Proof-of-Concept in a Murine Model of Treatment of Thrombotic Thrombocytopenic Purpura Using Engineered Red Blood Cells

Abstract

Thrombotic Thrombocytopenic Purpura (TTP) is caused by congenital or acquired deficiency of ADAMTS13, a metalloproteinase that cleaves von Willebrand Factor (vWF) multimers. Current treatments--plasma exchange and immunosuppression--are costly and associated with significant morbidity therefore, alternative strategies are needed. We developed the kitJak2 platform for producing genetically engineered lab-grown red blood cells (lgRBCs) as drug delivery vectors. We hypothesized that membrane-bound ADAMTS13 displayed on lgRBCs could provide a durable treatment for TTP. To test this, we engineered erythroid cells expressing both wild-type and mutant variants MDTCS fragments of ADAMTS13, conferring resistance to autoantibodies. Flow cytometry and FRET-based assays confirmed robust membrane expression and enzymatic activity. Importantly, mutant MDTCS variants retained catalytic activity in the presence of plasma from TTP patients, whereas wild-type variants were inhibited. For in vivo evaluation, we generated transgenic mice expressing MDTCS ADAMTS13 on their RBC membranes. These mice exhibited normal RBC half-lives and stable, catalytically active ADAMTS13 expression. Using a murine model of TTP--where ADAMTS13 knockout mice challenged with recombinant human vWF (rhvWF) develop thrombocytopenia and schistocytes--we demonstrated that transfusion of ADAMTS13-expressing RBCs significantly mitigated disease, preventing platelet loss and schistocyte formation. This confirms that membrane-bound MDTCS ADAMTS13 cleaves circulating rhvWF under physiological flow conditions in vivo. Finally, employing our KitJak2 platform, we generated human enucleated lgRBCs expressing high levels of catalytically active ADAMTS13. This novel work establishes proof-of-concept that membrane-anchored ADAMTS13-expressing lab- grown RBCs may offer a feasible and effective therapeutic approach for both congenital and acquired TTP.