Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.
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.
Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Vladimir S
VS
Trubetskoy
Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Jacob B
JB
Griffin
Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Anthony L
AL
Nicholas
Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Eric M
EM
Nord
Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Zhao
Z
Xu
Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Ryan M
RM
Peterson
Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Christine I
CI
Wooddell
Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
David B
DB
Rozema
Department of Chemistry, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Darren H
DH
Wakefield
Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
David L
DL
Lewis
Department of Biology, Arrowhead Pharmaceuticals, Inc., Madison, WI, USA.
Steven B
SB
Kanner
D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D055785:Gene Knockdown Techniques; D006514:Hepatitis B Surface Antigens / Q000097:blood / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism; D019694:Hepatitis B, Chronic / Q000378:metabolism / Q000628:therapy / Q000821:virology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism / Q000821:virology; D051379:Mice; D008813:Mice, Inbred ICR; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008822:Mice, Transgenic; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution
C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D006514:Hepatitis B Surface Antigens; D020135:Peptide Nucleic Acids; D034741:RNA, Small Interfering; D012367:RNA, Viral; D034743:RNA-Induced Silencing Complex