CRISPR-Cas9-Mediated Glucocorticoid Resistance in Virus-Specific T Cells for Adoptive T Cell Therapy Posttransplantation

Immunosuppression posttransplantation exposes patients to an increased risk for refractory viral infections as an impor-tant cause of morbidity and mortality. Protective T cell im-munity can be restored by adoptive T cell transfer, but ongoing immunosuppression limits efficacy of T cell re-sponses. In order to deliver protection against viral patho-gens and allow at the same time necessary steroid therapy, we generated glucocorticoid-resistant T cells by CRISPR-Cas9-mediated knockout of the glucocorticoid receptor in primary human virus-specific T cell products. Characteriza-tion of the T cell product revealed high efficiency of gluco-corticoid receptor knockout and high purity of virus-specific T cells. This tandem T cell engineering preserved protective T cell functionality, such as cytotoxicity, CD107a degranulation, proliferative capacity, and cytokine release patterns. Vi-rus-specific T cells with glucocorticoid receptor knockout were resistant to the suppressive effect of dexamethasone treatment on lymphocyte proliferation and cytokine secre-tion (tumor necrosis factor alpha [TNF-alpha], interleukin-4 [IL-4], IL-6, and sFas). Additionally, glucocorticoid receptor knockout cells remained sensitive to cyclosporine A treat-ment, thereby providing a rescue approach for patients in case of safety issues. This novel approach provides a thera-peutic option for the treatment of patients with viral infec-tions after transplantation who are receiving glucocorticoid therapy.