title: Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.
abstract: Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Sabine
authors.initials: S
authors.lastname: Sewing
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Boess
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Annie
authors.initials: A
authors.lastname: Moisan
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Cristina
authors.initials: C
authors.lastname: Bertinetti-Lapatki
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Minz
authors.affiliation: Roche Pharmaceutical Research and Early Development, Therapeutic Modalities, Roche Innovation Center Copenhagen A/S, Hørsholm, Denmark.
authors.forename: Maj
authors.initials: M
authors.lastname: Hedtjaern
authors.affiliation: Roche Pharmaceutical Research and Early Development, Therapeutic Modalities, Roche Innovation Center Copenhagen A/S, Hørsholm, Denmark.
authors.forename: Yann
authors.initials: Y
authors.lastname: Tessier
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Franz
authors.initials: F
authors.lastname: Schuler
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Singer
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: Adrian B
authors.initials: AB
authors.lastname: Roth
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D018920:Coculture Techniques; D015925:Cryopreservation; D016207:Cytokines / Q000096:biosynthesis; D004277:DNA, Single-Stranded / Q000633:toxicity; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000473:pathology*; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D009841:Oligonucleotides / Q000633:toxicity*; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D015203:Reproducibility of Results; D018675:Toxicity Tests
chemical_list: D016207:Cytokines; D004277:DNA, Single-Stranded; D035683:MicroRNAs; C544354:Mirn122 microRNA, mouse; D009841:Oligonucleotides; D004364:Pharmaceutical Preparations