Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor

Abstract

Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues.

Chemical modifications of nucleic acid drugs and their delivery systems for gene‐based therapy

“…PS gapmer ASOs conjugated with triantennary GalNAc (GalNAc‐ASOs, Figure B) was developed, and high binding affinity between asialoglycoprotein and GalNAc‐ASOs was observed . This strong interaction between ASGPR and GalNAc‐ASOs enabled efficient hepatocyte‐targeting cellular uptake mediated by ASGPR .…”

“…PS gapmer ASOs conjugated with triantennary GalNAc (GalNAc-ASOs, Figure 4B) was developed, and high binding affinity between asialoglycoprotein and GalNAc-ASOs was observed. 117 This strong interaction between ASGPR and GalNAc-ASOs enabled efficient hepatocyte-targeting cellular uptake mediated by ASGPR. 118 GalNAc-ASOs, as a hepatocyte-specific prodrug, was metabolized to release parent ASOs after its internalization into the liver, resulting in the dramatic reduction of target mRNA and protein levels of apolipoprotein C-III and TTR by approximately tenfold enhanced potency in contrast to the parent ASOs in mice.…”

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

“…hsiRNA hepatocyte internalization is facilitated by a phosphorothioate-modified, singlestranded overhang A defining feature of the hsiRNA constructs used in this study is the presence of a 5-nt singlestranded, phosphorothioate-modified (PS) overhang, which resembles the fully PS, singlestranded backbone of conventional antisense oligonucleotides (ASOs). Recent studies investigating ASO internalization by hepatocytes have established that the asialoglycoprotein receptor (ASGPR) contributes to the uptake of unconjugated PS ASOs 21,22 . Therefore, one of the hepatocyte uptake pathways for DCA-hsiRNA may be a receptor-mediated process that requires a single-stranded, PS-modified overhang.…”

Antisense Cancer Therapy: Do Antisense Oligonucleotides Hold Promise as a Cure for Cancer?

“…The most common treatments for cancer are a) surgery, b) chemotherapy, c) radiation therapy, and d) targeted therapy. antisense therapeutic research [9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Currently, over a hundred oligonucleotides are in clinical trials from 30 different pharmaceutical companies [23].…”