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Topical Gene Delivery to Murine Skin
Topical Gene Delivery to Murine Skin Wei Hong Yu, Mohammed Kashani-Sabet,* Denny Liggitt,† Dan Moore, Timothy D. Heath,‡ and Robert J. Debs California Pacific Medical Research Institute, San Francisco, California, U.S.A.; *Cutaneous Oncology Division, Department of Dermatology, University of California San Francisco, San Francisco, California, U.S.A.; †Department of Comparative Medicine, University of Washington, Seattle, Washington, U.S.A.; ‡School of Pharmacy, University of Wisconsin-Madison, Wisconsin, U.S.A.
We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations ≥0.25 µg per µl were required to achieve maximal
expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders. Key words: epidermis/gene therapy/hair follicles/plasmid DNA. J Invest Dermatol 112:370–375, 1999
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dermis, epidermis and hair follicles following topical application of cationic liposome:DNA complexes (CLDC) onto mouse skin previously treated with a depilatory cream (Alexander and Akhurst, 1995). Recently, delivery of DNA into the skin without carriers has also been reported. Naked DNA was introduced into the skin of experimental animals by direct injection (Furth et al, 1995; Hengge et al, 1995; Ciernik et al, 1996), by gene gun (Williams et al, 1991), or by using an electric pulse (Zhang et al, 1996). These approaches resulted in the expression of the transferred genes within the epidermis and dermis. Despite these encouraging results, little is known about the principles governing cutaneous gene transfer. To date, skin gene transfer protocols using naked DNA have relied on methods such as localized intradermal injection and electric pulse, which may have limited clinical relevance. Topical skin gene transfer techniques reported to date have required liposomes as a DNA carrier. Here, we report an approach that permits gene transfer and expression following topical application of naked plasmid DNA to mouse skin.
omatic gene therapy will be of great importance in the treatment of genetic and acquired diseases. The skin is an attractive target tissue for somatic gene transfer for several reasons: (i) it is the largest organ in the body; (ii) it is easily accessible; (iii) genetically modified skin can be easily monitored and, if necessary, removed; and (iv) the skin has been extensively characterized at both the cellular and the molecular levels (Epstein Jr., 1993). Furthermore, the promoter elements of tissue-specific genes, including keratin genes, have been identified and can be used to target the expression of genes delivered into the skin (Bailleul et al, 1990; Vassar and Fuchs, 1991). In addition to treating skin disorders, cutaneous gene delivery can be used to express gene products with systemic effects (Fenjves et al, 1989; Palmer et al, 1989; Alexander et al, 1995). Multiple approaches, both ex vivo and in vivo, have been developed to deliver genes into the skin. To date, most epidermal gene transfer studies have focused on ex vivo approaches. Genes encoding human apolipoprotein E and clotting factor IX have been stably introduced into cultured keratinocytes or skin fibroblasts (Palmer et al, 1989; Fenjves et al, 1989, 1994). The transfected cells were then grafted onto mice, and the gene products expressed by these modified cells were detected in the circulation. Genetically modified keratinocytes were able to reconstitute the epidermis after being transplanted to wound sites, suggesting that introducing genes encoding therapeutic proteins may facilitate wound healing (Vogt et al, 1994). In addition to ex vivo gene delivery, several methods have been developed to deliver cloned genes into the skin in vivo. Li and Hoffman applied a lac Z expression plasmid associated with noncationic liposome complexes onto mouse skin and detected the lac Z gene product in hair follicles (Li and Hoffman, 1995). Alexander and Akhurst showed β-galactosidase expression in the
MATERIALS AND METHODS Topical DNA delivery Eight week old ICR female mice (Simonson) were anesthetized in a glass bell jar with metofane (Pitman-Moore, Mundelein, IL). The hair of the dorsal surface was shaved using an electrical clipper (Butler, Union City, CA). The shaved dorsal skin was then either stripped by applying Blenderm tape (3M Health Care, St. Paul, MN) five times and/or brushed using a tooth brush with rounded firm nylon bristles. The number of brushing strokes varied from 50 to 200. Circled areas with a 1 cm diameter were marked around the treated skin site with an indelible stamp. Twenty microliters of naked plasmid DNA in sterile water or CLDC were pipetted onto the circled area and spread evenly using a flame sealed 1 ml plastic eppendorf pipette tip. Following stripping and/or brushing, the applied DNA was allowed to completely air dry on the treated skin, which took µ10 min. All of the applied solution was either absorbed or had evaporated by this time. Treated mice were sacrificed from 1 h to 14 d following application, and the treated skin was harvested, homogenized, and assayed for the product of the applied gene as described below.
Manuscript received February 3, 1998; revised July 14, 1998; accepted for publication November 6, 1998. Reprint requests to: Robert J. Debs, California Pacific Medical Research Institute, 2330 Clay St., San Francisco, CA 94115.
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0022-202X/99/$10.50 Copyright © 1999 by The Society for Investigative Dermatology, Inc.
