Complete but not partial reduction of plasmid CpG content reduces the inflammatory response associated with delivery of GL67/pDNA complexes to the mouse lung. (2006)

Pringle, I. A., Abdullah, S., Lawton, A. E., Varathalingam, A., Yew, N. S., Cheng, S. H., Gill, D. R. & Hyde, S. C.

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Nonviral gene therapy is being developed for Cystic Fibrosis (CF) lung disease. However, the level and duration of expression achieved from current vectors is poor and is associated with an level and duration of expression achieved from current vectors is poor and is associated with an detection of CpG dinucleotide motifs present in plasmid DNA by toll-like receptor 9. The most direct approach to overcome this, is to reduce or completely remove CpGs from plasmid vectors. In addition to our standard plasmids, we have developed a second generation of plasmids with a 40 50% reduction in CpGs. Furthermore, third generation plasmids have been constructed with zero CpG motifs. Luciferase expressing versions of all three generations of plasmid were complexed with Genzyme Lipid 67 and delivered to the mouse lung via nasal instillation (BALB/c, n=6-10, 80 µg pDNA/100µl) At 24-hours post-dosing, bronchoalveolar lavage fluid (BALF) was collected for analysis of total cells, IL-12, IFN-gamma and TNF-alpha. Compared to untreated controls, mice that received first generation (pCIKLux) complexes had elevated levels of IL-12, IFN-gamma and TNF-alpha and cell infiltrates. Delivery of second generation, CpG-reduced plasmids did not significantly reduce any of the four markers of lung inflammation relative to pCIKLux. However, following delivery of third generation, CpG-free plasmids, levels of inflammatory cytokines and numbers of cells in the BALF were indistinguishable from untreated control mice. Moreover, luciferase activity in the lung was 10-fold higher from these plasmids compared to first or second generation plasmids at 24-hours post-dosing with expression persisting for at least 60 days. Together, these results suggest that zero-CpG plasmids represent a significant advance in terms of safety and efficacy of nonviral delivery to the lung

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