Mucociliary clearance (MCC) is a primary defense mechanism of the human airway, and its dysfunction contributes to the pathophysiology of numerous airway diseases. In primary ciliary dyskinesia (PCD), mutations affecting multiciliated airway epithelial cells disrupt ciliary structure and function. As gene therapy progresses toward clinical application, a key question is how much functional restoration is needed to meaningfully repair MCC. We hypothesized that established structural and functional metrics, modeled to resemble ex vivo ciliated tissues, could define MCC performance in in vitro co-cultures consisting of CCDC40-mutant and wild-type (WT) human bronchial epithelial cells (HBECs). Genetic screening identified two pathogenic allelic variants: c.1620del (p.Ile541Serfs*2) and c.248del (p.Ala83Valfs*84). Co-cultures containing mutant:WT ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 were differentiated at an air–liquid interface for 28 days. Mutant cells were labeled with CellTracker Red and WT cells with CellTracker Green to document initial spatial distribution before and at airlift. We assessed cilia beat frequency (CBF), MCC efficiency, clearance patterning, cilia length, and epithelial cell-type composition. MCC efficiency declined progressively as the proportion of mutant cells increased, with a pronounced functional inflection between the 50:50 and 25:75 mutant:WT ratios. Impairment was characterized by slower clearance velocity, reduced directional coordination, and a sharp loss of overall clearance quality below ~50% WT ciliated cells. Cultures containing ≥90% WT ciliated cells demonstrated strong clearance per beat (CPB; ~8 μm/beat). As WT ciliation declined to ~70–60%, CPB decreased to ~6.8 μm/beat. CPB dropped steeply thereafter, reaching ~2.8 μm/beat at ~20% WT ciliated cells. These data were used to generate a modestly non-linear fit curve that predicts MCC functionality based on WT ciliation. Notably, in 4 of 5 biological replicates, WT CBF exhibited a modest compensatory increase with higher mutant cell proportions. Cilia length differed significantly between genotypes (WT ~7.35 μm; mutant ~5.54 μm). Increased goblet cell abundance was observed in cultures with higher mutant cell ratios, suggesting broader epithelial remodeling in response to ciliary dysfunction. Together, these findings identify a critical threshold of WT cells required to sustain effective MCC in vitro and provide quantitative insight into the degree of cellular correction needed for gene therapy targeting CCDC40-mutant PCD. More broadly, this work establishes a tractable framework for assessing how specific ciliary defects shape MCC dynamics, with potential applicability to other ciliated organ systems. This study was funded by NIH:NHLBI R01 HL153622 (awarded to ALR) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Fitzpatrick et al. (Fri,) studied this question.