Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2′-O-methyl (2′-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2′-methoxyethyl, 2′ constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2′-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5′ end of the gap or in the 3′-wing of a Gap 2′-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2′-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

中文翻译：

---

反义寡核苷酸中选定位置的甲磺酰-氨基磷酸酯核苷酸间键和 2'-O-甲基的组合增强了 RNaseH1 活性 PS-ASO 的性能

介导 RNase H1 对 RNA 靶标降解的反义寡核苷酸 (ASO) 被用作治疗各种疾病的药物。以前我们发现引入单个 2'- O-甲基（2'-OMe）修饰gapmer硫代磷酸酯（PS）ASO的中心脱氧核苷酸区域的第2位，其中末端的几个残基是2'-甲氧基乙基，2'约束乙基或锁定核酸，显着降低细胞毒性，仅对效力产生适度影响。最近，我们证明了用甲磺酰基-氨基磷酸酯 (MsPA) 键替换间隙中位置 2 或 3 的 PS 键也显着降低了毒性，而没有明显的效力损失，并增加了 ASO 的消除半衰期。在本研究中，我们评估了 MsPA 键和 2'-OMe 核苷酸组合对 PS ASO 性能的影响。我们发现在间隙 5' 端或间隙 2'-OMe PS ASO 的 3' 翼中的两个 MsPA 修饰显着增加了间隙位置 2 处带有 OMe 的 ASO 的活性，而不会改变安全性。在细胞和动物中用多个序列观察到这种效应。因此，MsPA 修饰提高了间隙中有 2'-OMe 的 PS ASO 的 RNase H1 切割率，显着降低了与细胞毒性相关的蛋白质的结合，并延长了消除半衰期。