Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes Garth A. Kinberger, Thazha P. Prakash, Jinghua Yu, Guillermo Vasquez, Audrey Low, Alfred Chappell, Karsten Schmidt, Heather M. Murray, Hans Gaus, Eric E. Swayze, Punit P. Seth ⇑ Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, United States article info Article history: Received 5 May 2016 Revised 24 May 2016 Accepted 27 May 2016 Available online xxxx Keywords: ASGR GalNAc ASO Delivery Hepatocytes abstract Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)—a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5–10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency. Ó 2016 Elsevier Ltd. All rights reserved. Antisense oligonucleotides bind their cognate mRNA in cells by Watson–Crick base pairing. Upon binding, they modulate function of the mRNA via one or more antisense mechanisms to produce a pharmacological effect. 1 Second generation ASOs are phosphoroth- ioate (PS) backbone modified with a central gap region of 7–14 DNA nucleotides flanked on either end with 2 0 -modified nucleotides. 2 The DNA gap region promotes cleavage of the tar- geted mRNA by RNase H1 mediated hydrolysis, 3 while the 2 0 -mod- ified nucleotides enhance ASO affinity for cognate RNA. 4 We recently showed that the potency of second generation ASOs for suppressing gene targets expressed in hepatocytes can be enhanced 10-fold by targeted delivery via the Asialoglycoprotein receptor (ASGR). 5,6 The ASGR is a C-type lectin which is abundantly expressed on hepatocytes of all mammals and regulates levels of plasma glyco- proteins terminating with sialic acid a2,6 galactose and N-acetyl galactosamine (GalNAc) sugars. 7,8 The functional receptor in mam- mals is comprised of two subunits (ASGR1 and ASGR2) which form a hetero-oligomeric complex with varying ratios (2–5:1). 9 The ASGR is internalized via clathrin mediated endocytosis in coated pits on the basolateral membrane of hepatocytes. 10 Upon internal- ization, the ligand–receptor complex is transported to endo-lyso- somal compartments. 11 Acidification of endosomal compartments promotes dissociation of the ligand–receptor complex. The recep- tor is recycled back to the plasma membrane while the cargo is sorted to lysosomes for degradation. Mice lacking the ASGR2 subunit are viable and fertile but express the ASGR1 subunit at reduced levels. 12 Cells expressing ASGR1 alone are capable of binding ligand but substantial binding is dependent on the level of expression of the protein. 13 In contrast, mice lacking the ASGR1 subunit do not bind ligand and do not express ASGR2 on the plasma membrane. 14 The ASGR1 possesses the carbohydrate recognition domain (CRD) for calcium mediated sugar binding 15 and the cytoplasmic signal for binding clathrin adaptor proteins within coated pits. 16 Elegant work by Lee showed that synthetic glycosides with branched tethers bind the ASGR with high affinity. 17,18 Binding affinity was dependent on the nature of the sugar (GalNAc > galac- tose), number of sugars (4 = 3 > 2 > 1) and the geometrical spacing between the sugar moieties. 19 Given the complexity of ASGR bind- ing and trafficking pathways, we wished to determine optimal valency of GalNAc sugars required for efficient ASGR-mediated delivery of ASOs to hepatocytes. In this manuscript, we show that second generation ASOs of different configurations can be effec- tively delivered to hepatocytes in mice using two and even a single GalNAc sugar. Our results suggest that the chemical features of http://dx.doi.org/10.1016/j.bmcl.2016.05.084 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail address: pseth@ionisph.com (P.P. Seth). Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Please cite this article in press as: Kinberger, G. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.084