An engineered EBV vector expressing human factor VIII and von Willebrand factor in cultured B-cells

BACKGROUND: Hemophilia A is a congenital disorder caused by a deficiency of the blood-clotting factor VIII (FVIII) and is an attractive candidate for gene therapy. Most of the studies have only explored the potential of hepatocytes and muscle cells as the targets for gene transfer. Attempts to transfer the genes into hematopoietic cells have so far been mostly unsuccessful due to inefficiency of most viral vectors to transduce these cells and the supposed inability of the cells to express FVIII. METHODS: We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry either the full-length FVIII cDNA or its B-domain-deleted form; a modified version of the vector that carries B-domain-deleted FVIII along with the von Willebrand factor (vWF) cDNA or the reporter gene DsRed2 was also used. All these vectors have been safety modified with viral thymidine kinase cDNA to transduce human B-cells in culture. RESULTS: Low-level expression of FVIII in the order of 5-8 ng FVIIIC/ml were observed in the cells stably transduced with full-length FVIII, while cells with the B-domain-deleted version expressed 8-16 ng FVIIIC/ml. Expression of vWF and B-domain-deleted FVIII resulted in a moderate expression of 18-30 ng FVIIIC/ml. Long-term expression for 12-16 weeks was observed in these cells regardless of selection pressure. CONCLUSIONS: These results support the development of an episomal engineered EBV vector for treatment of hemophilia A using the hematopoietic cells as a target for providing immediate secretion of functionally active product in the circulating bloodstream.