161. Novel Gene Transfer Strategy for Hemophilia A by Omental Implantaion of Autologous Endothelial Cell Sheet
Authors: Hideto Matsui, Masahi Noda, Midori Shima, Rie Utoh, Teruo Okano, Mitsuhiko Sugimoto
Journal: Molecular Therapy (2016)

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.