Molecular Therapy
Volume 24, Issue 3, March 2016, Pages 570-581
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Original Article
Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases

https://doi.org/10.1038/mt.2015.197Get rights and content
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Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.

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published online 05 January 2016

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The first three and the last three authors contributed equally to this work.