34. Transposon-Based Gene Therapy of Hemophilia A Targeting Endothelial Cells in Neonates

Abstract

Hemophilia A is an X-linked bleeding disorder caused by the absence of functional coagulation factor VIII (FVIII). Gene therapy is a promising strategy for the treatment of FVIII deficiency, as only small amounts of protein are needed to phenotypically correct the disorder. Obstacles to non-viral gene therapy of hemophilia A include the lack of prolonged expression and immune responses to the FVIII transgene in the form of neutralizing inhibitory antibodies. In an effort to overcome these obstacles we have utilized the Sleeping Beauty (SB) transposon system to facilitate gene insertion and promote long-term expression. To reduce the likelihood of generating inhibitory antibodies, gene therapy was conducted in neonatal mice, which are more likely to be tolerant of foreign protein expression. While FVIII is thought to be primarily synthesized in the liver, endothelial cells may also be a source for FVIII production. Here we targeted transgene expression to endothelial cells within the lung by a combination of gene delivery approach and a transposon that harbors an endothelial cell specific promoter (Liu et al., (2004) Mol. Ther. 10(1):97-105). Two groups of FVIII deficient mice (n=7) were treated with a transposon plasmid expressing FVIII (pMSZ-FVIII) and an expression plasmid encoding either a defective SB transposase (mutant), or a hyperactive transposase (HSB|[num]|17) (2:1 molar ratio). A total of 5 |[igrave]|g of plasmid DNA was complexed with polyethyleneimine (PEI) and injected via the superficial temporal vein in one-day-old mice (total volume 30 |[mu]|l). Chromogenic FVIII activity assays (Coatest) revealed |[sim]|80% of normal activity at day +3 after injection. Immunohistochemical studies at day +3 showed FVIII immunoreactivity in blood vessels within the lung and scattered throughout the liver. FVIII activity assays and Bethesda titers were monitored over the ensuing 6 months. The activity of FVIII in mice that received the hyperactive SB transposase reached a plateau of approximately 10% by 8 weeks and remained stable thereafter. The FVIII activity in mice receiving the mutant transposase fell to around 2|[ndash]|4%. Bethesda titers for both groups ranged from 0.2 to 0.8 BU, with the higher titers within the hyperactive transposase group. Immunohistochemistry at 24 weeks showed the presence of FVIII protein within endothelial cells of the lung, while there was no detectable immunoreactive protein within the liver. Phenotypic correction of the bleeding disorder was confirmed by tail bleeding times, which were significantly reduced in the gene therapy treated animals. These results suggest that endothelial cell targeting of transposon-based gene therapy can result in long-term correction of the bleeding disorder in hemophilia A.