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

Aristides Tagalakis, MM Mynue, Rositsa Ivanova, H Chen, CM Smith, AM Aldossary, LZ Rosa, Dale Moulding, Josephine Barnes, Konstantinos Kafetzis, Stuart Jones, Deborah Baines, Guy Moss, Christopher O'Callaghan, Robin McAnulty, Stephen Hart

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Abstract

Introduction Loss 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. Methods We 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 (V t), short circuit current (I sc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Results Nanocomplexes 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 V t, the amiloride-sensitive I sc 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%. Conclusion Nanoparticle-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.

Original languageEnglish
Pages (from-to)847-856
Number of pages10
JournalThorax
Volume73
Issue number9
Early online date10 May 2018
DOIs
Publication statusPublished - 1 Sep 2018

Keywords

  • ALI
  • nanoparticle
  • ENaC
  • cilia
  • ASL
  • cystic fibrosis
  • airway epithelia
  • lung physiology
  • airway epithelium
  • nebuliser therapy

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    Tagalakis, A., Mynue, MM., Ivanova, R., Chen, H., Smith, CM., Aldossary, AM., Rosa, LZ., Moulding, D., Barnes, J., Kafetzis, K., Jones, S., Baines, D., Moss, G., O'Callaghan, C., McAnulty, R., & Hart, S. (2018). Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung. Thorax, 73(9), 847-856. https://doi.org/10.1136/thoraxjnl-2017-210670