The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

中文翻译：

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迈向下一代反义寡核苷酸：甲磺酰氨基磷酸酯修饰提高了gapmer反义寡核苷酸的治疗指数和作用持续时间

PS修饰增强了gapmer反义寡核苷酸（ASO）的核酸酶稳定性和蛋白质结合特性，是支持RNaseH1活性的极少数修饰之一。我们评估了在gapmer PS ASO的DNA间隙和侧翼中引入立体随机和手性甲磺酰基-氨基磷酸酯（MsPA）键的影响，并表征了这些键对RNA结合、核酸酶稳定性、蛋白质结合、促炎谱、反义的影响在细胞和小鼠中的活性和毒性。我们表明，gapmer ASO 中的所有 PS 连接都可以用 MsPA 代替，而不会影响化学稳定性和 RNA 结合亲和力，但这些设计会降低活性。然而，用 MsPA 替换间隙中多达 5 个 PS 具有良好的耐受性，并且在适当的位置替换特定的 PS 连接能够大大降低免疫刺激和细胞毒性。MsPA 相对于 PS 改进的核酸酶稳定性转化为小鼠 ASO 作用持续时间的显着改善，这与增强的稳定 siRNA 设计相当。我们的工作强调了 PS 和 MsPA 链接的组合作为下一代化学平台，用于识别具有提高效力和治疗指数、降低促炎作用和延长作用持续时间的 ASO 药物。