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.

Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Figures

Figure 1.

piggyBac transposase requires the presence of the transposon to induce DNA double stranded breaks (DSBs) in human cells. Formation of DSBs by piggyBac transposase was quantified by determining the level of phosphorylated H2A.X at serine 139. HEK293 cells were transfected with either a vector carrying the transposase (pCMV-PB), a transposon (pT-CMV-eGFP) or both. Cells transfected with pUC19 were used as negative control and cells treated with 2.5 μM camptothecin served as positive control. Approximately 24 h after transfection histones were extracted and probed with anti-phospho-H2A.X. H1.0 was used as loading control. Quantitative analysis using ImageJ demonstrated a statistically significant increase (*) in pH2A.X levels after 2.5 μM camptothecin or transposase + transposon compared to control (N = 3–5 independent experiments, ANOVA with Bonferroni post-test).

Figure 2.

piggyBac does not mobilize endogenous human TR-like sequences. 5′ or 3′ native TRs were replaced with PCR amplified genomic sequences. (A) and (B) The colony count was used as a measure of stable transposition. Only the native 5′ (A) or 3′ (B) in presence of the transposase leads to efficient and stable transposition in HEK293 cells. Elimination of the TRs or the transposase led to significantly reduced transposition and serve as negative controls. Genomic sequences similar to the piggyBac TRS did not lead to efficient and stable transposition and were comparable to the negative controls (N = 3 independent experiments, ANOVA with Bonferroni post-test; *, statistically different from negative control). An excision assay was used to determine whether a transposon is recognized and excised out of the vector by the transposase. A transposon with native TR sequences was efficiently recognized and excised by the piggyBac transposase, so a PCR product was visible on the agarose gels shown (pTpB lanes). Transposons carrying the Genomic TR-like sequences were not recognized and excised from the transposon either in the 5′ (C) or 3′ (D) position.

Figure 3.

Residual transposase expression after ‘cis’ delivery. (A) In the ‘cis’ configuration, the transposase and transposon are on the same plasmid DNA vector. (B) HEK293 cells were transfected with a vector carrying both the transposon and the transposase. The transposon had a Kan/Neo selection gene and cells were passaged in G418 containing media to select for cells with stable transposon integrations. After 8 weeks of selection in G418 media, protein purified from single cell clones was analyzed by western blot for residual transposase expression. +CON(NE), nuclear extract from HEK293 cells transfected with PB transposase harvested at 48 h. +CON(HA.PB), whole cell extract from HEK293 cells transfected with HA tagged PB transposase harvested at 48 h. (C) A mixed population of polyclonal HEK293 cells had residual transposase expression at day 31. (D) Residual transposase expression after ‘cis’ delivery is also noted for the Sleeping Beauty (SB12) transposon system. Positive controls were whole cell extracts as described above.

Figure 4.

Lack of residual transposase expression after ‘trans’ delivery. (A) In the ‘trans’ configuration the transposase and transposon are supplied on separate DNA plasmid. (B) HEK293 cells were transfected with two separate vectors individually carrying the transposon and the transposase. The transposon had a Kan/Neo selection gene and cells were passaged in G418 containing media to select for cells with stable transposon integrations. Single cell clones had no residual transposase expression after 8 weeks of selection in G418 media. (B) A mixed population of HEK-293 cells had no residual transposase expression after approximately day 7.

Figure 5.

The effect of varying the transposon-to-transposase DNA ratio on backbone-free transposon integration. (A, top) Schematic of pCMV-eGFP-pT-Nori vector carrying an eGFP reporter on the backbone and a Kan/Neo selection gene in the transposon. (A, bottom) Flow cytometry for eGFP expression was used to evaluate for backbone-free transposon integration at transposon-to-transposase ratios of 1000 ng:1000 ng and 1000 ng:100 ng and 1000 ng:10 ng. (B, top) Schematics of vectors used. A linearized puromycin-resistance cassette was transfected with a neomycin resistant transposon and integration was measured evaluating for puromycin-resistant colonies after 2 weeks of selection in puromycin (B, bottom). A puromycin-resistant transposon was used as a positive control. ANOVA revealed pT-eGFP-puroR + PB was different from linear puroR. Linear puroR + PB ± pTpB was not statistically different from linear puroR alone (N = 3).

Figure 6.

Quantitation of transposon and backbone copy number and isolation of backbone-free piggyBac-modified human cells. pCMV-eGFP-pTNori and transposase plasmids were transfected into HEK-293 cells that were cultured in selection media to produce a population of cells carrying the PB transposon. The plasmid backbone contained eGFP and cells were either unsorted, eGFP-negative (-), or eGFP-positive (+). The number of copies of the transposon (black bars) and plasmid backbone (white bars) per haploid genome (copy of RNaseP) was determined by qPCR. There was no significant difference between groups with regard to the transposon copy number by ANOVA. There was a significant difference between the copies of plasmid backbone per haploid genome by ANOVA (P = 0.001). We found that both unsorted versus eGFP-negative (P < 0.05) and eGFP-negative versus eGFP-positive (P < 0.01) were significantly different while there was no significant difference between unsorted and eGFP-positive cells (Tukey HSD test).

Figure 7.

Evaluation of promoter activity and enhancer effects of PB TRs in human cells. The PB 5′TR has weak directional promoter activity only inside the transposon. The 3′TR has no detectable promoter activity in either orientation. There is no significant change in the activity of a CAG-driven luciferase reporter activity when placed inside the transposon (N = 3). Rev, the TR is flipped to the reverse orientation. The dashed arrow indicates the 5′TR, whereas the solid gray arrow indicates the 3′TR.

Figure 8.

Mutation of the cryptic splice site in the PB 5′TR has no effect on transposition. The 5′TR is 311 bp. The mutated non-splice version (nspTpB) contains a G to C mutation at position 73, a G to C mutation at position 149 and a T to A mutation at position 184. These mutations eliminate predicted splice sites derived from NetGene2 (http://www.cbs.dtu.dk/services/NetGene2/) and those proven in Wang et al. (36). (N = 3).

Figure 9.

Lack of observable selective advantage of primary cells modified with the PB gene delivery system. (A) Human foreskin fibroblast-1 (HFF-1) cells were transfected with either pT-CMV-eGFP or pT-CMV-eGFP.2A.eGFP. If PB transposon integration led to any selective advantage the population would be expected to become increasingly eGFP positive. A dual expression vector expression both an eGFP reporter and the human telomerase reverse transcriptase (hTERT) was used as a positive control. (B) HFF-1 cells transfected with only an eGFP reporter transposon had no selective advantage, whereas cells transfected with the positive control exhibited selective advantage. Shown is a representative of 3 independent experiments.