This is a grant by a new investigator who proposes to use the skin as a target tissue for gene therapy in diseases where a soluble factor is curative. The rationale for the experiments is that keratinocytes are secretory cells. The candidate heritable disorder for the proposed studies is Hemophilia A, caused by the deficiency of a secreted protein (factor VIII) that, when present even at low levels, is corrective. Preliminary experiments by Dr. Fakharzadeh show that skin transplants from transgenic mice over-expressing factor VIII maintain high levels of the coagulation factor in factor VIII deficient mice and the rate of clotting is improved. Successful completion of the planned experiments are critical for developing cutaneous factor VIII gene therapy for treating Hemophilia A. Strategies for optimizing factor VIII expression, delivery to the circulation, and activity are proposed in five subheadings in Aim 1. These include comparisons performed in factor VIII deficient mice: of human factor VIII levels generated by transgenes targetted to express the coagulation factor in basal or suprabasal epidermis; of the circulating levels of factor VIII following structural alterations (expression as a single peptide chain, as an inactivation resistant form, or as a species-specific form); and of the levels of factor VIII following stabilization by coexpressing factor VIII with vWF.
Aim 2 deals with the treatment of immunoincompetent knock-out mice following grafting of gene transduced human keratinocytes. The humoral response of naive knock-out mice to human factor VIII is characterized in Aim 3. First, an immunocompetent Factor VIII ko mouse will be generated that is compatible with the donor skin but not the product. Second, the humoral response (anti-factor VIII antibody formation and their inhibitory responses) are monitored in the immunocompetent ko model. Third, the cell-mediated response is characterized by monitoring factor VIII-reactive T lymphocytes, helper T cells, and cytotoxic T lymphocytes.
