Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis

Abstract

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis.

The epithelial sodium channel (ENaC) as a therapeutic target for cystic fibrosis

“…This may be dose-limiting for ENaC inhibiting drugs, mainly traditional small-molecule global ENaC blockers. However, newer approaches which either exploit innate regulatory mechanisms specific to the airway epithelial milieu [53], or tissue-specific targeting may enhance efficacy in the lung while simultaneously reducing systemic effects [54,55], thus minimizing the risk of hyperkalemia and other off-target drug effects. Furthermore, ongoing development of new small molecule direct ENaC inhibitors which do not cause hyperkalemia are promising (AZD5634; clinicaltrials.gov: NCT02950805).…”

“…While the β-subunit has been shown to be regulator of channel activity [6,58], the α-subunit appears to be critical for full channel function [77,78]. Thus, molecular knockdown of α-ENaC by multiple oligonucleotide therapies has repeatedly shown to reduce ENaC expression and channel activity in vitro and in vivo [55,79–84]. These approaches have the potential to overcome challenges which have limited the utility of other ENaC-targeted therapeutics such as specificity, durability, and efficacy.…”

“…Identifying siRNAs to inhibit ENaC in the airways selectively has been of great interest in the past decade, with several independent groups developing various siRNAs against ENaC [55,82,83,96], or regulators of ENaC [97]. The latter study utilized a large-scale screen to identify candidate genes which may be involved in the regulation of ENaC, particularly ENaC activators.…”

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

“…In the latter study, monthly aerosol administration of DNA/liposomes was sufficiently effective to produce significant improvements in lung function. Non-viral vectors are usually based on cationic lipids [16,17] or polymers [18], encapsulating plasmid DNA or siRNA species. Unlike most viral vectors, non-viral vectors lack an active mechanism to import their genome into the nucleus of targeted cells, although passive import features can be added by the incorporation of tissue-specific DNA nuclear import signals found in certain promoter sequences, including the lung-specific SP-C promoter [19].…”

“…Unlike most viral vectors, non-viral vectors lack an active mechanism to import their genome into the nucleus of targeted cells, although passive import features can be added by the incorporation of tissue-specific DNA nuclear import signals found in certain promoter sequences, including the lung-specific SP-C promoter [19]. Non-viral vectors can also be combined with targeting peptide moieties to promote cell specificity and targeting [17], although in doing so, some of the advantages of a non-viral vector begin to be eroded.…”