Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142, USA.
MGH Program in Membrane Biology, Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Nucleic Acids Res. 2020 Dec 2;48(21):11827-11844. doi: 10.1093/nar/gkaa670.
One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.
三价 N-乙酰半乳糖胺（GalNAc）缀合的 siRNA 的一个显著特点是沉默作用的持久性，可以在临床前物种和人类中持续数月。在这里，我们研究了支持这种延长的药理活性的基础生物学。我们发现，siRNA 在酸性细胞内隔室中的积累和稳定性对于长期活性至关重要。我们表明，功能性 siRNA 可以从这些隔室中释放出来，并在给药数周后加载到新生成的 Argonaute 2 蛋白复合物中，从而随着时间的推移持续进行 RNAi 活性。在脂质纳米颗粒或 GalNAc 缀合物中递送相同的 siRNA 以实现类似的敲低，但只有 GalNAc-siRNA 支持延长的活性持续时间，这说明了受体介导的 siRNA 转运在该过程中的重要性。综上所述，我们提供了几条证据表明，酸性细胞内隔室充当 GalNAc-siRNA 缀合物的长期储存库，是体内观察到的延长活性持续时间的主要贡献者。
