Comprehensive Structure-Activity Relationship of Triantennary N-Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes

J Med Chem. 2016 Mar 24;59(6):2718-33. doi: 10.1021/acs.jmedchem.5b01948. Epub 2016 Mar 9.


The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.

MeSH terms

  • Acetylgalactosamine / chemical synthesis*
  • Acetylgalactosamine / pharmacology*
  • Animals
  • Apolipoprotein C-III / drug effects
  • Drug Delivery Systems
  • Factor XI / drug effects
  • Hepatocytes / drug effects*
  • Humans
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Oligonucleotides, Antisense / chemical synthesis*
  • Oligonucleotides, Antisense / pharmacology*
  • Scavenger Receptors, Class B / biosynthesis
  • Scavenger Receptors, Class B / genetics
  • Structure-Activity Relationship


  • Apolipoprotein C-III
  • Oligonucleotides, Antisense
  • Scarb1 protein, mouse
  • Scavenger Receptors, Class B
  • Factor XI
  • Acetylgalactosamine