| Autor: |
Stefano Castellani, Pieter S. Hiemstra, Anna Diana, Pascal van der Wegen, Francesca Tilesi, Guelnihal Yueksekdag, Joseph Rosenecker, Jamil Aarbiou, Bob J. Scholte, Ruvalic M. Buijs-Offerman, Elena Copreni, Fiorentina Ascenzioni, Piera Assunta Fradiani, Annalucia Carbone, Massimo Conese |
| Rok vydání: |
2012 |
| Předmět: |
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| Zdroj: |
The Journal of Gene Medicine. 14:733-745 |
| ISSN: |
1099-498X |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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