Abstract Genetic modification of cytolytic T-lymphocytes (CTL) for enhancing their functional immunobiology is a promising immunotherapeutic approach for the treatment of cancer and infectious disease. CTLs modified to express a chimeric antigen receptor comprising an extracellular IL13 domain and cytoplasmic CD3 domain (IL13-zetakine) can be re-directed both in vitro and in animal models to target glioblastoma multiforme (GBM), which is characterized by high expression of IL13Ralpha2. Patient-derived IL13-zetakine/HyTK expressing CD8+ CTL clones have entered early stage clinical trials. However, their clinical application is frequently limited in this patient population by the pervasive use of dexamethasone, a potent glucocorticoid analogue employed in the management of cerebral edema. Thus iatrogenic dexamethasone-mediated T-cell functional anergy and apoptosis in these patients is a barrier to realizing the full clinical utility of this adoptive therapy strategy. We hypothesized that knocking out the expression of the glucocorticoid receptor would render therapeutic CTLs resistant to the effects of synthetic glucocorticoids, including dexamethasone. We therefore developed engineered zinc finger nucleases (ZFNs) to specifically disrupt the glucocorticoid receptor (GR) locus in the human genome. ZFNs include the cleavage domain of the restriction enzyme FokI linked to an engineered zinc finger DNA-binding domain and can be designed to cleave a predetermined site in the genome. Natural repair of such DNA breaks via the error-prone non-homologous end joining pathway results in the inactivation of the target gene at frequencies which permit the isolation of knock out clones. Employing adenovirally delivered and transiently expressed ZFNs targeting exon 3 of the human GR gene, we isolated IL13-zetakine+ CD8+T-cells containing a biallelically mutated GR locus. These cells were characterized by the absence of full length GR protein, lack of glucocorticoid hormone-induced gene regulation and resistance to glucocorticoid hormone-mediated immunosupression and apoptosis. Importantly, the ZFN-modified, glucocorticoid-resistant CTLs demonstrated zetakine re-directed cytolytic activity and tumor cell specificity in chromium release assays in vitro and in an orthotopic mouse model of GBM in vivo. These results indicate that glucocorticoid-resistant IL13-zetakine targeted CTLs should retain function in cancer patients receiving glucocorticoids. A clinical trial to test this hypothesis is currently under development.