Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells
Rafet Başar, May Daher, Nadima Uprety, Elif Gokdemir, Abdullah Alsuliman, Emily L. Ensley, Gonca Ozcan, Mayela Carolina Mendt, Mayra Hernandez Sanabria, Lucila Nassif Kerbauy, Ana Karen Nunez Cortes, Li Li, Pinaki P. Banerjee, Luis Muniz-Feliciano, Sunil Acharya, Natalie W. Fowlkes, Junjun Lu, Sufang Li, Stephan Mielke, Mecit Kaplan, Vandana Nandivada, Mustafa Bdaiwi, Alexander D. Kontoyiannis, Ye Li, Enli Liu, Sonny Ang, David Marín, Lorenzo Brunetti, Michael C. Gundry, Rolf Turk, Mollie S. Schubert, Garrett R. Rettig, Matthew McNeill, Gavin Kurgan, Mark A. Behlke, Richard E. Champlin, Elizabeth J. Shpall, Katayoun Rezvani
Abstract
Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.