Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy

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Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an "off-the-shelf" therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR-Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared with the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR-Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.

Original languageEnglish
Pages (from-to)3155-3175
Number of pages21
JournalMolecular Therapy : The Journal of the American Society of Gene Therapy
Issue number10
Early online date16 Jun 2022
Publication statusPublished - 5 Oct 2022

Bibliographical note

Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.


  • CRISPR-Cas9
  • Sleeping Beauty
  • T cell receptor
  • chimeric antigen receptor
  • leukemia
  • transposon


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