Hemophilia is an excellent candidate disease to be treated using gene therapy technology. Current treatment protocols call for inter venous injections of clotting factor preparations and thus the route of delivery of the factors to the circulation is not critical. In addition, studies suggest that restoration of 1% factor activity may be of significant clinical benefit. Dr. Morgan's laboratory has constructed retroviral vectors and adeno-associated virus vectors designed to produce the two major clotting factors associated with hemophilia, factor VIII and factor IX. Dr. Morgan's group has built several retroviral vectors designed to optimize factor IX gene expression in different cell types (including hematopoietic cells, fibroblasts and hepatocytes) using various murine leukemia virus enhancer/promoter elements. Results indicate that the enhancer/promoter elements from the SL3-3 and MPSV viruses were more efficient promoters in lymphocytes, while the HaMSV promoter was better in fibroblasts. Fibroblasts are an attractive target for potential clinical applications because they are easy to obtain and manipulate ex vivo and can be successfully transplanted in humans. Dr. Morgan's group has treated a small cohort of rabbits using an autologous fibroblast transplant procedure and found 2 of 10 animals produced 5-15 ng/ml of human factor IX in their plasma for >300 days. This result demonstrates that it is possible to obtain long-term gene expression in vivo from engineered fibroblasts. In addition to the emphasis on lymphocyte/bone marrow or fibroblast-based delivery and expression of factor IX, Dr. Morgan's laboratory is investigating alternative delivery systems, including selectively permeable membrane devices and direct in vivo gene transfer using adenoviruses. Dr. Morgan's section work on the construction of factor VIII expression vectors was initially hindered by low levels of gene expression. To circumvent this problem, the laboratory constructed retroviral vectors in which factor VIII was expressed from a vector containing an intron 5' to the factor VIII coding sequence. Results demonstrated that this approach increased both gene expression and vector titer. It is proposed to use these improved factor VIII vectors in a small animal model for factor VIII deficiency (a factor VIII knock-out mouse). Dr. Morgan's laboratory plans to pursue an both ex vivo (retrovirus based) and in vivo (adenovirus based) gene delivery system in this animal model.
