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Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

The adult Göttingen Minipig is an acknowledged model for safety assessment of antisense oligonucleotide (ASO) drugs developed for adult indications. To assess whether the juvenile Göttingen Minipig is also a suitable nonclinical model for pediatric safety assessment of ASOs, we performed an 8-week repeat-dose toxicity study in different age groups of minipigs ranging from 1 to 50 days of age. The animals received a weekly dose of a phosphorothioated locked-nucleic-acid-based ASO that was assessed previously for toxicity in adult minipigs. The endpoints included toxicokinetic parameters, in-life monitoring, clinical pathology, and histopathology. Additionally, the ontogeny of key nucleases involved in ASO metabolism and pharmacologic activity was investigated using quantitative polymerase chain reaction and nuclease activity assays. Similar clinical chemistry and toxicity findings were observed; however, differences in plasma and tissue exposures as well as pharmacologic activity were seen in the juvenile minipigs when compared with the adult data. The ontogeny study revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. These data indicate that the juvenile Göttingen Minipig is a promising nonclinical model for safety assessment of ASOs intended to treat disease in the human pediatric population.

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics. Keywords: kidney, nephrotoxicity, EGF, EGFR, PTEC, antisense, oligonucleotide, safety, preclinical

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.

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.

Over the last decade, single stranded oligonucleotides (ON) have gained increased attention as a new drug modality. Because the assessment of genotoxicity risk during early development of pharmaceuticals is essential, we evaluated the potential of locked nucleic acids (LNA)-ONs to induce DNA damage in L5178Y tk+/− cells both with the mouse lymphoma assay (MLA) and the micronucleus test (MNT). Further, the MLA was performed to assess gene and chromosome mutation over 3 and 24h (± metabolic activation). In addition, the MNT was performed to assess, in addition, a potential aneugenic liability. None of the experiments demonstrated a genotoxic effect for the five tested LNA-ONs. We further show data from four proprietary LNA-ONs tested in standard genotoxicity assays in vitro and partially in vivo, which were all negative. In addition, cellular and nuclear uptake of LNA-ONs in L5178Y tk+/− cells was demonstrated. Based on the results presented here as well as in the literature about other representatives of this class, we consider LNA-ONs as generally not DNA reactive and question whether genotoxicity testing of this class of ONs should be required. This is in line with recent recommendation made by the OSWG that extensively assessed the genotoxicity of oligonucleotides. Environ. Mol. Mutagen. 58:112-121, 2017. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]