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Intradermal DNA delivery ICR female mice were anesthetized and shaved as described above and circled areas 1 cm in diameter were marked. Plasmid DNA was injected intradermally into the marked area. Treated skin was harvested 24 h after injection as above. Chloramphenicol acetyltransferase (CAT) assay CAT assay was carried out as described previously (Liu et al, 1995) with the following modifications. In each reaction, 0.15 µCi of 14C-labeled chloramphenicol (Amersham, Arlington Heights, IL) and 5 µl of 20 mM acetyl coenzyme A stock solution in water (Sigma, St. Louis, MO) were added to samples in a final volume of 111 µl. The mixture was incubated at 37°C overnight. The CAT units in tissue extracts were quantitated using a phosphor-imager (Liu et al, 1997). Luciferase assay Skin samples were placed in 600 µl of lysis buffer (Promega Luciferase Assay System) and the luciferase assay was carried out as described previously (Liu et al, 1997). RLU were converted to amounts of luciferase using a standard curve for luciferase, as described previously (Liu et al, 1997). Plasmid construction and purification of plasmid DNA The construction of plasmid 4119 (CMV-CAT) has been described previously (Liu et al, 1995). The construction of plasmid p4241 (CMV-Luciferase) was described by Liu et al (1997). Plasmid DNA was purified as described previously (Liu et al, 1995).
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delivering genes to the skin by topical administration. Tape stripping is a commonly used method in order to disrupt the epidermal barrier and to enhance the delivery of macromolecules into the skin (Yang et al, 1995). After tape stripping, a CMV-CAT expression plasmid was applied to the stripped area. We found that CAT activity was higher in the skin samples stripped five times prior to DNA application compared with those stripped three times prior to DNA application (data not shown). This result suggested that abrasion of the skin prior to DNA application could improve cutaneous gene transfer and expression. In order to optimize this technique, we used a tooth brush with rounded nylon bristles to brush the skin after shaving the hair. Either CMV-CAT or CMVLuciferase plasmids were applied topically. Two different reporter genes were examined in order to provide a more complete picture of both the efficiency and the consistency of cutaneous gene transfer we achieved. Because the two gene products differ in their relative half-lives and intracellular localizations, we undertook this combined analysis. Figure 1 shows the levels of luciferase activity produced in mouse skin by topical application of DNA following different numbers of brushstrokes. Reporter gene activity was consistently at or near peak levels in mice treated with 100 brushstrokes. Therefore, 100 brushstrokes was used in subsequent experiments.
Preparation of cationic liposomes Liposomes containing either DOTIM (Solodin et al, 1995) or DOTAP (N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethyl-ammonium-methyl-sulfate, purchased from Avanti Polar-lipids, Alabaster, AL) in a 1:1 ratio with either DOPE (dioleoyl phosphatidylethanolamine, Avanti Polar-lipids) or cholesterol (Calbiochem, Los Angeles, CA) were prepared as described previously (Liu et al, 1997). The multilamellar vesicles (MLV) were made by resuspending the dried lipid film in 5% dextrose in water. The small unilamellar vesicles (SUV) were made by sonicating MLV liposome in a bath sonicator for 20 min. Preparation of DNA/cationic liposome complexes DNA/liposome complexes were prepared as described previously (Liu et al, 1995). Immunohistochemical analysis to detect CAT enzyme in mouse skin Twenty-four hours after topical application of DNA with 100 brush strokes, as described above, skin samples were taken and embedded in O.C.T. (Miles, Elkhart, IN) and quick frozen in isopentane chilled in liquid nitrogen. Frozen specimens were sectioned on a cryostat and collected onto clean, charged glass slides (Probe On Plus; Fisher Scientific, Pittsburgh, PA). Sections were dried for 24 h at room temperature and then fixed in 100% acetone at –20°C for 20 min. Following three 5 min washes in phosphate-buffered saline containing 0.2% Tween-20, the sections were blocked with 2% normal sheep serum in phosphate-buffered saline for 20 min and then followed by another six washes in phosphate-buffered saline containing 0.2% Tween-20. Sections were then incubated with sheep anti-CAT-digoxigenin labeled antibody (Boehringer Mannheim, Indianapolis, IN) at the appropriate dilution overnight at 4°C. The slides were then washed and incubated with alkaline phosphatase conjugated anti-digoxigenin Fab fragment (Boehringer) for 60 min at room temperature in the dark. Color was developed using a 5-bromo-4-chloro-3-indolyl/ nitroblue tetrazolium substrate kit (BCIP/NBT; Vector Labs, Burlingame, CA). Counterstains were avoided because they interfered with signal detection. Stained sections were then rinsed in distilled water, mounted, and examined using light microscopy. Controls included use of normal sheep serum to replace the primary antibody and skin sections transfected with CMV-Luciferase plasmid. Statistical methods Plots of the data showed significant heterogeneity of variance over treatment conditions with occasional outlier values. For this reason, comparisons between treatments were made using nonparametric tests. The Kruskal–Wallis one-way analysis of variance by ranks test was used for all comparisons (Kruskal and Wallis, 1952). The nonparametric test for trend (Cuzick, 1985) was used to test for a linear trend. For the data of Fig 5, we fit a function of the form y 5 b1(1 – b2x) to the natural log of the measurements to provide a smooth fit to the data. All tests and fits were carried out in Stata (Stata, Statistics/Data Analysis. Stata 702 University Drive East College Station, Texas).
RESULTS Genes can be topically delivered to and expressed in the skin Our goal was to develop a clinically relevant method for
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 5 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.
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We applied different concentrations of p4241 (CMV-Luciferase) plasmid DNA onto shaved mouse skin in order to determine the optimal plasmid concentration for gene delivery and expression. Luciferase activity was detected following application of DNA concentrations as low as 0.01 µg per µl (Fig 2), and increased with increasing concentrations of DNA up to 0.5 µg per µl. Luciferase activity plateaued at DNA doses from 0.25 to 1.0 µg per µl. DNA concentrations higher than 1.0 µg per µl produced less luciferase activity.
