Genetically engineered T cells represent a promising new approach to the treatment of cancer. Positive results in recent clinical trials exploiting T cells engineered to express chimeric antigen receptors (CARs) have highlighted the potential for these cell-based therapies in patients with B cell malignancies. To extend these successes to a broader range of tumor types may require additional T cell engineering beyond simple CAR addition, such as gene knockout and/or coupling deletion of a target gene with site-specific addition of a CAR transgene. To this end, we have developed a genome editing strategy for the simultaneous elimination of an endogenous target gene with site-specific addition of a CAR via homology-directed repair (HDR). To demonstrate the utility of this approach, we used a previously characterized megaTAL (an engineered nuclease created by the fusion of an engineered meganuclease and a transcription activator-like (TAL) -DNA binding domain) specific for the T cell receptor alpha constant region gene (TRAC). Delivery of this megaTAL to primary human T cells via mRNA electroporation results in efficient and specific disruption of the TRAC locus (Boissel et al, 2013). To achieve simultaneous target gene disruption with site-specific CAR transgene insertion, we designed an adeno-associated virus (AAV) vector for DNA template delivery encoding the CAR and flanked by regions of DNA homologous to the genome immediately surrounding the megaTAL cleavage site. Co-delivery of the megaTAL and AAV encoding a CD19-CAR with TRAC homology arms resulted in >50% CAR+TRAC- cells. In vitro assays of cytotoxicity and cytokine responses against CD19+ Nalm-6 cells confirmed that TRAC-targeted CD19-CAR T cells were comparable to CD19-CAR T cells generated by lentiviral transduction. Interestingly, a similar level of CAR T cell function was observed even though TRAC-targeted CD19-CAR T cells expressed lower amounts of the CAR, as determined by flow cytometry. Similar CAR integration efficiency and functional efficacy was observed using a TRAC-targeting AAV vector containing a distinct B cell maturation antigen (BCMA)-specific CAR. These findings demonstrate megaTAL-mediated targeted gene addition as a feasible, efficient, and potentially safer approach for generation of gene-edited CAR T cell product. Moreover, the ability to combine the disruption of a target gene with the site-specific integration of the CAR eliminates the need for randomly integrating viral vectors while satisfying the potential need for more complex genome-edited T cell products.

Citation Format: Baeckseung Lee, Wai-Hang Leung, Mark Pogson, Jordan Jarjour, Alexander Astrakhan. Efficient generation of CAR T cells by site-specific gene addition into the TRAC locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3756. doi:10.1158/1538-7445.AM2017-3756