Optimization of the piggybac transposon system for the sustained genetic modification of human T lymphocytes

Yozo Nakazawa, Leslie E. Huye, Gianpietro Dotti, Aaron E. Foster, Juan F. Vera, Pallavi R. Manuri, Carl H. June, Cliona M. Rooney, Matthew H. Wilson

Research output: Contribution to journalArticlepeer-review

78 Scopus citations

Abstract

Optimal implementation of adoptive T-cell therapy for cancer will likely require multiple and maintained genetic modifications of the infused T cells and their progeny so that they home to tumor sites and recognize tumor cells, overcome tumor immune evasion strategies, and remain safe. Retroviral vectors readily transduce T cells and integrate into the host cell genome, but have a limited capacity for multigene insertion and cotransduction and are prohibitively expensive to produce at clinical grade. Genetic modification of T cells using transposons as integrating plasmids is an attractive alternative because of the increased simplicity and cost of production. Of available transposons, piggyBac has the higher transposase activity and larger cargo capacity, and we now evaluate piggyBac for potential adoptive therapies with primary T cells. PiggyBac transposons mediated stable gene expression in approximately 20% of primary T cells without selection. Treatment and maintenance of T cells with interleukin-15 increased stable transgene expression up to approximately 40% and expression was sustained through multiple logs of expansion for over 9 weeks in culture. We demonstrate simultaneous integration of 2 independent transposons in 20% of T cells, a frequency that could be increased to over 85% by selection of a transgenic surface marker (truncated CD19). PiggyBac could also deliver transposons of up to 13kb with 10,000-fold expansion of transduced T cells in culture and finally we demonstrate delivery of a functional suicide gene (iCasp9). PiggyBac transposons may thus be used to express the multiple integrated transgenes that will likely be necessary for the broader success of T-cell therapy.

Original languageEnglish (US)
Pages (from-to)826-836
Number of pages11
JournalJournal of Immunotherapy
Volume32
Issue number8
DOIs
StatePublished - Oct 2009

Keywords

  • Gene transfer
  • Genetically modified T cells
  • T-cell immunotherapy
  • Transposons

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Cancer Research
  • Pharmacology

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