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et al, 1993; Alexander and Akhurst, 1995; Liu et al, 1997). These include DOTIM:cholesterol (MLV), DOTAP:DOPE (SUV), and DOTAP:cholesterol (MLV). As shown in Fig 4, when the DNA:lipid ratio (µg DNA:nmol total lipid) was ù1:1, the expression levels produced by topical application of CLDC containing either
Sites of gene expression We examined the expression of CAT in mouse skin 24 h following topical application of plasmid DNA using immunohistochemical analysis. As shown in Fig 3, CAT antigen was detected primarily in superficial keratinocytes within the superficial epidermis, as well as in hair follicles. The epidermis was intact and there was no significant inflammation recognized histologically in these sections. Keratin was removed from the majority of the epidermis following brushing and/or tape stripping, although some remnants persisted adjacent to hair follicles (Fig 3b). Cationic liposomes can inhibit gene expression in skin after topical delivery Cationic liposomes have been widely used as carriers to deliver DNA into animals both systemically (Zhu et al, 1993; Liu et al, 1995; Thierry et al, 1995) and locally (Alexander and Akhurst, 1995). Because different cationic liposome formulations can result in significantly different gene transfer efficiencies, we screened a variety of different cationic lipid formulations that have previously been shown to produce significant levels of gene expression following in vitro and/or in vivo administration (Philip
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 5 0.001 by the Kruskal–Wallis test), but there is no difference between the 0.01 and 0.05 µg per µl treatments (p 5 0.17 by rank-sum test).
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.
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luciferase (Fig 4) or CAT (data not shown) genes were comparable with those produced by application of DNA alone. With increasing lipid concentrations, however, reporter gene expression decreased significantly. Thus, although cationic liposomes significantly enhance transfection efficiency in vitro (Caplen et al, 1995) and in vivo following intravenous injection (Liu et al, 1995), aerosol (Stribling et al, 1992; Alton and Geddes, 1995) or intraperitoneal injection (Philip et al, 1993) of DNA, low DNA:lipid ratios inhibit reporter gene expression following topical cutaneous gene delivery. The presence of a vehicle does not alter cutaneous gene transfer efficiency In the clinical setting, lipophilic ointments such as petroleum jelly are frequently used as a vehicle to enhance drug penetration into the skin. In an attempt to improve the efficiency of reporter gene transfer and expression in the skin, we used a lipophilic vehicle in our topical application experiments. We applied plasmid DNA alone or DNA mixed with vehicles (Nivea cream, clobetasol propionate ointment, or petroleum jelly) onto shaved mouse skin without using either tape stripping or brushing. No CAT activity was detected 24 h after the topical application of plasmid DNA alone, DNA mixed with Nivea cream, or DNA mixed with clobetasol propionate ointment in the skin samples (data not shown). Petroleum jelly could not be mixed with DNA to yield a homogeneous solution. To test whether DNA application in a vehicle interfered with the transfection efficiency of genes in brushed skin, we applied petroleum jelly on mouse skin after plasmid DNA application, and found that the presence
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.
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of petroleum jelly did not inhibit the transfection efficiency (data not shown). Time course of gene expression in skin Skin samples were collected at different time points after topical application of DNA alone. Figure 5 shows the CAT activity in skin samples harvested from one through 48 h after DNA application. Significant levels of CAT activity were detected as early as 4 h after DNA application. CAT activity reached a peak at 16 h and remained at this level up to 72 h (Fig 5 and data not shown). Seven days after DNA application, significant levels of CAT were still detected in skin samples; however, by 14 d after DNA application, CAT activity had returned to baseline levels (data not shown). Because the halflife of CAT in this system is unknown, it is unclear whether the CAT activity present at the later time points represented ongoing gene expression versus residual enzyme trapped in the skin. Previously the half-life of CAT protein was found to be µ20 h in cultured cells (Philip et al, 1993), suggesting that prolonged CAT activity may be due to persistence of the gene product. Comparison of transfer efficiency following different routes We then compared the expression of Luciferase in the skin following different modes of DNA administration into the skin. Figure 6 shows that the Luciferase activity in the skin of mice treated with topical DNA application on abraded areas or with intradermal injection of DNA is substantially higher than that in the depilatory cream treated group; however, there was no significant difference in CAT activity in mice undergoing either topical application or intradermal injection, indicating that topical application of DNA following brushing is as efficient as intradermal
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 5 0.0001 by the Kruskal–Wallis test). CAT activity increases with time, but levels off at 16 h (p 5 0.46 for time 16 h).
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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 5 0.78), whereas each of these groups was significantly higher (p 5 0.001) than either the control (untreated) or the Nair cream groups by the Kruskal–Wallis test.
