Defining functional correction thresholds in primary ciliary dyskinesia for effective gene therapies

Abstract

ABSTRACT Rationale Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by defective motile cilia and impaired mucociliary clearance. Mutations in CCDC40 disrupt axonemal organization, resulting in dyskinetic or immotile cilia. While emerging therapies may restore function in only a subset of cells, the functional consequences of mixed populations of mutant and healthy cilia are not well understood. Objectives To determine how defined mixtures of CCDC40-deficient and wild-type ciliated cells influence mucociliary transport. Methods Human bronchial epithelial cells were combined at varying ratios of CCDC40-deficient and wild-type cells and differentiated to model heterogeneous epithelia. High-speed video microscopy and particle-tracking algorithms were used to assess ciliary motion and mucus transport and combined with electron microscopy to evaluate cilia ultrastructure. Measurements and Main Results Airway differentiation was largely preserved with marked ultrastructural defects observed in CCDC40 cells, including multiple central centrioles, absent inner dynein arms, and basal body misorientation. Ciliated surface coverage decreased, and goblet coverage increased with higher mutant representation. Mucociliary transport declined nonlinearly, with speeds dropping from ∼56 µm/s (100% WT) to ∼9 µm/s in 100% mutant cultures. Clearance-per-beat and flow coordination decreased sharply with rising mutant burden. Modeling revealed that transport efficiency was equivalent to that recorded in ex vivo human tissues and plateaued when ∼75% of the ciliated population was WT. Conclusions Together, these findings define a PCD-specific functional correction threshold and show that effective therapy must overcome the disruptive biomechanical and cellular influences of mutant epithelial cells, providing a quantitative benchmark to guide gene-therapy design and clinical translation.

The authors' abstract, as published at the source. European Respiratory Journal, 2026 · DOI ↗