Ursch Laurenz T, Müschen Jule S, Ritter Julia, Klermund Julia, Bernard Bettina E, Kolb Saskia, Warmuth Linda, Andrieux Geoffroy, Miller Gregor, Jiménez-Muñoz Marina, Theis Fabian J, Boerries Melanie, Busch Dirk H, Cathomen Toni, Schumann Kathrin
Technical University of Munich (TUM), School of Medicine and Health, Department of Preclinical Medicine, Institute for Medical Microbiology, Immunology and Hygiene, 81675 Munich, Germany.
Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, 79106 Freiburg, Germany.
Cell Rep Med. 2024 Dec 17;5(12):101846. doi: 10.1016/j.xcrm.2024.101846. Epub 2024 Dec 4.
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.
经CRISPR基因编辑的嵌合抗原受体（CAR）T细胞处于新型癌症治疗的前沿。然而，有几份报告描述了CRISPR诱导的染色体畸变的发生。到目前为止，提高T细胞产品基因组安全性的措施主要集中在CRISPR-Cas9系统的组件上，而较少关注T细胞内在特征，例如它们在T细胞受体（TCR）刺激后的大量扩增。在此，我们描述了原代人T细胞中插入缺失形成的驱动因素。T细胞活化和增殖速度的增加与更大的缺失相关。对未活化的T细胞进行编辑可降低大缺失的风险，但缺点是敲除效率降低。或者，添加小分子pifithrin-α以p53非依赖的方式限制大缺失、染色体易位和非整倍体，同时保持CRISPR基因编辑的T细胞（包括CAR T细胞）的功能。控制T细胞活化和pifithrin-α处理是提高CRISPR基因编辑的T细胞基因组完整性的易于实施的策略。
