Retrovirus-mediated transduction of porcine keratinocytes in organ culture

Direct transfer of new genetic information to keratinocytes in epidermis may prove effective in treating certain genodermatoses; however, current methods for in vivo gene transfer to skin do not lead to persistence of the transgene, The goal of this study was to explore direct gene transfer using retrovirus-mediated transduction. Retroviral vectors integrate a DNA copy of their genome into the host chromosome and therefore have the potential to effect a permanent gene therapy. To facilitate development of methods for in vivo transduction with retroviral vectors, a porcine skin organ culture model was constructed in which the denuded surface was repopulated with replicating keratinocytes fi om hair follicles and epidermal remnants, In situ transduction was carried out by topical application of two retrovirus vectors, MFGlacZ (10(7) blue forming units per mi) and LZRN pseudotyped with the G protein of vesicular stomatitis virus (VSV) (10(9) colony forming units per mi), each encoding the beta-galactosidase reporter gene and the latter encoding the neomycin phosphotransferase selectable gene. beta-galactosidase expressing cells were observed more frequently with LZRN than with MFGlacZ; however, transduction efficiency remained low in both instances. At equivalent titers, the VSV-G pseudotyped retroviral vector was shown to transduce porcine keratinocytes more efficiently than a similar vector with the amphotropic envelope, The number of beta-gal+ cells in organ culture could be increased by selection of LZRN-transduced cells in situ with G418, To achieve transduction of epidermis in vivo, these studies point out the importance of high titer retroviral vectors, pseudotyping with VSV-G protein, and in situ selection.