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

The comprehensive structure activity relation ships 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 GalAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GaINAc 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 GaINAc 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 clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.