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The aim of this study was to develop in\u00a0vitro assays to predict in\u00a0vivo metabolism of ASOs. Stability studies of ASOs with varying phosphorothioate\/phosphodiester (PS\/PO) content were conducted using nucleolytic matrices (snake venom 3\u2032\u2010exonuclease phosphodiesterase I [PDEI], mouse serum, and mouse liver homogenate) and analyzed by gel electrophoresis and liquid chromatography with ultraviolet and mass spectrometry detection (LC\u2010UV\/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\u2010methylcytidine nucleoside (<jats:sup>Me<\/jats:sup>C) on stability was observed. When the PO link was located after <jats:sup>Me<\/jats:sup>C (from 3\u2032 end), resistance toward nuclease hydrolysis increased, while the PO modification being before <jats:sup>Me<\/jats:sup>C had no protective effect. Employing nucleases and comparing stability data with matrices of animal origin holds promise for a future fast\u2010track assessment of drug stability, enabling the efficient selection of optimal drug candidates for subsequent in\u00a0vivo studies.<\/jats:p>","DOI":"10.1002\/prp2.70096","type":"journal-article","created":{"date-parts":[[2025,4,16]],"date-time":"2025-04-16T12:28:26Z","timestamp":1744806506000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["In Vitro Structure\u2013Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases"],"prefix":"10.1002","volume":"13","author":[{"given":"Jelena","family":"Lovri\u0107","sequence":"first","affiliation":[{"name":"DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&amp;D AstraZeneca  Gothenburg 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