Site-specific in vivo T cell engineering to express Chimeric Antigen Receptors

The ex vivo culturing and engineering of autologous T cells is cumbersome, time consuming and expensive. Here, we propose to develop methods for the in vivo engineering of CAR-T cells, obviating pre-conditioning and reducing timelines and expenses without risking GVHD or graft rejection. We use adeno associated viral vectors (AAV) coding for the CAR as well as for a nuclease targeting the CAR into the TCR alpha constant (TRAC) locus. Our AAV encodes “ARCUS”, a homing endonuclease derivative, with the CAR gene flanked by homologous arms. In order to control cell specific transduction of human T cells, we use DARPins (designed ankyrin repeat proteins) which target the human CD8 receptor. Uniquely, our design allows co-encapsulation of the CAR gene together with the nuclease gene in a single AAV to facilitate efficient in vivo T cell targeting. In vitro transduction of primary human T cells by the ARCUS-CAR AAV leads to CAR expression from the TRAC endogenous promoter in up to 10% of the cells. Furthermore, IFN-gamma secretion was elevated when CAR T cells are co- incubated with NALM6 cells expressing the CD19 target. In our current in vivo studies, we target human T cells in NSG mice that harbor leukemic B cells. Finally, we are developing a transgenic mouse, bearing the ARCUS site at the TRAC locus, to facilitate in vivo engineering in the immunocompetent setting. Site specific in vivo engineering of CAR T cells may revolutionize the safety, efficacy and scalability of cellular immunotherapies for hematological malignancies and beyond.