Topical Gene Delivery to Murine Skin Wei Hong Yu, Dan Moore, Robert J. Debs  Journal of Investigative Dermatology  Volume 112, Issue 3, Pages 370-375 (March 1999) DOI: 10.1046/j.1523-1747.1999.00513.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cutaneous gene delivery by topical application. After shaving, mouse skin was brushed and then 20 μl of 1 μg plasmid DNA per μl was applied topically as described in Materials and Methods. For each treatment group, DNA alone was applied to each of four individual sites on the dorsum of two individual mice. Samples were collected 24 h after DNA application and analyzed for luciferase activity as described in Materials and Methods. Statistical test results: Each brushing condition is significantly higher than control, based on the rank-sum test. (p = 0.0157, 0.0063, 0.0008, and 0.0008 for 50, 100, 150, and 200 brush strokes, respectively.) Data was tested using nonparametric tests. The Kruskal–Wallis test was used to compare three or more treatments; the rank-sum test was used for comparing pairs of treatments. Variances of measurements appear to increase with average of measurements, suggesting log transformation to achieve equal variance of measurements. Nevertheless, nonparametric tests were used to avoid making assumptions about the shape of the distribution. Journal of Investigative Dermatology 1999 112, 370-375DOI: (10.1046/j.1523-1747.1999.00513.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Luciferase activity following topical application of different concentrations of DNA. Twenty microliters of plasmid DNA at different concentrations was applied topically to the skin following shaving and 100 brushstrokes. Samples were collected 24 h after DNA application and assayed for luciferase activity. Statistical test results: The 0.01 and 0.05 μg per μl treatments are significantly lower than the other treatments (p = 0.001 by the Kruskal–Wallis test), but there is no difference between the 0.01 and 0.05 μg per μl treatments (p = 0.17 by rank-sum test). Journal of Investigative Dermatology 1999 112, 370-375DOI: (10.1046/j.1523-1747.1999.00513.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunohistochemical analysis of CAT enzyme in mouse skin following topical application of plasmid DNA. Multiple dark staining foci in (a) and (b) represent sites of CAT antigen accumulation as detected by immunohistochemistry. CAT antigen is recognizable within and on the superficial epidermis of CAT-treated mice (a, b). CAT antigen was predominately confined to superficial keratinocytes (b). A section of skin (c) from a control mouse that was stained at the same time demonstrates a lack of staining for CAT antigen. Tissues were collected 24 h after DNA application using 100 brushstrokes as described in Materials and Methods. (a, c) X175; (b) X900. Journal of Investigative Dermatology 1999 112, 370-375DOI: (10.1046/j.1523-1747.1999.00513.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Gene expression in the skin following topical delivery of DNA alone or CLDC. Twenty microliters of DNA alone or CLDC was applied topically. Mice were sacrificed 24 h after DNA application and assayed for luciferase activity. p4241 (1 μg per μl) was mixed with DOTAP:DOPE (SUV) (CLDC1) or DOTAP:cholesterol (MLV) (CLDC2) at 4:1 and 1:7 ratios, respectively. Statistical test results: Both CLDC groups at the 1:7 ratio are significantly lower than the DNA alone group (p < 0.001) for each by the Kruskal–Wallis test. The DNA alone and CLDC 4:1 groups did not differ significantly from each other. Journal of Investigative Dermatology 1999 112, 370-375DOI: (10.1046/j.1523-1747.1999.00513.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Time course of CAT gene expression following topical application of DNA. Mouse skin was shaved and brushed 100 times. Twenty microliters of 4119 plasmid DNA (1 mg per ml) was applied. Mice were sacrificed from 1 to 48 h after DNA application, and skin samples were assayed for CAT activity. Statistical test results: All treated groups were significantly higher than the control group (p = 0.0001 by the Kruskal–Wallis test). CAT activity increases with time, but levels off at 16 h (p = 0.46 for time 16 h). Journal of Investigative Dermatology 1999 112, 370-375DOI: (10.1046/j.1523-1747.1999.00513.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Comparison of gene transfer efficiency following different modes of cutaneous gene delivery. Twenty microliters of 4241 plasmid DNA (1 μg per μl) was injected intradermally or topically applied following shaving and brushing or shaving and treatment with a depilatory cream (Nair cream). Control: 20 μl of plasmid DNA applied to shaved, unbrushed skin. Samples were collected and assayed for luciferase activity 24 h after DNA application. Statistical test results: The 100X brushing and intradermal injection groups did not differ significantly from each other (p = 0.78), whereas each of these groups was significantly higher (p = 0.001) than either the control (untreated) or the Nair cream groups by the Kruskal–Wallis test. Journal of Investigative Dermatology 1999 112, 370-375DOI: (10.1046/j.1523-1747.1999.00513.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions