Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells

Jamil Aarbiou, Elena Copreni, Ruvalic M. Buijs-Offerman, Pascal van der Wegen, Stefano Castellani, Annalucia Carbone, Francesca Tilesi, Piera Fradiani, Pieter S. Hiemstra, Guelnihal Yueksekdag, Anna Diana, Joseph Rosenecker, Fiorentina Ascenzioni, Massimo Conese, Bob J. Scholte

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Background: Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. In the present study, we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. Methods: Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the α subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down-regulation was assayed by the real-time polymerase chain reaction (PCR), membrane potential assay, western blotting, short-circuit currents and fluid absorption. Off-target effects were investigated by a lab-on-a-chip quantitative PCR array. Results: Transduction to near one hundred percentage efficiency of H441, CFBE and HBEC-ALI was achieved by the addition of the LV vector before differentiation and polarization. Transduction resulted in the inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC-dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP-induced secretion responses was observed in HBEC-ALI. The production of interferon α and pro-inflammatory cytokine mRNA, indicating Toll-like receptor 3 or RNA-induced silencing complex mediated off-target effects, was not observed in HBEC-ALI transduced with this vector. Conclusions: We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis.

Original languageEnglish
Pages (from-to)733-745
Number of pages13
JournalJournal of Gene Medicine
Volume14
Issue number12
DOIs
Publication statusPublished - Dec 2012

Fingerprint

Sodium Channels
Small Interfering RNA
Epithelial Cells
Air
Cystic Fibrosis
RNA Interference
Down-Regulation
RNA-Induced Silencing Complex
Toll-Like Receptor 3
Epithelial Sodium Channels
Messenger RNA
Vesicular Stomatitis
Membrane Potentials
Interferons
Electrolytes
Real-Time Polymerase Chain Reaction
Glycoproteins
Western Blotting
Cytokines
Viruses

Keywords

  • Cell biology
  • Cystic fibrosis
  • Lentiviruses
  • Respiratory medicine
  • RNA interference

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Molecular Medicine
  • Genetics(clinical)
  • Drug Discovery

Cite this

Aarbiou, J., Copreni, E., Buijs-Offerman, R. M., van der Wegen, P., Castellani, S., Carbone, A., ... Scholte, B. J. (2012). Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells. Journal of Gene Medicine, 14(12), 733-745. https://doi.org/10.1002/jgm.2672

Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells. / Aarbiou, Jamil; Copreni, Elena; Buijs-Offerman, Ruvalic M.; van der Wegen, Pascal; Castellani, Stefano; Carbone, Annalucia; Tilesi, Francesca; Fradiani, Piera; Hiemstra, Pieter S.; Yueksekdag, Guelnihal; Diana, Anna; Rosenecker, Joseph; Ascenzioni, Fiorentina; Conese, Massimo; Scholte, Bob J.

In: Journal of Gene Medicine, Vol. 14, No. 12, 12.2012, p. 733-745.

Research output: Contribution to journalArticle

Aarbiou, J, Copreni, E, Buijs-Offerman, RM, van der Wegen, P, Castellani, S, Carbone, A, Tilesi, F, Fradiani, P, Hiemstra, PS, Yueksekdag, G, Diana, A, Rosenecker, J, Ascenzioni, F, Conese, M & Scholte, BJ 2012, 'Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells', Journal of Gene Medicine, vol. 14, no. 12, pp. 733-745. https://doi.org/10.1002/jgm.2672
Aarbiou, Jamil ; Copreni, Elena ; Buijs-Offerman, Ruvalic M. ; van der Wegen, Pascal ; Castellani, Stefano ; Carbone, Annalucia ; Tilesi, Francesca ; Fradiani, Piera ; Hiemstra, Pieter S. ; Yueksekdag, Guelnihal ; Diana, Anna ; Rosenecker, Joseph ; Ascenzioni, Fiorentina ; Conese, Massimo ; Scholte, Bob J. / Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells. In: Journal of Gene Medicine. 2012 ; Vol. 14, No. 12. pp. 733-745.
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AU - van der Wegen, Pascal

AU - Castellani, Stefano

AU - Carbone, Annalucia

AU - Tilesi, Francesca

AU - Fradiani, Piera

AU - Hiemstra, Pieter S.

AU - Yueksekdag, Guelnihal

AU - Diana, Anna

AU - Rosenecker, Joseph

AU - Ascenzioni, Fiorentina

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AU - Scholte, Bob J.

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