injection. Cutaneous transfer and expression of the β-galactosidase gene has previously been reported by topical application of CLDC following application of a depilatory cream (Alexander and Akhurst, 1995). In our experiments, however, use of a depilatory cream did not produce significant levels of transfection (Fig 6). DISCUSSION In this study, we have developed a method for topical skin gene delivery that utilizes naked plasmid DNA. The expression level of reporter genes in the skin following topical application of DNA was comparable with that following intradermal injection of DNA, indicating that topical application of DNA to the skin following mild abrasion is a useful method of cutaneous gene transfer and expression. High levels of target gene expression are desirable for producing a biologically significant phenotype in many cutaneous gene therapy approaches. Although the precise level of gene product required to achieve phenotypic change in the skin will vary with the gene delivered, the significant levels of reporter gene expression we have produced suggest the potential utility of topical cutaneous gene delivery for the treatment of cutaneous disorders. Furthermore, the expression level of reporter genes in the skin following our mode of topical administration is high compared with other methods of gene delivery (Fig 6). The much lower efficiency of depilatory cream mediated cutaneous gene delivery we observed may relate
to the different strains of mice, different age groups, and different depilatory creams used previously (Alexander and Akhurst, 1995). Surprisingly, gene transfer efficiency produced by topical application of DNA alone was equal to or greater than that produced by topical application of CLDC. This is in direct contrast to intravenous administration, where the use of cationic liposomes as a DNA carrier increases the expression level of reporter genes by several orders of magnitude (Liu et al, 1995). It is possible that DNA alone penetrates the epidermal barrier better than CLDC, or that the presence of the cationic lipid limits access of the complexed DNA to keratinocytes themselves within the cutaneous matrix. For example, DNA complexed with cationic liposomes may be sequestered in the lamellar bodies and unavailable for gene expression (Menon et al, 1992). Previously, naked DNA has been shown to be delivered and efficiently expressed following direct injection into muscle and tumor cells or liver (Malone et al, 1994; Takehara et al, 1996; Yang and Huang, 1996), as well as into skin (Furth et al, 1995; Hengge et al, 1995; Ciernik et al, 1996), without the need for a DNA carrier. The mechanisms controlling the topical delivery of naked plasmid DNA are poorly understood and need to be further investigated. One advantage of using plasmid DNA alone in gene delivery is that it circumvents potential host responses elicited by viral DNA carriers. It also may allow repeated application of the plasmid encoding the desired gene product to the same area in order to maintain expression levels. The advantage of injecting DNA into skin is that DNA can be repeatedly injected to the desired area to compensate for the loss of gene expression over time. This approach has significant potential for genetic immunization. Intradermal gene immunization has been reported to induce higher antibody titers than intramuscular administration (Raz et al, 1994). Potential disadvantages of this approach are that transfection via intradermal injection is not site specific and only very limited areas of the skin can be transfected per injection. Moreover, intradermal injection may transduce cells in the epidermis, dermis, and subcutis. Therefore, direct injection of DNA into skin may have limited clinical application in the treatment of epidermally based cutaneous disorders. The gene gun (Williams et al, 1991) has also been shown to deliver DNA-coated microprojectiles into skin efficiently; however, this approach may have its clinical limitations, especially when a large treatment area or frequent treatments are involved. Similar factors may limit the use of electric pulse (Zhang et al, 1996) and puncture-mediated gene transfer (Ciernik et al, 1996). The major advantage of topical cutaneous gene delivery is that it can produce efficient transfection largely limited to the epidermis, and can potentially transfect large areas of skin using a noninvasive method. This approach may prove useful in the treatment of many cutaneous disorders. Further studies are required to determine the preclinical utility of this model system. Support was provided by NIH grants DK45917, CA58914, DK49550, and HL53762 to R.J.D. M.K.S. is supported by a Leaders Society Clinical Career Development Award from the Dermatology Foundation.
REFERENCES Alexander MY, Akhurst RJ: Liposome-mediated gene transfer and expression via the skin. Hum Mol Genet 4:2279–2285, 1995 Alexander MY, Bidichandani SI, Cousins FM, Robinson CJM, Duffie E, Akhurst RJ: Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of hemophilia B. Hum Mol Genet 4:993–999, 1995 Alton EWFW, Geddes DM: Gene therapy for cystic fibrosis: a clinical perspective. Gene Therapy 2:88–95, 1995 Bailleul B, Surani MA, White S, et al: Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter. Cell 62:697–708, 1990 Caplen NJ, Alton EWFW, Middleton PG, et al: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nature Med 1:39–46, 1995 Ciernik IF, Krayenbuhl BH, Carbone DP: Puncture-mediated gene transfer to the skin. Hum Gene Ther 7:893–899, 1996
