551. The Use of Electroporation To Transfer Plasmids to the Lung Results in Reduced TLR9 Signaling and Inflammation

Abstract

Electroporation is an efficient and safe technique to deliver naked DNA to both cells in culture and in living animals. Application of brief square wave pulses to cells induces transient pores to open in the plasma membrane, allowing DNA to enter the cytoplasm directly. We have used this approach to deliver plasmid DNA to the murine lung and found high level gene expression with almost undetectable inflammation or lung injury. By contrast, cationic lipid mediated gene transfer routinely induces inflammatory responses characterized by increased levels of cytokines. This inflammatory response is caused by unmethylated CpG residues in plasmid DNA, which activate toll-like receptor 9 (TLR9). Since TLR9 has been reported to be located in endosomes, we hypothesized that plasmid DNA does not activate the TLR9 pathway when delivered by electroporation, because electroporation bypasses endocytosis. To test this, plasmids were transfected into HEK293-TLR9 cells and HEK293-Null cells, using electroporation or liposomes. Twenty- four hours later, expression of IL-8 was measured by qPCR. IL-8 mRNA increased more than 10-fold in Lipofectin transfected HEK293-TLR9 cells, but no significant increase was seen in electroporated cells. Neither method caused increases in IL-8 expression in HEK293-Null cells. Western blotting using an antibody against phosph-IRAK1 shows the activation of TLR signaling pathway after cells were transfected by lipofectin, but activation is greatly reduced following electroporation of the same plasmids. To follow this signaling in living animals, 20 μg plasmid DNA was transferred to the lungs of mice by tracheally delivered lipoplex or electroporation and activation of cytokine expression was measured 24 hours later by either qPCR or by Cytometric Bead Array to measure cytokine proteins in BALF. The levels of IL-6, IL-12, and IFN-γ were only slightly higher with electroporation gene transfer compared with DNA only and liposome only. By contrast, liposome/DNA complex caused more than 10-fold increase in these three cytokines compared to electroporation gene transfer. Taken together, these results suggest that electroporation-mediated transfer of DNA appears to bypass the TLR-9 pathway, accounting for the very low levels of inflammation seen in animals.