BC-mediated delivery of thromboprophylaxis

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No anti-thrombotic agent (ATA) is safe and effective in the many patients at a combined risk of acute thrombosis and bleeding, e.g., in the early post-surgery period. To address this unmet need, we develop drug delivery systems (DDS) executing two main functions: A) Block access of ATA to off- target sites, e.g., hemostatic plugs formed after surgery, while B) Optimize pharmacokinetics and deliver ATA into subsequent thrombi, where ATA is activated by thrombin. ATA fused with single- chain fragments (scFv) targeted to red blood cells (RBC) bind to these carriers that execute dual blocking/delivering function. Proof-of-concept is emerging in models of pre-existing and nascent clots in animals. Here we devise humanized scFv/ATA targeted to human RBC and will test them in a humanized microfluidic system (HMF), in transgenic (TG) mice expressing humanized target epitopes on blood cells, and in the perfusion of isolated human lungs. We will pursue three aims. Aim 1. RBC loading. We will characterize scFv/ATA loading onto RBC: A) Binding (copies/cell, on/off kinetics); B) Effect on RBC functionality, biocompatibility and biomechanics; and, B) Regulation of distribution of scFv/ATA between RBC in circulation. We also will characterize biomechanical factors modulating RBC/ATA delivery and effect on clot dynamics and structure, in particular, impact of RBC rigidification, caused by either drug loading or by intrinsic pathophysiological changes in patient's blood. Aim 2. Mechanistic insights. We will interrogate previously unrecognized yet critically aspects of the RBC/ATA workings, in particular their interaction with vascular endothelium and transfer of the drug cargo to these and other vascular cells. In this Aim we will use standard mouse in vivo models, microfluidic model and perfusion of isolated human lungs model. Aim 3. Appraisal of benefit/risk ratio. We are developing TM mice expressing human RBC determinants in mouse EBC, in order to study scFv/ATA loaded on "human RBC" in vivo: A) PK/BD, complement activation, phagocyte uptake and vascular adhesion of RBC/ATA in TG mice; B) Define the time window/extent of anti-thrombotic effect of human RBC/ATA in models of arterial vs venous thrombosis in TG mice; B) Affirm the safety of RBC/ATA. We will detect adversities of scFv/ATA including abnormalities of RBC. To defuse potential issues, if necessary, we will use more benign loading regimen. Together, these studies will advance mechanistic insights and clinical translation of a novel way to mitigate thrombosis in currently unprotected patients by providing a new and tractable approach to understanding thrombus development and a rational approach to deliver cell-directed therapeutics