Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells

Systemic treatment with antisense oligonucleotides is confounded by the dua l problems of potential: cytotoxicity of antisense oligonucleotides and car rier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the po or uptake of the transfection reagent and inadequate intracellular compartm entalization. Human skin epithelial cells, therefore, are attractive experi mental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which rep roducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol; for SZ95 sebocytes w as a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide rati o in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucle otide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 mug/ml) in a medi um without bovine pituitary extract over 4 hours. The uptake of the oligonu cleotide increased in the presence of the polycation and oligonucleotide mo lecules were localized in the cytoplasm of keratinocytes. Oligonucleotide t ransfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta -actin. Thus, catio nic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human, skin. Copyright (C) 2001 S. Karger AG, Basel.