Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia

Abstract

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.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

“…In addition, we found no measurable changes in blood chemistry or cell counts ( Supplementary Fig. 20), which is particularly important because thrombocytopenia (low platelet count) has been reported as a PS-mediated rare adverse event 13,14 .…”

“…The most advanced CNS-active ASO scaffold utilizes a mixed backbone configuration with at least 70-80% PS content. This modification maintains efficacy and distribution with minimal risk of toxic phenotypes 13,14 via mechanisms that are not fully understood 15 . PS modifications are also essential for clinically-advanced siRNA configurations 1,16 .…”

The Nonclinical Safety Profile of GalNAc-conjugated RNAi Therapeutics in Subacute Studies

“…Chemical modifications can substantially affect the toxicity profiles of oligonucleotides ( Frazier 2015 ; Stanton et al 2012 ). High PS content in single-stranded oligonucleotides has been linked to high protein binding that may result in hepatotoxicity or nephrotoxicity ( Brown et al 1994 ; Guvakova et al 1995 ; Rockwell et al 1997 ; Sewing et al 2017 ). In addition, immunostimulatory effects may be associated with high PS content and can include complement activation, vasculitis, and glomerulopathy, as well as coagulopathies such as target independent severe thrombocytopenias ( Engelhardt et al 2015 ; Ferdinandi et al 2011 ; Frazier 2015 ; Frazier et al 2014 ; Hartmann et al 1996 ; Henry et al 1997 , 2017 ; Senn, Burel, and Henry 2005 ; Crooke et al 2017 ).…”

Targeting RNA: A Transformative Therapeutic Strategy

“…Factors that might contribute to the specific toxicities observed with oligonucleotide therapeutics can include dose, treatment duration, target potency, specific chemical classes, sequence, or sequence motif, etc. The potential mechanisms responsible for serious adverse events, the preclinical discovery and screening strategies to reduce the liability of selected oligonucleotides, and the extrapolation of some of these effects from nonclinical studies to humans have been specifically reported elsewhere . The readers are encouraged to read those reports.…”

“…The potential mechanisms responsible for serious adverse events, the preclinical discovery and screening strategies to reduce the liability of selected oligonucleotides, and the extrapolation of some of these effects from nonclinical studies to humans have been specifically reported elsewhere. 15,16,73 The readers are encouraged to read those reports.…”