Complete but not Partial Reduction of Plasmid CpG Content Increases Transgene Expression and Eliminates the Inflammatory Response Associated with Delivery of Non-Viral Vectors to the Lung.

Hyde SC, Lawton AE, Varathalingam A, Cheng SH, Davies LA, Pringle IA, Gill DR

Pediatric Pulmonology, Vol 41, 310

Download

North American Cystic Fibrosis Conference, Denver, 2006

Non-viral gene therapy is being developed for Cystic Fibrosis (CF) lung disease. However, the level and duration of expression achieved from current vectors is modest and is associated with an inflammatory cytokine response in mice and CF subjects. The response is due in part to the 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, 80µg pDNA/100µl).

At 24-hours post-dosing, bronchoalveolar lavage fluid (BALF) was collected for analysis of total cells, IL-12, IFN-g and TNF-a. Compared to untreated controls, mice that received first generation (pCIKLux) complexes had elevated levels of IL-12, IFN-a, TNF-a and cell infiltrates (p=0.002).

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 (p=0.74). Moreover, luciferase activity in the lung was 10-fold higher (p=0.01) 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 non-viral delivery to the lung.