1078. Silencing of Transgene Expression Following Plasmid Based Delivery to Murine Skeletal Muscle Is Dose Dependent

Abstract

Plasmid based gene delivery to skeletal muscle is an attractive method for the expression of therapeutic proteins. However, the level of transgene expression decreases over time and may be an obstacle to achieving sustained therapeutic levels of proteins. To investigate the mechanism responsible for loss of expression over time, a standard plasmid backbone containing the luciferase gene driven by the CMV promoter was injected into the skeletal muscle of the hind limb of C57Bl/6 mice at either a high dose (125|[mu]|g) or a low dose (25|[mu]|g) followed by electroporation. Transgene expression was monitored over time using in situ CCCD imaging. At the end of the study the quantity of transgene DNA and luciferase mRNA in the muscle was measured by quantitative PCR. At early time points (day 3 and day 7) luciferase activity in the high dose group was 2.5- fold higher than in the low dose group. However, luciferase activity in mice that received the high dose fell 230-fold between day 7 and day 42, while in mice that received the low dose of the same plasmid it fell only 4-fold. Vector DNA levels in the injected muscles at the end of the study (day 84) were not significantly different between the two doses indicating that the 27-fold lower luciferase activity in the high dose group compared to the low dose group at day 84 was not due to differences in vector DNA levels but rather to a difference in gene expression. Indeed, the luciferase mRNA to vector DNA ratio was 9-fold higher in the low dose group than in the high dose group at this time point. In the same study, a plasmid containing the identical CMV-luciferase expression cassette flanked by matrix attachment sites (MARs) and placed into a plasmid backbone from which all CpG dinucleotides had been removed, was tested at the same doses. At the low dose the CpG-depleted/MARs plasmid exhibited a similar luciferase expression profile to that of the standard plasmid at the same dose. However, in the high dose group the CpG- depleted/MARs plasmid partially prevented the decrease in luciferase expression over time that was observed with the standard plasmid at the high dose. At 28, 42 and 84 days after plasmid injection the CpG- depleted/MAR plasmid expressed 10-, 13- and 9-fold higher luciferase activity respectively than the standard plasmid. Vector DNA levels in the muscle at day 84 were only 2-fold higher in the CpG-depleted/ MAR group than in the mice that received the standard plasmid. These results demonstrate that reducing the plasmid dose can result in a significant improvement in the duration of transgene expression, and that this effect was not due only to improved persistence of the plasmid DNA in the muscle. In addition, use of a CpG-free plasmid backbone plus MARs partially prevented the decline of transgene expression that occurred at the high plasmid dose, suggesting that the presence of CpG's and/or absence of MARs may contribute to loss of expression over time.