379. Delivery of Human Clotting Factors By Expression from B lymphocytes Genetically Engineered Using the Sleeping Beauty Transposon System

Hemophilia A and hemophilia B are X-linked, genetic disorders that are caused by defective or deficient coagulation factor VIII and IX, respectively, resulting in the inability to form blood clots and sustained bleeding after trauma or injury. Recombinant clotting factor protein is currently used to treat hemophilia at a high cost per patient. As a therapeutic approach for hemophilia, gene transfer has the potential to provide more consistent levels of circulating clotting factor over an extended period of time for more cost-effective treatment. Moreover, only modest levels of FVIII or FIX expression (2%-5% of normal) can improve clinical outcomes. We are using the Sleeping Beauty (SB) transposon system to engineer autologous human B cells for secretion of clotting factors as a cellular therapy for hemophilia. An in vitro system for expansion and differentiation of memory B cells into plasma cells has been developed. Plasma cells are suitable for sustained delivery of FVIII or FIX, since they secrete high levels of protein and may survive for years in vivo. For human FIX expression, we assembled an SB transposon with the human FIX coding sequence (codon-optimized with R338L mutation for enhanced potency) regulated by the CAGS promoter. Elevated levels of hFIX in cultures of B lymphoblastoid cells required co-electroporation of the hFIX transposon along with an SB transposase encoding plasmid, demonstrating the role of transposition in achieving extended hFIX expression. There have been remarkable advances recently in the treatment of hemophilia B by systemic AAV8-hFIX administration, but similar treatment of hemophilia A presents a significant challenge due to the size of the FVIII-encoding sequence and the complexity of the protein. We previously demonstrated B-domain deleted hFVIII expression and correction of clotting dysfunction in FVIII deficient mice by hydrodynamic delivery using the SB transposon system (Ohlfest et al, Blood 105: 2691, 2005). Current studies are focused on identifying conditions for effective hFVIII transposon delivery and long term expression in primary human B cells after Sleeping Beauty-mediated transposition. Results from these studies will be applicable to the development of a clinical protocol for treatment of human hemophilia by infusion of B cells genetically engineered using the Sleeping Beauty transposon system.