A Dual‐Enzyme–Responsive DNA‐Based Nanoframework Enables Controlled Co‐Delivery of CRISPR‐Cas9 and Antisense Oligodeoxynucleotide for Synergistic Gene Therapy

Abstract CRISPR‐Cas9 has demonstrated potential in inhibiting tumor growth by downregulating target gene expression, and the efficiency is expected to be enhanced with other nucleic acid drugs. Herein, a dual‐enzyme–responsive DNA‐based nanoframework (NF) that enables controlled co‐delivery of Cas9 ribonucleoprotein (RNP) and antisense oligodeoxynucleotide (ASO) for synergistic gene therapy is reported. NF is synthesized with acrylamide DNA (acDNA) as a cross‐linker and loading site. A ternary complex containing sgRNA, ASO, and Cas9 that bound to acDNA is designed. The phase transition property of NF allows to reversibly swell and aggregate thermo‐responsively for the high‐capacity loading of ternary complex. RNA is cleaved from DNA–RNA complex by the overexpressed ribonuclease H for the controlled release of Cas9 RNP, and the bifurcated DNA is cleaved by the overexpressed flap structure‐specific endonuclease 1 for the controlled release of overhanging ASO. The combination of gene editing with Cas9 RNP and gene silencing with ASO allows simultaneous manipulation of target gene in nucleus and its mRNA in cytoplasm, and this synergistic gene therapy has shown remarkable therapeutic effect in a breast cancer mouse model, making it a promising therapeutic strategy for future cancer treatment.