Structural Analysis of Human Argonaute-2 Bound to a Modified siRNA Guide.

Incorporation of chemical modifications into small interfering RNAs (siRNAs) increases their metabolic stability and improves their tissue distribution. However, how these modifications impact interactions with Argonaute-2 (Ago2), the molecular target of siRNAs, is not known. Herein we present the crystal structure of human Ago2 bound to a metabolically stable siRNA containing extensive backbone modifications. Comparison to the structure of an equivalent unmodified-siRNA complex indicates that the structure of Ago2 is relatively unaffected by chemical modifications in the bound siRNA. In contrast, the modified siRNA appears to be much more plastic and shifts, relative to the unmodified siRNA, to optimize contacts with Ago2. Structure-activity analysis reveals that even major conformational perturbations in the 3' half of the siRNA seed region have a relatively modest effect on knockdown potency. These findings provide an explanation for a variety of modification patterns tolerated in siRNAs and a structural basis for advancing therapeutic siRNA design.

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.

Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, ∼ 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.