A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon

Abstract

Nonviral vector systems are used increasingly in gene targeting and gene transfer applications. The piggyBac transposon represents an alternative integrating vector for in vivo gene transfer. We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically. We report that a novel hyperactive transposase generated higher transposition efficiency than a codon-optimized transposase in a human liver cell line. Hyperactive transposase-mediated reporter gene expression persisted at levels twice that of codon-optimized transposase in the livers of mice for the 6-month study. Of note, expression persisted in mice following partial hepatectomy, consistent with expression from an integrated transgene. We also used the hyperactive transposase to deliver the human α1-antitrypsin gene and achieved stable expression in serum. To determine the integration pattern of insertions, we performed large-scale mapping in human cells and recovered 60,685 unique hyperactive transposase-mediated insertions. We found that a hyperactive piggyBac transposase conferred an altered pattern of integration from that of insect piggyBac transposase, with a decreased frequency of integration near transcription start sites than previously reported. Our results support that the piggyBac transposon combined with the hyperactive transposase is an efficient integrating vector system for in vitro and in vivo applications.

piggyBac transposase tools for genome engineering

“…To quantitatively characterize PB R372A/K375A transposition in cells, we assayed the excision activity in a genetically manipulated HEK293 cell line (8). Briefly, Tol2 transposition was used to stably introduce a gene cassette carrying a cycle 3 GFP gene (Invitrogen) interrupted by a piggyBac transposon, termed GFP::PB, into the genome.…”

“…We used this genetically tractable system to isolate excision-hyperactive mutants of PB R372A/K375A . Similarly to the HEK293 GFP::PB assay described above, we used a fluorescence assay in yeast, in which PB promotes the precise excision of a piggyBac transposon from a fluorescent reporter gene mCherry, termed mCherry::PB (8). In this system, colonies are screened for increased mCherry expression to reflect increased excision.…”

“…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.…”

“…piggyBac has been used as a vector for reversible integration; however, reintegration of the transposon catalyzed by piggyBac (PB) transposase occurs in 40-50% of cells (14). To generate iPSCs without any genetic change, a PB mutant, which can promote only excision (Exc − mutations into a hyperactive version of PB (5,8,15) yields an Exc + Int − transposase whose excision frequency is five-to six-fold higher than that of wild-type PB. We speculate that the changed amino acids in the Exc + Int − mutant are likely positions of interaction between the transposase and the target DNA.…”

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

“…All have high transgenic potential and can mobilize large cargos (Rostovskaya et al, 2012;Wang et al, 2014), which is a considerable factor as compared with viral packaging systems. For safety considerations in gene therapy, SB seems to be the most ideal system because of its favorable integration profile: there is no obvious preference at the genomic level, and therefore the delivered transgene is integrated randomly, lowering the risk of undesirable insertional mutagenesis (Galvan et al, 2009;Grabundzija et al, 2010;Izsvák et al, 2010;Meir et al, 2011;Burnight et al, 2012;M.A. Li et al, 2013).…”