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A convenient method for the synthesis of several triantennary GalNAc clusters based on a nitromethanetrispropionic acid core was developed. The synthetic approach involves pentafluorophenolic ester intermediates which can be used in a one-pot, seven reaction procedure to quickly prepare a variety of triantennary GalNAc conjugated ASOs. The GalNAc clusters were conjugated to the 5'-end of an antisense oligonucleotide and evaluated for activity in primary mouse hepatocytes where they showed ∼10-fold improvement in activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5'-end of antisense oligonucleotide using phosphoramidite chemistry. Conjugation of the 5'-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5'-triantennary GalNAc cluster conjugated ASOs and helps understand the structure-activity relationship for targeting hepatocytes with oligonucleotide therapeutics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.