title: Sequence motifs associated with hepatotoxicity of locked nucleic acid--modified antisense oligonucleotides.
abstract: Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure-toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure-toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.
authors.affiliation: Drug Safety Research and Development, Worldwide Research and Development, Pfizer Inc., Cambridge, MA 02140, USA, Oligonucleotide Therapeutic Unit, Worldwide Research and Development, Pfizer Inc., Cambridge, MA 02139, USA and Attagene Inc., Morrisville, NC 27560, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Burdick
authors.forename: Simone
authors.initials: S
authors.lastname: Sciabola
authors.forename: Srinivasa R
authors.initials: SR
authors.lastname: Mantena
authors.forename: Brett D
authors.initials: BD
authors.lastname: Hollingshead
authors.forename: Robert
authors.initials: R
authors.lastname: Stanton
authors.forename: James A
authors.initials: JA
authors.lastname: Warneke
authors.forename: Ming
authors.initials: M
authors.lastname: Zeng
authors.forename: Elena
authors.initials: E
authors.lastname: Martsen
authors.forename: Alexander
authors.initials: A
authors.lastname: Medvedev
authors.forename: Sergei S
authors.initials: SS
authors.lastname: Makarov
authors.forename: Lori A
authors.initials: LA
authors.lastname: Reed
authors.forename: John W
authors.initials: JW
authors.lastname: Davis
authors.forename: Laurence O
authors.initials: LO
authors.lastname: Whiteley
mesh_terms: D000818:Animals; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008297:Male; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D059372:Nucleotide Motifs; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D011506:Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D011506:Proteins; D016159:Tumor Suppressor Protein p53; C477371:locked nucleic acid