CpG-Dependent Inflammatory Response after Delivery of Lipid/pDNA Complexes to Murine Lungs. (2007)

Bazzani, R. P., Varathalingam, A., Goonetilleke, N., Cheng, S. H., Pringle, I. A., Hyde, S. C. & Gill, D. R.

Molecular Therapy, 15, S388

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A potential problem of gene therapy is the induction of a host immune response after delivery of viral or non-viral gene transfer agents. Our work investigates non-viral vectors, in particular plasmid DNA (pDNA) complexed with Genzyme lipid GL67, administered to airways for the treatment of lung diseases like Cystic Fibrosis (CF). Alton et al. (Lancet 1999; 353:947) and Ruiz et al. (Hum Gene Ther 2001; 12:751) reported 'flu-like' symptoms in CF patients upon administration of GL67/pDNA complexes delivered via aerosol. Additionally, it has been shown that unmethylated CpG motifs present in bacterially derived pDNA have an immunostimulatory effect in mammalian cells. Previously, partial elimination of CpG motifs resulted in decreased levels of pro-inflammatory cytokines (Yew et al. Mol Ther 2000; 1:255). To further study this effect, we constructed pDNA vectors, varying in their content of unmethylated CpG-motifs, to assess the impact on pro-inflammatory cytokine levels. As a model we used intranasal instillation of GL67/pDNA complexes (80µg pDNA/100µl) into the lungs of female BALB/c mice. After 24h, bronchoalveolar lavage fluid (BALF) was taken and analysed by ELISA for TNF-alpha, IFN-gamma, IL-12p40 and IL-12p70, or using a high-throughput multiplex assay for a panel of 23 cytokines (Bio-Plex, Bio-Rad, Hemel Hempstead, UK). Administration of GL67/pDNA complexes with different CpG content had a significant impact on the production of host pro-inflammatory cytokines. Levels of TNF-alpha, IFN-gamma, IL12p40, IL-12p70 plus an additional 6 out of 23 cytokines (IL-6, RANTES, G-CSF, MCP-1, MIP-1beta, and MIP-1alpha) were significantly reduced after instillation with a CpG-free pDNA complex compared with a CpG-containing pDNA complex (p = 0.05, ANOVA/PLSD analysis). These results show that unmethylated CpG motifs in pDNA are associated with an acute inflammatory response resulting in increased expression of various cytokines in the lung. They also show that host immune response against non-viral gene transfer reagents can be minimised by the depletion of CpG-motifs in plasmid DNA. We propose that the depletion of CpG-motifs from clinical plasmid vectors may be an important step forward in improving the safety of many non-viral gene transfer agents

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