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M.H. Lundberg Slingsby et al.
generation ASO have the PS backbone and often include 2’- O-methoxyethyl (MOE) modifications, which can further reduce ASO degradation and increase affinity for the target mRNA.1
While several trials with ASO drugs have shown 2’MOE- containing ASO drugs to be well tolerated, there have been reports of adverse events including dose-dependent throm- bocytopenia, with two phenotypes having been described.8,9 Phenotype 1 is a moderate dose-dependent drop in platelet counts that is reversible with cessation of drug treatment.8,10 The Ionis integrated safety database con- cluded that certain sequences (3 out of 16 2’MOE ASO) were associated with phenotype 1, with dose-dependent moderate (>30%) declines in platelet count without an effect on bleeding risk.8 Phenotype 1 has also been observed in monkeys treated with 40% of the evaluated 2’MOE ASO, in which it was manifested as a consistent, repro- ducible decline of platelets over 4-6 weeks, with the platelet counts decreasing moderately (by 30-50%) and then remaining steady.11 Phenotype 2 is a sporadic severe drop in platelet count (clinical thrombocytopenia) and is often not reproducible (for the same 2’MOE ASO), but appears to be dose-dependent.11 Treatment with the 2’MOE ASO inot- ersen induced phenotype 2 in a few individuals who were predisposed, in relation to their underlying disease, and was shown to be dependent on platelet antibodies.12
This paper will focus on the underlying cause of the more prevalent phenotype 1.
Drug-induced thrombocytopenia can stem from decreased production of platelets in the bone marrow and/or increased destruction and clearance of platelets from peripheral blood.13 To examine the in vivo effects on platelet counts, monkeys were treated with the 2’MOE ASO 104838 for 12 weeks.10 Platelet counts decreased ~50% in all monkeys by day 30, and four out of five monkeys had decreases in platelet counts consistent with phenotype 1. The drops in platelet counts were attributed to a 60-80% increase in platelet sequestration in liver and spleen, caused by either increased phagocytosis of platelets or trapping of platelets on the reticuloendothelial surface of these organs.10 Thrombopoietin levels were not altered in the monkeys and bone marrow megakaryocyte morphology, cell density and maturation appeared normal. These findings suggested that the mild thrombocytopenia was due to increased platelet destruction or splenic sequestration and not to diminished platelet production).10
There have been few studies investigating the direct effects of ASO on human platelets. Flierl et al. showed that incubating human platelets with an oligonucleotide PS- ODN 2395 (without 2’MOE modifications), led to platelet activation (increased platelet P-selectin surface expres- sion).14 The same study established that the PS backbone modification of ODN 2395 is a significant driver of this drug’s effects on platelets, through binding and activation of the platelet receptor glycoprotein VI (GPVI), by enhanced GPVI receptor clustering/dimerization.14 Sewing et al. sup- ported these findings and demonstrated that ASO contain- ing locked nucleic acid modifications had reduced binding to GPVI and platelet activation.15 These studies did not include 2’MOE-modified ASO. Considering that most ASO in clinical use and in the drug development pipeline are 2’MOE ASO, it is imperative to understand how 2’MOE modifications affect platelet responses in humans. Furthermore, it remains unknown whether ASO affect pro- platelet production from megakaryocytes and to what
extent 2’MOE-modified PS-ASO share the platelet-activat- ing effects of PS-ODN 2395 on human platelets. In the cur- rent study, we therefore focused on investigating the in vitro effects of 2’MOE ASO on proplatelet production from human cord blood-derived megakaryocytes and studied the direct effects of 2’MOE ASO on human platelets as well as interactions with immune cells. Specifically, we tested 2’MOE ASO 104838, which is known to cause phenotype 1 platelet count reductions.10,16 We also included 2’MOE ASO 501861, with unknown effects on human platelets, and 2’MOE ASO 487660, which has not been associated with reductions in platelet counts in monkeys. CpG ASO 2395 and another CpG 2’MOE ASO 120704 were included for comparison for their known platelet-activating effects.10,14
Methods
Human and mouse megakaryocyte cultures and proplatelet analysis
Human cord blood-derived primary CD34+ cells were cultured and mature megakaryocytes were purified by magnetic bead sep- aration on day 11 as previously described.17 Mouse megakaryo- cytes were derived from fetal liver cultures extracted from CD-1 pregnant mice at day 13.5 of gestation.18 Human-derived, or mouse-derived megakaryocytes were plated on a 96-well half-area plate (Greiner Bio one 675101), followed by addition of ASO (5 mM) and imaged at hourly intervals for 24 h using an IncuCyte Live Cell Analysis System (IncuCyte Zoom). These images were ana- lyzed for the percentage of megakaryocytes producing pro- platelets, as well as the area containing proplatelets using Ilastik (version 1.3.0) and Cell Profiler (version 3.0.0) as described previ- ously.19
Blood collection
Blood was collected from healthy male and female human donors, after informed consent and institutional review board approval (2012P001526), in accordance with the Declaration of Helsinki. Washed platelets, platelet-rich plasma or whole blood was treated for 30 min (platelet studies) or 6 h (cytokine release experiments) with 1, 5 or 10 mM ASO. Previous studies have shown that ASO-induced activation of platelets is concentration- dependent.10,14,15 A therapeutically relevant dose of ASO, i.e., 300 mg, administered subcutaneously results in a Cmax (maximum concentration that a drug achieves after dosing) of 1-2 mM.20
Antisense oligonucleotides
ASO were synthesized at Ionis Pharmaceuticals, Inc. (Carlsbad, CA, USA), purified by reverse-phase high-performance liquid chromatography and formulated in 10 mM HEPES. Five ASO (all with a PS backbone) were included: three with 2’-MOE sequences; 487660 (no effects on platelet count), 104838 (associated with phe- notype 1),8,10 501861 (effects unknown) and two with CpG sequences; 120704 and ODN 2395 (known to activate platelets)10,14 (Table 1). More details of the Methods can be found in the Online Supplementary Material.
Results
Proplatelet formation from human cord blood or murine fetal liver-derived megakaryocytes is not inhibited by 2’MOE or CpG ASO treatment
To investigate whether the ASO induced thrombocy-
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haematologica | 2022; 107(2)