In Vivo Site-Specific T Cell Engineering Allows Complete Remission of Human Leukemia in Mice

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 is cumbersome, time-consuming, and expensive. Here, we 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. Uniquely, our design allows co-encapsulation in a single AAV of the CAR gene, flanked by homology arms, together with a cassette coding 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, in the capsid of our AAV, a designed ankyrin repeat protein (DARPin) targeting the human CD8 receptor. Importantly, we demonstrate both ex vivo and in vivo engineering of primary human T cells with our single AAV in mice xenotransplanted with a human leukemia. In particular, subsequent to 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 ~12% of blood CD8+ T cells. When applying IV injections, complete remission of the human leukemia was achieved. Site-specific in vivo engineering of CAR T cells may revolutionize the safety, efficacy, and scalability of cellular immunotherapies for hematological malignancies and beyond.