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What's this?

Human Molecular Genetics Pages 1761-1767  
Gene delivery to the epidermis
The Epidermis As A Target Organ
Models For Epidermal Gene Expression
   Ex vivo and in vivo epidermal transduction
   Transgenic models
Gene Transfer Systems
   Viral methods of epidermal gene transfer
   Non-viral methods of epidermal gene transfer
   Duration of gene expression
Candidate Conditions For A Gene Therapy Approach Via The Epidermis
   Inherited skin disorders
   Wound healing
   Systemic disorders
   Cutaneous immunomodulation
   Squamous cell carcinoma
   Melanoma
Future Developments
Acknowledgements
References

Gene delivery to the epidermis

Gene delivery to the epidermis

Alison H. Trainer*, M. Yvonne Alexander+

Department of Medical Genetics, Glasgow University, Yorkhill Hospital, Glasgow G3 8SJ, UK

Received May 2, 1997

Epidermal gene delivery techniques are being developed as an experimental approach to understanding the pathogenesis of skin disorders and for developing therapeutic strategies for the treatment of disease. This technology is being evaluated in many clinical trials in the treatment of disorders such as cutaneous melanoma and skin wounding, with 20% of all gene therapy protocols being applied in the field of dermatology. This review focuses on recent advances in the development of gene transfer technology to the epidermis, describing the diseases that may be amenable to treatment by use of these strategies. We will discuss the advantages and limitations of the currently used techniques and the future prospects for gene therapy via the epidermis.

THE EPIDERMIS AS A TARGET ORGAN

The epidermis has compelling appeal as a target tissue for gene therapy, due primarily to its accessibility. In vivo gene delivery is feasible, as is monitoring the genetically modified region, and the possibility of surgical removal of aberrant tissue (1).

The majority of the epidermis is comprised of keratinocytes, of which there are two major compartments, the basal and suprabasal layers. In the human skin, the suprabasal compartment is divided into multiple layers, the stratum spinosum, stratum granulosum and outer stratum corneum.

Undifferentiated, proliferating keratinocytes reside in the basal layer. Loss of their proliferative capacity is concomitant with a process of upward movement and terminal differentiation. Keratinocytes can be classified into three types with respect to their clonal proliferative capacity: (i) holoclones or stem cells with extensive growth capacity; (ii) differentiated paraclones with a limited growth capacity; and (iii) intermediate meroclones, which are thought to constitute long lived progenitor cells in vivo (2). Ideally, epidermal gene targeting would involve integration of an exogenous gene into the genome of holoclone keratinocytes, whose progeny form both a self-renewing population of stem cells allowing long-term expression, and a more differentiated suprabasal population of keratinocytes. However, targeting of meroclones might be of therapeutic value, in the same way that T cells are used in the treatment of blood disorders (3). Although both these cells have a finite lifespan, they might be easier to target due to their greater number and higher proliferative rate compared with the true stem cells.

Keratinocyte terminal differentiation involves the sequential expression of many proteins including keratins, integrins and involucrin. Keratins 5 and 14 are expressed by basal epidermal cells in conjunction with the integrins (4). During differentiation, these genes are down-regulated and keratins 1 and 10 are upregulated in the suprabasal layers. Involucrin expression occurs in the outer cornified layer. The regulatory sequences of these proteins have been well documented (5,6) and their use allows targeting of gene expression to the suprabasal as well as basal compartments in the epidermis.

Through the use of the different keratin promoters, keratinocytes have been shown to be amenable to the expression of exogenous genes, such as the coagulation cascade protein Factor IX (7) and growth hormone (8), indicating that keratinocytes have the capability to modify these exogenous gene products correctly at a post-translational level. Furthermore, the epidermis has been shown to have a secretory capability, allowing recombinant gene products expressed in the epidermis to be secreted into the circulation, indicating its potential for the treatment of systemic disorders.

MODELS FOR EPIDERMAL GENE EXPRESSION

Ex vivo and in vivo epidermal transduction

Preliminary keratinocyte gene therapy protocols focused on in vitro epidermal transduction, as primary human keratinocyte cultures are very amenable to genetic modification (9), can be massively expanded in culture and surgically grafted back to the patient. However, an in vivo approach to epidermal gene delivery is advantageous technically, clinically and economically, as it obviates both the need for in vitro cell culture, with its inherent cost and increase in cellular manipulation, and also circumvents surgery.

