Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)-deficient plasma by an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor-initiated fibrin formation and tissue-plasminogen activator-induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombin-Solulin complex were determined to be 1.53 and 0.02/μM/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses.

Abstract Abstract 1299 Poster Board I-321 The current most serious treatment-related complication in hemophilia A is the development of antibodies to FVIII following exposure to FVIII replacement therapy. We have previously demonstrated that the injection of syngeneic mesenchymal stromal cells (MSCs) into hemophilia A mice with FVIII antibodies lead to a robust decrease in FVIII specific IgG levels because the secretome of MSCs suppresses plasma cell immunoglobulin production, induces plasmablast proliferation, and leads to IL-10-mediated blockade both in vitro and in vivo. Therefore, we wanted to pursue this study in a large animal model. In the current study, we harvested MSCs from bone marrow aspirates obtained from 2 hemophilia A dogs with both human and canine FVIII inhibitors. The isolated MSCs were expanded ex vivo for the infusions. Prior to the infusions, we performed in vitro characterization of the canine MSCs and found that the cells secrete a wide range of cytokines and chemokines including CC chemokine ligand 2 (CCL2) and CCL2 that has been cleaved to its truncated inhibitory form by proteolysis with matrix metalloproteinases (MMPs). These results are identical to those seen in the previous mouse experiments. Before the MSC infusions, the hemophilia A dogs with inhibitors were immunized by multiple intravenous infusions of both human FVIII and canine cryoprecipitate to further boost the development of FVIII antibodies. Expanded autologus MSCs (4.5-6.0×106 cells/Kg) were infused intravenously into the first dog twice, with a month in between each injection. After the second infusion, the titer of anti-human FVIII inhibitory activity was significantly decreased from 6.2 to 1.9 BUs and a transient but significant decrease from 3.5 to 2.2 BUs against canine FVIII. Second MSC infusion trial is currently ongoing in a second dog. In parallel with in vivo study, the inhibitory response mediated by MSCs on the FVIII antibody producing B cells was assessed by ELISPOT assay. In conclusion, MSCs may play an immunosuppressive role in modulating Immunoglobulin production by plasma cells via MMP processing of paracrine CCL2 secretion. This may represent a novel cellular therapy for attenuation of pathological humoral responses such as anti-FVIII antibody development. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3578 Poster Board III-515 Ex vivo delivery of therapeutic transgenes provides an additional level of safety as it avoids systemic administration of viral vectors. Our group has shown that autologous blood outgrowth endothelial cells (BOECs) transduced with a lentiviral vector delivery system containing the FVIII transgene is a promising gene therapy strategy for hemophilia A. We have shown that subcutaneous implantation of factor (F) VIII-expressing BOECs in a murine model of hemophilia A can produce therapeutic levels of FVIII that are sustained for more than 6 months. However, to improve the levels of FVIII expression and cell viability we wanted to evaluate the omentum as an alternative site for BOEC implantation. Initially this strategy was evaluated in two normal dogs. One and three months after delivery of the cells, immunostaining of biopsies from the injection sites showed the presence of viable cells expressing FVIII and evidence of neovascularization. To evaluate the efficacy of this strategy, a hemophilia A dog received 5×108 transduced autologous BOECs that expressed high levels of FVIII in vitro (1.5 IU/106 cells/24hrs). We used autologous fibrinogen as a vehicle for the cells along with canine endothelial growth factors (VEGF and bFGF). For the implantation procedure the dog received prophylaxis with canine cryoprecipitate transfusions. FVIII antigen levels (FVIII:Ag) of between 20 and 50 ng/mL continue to be detected in the plasma 8 months post-implantation, indicating that these cells remain viable and express persistent high levels of FVIII over an extended period of time. However, two weeks after the procedure, the dog developed an anti-FVIII immune response comprising both inhibitory and non-inhibitory antibodies, and therefore no FVIII coagulant activity (FVIII:C) was detected. With a view to preventing the development of the anti-FVIII immune response, we used immunosuppression with cyclophosphamide in two additional hemophilia A dogs. Each of these dogs received 5 × 108 transduced autologous BOECs. In place of fibrinogen, these cells were delivered in a gel comprised of synthetic, heparin-binding peptide-amphiphiles (HBPA) and heparan sulfate, along with canine VEGF and bFGF. The peptide gel prolongs the activity of these growth factors and protects them from proteolysis, enhancing their angiogenic activity. HBPA gel has been shown to increase vascularization of cell transplant sites, which should improve BOEC survival in the omentum. The procedure of implanting the genetically modified BOECs was completed without complications in all hemophilia A dogs and we are continuing to evaluate the efficacy of this strategy. Disclosures: Hulvat: Nanotope Inc.: Employment.

AV513 is a select fucoidan, a sulfated polysaccharide of botanical origin. It inhibits tissue factor pathway inhibitor (TFPI) activity and accelerates clotting of human hemophilia A and B plasma. In prior work, subcutaneous administration of AV513 to mice with hemophilia A improved hemostasis. The current studies were designed to evaluate potential efficacy and safety in dogs with hemophilia A (hemophilia A dogs) with minimally increased hemostasis after adenoassociated viral-FVIII gene transfer and in treatment-naive severe hemophilia A dogs. AV513 administered subcutaneously to low-FVIII dogs for multiple weeks improved hemostasis as exhibited in thromboelastography (TEG) and cuticle bleeding time (CBT) tests. Moreover, AV513 administered orally to AAV-FVIII dogs and treatment-naive severe hemophilia A dogs for a multiweek dose-escalating period yielded correction to normal ranges in both TEG and CBT end points at 5 to 15 mg/kg and 15 to 20 mg/kg dose levels, respectively. In all 3 separate studies, throughout their duration, AV513 was well tolerated by the dogs without any adverse events. Additional pharmacologic characterization of AV513 included intravenous pharmacokinetic analysis in rats. In summary, the combination of safety and efficacy in 2 global tests of hemostasis in the hemophilia A dog model indicate that further evaluation of AV513 as a hemostatic agent in hemophilia A patients is warranted.

To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1-5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here we report the long term efficacy and safety of AAV-cFVIII vectors of serotypes 2/5/6/8 in both hemophilia A mice and dogs. AAV6- and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared to AAV2 and AAV5. However, supra-physiological cFVIII levels correlated with the formation of cFVIII neutralizing antibodies in these mice. However, liver first report demonstrating multi-year therapeutic efficacy and safety of multiple AAV-cFVIII vectors hemophilia basis human studies.

One of the major limitations to the use of adeno‐associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial. More than 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable AAV producer cell line grown in serum‐free media will meet the needs for the manufacture of AAV gene therapeutics.