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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Schmidt K,Prakash TP,Donner AJ,Kinberger GA,Gaus HJ,Low A,Østergaard ME,Bell M,Swayze EE,Seth PP

doi

10.1093/nar/gkx060

subject

Has Abstract

pub_date

2017-03-17 00:00:00

pages

2294-2306

issue

5

eissn

0305-1048

issn

1362-4962

pii

2967000

journal_volume

45

pub_type

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