Inflammation plays a major role in the progressive pathology of cystic fibrosis (CF), and is generally thought to be a response to increased microbial colonization of CF lungs. However, recent studies involving normal and CFTR-mutant ferrets raised under broad-spectrum antibiotics show robust inflammation in the CF lung despite the absence of bacterial pathogens. Moreover, while the revolutionary class of CFTR modulators improve lung function and reduce exacerbations, they are less successful in mitigating inflammation of the CF lung. These findings raise the possibility that inflammation, and perhaps other pathogenic features of CF, are maintained by elements that emerge in the disease but then drive progression independent of CFTR activity. An analogous scenario may be operating in chronic obstructive pulmonary disease (COPD), where inflammation and disease progression continues despite smoking cessation. In COPD, recent studies have shown a strong correlation (p<10-16) between the emergence of pro-inflammatory, small airway epithelial cells and the disease itself. These pathogenic variants are also present, albeit at low levels, in control patients without COPD and in fetal lung. A similar analysis of CF lungs has revealed them to be inundated by pathogenic stem cell variants highly related to those seen in COPD, along with two novel, hyperinflammatory variants not previously identified in COPD lungs. We hypothesize that these CF stem cell variants play key roles in the progression of CF, and represent pathogenic elements of this disease triggered by, and yet independent of, the CFTR genotype. To test this hypothesis and extend our understanding of the potential significance of these variants in CF disease processes, we will, in three specific aims, 1) identify key inflammatory drivers in the three, hyperinflammatory human CF variants using CRISPR-Cas9-directed mutations and xenograft models, 2) test the dependence of the pro-inflammatory phenotype of these three variants found in CF patients on CFTR activity using gene complementation and CFTR-modulating drugs such as ivacaftor, elexacaftor, and tezacaftor, and 3) exploit our recently developed methods for cloning ferret airway stem cells to determine the dynamics of the pathogenic variants in a ferret conditional model of CF progression. We anticipate that these studies will provide context and insight into the contributions of variant stem cells that dominate CF lungs, assess the impact of the new CF therapeutics on the pathogenic features of these cells, as well as identify nodal genes in the inflammatory signatures of these variants whose suppression could be of therapeutic benefit to these patients.