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Cuzick J: A Wilcoxon-type test for trend. Stat Med 4:87–90, 1985 Epstein EH Jr: The morbid cutaneous anatomy of the human genome. Arch Dermatol 129:1417–1423, 1993 Fenjves ES, Gordon DA, Pershing LK, Williams DL, Taichman LB: Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: Implications for epidermal function and gene therapy. Proc Natl Acad Sci USA 86:8803–8807, 1989 Fenjves ES, Smith J, Zaradic S, Taichman LB: Systemic delivery of secreted protein by grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy. Hum Gene Ther 5:1241–1248, 1994 Furth PA, Shamay A, Hennighausen L: Gene transfer into mammalian cells by jet injection. Hybridoma 14:149–152, 1995 Hengge UR, Chan EF, Foster RA, Walker PS, Vogel JC: Cytokine gene expression in epidermis with biological effects following injection of naked DNA. Nature Genet 10:161–166, 1995 Kruskal WH, Wallis WA: Use of ranks in one-criterion variance analysis. J Amer Statist Assoc 47:583–621, 1952 Li L, Hoffman RM: The feasibility of targeted selective gene therapy of the hair follicle. Nature Med 7:705–706, 1995 Liu Y, Liggitt D, Zhong W, Tu G, Gaensler K, Debs R: Cationic liposome-mediated intravenous gene delivery. J Biol Chem 270:24864–24870, 1995 Liu Y, Mounkes LC, Liggitt DH, Brown CB, Solodin I, Heath TD, Debs RJ: Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nature Biotechnol 15:167–173, 1997 Malone RW, Hickman MA, Lehmann-Bruinsma K, Sih TR, Walzem R, Carlson DM, Powell JS: Dexamethasone enhancement of gene expression after direct hepatic DNA injection. J Biol Chem 269:29903–29907, 1994 Menon GK, Ghadially R, Williams ML, Elias PM: Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation. Br J Dermatol 126:337–345, 1992 Palmer TD, Thompson AR, Miller AD: Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B. Blood 73:438–445, 1989 Philip R, Liggitt D, Philip M, Dazin P, Debs R: In vivo gene delivery. Efficient transfection of T lymphocytes in adult mice. J Biol Chem 268:16087–16090, 1993
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Raz E, Carson DA, Parker SE, et al: Intradermal gene immunization: The possible role of DNA uptake in the induction of cellular immunity to viruses. Proc Natl Acad Sci USA 91:9519–9523, 1994 Solodin I, Brown C, Bruno M, Chow C, Jang E-H, Debs R, Heath T: High efficiency in vivo gene delivery with a novel series of amphilic imadazolinium compounds. Biochemistry 34:13537–13544, 1995 Stribling R, Brunette E, Liggitt D, Gaensler K, Debs RJ: Aerosol gene delivery in vivo. Proc Natl Acad Sci USA 89:11277–11281, 1992 Takehara T, Hayashi N, Yamamoto M, Miyamoto Y, Fusamoto H, Kamada T: In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA. Hum Gene Ther 7:589–593, 1996 Thierry AR, Iskandar YL, Bryant JC, Rabinovich P, Gallo RC, Mahan LC: Systemic gene therapy: biodistribution and long-term expression of a transgene in mice. Proc Natl Acad Sci USA 92:9742–9746, 1995 Vassar H, Fuchs E: Transgenic mice provide new insights into the role of TGFalpha during epidermal development and differentiation. Genes Dev 5:714– 727, 1991 Vogt PM, Thompson S, Andree C, et al: Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis. Proc Natl Acad Sci USA 91:9307–9311, 1994 Williams RS, Johnston SA, Riedy M, DeVit MJ, McElligott SG, Sanford JC: Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci USA 88:2726–2730, 1991 Yang J-P, Huang L: Direct gene transfer to mouse melanoma by intratumor injection of free DNA. Gene Therapy 3:542–548, 1996 Yang L, Mao-Qiang M, Taljebini M, Elias PM, Feingold KR: Topical stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment. Br J Dermatol 133:679– 685, 1995 Zhang L, Li L, Hoffman GA, Hoffman RM: Depth-targeted efficient gene delivery and expression in the skin by pulsed electric field: an approach to gene therapy of skin aging and other diseases. Biochem Biophys Res Commun 220:633– 636, 1996 Zhu N, Liggitt D, Liu Y, Debs R: Systemic gene expression after intravenous DNA delivery into adult mice. Science 261:209–211, 1993
We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations ≥0.25 µg per µl were required to achieve maximal
expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders. Key words: epidermis/gene therapy/hair follicles/plasmid DNA. J Invest Dermatol 112:370–375, 1999
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dermis, epidermis and hair follicles following topical application of cationic liposome:DNA complexes (CLDC) onto mouse skin previously treated with a depilatory cream (Alexander and Akhurst, 1995). Recently, delivery of DNA into the skin without carriers has also been reported. Naked DNA was introduced into the skin of experimental animals by direct injection (Furth et al, 1995; Hengge et al, 1995; Ciernik et al, 1996), by gene gun (Williams et al, 1991), or by using an electric pulse (Zhang et al, 1996). These approaches resulted in the expression of the transferred genes within the epidermis and dermis. Despite these encouraging results, little is known about the principles governing cutaneous gene transfer. To date, skin gene transfer protocols using naked DNA have relied on methods such as localized intradermal injection and electric pulse, which may have limited clinical relevance. Topical skin gene transfer techniques reported to date have required liposomes as a DNA carrier. Here, we report an approach that permits gene transfer and expression following topical application of naked plasmid DNA to mouse skin.
