Multistage Sensitive NanoCRISPR Enable Efficient Intracellular Disruption of Immune Checkpoints for Robust Innate and Adaptive Immune Coactivation

Abstract Synergistic immune checkpoints blockade (ICB) has the potential to surmount the heterogeneity of tumors that are resistant to conventional therapeutics and result in better treatment outcomes than monotherapy targetting of to a single immune checkpoint. However, current stategies to combining multi‐target ICB are mainly focused on blockades on the cell surface which are incomplete, temporary, and inefficient. Here, a multistage sensitive nanocomplex (MUSE) loaded with programmed death‐ligand 1/CD47 multiple targeting clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) (MT‐CRISPR/Cas9) system for complete intracellular immune checkpoint blockade are conducted. The core‐shell structure not only endows the MUSE with prolonged circulation capacity and precise tumor recognition, but also facilitates cell uptake and endosomal escape when confronted with the tumor extra/intracellular environment, leading to efficient delivery of MT‐CRISPR/Cas9 payloads with minimal toxity. It is demonstrated that the MUSE treatment in tumor models can activate robust CD8 + T‐cells and M1 macrophages‐mediated adaptive and innate anti‐tumor immune responses and trigger a persistent immune memory effect, which results in significantly inhibition of tumor growth and improved survival rate with almost undetectable off‐target delivery effect. Taken together, the MUSE‐nanoCRISPR system opens a new avenue for combinative blockade of multiple immune checkpoints, in turn bringing clinical benefits for oncological treatment.