University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
Cell. 2023 Oct 12;186(21):4567-4582.e20. doi: 10.1016/j.cell.2023.08.041. Epub 2023 Oct 3.
CRISPR-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 targeted chromosome, including in preclinical 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 (NCT03399448), 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.
CRISPR-Cas9 基因组编辑技术使先进的 T 细胞疗法成为可能，但靶向染色体偶尔丢失仍然是一个安全隐患。为了研究 Cas9 诱导的染色体丢失是否是一种普遍现象，并评估其临床意义，我们在原代人 T 细胞中进行了系统分析。阵列和池 CRISPR 筛选表明，染色体丢失具有普遍性，可以导致靶向染色体的部分和全部丢失，包括临床前嵌合抗原受体 T 细胞。在培养中，具有染色体丢失的 T 细胞持续存在数周，这意味着有可能干扰临床应用。在我们的 Cas9 工程 T 细胞的首次人体临床试验（NCT03399448）中采用了一种改良的细胞制造工艺，该工艺减少了染色体丢失，同时在很大程度上保留了基因组编辑的效果。在该方案中观察到的 p53 表达与染色体丢失的保护相关，这表明在临床上减轻这种遗传毒性的 T 细胞工程的机制和策略。