omatic gene therapy will be of great importance in the treatment of genetic and acquired diseases. The skin is an attractive target tissue for somatic gene transfer for several reasons: (i) it is the largest organ in the body; (ii) it is easily accessible; (iii) genetically modified skin can be easily monitored and, if necessary, removed; and (iv) the skin has been extensively characterized at both the cellular and the molecular levels (Epstein Jr., 1993). Furthermore, the promoter elements of tissue-specific genes, including keratin genes, have been identified and can be used to target the expression of genes delivered into the skin (Bailleul et al, 1990; Vassar and Fuchs, 1991). In addition to treating skin disorders, cutaneous gene delivery can be used to express gene products with systemic effects (Fenjves et al, 1989; Palmer et al, 1989; Alexander et al, 1995). Multiple approaches, both ex vivo and in vivo, have been developed to deliver genes into the skin. To date, most epidermal gene transfer studies have focused on ex vivo approaches. Genes encoding human apolipoprotein E and clotting factor IX have been stably introduced into cultured keratinocytes or skin fibroblasts (Palmer et al, 1989; Fenjves et al, 1989, 1994). The transfected cells were then grafted onto mice, and the gene products expressed by these modified cells were detected in the circulation. Genetically modified keratinocytes were able to reconstitute the epidermis after being transplanted to wound sites, suggesting that introducing genes encoding therapeutic proteins may facilitate wound healing (Vogt et al, 1994). In addition to ex vivo gene delivery, several methods have been developed to deliver cloned genes into the skin in vivo. Li and Hoffman applied a lac Z expression plasmid associated with noncationic liposome complexes onto mouse skin and detected the lac Z gene product in hair follicles (Li and Hoffman, 1995). Alexander and Akhurst showed β-galactosidase expression in the
MATERIALS AND METHODS Topical DNA delivery Eight week old ICR female mice (Simonson) were anesthetized in a glass bell jar with metofane (Pitman-Moore, Mundelein, IL). The hair of the dorsal surface was shaved using an electrical clipper (Butler, Union City, CA). The shaved dorsal skin was then either stripped by applying Blenderm tape (3M Health Care, St. Paul, MN) five times and/or brushed using a tooth brush with rounded firm nylon bristles. The number of brushing strokes varied from 50 to 200. Circled areas with a 1 cm diameter were marked around the treated skin site with an indelible stamp. Twenty microliters of naked plasmid DNA in sterile water or CLDC were pipetted onto the circled area and spread evenly using a flame sealed 1 ml plastic eppendorf pipette tip. Following stripping and/or brushing, the applied DNA was allowed to completely air dry on the treated skin, which took µ10 min. All of the applied solution was either absorbed or had evaporated by this time. Treated mice were sacrificed from 1 h to 14 d following application, and the treated skin was harvested, homogenized, and assayed for the product of the applied gene as described below.
Manuscript received February 3, 1998; revised July 14, 1998; accepted for publication November 6, 1998. Reprint requests to: Robert J. Debs, California Pacific Medical Research Institute, 2330 Clay St., San Francisco, CA 94115.
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Intradermal DNA delivery ICR female mice were anesthetized and shaved as described above and circled areas 1 cm in diameter were marked. Plasmid DNA was injected intradermally into the marked area. Treated skin was harvested 24 h after injection as above. Chloramphenicol acetyltransferase (CAT) assay CAT assay was carried out as described previously (Liu et al, 1995) with the following modifications. In each reaction, 0.15 µCi of 14C-labeled chloramphenicol (Amersham, Arlington Heights, IL) and 5 µl of 20 mM acetyl coenzyme A stock solution in water (Sigma, St. Louis, MO) were added to samples in a final volume of 111 µl. The mixture was incubated at 37°C overnight. The CAT units in tissue extracts were quantitated using a phosphor-imager (Liu et al, 1997). Luciferase assay Skin samples were placed in 600 µl of lysis buffer (Promega Luciferase Assay System) and the luciferase assay was carried out as described previously (Liu et al, 1997). RLU were converted to amounts of luciferase using a standard curve for luciferase, as described previously (Liu et al, 1997). Plasmid construction and purification of plasmid DNA The construction of plasmid 4119 (CMV-CAT) has been described previously (Liu et al, 1995). The construction of plasmid p4241 (CMV-Luciferase) was described by Liu et al (1997). Plasmid DNA was purified as described previously (Liu et al, 1995).
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delivering genes to the skin by topical administration. Tape stripping is a commonly used method in order to disrupt the epidermal barrier and to enhance the delivery of macromolecules into the skin (Yang et al, 1995). After tape stripping, a CMV-CAT expression plasmid was applied to the stripped area. We found that CAT activity was higher in the skin samples stripped five times prior to DNA application compared with those stripped three times prior to DNA application (data not shown). This result suggested that abrasion of the skin prior to DNA application could improve cutaneous gene transfer and expression. In order to optimize this technique, we used a tooth brush with rounded nylon bristles to brush the skin after shaving the hair. Either CMV-CAT or CMVLuciferase plasmids were applied topically. Two different reporter genes were examined in order to provide a more complete picture of both the efficiency and the consistency of cutaneous gene transfer we achieved. Because the two gene products differ in their relative half-lives and intracellular localizations, we undertook this combined analysis. Figure 1 shows the levels of luciferase activity produced in mouse skin by topical application of DNA following different numbers of brushstrokes. Reporter gene activity was consistently at or near peak levels in mice treated with 100 brushstrokes. Therefore, 100 brushstrokes was used in subsequent experiments.
