1118. A Skin Gene Therapy Approach to Treat Hypertension Using Long-Term Expression and Systemic Delivery of Atrial Natriuretic Peptide (ANP) from the Skin

In order to deliver therapeutic molecules systemically to treat cancers, skin diseases or systemic diseases, skin is a very attractive organ for gene therapy targeting. Because skin is renewable tissue, the ability to achieve long-term stable expression of a therapeutic gene requires targeting keratinocytes stem cells (KSC). Since unique specific extracellular markers to isolate living KSC do not exist, we have developed a system to select in vivo for the therapeutic gene using a bicistronic retroviral vector expressing the desired therapeutic gene and a selectable marker gene (MDR1) combined with topical colchicine treatment of the skin. Previous studies demonstrate in vivo enrichment for human MDR1-expressing keratinocytes that have been grafted to immunocompromised mice and treated with topical colchicine. As a model to validate our system we have chosen ANP as a therapeutic gene to treat hypertension. ANP is 126 amino acids (aa) peptide hormone synthetized mainly in cardiomyocytes of the heart as an inactive precursor (pro-ANP). Pro-ANP is secreted and converted into an active ANP (28 aa) by corin receptor in response to volume expansion and pressure overload. In target tissues, active ANP induce a signal transduction pathway in a receptor-dependent manner to decrease blood pressure. Although studies have demonstrated that ANP infusion can decrease blood pressure in hypertensive patients and animal models, stable long-term expression of ANP from a targeted tissue such as skin will be required for clinical application. We have now constructed and produced a retroviral vector containing ANP and MDR1 genes with a viral titer 104-105 cfu/ml, similar to titers obtained in other studies using MDR1 in a bicistronic system. FACS analysis using an anti-MDR antibody, shows that up to 46.4% and 11.5% of transduced-fibroblasts (Fb) or keratinocytes (Kc), respectively, express MDR1. Using a more sensitive rhodamine exclusion assay, we also demonstrate higher percentage of transduced-Fb and Kc expressing a functional MDR1 in the plasma membrane. We anticipate that colchicine selection will increase the percentage of ANP-expressing keratinocytes and human skin has been engineered in vitro with genetically modified Fb and Kc. Using an immuno-radiometric assay specific for ANP these bioengineered rafts are able to secrete significant levels of ANP into the raft culture media. We are currently grafting the genetically modified human skin onto immunocompromised mice to determine if the human ANP can be detected systemically in the mice and is able to decrease their systemic blood pressure.