For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2′-deoxy-2′-fluoro (2′-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2′-F-monomer metabolites was low and transient in rats and humans. In vitro, 2′-F-nucleoside 5′-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2′-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2′-F-monomers were observed after weekly administration of two GalNAc–siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2′-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

中文翻译：

---

GalNAc-siRNA结合物中2'-脱氧-2'-氟核苷酸的安全性评估

对于寡核苷酸治疗，糖-磷酸主链的化学修饰经常用于赋予类似药物的特性。由于尚不知道天然存在2'-脱氧-2'-氟（2'-F）核苷酸，因此当将它们用于相同序列的两个短干扰RNA（siRNA）的repusiran和ALN-TTRSC02时，评估了它们的安全性但与N-乙酰半乳糖胺（GalNAc）配体缀合的化学修饰方式和代谢稳定性不同，可靶向递送至肝细胞。在大鼠和人类中，暴露于2'-F-单体代谢物的程度很低且短暂。体外，2'-F-核苷5'-三磷酸酯既不是抑制剂也不是人类聚合酶的优选底物，并且未观察到专性或非专性链终止。在高浓度的2'-F-核苷中，通常在体内无法获得的细胞类型的子集中，在体外观察到对细胞活力和线粒体DNA的适度影响。在大鼠中每周服用两种GalNAc-siRNA偶联物约2年后，未观察到对线粒体的明显功能性影响，也未观察到2'-F-单体的大量积累。综上所述，结果支持以下结论：2'-F核苷酸可以安全地用于代谢稳定的治疗性GalNAc-siRNA的设计，具有良好的功效和延长的活性持续时间，可实现低剂量和不频繁给药。