Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA.
Nucleic Acids Res. 2019 Jun 20;47(11):5465-5479. doi: 10.1093/nar/gkz247.
Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.
硫代磷酸酯修饰的反义寡核苷酸（PS-ASO）与许多血浆、细胞表面和细胞内蛋白相互作用，从而控制其治疗特性。鉴于 PS 骨架对于与蛋白相互作用的重要性，我们系统地用电荷中性的烷基膦酸酯键取代了有毒 ASO 中的阴离子 PS 键。烷基膦酸酯的定点掺入改变了 RNaseH1 的切割模式，但总体切割率和在细胞和小鼠中针对靶基因的活性仅受到轻微影响。然而，即使将位于 DNA 缺口 5'-侧的 PS 键中的一个或两个替换为烷基膦酸酯，也会降低或消除几种肝毒性 gapmer ASO 的毒性。毒性降低伴随着核仁斑点蛋白 P54nrb 的核仁定位缺失、细胞中 P21 mRNA 水平升高和 caspase 激活的缺失，以及小鼠的肝毒性缺失。这些观察结果对于几种 ASO 与多个基因靶标进一步证明了这些观察结果的普遍性。我们的结果增加了可以在缺口区域中使用的结构修饰类型，以提高 ASO 的安全性，并深入了解 PS ASO 蛋白相互作用的生物化学。
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