Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. Copyright (C) 2004 John Wiley Sons, Ltd

Quantitative analysis of α-L-iduronidase expression in immunocompetent mice treated with the Sleeping Beauty transposon system.

The Sleeping Beauty transposon system, a non-viral, integrating vector that can deliver the alpha-L-iduronidase-encoding gene, is efficient in correcting mucopolysaccharidosis type I in NOD/SCID mice. However, in previous studies we failed to attain reliable long-term alpha-L-iduronidase expression in immunocompetent mice. Here, we focused on achieving sustained high-level expression in immunocompetent C57BL/6 mice. In our standard liver-directed treatment we hydrodynamically infuse mice with plasmids containing a SB transposon-encoding human alpha-L-iduronidase, along with a source of SB transposase. We sought to 1) minimize expression of the therapeutic enzyme in antigen-presenting cells, while avoiding promoter shutdown and gender bias, 2) increase transposition efficiency and 3) improve immunosuppression. By using a liver-specific promoter to drive IDUA expression, the SB100X hyperactive transposase and transient cyclophosphamide immunosuppression we achieved therapeutic-level (>100 wild-type) stabilized expression for 1 year in 50% of C57BL/6 mice. To gain insights into the causes of variability in transgene expression, we quantified the rates of alpha-L-iduronidase activity decay vis-a-vis transposition and transgene maintenance using the data obtained in this and previous studies. Our analyses showed that immune responses are the most important variable to control in order to prevent loss of transgene expression. Cumulatively, our results allow transition to pre-clinical studies of SB-mediated alpha-L-iduronidase expression and correction of mucopolysaccharidosis type I in animal models

Summary of quantitative evaluation of transgene expression in mice following hydrodynamic delivery to the liver.

<p>Three decay rates can account for all of the profiles of expression that we have observed under about 128 different combinations of experimental variables. These decay rates are: <i>initial</i> (i), associated with promoter silencing [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#B8" target="_blank">8</a>], green line; <i>rapid</i> (ii), associated with loss of plasmids and transgenes, red line; and <i>gradual</i> (iii), blue line. <i>Stabilized</i> expression (iv), black line, at levels <u>></u>100WTdeemed therapeutic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#B8" target="_blank">8</a>] was attained only with LSP, transposition using SB 100X transposase and a transient 4-dose immunosuppression with CP administered intraperitoneally around the time of hydrodynamic delivery. We hypothesize that the delayed loss of expression (red dashed lines, mice 25 and 27 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#pone-0078161-g002" target="_blank">Figure 2F</a>), that has the same half-life of <3 days, is rapid as (ii). </p

Expression of transgenic hIDUA in C57BL/6 mice.

<p>Mice aged 12 weeks were treated with 5μg or 25μg pKT2/LSP-IDUA ± pCMV-SB100X at 5:1 and 10:1 weight ratio, respectively. Plasma IDUA activity in untreated controls (WT) was 10±4 nmol/ml/hr. The dashed double-lines indicate the therapeutic level [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#B8" target="_blank">8</a>]. Arrows indicate expression profiles of +SB-treated mice with gradual or incomplete loss of IDUA activity. Asterisks indicate rapid decay in CP-immunosuppressed mice. Numbers indicate mouse IDs as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#pone-0078161-t001" target="_blank">Table <b>1</b></a>.</p

Expression of IDUA from different promoters in NOD/SCID MPS I mice.

<p><b>A</b>. Vectors used in this study. The T2 <i>SB</i> transposon vectors (inverted arrowheads) contained either a “ubiquitous” CAGGS promoter or the liver-specific promoter (LSP) ApoEHCRhAAT. pKT2/ApoEHCRhAAT-hIDUA and pT2/miniCAGGS-hIDUA have different backbones and the selectable markers, <i>ka</i>n and <i>amp</i>, respectively. <b>B</b>. Kinetics of hIDUA expression from LSP and CAGGS promoters in NOD.129(B6)-Prkdc^scidIDUA^tm1Clk/J (NOD/SCID) mice. Symbols: +SB11 females, filled red circles and solid lines; –SB females, open red circles and dotted lines; +SB11 males, filled blue squares and solid lines; –SB males, open blue squares and dotted lines. Each point represents the mean hIDUA activity ± SD, n=6. The CAGGS graph in panel <b>B</b>, is from Figure 1d of [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#B8" target="_blank">8</a>]. <b>C</b>. Comparison of transposition efficiency of SB11 and SB100X transposases in C57BL/6 mice by the excision product (EP) assay. 2μg of either pCMV-SB11 or pCMV-SB100X transposase-encoding plasmid was co-delivered with 15μg of pT2/CAGGS-GUSB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078161#B7" target="_blank">7</a>] transposon plasmid. DNA for excision assay was isolated from livers 5 days p.i. </p

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

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