Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo

Abstract

We characterized a recently developed hyperactive piggyBac (pB) transposase enzyme [containing seven mutations (7pB)] for gene transfer in human cells in vitro and to somatic cells in mice in vivo. Despite a protein level expression similar to that of native pB, 7pB significantly increased the gene transfer efficiency of a neomycin resistance cassette transposon in both HEK293 and HeLa cultured human cells. Native pB and SB100X, the most active transposase of the Sleeping Beauty transposon system, exhibited similar transposition efficiency in cultured human cell lines. When delivered to primary human T cells ex vivo, 7pB increased gene delivery two-to threefold compared with piggyBac and SB100X. The activity of hyperactive 7pB transposase was not affected by the addition of a 24-kDa N-terminal tag, whereas SB100X manifested a 50% reduction in transposition. Hyperactive 7pB was compared with native pB and SB100X in vivo in mice using hydrodynamic tail-vein injection of a limiting dose of transposase DNA combined with luciferase reporter transposons. We followed transgene expression for up to 6 months and observed approximately 10-fold greater long-term gene expression in mice injected with a codon-optimized version of 7pB compared with mice injected with native pB or SB100X. We conclude that hyperactive piggyBac elements can increase gene transfer in human cells and in vivo and should enable improved gene delivery using the piggyBac transposon system in a variety of cell and gene-therapy applications.

Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency ofSleeping BeautyandpiggyBacDNA Transposons

“…3B), in contrast to the strong OI results reported previously (Grabundzija et al, 2010). On the other hand, mPB showed a tendency toward slightly higher OI at higher transposase dose (especially at 10:20; see and also in line with some previous data (Grabundzija et al, 2010) but not with others (Doherty et al, 2012).…”

“…When examining the PB systems, in spite of exhibiting the highest excision activity in HEK-293 cells, the transgenic rate of the hyPB variant was similar to that of mPB even at different transposase doses, and it was lower than that of SB100X ( Fig. 4A and B), which is not in agreement with some previous results (Doherty et al, 2012). Taken together, our results pointed out that despite the higher detectable excision rate of DNA transposons, HEK-293 cells seemed to be less permissive for stable genomic integration of transgenes, resulting in lower transgenic rates after transposition.…”

“…There are only a few studies addressing systematic comparisons of available transposon-based systems, and they often concentrate on a limited number of cell lines, mainly tumor cell lines, that may not be representative for all cell types or for in vivo applications (Grabundzija et al, 2010;Huang et al, 2010;Doherty et al, 2012;Sharma et al, 2012). Moreover, specific interactions of the transposase proteins with endogenous cell type-specific factors may also influence the overall outcome of transposition, an important aspect that also requires further investigations.…”

piggyBacTransposon

“…All seven amino acid substitutions that were found in the five mutants were then combined into one coding sequence and the resulting transposase, named hyPBase, showed increases of approximately 20-fold in excision and 10-fold in integration in mouse ES cells (45). The hyPBase was also able to increase transposition efficiency in human cells (67). The mechanisms by which these mutations alter transposition activity remain elusive.…”

piggyBac transposase tools for genome engineering

“…piggyBac is also a DNA transposon and a promising alternative to Sleeping Beauty. piggyBac, originally isolated from the cabbage looper moth Trichoplusia ni genome (3), has a large cargo size (4), is highly active in many cell types, and mediates long-term expression in mammalian cells in vivo (5)(6)(7)(8)(9)(10). piggyBac is also distinguished by its ability to excise precisely (11), thus restoring the donor site to its pretransposon insertion sequence.…”