Genome-wide Mapping of PiggyBac Transposon Integrations in Primary Human T Cells

Abstract

The piggyBac transposon system represents a promising non-viral tool for gene delivery and discovery, and may also be of value for clinical gene therapy. PiggyBac is a highly efficient integrating vector that stably transfects (~40%) of primary human T cells for potential adoptive immunotherapy applications. To evaluate the potential genotoxicity of piggyBac, we compared 228 integration sites in primary human T cells to integrations in two other human derived cell lines (HEK293 and HeLa) and randomly simulated integrations into the human genome. Our results revealed distinct differences between cell types. PiggyBac had a non-random integration profile and a preference for transcriptional units (~50% into RefSeq genes in all cell types), CpG islands (18% in T cells and 8% in other human cells), and transcriptional start sites (TSS) (< 5kb, 16–20% in all cell types). PiggyBac also preferred TTAA but not AT rich regions of the human genome. We evaluated the expression of mapped genes into which piggyBac integrated, and found selection of more active genes in primary human T cells compared to other human cell types, possibly due to concomitant T cell activation during transposition. Importantly, we found that in comparison to what has been reported for gammaretroviral and human lenitviral vectors, piggyBac had decreased integration frequency into or within 50kb of the TSS of known proto-oncogenes. Hence the piggyBac non-viral gene delivery system appears to represent a promising gene transfer system for clinical applications using human T lymphocytes.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…As an alternative to random transgene integration, piggyBac or sleeping beauty-mediated transposition can be used, where transgene integration into highly transcribed regions of the host genome is favoured. This leads to a generally higher rate of transgene transcription compared to the random integration of plasmids (Ding et al, 2005;Galvan et al, 2009;Wilson et al, 2007). Consequently, higher q p and stability compared to stable producer clones obtained by random integration have been achieved with MAb titers of up to 7.6 g/L Rajendra et al, 2016).…”

Modification of a Popular Syngeneic Murine Mammary Tumor Model for Immunotherapy Studies

“…Part of the reason for such a high efficiency in multiple gene co-integration is probably due to high copy number of gene integration during piggyBac-mediated gene transduction. It has been estimated that, by average, there are 50 copies of gene integration at multiple chromosome foci when using the piggyBac gene delivery [14,15].…”

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

“…Nevertheless, the hyperactive transposases are definitely the method of choice both for insertional mutagenesis and for general gene delivery purposes. Our results also indicate that in terms of efficiency, the PB system seemed to outweigh SB100X in human embryonic stem cells, although its reported nonrandom integration profile (Wilson et al, 2007;Wang et al, 2008;Galvan et al, 2009;Liang et al, 2009) should be considered when such experiments are designed for gene therapy purposes.…”

“…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).…”