Preparation of cationic liposomes Liposomes containing either DOTIM (Solodin et al, 1995) or DOTAP (N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethyl-ammonium-methyl-sulfate, purchased from Avanti Polar-lipids, Alabaster, AL) in a 1:1 ratio with either DOPE (dioleoyl phosphatidylethanolamine, Avanti Polar-lipids) or cholesterol (Calbiochem, Los Angeles, CA) were prepared as described previously (Liu et al, 1997). The multilamellar vesicles (MLV) were made by resuspending the dried lipid film in 5% dextrose in water. The small unilamellar vesicles (SUV) were made by sonicating MLV liposome in a bath sonicator for 20 min. Preparation of DNA/cationic liposome complexes DNA/liposome complexes were prepared as described previously (Liu et al, 1995). Immunohistochemical analysis to detect CAT enzyme in mouse skin Twenty-four hours after topical application of DNA with 100 brush strokes, as described above, skin samples were taken and embedded in O.C.T. (Miles, Elkhart, IN) and quick frozen in isopentane chilled in liquid nitrogen. Frozen specimens were sectioned on a cryostat and collected onto clean, charged glass slides (Probe On Plus; Fisher Scientific, Pittsburgh, PA). Sections were dried for 24 h at room temperature and then fixed in 100% acetone at –20°C for 20 min. Following three 5 min washes in phosphate-buffered saline containing 0.2% Tween-20, the sections were blocked with 2% normal sheep serum in phosphate-buffered saline for 20 min and then followed by another six washes in phosphate-buffered saline containing 0.2% Tween-20. Sections were then incubated with sheep anti-CAT-digoxigenin labeled antibody (Boehringer Mannheim, Indianapolis, IN) at the appropriate dilution overnight at 4°C. The slides were then washed and incubated with alkaline phosphatase conjugated anti-digoxigenin Fab fragment (Boehringer) for 60 min at room temperature in the dark. Color was developed using a 5-bromo-4-chloro-3-indolyl/ nitroblue tetrazolium substrate kit (BCIP/NBT; Vector Labs, Burlingame, CA). Counterstains were avoided because they interfered with signal detection. Stained sections were then rinsed in distilled water, mounted, and examined using light microscopy. Controls included use of normal sheep serum to replace the primary antibody and skin sections transfected with CMV-Luciferase plasmid. Statistical methods Plots of the data showed significant heterogeneity of variance over treatment conditions with occasional outlier values. For this reason, comparisons between treatments were made using nonparametric tests. The Kruskal–Wallis one-way analysis of variance by ranks test was used for all comparisons (Kruskal and Wallis, 1952). The nonparametric test for trend (Cuzick, 1985) was used to test for a linear trend. For the data of Fig 5, we fit a function of the form y 5 b1(1 – b2x) to the natural log of the measurements to provide a smooth fit to the data. All tests and fits were carried out in Stata (Stata, Statistics/Data Analysis. Stata 702 University Drive East College Station, Texas).
RESULTS Genes can be topically delivered to and expressed in the skin Our goal was to develop a clinically relevant method for
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 5 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.
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We applied different concentrations of p4241 (CMV-Luciferase) plasmid DNA onto shaved mouse skin in order to determine the optimal plasmid concentration for gene delivery and expression. Luciferase activity was detected following application of DNA concentrations as low as 0.01 µg per µl (Fig 2), and increased with increasing concentrations of DNA up to 0.5 µg per µl. Luciferase activity plateaued at DNA doses from 0.25 to 1.0 µg per µl. DNA concentrations higher than 1.0 µg per µl produced less luciferase activity.
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et al, 1993; Alexander and Akhurst, 1995; Liu et al, 1997). These include DOTIM:cholesterol (MLV), DOTAP:DOPE (SUV), and DOTAP:cholesterol (MLV). As shown in Fig 4, when the DNA:lipid ratio (µg DNA:nmol total lipid) was ù1:1, the expression levels produced by topical application of CLDC containing either
Sites of gene expression We examined the expression of CAT in mouse skin 24 h following topical application of plasmid DNA using immunohistochemical analysis. As shown in Fig 3, CAT antigen was detected primarily in superficial keratinocytes within the superficial epidermis, as well as in hair follicles. The epidermis was intact and there was no significant inflammation recognized histologically in these sections. Keratin was removed from the majority of the epidermis following brushing and/or tape stripping, although some remnants persisted adjacent to hair follicles (Fig 3b). Cationic liposomes can inhibit gene expression in skin after topical delivery Cationic liposomes have been widely used as carriers to deliver DNA into animals both systemically (Zhu et al, 1993; Liu et al, 1995; Thierry et al, 1995) and locally (Alexander and Akhurst, 1995). Because different cationic liposome formulations can result in significantly different gene transfer efficiencies, we screened a variety of different cationic lipid formulations that have previously been shown to produce significant levels of gene expression following in vitro and/or in vivo administration (Philip
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 5 0.001 by the Kruskal–Wallis test), but there is no difference between the 0.01 and 0.05 µg per µl treatments (p 5 0.17 by rank-sum test).
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.
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luciferase (Fig 4) or CAT (data not shown) genes were comparable with those produced by application of DNA alone. With increasing lipid concentrations, however, reporter gene expression decreased significantly. Thus, although cationic liposomes significantly enhance transfection efficiency in vitro (Caplen et al, 1995) and in vivo following intravenous injection (Liu et al, 1995), aerosol (Stribling et al, 1992; Alton and Geddes, 1995) or intraperitoneal injection (Philip et al, 1993) of DNA, low DNA:lipid ratios inhibit reporter gene expression following topical cutaneous gene delivery. The presence of a vehicle does not alter cutaneous gene transfer efficiency In the clinical setting, lipophilic ointments such as petroleum jelly are frequently used as a vehicle to enhance drug penetration into the skin. In an attempt to improve the efficiency of reporter gene transfer and expression in the skin, we used a lipophilic vehicle in our topical application experiments. We applied plasmid DNA alone or DNA mixed with vehicles (Nivea cream, clobetasol propionate ointment, or petroleum jelly) onto shaved mouse skin without using either tape stripping or brushing. No CAT activity was detected 24 h after the topical application of plasmid DNA alone, DNA mixed with Nivea cream, or DNA mixed with clobetasol propionate ointment in the skin samples (data not shown). Petroleum jelly could not be mixed with DNA to yield a homogeneous solution. To test whether DNA application in a vehicle interfered with the transfection efficiency of genes in brushed skin, we applied petroleum jelly on mouse skin after plasmid DNA application, and found that the presence
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.
