Autologous chimeric antigen receptor T (CAR-T) cell therapy has demonstrated therapeutic effectiveness in hematologic malignancies and autoimmune diseases. However, the manufacturing complexity and the requirement for lymphodepletion have hindered its widespread clinical application. Engineering human T cells in vivo holds promise to conquer these limitations but requires effective T cell-targeted CAR delivery with demonstrated safety. Here, we show that an engineered AAV6 variant, AAV6-M2, enables in vivo CAR expression in human T cells following systemic administration in a Humanized Immune System (HIS) mouse model. AAV6-M2-CD19CAR turned up to 77.5% of human CD8+ T cells into CAR-T cells across multiple... More
Autologous chimeric antigen receptor T (CAR-T) cell therapy has demonstrated therapeutic effectiveness in hematologic malignancies and autoimmune diseases. However, the manufacturing complexity and the requirement for lymphodepletion have hindered its widespread clinical application. Engineering human T cells in vivo holds promise to conquer these limitations but requires effective T cell-targeted CAR delivery with demonstrated safety. Here, we show that an engineered AAV6 variant, AAV6-M2, enables in vivo CAR expression in human T cells following systemic administration in a Humanized Immune System (HIS) mouse model. AAV6-M2-CD19CAR turned up to 77.5% of human CD8+ T cells into CAR-T cells across multiple organs six weeks post-AAV injection. In HIS mice exhibiting systemic lupus erythematosus-like symptoms, AAV6-M2-CD19CAR treatment effectively depleted B cells in both peripheral blood and tissues, accompanied by ameliorated lupus pathologies. Importantly, systemic delivery of AAV6-M2 resulted in significant liver de-targeting, with viral genome levels in the liver reduced by over two orders of magnitude in both mice and cynomolgus macaques compared to wild-type AAV. Through CRISPR screening, cryo-EM structural analysis, and molecular docking, we identified CD62L as a key mediator of AAV6-M2's enhanced transduction to human T cells, enabling CAR delivery without the need for prior T cell activation. These findings establish that AAV-mediated CAR delivery can generate functional human CAR-T cells in vivo, providing mechanistic insights into the selective targeting of T cells. This work highlights engineered AAV vectors as a promising platform for in vivo CAR-T therapy and expands the therapeutic landscape of AAV beyond inherited diseases.
