1042. RNA as a Source of Transposase for Sleeping Beauty -Mediated Transposition and Long-Term Expression in Somatic Cells and Tissues
Authors: Andrew C. Wilber, Joel L. Frandsen, Lalitha R. Belur, Paul R. Score, Jennifer L. Geurts, David A. Largaespada, Perry B. Hackett, R. Scott McIvor
Journal: Molecular Therapy (2004)

The Sleeping Beauty (SB) transposon system has been demonstrated to mediate gene insertion and long-term expression, suggesting the potential application of this plasmid based system for non-viral gene therapy. All reported studies of SB-mediated gene transfer to somatic cells have supplied the transposase by expression of co-injected plasmid DNA. Although successful for both in vitro and in vivo transposition, there is a lack of control with respect to the transient and long-term levels of transposase expressed in a target cell. There is also the potential for genomic integration of the transposase encoding DNA, resulting in sustained transposase expression and potential destabilizing effects on the integrated transgene or host genome integrity. We address these problems by supplying the transposase-encoding molecule in the form of mRNA. Rapid, high pressure injection of m7G-capped luciferase (Luc) mRNA through the tail vein of mice resulted in efficient luciferase expression with respect to a DNA element where the Caggs promoter controlled luciferase transcription. Using this system as a model for efficient gene transfer in the liver, we have taken steps to optimize delivery/expression of the mRNA by generating constructs to promote stability and translation. Real-time whole body in vivo imaging of Luc mRNA translation was conducted to measure the effect of an IRES and UTR sequences on luciferase expression. These studies determined that incorporation of 5' and 3' UTR elements from the Xenopus -globin gene were essential to achieve peak luciferase expression levels from an otherwise unprotected transcript. Co-delivery of these mRNAs with an inhibitor of or a substrate for endogenous RNases provided luciferase expression sufficient to mimic levels achieved using promoters known to provide optimum transposase expression for in vivo transposition. Using this genetic arrangement, we have co-delivered SB transposase mRNA with a transposon encoding the Luc gene (driven by the Caggs promoter) to the liver of transgenic mice in which transcription of Cre recombinase is under the control of an interferon response element. To distinguish between plasmid-mediated expression and expression mediated by transposition, we have engineered this transposon encoding plasmid with lox-P sites positioned such that Cre-mediated recombination excises a sequence which includes the Caggs promoter, unless it has been segregated away from the plasmid by the SB transposase (Score et al., Mol. Ther. 7: S9, 2003). We found that pIpC induction of Cre recombinase caused a 1600-fold reduction in luciferase expression in animals infused with the transposon alone. While co-delivery of transposase mRNA resulted in a moderate degree of inadvertent Cre induction, we observed only a 45-fold reduction in luciferase expression following analogous pIpC administration. This remaining luciferase expression is most likely attributable to SB mediated transposition. These results provide the first evidence for use of SB transposase mRNA to mediate transposition and long-term expression in an in vivo setting.