Thursday, November 21, 2024
11/21/2024
Project Summary The long-term objective of this proposal is to develop first-in-class, best-in-class noncoding RNA-based gene therapeutics with a novel mechanism of action, to improve standard of care (SOC) for a broad range of blood and vascular diseases with unmet clinical need: management of thrombotic risk, and management of bleeding disorders. The basis for these therapeutics is our discovery of efficient, tissue-specific, tunable, temporally-controlled, non-integrative gene suppression in platelets and megakaryocytes (MKs) in vivo, via tissue-selective transfection with ectopic naked double-stranded (ds) short inhibitory RNAs (siRNAs) harboring modified nucleotides, following intravenous or subdermal administration in mice. We have shown for the first time that platelets and MK can be transfected in vivo, selectively and at high efficiency by these approaches, resulting in suppression of target gene expression, altered gene-dependent functions restricted to platelets and MK, and antithrombotic and antibleeding effects. SOC for thrombosis and bleeding management continues to be plagued with undesirable counter-effects: increased risk of bleeding with antithrombotics, and generally poor management of bleeding disorders with blunt, untargeted treatments that increase risk for thrombosis and other morbidities. The unique ability of MK and platelets to internalize and utilize naked ds siRNAs - 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 unprecedented opportunity to develop new antiplatelet drugs via an entirely novel approach to disease management. The specific goals of this proposal are: 1) to select and characterize naked siRNA oligonucleotide therapeutics with high efficacy for antithrombotic treatments with improved hemostatic protection compared to drug blockade; 2) to select and characterize naked siRNA oligonucleotide therapeutics for antibleeding treatments with improved efficacy over SOC; 3) to determine the timing and efficiencies of alternative drug administration routes. In Aim 1 we will use in vivo mouse models to select and characterize lead siRNA compounds for strongest antithrombotic effects, and determine the boundaries of dosing and timing that do not cause unwanted bleeding (therapeutic windows). We will test two candidate siRNAs: targeting P2y12, the primary purinergic (ADP) receptor on platelets that is the molecular target of current frontline antiplatelet drugs, and siRNA targeting PAR4, the low-affinity thrombin receptor in human platelets that represents a frontier of antiplatelet therapeutic targeting. We will also determine efficacies of intravenous and subdermal administration routes in this aim. In Aim 2 we will target endogenous negative regulators of platelet reactivity to select lead siRNAs for antibleeding therapeutics, including testing benefit in mouse models of acquired and inherited bleeding disorders. Together, these studies will firmly establish and deeply characterize lead siRNA compositions for pre-clinical development of novel antiplatelet therapeutics.
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