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Gene Therapy (2007) 14, 775780 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt
SHORT COMMUNICATION
Electroporation-mediated transfer of plasmids to the lung results in reduced TLR9 signaling and inammation
R Zhou1, JE Norton1, N Zhang2 and DA Dean1
1Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA and
2Division of Allergy-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Electroporation can deliver DNA efciently and safely to tissues in live animals, including the lung where it causes little inammation or lung injury. In contrast, cationic lipid-mediated gene transfer has been shown to induce an inammatory response caused by unmethylated plasmid CpG residues, which activate the toll-like receptor (TLR9) signaling pathway. As TLR9 is located in the endosomal/ lysosomal compartment, we hypothesized that plasmids do not activate TLR9 during electroporation because they enter the cytoplasm directly through transient pores in the plasma membrane. To test this, plasmids were transfected into kidney epithelial cells overexpressing TLR9 (HEK293-TLR9+) and cells lacking TLR9 (HEK293-TLR9-null). Interleukin (IL)-8 expression, an indicator of TLR9 activation, increased more than 10-fold at 24 h
post-liposome transfection in HEK293-TLR9+ cells, but showed no signicant increase in electroporated cells, compared with untransfected cells. In vivo liposome-mediated gene transfer caused increases in IL-6, IL-12, tumor necrosis factor a and interferon g in mouse bronchial alveolar lavage uid, whereas the levels of these cytokines were more than 10-fold lower by comparison following electroporation. Depletion of alveolar macrophages suggested that this inammatory response is mediated by resident pulmonary epithelial cells. These results suggest that electroporation-mediated gene transfer bypasses the TLR-9 pathway, thus accounting for the low levels of inammation seen with this approach. Gene Therapy (2007) 14, 775780. doi:10.1038/sj.gt.3302936; published online 8 March 2007
Keywords: electroporation; liposomes; lipoplex; inammation; gene delivery; nonviral vector
Toll-like receptors (TLRs) play a critical role in the early innate immune response to invading pathogens by recognizing pathogen-associated molecular patterns. Stimulation of these TLRs activates signaling cascades that lead to induction of a number of pro-inammatory cytokines, including interleukin (IL)-6, IL-8, IL-12, tumor necrosis factor (TNF)-a and interferon (IFN)-g.1 Ten TLRs have been identied in humans and 13 have been identied in mice. Among these TLRs, TLR9 recognizes unmethylated CpG motifs in bacterial DNA,2...