Duration of Expression and Activity of Sleeping Beauty Transposase in mouse liver following hydrodynamic DNA delivery

Abstract

The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that "cuts" the transposon from a plasmid and "pastes" it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts.

Gene Expression in Lung and Liver After Intravenous Infusion of Polyethylenimine Complexes of Sleeping Beauty Transposons

“…The subsequent slower loss of expression is most probably due to epigenetic silencing of integrated or unintegrated plasmids, coupled with slow cellular turnover and loss of unintegrated plasmids. This interpretation is consistent with what we find for expression of the Sleeping Beauty gene after hydrodynamic delivery (Bell et al, 2006). PEI-mediated toxicity in the lung is supported by the results from our quantification of excision products (Table 2).…”

“…PEI-mediated toxicity in the lung is supported by the results from our quantification of excision products (Table 2). Normally, excision products are stable in the liver (Bell et al, 2006;Aronovich et al, 2007Aronovich et al, , 2009; but, as can be seen in the lower right quadrant of Table 2, over 7 days the number of excision products in the lung decreases more than 10-fold on a per-cell basis, which could be the result of cell death in those cells that took up PEI=DNA complexes. After 2-4 weeks, we observed that luciferase expression stabilized in both lung and liver when transposons were delivered with a source of transposase, which is consistent with expression from transgenes that have transposed into chromatin in the lungs (Belur et al, 2003;L.…”

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

“…In plasmids, reconstitution of the TTAA site posttransposition should result in the recircularization of the remaining linear backbones. Similar recircularization mechanisms have been described in systems other than pB (28,29). Recircularized plasmids remain in the nucleus and are thought to be degraded over time.…”

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery ofSleeping BeautyTransposons: Implications for Non-Viral Gene Therapy of Systemic Disease

“…13,19,60 This suggests that 70-80% of plasmids in the liver are lost, but those that enter nuclei and are expressed are retained at a higher rate. In immunocompetent mice, further loss of activity/transfected cells occurs with the onset of adaptive immune responses.…”