Pediatric Pulmonology, 42 S30 307
The North American Cystic Fibrosis Conference, Anaheim, 2007
Gene therapy for Cystic Fibrosis lung disease will likely require long-term transgene expression; however, in the lung, many gene transfer agents have resulted in only transient gene expression. Previously we have shown that the choice of enhancer/promoter elements has a strong influence on the duration of reporter gene expression following delivery of non-viral vectors to the mouse lung (Gill et al., 2001, Gene Ther, 8, 1539). However, it is also possible that other elements of plasmid vector design play an important role. Using a clinically relevant mouse lung aerosol model, we have studied the effect of varying the plasmid CpG-dinucleotide content on the duration of expression from plasmids that had an identical enhancer/promoter sequence. Firstly we constructed a CpG-free plasmid vector containing a synthetic CpG-free luciferase gene under the transcriptional control of the human CMV enhancer/EF1? promoter. Secondly, we constructed a similar vector containing an identical enhancer and promoter but with a small number of CpG motifs, in the luciferase gene (97 CpGs) and in the backbone sequence (52 CpGs). We then investigated the level and duration of transgene expression following aerosol delivery of these plasmids complexed with the Genzyme lipid GL67 to the lungs of BALB/c mice (2.5 mg/ml pDNA, 6mM GL67, 10ml, Pari LC+ Nebuliser). Total lung extracts were assayed for luciferase activity at days 1, 2, 7, 14 & 28 (n=6 per time-point). The CpG-containing plasmid initially directed approximately 2-fold higher levels of reporter gene expression than the CpG-free plasmid (p<0.05 both days 1 and 2 post-delivery). However, while expression from the CpG-containing plasmid subsequently fell slowly to background levels, expression from the CpG-free plasmid increased 2-fold to day 7, and remained at the peak levels observed with the CpG-containing plasmid until the end of the experiment at day 28 (CpG-free plasmid 4-fold, 16-fold and 50-fold higher expression at days 7, 14 and 28 post-delivery respectively; p<0.05 for each). These data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is dependent by the sequence of the plasmid backbone. One explanation for this observation is that a specific, but as yet unidentified, sequence exists in the CpG containing backbone that triggers transcriptional silencing. Alternatively, the well-described host inflammatory response to CpG-containing DNA, may be involved and the CpG content of the vector could be responsible for these differences. We are currently evaluating the potency and duration of effect of CFTR expressing, CpG-free, plasmids in late-stage pre-clinical studies prior to the initiation of a further round of clinical studies of non-viral gene transfer in CF patients.