Background: Inherited primary ciliary dyskinesia (PCD) is rare (1:7,500) and heterogeneous (>50 genes). Abnormal cilia preclude mucociliary clearance causing respiratory symptoms, multiple infections quickly leading to bronchiectasis if untreated. Primary airway cell senescence and limited patients? samples hinders experimental modelling of environmental factors (e.g., infection) and therapeutics.

Aim: To produce a BCi-NS1.1 DNAH5 knock out (KO) ?static PCD model? to circumvent need for primary samples.

Method: The human bronchial epithelial BCi-NS1.1 cell line (hTERT immortalized) differentiates and widespread ciliates at air-liquid interface (ALI). CRISPR/Cas9 gene editing of DNAH5 exon 2 was carried out by microfluidic injection of single cell nuclei. PCR-Sanger sequencing of clone gDNA confirmed bi-allelic indels causing functional KO. BEAT-PCD advocated ERS PCD diagnostics [1] were undertaken on expanded cell lines on ALI-cultures from week 4.

Results: Original BCi-NS1.1 cultures were 52% ciliated (normal CBF=14 Hz, SD±3) and ciliary beat pattern coordinated/ some rotating. Ultrastructure was normal (>90% microtubules and >96% dynein arms), 46-PCD gene panel negative and anti-DNAH5 positive by immunofluorescence (IF) labelling. Proteomics identified 39 cilia proteins including DNAH5. CRISPR editing achieved a BCi-NS1.1 DNAH5 KO model with widespread static cilia, class 1a TEM defects (68% outer and 62% inner dynein arms were absent or short) and absent anti-DNAH5 IF labelling in cilia.

Conclusion: This ?static? PCD model will allow scaled-up infection, treatment and molecular therapy research.

[1] Shoemark A. et al., Eur Respir J. doi: 10.1183/13993003.01066-2019