Lentiviral-Mediated ENaCα Knockdown, as a Treatment for Cystic Fibrosis Lung Disease

Harding-Smith RE, Gill DR, Hyde SC

Molecular Therapy

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The American Society of Gene and Cell Therapy Annual Conference, Salt Lake City, 2013

Cystic Fibrosis (CF) is a life threatening monogenic disorder caused by mutations in the Cystic Fibrosis transmembrane regulator (CFTR) gene. CFTR is an epithelial chloride channel that regulates the activity of the epithelial sodium channel (ENaC) in order to maintain the ionic balance of airway epithelial surfaces.

However, in the CF lung, the lack of functional CFTR leads to ENaC hyper-activity and concomitant reduction in airway surface liquid (ASL) volume. This in turn leads to impaired mucociliary clearance and repeated rounds of bacterial lung infections, manifesting as CF lung disease. Thus, strategies designed to inhibit the excessive ENaC function in CF may result in clinical benefit. One such strategy, RNA interference (RNAi), is mediated by short, double-stranded RNA molecules, and 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. We sought to combine these technologies by developing short hairpin RNA (shRNA) expression vectors capable of directing long-lasting in vivo knockdown of ENaCα mRNA. These expression vectors are designed to allow easy insertion of various promoter elements, transgenes and RNAi constructs for knockdown optimisation. Non-viral delivery of these vectors to human and mouse cell lines resulted in significant knockdown of ENaCα (61.4%, p<0.001), however, this method did not result in a long-lasting effect. Here we have tested a panel of shRNA molecules, with 100% sequence identity to human ENaCα, in a VSV-G pseudotyped Lentiviral shRNA expression system.

The efficacy of these viruses was evaluated 72 hours post-transduction, in A549 (human lung carcinoma) cells, and highly efficient knockdown of hENaCα (>80%, p<0.05) was observed. In conclusion, ENaCα-targeted shRNA Lentiviral vectors can be used to knockdown human ENaCα mRNA. Further work in primary cell cultures, grown at the air-liquid interface, is needed to mimic lung morphology and to assess the functional consequences of inhibiting ENaC following shRNA vector delivery.