Journal
JOURNAL OF IMMUNOTHERAPY
Volume 32, Issue 8, Pages 837-844Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/CJI.0b013e3181b2914c
Keywords
transposon; T cell immunotherapy; genetically modified T cells; integration sites
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Funding
- NCI NIH HHS [P50 CA126752-01, P50 CA126752] Funding Source: Medline
- NHLBI NIH HHS [T32 HL092332-07, T32HL092332, T32 HL092332] Funding Source: Medline
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The piggyBac transposon system represents a promising nonviral 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 (approximately 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 2 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 nonrandom integration profile and a preference for transcriptional units (approximately 50% into RefSeq genes in all cell types), CpG islands (18% in T cells and 8% in other human cells), and transcriptional start sites ( < 5 kb, 16% to 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 with 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 50 kb of the transcriptional start sites of known proto-oncogenes. Hence the piggyBac nonviral gene delivery system seems to represent a promising gene transfer system for clinical applications using human T lymphocytes.
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