Contribution of Plasmid DNA to Inflammation in the Lung after Administration of Cationic Lipid:pDNA Complexes

Abstract

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

“…[39][40][41] Therefore, there may be considerable utility in finding an intravenous formulation for PEI which can deliver small quantities of plasmid not eliciting inflammatory responses, can be administered in lower volumes and infusion rates, and is effective in the presence of physiological salt and protein concentrations. The addition of an appropriate concentration of soluble protein such as serum albumin to PEI and plasmid DNA in PBS meets these needs for transfection of lung tissue via intravenous administration.…”

Bacterial DNA is implicated in the inflammatory response to delivery of DNA/DOTAP to mouse lungs

“…It is more likely that other factors involved in the inflammatory cascade in the mice are unaffected by the methylation status of the plasmid DNA and may play a more important role in the overall inflammatory environment. In the period during which this manuscript was under revision a paper was published 6 describing similar studies in mice with analysis at day 1 after administration. They also noted that despite differences in cytokine levels between MpDNA:lipid and UMpDNA:lipid treated animals there was no significant difference in the inflammation score between the two groups when histopathological analysis was performed.…”

Chitosan as a nonviral gene delivery system. Structure–property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo

“…44,45 The second-generation cationic lipid GL-67 displayed a higher transgene expression in an initial study, 46 but this result has not been confirmed in later studies, where maximal gene expressions comparable to those of DOTMA and UPC have been reported. 47 The high efficiency of PEI may be due to its high buffering capacity in the endosomal compartment, which leads to positively charged ions being trapped by the amines in PEI, enhanced osmolarity and subsequent endosomal rupture and escape into the cytoplasm. 18,27 The high efficiency may also be due to enhancement of transgene transport from the cytoplasm to the nucleus.…”