Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Abstract

IntroductionLoss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport.MethodsWe investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI). Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections.ResultsNanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%.ConclusionNanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Peripheral localization of the epithelial sodium channel in the apical membrane of bronchial epithelial cells

“…Therefore, ENaC represents an important therapeutic target. Its inhibition with small molecules or other therapeutic agents could bring a beneficial effect to all CF patients, irrespective of their mutations, and possibly to patients with other respiratory diseases (Caci et al., ; Coote et al., ; Hirsh et al., ; Scott et al., ; Shei, Peabody, Kaza, & Rowe, ; Tagalakis et al., ).…”

“…ENaC is considered to be a key protein in the pathogenesis of the (Caci et al, 2009;Coote et al, 2015;Hirsh et al, 2008;Scott et al, 2017;Shei, Peabody, Kaza, & Rowe, 2018;Tagalakis et al, 2018).…”

Silencing E3 Ubiqutin ligase ITCH as a potential therapy to enhance chemotherapy efficacy in p53 mutant neuroblastoma cells

“…In our experiments, we used nanoparticles as a carrier system to deliver the ITCH siRNA in vivo. This was based on our previous findings that the receptor-targeted liposome-peptide-siRNA nanoparticle is an efficient delivery system for ex vivo and in vivo applications [49][50][51][52][53] . The formulation of the nanoparticles contains peptide ME27 which can mediate receptor-specific targeting via its interaction with integrin αvβ3 and αvβ5 on cells.…”

“…Transfection with nanoparticles in vitro. Nanoparticles were formulated as a mixture of Lipid:peptide:siRNA, as described previously 52,57 . In this study, we pre-formulated 3 different liposomes with different concentrations of PEG; 1) DOTMA/DOPE (DD), containing no PEG; 2) AT-1, DD containing 1% PEG; and 3) GK27, DD which contained 5% PEG.…”

Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis

“…The authors showed the promising chance to use this approach as co-adjuvant therapy for CF. Tagalakis et al (2018) developed siRNA nanocomplexes that could mediate silencing of airway epithelial sodium channel (ENaC) with functional correction of epithelial ion transport. Receptor-targeted nanocomplexes (RTNs) are made of cationic liposomes and targeting peptide mixed with siRNA.…”