Modulation of TCR stimulation and pifithrin-a improves the genomic safety profile of CRISPR- engineered human T cells

Laurenz T. Ursch,  Jule S. Müschen,  Julia Ritter,  Julia Klermund,  Bettina E. Bernard,  Saskia Kolb, Linda Warmuth,  Geoffroy Andrieux,  Gregor Miller,  Marina Jiménez-Muñoz, Fabian J. Theis, Melanie Boerries, Dirk H. Busch,  Toni Cathomen and Kathrin Schumann (2024 Dec 17) Modulation of TCR stimulation and pifithrin-a improves the genomic safety profile of CRISPR-engineered human T cells. Cell Reports Medicine 5, 101846. DOI: 10.1016/j.xcrm.2024.101846 (Projects B09, B16)

Summary cited directly from the article:

CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.