Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing

• Published in: Frontiers in immunology 2018-11, Vol.9, p.2503-2503
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: adoptive cell therapy (ACT); DsiRNA; electroporation; Immunology; RNA transfection; TCR-gene transfer
• ISSN: 1664-3224; 1664-3224
• DOI: 10.3389/fimmu.2018.02503

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 mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and 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 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.