Endowing T cells with a synthetic chimeric antigen receptor (CAR) helps to better mediate tumor rejection.1 Moreover, directing CARs to target CD19 can further enhance therapeutic potency and could even lead to complete remission in patients with chemo refractory or relapsed B-cell malignancies.2 Since conventional generation of CAR T cells - via randomly integrating vectors - can lead to undesired genetic outcomes (e.g. variegated transgene expression, transcriptional silencing, oncogenic transformation, etc.)3-7, Justin Eyquem and his colleagues in the group of Michel Sadelain sought to deliver a CAR to the T-cell receptor α constant (TRAC) locus by harnessing the genome editing power of CRISPR/Cas9.8
Leveraging TLA analysis for the in-depth genetic characterization of TRAC-CAR T cells
By designing CAR-specific primers, our TLA genome-wide coverage plot revealed sequence coverage at a specific position on chromosome 14. This confirmed that a single integration event had occurred at the intended site.8
Additionally, a second TLA strategy was employed whereby TLA primers were placed adjacent to the integrated sequence (i.e., left homology arm). This approach allowed us to generate sequence information across the wild-type (WT) allele, to measure faithful and unfaithful homologous recombinations (HR). In fact, by comparing coverage between ITR and CAR integration at the 5’ and 3’ ends of the TRAC homology arms locus, we found that 80% of the targeted alleles remained WT while 20% had an integration. Among the latter, 75% had perfect HR while 25% did not.8
TRAC-CAR T cells outperform conventional CAR T cells
All in all, the scientists were able to create more-potent CAR T cells by targeting a CAR coding sequence to the TRAC locus and placing it under the control of endogenous regulatory elements. In a mouse model of acute lymphoblastic leukemia, their edited cells markedly outperformed retroviral-CAR T cells by displaying greater persistence in the bone marrow and tumors. Furthermore, their protocol also showed uniform CAR expression in human peripheral blood T cells.
In summary, the researchers were able to demonstrate that their strategy and engineering led to CAR T cells that (1) avert tonic CAR signaling (2) establish effective internalization and re-expression of the CAR following single or repeated exposure to antigen, (3) display delayed effector T-cell differentiation and (4) are less prone to exhaustion. Overall, their generated CAR T cells proved to be more endurant in their fight against tumor cells than conventional ones.
According to Sadelain, “it should be noted that because T cells engineered at the TRAC locus are devoid of their own TCR, they cannot engage in graft-versus-host responses; thus, in addition to generating more potent T cells for autologous application, this approach will also facilitate the safe use of CAR T cells in the post-transplant setting.”9
These findings underscore the immense potential of genome editing to design the next generation of CAR T therapies and advance immunotherapies.
Confirming targeted gene delivery with TLA
Thanks to the unique capabilities of our proprietary technology, researchers regularly turn to our TLA-based solutions to:
- Identify the genomic position(s) of integration site(s)
- Assess whether structural changes, surrounding integration site(s), may have accompanied their genetic engineering
- Reliably detect any potential single nucleotide as well as structural variants, which can affect the integrity of their vector
Here, we have also demonstrated the ability of TLA to quantify the number of correct (and incorrect) integration events at the intended site. As a result, TLA is also suitable to evaluate the specificity (i.e., accuracy and efficiency) of targeted integrations.
To see how our TLA fares against conventional technologies, check out this comparison table. If you want a more comprehensive overview of TLA, to better understand the relevance of our analysis in the up- and downstream manufacturing processes of cell and gene therapy products, click below to watch our latest webinar.
Webinar
Improved genetic QC for viral vector and ATMP manufacturing
Recorded 7 April 2022
References
[1] Jensen, M. C. & Riddell, S. R. Designing chimeric antigen receptors to effectively and safely target tumors. Curr. Opin. Immunol. 33, 9–15 (2015).
[2] Sadelain, M. CAR therapy: the CD19 paradigm. J. Clin. Invest. 125, 3392–3400 (2015).
[3] Sadelain, M. & Mulligan, R. C. Efficient retroviral-mediated gene transfer into murine primary lymphocytes. Ninth International Immunology Congress, Budapest. 88:34. (1992).
[4] Wang, X. & Rivière, I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol. Ther. Oncolytics. 3, 16015 (2016).
[5] Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005).
[6] Rivière, I., Dunbar, C. E. & Sadelain, M. Hematopoietic stem cell engineering at a crossroads. Blood 119, 1107–1116 (2012).
[7] von Kalle, C., Deichmann, A. & Schmidt, M. Vector integration and tumorigenesis. Hum. Gene Ther. 25, 475–481 (2014).
[8] Eyquem, J., Mansilla-Soto, J., Giavridis, T. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017). https://doi.org/10.1038/nature21405
[9] Killock, D. Start your engineering — CARs take to the TRAC. Nat Rev Clin Oncol 14, 198 (2017). https://doi.org/10.1038/nrclinonc.2017.39
