A novel inducible vector called the gene switch has been developed(O'Malley et al.). This gene-switch relies on the finding that a carboxy-terminal deletion of the human progesterone receptor (PR) fails to bind progesterone but will bind progesterone antagonists. By replacing the DNA binding domain of this mutant PR with that of the yeast transcription activator GAL4, the possibility of activating endogenous genes containing the progesterone response element is eliminated. Since GAL4 activated genes do not exist in mammalian cells, this regulator specifically activates reporter constructs that have been engineered to contain GAL4 binding sites. This expression system was originally designed to be turned on and off by a systemic dose of antiprogesterone. In these studies, the antiprogesterone(mifepristone) is applied topically reducing the risk of adverse effects.

To develop a bigenic mouse that expresses the transgene exclusively in the skin, the epidermal specific Loricrin and Keratin-14 promoters were utilized to limit expression of the chimeric regulator. The reporter proteins were put under the control of the minimal thymidine kinase promoter containing GAL4 binding sites. Two lines of transgenic mice, each containing half the gene-switch system, were then mated.

The resultant complete gene-switch bigenic mice are exposed to topical mifepristone. The dorsal skin is shaved and 100 μl of various concentrations of mifepristone (dissolved in ethanol) applied. We have shown by RT-PCR and RNase Protection assay that the reporter genes are not expressed until mifipristone is applied to the skin surface. Expression reached maximal levels at 12 hours and lasted at least 24 hours with a single application. Mifepristone was then applied daily to induce high expression levels. The maximal topical dose of mifepristone was 1μg/mouse (about 30μg/kg).

We have placed apolipoprotein E (apoE), TGF-β, T-Antigen and growth hormone (GH) target genes in this gene switch system. Inducible and keratinocyte-specific expression of two of the four reporter proteins has been achieved as evidenced by Western and imunoflorescence data and by physiologic changes associated with the T-Antigen and TGF-β proteins. The bigenic apoE and GH producing mice are currently under investigation.

The ability to control the “dose” of therapeutic protein with this system has major advantages for gene therapy development in epidermis. We speculate that by secreting these products from skin, regulated systemic gene therapy may be possible for a wide variety of diseases.