Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells.
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.
Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands.
Jamil
J
Aarbiou
Elena
E
Copreni
Ruvalic M
RM
Buijs-Offerman
Pascal
P
van der Wegen
Stefano
S
Castellani
Annalucia
A
Carbone
Francesca
F
Tilesi
Piera
P
Fradiani
Pieter S
PS
Hiemstra
Guelnihal
G
Yueksekdag
Anna
A
Diana
Joseph
J
Rosenecker
Fiorentina
F
Ascenzioni
Massimo
M
Conese
Bob J
BJ
Scholte
D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism* / Q000821:virology; D053503:Epithelial Sodium Channels / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D023061:Gene Order; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000821:virology; D014161:Transduction, Genetic
D053503:Epithelial Sodium Channels; D034741:RNA, Small Interfering