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

Frontiers in Immunology

Fumihiro Fujiki¹  Evelien L. J. M. Smits¹  Herman Goossens¹  Haruo Sugiyama³  Johan M. J. Van den Bergh⁴  Viggo Van Tendeloo⁴  Zwi N. Berneman⁵  Eva Lion⁶  Hans De Reu⁷  Diana Campillo-Davo⁷ 

[1] Infectious Disease Institute (VAXINFECTIO), University of Antwerp, Antwerp, Belgium;Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium;;Center for Cell Therapy &Department of Cancer Immunology, Osaka University Graduate School of Medicine, Osaka, Japan;Division of Clinical Biology, Antwerp University Hospital, Edegem, Belgium;Faculty of Medicine and Health Sciences, Center for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium;;Faculty of Medicine and Health Sciences, Vaccine &

关键词: TCR-gene transfer;    electroporation;    adoptive cell therapy (ACT);    DsiRNA;    RNA transfection;   

DOI  :  10.3389/fimmu.2018.02503

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown