(348g) Regulated Production of Biologically Active Insulin from Engineered Human Skin Substitutes for Treatment of Diabetes

Diabetes is characterized by insufficient or absolute lack of insulin production from the pancreatic β-cells to mediate glucose uptake in the body. One approach to overcome this problem is to genetically modify non-β cells to produce insulin. Previously we have used an optimized retroviral gene transfer protocol to modify human epidermal keratinocytes to produce insulin. To further improve insulin production, we examined the effect of mutations on different parts of the proinsulin sequence. We found that an H to D mutation at the 10th amino acid on the B-chain of proinsulin increased insulin production by 10-fold yielding 50 pmol insulin/10⁶ cells/day. Here, we extended our study to obtain a regulatable system through the use of a tetracycline regulatable vector (Tet-on). We found that insulin secretion by keratinocytes was dependent on the concentration of doxycycline (Dox) in the medium and was maximum at a 2-5 mg/ml Dox. Genetically modified keratinocytes were used to engineer skin equivalents demonstrating that regulated insulin secretion did not affect the program of epidermal differentiation and stratification. Finally, gene modified bioengineered skin secreted insulin in a regulatable manner upon addition of tetracycline. Our results demonstrated that epidermal keratinocytes can be modified to deliver insulin in a controllable manner and that genetically modified bioengineered skin has the potential to serve as an alternative therapy for diabetes.