SUMMARY: PROJECT 3 There are striking challenges to the diagnosis, care, and treatment of individuals with primary ciliary dyskinesia (PCD). These hurdles are implicit to the fundamental biology of PCD. Compared to cystic fibrosis, a monogenic airway disease, PCD is caused by mutations in >50 genes that encode proteins involved in ciliogenesis and that result in impaired mucociliary clearance. Unlike CF, where 90% of individuals carry at least one copy of the same variant, the genetic landscape of PCD is heterogeneous, both in terms of number of genes and variants within those genes. In CF, we now have highly effective therapies for those 90% of individuals. There are no precision therapies for PCD, nor do they seem close. A paradigm shift is required to enable the development of those therapies. In this proposal, we develop methods to generate a panel of PCD airway cells using induced pluripotent stem cells (iPSCs). We do so using our established methods to generate a self-renewing source of airway basal cells (iBCs) from iPSCs capable of differentiating into multiciliated cells (MCCs). We present preliminary data, demonstrating our ability to efficiently knock out or precisely edit genes in iBCs and phenotype the ciliary defects that result. We test the hypothesis that this iBC platform can detect the pathogenicity of PCD mutations and predict the pathogenicity of variants of uncertain significance. To do so we focus on 5 high priority PCD genes (Aim 1). We take the novel approach of directly inserting VUS (5 per gene) into a gold- standard iBC line to determine if they lead to defects in cilia ultrastructure and or function (Aim 2). In aim 3, we address the intriguing question of why mutations in certain genes (CCDC39 and CCDC40) lead to more severe lung disease. We test the hypothesis that these genes encode for proteins with broader biological roles and their loss leads to cell intrinsic defects and not simply loss of cilia function. Finally, in aim 4 we perform proof-of concept experiments to test the therapeutic potential of iBCs as an autologous, gene-corrected cell-based therapy approach in xenogeneic transplantation studies in NSG mice.