广泛应用于基因功能调控的诱导活化反义寡核苷酸已经成为常见的研究手段。其中，被阻抑的环状反义寡核苷酸（cASOs）通过保持特定拓扑结构，在一定期间内抑制其与靶基因的相互作用。通过插入能够响应细胞特异性内源性刺激的连接物，它们可以成为对特定类型的癌细胞具有低无靶效应的有效工具和潜在治疗药物。在本研究中，我们通过将线性反义寡核苷酸的两个末端通过降解酶B（CB）底物肽（Gly-Phe-Leu-Gly [GFLG]）连接，制备了酶活化的cASOs，CB可以有效去除其阻抑作用。CB活化的cASOs应用于CB富集的PC-3肿瘤细胞中，在mRNA和蛋白水平上成功击败了两种与疾病相关的内源基因，但对于CB缺乏的人脐静脉内皮细胞（HUVECs）中的基因靶向敲除效果较小。此外，与线性对应物相比，cASOs在免疫刺激方面显示出降低的非特异性反应。进一步的体内研究表明，CB活化的cASOs通过下调翻译调控的肿瘤蛋白（TCTP）在PC-3肿瘤模型小鼠中达到了有效的抑制肿瘤的效果。本研究应用内源酶活化的cASOs用于肿瘤模型小鼠的抗肿瘤治疗中，展示了一种有前景的刺激响应性cASO策略，用于特异性细胞基因敲除及ASO前药开发。© 2023 作者

Trigger-activatable antisense oligonucleotides have been widely applied to regulate gene function. Among them, caged cyclic antisense oligonucleotides (cASOs) maintain a specific topology that temporarily inhibits their interaction with target genes. By inserting linkers that respond to cell-specific endogenous stimuli, they can be powerful tools and potential therapeutic agents for specific types of cancer cells with low off-target effects on normal cells. Here, we developed enzyme-activatable cASOs by tethering two terminals of linear antisense oligonucleotides through a cathepsin B (CB) substrate peptide (Gly-Phe-Leu-Gly [GFLG]), which could be efficiently uncaged by CB. CB-activatable cASOs were used to successfully knock down two disease-related endogenous genes in CB-abundant PC-3 tumor cells at the mRNA and protein levels but had much less effect on gene knockdown in CB-deficient human umbilical vein endothelial cell (HUVECs). In addition, reduced nonspecific immunostimulation was found using cASOs compared with their linear counterparts. Further in vivo studies indicated that CB-activatable cASOs showed effective tumor inhibition in PC-3 tumor model mice through downregulation of translationally controlled tumor protein (TCTP) protein in tumors. This study applies endogenous enzyme-activatable cASOs for antitumor therapy in tumor model mice, which demonstrates a promising stimulus-responsive cASO strategy for cell-specific gene knockdown upon endogenous activation and ASO prodrug development.© 2023 The Author(s).