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of petroleum jelly did not inhibit the transfection efficiency (data not shown). Time course of gene expression in skin Skin samples were collected at different time points after topical application of DNA alone. Figure 5 shows the CAT activity in skin samples harvested from one through 48 h after DNA application. Significant levels of CAT activity were detected as early as 4 h after DNA application. CAT activity reached a peak at 16 h and remained at this level up to 72 h (Fig 5 and data not shown). Seven days after DNA application, significant levels of CAT were still detected in skin samples; however, by 14 d after DNA application, CAT activity had returned to baseline levels (data not shown). Because the halflife of CAT in this system is unknown, it is unclear whether the CAT activity present at the later time points represented ongoing gene expression versus residual enzyme trapped in the skin. Previously the half-life of CAT protein was found to be µ20 h in cultured cells (Philip et al, 1993), suggesting that prolonged CAT activity may be due to persistence of the gene product. Comparison of transfer efficiency following different routes We then compared the expression of Luciferase in the skin following different modes of DNA administration into the skin. Figure 6 shows that the Luciferase activity in the skin of mice treated with topical DNA application on abraded areas or with intradermal injection of DNA is substantially higher than that in the depilatory cream treated group; however, there was no significant difference in CAT activity in mice undergoing either topical application or intradermal injection, indicating that topical application of DNA following brushing is as efficient as intradermal
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 5 0.0001 by the Kruskal–Wallis test). CAT activity increases with time, but levels off at 16 h (p 5 0.46 for time 16 h).
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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 5 0.78), whereas each of these groups was significantly higher (p 5 0.001) than either the control (untreated) or the Nair cream groups by the Kruskal–Wallis test.
injection. Cutaneous transfer and expression of the β-galactosidase gene has previously been reported by topical application of CLDC following application of a depilatory cream (Alexander and Akhurst, 1995). In our experiments, however, use of a depilatory cream did not produce significant levels of transfection (Fig 6). DISCUSSION In this study, we have developed a method for topical skin gene delivery that utilizes naked plasmid DNA. The expression level of reporter genes in the skin following topical application of DNA was comparable with that following intradermal injection of DNA, indicating that topical application of DNA to the skin following mild abrasion is a useful method of cutaneous gene transfer and expression. High levels of target gene expression are desirable for producing a biologically significant phenotype in many cutaneous gene therapy approaches. Although the precise level of gene product required to achieve phenotypic change in the skin will vary with the gene delivered, the significant levels of reporter gene expression we have produced suggest the potential utility of topical cutaneous gene delivery for the treatment of cutaneous disorders. Furthermore, the expression level of reporter genes in the skin following our mode of topical administration is high compared with other methods of gene delivery (Fig 6). The much lower efficiency of depilatory cream mediated cutaneous gene delivery we observed may relate
to the different strains of mice, different age groups, and different depilatory creams used previously (Alexander and Akhurst, 1995). Surprisingly, gene transfer efficiency produced by topical application of DNA alone was equal to or greater than that produced by topical application of CLDC. This is in direct contrast to intravenous administration, where the use of cationic liposomes as a DNA carrier increases the expression level of reporter genes by several orders of magnitude (Liu et al, 1995). It is possible that DNA alone penetrates the epidermal barrier better than CLDC, or that the presence of the cationic lipid limits access of the complexed DNA to keratinocytes themselves within the cutaneous matrix. For example, DNA complexed with cationic liposomes may be sequestered in the lamellar bodies and unavailable for gene expression (Menon et al, 1992). Previously, naked DNA has been shown to be delivered and efficiently expressed following direct injection into muscle and tumor cells or liver (Malone et al, 1994; Takehara et al, 1996; Yang and Huang, 1996), as well as into skin (Furth et al, 1995; Hengge et al, 1995; Ciernik et al, 1996), without the need for a DNA carrier. The mechanisms controlling the topical delivery of naked plasmid DNA are poorly understood and need to be further investigated. One advantage of using plasmid DNA alone in gene delivery is that it circumvents potential host responses elicited by viral DNA carriers. It also may allow repeated application of the plasmid encoding the desired gene product to the same area in order to maintain expression levels. The advantage of injecting DNA into skin is that DNA can be repeatedly injected to the desired area to compensate for the loss of gene expression over time. This approach has significant potential for genetic immunization. Intradermal gene immunization has been reported to induce higher antibody titers than intramuscular administration (Raz et al, 1994). Potential disadvantages of this approach are that transfection via intradermal injection is not site specific and only very limited areas of the skin can be transfected per injection. Moreover, intradermal injection may transduce cells in the epidermis, dermis, and subcutis. Therefore, direct injection of DNA into skin may have limited clinical application in the treatment of epidermally based cutaneous disorders. The gene gun (Williams et al, 1991) has also been shown to deliver DNA-coated microprojectiles into skin efficiently; however, this approach may have its clinical limitations, especially when a large treatment area or frequent treatments are involved. Similar factors may limit the use of electric pulse (Zhang et al, 1996) and puncture-mediated gene transfer (Ciernik et al, 1996). The major advantage of topical cutaneous gene delivery is that it can produce efficient transfection largely limited to the epidermis, and can potentially transfect large areas of skin using a noninvasive method. This approach may prove useful in the treatment of many cutaneous disorders. Further studies are required to determine the preclinical utility of this model system. Support was provided by NIH grants DK45917, CA58914, DK49550, and HL53762 to R.J.D. M.K.S. is supported by a Leaders Society Clinical Career Development Award from the Dermatology Foundation.
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