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Efficient non-viral T cell engineering by Sleeping Beauty minicircles diminishing DNA toxicity and miRNAs silencing the endogenous TCR

Item Type:Article
Title:Efficient non-viral T cell engineering by Sleeping Beauty minicircles diminishing DNA toxicity and miRNAs silencing the endogenous TCR
Creators Name:Clauss, J. and Obenaus, M. and Miskey, C. and Ivics, Z. and Izsvák, Z. and Uckert, W. and Bunse, M.
Abstract:Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells which we found to cause adverse effects. T cell viability was decreased in a dose-dependent manner and DNA-transfected T cells showed a delayed response upon T cell receptor (TCR) stimulation with regard to blast formation, proliferation and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and IFN-β upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, we were able to reduce the amount of total DNA required and achieved stable expression of therapeutic TCRs in more than 50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additonally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains and enhanced antigen-specific T cell functionality. Our approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.
Keywords:Immunotherapy, TCR Gene Therapy, T-Cell Engineering, Transposon, Sleeping Beauty, Minicircle Vector
Source:Human Gene Therapy
ISSN:1043-0342
Publisher:Mary Ann Liebert (U.S.A.)
Volume:29
Number:5
Page Range:569-584
Date:May 2018
Official Publication:https://doi.org/10.1089/hum.2017.136
PubMed:View item in PubMed

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