title: Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.
abstract: Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity.
authors.affiliation: 1 Drug Safety Research and Development, Pfizer, Inc. , Groton, Connecticut.
authors.forename: Satoko
authors.initials: S
authors.lastname: Kakiuchi-Kiyota
authors.affiliation: 2 Drug Safety Research and Development, Pfizer, Inc. , Andover, Massachusetts.
authors.forename: Lawrence O
authors.initials: LO
authors.lastname: Whiteley
authors.affiliation: 1 Drug Safety Research and Development, Pfizer, Inc. , Groton, Connecticut.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Ryan
authors.affiliation: 1 Drug Safety Research and Development, Pfizer, Inc. , Groton, Connecticut.
authors.forename: Nagappan
authors.initials: N
authors.lastname: Mathialagan
mesh_terms: D000818:Animals; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000235:genetics; D040081:Protein Array Analysis; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid