Cycloalkane Incorporation Into the 2′,4′-Bridge of Locked Nucleic Acid: Enhancing Nuclease Stability, Reducing Phosphorothioate Modifications, and Lowering Hepatotoxicity in Antisense Oligonucleotides

2'-<i>O</i>,4'-<i>C</i>-Methylene-bridged nucleic acid (2',4'-BNA), also known as locked nucleic acid (LNA), is widely used to modify antisense oligonucleotides (ASOs) because it significantly enhances their ability to form duplexes with target RNAs, thereby boosting ASO activity. However, 2',4'-BNA/LNA exhibits only moderate nuclease stability, necessitating additional modifications, such as phosphorothioate (PS) linkages. Our previous studies demonstrated that oligonucleotides modified with 2'-<i>O</i>,4'-<i>C</i>-spirocyclopropylene-bridged nucleic acid (scpBNA) retain duplex-forming abilities similar to those of 2',4'-BNA/LNA, while showing significantly improved nuclease stability. In the present study, we introduce 2'-<i>O</i>,4'-<i>C</i>-spirocyclopentylene-bridged nucleic acid (scpBNA2), which offers even greater nuclease stability than scpBNA. We synthesized scpBNA2 phosphoramidites containing either thymine or 5-methylcytosine nucleobases and incorporated them into gapmer-type ASOs. Both scpBNA- and scpBNA2-modified ASOs exhibited antisense activity comparable to that of their 2',4'-BNA/LNA-modified counterparts, maintaining activity even with reduced PS modifications in the scpBNA- or scpBNA2-modified regions. Furthermore, hepatotoxicity observed with 2',4'-BNA/LNA-modified ASOs was significantly reduced when 2',4'-BNA/LNA was replaced with either scpBNA or scpBNA2. Overall, these findings underscore the potential of scpBNA and scpBNA2 for use in therapeutic ASOs.