Targeting of specific miR-145-5p or miR-223-3p binding sites restores CFTR activity in CF BECs

Background. Accumulating evidence indicates that CFTR modulators can be effective for CF sufferers. However, in vivo the microRNAs that regulate CFTR are increased in the CF lung milieu and this can nullify the beneficial effects of CFTR modulators. Methods. A panel of target site blockers (TSBs) designed against the CFTR 3’untranslated region (UTR) were tested for their ability to increase CFTR expression by CFTR 3’UTR luciferase assay and western blot. The effect of two lead TSBs on CFTR activity were tested alone or in combination with selected CFTR modulators in four separate p.Phe508del/p.Phe508del in vitro and ex vivo models (CFBE41o-, Cufi-1, primary bronchial epithelial cells, iPSC-derived lung organoids). Respirable poly-lactic-co-glycolic acid (PLGA) nanoparticles encapsulating the TSBs were formulated and tested. Findings. TSBs that target the miR-145-5p or miR-223-3p binding sites at positons 298-305 and 166-173 in the CFTR 3’UTR, respectively, increased CFTR expression and CFTR anion channel activity, and enhance the effects of Lumacaftor/Ivacaftor or Lumacftor/Tezacaftor in CF BECs. PLGAs encapsulating the TSBs promote CFTR expression in primary BECs and retain their biophysical characteristics following nebulization. Interpretation. Alone or in combination with CFTR modulators, CFTR-targeting TSBs encapsulated in PLGA nanoparticles and nebulized to the lung could overcome microRNA-mediated inhibition of CFTR in CF bronchial epithelium. This strategy represents a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung. 12 samples of primary bronchial epithelial cells were analysed, six from children with cystic fibrosis and six from children without cystic fibrosis. Each sample was treated as a separate biological replicate and microRNAome expression was compared between cystic fibrosis and non-cystic fibrosis groups. Analysis was performed on all samples by RT-PCR with single technical replicates.