Cathepsin B-activatable cyclic antisense oligonucleotides for cell-specific target gene knockdown in vitro and in vivo

Zhongyu Wang; Xinli Fan; Guanqun Mu; Xiaoran Zhao; Qian Wang; Jing Wang; Xinjing Tang

doi:10.1016/j.omtn.2023.07.022

Cathepsin B-activatable cyclic antisense oligonucleotides for cell-specific target gene knockdown in vitro and in vivo

Mol Ther Nucleic Acids. 2023 Jul 25:33:548-558. doi: 10.1016/j.omtn.2023.07.022. eCollection 2023 Sep 12.

Authors

Zhongyu Wang¹, Xinli Fan¹, Guanqun Mu¹, Xiaoran Zhao¹, Qian Wang¹, Jing Wang¹, Xinjing Tang^{1

2}

Affiliations

¹ 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.
² State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu 210023, People's Republic of China.

Abstract

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.

Keywords: MT: Oligonucleotides: Therapies and Applications; TCTP; caged antisense oligonucleotide; cathepsin B; cell selectivity; circular oligonucleotide; enzyme caging; gene knockdown.