Conjugation of the synthetic triantennary N-acetyl-D-galactosamine (GalNAc) ligand to siRNA mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake of the siRNA and leading to RNAi-mediated gene silencing. Susceptibility of the natural β-glycosidic bond to glycosidases results in rapid cleavage of the sugar moiety from the triantennary GalNAc ligand following ASGPR-mediated uptake into hepatocytes. In an effort to improve the metabolic stability of the glycosidic bond and to understand the impact of slower metabolism of the GalNAc moiety and the roles of other atoms at the anomeric linkage on the gene silencing activity of GalNAc-siRNA, we designed and introduced a general synthetic method to synthesize several novel GalNAc ligands with metabolically stable S-, C-, and N-glycosides with both α- and β-anomeric linkages and prepared siRNA conjugates. Because of their resistance to degradation both in plasma and in liver, these conjugates have prolonged stability relative to the canonical β-anomeric O-glycosidic design. The ligands with anomeric linkages have similar affinity for ASGPR as the original ligands and do not affect the performance of the conjugated siRNA. Importantly, the stabilized triantennary ligands described in this study do not impair loading of the antisense strand into the RNA-induced silencing complex as silencing activity was maintained both in vitro and in vivo.

Metabolically stable anomeric linkages containing GalNAc-siRNA conjugates: An interplay among ASGPR, glycosidase, and RISC pathways

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ACS Fall 2022

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