Developing methods of gene therapy for hemophilia A

Hemophilia A is an X-linked bleeding disorder for which there is currently no cure. While effective treatments do exist, they are not without complications. Many hemophiliacs still suffer from routine bleeding episodes resulting in disability and even death.; Gene therapy is a promising new technology which may be capable of treating hemophilia without the complications associated with current forms of treatment. We evaluated three distinct approaches to gene therapy to determine their feasibility for clinical application to hemophilia A.; The first approach employed a single-stranded DNA oligonucleotide (ssDO) designed to trigger a nucleotide exchange event at a targeted allele. Our results indicate that ssDO-mediated gene correction can occur in human hepatocytes. In addition, we show that conversion does not require several key enzymes of traditional DNA repair, and may involve an alternate mechanism of action. Overall, the efficiency of nucleotide conversion was low, and our results do not currently support the extended use of this technology to clinical application.; We also evaluated the newly developed helper-dependent adenoviral vector system (HD-Ad). HD-Ad vectors were constructed encoding the canine FVIII B-domain deleted cDNA (cFVIIIΔB) under the control of either a ubiquitously expressed viral promoter or a tissue-restricted mammalian regulatory element. Vectors were tested in both mouse and canine models of hemophilia A. Our results show that physiological levels of FVIII could be achieved in the absence of transgene-specific immunity in both species. In mice, the tissue-restricted vector was capable of mediating sustained FVIII activity, at normal levels, for the lifetime of the animal. In the dogs, high doses of HD-Ad (>1 × 10 12 vp/kg) were required to attain therapeutic levels of FVIII. At these doses, transient hepatotoxicity and thrombocytopenia were observed, and FVIII activity diminished to near undetectable levels over the 3 months following vector administration. These results are significant however, as they demonstrate that FVIII gene transfer can be carried out in a large animal model of hemophilia A without provoking neutralizing FVIII antibody (inhibitor) formation.; Finally, we assessed the use of an HIV-1 based lentiviral vector system for FVIII gene transfer to neonatal hemophiliac mice. Three day old animals were administered vectors by intraperitoneal injection. Levels of FVIII activity ranging from 10–400 mU/mL were achieved in 28.6% of treated mice, and sustained for over 6 weeks. Although there was a high incidence of inhibitor formation, this is the first report to demonstrate that lentiviral vector gene transfer can mediate FVIII expression in the absence of inhibitor development.