Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system

Efficient gene delivery to the skin is important for gene therapy of skin d iseases and in-depth biologic studies of epidermis. In this report, we inve stigated three nonviral transfection systems for gene transfer in cultured human keratinocytes and organotypic cultures. SuperFect is a highly branche d polycationic transfection reagent, PrimeFector a polycationic liposome co mpound, and the AVET (adenovirus-enhanced transferrin-mediated) system cons ists of a ternary complex of biotinylated chemically inactivated adenovirus noncovalently complexed with plasmid DNA and polylysine-transferrin. After AVET transfection of cultured keratinocytes with pCI beta gal, a CMV/beta- galactosidase reporter plasmid, 28.8% +/- 1.4% of the cells were stained bl ue. SuperFect was about 2-fold less efficient, whereas Primefector did not transfect keratinocytes. Similar results were obtained when transfection ef ficiencies were measured by enzyme assays. Addition of holotransferrin to t he culture medium or replacement of polylysine-transferrin by polylysine in the ternary complex did not affect the transfection efficiency. Using AVET complexes without adenovirus, however, strongly diminished gene delivery. This indicates that the AVET complex is taken up by an adenovirus receptor. Separation of AVET/pCI beta gal transfected keratinocytes by adhesion to c ollagen IV into two fractions (rapidly and slowly adhering cells) showed th at the latter were transfected at a 3-fold higher level. Therefore, it seem s that putative stem cells adhering rapidly to collagen IV are not efficien tly transfected by AVET. AVET-transfected keratinocytes derived from kerati nocyte trans- glutaminase negative lamellar ichthyosis patients with a CMV- TGK expression plasmid showed that it is possible to reach a level of total enzyme activity similar to that found in cultured keratinocytes from norma l individuals. In organotypic cultures from outer root sheath cells AVET tr ansfection was not successful, which might be due to the presence of the co rnified layer or inaccessibility of the adenovirus receptor. In summary, th e AVET system provides a powerful tool for transient in vitro transfection of keratinocytes.