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

Connor A Tsuchida; Nadav Brandes; Raymund Bueno; Marena Trinidad; Thomas Mazumder; Bingfei Yu; Byungjin Hwang; Christopher Chang; Jamin Liu; Yang Sun; Caitlin R Hopkins; Kevin R Parker; Yanyan Qi; Laura Hofman; Ansuman T Satpathy; Edward A Stadtmauer; Jamie H D Cate; Justin Eyquem; Joseph A Fraietta; Carl H June; Howard Y Chang; Chun Jimmie Ye; Jennifer A Doudna

doi:10.1016/j.cell.2023.08.041

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

Cell. 2023 Oct 12;186(21):4567-4582.e20. doi: 10.1016/j.cell.2023.08.041. Epub 2023 Oct 3.

Authors

Connor A Tsuchida¹, Nadav Brandes², Raymund Bueno², Marena Trinidad³, Thomas Mazumder², Bingfei Yu⁴, Byungjin Hwang², Christopher Chang⁵, Jamin Liu⁶, Yang Sun², Caitlin R Hopkins⁷, Kevin R Parker⁸, Yanyan Qi⁹, Laura Hofman¹⁰, Ansuman T Satpathy¹¹, Edward A Stadtmauer¹², Jamie H D Cate¹³, Justin Eyquem¹⁴, Joseph A Fraietta⁷, Carl H June¹⁵, Howard Y Chang¹⁶, Chun Jimmie Ye¹⁷, Jennifer A Doudna¹⁸

Affiliations

¹ 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.
² Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
⁴ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA; Parker Institute for Cancer Immunotherapy, Stanford University School of Medicine, Stanford, CA, USA.
⁵ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁶ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA.
⁷ Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA.
⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA; Graduate School of Life Sciences, Utrecht University, Utrecht, the Netherlands.
¹¹ Parker Institute for Cancer Immunotherapy, Stanford University School of Medicine, Stanford, CA, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
¹² Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
¹³ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
¹⁴ Department of Medicine, University of California, San Francisco, San Francisco, CA, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
¹⁵ Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
¹⁶ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA; Parker Institute for Cancer Immunotherapy, Stanford University School of Medicine, Stanford, CA, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.
¹⁷ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA; Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA; Institute for Computational Health Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA, USA. Electronic address: jimmie.ye@ucsf.edu.
¹⁸ 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; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA; Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA; Department of Chemistry, University of California Berkeley, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA. Electronic address: doudna@berkeley.edu.

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.

Keywords: CAR T cells; CRISPR screen; CRISPR-Cas9; DNA repair; T cells; aneuploidy; chromosome loss; clinical trial; genome editing; immunoengineering.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Cas Systems* / genetics
Chromosome Aberrations*
Chromosomes
Clinical Trials as Topic
DNA Damage
Gene Editing* / methods
Humans
T-Lymphocytes*

Associated data

ClinicalTrials.gov/NCT03399448

Abstract

Publication types

MeSH terms

Associated data

Grants and funding