The mechanism of thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides. (June 2023)

• Record Type: Journal Article
• Authors: Harada, Kosuke; Furukawa, Hideki; Kohara, Hiroshi; Nishimura, Koki; Komori, Hisakazu; Okai, Yoshiko; Arai, Yuta; Yamamoto, Yuhei; Yamamoto, Katsuhiko; Imanishi, Akio; Shinozawa, Tadahiro
• Is Part Of: Toxicology in vitro. Volume 89(2023)
• Publication Date: 2023-06
• Subjects: Platelet aggregation -- Antisense oligonucleotides -- Aggregates -- Plasma -- Cholesterol ligand -- Thrombocytopenia; ASO antisense oligonucleotides -- BSA bovine serum albumin -- Chol-ASO cholesterol-conjugated antisense oligonucleotides -- DLS dynamic light scattering -- FSC forward scatter characteristics -- GPVI glycoprotein VI -- Kd dissociation constant -- MADLS® multi-angle dynamic light scattering -- PBS phosphate-buffered saline -- PRP platelet-rich plasma -- SSC side scatter characteristics -- TEM transmission electron microscopy; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2023.105569
• Languages: English
• ISSNs: 0887-2333

Abstract

Abstract: In this study, we investigated thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides (Chol-ASO). First, we evaluated platelet activation induced by Chol-ASO in mice by flow cytometry after administration of platelet-rich plasma (PRP). An increase in the number of large particle-size events with platelet activation was detected in the Chol-ASO-treated group. In a smear study, numerous platelets were observed to attach to nucleic acid-containing aggregates. A competition binding assay showed that the conjugation of cholesterol to ASOs increased their affinity for glycoprotein VI. Platelet-free plasma was then mixed with Chol-ASO to form aggregates. The assembly of Chol-ASO was confirmed by dynamic light scattering measurements in the concentration range in which the formation of aggregates with plasma components was observed. In conclusion, the mechanism by which Chol-ASOs causes thrombocytopenia is proposed to be as follows: (1) Chol-ASOs form polymers, (2) the nucleic acid portion of the polymers interacts with plasma proteins and platelets, which cross-links them to form aggregates, and (3) platelets bound to aggregates become activated, resulting in platelet aggregation, leading to a decrease in platelet count in vivo. The details of the mechanism revealed in this study could contribute to creating safer oligonucleotide therapies without the risk of thrombocytopenia. Highlights: Cholesterol-conjugated antisense oligonucleotides (Chol-ASO) may cause thrombocytopenia. Chol-ASO binds to itself to form homopolymers We show that platelets attach to Chol-ASO aggregates, leading to a decrease in platelet count in mice