| Cancer immunotherapy aims to enhance the immune system to eliminate tumor cells,which has achieved major breakthroughs and promises to revolutionize the cancer treatment.Since cancer immunotherapy was selected by Science as breakthrough of the year 2013,immune checkpoint blockade therapy has been in the spotlight,and multiple PD-1/PD-L1blocking antibodies have been approved by the FDA for clinical use.However,tumor cells have evolved multiple mechanisms to evade immune surveillance,such as defective presentation of antigen,upregulation of immune checkpoints,and recruitment of immunosuppressive cells,hindering the antitumor immune response of immune cells.The most representative example is PD-L1,which is highly expressed in various malignant tumors and can bind to PD-1 on activated T cells to deplete T cells and ultimately achieve tumor immune escape.Therefore,blocking the PD-1/PD-L1 pathway can relieve immunosuppression and reactivate T cell-mediated antitumor responses.Antibodies used clinically or small interfering RNA(si RNA)used in basic research block immune checkpoint pathways for tumor immunotherapy,but both blockade strategies can only temporarily block the PD-1/PD-L1 interaction.To prevent tumor immune escape more effectively,permanently disrupting the PD-1/PD-L1 interaction promises to address this conundrum at the source.Accumulating evidence has demonstrated that some tumors have insufficient tumor infiltration of T cells and an immunosuppressive tumor microenvironment,known as“cold tumors”,and these traits are critical factors that induce a low response rate to anti-PD-1/PD-L1 antibody-based therapies.Thus,enhancing PD-1/PD-L1blocking efficacy and further combining with reshape“cold”tumor microenvironments into“hot”microenvironments has become a promising strategy.Based on the above considerations,we designed a selectively activated CRISPR/Cas9system to improve the insufficiency of current immune checkpoint blockade(ICB)therapy and study its antitumor effect.The main research content of this study is divided into the following two parts:(1)We developed a red light-activated CRISPR/Cas9 system for genomic disruption of the PD-L1 gene to achieve permanent blockade of the PD-1/PD-L1 pathway.This system was constructed by using a photoactivated self-degradable polyethyleneimine(PEI)derivative and the plasmid p CP(expression of the Cas9 protein and single-guide RNA targeting PD-L1).Under light irradiation,this photoswitched CRISPR/Cas9 system efficiently genetically disrupted the PD-L1 gene in not only bulk cancer cells but also cancer stem-like cells.Mouse tumor model results show that the red light-activated CRISPR/Cas9 system significantly increased the infiltration of CD8~+T cells into tumor tissue,leading to effective activation of a T cell-mediated antitumor response against cancer cells and cancer stem-like cells.This study provides an alternative strategy to block the PD-1/PD-L1 pathway for efficacious immune checkpoint therapy.(2)We propose a specific promoter-driven CRISPR/Cas9 system,F-PC/p HCP,that achieves permanent genomic disruption of PD-L1 and elicits a multifaceted anticancer immune response to potentiate immunotherapy.This system consists of a chlorin e6-encapsulated fluorinated dendrimer(F-PC)and HSP70 promoter-driven CRISPR/Cas9(p HCP).F-PC/p HCP under 660 nm laser activated the HSP70 promoter and enabled the specific expression of the Cas9 protein to disrupt PD-L1 at the genomic level,preventing immune escape.Moreover,F-PC/p HCP also induced immunogenic cell death(ICD)of tumor cells and reprogrammed the immunosuppressive tumor microenvironment.Overall,this specific promoter-driven CRISPR/Cas9 system showed great anticancer efficacy and,more importantly,stimulated an immune memory response to inhibit distant tumor growth and lung metastasis.This CRISPR/Cas9 system represents an alternative strategy for ICB therapy as well as enhanced cancer immunotherapy. |
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