Molecular Therapy, 15 S1 S130
The American Society of Gene Therapy Annual Conference, Seattle, 2007
Gene therapy for Cystic Fibrosis lung disease will likely require long-term transgene expression; however, in the lung, many gene transfer agents only result in transient gene expression. Previously we have shown that the choice of promoter 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 plasmid CpG content on the duration of expression from plasmids which had an identical enhancer/promoter sequence.
Firstly we constructed a CpG-free plasmid vector containing a synthetic CpG-free luciferase gene driven by a human CMV enhancer/EF1alpha 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) and in the backbone sequence (52). 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). Total lung extracts were assayed for luciferase activity at days 1, 2, 7 & 14 (n=6 per time-point). The CpG-containing plasmid gave initially high levels of expression at day 1 and 2 post-delivery, which had fallen four-fold by day 7, and 20-fold to background levels by day 14. In contrast, the CpG-free plasmid gave broadly similar levels of expression at day 1 and 2 but in this case the level of expression had risen two-fold by day 7 and persisted at this level to day 14 post-delivery.
These data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is also affected by the sequence of the plasmid backbone.
One explanation is that a specific, but as yet unknown, sequence exists in the plasmid backbone which triggers the silencing of this promoter. However, it seems increasingly likely that a major factor in this relationship is the CpG content of the vector as only when CpG-free vectors are used do we observe long-term expression from pDNA in the mouse lung.
It is possible that the relative levels and distribution of CpG motifs throughout the plasmid may impact on the promoter activity affecting subsequent duration of expression.