Correction of ADAMTS13 Deficiency by AAV-Mediated Gene Transfer of a C-Terminal Truncated ADAMTS13 Variant In a Murine Model

Abstract 251

ADAMTS13 is primarily synthesized in hepatic stellate cells. Plasma ADAMTS13 concentration in humans ranges from 0.5 to 1.2 μ g/ml. ADAMTS13 cleaves von Willebrand factor (VWF) after the Tyr¹⁶⁰⁵ at the central A2 domain. This proteolytic cleavage is essential for removing newly released ultra large (UL) VWF from endothelial cell surface and further reducing the size of the released UL-VWF multimers in circulation under high flow shear stress. Deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome. Approximately 5–10% of all TTP cases are caused by a genetic deficiency of ADAMTS13 and known as hereditary TTP (or Upshaw-Schulman syndrome). Patients with this syndrome may present as neonates or during early childhood with unexplained jaundice, thrombocytopenia, and microangiopathic hemolytic anemia. If left untreated, central nervous system abnormality, chronic renal insufficiency and end-stage renal failure may develop in some cases. To date, the only treatment for hereditary TTP is intermittent infusion of fresh frozen plasma. The complications associated with administration of plasma including adverse events with central line placement, bacterial infections, chronic hepatitis C and allergic reactions to plasma proteins remain problematic, particularly in pediatric population. To develop a better therapy, we explored expression of an ADAMTS13 variant with adeno associated virus (AAV) serotype 2-mediated gene transfer in Adamts13^-/- mice. We showed that a single injection of AAV2 encoding an ADAMTS13 variant truncated after the spacer domain (i.e. construct S), either under a ubiquitous promoter, elongation factor 1a (EF1a), or under a liver-specific promoter, human alpha 1-anti-trypsin (hAAT), at the vector doses between 1.3×10¹² and 1×10¹³ vg/kg, resulted in a sustained expression of recombinant S for approximately 19 weeks analyzed. The maximal levels of plasma ADAMTS13 activity were between 1.0 and 1.6 units/ml at 4–6 weeks of post vector administration. The ADAMTS13 activity was determined in murine plasma by cleavage of a murine-specific recombinant fluorescein-labeled VWF73 peptide (rF-mVWF73). The pooled murine plasma from wild-type (C57BL/6) mice was used for calibration, defined as having 1.0 unit/ml. Plasma VWF multimers were assessed at 2, 4, 8, 12, 16 and 19 weeks after the vector administration by 1% agarose gel electrophoresis and Western blotting. Our results showed that UL-VWF multimers were present in plasma of Adamts13^-/- mice, but not in wild-type mice. The plasma UL-VWF multimers were, however, dramatically reduced or eliminated in Adamts13^-/-mice expressing recombinant S. The size of VWF multimers appeared to be correlated with plasma ADAMTS13 activity. As expected, a real-time PCR analysis demonstrated that the transgene S mRNA was detected in the liver, lung, kidney, spleen, brain and heart after the injection of AAV2-EF1a-S, but restricted to the liver and heart after the injection of AAV2- hAAT-S. To assess the minimal AAV vector dose required to produce therapeutic levels of ADAMTS13 activity, we injected AAV2-EF1a-S and AAV2-hAAT-S at lower doses, i.e. 4×10¹¹ and 1.3×10¹¹vg/kg. The maximal levels of ADAMTS13 activity at the vector dose of 1.3×10¹¹ vg/kg were detected 4–6 weeks after vector injection, which were 0.2 units/ml and 0.3 units/ml for AAV2-EF1a-S and AAV2-hAAT-S, respectively. However, the maximal levels of ADAMTS13 activity in Adamts13^-/- mice receiving AAV2-EF1a-S and AAV2-hAAT-S at the vector dose of 4×10¹¹ vg/kg reached 1.0 unit/ml and 1.4 unit/ml, respectively. These levels of plasma ADAMTS13 activity were comparable with those observed in wild type mice and in Adamts13^-/- mice receiving higher doses of vector. Our results suggest that a vector dose of 4×10¹¹ vg/kg appears to be sufficient to transduce the therapeutic levels of ADAMTS13 expression. In summary, we have demonstrated a successful correction of ADAMTS13 deficiency in a murine model using recombinant AAV2 encoding a C-terminal truncated ADAMTS13 variant S. Our ongoing study is to determine whether the expressed ADAMTS13 variant is efficacious to prevent Shiga toxin-induced TTP in Adamts13^-/- and disease-susceptible strain (CAST/Ei). The results of our study may provide a molecular basis for a rational design of a gene therapy approach for hereditary TTP.