Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses

Nucleic Acids Res. 2017 Feb 17;45(3):1469-1478. doi: 10.1093/nar/gkw828.

Abstract

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.

MeSH terms

  • Animals
  • Argonaute Proteins / genetics
  • Argonaute Proteins / metabolism
  • Female
  • Gene Knockdown Techniques
  • Hepatitis B Surface Antigens / blood
  • Hepatitis B Surface Antigens / genetics
  • Hepatitis B virus / genetics
  • Hepatitis B virus / metabolism
  • Hepatitis B, Chronic / metabolism
  • Hepatitis B, Chronic / therapy
  • Hepatitis B, Chronic / virology
  • Humans
  • Kinetics
  • Liver / metabolism
  • Liver / virology
  • Mice
  • Mice, Inbred ICR
  • Mice, Inbred NOD
  • Mice, SCID
  • Mice, Transgenic
  • Peptide Nucleic Acids / genetics
  • Peptide Nucleic Acids / metabolism
  • Phosphorylation
  • RNA Interference*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • RNA-Induced Silencing Complex / genetics
  • RNA-Induced Silencing Complex / metabolism
  • Tissue Distribution

Substances

  • Ago2 protein, mouse
  • Argonaute Proteins
  • Hepatitis B Surface Antigens
  • Peptide Nucleic Acids
  • RNA, Small Interfering
  • RNA, Viral
  • RNA-Induced Silencing Complex