Mitigation of Chromosome Loss in Clinical CRISPR-Cas9-Engineered T Cells

• DOI: 10.2139/ssrn.4380781
• Publication Date: 2023-03-08T20:31:15Z
• Authors: Connor Tsuchida; Nadav Brandes; Raymund Bueno; Marena Trinidad; Thomas Mazumder; Bingfei Yu; Byungjin Hwang; Christopher Chang; Jamin Liu; Yang Sun; Caitlin Hopkins; Kevin Parker; Yanyan Qi; Ansuman T. Satpathy; Edward Stadtmauer; Jamie Cate; Justin Eyquem; Joseph A. Fraietta; Carl June; Howard Chang; Jimmie Ye; Jennifer Doudna

SummaryCRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells,1dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.