Optimization of CFTR-mRNA transfection in human nasal epithelial cells

Background: Cystic fibrosis (CF) is the most common life-threatening inherited disease in the Caucasian population. It is caused by genetic defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR), a cAMP regulated chloride-bicarbonate channel mainly located in the apical membrane of polarized epithelial cells. CFTR is proposed to regulate other proteins, including the epithelial sodium channel (ENaC). Recently, we successfully restored chloride current in CFTR deficient human airway epithelial cells using wtCFTR-mRNA transfection compared to non-CF cells showing similar values. The present study aimed to optimize the wtCFTR-mRNA transfection procedures in primary cultured human nasal epithelial (HNE) cells. Methods: Dose and time dependence experiments were performed. In addition, we investigated the possible impact of the wtCFTR-mRNA transfection on ENaC function in transepithelial measurements. We reduced the wtCFTR-mRNA dose stepwise and determined the minimal concentration of 0.6 μg/cm, which is needed for the most efficient restoration of CFTR function. Furthermore, CFTR expression was evaluated 24, 48 and 72 h after transfection. Results: Using the minimal concentration of 0.6 μg/cm wtCFTR-mRNA we confirmed a positive functional CFTR restoration over a period of 72 h. Biochemical analyses confirmed these findings. Furthermore, we could not find any significant effect on ENaC after the recovery of CFTR by wtCFTR-mRNA transfection. Conclusions: Our data show that wtCFTR-mRNA transfection is an encouraging alternative “gene” therapy in human primary culture.