Investigating the Pharmacodynamic Durability of GalNAc–siRNA Conjugates

Authors: C.R. Brown, S. Gupta, J. Qin, T. Racie et. al.

Journal: Nucleic Acids Research

DOI: 10.1093/nar/gkaa670

Publication - Abstract

August 18, 2020


In this paper Alnylam Pharmaceuticals, developer of RNAi therapeutics ONPATTRO® and GIVLAARI®, investigated  the extended duration of N-acetylgalactosamine silencing RNA (GalNAc-siRNA) activity observed in vivo. Using the advanced Enhanced Stabilization Chemistry (ESC) design of GalNAc-siRNA, they performed in vitro studies with naked GalNAc-siRNAs and in vivo rodent studies with naked GalNAc-siRNAs and GalNAc-siRNAs in lipid nanoparticles (LNP). 
They found that "acidic intracellular compartments are long-term depot" for GalNAc–siRNA conjugates and are responsible for the long-lasting activity observed in vivo, effectively precluding subcutaneous site of injection as a depot for siRNA. In addition, the mode of GalNAc-siRNAs delivery was found to have a significant impact on siRNA efficacy and duration, with LNP delivery requiring a lower dose to achieve a similar knockdown effect as naked GalNAc-siRNAs. The LNPs used in this study were prepared using NanoAssemblr technology and had a mean diameter of ∼65 nm with excellent homogeneity (PDI = 0.05). 

These findings demonstrate that an improved siRNA LNP drug design can minimize degradation, amplifying target knockdown and duration of effect.


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.

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