Engineering CRISPR System‐Based Bacterial Outer Membrane Vesicle Potentiates T Cell Immunity for Enhanced Cancer Immunotherapy

Abstract Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment but only benefits a subset of patients because of insufficient infiltration and inactivation of effector T cells. Bacterial outer membrane vesicles (OMVs) can activate immunity and deliver therapeutic agents for immunotherapy. However, efficiently targeting and packaging therapeutic molecules into OMVs remains challenging. Here, the engineered E. coli BL21‐derived OMVs enable the packaging of multiple genes, resulting in a 7‐fold increase in DNA enrichment efficiency and gene silencing in vitro. Moreover, the engineered OMVs carrying genes encoding CXCL9 and IL12 (OMV‐C9I12) reprogram tumor cells to secrete these factors, significantly enhancing T‐cell chemotaxis and activation. More importantly, this system markedly inhibits tumors, extends survival, and synergizes with anti‐PD‐1/PD‐L1 therapy in murine MB49 and B16F10 tumor models. Single‐cell RNA sequencing (scRNA‐seq) further reveals significant upregulation of T‐cell chemotaxis and activation‐related pathways following OMV‐C9I12 treatment. Finally, OMV‐C9I12 potentiates T cell‐mediated immunotherapy and suppresses the growth of bladder and breast cancer tumors in humanized mouse models. These findings highlight the potential of this engineered OMV platform for cancer gene therapy and provide novel strategies to overcome resistance to immunotherapy.