The Sleeping Beauty (SB) transposon system can direct the integration of precise DNA sequences into human and mouse genomes. The SB vector system has two components: 1) a transposon containing a genetic cargo and 2) the catalytic transposase enzyme that mediates the cut-and-paste reaction from a plasmid into a vertebrate chromosome. In most cases, the source of SB transposase has been a gene that can integrate into chromosomes via illegitimate recombination. There is a concern that random integration and extended expression of a transposase gene might lead to remobilization of inserted transposons in the same genomes. Consequently, we have examined the relative levels of SB transposon genes and the duration of expression of SB transposase when delivered via plasmids to cells in livers of mice. The SB11 transposase gene, under direction of a ubiquitin (Ub) promoter was expressed from either of two cis plasmids, pT2/mCAGGS-Luc//Ub-SB11 or pT2/mCAGGS-IDUA//Ub-SB11, which express luciferase (Luc) and α-L-iduronidase (IDUA), respectively, under direction of an mCAGGS promoter. The lysosomal enzyme, IDUA, escapes from cells whereas Luc is confined to the cytoplasm. Consequently, they may elicit different immunological responses that could affect duration of expression of SB11. We used hydrodynamic, tail-vein injection to deliver these constructs to the livers of WT C57Bl/6 mice. The injections were evaluated 24-hr post injection for expression of Luc using the Xenogen™ imaging system to evaluate efficiency of gene delivery. Mice were re-imaged and euthanized at 1, 2, 3, 4, 5, 6, 7, 10, 14, 17 days and 3, 4, 6, 8, 10, 12, 14, 16, 20, 24 weeks. Expression of SB transposase was measured by western blotting over several weeks using anti-SB monoclonal antibodies, by RT-PCR, excision-PCR and, for finer resolution, by immunohistochemical staining for SB11 using a monoclonal antibody. The presence of the SB11 and either Luc or IDUA was measured by QPCR. Our results indicate that we lose detectable SB transposase protein expression within the first few weeks. We also saw, using PCR, loss of DNA and RNA in the IDUA-injected mice, which suggested an immunological response was eliminating transgenic liver cells (see Aronovich et al. abstract). We conclude that long-term residual SB transposase activity in livers of mice is below minimal detection and, accordingly, that delivery of the gene for the transposase does not represent a measurable risk. Supported by NIH (PO1-HD32652), the Arnold and Mabel Beckman Foundation, and a fellowship grant from the National MPS Society, USA (ELA).
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Bell et al. (2006) studied this question.