Guanine base modifications in antisense oligonucleotides mitigate acute central nervous system toxicity

Antisense oligonucleotides (ASOs) are recognized as promising therapeutic agents for central nervous system (CNS) diseases. However, neurotoxicity induced by ASOs via intrathecal administration poses a major limitation for clinical use. Several approaches have been reported to mitigate CNS toxicity; however, the effects of chemical modifications to nucleic acid bases on toxicity remain poorly understood. In this study, we investigated the effects of hypoxanthine substitution for other nucleobases in neurotoxic gapmer ASOs on toxicity and activity using in vitro and in vivo assays. Similarly, guanine modifications were evaluated for their influence on CNS toxicity and efficacy following intracerebroventricular injection in mice. We first found that substituting guanine with hypoxanthine mitigated neurotoxicity, whereas substituting adenine or cytosine with hypoxanthine exacerbated it. In contrast, all four types of hypoxanthine-substitutions reduced the binding affinity for target RNA and decreased in vivo silencing efficacy. We next identified several guanine modifications that alleviated neurotoxicity. In particular, 7-deazaguanine modification reduced CNS toxicity while maintaining the silencing activity of the ASOs. Our findings provide useful insights into nucleobase-dependent neurotoxicity and suggest a promising strategy involving guanine modifications to mitigate ASO-induced neurotoxicity without compromising therapeutic efficacy for the treatment of CNS diseases.