needed before phage therapy becomes the standard of care for treatment of multidrug-resistant infections in CF.

Background: SPL84 is an antisense oligonucleotide drug targeting the CF splicing mutation 3849 +10 kb C-to-T (3849).Full rescue of CFTR activity was demonstrated in human bronchial epithelial cells (hBECs) and human nasal epithelial cells (hNECs) from people who were homozygous and heterozygous for the 3849 +10 kb C-to-T mutation after basal exposure of SPL84 [1].Because SPL84 is an inhaled drug, it reaches the lung epithelial cells through the apical side.To model the in vivo exposure, we analyzed the penetration and effect of SPL84 through the apical side of welldifferentiated hBECs.Methods: hBECs grown in air-liquid interface conditions were treated with SPL84 through the apical side of the cells.Analysis of SPL84 penetration into the epithelial cells and nuclei was analyzed by imaging of Cy5-labled SPL84 using a confocal microscope.Cell nuclei penetration was analyzed by imaging the optical planes positive for nuclear staining (4′,6-diamidino-2phenylindole).Analysis of the function of SPL84 after apical treatment was performed by analyzing CFTR splicing modulation using reverse-transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR and CFTR activity using the Ussing Chambers system.Results: To analyze SPL84 penetration through mucus to the cells, hBECs were grown in PneumaCult medium to generate a pseudo-stratified epithelial layer with a mixture of ciliated and mucus-secreting epithelial cells, leading to secretion of a mucus layer at the apical side of the cells.Cy5-labeled SPL84 was shown to penetrate rapidly through the mucus layer into the epithelial cells and to penetrate efficiently into the epithelial cell nuclei, where it must act to modulate CFTR splicing.Apical treatment of SPL84 in cells from a person homozygous for the 3849-mutation led to correction of the splicing defect and rescue of CFTR activity.Conclusions: The efficient penetration of SPL84 through the mucus layer from the apical side of the cells into the cell nuclei, leading to rescue of CFTR activity, models the exposure in the person's lungs to the inhaled drug and highlights the potential of SPL84, currently in a Phase 1/2 clinical study, to provide a significant clinical benefit for people with the 3849 mutation.