Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical...

Antisense oligonucleotides (ASOs) represent an attractive therapeutic approach for CNS disorders. However, ASO-induced neurotoxicity, especially late-onset adverse events, remains a crucial issue, leading to failures in clinical applications. This study aims to determine the neurological features and molecular mechanisms of the late-onset neurotoxicity and provide strategies to overcome this toxicity. We initially established neurobehavioral assays of rodent neurotoxicity with intracerebroventricular and intrathecal injections of various gapmer-type ASOs and a neuronal cytotoxicity analysis. Through both in vitro and in vivo assessments, we identified a site-specific chemical modification, 5'-cyclopropylene (5'-CP), that significantly reduced late-onset neurotoxicity without compromising knockdown activity, providing useful insights into structure-toxicity and structure-activity relationships in ASOs targeting CNS. Additionally, we revealed a toxicity-related mechanism as an elevation of p53-regulated transcripts and paraspeckle protein mislocalization in neuronal cells, which is alleviated through the chemical modifications. Our findings provide mechanistic insights into late-onset ASO-induced neurotoxicity and highlight the potential of optimized chemical modifications to expand the therapeutic window for clinical applications targeting intractable neurological diseases.