Lentiviral shRNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells

BACKGROUND: Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. Here we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. METHODS:Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a VSV-G pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the alpha subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down regulation was assayed by real time PCR, membrane potential assay, western blotting, short-circuit currents, and fluid absorption. Off-target effects were investigated by a lab-on-a-chip QPCR array. RESULTS:Transduction to near one hundred percent efficiency of H441, CFBE and HBEC-ALI was achieved by addition of the LV vector prior to differentiation and polarization. Transduction resulted in inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP induced secretion responses was observed in HBEC-ALI. Production of interferon alpha and pro-inflammatory cytokine mRNA, indicating TLR3 or RISC mediated off-target effects, were not observed in HBEC-ALI transduced with this vector. CONCLUSIONS:We have established a generic method to study the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Downregulation of ENaCα by lentiviral shRNA expression vectors as shown here in the absence of off-target effects has potential therapeutic value in the treatment of cystic fibrosis