Sleeping Beauty-Mediated Gene Therapy for Hemophilia A

DESCRIPTION (provided by applicant): Hemophilia A is a bleeding disorder caused by the absence of clotting factor VIII (FVIII). This disease is inherited in an X-linked recessive manner and it is estimated that there are 15,000 affected individuals in the U.S. Treatment of Hemophilia A has been greatly improved over the last 10 years with the availability of recombinant FVIII (recombinate). However, the high expense of this treatment (as much as $100,000 per year in severe cases) and variable levels of FVIII maintained in the circulation compromise the effectiveness of this therapy. FVIII gene therapy presents a therapeutic alternative for hemophilia A, which would provide more consistent levels of circulating FVIII and thus more efficacious and cost-effective treatment of the disease. Here the investigators propose development of the Sleeping Beauty (SB) transposon system for non-viral FVIII gene transfer and expression in the liver as a therapeutic approach for hemophilia A. The approach is based on the studies that demonstrate long-term, SB transposon-mediated gene expression in the liver and lung. It is hypothesized that the FVIII gene can similarly be delivered to the liver of experimental animals, providing long-term and curative expression of human FVIII. Two Specific Aims are proposed to address this prediction. In SPECIFIC AIM 1, several SB transposons will be assembled which are designed to introduce and express the human FVIII gene under transcriptional regulation of several different strong promoters. Constructs that contain both transposon and transposase functions on the same plasmid will be made. FVlll transposition and expression functions will first be tested in cultured human cells (HEK 293 and Huh7 hepatoma). In SPECIFIC AIM 2, the FVIII transposon constructs tested in Aim 1 will be introduced into the livers of FVIII-deficient knock-out mice by intravenous administration, subsequently testing these animals for long-term expression of FVIII in the circulation with improved clotting of tested blood and decreased bleeding. Results from these experiments will support subsequent scale-up and translation of the Sleeping Beauty transposase system for treatment of hemophilia A, to be proposed as part of a phase II SBIR study. Technological Innovation: Sleeping Beauty is a novel gene transfer system with potential application in gene therapy. The market size for the inherited-diseases market niche, to which this technology applies, is about to $5.8 billion