Targeting aENaC with an epithelial RNAi trigger delivery platform for the treatment of cystic fibrosis

Objectives: The development of inhaled epithelial sodium channel (ENaC) inhibitors has been limited by their short duration of action and renal side effects. To enable durable, renal-sparing therapeutic inhibition, we have utilized Arrowhead’s Targeted RNAi Molecule (TRiM^TM) technology to develop epithelial-targeted conjugates comprised of an optimized RNAi trigger against αENaC paired with a ligand (EpL) to the epithelial integrin αvβ6. Methods: Uptake of Cy3-labeled EpL conjugates was evaluated in primary epithelial cells and in vivo. In knockdown studies, rats received intratracheal or inhaled aerosol doses of conjugate; ENaC mRNA and protein expression was analyzed by qPCR and immunohistochemistry. Results: αvβ6 ligands facilitated receptor-mediated, epithelial cell selective uptake of conjugates in vivo. EpL-RNAi conjugates produced deeper, isoform-selective whole-lung αENaC mRNA knockdown at lower exposures than trigger alone and correlated with nearly complete elimination of airway epithelial αENaC protein expression. EpL-RNAi activity was durable, maintaining knockdown for >3 weeks after a single dose and was well-tolerated with no evidence of renal activity. Furthermore, aerosol inhalation studies confirmed that knockdown could be achieved following a single deposited dose of 0.15 mg/kg. Conclusion: EpL-RNAi conjugates employing αvβ6 ligands improve functional delivery of αENaC RNAi triggers to the pulmonary epithelium, producing deep, durable whole-lung mRNA knockdown and reduced airway protein expression. Studies are in progress to define the effects of αENaC knockdown on airway hydration, mucociliary clearance and disease progression in animal models of CF lung disease.