Roth et al. developed a rapid genome-targeting system that involves co-electroporation of CRISPR-Cas9 ribonucleoprotein complexes and short or long, double- or single-stranded DNA. They successfully demonstrated this strategy in human primary T cells, targeting up to three different genes simultaneously. They also utilized this system to correct IL2RA mutations in cells from patients with an autoimmune disease. Finally, they applied this method to replace the endogenous TCR locus with a new, tumor-targeting TCR, resulting in cells that led to antitumor response in vitro and in vivo.
Decades of work have aimed to genetically reprogram T cells for therapeutic purposes(1,2) using recombinant viral vectors, which do not target transgenes to specific genomic sites(3,4). The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair(5,6). Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
Author Info: (1) Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA. Biomedical Sciences Graduate Program, University of California, San Francis
Author Info: (1) Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (2) Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. (3) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (4) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (5) UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. (6) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (7) Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (8) Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. (9) Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. (10) Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA. (11) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (12) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (13) Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. (14) Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. (15) HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute, Frederick, MD, USA. (16) Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA. (17) Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA. (18) Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, USA. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. (19) Section of Adult and Pediatric Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Pediatrics, The University of Chicago, Chicago, IL, USA. (20) Department of Human Genetics, The University of Chicago, Chicago, IL, USA. (21) Department of Human Genetics, The University of Chicago, Chicago, IL, USA. (22) Takara Bio USA, Inc, Mountain View, CA, USA. (23) Takara Bio USA, Inc, Mountain View, CA, USA. (24) Takara Bio USA, Inc, Mountain View, CA, USA. (25) Chan Zuckerberg Biohub, San Francisco, CA, USA. (26) Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA. (27) Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA. (28) Takara Bio USA, Inc, Mountain View, CA, USA. (29) Takara Bio USA, Inc, Mountain View, CA, USA. (30) HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute, Frederick, MD, USA. Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA. (31) Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA. (32) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. (33) Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA. (34) Chan Zuckerberg Biohub, San Francisco, CA, USA. (35) UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. (36) Department of Pediatrics, Pathology, Yale School of Medicine, New Haven, CT, USA. (37) Section of Adult and Pediatric Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Pediatrics, The University of Chicago, Chicago, IL, USA. (38) Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, USA. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. (39) Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA. (40) Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, USA. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. (41) Division of Immunology and Allergy, The Children's Hospital of Philadelphia, Philadelphia, PA, USA. Department of Pediatrics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. (42) Departments of Immunobiology and Internal Medicine, Yale University, New Haven, CT, USA. (43) Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. Department of Surgery, University of California, Los Angeles, Los Angeles, CA, USA. Department of Medical and Molecular Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA. Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA. (44) Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA. Chan Zuckerberg Biohub, San Francisco, CA, USA. (45) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. alexander.marson@ucsf.edu. UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu. Chan Zuckerberg Biohub, San Francisco, CA, USA. alexander.marson@ucsf.edu. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
