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Published on: July 22, 2019
Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells
Connor A Tsuchida1, Nadav Brandes2, Raymund Bueno2
1University 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.
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 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.
Insights
CRISPR-Cas9 gene editing can cause chromosome loss in T cells, a safety concern for therapies. A new manufacturing process and p53 expression reduce this genotoxicity in clinical applications.
Area of Science:
- Genomics
- Cell Biology
- Immunotherapy
Background:
- CRISPR-Cas9 genome editing advances T cell therapies.
- Chromosome loss is a potential safety concern in CRISPR-Cas9 applications.
Purpose of the Study:
- To determine if Cas9-induced chromosome loss is widespread.
- To assess the clinical relevance of chromosome loss.
- To identify strategies for mitigating chromosome loss.
Main Methods:
- Systematic analysis in primary human T cells.
- Arrayed and pooled CRISPR screens.
- Evaluation of a modified cell manufacturing process in a clinical trial (NCT03399448).
Main Results:
- Chromosome loss is generalizable across the genome, affecting targeted chromosomes.
- T cells with chromosome loss persist in culture, posing clinical challenges.
- A modified manufacturing process and p53 expression significantly reduced chromosome loss.
Conclusions:
- Cas9-induced chromosome loss is a significant genotoxic risk in T cell engineering.
- Modified manufacturing processes and p53 expression offer strategies to mitigate this risk.
- These findings are crucial for the safe clinical application of CRISPR-Cas9 engineered T cell therapies.
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