CpG depletion results in increased duration of gene expression from plasmid DNA vectors in vivo.

Lawton AE, Davies LA, Yew NS, Cheng SH, Gill DR, Hyde SC, McLachlan G, Collie DD

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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.