Effect of Methylated Adenine in Plasmid DNA on Transgene Expression in Mice

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Cationic lipid-mediated transfer of DNA is promising in gene therapy. However, one disadvantage with this approach is the induction of an inflammatory response, which may decrease transgene expression. Recently, we found that plasmid DNA containing N⁶-methyladenine (N⁶-MeA), a bacterium-specific modified base, induced cytokine twice as efficiently as plasmid DNA without N⁶-MeA, when complexed with cationic lipids. Thus, plasmid DNA without N⁶-MeA might express a transgene more efficiently than that containing N⁶-MeA in vivo. To evaluate the effects of adenine methylation on transgene expression in vivo, we injected luciferase-encoding plasmid DNA, complexed with cationic lipids or a cationic polymer, intravenously into mice. When the plasmid DNA-cationic lipid complexes were injected, the luciferase expression from the methylated and unmethylated plasmids was similar, although cytokine was more efficiently elicited by the methylated DNA than the unmethylated DNA. Hydrodynamics-based injections of plasmid DNA–cationic polymer complexes did not induce cytokine, and the luciferase expression from the unmethylated plasmid was slightly lower than that from the methylated plasmid DNA. These results suggest that the presence of N⁶-MeA did not reduce transgene expression in vivo.