Sequence-specific inhibition of gene expression in intact human skin by epicutaneous application of chimeric antisense oligodeoxynucleotides

Targeted and selective inhibition of keratinocyte gene expression in human epidermis could be an efficient and safe pharmacologic approach in many ski n diseases. In this study we investigated whether topical application of an tisense oligodeoxynucleotides (ODN) on intact human skin can be used to inh ibit expression of a gene in the differentiated compartment of the epidermi s. We applied a variety of 20-mer antisense and control ODN designed to hyb ridize to different regions on the mRNA of the inducible epidermal proteina se inhibitor skin-derived antileukoproteinase (SKALP)/elafin that was used as a model target gene. When nuclease-resistant fully phosphorothioate ODN were applied to explant cultures of human skin, they were found to be eithe r ineffective at low doses or severely toxic at higher doses which could be attributed to the extremely high degree of protein binding found with this type of ODN. When chimeric ODN with a phosphodiester core and phosphorothi oate 5' and 3' ends were applied to intact skin, no toxicity was noted. One of the tested chimeric ODN, that exhibit only minor protein binding, was r ound to inhibit SKALP expression at the protein level in a dose-dependent m anner. The observed inhibition on SKALP expression levels was specific as e valuated by application of strict criteria. Sequence specificity was assess ed by the addition of sense and scrambled ODN which were ineffective. Furth ermore the expression levels of three other differentiation-related genes ( involucrin, cytokeratin 16, and secretory leukocyte proteinase inhibitor) w ere not affected, indicating that the inhibition was gene specific. Confoca l laser scanning analysis of fluorescently labeled ODN confirmed that these molecules can easily penetrate the epidermis and localize in the cytoplasm of differentiated keratinocytes. We conclude that topical application of a ntisense ODN can be used to modulate epidermal gene expression, and could p otentially be useful to inhibit expression of genes that are relevant in sk in diseases.