Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

Abstract

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,1 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.

Tumor specific TGF-β insensitive CD8 + T cells augments the antitumor effect through inhibition of epithelial-mesenchymal transition in CD 105 + renal carcinoma stem cells

“…Redirect and reprogram T cells to enhance the T cell toxicity ability is also important in the process of tumor rejection. Eyquem J et al reported that using the CRISPR/Cas9 genome editing technique targeting the CAR to the TRAC locus in T cell could avert tonic CAR signaling and establish effective internalization and re-expression of the CAR following delaying effector T-cell differentiation and exhaustion [34]. Zheng F et al reported that combined α-CTLA-4/α-PD-L1 and CSC-DC vaccine could enhance the number of circulating functional CD8 + T cells, enhanced CSC-DC vaccine-induced host immune responses [35].…”

The Dawn of In Vivo Gene Editing Era: A Revolution in the Making

“…In a first-in-human clinical trial of Cas9-engineered T cells, a modified cell manufacturing process has shown dramatically reduced chromosome loss while essentially preserving genome editing efficacy. Expression of p53 correlates with protection from chromosome loss observed, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic These recent findings need closer attention to the safety issues in gene editing [184].…”

A benchmark of computational methods for correcting biases of established and unknown origin in CRISPR-Cas9 screening data

“…Several studies have shown that the activity of the Cas9 enzyme may lead to undesired ontarget edits in genomic regions proximal to the targeted gene, also known as "proximity bias", ranging from kilobase-scale deletions to whole chromosome truncation [36][37][38]49,50]. While many efforts have been put forward to mitigate the off-target effects of the Cas9 enzyme, the phenomenon of chromosome loss remains a concern and it is not fully understood.…”