SBIR-STTR Award

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

Thesaurus Terms:
coagulation factor VIII, gene expression, gene targeting, gene therapy, hemophilia A, technology /technique development, transposon /insertion element liver, liver circulation, recombinant protein biotechnology, expression cloning, laboratory mouse

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. There are 15,000 affected individuals in the U.S. Treatment of Hemophilia A is based on frequent delivery 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 represents a more efficacious and cost-effective treatment of the disease. We propose continued development of the Sleeping Beauty (SB) transposon system in a Phase II study of non-viral FVIII-gene transfer and expression in the liver as a therapeutic approach for hemophilia A. This approach is based on results from our Phase I studies of long-term, SB transposon-mediated FVIII gene expression in livers of FVIII-deficient mice. We hypothesize that the FVIII gene can similarly be delivered and expressed in the livers of larger animals (ultimately, humans), providing long-term and curative expression of FVIII. These goals are addressed in four Specific Aims. In Aim 1 we will test for SB-mediated long-term expression of reporter genes and the FVIII gene in the livers of mice after delivery of transposon and transposase components directly to the hepatic circulation via retroductal delivery. In Aim 2 we will determine the efficacy of using DNA, DNA-polycation complexes or other DNA complexes for delivery of transposons to liver. These conditions will then be used in Aim 3 to test for SB-mediated long-term expression of reporter genes in the livers of dogs as a large animal model for in vivo gene transfer. In Aim 4, results from the experiments in normal dogs will be used to evaluate the SB system for transposition and long-term expression of the canine FVIII gene in a dog model of hemophilia A, testing for improved clotting and correction of the bleeding disorder. At every stage we will examine animal tissues for histopathological indications of adverse events. Results from these preclinical studies will position DGI for submission of an Investigational New Drug application (IND) to the FDA, with subsequent initiation of a clinical trial testing the effectiveness of the SB system FVIII gene therapy in human subjects. 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 $5.8 billion