Adamts13-Cultured Red Blood Cells to Treat Thrombotic Thrombocytopenic Purpura

Red Blood Cells (RBCs) have long been considered a potentially useful means of delivering drugs to the circulation because delivery through therapeutic RBCs as compared to direct injection in the plasma can lengthen the half-life of the therapeutic agent in the circulation, spatially restrict the drugs to the lumen of the cardio-vascular system, and shield the drug from the immune system. Despite some progress, loading the cells with therapeutically useful cargo remains technically challenging. We have recently developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into unlimited numbers of enucleated cultured RBCs. This provides an ideal method to produce therapeutic RBCs since iPSCs can be genetically manipulated with powerful CRISPR-based technologies. ADAMTS13, whose deficiency is responsible for congenital and acquire Thrombotic Thrombocytopenic Purpura (TTP) is a good target as a therapeutic that could be delivered through drug-carrying RBCs because large amounts of plasma concentrate, or more recently recombinant proteins, are necessary to treat TTP. We report here we have produced engineered erythroid cells that contains globin-LCR driven ADAMTS13 fusion transgenes inserted at safe harbor AAVS1, and that these cells express a membrane bound version of an inhibitor-resistant version of ADAMTS13. We show using flow cytometry that the fusion protein is express at high levels, and using a FRET assay that detect cleavage of the von Willebrand cognate site, that the membrane bound ADAMTS13 is enzymatically active. Comparison of enzymatic activity with plasma concentrate suggests that about 50 billion engineered ADAMTS13-cRBCs would be sufficient to deliver an amount of ADAMTS13 equivalent to 2 liters of plasma concentrate. This suggests that a transfusion of about 10 mL of ADAMTS13-RBCs could be therapeutic for congenital and acquired TTP. The number of cRBCs necessary to treat even a few patients is very large. This has been considered a major obstacle to the development of treatment based on in vitro produced RBCs because of the volume of culture that is necessary to produce the cells. We also report that we have developed a culture method based on holo-fiber bioreactors that allows the production of cRBCs at a density of 5.10⁸ cell/mL which is sufficient to produce enough cells to performed small clinical trials.