RETROVIRUS-MEDIATED TRANSDUCTION OF PORCINE KERATINOCYTES IN ORGAN-CULTURE

Direct transfer of new genetic information to keratinocytes in epiderm is may prove effective in treating certain genodermatoses; however, cu rrent methods for in vivo gene transfer to skin do not lead to persist ence of the transgene, The goal of this study was to explore direct ge ne transfer using retrovirus-mediated transduction. Retroviral vectors integrate a DNA copy of their genome into the host chromosome and the refore have the potential to effect a permanent gene therapy. To facil itate development of methods for in vivo transduction with retroviral vectors, a porcine skin organ culture model was constructed in which t he denuded surface was repopulated with replicating keratinocytes fi o m hair follicles and epidermal remnants, In situ transduction was carr ied 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 e ncoding the neomycin phosphotransferase selectable gene. beta-galactos idase expressing cells were observed more frequently with LZRN than wi th MFGlacZ; however, transduction efficiency remained low in both inst ances. At equivalent titers, the VSV-G pseudotyped retroviral vector w as shown to transduce porcine keratinocytes more efficiently than a si milar vector with the amphotropic envelope, The number of beta-gal+ ce lls in organ culture could be increased by selection of LZRN-transduce d cells in situ with G418, To achieve transduction of epidermis in viv o, these studies point out the importance of high titer retroviral vec tors, pseudotyping with VSV-G protein, and in situ selection.