Platelet-Targeted Self-Amplifying mRNA for Safe, Long-Acting Thromboprophylaxis.

Abstract

Thrombosis is a major contributor to morbidity and mortality in cardiovascular diseases. Although current antiplatelet therapies reduce thrombotic complications, they are associated with systemic bleeding complications. We previously developed a single-chain variable fragment (scFv) that specifically binds and blocks the activated platelet integrin αIIbβ3 (GPIIb/IIIa [glycoprotein IIb/IIIa]), preventing thrombosis without impairing hemostasis. We hypothesized that an mRNA-based therapeutic encoding scFv_αIIbβ3 would offer prolonged expression and enhanced therapeutic durability while maintaining a favorable safety profile. We designed and synthesized both conventional mRNA and self-amplifying mRNA constructs encoding scFv_αIIbβ3. The constructs were first validated in vitro by confirming their expression of a functional scFv_αIIbβ3. For in vivo experiments, mRNAs were encapsulated in a novel lipid nanoparticle formulation comprising of γ-oryzanol and DLin-KC2-DMA (OryKL) for systemic delivery. Their biosafety was assessed by a series of biochemical and histological examinations. The murine FeCl₃-induced arterial thrombosis model was used to assess the preventive effect of scFv_αIIbβ3-encoding mRNAs on thrombus formation. In vitro assays confirmed the efficient expression and secretion of a functional scFv_αIIbβ3 that selectively bound to and blocked activated GPIIb/IIIa, thereby inhibited platelet aggregation. In mice with induced thrombosis, OryKL-delivered scFv_αIIbβ3 mRNA significantly prolonged occlusion time at 24 hours posttreatment, while self-amplifying mRNA provided sustained thromboprotection for over 7 days. Tail bleeding times were unchanged across all groups, and no systemic toxicity or histopathologic abnormalities were observed. Our findings demonstrate that scFv_αIIbβ3 mRNA and self-amplifying mRNA therapeutics enable safe, effective, and long-acting antithrombotic protection in vivo, offering a promising strategy for thromboprophylaxis that prevents the bleeding risks associated with current antiplatelet therapies.