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Platelet Biology & its Disorders
Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates
Martina H. Lundberg Slingsby,1,2 Prakrith Vijey,2 I-Ting Tsai,1,2 Harvey Roweth,2 Genevieve Couldwell,2 Adrian R. Wilkie,1,2 Hans Gaus,3 Jazana M. Goolsby,2 Ross Okazaki,2 Brooke E. Terkovich,2 John W. Semple,4,5 Jonathan N. Thon,2 Scott P. Henry,3 Padmakumar Narayanan3 and Joseph E. Italiano Jr.1,2
1Vascular Biology Program, Department of Surgery, Boston Children’s Hospital, Boston, MA, USA; 2Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; 3Nonclinical Development, Ionis Pharmaceuticals Inc., Carlsbad, CA, USA; 4Departments of Pharmacology and Medicine, University of Toronto, Toronto, Ontario, Canada and 5Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden
ABSTRACT
Antisense oligonucleotides (ASO) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2’MOE) ASO have shown dose- and sequence-specific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytope- nia. This article focuses on the underlying cause of the more common phe- notype 1, investigating the effects of ASO on platelet production and platelet function. Five phosphorothioate ASO were studied: three 2’MOE sequences; 487660 (no effects on platelet count), 104838 (associated with phenotype 1), and 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord blood- derived megakaryocytes were treated with these ASO to study their effects on proplatelet production. Platelet activation (determined by surface P- selectin) and platelet-leukocyte aggregates were analyzed in ASO-treated blood from healthy human volunteers. None of the ASO inhibited pro- platelet production by human megakaryocytes. All the ASO were shown to bind to the platelet receptor glycoprotein VI (KD ~0.2-1.5 mM). CpG ASO had the highest affinity to glycoprotein VI, the most potent platelet-activat- ing effects and led to the greatest formation of platelet-leukocyte aggre- gates. 2’MOE ASO 487660 had no detectable platelet effects, while 2’MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYK- dependent formation of platelet-leukocyte aggregates. Donors with higher platelet glycoprotein VI levels had greater ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and platelet-leuko- cyte aggregates may explain phenotype 1 (moderate drops in platelet count). Platelet glycoprotein VI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.
Introduction
Antisense oligonucleotides (ASO) are short, synthetic, single-stranded DNA mole- cules (between 8 and 50 nucleotides) that bind to specific mRNA segments through Watson-Crick base pairing.1 They are designed to modulate targeted mRNA by inter- fering with its function or promoting its degradation, ultimately altering protein expression.1,2 ASO offer therapeutic opportunities for treating rare genetic diseases such as spinal muscular atrophy (nusinersen),3,4 hereditary transthyretin amyloidosis (inotersen)5 and homozygous familial hypercholesterolemia (mipomersen).6 There has been a surge in ASO entering clinical trials for a wide range of diseases, which has been attributed to improvements in chemical modifications of ASO.7 Incorporating a phosphorothioate (PS)-containing backbone to the ASO increased the stability and resistance of the oligonucleotides to nucleolytic degradation.1 Second-
Ferrata Storti Foundation
Haematologica 2022 Volume 107(2):519-531
Correspondence:
MARTINA LUNDBERG SLINGSBY
martina.slingsby@gmail.com
Received: June 22, 2020. Accepted: February 2, 2021. Pre-published: February 11, 2021.
https://doi.org/10.3324/haematol.2020.260059 ©2022 Ferrata Storti Foundation
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