Abstract
Hemophilia A (HA) is an X-linked genetic disorder which currently affects over 400,000 people worldwide. There are three clinical levels of HA: mild, moderate or severe with an increase in severity corresponding to a decrease in levels of circulating clotting protein factor VIII. A diagnosis of “Severe” HA means that the patient has <1% of normal circulating protein factor VIII (100-200 ng/ml). HA is the result of a mutation in the F8 gene, which, depending on the type and size of the mutation, may render a person incapable of producing factor VIII entirely, or the patient will produce non-functional factor VIII. Factor VIII is secreted primarily by sinusoidal endothelial cells in the liver, and other organs, and plays a vital role in stabilizing the forming clot during early wound healing. Without functional factor VIII patients will experience delayed wound response, prolonged or spontaneous bleeding, or renewed bleeding after injuries. There is presently no cure for hemophilia A, and current therapeutic procedures involve frequent injections (every 2-3 days) of recombinant factor VIII, which may be cost prohibitive, and cause fluctuating levels of the protein in the blood. Promising advances have been made recently using cell and gene therapy as treatments; however, the sustainability of these treatments is still unknown. The goal of this project was to test secretion of factor VIII by induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) in vivo for the treatment of hemophilia A. First, we cultured the iPSCs derived from an HA patient and differentiated these cells to endothelial cells. . iPSCs were chosen because they can be generated from any adult cell and provide an autologous source of cells for therapeutics. Since the HA patient’s cells were not able to secrete factor VIII, we next transduced these HA-iPSC-derived endothelial cells (HA-iPSC-ECs) with lentiviral vectors encoding a functional copy of the F8 gene, the luciferase gene (LUC) and green fluorescent protein (GFP). Flow cytometry analysis of these cells revealed that 90% of the cells were transduced. The expression of F8 in these cells was confirmed by quantitative PCR and ELISA. Furthermore, we showed that these transduced HA-iPSC-ECs successfully engrafted over 16 weeks in an immune deficient mouse model using bioluminescent imaging and immunostaining of the transplanted cells. Finally, we utilized a murine model of HA to test the ability of the transduced HA-iPSC-ECs to functionally reduce bleeding time. We found that the transduced HA-iPSC-ECs would persistently secrete functional factor VIII and ameliorate the symptoms of hemophilia A in the HA murine model. Our study suggests that patient specific iPSC-derived endothelial cells hold a great potential for treating HA.