Sunday, May 18, 2025
5/18/2025
Short inhibitory RNAs in platelet and megakaryocyte function - Project Summary The goals of this proposal are: 1) to investigate siRNA-mediated in vivo knockdown of megakaryocyte (MK) / platelet P2y12 as antithrombotic with improved hemostatic protection compared to drug blockade; 2) to investigate siRNA-mediated in vivo knockdown of MK / platelet negative regulators IP (PGI2 receptor) and Gαs in platelet function and hemostasis; 3) to determine the mechanistic basis for selective uptake and utilization of naked double-stranded (ds) small non-coding (nc) RNAs by MK / platelets ex vivo and in vivo. We have shown for the first time that platelets and MK can be transfected in vivo, selectively and at high efficiency, by intravenous or subdermal administration of naked (unencapsulated, unconjugated) ds short inhibitory RNAs (siRNAs), resulting in suppression of target gene expression and altered gene-dependent functions restricted to platelets and MK. The unique ability of MK and platelets to internalize and utilize naked ds ncRNAs - without encapsulation in liposomes or other nanoparticle structures that eliminate tissue targeting specificity, and without covalent targeting moieties that greatly reduce silencing efficiency – provides the opportunity both to study and to manipulate gene expression specifically in MK and platelets. Thus, we have established a highly novel, tractable system of MK and platelet reprogramming in vivo by gene silencing selectively in the MK / platelet lineage. This proposal aims both to exploit, and to study, this tissue-selective siRNA-mediated gene knockdown approach. In Aim 1 we will focus on siRNA-mediated in vivo knockdown of P2y12, the primary purinergic (ADP) receptor on platelets that is the molecular target of current frontline antiplatelets such as clopidogrel, cangrelor and ticagrelor. While these drugs can be effective antithrombotics, they are also associated with risk of clinical bleeding ranging from moderate to severe including intracranial hemorrhage, as a result of total pharmacological blockade of P2y12 that compromises hemostasis. We will test the hypothesis that reduced P2y12 expression by in vivo knockdown is sufficient to modulate thrombosis without compromising hemostasis. We will further exploit this approach to investigate P2y12 expression during MK / platelet development. In Aim 2 we will employ in vivo platelet-selective siRNA transfection to investigate the roles of the IP and Gαs pathways in platelet reactivity and hemostasis. In both Aims 1 and 2, we will investigate the hypothesis that dynamic control of expression of these key regulatory proteins across the circulating platelet lifespan contributes essentially to diversity in platelet function among the population, with important functional outcomes. This hypothesis represents a novel fundamental aspect of platelet molecular physiology with strong potential impact. In Aim 3 we will investigate the hypothesis that the unique uptake and utilization mechanisms of naked mi/siRNAs by MK and platelets derive from specialized fluid-phase endocytosis. Together, these studies will elucidate several related pathways highly relevant to platelet physiology and disease, and will form the basis for pre-clinical development of novel antiplatelet therapeutics.
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