Electroporation-mediated transfer of plasmids to the lung results in reduced TLR9 signaling and inflammation.

Electroporation can deliver DNA efficiently and safely to tissues in live animals, including the lung where it causes little inflammation or lung injury. In contrast, cationic lipid-mediated gene transfer has been shown to induce an inflammatory 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 significant increase in electroporated cells, compared with untransfected cells. In vivo liposome-mediated gene transfer caused increases in IL-6, IL-12, tumor necrosis factor alpha and interferon gamma in mouse bronchial alveolar lavage fluid, whereas the levels of these cytokines were more than 10-fold lower by comparison following electroporation. Depletion of alveolar macrophages suggested that this inflammatory 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 inflammation seen with this approach.