Reshaping tumor immune microenvironment through ROS-responsive prodrug polyplexes via synergistic effect of CRISPRi system and epigenetic inhibitor for breast cancer therapy

Engagement of programmed death-ligand 1 (PD-L1) on tumor cells with its receptor PD-1 on immune cells can transmit an inhibitory signal to induce immune evasion. Although the immune checkpoint inhibitor PD-L1 antibody has shown antitumor capability in clinical treatment, its wide clinical application still faces several side effects and individual selectivity. In our research, we utilized the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system to suppress PD-L1 expression on breast cancer cells (4T1) and combined it with epigenetic inhibitor azacytidine (AZA) for enhanced cancer immunotherapy. Reactive oxygen species (ROS)-responsive poly(β-amino ester) (PBAE)-S-AZA cationic polymeric prodrug was fabricated, which could complex with CRISPRi plasmids to form the composite polyplexes via electrostatic interaction. The composite polyplexes could be taken up by tumor cells with high efficiency, followed by plasmid release with the cooperation of PBAE. The CRISPRi plasmids could lead to PD-L1 downregulation in tumor cells, leading to obvious relief of immune checkpoint blockade. In the meantime, the epigenetic inhibitor AZA was also released from the polyplexes due to the high intracellular ROS level, thereby enhancing the efficacy of immunotherapy via elevating MHC class I expression, enhancing antigen presentation, and inducing dendritic cell (DC) maturation. The ROS-responsive polyplexes helped to realize the combination of genome editing, immunotherapy, and epigenetic regulation. It will provide an effective platform for promoting antitumor treatment and precision medicine.