Immune checkpoint blockade (ICB) is an attractive option in cancer therapy, but its efficacy is still less than expected due to the transient and incomplete blocking and the low responsiveness. Herein, an unprecedented programmable unlocking nano-matryoshka-CRISPR system (PUN) targeting programmed cell death ligand 1 (PD-L1) and protein tyrosine phosphatase N2 (PTPN2) is fabricated for permanent and complete and highly responsive immunotherapy. While PUN is inert at normal physiological conditions, enzyme-abundant tumor microenvironment and preternatural intracellular oxidative stress sequentially trigger programmable unlocking of PUN to realize a nano-matryoshka-like release of CRISPR/Cas9. The successful nucleus localization of CRISPR/Cas9 ensures the highly efficient disruption of PD-L1 and PTPN2 to unleash cascade amplified adaptive immune response via revoking the immune checkpoint effect. PD-L1 downregulation in tumor cells not only disrupts PD-1/PD-L1 interaction to attenuate the immunosurveillance evasion but also spurs potent immune T cell responses to enhance adaptive immunity. Synchronously, inhibition of JAK/STAT pathway is relieved by deleting PTPN2, which promotes tumor susceptibility to CD8⁺ T cells depending on IFN-γ, thus further amplifying adaptive immune responses. Combining these advances together, PUN exhibits optimal antitumor efficiency and long-term immune memory with negligible toxicity, which provides a promising alternative to current ICB therapy.

中文翻译：

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可编程解锁 Nano-Matryoshka-CRISPR 精确逆转免疫抑制，释放级联放大的适应性免疫反应

免疫检查点阻断（ICB）是癌症治疗中一个有吸引力的选择，但由于短暂且不完全的阻断以及低反应性，其疗效仍然低于预期。在此，制造了一种前所未有的可编程解锁纳米套娃-CRISPR系统（PUN），其靶向程序性细胞死亡配体1（PD-L1）和蛋白酪氨酸磷酸酶N2（PTPN2），用于永久、完整和高响应的免疫治疗。虽然 PUN 在正常生理条件下呈惰性，但酶丰富的肿瘤微环境和异常的细胞内氧化应激会依次触发 PUN 的可编程解锁，从而实现 CRISPR/Cas9 的纳米套娃式释放。CRISPR/Cas9 的成功核定位确保了 PD-L1 和 PTPN2 的高效破坏，通过撤销免疫检查点效应来释放级联放大的适应性免疫反应。肿瘤细胞中的 PD-L1 下调不仅破坏 PD-1/PD-L1 相互作用以减弱免疫监视逃避，而且还刺激有效的免疫 T 细胞反应以增强适应性免疫。同时，通过删除PTPN2来缓解对JAK/STAT通路的抑制，从而促进肿瘤对依赖于IFN- γ的CD8 ⁺ T细胞的易感性，从而进一步放大适应性免疫反应。将这些进步结合在一起，PUN 表现出最佳的抗肿瘤效率和长期免疫记忆，且毒性可忽略不计，为当前 ICB 疗法提供了一种有前景的替代方案。