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Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo.

Authors :
Griesenbach, Uta
Kitson, Chris
Garcia, Sara Escudero
Farley, Raymond
Singh, Charanjit
Somerton, Luci
Painter, Hazel
Smith, Rbecca L.
Gill, Deborah R.
Hyde, Stephen C.
Yu-Hua Chow
Hu, Jim
Gray, Mike
Edbrooke, Mark
Ogilvie, Varrie
MacGregor, Gordon
Scheule, Ronald K
Cheng, Seng H.
Caplen, Natasha J.
Alton, Eric W. F. W.
Source :
Respiratory Research. 2006, Vol. 7, p26-15. 15p.
Publication Year :
2006

Abstract

Background: The cationic lipid Genzyme lipid (GL) 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Methods: Anti-lacZ and ENaC (epithelial sodium channel) siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. Results: In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. Conclusion: This study suggests that although siRNAs and asODNs can be developed to inhibit gene expression in culture systems and certain organs in vivo, barriers to nucleic acid transfer in airway epithelial cells seen with large DNA molecules may also affect the efficiency of in vivo uptake of small nucleic acid molecules. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
14659921
Volume :
7
Database :
Academic Search Index
Journal :
Respiratory Research
Publication Type :
Academic Journal
Accession number :
30737531
Full Text :
https://doi.org/10.1186/1465-9921-7-26