Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases
Mark J Osborn1,2,3, Beau R Webber1, Friederike Knipping4,5
1Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
Abstract:
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.
Insights
Researchers improved adoptive immunotherapy by disrupting T-cell receptors (TCRs) in donor T-cells. This enhances safety and consistency for next-generation cancer treatments using gene editing tools like megaTAL and CRISPR/Cas9.
Area of Science:
- Immunology
- Gene Therapy
- Oncology
Background:
- Current adoptive immunotherapy relies on autologous T-cells, leading to variable potency due to patient-specific T-cell properties.
- Allogeneic T-cells from healthy donors offer more consistent therapeutic potential but face challenges from endogenous T-cell receptors (TCRs) causing off-tumor, anti-host reactivity.
Purpose of the Study:
- To evaluate TCR-α-targeted nucleases for disrupting endogenous TCR expression in primary human T-cells.
- To identify efficient and safe gene editing tools for producing allogeneic cellular substrates for advanced immunotherapies.
Main Methods:
- Assessed three TCR-α-targeted nucleases for their ability to disrupt T-cell receptor expression in human T-cells.
- Optimized reagent delivery conditions and evaluated off-target cleavage.
- Utilized megaTAL and CRISPR/Cas9 for a translatable manufacturing process.
Main Results:
- MegaTAL and CRISPR/Cas9 demonstrated high disruption efficiency of T-cell receptor expression.
- These reagents showed low toxicity and minimal off-target cleavage.
- Successful application in a translatable manufacturing process for cellular immunotherapy substrates.
Conclusions:
- TCR-α-targeted nucleases, particularly megaTAL and CRISPR/Cas9, are effective for disrupting endogenous TCRs in human T-cells.
- This approach enables the generation of safer allogeneic cellular substrates for next-generation immunotherapies.
- The optimized methods provide a foundation for translatable manufacturing of improved cancer treatments.
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