In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases

The primary degradation pathway for antisense oligonucleotides (ASOs) involves endonuclease and exonuclease-mediated breakdown. The effect of various chemical modifications on the stability of oligonucleotides has not been systematically investigated. The aim of this study was to develop in vitro assays to predict in vivo metabolism of ASOs. Stability studies of ASOs with varying phosphorothioate/phosphodiester (PS/PO) content were conducted using nucleolytic matrices (snake venom 3'-exonuclease phosphodiesterase I [PDEI], mouse serum, and mouse liver homogenate) and analyzed by gel electrophoresis and liquid chromatography with ultraviolet and mass spectrometry detection (LC-UV/MS). Both sequence composition and backbone chemistry play important roles in influencing the stability of ASOs. Nucleolytic sensitivity observed with PDEI and mouse serum shows that ASOs with one PO modification in the backbone have higher stability than ASOs having two or three PO links, and with a lower PO content, the sequence has a larger influence on stability. Furthermore, the impact of a 5-methylcytidine nucleoside (^MeC) on stability was observed. When the PO link was located after ^MeC (from 3' end), resistance toward nuclease hydrolysis increased, while the PO modification being before ^MeC had no protective effect. Employing nucleases and comparing stability data with matrices of animal origin holds promise for a future fast-track assessment of drug stability, enabling the efficient selection of optimal drug candidates for subsequent in vivo studies.