Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo.
In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult. One solution to enhance expression would be to select for cells expressing both the desired gene and a linked selectable marker gene in a bicistronic vector. As a potential target tissue, the skin is easily accessible for safe topical application of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes. To test the feasibility of such an approach, a skin raft culture model was developed. Human keratinocytes were transduced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine. While growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml). Colchicine treatment increased the percentage of keratinocytes expressing MDR to almost 100% in raft cultures, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differentiated epidermis. This model suggests that topical selection for MDR-expressing keratinocytes in vivo should be feasible without hampering the biologic integrity of skin. Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for skin gene therapy.
Pfützner
W
W
Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1908, USA. Jonvogel@Box-j.nih.gov
Hengge
U R
UR
Joari
M A
MA
Foster
R A
RA
Vogel
J C
JC
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003078:Colchicine / Q000494:pharmacology; D046508:Culture Techniques; D005240:Feasibility Studies; D015870:Gene Expression; D018014:Gene Transfer Techniques; D019450:Genes, MDR; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D008957:Models, Genetic; D012867:Skin / Q000166:cytology / Q000187:drug effects; D013552:Swine; D013556:Swine, Miniature; D014162:Transfection
D003078:Colchicine