Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
The adhesive protein von Willebrand factor (VWF) plays an essential role in physiological hemostasis, mediating platelet adhesion and aggregation under high shear stress conditions.[1][1],[2][2] The VWF-cleaving protease ADAMTS13 precisely down-regulates VWF activity to avoid pathological
The development of neutralizing antibodies (inhibitors) directed against Factor VIII (FVIII) is currently the most significant complication associated with FVIII replacement therapy in hemophilia A patients and presents a considerable obstacle to successful gene therapy strategies. Induction of immunological tolerance (ITI) to FVIII involves frequent intravenous infusions of FVIII according to various therapeutic regimens, and has been successful in many inhibitor-positive patients. However, ITI is not successful in all patients and it requires frequent intravenous access and is very expensive. Alternative methods of inducing tolerance to the FVIII protein may prove beneficial especially if this can be achieved prior to inhibitor development. With a view to utilizing the potential of neonatal immune-privilege, we administered canine-FVIII expressing lentiviral vectors prior to FVIII protein challenge. The lentiviral vector, hAAT-cFVIII-LV, contained a B domain-deleted canine FVIII transgene under the control of a liver specific promoter. 1x108 Transducing Units (TU)/mouse were administrated to 0-1 day old Balb/c hemophilia A neonates either intravenously (IV), intraperitoneally (IP) or subcutaneously (SC). Neutralizing anti- canine FVIII antibodies developed in all mice treated with IP or SC injection, In contrast, FVIII activity (range <10-1586.7mU/mL) was detected for greater than 3 months in 8/13 mice treated intravenously and all mice failed to develop high-titer neutralizing anti-FVIII antibodies. To evaluate long-term tolerance induction following this protocol, 3 of these mice were challenged, 52 weeks post-hAAT- cFVIII-LV treatment, with four intravenous infusions of 0.2ug recombinant canine FVIII (rcFVIII) protein (80U/kg). Splenocytes were isolated, cultured and in vitro cytokine production and T cell proliferation, was assessed. Treated mice injected with rcFVIII remained inhibitor negative and this tolerance was accompanied by increased in vitro T cell production of IL-10 in parallel with decreased production of IL-6 and IFN-γ in vitro. The splenocytes from these tolerant mice are capable of transferring tolerance to naïve recipient hemophilia A Balb/c mice indicating that the mechanism underlying this phenomenon likely involves regulatory T lymphocytes.
Preclinical and clinical studies using adeno-associated viral (AAV) vectors for hemophilia B showed that the safety profile is vector dose-dependent and that immune responses to AAV-capsid proteins with subsequent hepatocyte toxicity required transient immunosuppression for sustained transgene expression. However, there still remain concerns over the safety of systemic vector injection. Potential side effects include adverse immunological reactions, vector-mediated cytotoxicity, germ-line transmission, and insertional oncogenesis. Moreover, especially in hemophilia A, an alternative transgene delivery approach may be necessary due to the large size of the factor VIII (FVIII) cDNA. Blood outgrowth endothelial cells (BOECs) are considered to be an attractive candidate to treat hemophilia A, because BOECs express von Willebrand factor, which is known to act as a carrier protein for FVIII and prevents its proteolytic degradation. We previously demonstrated that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 300 days, in which lentivirally-engineered blood outgrowth endothelial cells (BOECs) sheet were implanted subcutaneously (Tatsumi K et. al. PLoS One 2013 8(12):e83280). While this new technology is effective and safe in small animal such as mouse (20-25g body weight), the major challenge is in applying this functioning procedure in the patients with hemophilia A. For this purpose, the current study focus on an assessment of the safety of cell sheet implantation in canine models as the first step toward gene therapy in clinic. GFP-transduced BOECs were cultured on temperature-responsive poly (N-isopropylacrylamide) (PIPAAm)-grafted dish to engineer BOECs sheet. This dish allows for simple detachment of the cultured cells without the use of proteolytic enzymes such as trypsin and enables us to harvest the cell sheet as a contiguous monolayer that retains its native intercellular communications and intracellular microstructure, which are indispensable for normal cellular function. When the cultured BOECs reached confluency, the cultured cells were detached from the PIPPAm dish as a uniformly connected tissue sheet by lowering the culture temperature to 20°C for 30 minutes. Under general anesthesia using isoflurane, beagle dog (10-12Kg body weight) was placed on the operation table. To exteriorize the greater omentum, an abdominal ventral midline incision was made and 20 BOECs sheets in total were implanted. Upon completion, the omentum was placed back into the abdomen and the incision was closed. For the assessment of GFP expression, the liver samples were collected by open liver biopsy both 30 and 120 days after sheet implantation. We confirmed that implanted BOECs were differentiated into mature endothelial cells and contribute to new blood vessel formation by histological examination. In conclusion, tissue engineering approach by omental endothelial cell sheet implantation are viable for persistent tissue survival and providing therapeutic values in canine model. This novel ex vivo gene transfer strategy can provide the safe and efficacious delivery of FVIII in hemophilia A. Now, we are conducting FVIII gene transfer by cell sheet implantation in canine hemophilia A model.
