Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor.
Nucleic Acids Res
; 45(5): 2294-2306, 2017 03 17.
Article
em En
| MEDLINE
| ID: mdl-28158620
ABSTRACT
Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs â¼30-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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Acetilgalactosamina
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Bioensaio
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DNA
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DNA de Cadeia Simples
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Oligonucleotídeos Antissenso
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Receptor de Asialoglicoproteína
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Oligonucleotídeos Fosforotioatos
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos