RT Journal Article
SR Electronic
T1 Reprogramming human T cell function and specificity with non-viral genome targeting
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 183418
DO 10.1101/183418
A1 Theodore L. Roth
A1 Cristina Puig-Saus
A1 Ruby Yu
A1 Eric Shifrut
A1 Julia Carnevale
A1 Joseph Hiatt
A1 Justin Saco
A1 Han Li
A1 Jonathan Li
A1 Victoria Tobin
A1 David Nguyen
A1 Andrea M. Ferris
A1 Jeff Chen
A1 Jean-Nicolas Schickel
A1 Laurence Pellerin
A1 David Carmody
A1 Gorka Alkorta-Aranburu
A1 Daniela Del Gaudio
A1 Hiroyuki Matsumoto
A1 Montse Morell
A1 Ying Mao
A1 Rolen Quadros
A1 Channabasavaiah Gurumurthy
A1 Baz Smith
A1 Michael Haugwitz
A1 Stephen H. Hughes
A1 Jonathan Weissman
A1 Kathrin Schumann
A1 Andrew P. May
A1 Alan Ashworth
A1 Gary Kupfer
A1 Siri Greeley
A1 Rosa Bacchetta
A1 Eric Meffre
A1 Maria Grazia Roncarolo
A1 Neil Romberg
A1 Kevan C. Herold
A1 Antoni Ribas
A1 Manuel D. Leonetti
A1 Alexander Marson
YR 2017
UL http://biorxiv.org/content/early/2017/12/07/183418.abstract
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.