Abstract
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 α and interferon γ 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.
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References
Kaisho T, Akira S . Toll-like receptor function and signaling. J Allergy Clin Immunol 2006; 117: 979–987.
Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 740–745.
Audouy SA, de Leij LF, Hoekstra D, Molema G . In vivo characteristics of cationic liposomes as delivery vectors for gene therapy. Pharm Res 2002; 19: 1599–1605.
Yew NS, Scheule RK . Toxicity of cationic lipid–DNA complexes. Adv Genet 2005; 53PA: 189–214.
Suzuki Y, Wakita D, Chamoto K, Narita Y, Tsuji T, Takeshima T et al. Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity. Cancer Res 2004; 64: 8754–8760.
Tan Y, Li S, Pitt BR, Huang L . The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo. Hum Gene Ther 1999; 10: 2153–2161.
Emerson M, Renwick L, Tate S, Rhind S, Milne E, Painter HA et al. Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid–DNA complexes to ovine lung segments. Mol Ther 2003; 8: 646–653.
Pringle IA, Raman S, Sharp WW, Cheng SH, Hyde SC, Gill DR . Detection of plasmid DNA vectors following gene transfer to the murine airways. Gene Therapy 2005; 12: 1206–1214.
McLachlan G, Stevenson BJ, Davidson DJ, Porteous DJ . Bacterial DNA is implicated in the inflammatory response to delivery of DNA/DOTAP to mouse lungs. Gene Therapy 2000; 7: 384–392.
Yew NS, Cheng SH . Reducing the immunostimulatory activity of CpG-containing plasmid DNA vectors for non-viral gene therapy. Expert Opin Drug Deliv 2004; 1: 115–125.
Yew NS, Zhao H, Przybylska M, Wu IH, Tousignant JD, Scheule RK et al. CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 2002; 5: 731–738.
McMahon JM, Wells KE, Bamfo JE, Cartwright MA, Wells DJ . Inflammatory responses following direct injection of plasmid DNA into skeletal muscle. Gene Therapy 1998; 5: 1283–1290.
Reyes-Sandoval A, Ertl HC . CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol Ther 2004; 9: 249–261.
Dean DA, Machado-Aranda D, Blair-Parks K, Yeldandi AV, Young JL . Electroporation as a method for high-level non-viral gene transfer to the lung. Gene Therapy 2003; 10: 1608–1615.
Machado-Aranda D, Adir Y, Young JL, Briva A, Budinger GRS, Yeldandi A et al. Gene transfer of the Na+,K+-ATPase β1 subunit using electroporation increases lung liquid clearance in rats. Am J Respir Crit Care Med 2005; 171: 204–211.
Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004; 5: 190–198.
Leifer CA, Kennedy MN, Mazzoni A, Lee C, Kruhlak MJ, Segal DM . TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 2004; 173: 1179–1183.
Schmausser B, Andrulis M, Endrich S, Lee SK, Josenhans C, Muller-Hermelink HK et al. Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection. Clin Exp Immunol 2004; 136: 521–526.
Ahmad-Nejad P, Hacker H, Rutz M, Bauer S, Vabulas RM, Wagner H . Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol 2002; 32: 1958–1968.
Hacker H, Mischak H, Miethke T, Liptay S, Schmid R, Sparwasser T et al. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. Embo J 1998; 17: 6230–6240.
Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ et al. Theory and in vivo application of electroporative gene delivery. Mol Ther 2000; 2: 178–187.
Templeton NS, Lasic DD, Frederik PM, Strey HH, Roberts DD, Pavlakis GN . Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol 1997; 15: 647–652.
Maus UA, Koay MA, Delbeck T, Mack M, Ermert M, Ermert L et al. Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice. Am J Physiol Lung Cell Mol Physiol 2002; 282: L1245–L1252.
Suzuki K, Suda T, Naito T, Ide K, Chida K, Nakamura H . Impaired toll-like receptor 9 expression in alveolar macrophages with no sensitivity to CpG DNA. Am J Respir Crit Care Med 2005; 171: 707–713.
Golzio M, Teissie J, Rols MP . Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 2002; 99: 1292–1297.
Sukharev SI, Klenchin VA, Serov SM, Chernomordik LV, Chizmadzhev Yu A . Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys J 1992; 63: 1320–1327.
Li S, Wu SP, Whitmore M, Loeffert EJ, Wang L, Watkins SC et al. Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors. Am J Physiol 1999; 276: L796–L804.
Yasuda K, Kawano H, Yamane I, Ogawa Y, Yoshinaga T, Nishikawa M et al. Restricted cytokine production from mouse peritoneal macrophages in culture in spite of extensive uptake of plasmid DNA. Immunology 2004; 111: 282–290.
Yasuda K, Ogawa Y, Yamane I, Nishikawa M, Takakura Y . Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways. J Leukoc Biol 2005; 77: 71–79.
Gill DR, Smyth SE, Goddard CA, Pringle IA, Higgins CF, Colledge WH et al. Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Therapy 2001; 8: 1539–1546.
Acknowledgements
We thank Gokhan Mutlu, John Christman and Amy Bellmeyer for helpful discussions and technical advice. This work was supported in part by grants HL59956, HL71643 and HL81148 from the National Institutes of Health.
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Zhou, R., Norton, J., Zhang, N. et al. Electroporation-mediated transfer of plasmids to the lung results in reduced TLR9 signaling and inflammation. Gene Ther 14, 775–780 (2007). https://doi.org/10.1038/sj.gt.3302936
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DOI: https://doi.org/10.1038/sj.gt.3302936
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