Metabolically Stable Anomeric Linkages Containing GalNAc–siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways

Conjugation
of synthetic triantennary N-acetyl-d-galactosamine
(GalNAc) to small interfering RNA (siRNA) mediates
binding to the asialoglycoprotein receptor (ASGPR) on the surface
of hepatocytes, facilitating liver-specific uptake and siRNA-mediated
gene silencing. The natural β-glycosidic bond of the GalNAc
ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands
with S-, and C-glycosides with both
α- and β-anomeric linkages, N-glycosides
with β-anomeric linkage, and the O-glycoside
with α-anomeric linkage were synthesized and conjugated to siRNA
either on-column during siRNA synthesis or through a high-throughput,
post-synthetic method. Unlike natural GalNAc, modified ligands were
resistant to glycosidase activity. The siRNAs conjugated to newly
designed ligands had similar affinities for ASGPR and similar silencing
activity in mice as the parent GalNAc–siRNA conjugate. These
data suggest that other factors, such as protein–nucleic acid
interactions and loading of the antisense strand into the RNA-induced
silencing complex (RISC), are more critical to the duration of action
than the stereochemistry and stability of the anomeric linkage between
the GalNAc moiety of the ligand conjugated to the sense strand of
the siRNA.