Cas9-edited immune checkpoint blockade PD-1 DNA polyaptamer hydrogel for cancer immunotherapy.

Immune checkpoint inhibitors have been widely studied in immunotherapy. Although antibodies have been more widely used to block immune checkpoints, DNA aptamers have unique advantages for this purpose. Here, we designed a DNA polyaptamer hydrogel that can be precisely cut by Cas9/sgRNA for programmed release of an immune checkpoint-blocking DNA aptamer. As a representative immune checkpoint inhibitor, we used a PD-1 DNA aptamer. Rolling-circle amplification was used to generate a hydrogel comprising DNA with PD-1 aptamer and an sgRNA-targeting sequence. When mixed with Cas9/sgRNA, the PD-1 DNA aptamer hydrogel (PAH) lost its gel property and liberated the PD-1 aptamer sequence. The precise Cas9/sgRNA-mediated release of the PD-1 DNA aptamer, which was confirmed by gel electrophoresis, was found to effectively activate the cytokine-secretion function of splenocytes. In vivo, molecular imaging revealed that PD-1 DNA polyaptamer hydrogel co-injected with Cas9/sgRNA (Cas9/PAH) remained at the injection site longer than free aptamer and yielded significantly higher antitumor effects and survival than hydrogel or free aptamer. Moreover, increased immune cell filtration was observed at tumor tissues treated with Cas9/PAH. These results suggest that our Cas9/sgRNA-edited immune checkpoint-blocking aptamer hydrogel has strong potential for anticancer immunotherapy.