A Smart DNA Nanoassembly Containing Multivalent Aptamers Enables Controlled Delivery of CRISPR/Cas9 for Cancer Immunotherapy

Abstract CRISPR/Cas9 system is promising for the reversal of tumor immunosuppression in immunotherapy, but the controlled delivery of CRISPR/Cas9 remains challenging. Herein, the study reported a smart DNA nanoassembly containing multivalent aptamers, realizing the controlled delivery of Cas9/sgRNA ribonucleoprotein (RNP) for enhanced cancer immunotherapy. A single‐stranded DNA complementary to sgRNA in the Cas9/sgRNA RNP can initiate a cascade‐clamped hybridization chain reaction (C‐HCR) to wrap the Cas9/sgRNA RNP up in the DNA nanoassembly. After selective internalization of DNA nanoassembly by cancer cells, Cas9/sgRNA RNP is released to cytoplasm in response to endogenous RNase H and enters the nuclei to knock out β‐catenin. The expression of the programmed death‐ligand one gene is effectively suppressed, and the immunosuppressive tumor microenvironment is reprogrammed. Meanwhile, the migration of cancer cells is inhibited, and the apoptosis of cancer cells is promoted. In a breast cancer mouse model, the administration of DNA nanoassembly effectively increased the infiltration of CD8⁺ T cells, eventually achieving high therapeutic efficacy.