Proof-of-Concept in a Murine Model of Treatment of Thrombotic Thrombocytopenic Purpura Using Engineered Red Blood Cells Expressing Fragments of ADAMTS13 on Their Membrane

Thrombotic Thrombocytopenic Purpura (TTP) is due to congenital or acquired deficiency in A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13 (ADAMTS13), a metalloproteinase responsible for the cleavage of the pro-coagulant glycoprotein von Willebrand Factor (vWF). Lack of ADAMTS13 activity causes induction of systemic platelet aggregation and microthrombi in small blood vessels by vWF multimers. Current treatment of TTP consists of daily plasma exchange and immune suppression but is costly and associated with high morbidity. A recombinant ADAMTS13 treatment for the congenital form is in advanced clinical trials but alternative treatment methodologies remain needed.

We have developed methods to generate genetically engineered cultured red blood cells (cRBCs) from iPSCs which can be used as drug delivery vectors. We hypothesize that generating cRBCs with membrane-bound variants of ADAMTS13 could be a potential method of treatment for TTP. We have inserted at AAVS1, in human erythroid cells, constructs that contain globin-LCR driven MDTCS ADAMTS13 fusion transgenes with or without gain-of function mutations that confer resistance to ADAMTS13 auto-antibodies. We have shown using flow cytometry and a FRET assay that these erythroid cells express these fusion proteins at high levels and that membrane bound ADAMTS13 is enzymatically active. We have also shown that, as expected, the enzymatic activity of erythroid cells carrying the MDTCS fragment of ADAMTS13 with gain-of-function mutations is resistant to inhibition by plasma from TTP patients, while the wildtype version of the same fragment is not.

To obtain an in vivo proof-of-concept that such erythroid cells can prevent TTP, we have generated rosa26 knock-ins and transgenic mice expressing the wildtype or gain-of-function variants of MDTCS ADAMTS13 and observed that RBCs from these animals have normal half-lives, and, as shown via FACS and FRET assays, express significant amounts of catalytically active ADAMTS13 on their RBC membranes.

ADAMTS13 knock-out mice inoculated with large amounts of recombinant human vWF (rhvWF) have been shown to exhibit typical TTP symptoms including platelet drops, schistocytes formation and cardiac abnormalities providing a useful murine model for TTP. To determine if ADAMTS13 expressing RBCs could be used to prevent TTP symptoms, we transfused ADAMTS13 knock-out mice with either wild-type or ADAMTS13-RBCs up to 24 hours prior to inoculation with rhvWF. Mice transfused with ADAMTS13-RBCs displayed a significant reduction of ADAMTS13 symptoms including drastically reduced platelet drops, lack of schistocytes formation and absent cardiac abnormalities. We conclude that the membrane-bound MDTCS fragment of ADAMTS13 is capable of cleaving the full-length rhvWF in the presence of flow, in vivo.

The above data provide a proof-of-concept that RBCs expressing membrane-bound ADAMTS13 fusion proteins can be used as a therapeutic for congenital and acquired TTP. Experiments are in progress to determine in vivo, the TTP antibody resistance of these cells in the presence of anti-ADAMTS13 auto-antibodies from TTP patients.