AUTHOR=Campillo-Davo Diana , Fujiki Fumihiro , Van den Bergh Johan M. J. , De Reu Hans , Smits Evelien L. J. M. , Goossens Herman , Sugiyama Haruo , Lion Eva , Berneman Zwi N. , Van Tendeloo Viggo TITLE=Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing JOURNAL=Frontiers in Immunology VOLUME=9 YEAR=2018 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2018.02503 DOI=10.3389/fimmu.2018.02503 ISSN=1664-3224 ABSTRACT=

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.