Complexities in CFTR-expressing epithelial cells of the conducting human and mouse airway were recently revealed by single-cell RNA sequencing (scRNAseq). These studies identified the ionocyte, an infrequent cell type that expresses the majority of CFTR in the proximal airway and submucosal gland ducts. They also showed that the repertoire of ion channels in the ionocyte is uniquely suited to potentially regulate airway pH. However, whether only ionocytes contribute to innate immunity and clearance in the airways remains to be determined; it is possible that other airway cell types, such as ciliated cells, express CFTR at levels that are below the detection limits of scRNAseq yet are functionally important in the pathogenesis of CF lung disease. Deeper knowledge of CFTR cellular physiology in the airway will greatly enhance our understanding of CF pathogenesis, while also informing the cellular targets for gene therapy of CF lung disease. The proposed project will focus on understanding the cellular functions of CFTR in ionocytes and ciliated cells, and whether CFTR expression in each of these cell types is required or sufficient to prevent CF lung disease. Our hypotheses will be tested in genetic ferret models, a species that more accurately reflects human CF lung disease than mice. These studies are possible because of the creation of several new genetic ferret models that can conditionally (in specific cell types) inactivate CFTR expression on a wild-type (WT) background or reactivate CFTR expression on a CF background. Both of these strategies use CreERT2 technologies and enable lineage tracing of the targeted cells in vivo using a fluorescent Cre-reporter. Additionally, we propose to generate a new genetic ferret model in which CFTR is overexpressed specifically in ciliated cells, to test whether high-level ectopic expression therein is sufficient to protect from CF lung disease. Key goals of this project are to: 1) define the contributions of CFTR expression in ionocytes and ciliated cells to CF lung pathogenesis, 2) determine the half-lives of ionocytes and ciliated cells in the CF and non-CF airway, and 3) determine the extent to which CFTR expression in ionocytes and ciliated cells contributes to the regulation of disease-relevant features of the airway surface liquid (ASL) (i.e. volume, antibacterial activity, chloride and bicarbonate transport, and pH) and to mucociliary clearance. Each of these goals draws on the unique ability of the Engelhardt laboratory to genetically engineer transgenic ferrets to temporally regulate CFTR expression in specific cell types and to test these models for CF-relevant functional endpoints in vivo and in vitro. The proposed study is the first to use state-of-art functional genetic approaches in a non-mouse species to tackle difficult cell biology questions relating to CFTR function in the airway and CF lung pathogenesis. This research is expected to significantly enhance the field's ability to develop effective genetic therapies for CF lung disease.