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.

项目摘要粘膜校利清除是预防慢性气道疾病的重要功能。在健康的肺中，多个 Motile纤毛同步跳动以将吸入颗粒和粘液从气道中输出。粘毛当动向纤毛功能受到损害时，就会出现清除，并且是许多继承的基本特征获得的呼吸道疾病，包括原发性睫状运动障碍（PCD），哮喘，慢性支气管炎和囊性纤维化（CF）。由于纤毛纤毛是复杂且高度专业的细胞器，因此很大一部分基因，所以许多尚未发现的东西，可能会导致各种形式的PCD，其中可能不存在纤毛，减少了数字或缺失的关键结构，可以使有效的，协调的电动中风。这个宽广的广度病理使诊断变得困难，需要高度专业的专业知识来解释电子显微照片和睫状节频率，治疗主要是症状。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疗法的发展受到缺乏的阻碍可再现的患者特异性体外模型，以研究控制人类多毛的分子机制单元格（MCC）规范和功能。此应用程序通过利用该实验障碍来解决我们的新颖的体外人类系统，以系统地识别因果突变和信号传导机制基本的遗传和获得形式的睫状功能障碍。我们的专业知识是独特的纤毛生成，基因编辑（CRISPR/CAS9）和人IPSC完成以下特定目的：（ AIM 1）评估MCC与IPSC的分化，并产生完整的人类MCC转录组；（目标2）评估并纠正源自DNAH5突变PCD患者IPSC的肺上皮细胞中的睫状功能障碍；（AIM 3）识别并评估当前未鉴定的PCD患者中新型缺陷的纤毛基因型致病基因突变。这项创新提案的预期总体影响是获得机械使用强大的体外模型了解人类MCC规范和功能，其中直接对照和PCD患者细胞之间的比较将使人们更好地了解已知的人导致睫状功能障碍的基因。此外，这种实验方法将为鉴定新型PCD致病突变，从而为机制提供了重要的新见解具有纤毛功能障碍为特征的基本遗传和获得的疾病。拟议的研究是创新性，因为我们将利用人类IPSC方法来确定MCC分化的关键调节剂。来自PCD患者的人IPSC衍生的MCC的系统比较将导致功能验证已知和新颖的病因突变，同时解决了可再现和定义的人类的迫切需求在其中进行这些实验的模型系统。这些研究应导致快速发展新的治疗疗法和PCD的更好的诊断/基因检测到诊所。