性纤毛功能障碍。我们拥有独特的专业知识纤毛发生、基因编辑（CRISPR/Cas9）和人类 iPSC 来完成以下具体目标：（目标 1）评估 MCC 与 iPSC 的分化并生成完整的人类 MCC 转录组；（目标2）评估和纠正源自 DNAH5 突变 PCD 患者 iPSC 的肺上皮细胞的纤毛功能障碍；（目标 3）识别和评估目前尚未发现的 PCD 患者中新的有缺陷的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化使用稳健的体外模型了解人类 MCC 规格和功能，其中直接对照细胞和 PCD 患者细胞之间的比较将有助于更好地了解已知的人类细胞导致纤毛功能障碍的基因。此外，这种实验方法将为鉴定导致 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性和获得性疾病。拟议的研究是创新性的，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。对来自 PCD 患者的人 iPSC 衍生的 MCC 进行系统比较将导致功能验证已知和新颖的致病突变，同时满足可重复和明确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进展临床 PCD 的新疗法和更好的诊断/基因测试。