Although the majority of in vivo studies utilise murine epidermis, Hengge et al. recently suggested that this may not be the best model for clinical studies of gene delivery (10). When compared with human skin (Fig. 1), murine skin has a thin epidermis with fewer layers of keratinocytes and a thinner dermis populated with numerous hair follicles (Fig. 2C). Exogenous gene expression tends to involve both epidermal and dermal tissue, in contrast to the more specific epidermal expression seen in both human and porcine skin.


Figure 1 Human skin from forearm stained with haematoxylin and eosin. Note the thick cornified layer and thicker epidermal layer compared with the mouse skin in Figure 2.


Figure 2 Histological analysis of transfected mouse skins. Mouse dorsal skins were transfected with 10 [mu]g K5-driven [beta]-galactosidase DNA, and the skins were stained for [beta]-galactosidase activity 48 h later. (A) Telogen phase of the hair cycle, with follicular staining predominantly in the hair bulb. (B) Anagen phase skin with more widespread keratinocyte staining. (C) Control, mock transfected skin, note the thinner, epidermal layer of the mouse skin, as compared with human skin in Figure 1. (D) [beta]-galactosidase activity in the ear of a transgenic mouse, containing a K5-driven [beta]-galactosidase gene, with a more restricted staining pattern in the hair follicle. e, epidermis; f, dermal fibroblast; g, hair germ; h, hair follicle.

Transgenic models

Transgenic technology is a very powerful system allowing (i) the study of specific gene functions during development, (ii) the production of clinically significant mouse models (11), and (iii) the examination of the in vivo efficacy of gene expression constructs that may be of potential therapeutic value (12). Keratin promoters have been employed to express a variety of exogenous genes in the epidermis of transgenic mice (8,13,14). These studies indicate that they drive long-term, high levels of epidermal-specific gene expression, suggesting an important role for keratin regulatory sequences in targeting in vivo epidermal-specific gene expression.

GENE TRANSFER SYSTEMS

Many techniques have been evaluated for efficient epidermal gene transfer. These approaches may be classified broadly into two distinct categories, viral and non-viral.

Viral methods of epidermal gene transfer

Retroviruses. Retrovirus-mediated gene transfer was the first delivery system to be used and has been reviewed elsewhere (15). Their use in epidermal-directed gene delivery has been restricted to ex vivo approaches due to their inability to transduce non-proliferating cells (16,17).

Adenoviruses. The use of adenoviral vectors has additional advantages over retroviral vectors (18,19). Work by Setoguchi et al. has shown that recombinant adenovirus is not only effective in gene delivery in vitro but also in vivo (20). When injected subcutaneously, gene expression was seen in both the epidermis and dermis, encompassing keratinocytes, sebaceous glands, smooth muscle cells, fibroblasts and adipocytes. However, recent work has suggested that the presence of tissue macrophages may limit the use of this virus as an in vivo gene vector (21).

Vaccinia viruses. Vaccinia viruses have also been investigated in vivo. Subcutaneous injection of recombinant vaccina virus produced expression in the skin, although additional expression was noted in liver, spleen, kidney and lung indicating a worrying lack of tissue specificity even after local application (22).

Non-viral methods of epidermal gene transfer

Methods of epidermal targeting of `naked' DNA by direct penetration in vivo have included intradermal-injection (10,23), puncture-mediated gene transfer using high frequency oscillating fibres (24), and particle-bombardment (gene gun) transfer, which involves inert gold or tungsten particles being coated with DNA and delivered with a high pressure discharge into the skin to a depth dependent on the force applied (25-27). In addition, the topical application of DNA complexed with cationic liposome is effective in targeting expression to the epidermis (28).

Direct `naked' DNA gene transfer. The mechanism by which keratinocytes take up naked DNA is not understood, although it may be related to uptake by semi-permanent membrane vesicles, as suggested by Dowty et al. in myocytes (29). There is evidence that plasmid DNA is not integrated into the cellular genome, remaining instead in a non-replicating episomal form (23,30).

