Mucus obstruction is a key pathogenic step in many major human ainways diseases, including COPD and CF. As part of the drug discovery process, it is extremely valuable to have a small animal model that will accelerate the transition from in vitro studies of target identification/validation to eariy proof-of-concept studies in humans. The (3ENaC mouse has been engineered to exhibit many of the features of muco- obstructive human airways diseases, expressing a disease-initiating transgene (PENaC) that drives ainA/ay surface dehydration. The ainways dehydration of the PENaC mouse triggers a sequence of intraluminal airways mucus plaque/plug formation, airways inflammation with macrophage activation, ain/vays remodeling, bacterial infection, and emphysema. In Project II, we propose to contribute to the tPPG goals and needs for drug development as follows: 1) test in vivo the novel biophysical/biochemical formulation of a two-gel mucus clearance system, focusing on relationships between the rate of delivery of hypertonic saline (HS) and mucus clearance responses; 2) develop novel measures of mucus properties that will serve as validated biomarkers to predict the magnitude of the ain/vay mucus burden, i.e., our therapeutic target, utilizing PENaC mouse lines of differing ainways Na+ transport/ainway surface dehydration; 3) test the hypothesis that secreted mucins (MUC5AC, MUCSB) are therapeutic targets with favorable risk:benefit ratios by genetic studies of MUC5AC and MUCSB -/- crosses with PENaC mice and pharmacologic (novel ketolides/macrolides) approaches; and 4) test the hypothesis that bacterial infection of mucus-obstructed ainways can be prevented or reversed by hydration/mucolytic therapies. Thus, Project II should provide important in vivo data as to the validity of our novel two-gel model of mucus transport, identify biomarkers for pharmacodynamic studies of mucus modification to speed drug development in both animals and the clinic, and assist Core A to identify relevant targets and novel therapies of muco-obstructive lung disease. RELEVANCE (See instructions): Muco-obstructive airways diseases affect > 1S M Americans, both COPD and CF subjects. Drug development has been slow, due to lack of theoretical understanding of mucus clearance in health and how it fails in disease, and lack of a small animal model for the drug development process. Project II will test in such a model important concepts, and identify biomarkers, and targets, and indications for such therapy. Project II has the potential to greatly improve the treatment of patients with this unmet medical need. PROJECT/PERFORIVIANCE SITE(S) (if additional space is needed, use Project/