Introduction: Circular RNAs (circRNAs) are novel non-coding RNAs that regulate gene expression in several human diseases, however their role in cystic fibrosis (CF) has not been explored yet.

Aims: We hypothesize that circRNAs contribute to the pathophysiology of CF. Here we aim to assess their expression and function in CF bronchial epithelium to explore their therapeutic potential in combination with CFTR modulator therapies.

Methods: Three pairs of CF vs non-CF cell lines were studied, CFBE41o- (p.Phe508del/p.Phe508del) vs 16HBE14o-; IB3 (p.Phe508del/p.Trp1282X) vs S9; Cufi-1 (p.Phe508del/p.Phe508del) vs Nuli-1. CircRNA expression was assessed using Arraystar Human Circular RNA Microarray, followed by qRT-PCR validation in CF primary bronchial epithelial cells and lung organoids. Biochemical analyses assessed their circularity and sub-cellular localisation. Functional studies were carried out using siRNAs and over-expression plasmids.

Results: A total of 108 circRNAs were significantly downregulated in CF vs non-CF bronchial epithelial cell lines, while 50 were upregulated. qRT-PCR analyses confirmed downregulation of three circRNAs in primary bronchial epithelial cells, one of which was downregulated in the organoids too. Biochemical analyses confirmed they expressed primarily in the cytoplasm. Overexpression of hsa_circ_0002465, alone or in combination with the newest CFTR modulator Trikafta, increased CFTR anion channel activity in CFBE41o- cells.

Conclusions: Three cytoplasmic circRNAs are downregulated in CF bronchial epithelium. Restoring the expression of hsa_0002465 improves CFTR channel activity, suggesting it could have therapeutic potential as adjuvant of CFTR modulators.