Wang Zhongyu, Fan Xinli, Mu Guanqun, Zhao Xiaoran, Wang Qian, Wang Jing, Tang Xinjing
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences and Chemical Biology Center, Peking University, No. 38, Xueyuan Road, Beijing 100191, People's Republic of China.
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu 210023, People's Republic of China.
Mol Ther Nucleic Acids. 2023 Jul 25;33:548-558. doi: 10.1016/j.omtn.2023.07.022. eCollection 2023 Sep 12.
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 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.
触发激活型反义寡核苷酸已被广泛应用于调节基因功能。其中，笼状环状反义寡核苷酸（cASOs）保持特定的拓扑结构，暂时抑制它们与靶基因的相互作用。通过插入对细胞特异性内源性刺激有反应的连接子，它们可以成为对正常细胞具有低脱靶效应的特定类型癌细胞的强大工具和潜在治疗剂。在这里，我们通过组织蛋白酶B（CB）底物肽（甘氨酸-苯丙氨酸-亮氨酸-甘氨酸[GFLG]）将线性反义寡核苷酸的两个末端连接起来，开发了酶激活型cASOs，其可被CB有效解笼。CB激活型cASOs用于在CB丰富的PC-3肿瘤细胞中在mRNA和蛋白质水平成功敲低两个疾病相关内源性基因，但对CB缺乏的人脐静脉内皮细胞（HUVECs）中的基因敲低影响小得多。此外，与线性对应物相比，使用cASOs发现非特异性免疫刺激减少。进一步的研究表明，CB激活型cASOs通过下调肿瘤中的翻译控制肿瘤蛋白（TCTP）蛋白，在PC-3肿瘤模型小鼠中显示出有效的肿瘤抑制作用。本研究将内源性酶激活型cASOs应用于肿瘤模型小鼠的抗肿瘤治疗，这证明了一种有前景的刺激响应性cASO策略，用于内源性激活后的细胞特异性基因敲低和ASO前药开发。
