Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Abstract

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.

Temporal self-regulation of transposition through host-independent transposase rodlet formation

“…For TcBuster , transposition was essentially over by the time the protein was detectable by western blot while for piggyBac , detectable protein expression overlapped with colony formation at the 12, 16 and 24 h timepoints. We previously analyzed piggyBac protein expression over the week following transfection and found protein expression slowly dropped after the 48-h peak (43). Different transposases are expected to reach this ‘point of no return’ earlier or later due to their solubility properties, providing an important factor by which we may be able to increase transposase activity in our favor for gene transfer or mutagenesis.…”

Endogenous Transposase Source in Human Cells Mobilizes piggyBac Transposons

“…Thus, an exogenously supplied T. ni PB transposase could mobilize some of the human PB-like transposons. This possibility was experimentally addressed by Saha et al, 27 who found that expression of the insect PB transposase did not mobilize endogenous, human PB-like sequences, and did not lead to an increase in DNA double-strand breaks in the human genome. Second, the endogenous, domesticated PGBD1-5 proteins could excise and/or remobilize a transgene flanked by the ITRs of a PB vector (Figure 1b).…”

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

“…, the plasmid contains both transposase and the transposon) the probability that the transposase fragment will be integrated into the host cell genome may be increased, possibly through the actions of the host DNA repair mechanism. 6,33 To determine the incidence of plasmid backbone integration of the minimal piggyBac vector, we developed variants that contained two distinct reporter markers, mCherry in the transposon and eGFP in the transposase fragment of the plasmid. Adding these two fluorescent markers increased the size of the vectors, a change that caused a slight decrease in transfection efficiency, but this modification allowed us to discriminate between cells that had integrated the transposase fragment (the nontransposon part of the plasmid) from cells that had integrated only the (desired) transgene.…”

“…3,4,5 Full-length piggyBac transposons contain long terminal repeats, however, and the enhancers and promoters embedded within those terminal repeats can lead to activation of host cell proto-oncogenes. Promoter activity in mammalian cells has been detected in a sequence within the 5′' terminal repeat of full-length piggyBac transposons 5,6 while other studies have demonstrated enhancer activity in the 3′-terminal domain. 7 Unfortunately, while the promoters that drive the delivered transgene can be insulated from the host genome, 8 the promoters and enhancers within the transposon's terminal repeats cannot be insulated without interfering with the ability of the transposon to integrate and express.…”

“…, excluding the transgene of interest) are only 98 base pairs making it the smallest eukaryotic transposon developed to date. Because the known native transposon promoters and enhancers that reside in these long terminal sequences and which can interfere with cellular pathways after transposon integration 5,6,7 have been removed from the delivered fragment, this minimal piggyBac gene delivery system is potentially safer and may pose less of an oncogenic risk than other transposons and retroviruses.…”