Cationic liposome. Entry of the DNA-liposome complex into the cell is thought to involve endocytosis (28). Recent work by Li and Hoffman demonstrated hair follicle-specific gene expression following topical application of a DNA-liposome complex (31). This is in contrast to our own study (32) in which gene expression was seen in dermal fibroblasts, follicular keratinocytes and a minority of epidermal interfollicular keratinocytes, as shown in Figure 2, indicating a wider pattern of dermal and epidermal gene expression. This difference in uptake and/or expression may be partly attributable to differences in (i) hair cycle at the time of treatment; our study involved skin in the anagen, proliferative phase of hair cycle, in contrast to the resting, telogen phase used by Li and Hoffman or (ii) differences in the liposome formulation used. Both factors have been shown to be important in liposome-mediated gene transfer (33). Pre-treatment of the skin by tape-stripping (34) or the use of a commercial depilatory cream, as in our study, may cause disruption of disulphide bonds in the stratum corneum, allowing greater access of the DNA-liposome complex to the epidermis. Disruption of the stratum corneum by epidermal enzymes (35) may have potential for increasing liposome uptake in the future.

Duration of gene expression

In non-viral DNA delivery, gene expression has been found to be transient. In general, the gene product was detected for [sim]7 days (24,34). In certain cases, the gene product was found to be present for longer, but mRNA was not detected after 7 days (23), suggesting that gene expression may also be dependent on the stability of the gene product. This transient gene expression is in contrast to the longer term gene expression obtained by in vivo injection of skeletal muscle (30), and suggests that either holoclone keratinocytes have not been targeted, or that the episomal expression vector is lost or down-regulated during progeny segregation.

Incorporating a viral origin of replication into a plasmid vector, thereby allowing episomal plasmid replication, has been shown to increase durability of gene expression (36). Evidence for transduction of the holoclone keratinocytes has been cited by Xiao et al. using cottontail rabbit papillomavirus (CRPV) (37). This vector contains an origin of DNA replication, remains in a non-replicating episomal form in keratinocytes, and causes cutaneous papillomas. By means of particle bombardment delivery, CRPV was delivered to the epidermis and the papillomas formed were monitored for 8 weeks. The presence of CRPV DNA within each papilloma was demonstrated, although no evidence of gene expression was given. There was no direct evidence of stem cell transduction.

The use of viral vectors to transfect keratinocyte cell lines has been shown to achieve longer expression patterns although when transplanted, the expression pattern may be more transient (17,38). As retroviruses integrate into the genome, the latter work indicates that the gene is either not being targeted to holoclone keratinocytes, or that viral-mediated gene expression may be down-regulated due to methylation (39). The major deficiency in epidermal mediated gene therapy at present is the inability to produce prolonged, high level gene expression in vivo necessary for the treatment of many of the conditions discussed below.

CANDIDATE CONDITIONS FOR A GENE THERAPY APPROACH VIA THE EPIDERMIS

Inherited skin disorders

Recent progress in molecular biology has allowed a greater understanding of a number of inherited skin diseases, namely the genodermatoses in which keratin mutations have been identified (40).

Lamellar ichthyosis (LI) is a recessive, X-linked disorder and is associated with a defect in transglutaminase (Tgase1), an enzyme involved in the formation of the cornified epithelium barrier. Choate et al. have shown that in vitro retroviral transduction of primary keratinocytes taken from affected LI patients could restore defective involucrin cross-linking, and when this genetically-modified epidermis was transplanted onto immunodeficient mice the function of the cutaneous barrier was restored (41,42).

Xeroderma pigmentosa (XP), an autosomal recessive disorder, is caused by a defect in the XP gene and it has been shown that viral-mediated expression of XP in fibroblasts, complements the DNA repair deficiency of primary skin fibroblasts isolated from these patients (43).

X-linked ichthyosis is caused by a deficiency in steroid sulphatase (STS) leading to accumulation of cholestrol sulphate and resulting in abnormal scaling skin. Transfection in vitro with the gene encoding STS leads to increased cell maturation and partial correction of the phenotype (44).

