Developing in vitro models to elucidate the mechanisms of toxicity of locked nucleic acid gapmer antisense oligonucleotides.
type: CreativeWork
description: Antisense oligonucleotides are an emerging class of RNA therapeutics that have recently received several approvals from the clinic. Antisense oligonucleotides act at the RNA level to either degrade target RNA, or to regulate splicing of a particular gene, and have been applied to treat a variety of diseases including Duchenne muscular dystrophy (DMD), and familial chylomicronemia syndrome (FCS). Advanced chemical modifications have driven the field forward, allowing RNA-based drugs to be better tolerated in patients, reducing immune activation and increasing potency. However, these modifications have also shown various toxicities, including hepatotoxicity. Mechanisms of ASO-induced toxicity are still not fully understood, but ASO-protein interactions have been shown to play a vital role in the tolerability of ASOs. This study investigated the safety of antisense oligonucleotides by developing a toolbox of in vitro assays for assessing liver toxicity. HepG2/C3A cells were used as a cellular model to categorise the toxicity of tool ASOs and identify apoptosis as the main mechanism of cell death. Orthogonal organic phase separation (OOPS) was used to characterise the RNA-binding proteome (RBPome) of HepG2/C3As for the first time. In addition, OOPS and biotin pull-downs were used to characterise changes in the RBPome with ASOs, and identify direct interactors. Finally, secondary structure formation was identified as a key attribute that correlated with toxic ASOs. A novel, high-throughput assay was developed to rapidly assess the secondary structure formation of ASOs, allowing for the screening of potentially toxic ASOs. Using the lessons learnt from these experiments, a toxic ASO was mutated to become non-toxic, and vice versa. In addition, the mechanisms behind this secondary structure toxicity were interrogated using transcriptomic profiling, and the TNF-α signalling pathway was shown to contribute to the toxicity measured with these ASOs. Further work is required to…
abstract: Antisense oligonucleotides are an emerging class of RNA therapeutics that have recently received several approvals from the clinic. Antisense oligonucleotides act at the RNA level to either degrade target RNA, or to regulate splicing of a particular gene, and have been applied to treat a variety of diseases including Duchenne muscular dystrophy (DMD), and familial chylomicronemia syndrome (FCS). Advanced chemical modifications have driven the field forward, allowing RNA-based drugs to be better tolerated in patients, reducing immune activation and increasing potency. However, these modifications have also shown various toxicities, including hepatotoxicity. Mechanisms of ASO-induced toxicity are still not fully understood, but ASO-protein interactions have been shown to play a vital role in the tolerability of ASOs. This study investigated the safety of antisense oligonucleotides by developing a toolbox of in vitro assays for assessing liver toxicity. HepG2/C3A cells were used as a cellular model to categorise the toxicity of tool ASOs and identify apoptosis as the main mechanism of cell death. Orthogonal organic phase separation (OOPS) was used to characterise the RNA-binding proteome (RBPome) of HepG2/C3As for the first time. In addition, OOPS and biotin pull-downs were used to characterise changes in the RBPome with ASOs, and identify direct interactors. Finally, secondary structure formation was identified as a key attribute that correlated with toxic ASOs. A novel, high-throughput assay was developed to rapidly assess the secondary structure formation of ASOs, allowing for the screening of potentially toxic ASOs. Using the lessons learnt from these experiments, a toxic ASO was mutated to become non-toxic, and vice versa. In addition, the mechanisms behind this secondary structure toxicity were interrogated using transcriptomic profiling, and the TNF-α signalling pathway was shown to contribute to the toxicity measured with these ASOs. Further work is required to…
author: Fulton, Alex
language: eng
keywords: Antisense oligonucleotides, Liver toxicity, RNA binding proteins
license: https://www.repository.cam.ac.uk/bitstreams/fcdd9c99-2b4b-4472-9f78-29a02b43815a/download
license: http://purl.org/NET/rdflicense/allrightsreserved
date published: 2025-09-28
date modified: 2026-07-22T22:24:08Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.125331