Long-Term Expression of ADAMTS13 at Therapeutic Levels in Mice by In Utero Administration of Lentiviral Vector Encoding the Human ADAMTS13 Gene.
Authors: Niiya Masami , Endo Masayuki , Zoltick Philip , Flake Alan W. , Zheng X. Long 
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP), characterized by profound thrombocytopenia and microangiopathic hemolytic anemia, may occur in neonates, children and adults. Two types of TTP: hereditary and acquired, have been described. Hereditary TTP is caused by mutations in the ADAMTS13 gene, whereas the acquired TTP is mainly caused by autoantibodies against ADAMTS13 protease. Despite of low incidence of hereditary TTP, patients with hereditary TTP tend to recur or relapse after an initial episode, which may result in sustained neurological and/or renal complications or death. Therefore, early diagnosis and treatment of hereditary TTP are crucial to prevent catastrophic complications. Gene therapy may provide an attractive alternative therapeutic approach to plasma infusion or exchange for long-term correction of ADAMTS13 deficiency. Lentiviral vector encoding the full-length human ADAMTS13 gene tagged with V5-His at the carboxyl terminus and an enhanced green fluorescent protein (eGFP) reporter or PBS was administered to day 14 mouse fetuses (C57/BL6) via direct injection into yolk sac vessels. The blood was collected into sodium citrate at birth, 1, 4, 8, 12, 18, 24, 28 and 36 weeks following the injection. The plasma ADAMTS13 activity, VWF antigen, VWF activity and VWF multimers were determined by FRETS-VWF73, enzyme-linked immunoassay, type III collagen binding and agarose gel electrophoresis, respectively. The transduction of lentiviral vector and expression of human ADAMTS13 in vivo were determined by direct visualization of eGFP reporter by fluorescence stereomicroscopy and immunohistochemistry with monoclonal anti-V5 antibody. We showed that liver appeared to be the predominant organ expressing human ADAMTS13 and eGFP reporter. Other organs including brain, heart, kidney, lung, intestines and testes were also positive for the transgene products, particularly in the vessels. Full-length human ADAMTS13-V5-His (~195kDa) protein was detectable in mouse plasma after immunoprecipitation with mouse anti-V5 and Western blot with rabbit anti-ADAMTS13 IgG. The plasma ADAMTS13 activity in lentiviral vector-injected mice (n=8) was approximately 15~20% higher on average than that in the buffer-injected controls (n=8). The elevated levels of ADAMTS13 persisted for at least 9 months tested. Furthermore, the ratio of VWF activity to VWF antigen levels and the multimer sizes in the lentiviral vector-injected mice were significantly smaller than those in the PBS-injected control. The mice overexpressing human ADAMTS13 had normal complete blood count and did not show signs of spontaneous bleeding. To our surprise, these mice exhibited a significant prolongation of occlusion time in the carotid arteries after 10% FeCl3 injury (28.6 ± 13.0 min, n=8) compared to the control mice (8.5 ± 1.9 min, n=8) (p<0.001). These data demonstrate for the first time that long-term expression of human ADAMTS13 is achieved in mice by in utero administration of lentiviral vector and that the over expression of human ADAMTS13 may offer a systemic anti-thrombotic effect in vivo. Our study supports the feasibility of successful gene therapy for hereditary TTP and/or other arterial thrombotic disorders that are mediated by “unusually large” or “hyperactive” VWF.