A novel treatment for Thrombotic Thrombocytopenic Purpura

Thrombotic Thrombocytopenic Purpura (TTP)

TTP, a difficult to treat coagulation disorder: TTP is a rare disorder that can be diagnosed by the presence of micro-angiopathic hemolytic anemia, schistocytes, and thrombocytopenia in the absence of other likely etiologies.

Twenty years ago multiple investigators demonstrated that TTP was associated with the presence of ultra-large VWF multi-mers that was caused by the deficiency of a plasma factor. This plasma factor which was first demonstrated to be a metalloprotease by Tsai et al. and independently by Furlan et al. was eventually sequenced and identified as ADAMTS13.

Positional cloning by the Tsai, Ginsburg, and Bouhassira labs shortly after, demonstrated that ADAMTS13 was also responsible for the congenital form of TTP. Since then the interaction between ADAMTS13 and VWF have been characterized in details which has led to our current under-standing of TTP.

Briefly, low ADAMTS13 activity decreases the normal cleavage rate of Von Willebrand factor (VWF), which therefore accumulates in its high molecular weight form. These high molecular weight VWF molecules, unfold in the presence of shear stress in the circulation and interact with the vessel walls and platelets promoting thrombi formation in the absence of injury, which can lead to life‚Äźthreatening microvascular thrombosis and the clinical manifestations of TTP.

Auto-antibodies:The idiopathic form of TTP has an incidence of about 1/250,000 per year and is caused by auto-antibodies that inactivate ADAMTS13. Anti-ADAMTS13 auto-antibodies can either inhibit the proteolytic activity, enhance the clearance, or disturb the interaction with physiologic binding partners of ADAMTS13. Importantly, epitope mapping revealed that in more than 80% of the patients, these antibodies recognized regions of ADAMTS13 that are outside of the catalytic site, often in the spacer regions. This suggested that it might be possible to develop forms of ADAMTS13 that remain catalytically active but that are not inhibited by the most common au-to-antibodies.


Indeed the Tsai and Bouhassira labs tested a variety of ADAMTS13 truncation mutants and reported several variants that were not inhibited by patients auto-antibodies but that retained significant VWF cleaving activity. More recently, Jian et al. genetically engineered gain-of-function ADAMTS13 variants containing amino-acid changes that confer resistance to some of the most common auto-antibodies.

Development of therapeutic product resistant to auto-antibodies is important because the current treatment for id-iopathic TTP relies on plasma exchange requiring infusion of 2 to 4 liters of concentrate for up to several weeks. Plasma exchange, complemented or not with rituximab,62 an anti-CD20 Ab that suppresses the production of autoantibodies, or with Caplacizumab,63 a nanobody of VWF that blocks VWF-platelet aggregation, is a life-saving but cumbersome procedure that has significant toxicity (mostly allergies), a high number of relapses, and a 10-20% rate of mortality.

Congenital TTP represents about 5% of all TTP cases. The penetrance is very high (90%) but the age of onset and the severity and frequency of the episodes vary between patients, because most affected individuals are compound heterozygous and exhibit variable levels of residual ADAMTS13 activity. Congenital TTP is treated in a similar manner as the idiopathic form but with lower doses of plasma.

Recombinant ADAMTS13 has been approved to treat congenital TTP. This approach could also potentially be used to treat the idiopathic form. However, in the absence of an antibody resistant form of recombinant ADAMTS13 with a long half-life, infusion of very large amounts of the proteins will be required in order to saturate the auto-antibodies, since plasma exchange works in large part by removing the auto-antibodies.

Animal models of TTP: Shortly after the cloning of ADAMTS13, germline knockouts mice were generated but only exhibited a mild VWF-platelet interaction phenotype. Desch et al. obtained a phenotype similar to human congenital TTP in CASA mice by injecting phenotype.shiga toxin. A more recent model developed by Schivitz et al.involves inducing TTP-like symptoms in C57/Bl6 ADAMTS13KO mice by injecting large amounts of recombinant VWF . This model is experimentally more feasible than the CASA model and results in thrombocytopenia, schistocyte formation, anemia, weight loss, high LDH, and histological evidence of thrombosis. Acquired TTP has been modeled in wild-type mice and baboons by injecting anti-ADAMTS13 antibodies.

ADAMTS13 engineered RBCs


Red Blood Cells (RBCs) have been considered a potential game-changer in drug delivery due to their ability to lengthen the half-life of therapeutic agents in circulation, spatially restrict drugs to the cardiovascular system, and protect them from the immune system. However, loading the cells with therapeutic cargo remains a technical challenge.

Our team has developed PSC-RED, a chemically-defined, scalable method to differentiate induced pluripotent stem cells (iPSCs) into unlimited numbers of enucleated RBCs, providing an ideal method for producing therapeutic RBCs as iPSCs can be genetically modified using powerful CRISPR-based technologies.

ADAMTS13, which is deficient in congenital and acquired Thrombotic Thrombocytopenic Purpura (TTP), is a good target for therapeutic delivery through drug-carrying RBCs because large amounts of plasma concentrate or recombinant proteins are necessary to treat TTP.

We have produced engineered iPSCs that express a globin-LCR driven ADAMTS13 fusion transgene inserted at the safe harbor AAVS1 site. These cells can differentiate into RBCs that express a membrane-bound version of an inhibitor-resistant ADAMTS13. The fusion protein was shown to be expressed at high levels and highly active using flow cytometry and a FRET assay that detects cleavage of a peptide that mimics the von Willebrand cognate site. Comparison with plasma concentrate shows that 50 billion engineered ADAMTS13-cRBCs would be sufficient to deliver an amount of ADAMTS13 equivalent to 2 liters of plasma concentrate, suggesting that a transfusion of 10 mL of ADAMTS13-RBCs could be therapeutic for congenital and acquired TTP.

A major obstacle in the development of treatments based on in vitro-produced RBCs is the large volume of culture necessary to produce the cells. We have developed a culture method based on holofiber bioreactors that allows the production of cRBCs at a density of 5.108 cells/mL, which is sufficient to perform small clinical trials.

We are currently this approach in a mouse model.