The goal of this laboratory is to develop methods for stable introduction and expression of genes in miniature swine keratinocytes for gene therapy purposes. The in vivo approach directly introduces the gene into epidermis while in our ex vivo approach, we first isolate keratinocytes, insert the desired gene while in tissue culture, and graft the genetically modified keratinocytes back onto the donor. We have developed a novel in vivo approach for directly introducing and expressing the genes in epidermis by injecting naked plasmid DNA into the dermis underlying the epidermis. The naked DNA traverses the dermal-epidermal junction and is absorbed and expressed by keratinocytes throughout the epidermis. A quantitative assay for betaGal enzyme expression in epidermis shows that a dose response relationship exists for increasing concentrations of injected naked plasmid DNA. However, mRNA expression of injected plasmid DNA is only transient, probably due to disappearance and lack of integration of plasmid DNA over time as indicated by Southern analysis. Functional utility of this direct in vivo approach was demonstrated by eliciting a neutrophilic infiltrate following injection of an IL-8 plasmid into the skin. Ongoing investigations indicate that DNA injected into organ cultures of human skin is also expressed in the epidermis, while mouse skin expressed injected DNA in epidermis, dermis and underlying muscle. Currently, direct injection of DNA into the skin is being used to develop DNA vaccines for Leishmania and to express biological response modifier genes, such as interferon-alpha, for treatment of papillomas. The ex vivo approach can ensure that a much higher percentage of the keratinoctyes have the desired gene stably integrated into the genome, ensuring persistent expression. Ex vivo approaches utilizing topical selection for keratinocytes containing the multi-drug resistance (MDR) gene are also being pursued. We have not yet successfully grafted and topically selected genetically modified (MDR-gene) keratinocytes onto a donor pig, but have made progress in several of the required steps. The MDR gene has been transduced into primary pig keratinocytes using defective retroviral vectors and we have determined the appropriate colchicine formulations and dose/concentrations for in vivo topical selection. Assays which can immediately assess retroviral transduction efficiency in keratinocytes have been developed. We now know how to prepare pig dermis as a substrate for growing pig keratinocytes in vitro prior to grafting back to the donor pig. Finally, we have begun to investigate how a POU transcription factor, that is specifically expressed in epidermis, might influence and regulate epidermal differentiation.
