Correction of Murine Adamts13 Deficiency by Hematopoietic Progenitor Cell-Mediated Gene Therapy

ADAMTS13, a metalloprotease that cleaves von Willebrand factor (vWF), is primarily synthesized in liver, endothelial cells and megakaryocytes/platelets. It circulates in plasma as an active protease with concentrations between 0.5 and 1.0 μg per milliliter of plasma. Proteolytic cleavage of endothelial cell bound and plasma vWF by ADAMTS13 is critical for maintaining normal hemostasis. Inability to cleave newly released unusually large vWF as a result of hereditary or acquired deficiency of ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Although plasma infusion or exchange is a proven effective therapy for TTP, the life-long plasma infusion for hereditary TTP is not without complications. In search for a better therapy, we performed an autologous transplantation of hematopoietic progenitor cells (HPCs) for correcting Adamts13 deficiency in a mouse model. The bone marrows were harvested from Adamts13^−/− mice (C57BL6/129Sv) at 6–8 weeks of age and HPCs (CD48⁻/CD150⁺) were purified to a greater than 90% of purity in all cases using serial immune affinity depletion and enrichment techniques. The purified HPCs were transduced in culture with a self-inactivated lentiviral vector either encoding GFP or GFP plus murine full-length Adamts13 at 100 × MOI (multiplicity of infection) for 14–16 hours. The transduced HPCs (1 × 10⁵) mixed with freshly prepared non-transduced bone marrow mononucleated cells (2 × 10⁵) were then infused via tail vein into Adamts13^−/− mice (recipients ) after lethal irradiation (450 cGy, twice, 3 h apart, at a dose rate of 2.2 cGy per minute). Peripheral blood was collected at 1, 3, and 5 months of post-transplantation for assessing the bone marrow chimerism and Adamts13 activity. The GFP positive cells were determined by flow cytometry and Adamts13 activity was determined by FRETS-vWF73 and GST-vWF73 peptides as described previously. We showed that all ten mice transplanted with the transduced HPCs encoding GFP plus murine Adamts13 exhibited persistent levels of plasma Adamts13 proteolytic activity, ranging from 10% to 60% of pooled normal murine plasma (PNP), despite low levels of bone marrow chimerism based on the percentage of GFP-positive mononucleated cells (~10–40%) in the peripheral blood. The expressed Adamts13 in plasma was able to cleave process unusually large vWF, resulting in a reduction of vWF multimers sizes compared with those in Adamts13^−/− mice transplanted with HPCs transduced with the vector encoding GFP. Moreover, the levels of plasma Adamts13 appeared to be sufficient to protect mice against ferric chloride-induced carotid arterial thrombosis. The carotid arterial occlusion times for Adamts13^−/− mice which did not undergo bone marrow transplantation were 5.6 ± 1.6 min, similar to those transplanted with HPCs transduced with the vector encoding GFP (4.3 ± 0.8 min) (mean ±SD) (p>0.05). However, the carotid arterial occlusion times were significantly prolonged in Adamts13^−/− mice transplanted with the vector encoding GFP plus murine Adamts13 (15.5 ± 6.8 min). The differences between the experimental group and the control groups were all statistically very significant (p<0.01). The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional Adamts13 metalloprotease. This success may provide basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.