Vasquez Guillermo, Migawa Michael T, Wan W Brad, Low Audrey, Tanowitz Michael, Swayze Eric E, Seth Punit P
Department of Medicinal Chemistry, Ionis Pharmaceuticals, Carlsbad, California, USA.
Nucleic Acid Ther. 2022 Feb;32(1):40-50. doi: 10.1089/nat.2021.0064. Epub 2021 Oct 25.
The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and -isopropyl and -tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.
硫代磷酸酯（PS）键是治疗性寡核苷酸的重要组成部分。间隙mer反义寡核苷酸（ASO）的DNA区域中的PS支持RNaseH1活性并增强核酸酶稳定性。PS还促进与血浆、细胞表面和细胞内蛋白质的结合，这有利于PS ASO的组织分布、细胞摄取和内体逃逸。我们最近表明，用甲氧基丙基膦酸酯（MOP）键对DNA间隙中的PS进行位点特异性替换可以提高间隙mer ASO的治疗指数。在本文中，我们探索了18种基于磷和非磷的中性主链修饰，以确定增强治疗指数的中性键的构效关系。用其他烷基膦酸酯和磷酸三酯键取代MOP可提高治疗指数，但这些键在寡核苷酸从合成后的固体支持物上脱保护过程中易受化学降解。用非磷键取代MOP可提高化学稳定性，但这些键以核苷酸二聚体的形式引入ASO中，这限制了它们的通用性。总体而言，相对于MOP，异丙基和异丁基膦酸酯以及异丙基和四氢呋喃糖基磷酸三酯、缩醛和C3-酰胺等键在小鼠中显示出改善的活性。我们的数据表明，任何中性主链键的位点特异性掺入都可以提高治疗指数，但键的大小、疏水性和RNA结合亲和力会影响ASO活性。
