165. Long-Term Factor VIII Expression and Phenotypic Correction in a Murine Model of Hemophilia A Achieved by Immunosuppression and Non-Viral Gene Transfer Using the Sleeping Beauty Transposon

Abstract

Hemophilia A is an X-linked recessive genetic disorder that is characterized by sustained bleeding after trauma or injury. Currently, patients are treated by self-administration of recombinant factor VIII protein (FVIII), but this treatment is expensive and cumbersome. Gene therapy using viral vectors has been successfully used to correct the bleeding disorder in FVIII deficient mice and dogs. However, anti-viral immune responses, problems with scale-up, and insertional mutagenesis, due to a predilection for most integrating viruses to insert near genes, all raise safety and practicality questions for viral vectors. Therefore we used the Sleeping Beauty (SB) transposon system, which is delivered as plasmid DNA, to integrate a B-domain deleted FVIII genes into the chromosomes of C57BL/6J-F8−/− mice and correct the phenotype. The transposon plasmid was co-delivered with a Ubiquitin 3 promoter-regulated SB 10 transposase expression plasmid using the “hydrodynamic” method. Plasma FVIII levels were assayed in treated mice by ELISA, COAT test, and APTT clotting assay. Long-term FVIII expression (>9 weeks at present) required delivery of 280 micrograms of plasmid DNA, co-delivery of Ub-SB10 plasmid, and immunosuppression by bi-monthly 150 mg/kg cyclophosphamide injections or immunotolerization by FVIII (RefactoTM) injection in C57BL/6J-F8−/− neonates. Without immunosupression or tolerization, anti-FVIII antibody was detected in treated mice using the Bethesda assay. Transposon excision products were detected in DNA extracted from the liver of mice treated with transposon and transposase, providing evidence for transposition. Transposon insertion site analyses in hepatocytes from treated mice are ongoing. The SB transposon system thus provides non-viral means for obtaining stable FVIII gene insertion and expression for the treatment of hemophilia A.