Epithelial Sodium Channel Inhibition in Primary Human Bronchial Epithelia by Transfected siRNA

Abstract

Na(+) absorption and Cl(-) secretion are in equilibrium to maintain an appropriate airway surface fluid volume and ensure appropriate mucociliary clearance. In cystic fibrosis, this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in the absence of functional CFTR protein, which in turn results in deficient cAMP-dependent Cl(-) secretion and predominant Na(+) absorption. It has been suggested that down-regulation of the epithelial sodium channel, ENaC, might help to restore airway hydration and reverse the airway phenotype in patients with cystic fibrosis. We used an siRNA approach to analyze the possibility of down-regulating ENaC function in bronchial epithelia and examine the resulting effects on fluid transport. siRNA sequences complementary to each of the three ENaC subunits have been used to establish whether single subunit down-regulation is enough to reduce Na(+) absorption. Transfection was performed by exposure to siRNA for 24 hours at the time of cell seeding on permeable support. By using primary human bronchial epithelial cells we demonstrate that (1) siRNA sequences complementary to ENaC subunits are able to reduce ENaC transcripts and Na(+) channel activity by 50 to 70%, (2) transepithelial fluid absorption decreases, and (3) these functional effects last at least 8 days. A decrease in ENaC mRNA results in a significant reduction of ENaC protein function and fluid absorption through the bronchial epithelium, indicating that an RNA interference approach may improve the airway hydration status in patients with cystic fibrosis.

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

“…However, the pseudostratified epithelium presents significant barriers to the delivery of nucleic acid based reagents such as plasmids (17, 65), doublestranded oligonucleotides (9,23,36,50), and single-stranded oligonucleotides (9, 23). It is evident that physical barriers, airway secretions, and host defense mechanisms (9,17,23,49,62) significantly hinder the delivery and efficacy of the siRNA reagents. The mucosal surface of the airways and the alveoli elicits immunogenic responses to RNA interference (RNAi) oligonucleotides that confound results (13,31,35,45,54), and some animal studies attempting to deliver siRNAs to the respiratory tract have reported little efficacy (23).…”

“…However, the pseudostratified epithelium presents significant barriers to the delivery of nucleic acid based reagents such as plasmids (17, 65), doublestranded oligonucleotides (9,23,36,50), and single-stranded oligonucleotides (9, 23). It is evident that physical barriers, airway secretions, and host defense mechanisms (9,17,23,49,62) significantly hinder the delivery and efficacy of the siRNA reagents.…”

Changing the Paradigm – Treating the Basic Defect in Cystic Fibrosis

“…This is carried out by small sequences of 20 to 22 bases called Small-interfering RNA (siRNA) that are complementary to the target mRNA. Caci et al [34] showed this approach to be useful in inhibiting the mRNA for all 3 ENaC subunits leading to significant reduction in number of functional ENaC on respiratoary epithelium, thereby impacting fluid absorption. Further, Clark et al [35] reported the effectiveness of GSK2225745, which was designed as a siRNA targeting the alpha subunit of ENaC channel in primary cell assays, but initial attempts at delivering this oligonucleotide in the lungs or nasal cavities of mice did not show any significant change in ENaC function.…”

Ion transporting proteins of human bronchial epithelium

“…The channel is also thought to play an important role in cell volume regulation (Caci et al, 2009). ENaC channel plays crucial Table I.…”