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

T cells engineered to express chimeric antigen receptors (CAR-T cells) show great promise in treating hematological malignancies. However, the ex vivo culturing and engineering of autologous T cells are cumbersome, time-consuming, and expensive. Here, we develop a method for the in vivo engineering of CAR-T cells, eliminating the need for pre-conditioning while reducing timelines and expenses without risking GVHD or graft rejection. We use adeno-associated viral vectors (AAV) encoding for the CAR as well as for a nuclease targeting the CAR into the TCR alpha constant (TRAC ) locus. Uniquely, our design allows co-encapsulation of the CAR gene within a single AAV, flanked by homology arms, together with a cassette encoding for the “ARCUS” nuclease, an engineered derivative of the I-CreI homing endonuclease targeting the TRAC locus. Furthermore, to efficiently and specifically target cytotoxic T cells in vivo, we incorporate a designed ankyrin repeat protein (DARPin) into the capsid of our AAV, enabling targeting of the human CD8 receptor. Importantly, we demonstrate both ex vivo and in vivo engineering of primary human T cells with a single AAV in mice xenotransplanted with human leukemia. In particular, Following IP injection, in vivo-engineered CAR T cells with the expected CD8+, CD3-, CAR+ phenotype expand in the leukemia bearing mice, reaching levels of up to ~10% of blood CD8+ T cells. Site-specific in vivo engineering of CAR T cells may revolutionize the safety, efficacy and scalability of cellular immunotherapies for hematological malignancies and beyond.