Reprogramming human T cell function and specificity with non-viral genome targeting

  • Nature. 2018 Jul;559(7714):405-409. doi: 10.1038/s41586-018-0326-5.
Theodore L Roth  1  2  3  4  5 ,  Cristina Puig-Saus  6 ,  Ruby Yu  3  4  5 ,  Eric Shifrut  3  4  5 ,  Julia Carnevale  7 ,  P Jonathan Li  3  4  5 ,  Joseph Hiatt  1  2  3  4  5 ,  Justin Saco  6 ,  Paige Krystofinski  6 ,  Han Li  8  9 ,  Victoria Tobin  3  4  5 ,  David N Nguyen  3  4  5 ,  Michael R Lee  4 ,  Amy L Putnam  4 ,  Andrea L Ferris  10 ,  Jeff W Chen  11 ,  Jean-Nicolas Schickel  11 ,  Laurence Pellerin  12  13 ,  David Carmody  14 ,  Gorka Alkorta-Aranburu  15 ,  Daniela Del Gaudio  15 ,  Hiroyuki Matsumoto  16 ,  Montse Morell  16 ,  Ying Mao  16 ,  Min Cho  17 ,  Rolen M Quadros  18 ,  Channabasavaiah B Gurumurthy  18 ,  Baz Smith  16 ,  Michael Haugwitz  16 ,  Stephen H Hughes  10  11 ,  Jonathan S Weissman  8  9 ,  Kathrin Schumann  3  4  5 ,  Jonathan H Esensten  19 ,  Andrew P May  17 ,  Alan Ashworth  7 ,  Gary M Kupfer  20 ,  Siri Atma W Greeley  14 ,  Rosa Bacchetta  12  13 ,  Eric Meffre  11 ,  Maria Grazia Roncarolo  12  13 ,  Neil Romberg  21  22 ,  Kevan C Herold  23 ,  Antoni Ribas  6  24  25  26 ,  Manuel D Leonetti  8  9  17 ,  Alexander Marson  27  28  29  30  31  32
Affiliations
  • 1. Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
  • 2. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
  • 3. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
  • 4. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
  • 5. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • 6. Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA.
  • 7. UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
  • 8. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
  • 9. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.
  • 10. HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute, Frederick, MD, USA.
  • 11. Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA.
  • 12. Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, USA.
  • 13. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
  • 14. Section of Adult and Pediatric Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Pediatrics, The University of Chicago, Chicago, IL, USA.
  • 15. Department of Human Genetics, The University of Chicago, Chicago, IL, USA.
  • 16. Takara Bio USA, Inc, Mountain View, CA, USA.
  • 17. Chan Zuckerberg Biohub, San Francisco, CA, USA.
  • 18. Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
  • 19. Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA.
  • 20. Department of Pediatrics, Pathology, Yale School of Medicine, New Haven, CT, USA.
  • 21. Division of Immunology and Allergy, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
  • 22. Department of Pediatrics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
  • 23. Departments of Immunobiology and Internal Medicine, Yale University, New Haven, CT, USA.
  • 24. Department of Surgery, University of California, Los Angeles, Los Angeles, CA, USA.
  • 25. Department of Medical and Molecular Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA.
  • 26. Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA.
  • 27. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 28. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 29. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. [email protected].
  • 30. UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 31. Chan Zuckerberg Biohub, San Francisco, CA, USA. [email protected].
  • 32. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. [email protected].
PMID: 29995861 DOI: 10.1038/s41586-018-0326-5
Abstract

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using Recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. 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 repair5,6. 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.