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

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 beta-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. A smart DNA nanoassembly containing multivalent aptamers that controllably delivers Cas9/sgRNA ribonucleoprotein (RNP) is reported. The DNA nanoassembly is selectively internalized by tumor cells, releases Cas9/sgRNA RNP in response to endogenous RNase H, and executes effective gene editing, which results in the reversal of immunosuppression and inhibition of cell migration, achieving a high therapeutic efficacy of breast cancer.image