875. Development of Human Skin Gene Therapy for Systemic In Vivo Delivery of Atrial Natriuretic Peptide (ANP) To Treat Hypertension

Skin is a very attractive organ for targeted gene therapy to systemically deliver therapeutic proteins to treat cancers, skin diseases or systemic diseases. Since skin is a renewable tissue, achieving long-term stable expression of a therapeutic gene requires targeting keratinocytes stem cells (KSC), but specific extracellular markers to isolate living pure KSC populations do not exist. We have therefore developed an in vivo system using a bicistronic retroviral vector expressing the desired therapeutic gene and a linked selectable marker, multi-drug resistant gene (MDR). Topical colchicine treatment can be used to select and enrich for cells expressing the bicistronic vector. As a model to validate our system we have chosen ANP as a therapeutic gene to treat systemic 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, called |[alpha]|-ANP) by corin receptors located on the cell surface membrane of cardiomyocytes in response to volume expansion and pressure overload. In target tissues, |[alpha]|-ANP induces a signal transduction pathway in a receptor-dependent manner to decrease blood pressure. Infusion of ANP can decrease blood pressure in hypertensive patients and animal models, but stable long-term expression of ANP is required for clinical application. We have now constructed and produced a retroviral vector containing linked ANP and MDR genes with a viral titer 104|[minus]|105 cfu/ml. Human skin equivalent (HSE) expressing ANP/MDR have been engineered in vitro and are able to differentiate and stratify normally and have normal histology, similar to normal human skin. Prior to colchicine selection, up to 33% and 39% of transduced human fibroblasts and human keratinocytes, respectively, contain and express a functional MDR marker protein. The ANP expression by bioengineered HSE was confirmed by immunofluorescence labeling and by a radio-immuno assay (RIA) specific for ANP. The RIA shows that HSE expressing ANP were able to secrete high levels of ANP, |[sim]|2 400 pg/ ml/4h, into the culture media. Moreover, cGMP assay suggest that the HSE secretes the desired pro-ANP form rather than the |[alpha]|-ANP form, which have a very short half-life (2 to 5 min) and can be degraded by neutral endopeptidase present in the skin. Taken together, these results suggest that grafting genetically-modified HSE expressing ANP in immunocompromised mice maybe able to produce sufficient ANP, systemically, to decrease blood pressure in the recipient mice.