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Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.
Trubetskoy, Vladimir S; Griffin, Jacob B; Nicholas, Anthony L; Nord, Eric M; Xu, Zhao; Peterson, Ryan M; Wooddell, Christine I; Rozema, David B; Wakefield, Darren H; Lewis, David L; Kanner, Steven B.
Afiliación
  • Trubetskoy VS; Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Griffin JB; Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Nicholas AL; Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Nord EM; Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Xu Z; Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Peterson RM; Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Wooddell CI; Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Rozema DB; Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Wakefield DH; Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Lewis DL; Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
  • Kanner SB; Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Nucleic Acids Res ; 45(3): 1469-1478, 2017 02 17.
Article en En | MEDLINE | ID: mdl-28180327
The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Interferencia de ARN Idioma: En Revista: Nucleic Acids Res Año: 2017 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Interferencia de ARN Idioma: En Revista: Nucleic Acids Res Año: 2017 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido