566. Silencing of Plasmid DNA after Hydrodynamics-Based Transfection in Mice

Abstract

Top of pageAbstract Non-viral vectors are promising tools for gene therapy because of their safety profile and are expected to be alternative to viral vectors. However, gene expression efficiency of non-viral vectors is lower than that of viral vectors especially in vivo. This low expression of non-viral vectors is mainly thought to be due to insufficient transgene delivery into the nucleus of target cells, and many efforts have been made to improve the gene delivery. Although establishment of such delivery strategy is one of the most important issues for application of non-viral vectors, delivery of transgenes into the nucleus is not an endpoint of gene therapy. The final goal of non-viral gene therapy is to achieve the persistent and properly regulated expression of transgenes in the target nucleus without disrupting host's nuclear homeostasis. However, it has not been precisely revealed how tansgenes, especially transgenes on episomes, behave in the nucleus. In order to elucidate the fate of plasmid DNA (pDNA) after delivery into the nucleus in vivo, we investigated the time course of gene expression and the copy number of intranuclear pDNA after pDNA transfer to the liver. Here we report a gene silencing phenomenon of a plasmid DNA after delivery into the nucleus. In this study, a pDNA construct encoding luciferase driven by the cytomegalovirus (CMV) promoter was transfected into BALB/c mice using hydrodynamics-based injection, and luciferase expression was monitored for a week. The copy number of intranuclear pDNA was measured by quantitative real-time PCR. In agreement with some previous reports, luciferase expression peaked at 8 hours post injection, and then rapidly reduced (half-life was approximately 10 hours). On the other hand, the nuclear pDNA was gradually eliminated with a half-life of approximately 42 hours, about 4 times as long as that of luciferase expression. Next, the ratio of luciferase activity to the copy number of nuclear pDNA was calculated to directly compare the expression ability of one pDNA molecule at various time points. This ratio peaked at 24 hours post injection, and remarkably, decreased more than 100-fold within a few days. These results suggest that the pDNA was qualitatively changed and rapidly inactivated after delivery into the nucleus. Currently, we are examining the mechanism of such pDNA silencing.