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Title
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Efficient and non-genotoxic RNA-based engineering of human T cells using tumor-specific T cell receptors with minimal TCR mispairing
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Author
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Abstract
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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 α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that sequential double 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 sequential double 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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Language
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English
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Source (journal)
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Frontiers in immunology. - Place of publication unknown
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Publication
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Place of publication unknown
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2018
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ISSN
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1664-3224
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DOI
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10.3389/FIMMU.2018.02503
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Volume/pages
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9
(2018)
, 14 p.
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Article Reference
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2503
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ISI
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000449420000001
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Pubmed ID
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30464762
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Medium
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E-only publicatie
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Full text (Publisher's DOI)
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Full text (open access)
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