Knockdown of ENaCα, as a treatment for Cystic Fibrosis lung disease

Harding-Smith RE, Gill DR, Hyde SC

Molecular Therapy, 19 S1 Abstract


The American Society of Gene and Cell Therapy Annual Conference, Seattle, 2011

Cystic Fibrosis (CF) is a life threatening monogenic disorder caused by mutations in the Cystic Fibrosis transmembrane regulator (CFTR) gene. CFTR, an epithelial chloride channel, also regulates the activity of the epithelial sodium channel (ENaC). Together, CFTR and ENaC control the ionic balance of epithelial surfaces. In the CF lung, the lack of functional CFTR and concomitant ENaC hyper-activity, leads to reduced airway surface liquid (ASL) volume, which in turn leads to impaired mucociliary clearance and repeated rounds of bacterial lung infections and CF lung disease.

Thus, strategies designed to inhibit ENaC function may result in clinical benefit. RNAi interference, mediated by short, double-stranded RNA molecules, can be used to target complementary mRNA sequences for degradation via the RNA Induced Silencing Complex (RISC). ENaC is a heterotrimeric protein comprised of 3 subunits: α, β and γ. While ENaCβ and γ act to maximise channel activity, ENaCα is critical for channel function and is the most highly expressed subunit in human airway epithelial cells. Previously, we have demonstrated that ENaCα-targeted siRNA molecules inhibit the expression of ENaCα mRNA in murine and human cells grown in vitro and following in vivo delivery to the mouse lung. In parallel, we have developed plasmid DNA-based non-viral vectors that direct efficient and long-lasting transgene expression in the murine and human lung.

Here we sought to combine these technologies by developing shRNA expression vectors capable of directing long-lasting in vivo knockdown of ENaCα mRNA levels. To this end, we designed a panel of shRNA molecules with a high degree of sequence identity to both human and murine ENaCα. Two alternative expression cassette designs have been evaluated to direct expression of these shRNAs: a simple polIII promoter/terminator driven shRNA, and a polII promoter expression system where the shRNA is flanked by endogenous human microRNA-30 (mir-30) sequences.

The efficacy of the shRNA expression plasmids was evaluated in vitro using A549 (human lung carcinoma) and M1 (mouse kidney) cells. The plasmids were used to transfect cells with a standardised procedure utilising Lipofectamine2000 as the gene transfer agent. Total RNA was extracted 48 hours post-treatment and ENaCα (human or mouse appropriately) mRNA expression evaluated by quantitative real-time TaqMan RT-PCR using the ∆∆Ct method with GAPDH mRNA levels acting as normaliser. Significant knockdown of human and murine ENaCα (P<0.05 Dunnett’s Multiple Comparison Post Hoc test after significant ANOVA) was observed; the majority of shRNA expression vectors showed typical knockdown efficiencies of 70-80%.

The most potent shRNA expression vectors are being assessed in the mouse lung for their ability to knockdown ENaCα mRNA expression in vivo. In conclusion, ENaCα targeted shRNA can be used to knockdown both human and mouse ENaCα mRNA. Further work is needed to assess the functional consequences of inhibiting ENaC following shRNA vector delivery to the lung.