Abstract

Multiple antisense oligonucleotides (ASOs) have been developed and some approved for various indications. However, in toxicological assessment and clinical studies a dose- and sequence- dependent thrombocytopenia (TP) of varied severity has been reported. This has been mainly attributed to pro-inflammatory and platelet activating effects of the ASOs, leading to enhanced platelet sequestration. Accordingly, access to reliable and high throughput platelet function assays would aid safety assessment of ASOs earlier in development. Important reference agents are ODN2395, known to stimulate platelets in vitro, and inotersen, an ASO that produced TP in toxicology and clinical studies. We have used these compounds to develop and validate test systems suitable for screening for effects of ASOs on platelet function. Effects of the two compounds were assessed directly and in combination with collagen related peptide (CRP). Both platelet aggregation, using platelets in plasma and platelets isolated in protein-free buffer, and platelet activation in whole blood were used. All three assays showed an increase in potency of CRP in the presence of ODN2395 and inotersen. Effects of the compounds quantified by EC50 shift suggest that aggregation in washed platelets provides the most sensitive assay to investigate the effects of ASOs on platelets, followed by aggregation in platelet rich plasma. The platelet activation assay appeared to be the least sensitive. However, since it is performed in whole blood on fixed samples, it can be easily applied to testing ex vivo in samples from pre-clinical and clinical studies of ASOs.Platelet function testing can be used to de-risk selection and progression, or as a tool to identify individuals at risk of ASO-induced TP. Availability of a range of sensitive, reproducible, plate-based test systems allows platelet function testing to be considered as part of a screening strategy.