Acyclic artificial nucleic acid substitution expands the safe design space of gapmer ASOs by preventing P54nrb mislocalization

Abstract Gapmer-type antisense oligonucleotides (Gapmers) are promising therapeutic agents. However, their clinical potential is frequently limited by off-target toxicities. To address this issue, Gapmers have been optimized by modifying the ribose moiety or internucleotide linkage, but toxicity has not always been reduced. The toxicity is due to the unintended interactions between the ribose-type modified nucleic acids with phosphorothioate backbones and the endogenous proteins. We therefore hypothesized that the use of acyclic nucleic acids, which possess an entirely distinct structure to ribose, would solve the aforementioned issue. In this study, we demonstrate that the incorporation of an acyclic analog, serinol nucleic acid (SNA) or L-threoninol nucleic acid (L-aTNA), provides an alternative approach. Notably, substitution with SNA or L-aTNA effectively mitigated toxicity, even when conventional 2′-O-methyl modification was unsuccessful. This approach reduced cytotoxicity across multiple Gapmer sequences and designs in a position-dependent manner. Mechanistically, our investigation into these acyclic nucleic acids revealed that reduced toxicity was associated with suppression of P54nrb protein mislocalization. Furthermore, representative SNA- or L-aTNA-modified Gapmers exhibited markedly reduced hepatotoxicity in vivo. Collectively, these findings suggest that acyclic nucleic acids have potential as a useful chemical strategy for the development of safer Gapmer therapeutics.