Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs

An inflammatory response is invariably associated with administration of ge ne transfer complexes composed of cationic lipids and plasmid DNA (pDNA). I n the lung, an influx of neutrophils and elevated levels of several proinfl ammatory cytokines such as TNF-alpha, IFN-gamma, IL-6, and IL-12 characteri ze this dose-dependent response. The induction of these cytokines was shown previously to be due in part to the presence of unmethylated CpG dinucleot ides in the bacterially derived pDNA. We have eliminated 270 of 526 CpG din ucleotides in a reporter plasmid (pCFA-CAT) and tested the inflammatory res ponse to cationic lipid:pDNA complexes containing the modified vector (pGZA -CAT) after intravenous (iv) or intranasal (i.n.) delivery into BALB/c mice . Compared to the unmodified vector, the CpG-reduced pGZA-CAT was found to be significantly less immunostimulatory, as the levels of IL-72, IFN-gamma, and IL-6 in the serum 24 h after iv delivery were reduced by 40 to 75%. Si milar reductions in cytokine levels were also observed in the bronchoalveol ar lavage fluids (BALF) after i.n. administration, while the levels of repo rter gene expression were not affected by the modifications. We have also i nvestigated known inhibitors of the CpG signaling pathways in order to decr ease the inflammatory response. Two such inhibitors, chloroquine and quinac rine, greatly reduced the induction of IL-12 from mouse spleen cells in vit ro and inhibited cytokine production in the lung by approximately 50% witho ut affecting gene expression. These results illustrate that use of a less i mmunostimulatory pDNA vector or inhibitors of CpG immunostimulation can red uce significantly the toxicity associated with cationic lipid:pDNA complexe s thereby increasing the therapeutic index of this synthetic gene transfer vector.