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

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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.