In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases
ABSTRACT 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...
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2025-06-01
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| Series: | Pharmacology Research & Perspectives |
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| Online Access: | https://doi.org/10.1002/prp2.70096 |
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| author | Jelena Lovrić Jingjing Yan XueQing Li Tony Karlsborn Mattias Bood Anders Dahlén Constanze Hilgendorf Deepak Kumar Bhatt |
| author_facet | Jelena Lovrić Jingjing Yan XueQing Li Tony Karlsborn Mattias Bood Anders Dahlén Constanze Hilgendorf Deepak Kumar Bhatt |
| author_sort | Jelena Lovrić |
| collection | DOAJ |
| description | ABSTRACT 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. |
| format | Article |
| id | doaj-art-1cd34f7147cc451bbb571e20293c603d |
| institution | DOAJ |
| issn | 2052-1707 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | Pharmacology Research & Perspectives |
| spelling | doaj-art-1cd34f7147cc451bbb571e20293c603d2025-08-20T02:39:32ZengWileyPharmacology Research & Perspectives2052-17072025-06-01133n/an/a10.1002/prp2.70096In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and NucleasesJelena Lovrić0Jingjing Yan1XueQing Li2Tony Karlsborn3Mattias Bood4Anders Dahlén5Constanze Hilgendorf6Deepak Kumar Bhatt7DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenDMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenDMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenDMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenCell, Gene and RNA Therapeutics, Discovery Sciences, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenCell, Gene and RNA Therapeutics, Discovery Sciences, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenDMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenDMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D AstraZeneca Gothenburg SwedenABSTRACT 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.https://doi.org/10.1002/prp2.70096ASOmetabolic stabilitynuclease |
| spellingShingle | Jelena Lovrić Jingjing Yan XueQing Li Tony Karlsborn Mattias Bood Anders Dahlén Constanze Hilgendorf Deepak Kumar Bhatt In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases Pharmacology Research & Perspectives ASO metabolic stability nuclease |
| title | In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases |
| title_full | In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases |
| title_fullStr | In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases |
| title_full_unstemmed | In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases |
| title_short | In Vitro Structure–Activity Relationship Stability Study of Antisense Oligonucleotide Therapeutics Using Biological Matrices and Nucleases |
| title_sort | in vitro structure activity relationship stability study of antisense oligonucleotide therapeutics using biological matrices and nucleases |
| topic | ASO metabolic stability nuclease |
| url | https://doi.org/10.1002/prp2.70096 |
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