PT - JOURNAL ARTICLE AU - Theodore L. Roth AU - Cristina Puig-Saus AU - Ruby Yu AU - Eric Shifrut AU - Julia Carnevale AU - Joseph Hiatt AU - Justin Saco AU - Han Li AU - Jonathan Li AU - Victoria Tobin AU - David Nguyen AU - Andrea M. Ferris AU - Jeff Chen AU - Jean-Nicolas Schickel AU - Laurence Pellerin AU - David Carmody AU - Gorka Alkorta-Aranburu AU - Daniela Del Gaudio AU - Hiroyuki Matsumoto AU - Montse Morell AU - Ying Mao AU - Rolen Quadros AU - Channabasavaiah Gurumurthy AU - Baz Smith AU - Michael Haugwitz AU - Stephen H. Hughes AU - Jonathan Weissman AU - Kathrin Schumann AU - Andrew P. May AU - Alan Ashworth AU - Gary Kupfer AU - Siri Greeley AU - Rosa Bacchetta AU - Eric Meffre AU - Maria Grazia Roncarolo AU - Neil Romberg AU - Kevan C. Herold AU - Antoni Ribas AU - Manuel D. Leonetti AU - Alexander Marson TI - Reprogramming human T cell function and specificity with non-viral genome targeting AID - 10.1101/183418 DP - 2017 Jan 01 TA - bioRxiv PG - 183418 4099 - http://biorxiv.org/content/early/2017/12/07/183418.short 4100 - http://biorxiv.org/content/early/2017/12/07/183418.full AB - Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2–5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, 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 through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.