Journal
FRONTIERS IN IMMUNOLOGY
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fimmu.2018.02503
Keywords
TCR-gene transfer; electroporation; adoptive cell therapy (ACT); DsiRNA; RNA transfection
Categories
Funding
- Belgian Foundation against Cancer (Stichting tegen Kanker) [FAF-C/2016/764]
- Fund for Scientific Research Flanders (FWO Vlaanderen) [G.0535.18N]
- Foundation against Cancer (Stichting tegen Kanker)
- Gilead Sciences
- Kaushik Bhansali Fund
- Methusalem grant
- DOC-PRO Ph.D. grant of the Special Research Fund (BOF) of the University of Antwerp
- JSPS KAKENHI Grant [JP26430162]
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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 a and beta 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.
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