TRANSPLANTATION OF RETROVIRUS-TRANSDUCED CANINE KERATINOCYTES EXPRESSING THE BETA-GALACTOSIDASE GENE

We studied transplantation of retrovirus vector transduced canine kera tinocytes to determine whether keratinocytes could persist and express the transferred gene after superficial transplantation to full thickn ess wounds of dogs, a large random-bred model for gene transfer studie s. Canine keratinocytes were transduced by co-cultivation with PA317 r etrovirus packaging cells which produced helper-free amphotropic retro viral vectors (LZSN and LNPOZ) encoding the genes for beta-galactosida se (beta-gal) and neomycin phosphotransferase (neo). Efficient transfe r and expression of the two genes could be demonstrated in confluent k eratinocyte cultures for both vectors. When transduced keratinocytes w ere grown in organotypic cultures on a collagen matrix containing auto logous dermal fibroblasts at the air-liquid interface, the cultures sh owed well-organized and defined epidermal cell layers and several mark ers of terminal differentiation, including the presence of keratohyali n granules and a multilayered stratum corneum. To determine whether th e transferred beta-gal gene was also expressed in vivo, we performed a utologous transplantation of transduced keratinocytes onto full-thickn ess wounds of dogs. beta-Gal expressing keratinocytes could be demonst rated in situ in the regenerating epidermis 2 weeks after transplantat ion. We conclude that keratinocytes can be efficientyl transduced by r etroviral vectors, that retroviral transduction does not interfere wit h proliferation or differentiation, and that transduced keratinocytes express the transferred gene after transplantation to full-thickness s kin wounds of dogs. Keratinocytes thus seem to be good target cells fo r gene therapy.