Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells [CART]. Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells [CART]

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 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, dramatically 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. Overall design: Primary human T cells were electroporated with Cas9 RNP (Cas9 protein + one of three different gRNAs) in an arrayed format. Recombinant AAV6 encoding a chimeric antigen receptor (CAR) resulted in homology-directed repair. Genome edited T cells were barcoded using hashtag reagents before pooling and performing 10x Genomics 3' scRNA-seq.

CRISPR-Cas9基因组编辑（CRISPR-Cas9 genome editing）已为先进T细胞疗法的发展提供关键支撑，但靶向染色体的偶发性丢失仍是一项不容忽视的安全隐患。为探究Cas9诱导的染色体丢失是否为普遍现象并评估其临床意义，我们对原代人T细胞开展了系统性分析。阵列式与混合池式CRISPR筛选结果显示，染色体丢失在全基因组范围内均可发生，既可造成染色体局部片段丢失，也会引发整条染色体的完全丧失，该现象在临床前嵌合抗原受体T细胞（chimeric antigen receptor T cells, CAR-T细胞）中亦存在。发生染色体丢失的T细胞可在体外培养体系中存活数周，提示其具备干扰临床应用的潜在风险。我们在首个人类Cas9工程化T细胞临床试验中采用的改良细胞生产工艺，可在大幅保留基因组编辑效率的同时，显著降低染色体丢失的发生率。p53的表达与该工艺中观察到的染色体丢失防护效应相关，这既为阐释该遗传毒性的作用机制提供了线索，也为T细胞工程改造提供了可缓解临床遗传毒性的可行策略。实验整体设计如下：将Cas9核糖核蛋白复合物（Cas9蛋白+三种不同向导RNA（guide RNA, gRNA））以阵列式方式电穿孔转染至原代人T细胞中。编码嵌合抗原受体（chimeric antigen receptor, CAR）的重组腺相关病毒6型（adeno-associated virus 6, AAV6）可介导同源定向修复（homology-directed repair, HDR）。基因组编辑后的T细胞使用标签试剂进行条形码标记，随后进行混合并开展10x Genomics 3'端单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。