Article Text

Download PDFPDF

1221 Comparison of TcBusterTM non-viral transposon system to lentiviral transduction in primary human T cells
  1. Ellie Mews,
  2. Sophie Boisjolie-Gair,
  3. Dalton Smedley,
  4. HaeLee Ahn,
  5. Rebecca Haugen,
  6. Nicole Larson,
  7. Ariel Miller,
  8. Dane Rasmussen,
  9. Xiaobai Patrinostro and
  10. David Hermanson
  1. Bio-Techne, Minneapolis, MN, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background TcBusterTM is a commercially available hyperactive transposase that efficiently integrates poly-cistronic CAR constructs into primary human immune cells, overcoming many of the challenges associated with viral delivery. The use of viral vectors to generate these therapeutics has been the primary mechanism for payload delivery allowing relatively high efficiency of target editing with limited adverse effects. However, these viral vectors suffer from limited cargo size, risk of immunogenicity, and are both costly and difficult to produce. Non-viral transposase systems now rival viral gene editing, delivering equivalent efficiency with a much more cost-effective workflow.

Methods A range of different plasmid sizes, all containing a CD19 CAR, were inserted into primary human T cells from 3 healthy donors using either TcBusterTM or the equivalent lentiviral comparison. Flow cytometry was used to evaluate the efficiency of cargo insertion and resulting T cell phenotypes. The activity and anti-cancer efficacy of the engineered CAR T cells was evaluated following co-culture with Nalm-6 – luciferase target cells by luciferase assay and supernatants were collected for cytokine quantification on the Ella, an automated multi-analyte ELISA system.

Results TcBusterTM achieved similar integration efficiencies for smaller cargo sizes into primary human T cells to that of lentivirus; however, the transposase outperformed lentivirus when inserting larger poly-cistronic CAR constructs. The viability and fold expansion of the modified T cells was similar between the two technologies; however, the TcBusterTM transposed cells generated a larger population of memory T cells (stem cell memory and central memory) compared to their transduced counterparts. CD19 CAR T cells generated with TcBusterTM were similarly cytotoxic against Nalm-6 target cells to that of lentivirus transduced T cells but distinct differences were observed in the cytokine secretion profiles from each system.

Conclusions Here, we compared the generation, activity, and efficacy of CAR T cells produced using either TcBusterTM, a non-viral transposon system, or lentivirus. While this work shows that there are many similarities between transposed and lentivirus modified CAR T cells, we highlight some key strengths when engineering with the TcBusterTM system, such as the ability to integrate larger cargos and more robust memory cell populations.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.