Abstract Rationale Primary Ciliary Dyskinesia (PCD) is a hereditary genetic disease characterized by impaired ciliary function, leading to severely reduced or absent mucociliary clearance (MCC) and lung damage. Variants in the DNAI1 gene are an established cause of PCD. In patients with biallelic DNAI1 mutations, high speed video microscopy (HSVM) reveals predominately immotile cilia. Currently, no approved therapies directly target the underlying genetic defects of PCD. RCT110 is a DNAI1 mRNA-based therapy developed for inhalation to treat DNAI1 PCD, currently undergoing clinical trials.Three-Dimensional Explant Spheroids (3DE spheroids) is an ex vivo model where original nasal epithelial cells rapidly (within 4 hours) form fully differentiated spheroids with a ciliated outer surface accessible for ciliary function analysis and spheroid motion. The effect of RCT1100 in 3DE spheroids from DNAI1 PCD patients was assessed. Methods 3DE spheroids were generated from nasal brushings obtained from five PCD patients with confirmed biallelic DNAI1 defects. RCT1100 was administered directly into the wells containing spheroids three times a week over two weeks at the following concentrations: 1.25, 2.5, 5, 10 and 20µg/mL. One well per patient was left untreated as a control. Spheroid rotation and rolling was quantified as the percentage of rolling spheroids and their movement speed. Motion was recorded at 10x magnification and 25 frames per second using an Olympus IX83 microscope, and assessed continuously throughout the two weeks treatment period. After six doses the spheroids were harvested for post-treatment ciliary beat frequency (CBF) analysis by High-Speed Video Microscopy (HSVM). Results Treatment of 3DE spheroids with RCT1100 was successful in 3 of 5 patients (60%), while spheroids from 2 patients (40%) were lost due to infection in the wells.HSVM demonstrated a significant increase in CBF after six doses. Median CBF more than doubled, increasing by 107.85% across doses, from 2.8-3.5 Hz pre-treatment to 6.7-6.8 Hz post-treatment (Figure 1) 3DE Spheroid movement:Enhanced ciliary motility resulted in pronounced changes in both spheroid rolling and rotation after six doses.Prior to treatment, the mean movement-speed was 0.1113 µm/sec. The greatest improvement was observed at 10ug/mL after six doses, with mean movement speed of 3.868 µm/sec.Additionally, over 80% of spheroids treated with 10µg/mL displayed a distinct rolling movement pattern. Conclusion This pilot ex vivo study demonstrates robust restoration of ciliary function in 3DE spheroids derived from nasal epithelial cells of DNAI1 PCD patients following RCT110 treatment, supporting its potential as a targeted therapy for DNAI1-related PCD. This abstract is funded by: ReCode Therapeutics
Nygaard et al. (Fri,) studied this question.