British Society of Gene Therapy Conference, London, 2006
Gene therapy for Cystic Fibrosis lung disease will likely require long-term gene expression; however, in the lung, many gene transfer agents lead to transient gene expression. We have investigated the effects of CpG minimisation on the efficacy and duration of non-viral transgene expression following aerosol delivery of plasmid complexed with 25KDa Polyethylenimine (PEI) to the lungs of BALB/c mice and sheep. A variety of promoter/enhancer sequences were tested in the context of either a standard CpG-rich, or CpG-reduced plasmid backbone. The use of a CMV enhancer/promoter or a CpG-depleted CMV derivative both resulted in transient reporter (luciferase) activity, which fell to background within 7 days irrespective of the CpG status of the plasmid backbone. When the human polyubiquitin (UbC) promoter was tested in the context of a CpG-reduced backbone, luciferase activity was maintained at day 1 levels for at least 28 days (p=0.7459), and up to 56 days in a separate study. Importantly, luciferase expression from the UbC promoter in a CpG-reduced backbone was 10-fold higher than a CpG-rich backbone at day 28 (p=0.0001). To further boost expression levels, hybrid CMV enhancer/polyubiquitin promoters were tested. Iin the context of a CpG-reduced backbone, luciferase activity from the CUCI promoter (CMV enhancer/UbC promoter) in mice was detectable at 28 days post-dose (p=0.0082). Luciferase activity from the CUBI promoter (CMV enhancer/UbB promoter) was five-fold higher than CMV (p=0.0001) in mice. Expression from the CUBI promoter was also seen in the sheep lung at day 1 and was detectable at 8 days post-dose. When delivered to the sheep lung as a DNA/PEI aerosol (n=4), luciferase expression from both the CUCI and CUBI promoters was on average 5- to 10-fold higher than CMV at day 1, with expression still detectable at day 8.