In vivo T cell engineering

Ex vivo culturing and engineering of autologous T cells is cumbersome, time consuming and expensive. Banking of allogeneic CAR-T cells can save time and money, but complex manipulations may be required to alleviate concerns of graft-vs-host disease (GVHD) and graft rejection. In addition, the adoptive transfer of either autologous or allogeneic CAR-T cells often requires pre-conditioning chemotherapy that may have both short and long term adverse effects. We thus 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 or an integrase targeting the CAR into the TCR alpha constant (TRAC) locus. Similar targeting schemes ex vivo, were previously shown to reduce the variegated expression and premature T cell exhaustion associated with the commonly used integration techniques for CAR genes (retrovectors, lentivectors or transposons). Uniquely, our design allows co-encapsulation of the CAR gene together with the nuclease or integrase gene in a single AAV to facilitate efficient in vivo T cell targeting. In particular, our AAV will encode “ARCUS”, an engineered derivative of the I-CreI homing endonuclease, with the CAR gene flanked by homologous arms. Alternatively, our AAV may encode the integrase of the E. coli phage HK022 with the CAR gene flanked by appropriate attP recognition sites. Successful in vivo engineering of CAR T cells may revolutionize the safety, efficacy and scalability of cellular immunotherapies for hematological malignancies and beyond.