Targeted mRNA restoration of ciliary function in DNAI1-related primary ciliary dyskinesia: ex vivo rescue in patient-derived nasal spheroids—a proof-of-concept study

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, defective mucociliary clearance, and progressive lung disease. Pathogenic variants in the DNAI1 gene are a well-known cause of PCD. Currently, no approved therapies address the underlying genetic defect. RCT1100 is an inhaled mRNA therapy encoding DNAI1 currently under clinical development. This proof-of-concept study aims to evaluate the functional effects of RCT1100 in a rapid three-dimensional explant spheroid (3DE-S) model comprising apical-out, primary nasal epithelial cells derived from patients with DNAI1-PCD. 3DE-S were generated from nasal brushings of five patients with confirmed biallelic disease causing variants in DNAI1 . RCT1100 was administered from day 5 directly to culture wells three times weekly for 2 weeks. Spheroid motility was assessed throughout treatment by quantifying the proportion of moving spheroid rolling and their movement speed. Following six doses, spheroids were harvested for high-speed video microscopy for assessment of ciliary beat frequency. Evaluable data were obtained from three of five patient samples: two samples were excluded one due to contamination and one as a result of technical difficulties when producing videos for high-speed video microscopy. After six doses of RCT1100, ciliary beat frequency increased from a baseline range of 2.8–3.5 Hz to 6.7–6.8 Hz ( p = 0.033) postharvesting. Mean spheroid movement speed increased from 0.11 µm/s to 3.87 µm/s ( p = 0.0002) following dosing with 10 µg/mL RCT1100, with more than 80% of spheroids exhibiting coordinated rolling motion pattern. The 3DE-S model provides a feasible platform for assessing targeted therapies in PCD. In this proof-of-concept study, RCT1100 was associated with functional improvements in DNAI1-derived spheroids, warranting further investigation in larger studies and supporting the use of spheroid-based systems as translational tools for precision medicine in PCD. Trial registration EU Clinical Trials Register [EUCTR]: 2023–510521-14-00, ClinicalTrials.gov ID: NCT06633757, registration date 07-oct-2024.