Generating chimeric antigen receptor (CAR) T cells in vivo offers a promising avenue for overcoming barriers limiting the accessibility of ex vivo manufactured CAR T therapy, including high costs and long production time. However, efforts so far have relied on transient expression, which has limited durability, or on random integration of DNA payloads, which carries safety risks. Writing in Nature, Nyberg et al. report a delivery method for site-specific transgene integration to generate CAR T cells in vivo.

The authors used a two-component system: an enveloped delivery vehicle (EDV) carrying Cas9 protein and guide RNA targeting the T cell receptor-α constant gene TRAC, and an adeno-associated virus (AAV) carrying the homology-directed repair template encoding a CAR. They then optimized the EDV for T cell delivery and activation by surface-expressing anti-CD3 and evolved the AAV to improve resistance to neutralizing antibodies. Administering the optimized EDV/AAV system to a mouse model of leukemia resulted in complete B cell depletion and outperformed ex vivo-generated CAR T cells. Notably, the approach also effectively delivered an anti-B7H3 CAR construct, targeting an antigen expressed across various solid tumors, in a mouse model of sarcoma.