Marika Drescher

Background: Air-liquid interface (ALI) cultures of patient-derived primary nasal epithelial cells (pNEC) can support diagnostics and in vitro phenotyping of primary ciliary dyskinesia (PCD); however, low cell yield remains a limitation. We aimed to establish and optimize conditionally reprogrammed cell (CRC) technology for pNEC from individuals with PCD and use it as a diagnostic tool for inconclusive cases and for pharmacological testing. Methods: pNEC from 36 individuals (8 healthy controls (HC), 26 PCD, 2 PCD candidates) were expanded using CRC and differentiated at ALI. Ciliary beat frequency (CBF), mucociliary transport velocity (MCTV), and culture morphology were assessed. Further, we assessed the effect of the β-receptor agonist clenbuterol on cilia function in pNEC of HC, PCD (DNAH11, CCDC40) and PCD candidates harboring biallelic DNAH1 variants. Results: CRC expansion demonstrated a >90% success rate, yielding high cell counts (mean 9.65 × 10⁶). Differentiation at ALI resulted in pseudostratified ciliated pNEC cultures. CBF was comparable between native and cultured pNEC. MCTV was significantly reduced in PCD vs. HC cultures (p < 0.0001). Clenbuterol significantly increased CBF in HC and DNAH1, but not in CCDC40 or DNAH11 cultures. MCTV was significantly increased in HC by clenbuterol treatment. Conclusions: CRC technology provides a scalable platform for the expansion of pNEC that recapitulate in vivo ciliary function. By enabling genotype-specific pharmacological screening, CRC may serve as a tool for functional diagnostics and precision medicine in PCD.

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations.