Repeated siRNA application is a precondition for successful mRNA γENaC knockdown in the murine airways

The volume of the airway surface liquid is regulated by Na+ absorption and Cl− secretion by the respiratory epithelium. In cystic fibrosis, Na+ hyperabsorption caused by the absence of functional CFTR protein leads to an altered airway surface liquid composition and finally to a deteriorated mucociliary clearance. It has been suggested that down regulation or inhibition of the amiloride-sensitive epithelial Na+ channel (ENaC) could restore the disrupted airway hydration. Therefore, targeting ENaC by RNA interference could be of therapeutic relevance. In this context, we investigated whether RNAi could lead to a reduction in γENaC expression in epithelia in vitro and in vivo in mice. Transfection of cells with specific siRNA sequences for γENaC subunit reduced expression to ∼10% relative to control. For in vivo experiments, siRNA sequences specific for the γENaC subunit were administered to the murine nasal cavity and, 72 h later the animals were killed. In the first approach, only a single application of naked siRNA was given. In the second approach, repeated siRNA applications were performed. The single application of siRNA sequences had no effect on mRNA content of the targeted γENaC subunit, whereas repeated siRNA application resulted in a significant reduction in γENaC mRNA in the respiratory tissue. We conclude that repeated siRNA application is necessary for γENaC knockdown in the murine airways.