The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo

We have previously reported that intravenous administration of cationic lip id-protamine-DNA complexes (LPD) induces production of large quantities of proinflammatory cytokines that are toxic and cause inhibition of transgene expression. Cytokine induction appears to be mediated by the unmethylated C pG sequences since methylation of plasmid DNA significantly decreases the c ytokine levels. In this study, the inhibitory role of CpG in lipid-mediated gene transfer was further investigated using chemically well-defined, CpG- containing oligodeoxynucleotides (ODNs), Injection (intravenous) of ODNs fo rmulated in LPD into mice triggered production of proinflammatory cytokines including interferon gamma and TNF-alpha. The potency of CpG-containing OD Ns in cytokine induction was affected by its flanking sequences and was sig nificantly reduced when CPG was methylated, Preinjection of ODN-containing LPD led to inhibition of transgene expression in lungs after a subsequent i njection of LPD containing plasmid expression vector with luciferase gene. The degree of inhibition correlated with the levels of ODN-triggered cytoki nes. Finally, intraperitoneal injection of dexamethasone suppressed LPD-ind uced cytokine production, and led to significantly higher levels of transge ne expression on both first and second injection. These studies suggest tha t mutation of potent CpG motifs in plasmid DNA together with the use of imm une suppression agent may represent an effective approach to improve cation ic lipid-mediated gene transfer to the lung.