In addition, other skin disorders which may be amenable to treatment in the future are epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EHK). Both these disorders are due to mutations in keratin genes. Mutations in basal keratins, K5 and K14, cause the intra-epidermal blistering and collapse of the keratin filament network characteristic of EBS (40), whilst EHK is due to mutations in the suprabasal keratins, K1 and K10 (11).

The major limitation in treatment of these disorders is their generalised nature, necessitating treatment of the entire skin. In addition, the keratin mutations tend to be dominant and therefore difficult to rectify by supplementive gene therapy due to the possibility of a dominant-negative effect. A focal disease, such as psoriasis, would be more amenable to treatment. It may be feasible to use keratin regulatory elements which are induced in epidermal hyperproliferation (45) to target expression of an inhibitor of keratin proliferation such as transforming growth factor [beta] (TGF-[beta]).

Wound healing

Applications also encompass acute and chronic wound healing. Severe burns and chronic ulcers have been treated using keratinocyte-containing skin substitutes. These skin substitutes can be enhanced by genetic modification. Exogenous expression of an insulin-like growth factor has been shown to promote keratin growth in vitro and stimulate proliferation in vivo, without altering epidermal differentiation (46). In addition, it has been shown in the in vivo situation, that expression of exogenous epidermal growth factor in the skin increases wound healing by 20% (47). Another area of recent interest, which may lead to future in vivo treatment modalities, is the modulation of TGF-[beta] levels in preventing scarring and fibrosis during wound healing (48).

Systemic disorders

Gene transfer into primary culture has demonstrated the ability of keratinocytes to express and secrete recombinant proteins, such as the coagulation cascade factor IX (49) and growth hormone (38). Biological activity of some of these proteins has been definitively proven (7,8). This would indicate that the epidermis has a potential role as a secretory organ for systemic disorders, including serum protein deficiencies. Transgenic mouse models have shown that recombinant protein secretion into the circulation occurs from both the basal and suprabasal epidermal compartments, driven by K14 or K10 promoters, respectively (8,13).

Studies have strongly suggested that transgenes driven by foreign promoters do not show sustained transgene expressionwhen transplanted in vivo (17,38,49), but more sustained expression has been demonstrated by Wang et al. using a tissue specific K14 promoter (8). The keratin genes are expressed at very high levels in keratinocytes, keratin constituting 50-90% of the total protein content of the keratinocyte, and thus high levels could theoretically be obtained.

There are limitations to this approach, as careful modulation of gene expression is not yet possible. Conversely, it may be possible to use the epidermis as a `metabolic waste disposal unit' for diffusible toxic products in the treatment of conditions such as ornithine transcarbamylase deficiency or other metabolic disorders.

Cutaneous immunomodulation

Although current modes of in vivo gene delivery to the epidermis produce transient, low expression of exogenous gene products, these methods have been successful in stimulating the sensitive immune system present in the epidermis (50).

Genetic vaccination. Classical vaccination involves the use of either live, attenuated viral vaccines with the possibility of reversion to a virulent, pathological phenotype, or killed vaccines which elicit a less effective immune response. The dermal injection of `naked' DNA encoding an antigenic epitope has been shown to invoke an immune response, suggesting this as a possible alternative means of vaccination (51).

Ear-targeted expression of a gene encoding a bacterial antigen was shown by Lai et al. to be effective as a vaccine, producing both a humoral and cytotoxic mediated immune response (52). More recently, epidermal and dermal expression of a gene encoding a mutant p53 peptide sequence was also found to elicit a cellular immune response, albeit to a lesser extent than direct delivery of the peptide itself (24). Interestingly, in this study the sequence encoding the adenovirus E3 leader peptide was used to facilitate endoplasmic reticulum targeting of the gene product and loading of MHC class I with the peptide.

A recent novel approach to vaccination indicated that the transfer of human alpha-1 antitrypsin mRNA directly to the epidermis results in translation of a protein capable of eliciting a humoral immune response. The use of mRNA provides short term gene expression and reduces the possibility of insertional mutagenesis (53).

This body of genetic vaccination work could have significant implications for the use of epidermal gene expression in the correction of inherited skin disease and systemic disorders, suggesting as it does, that any epidermal-targeted gene product can elicit an immune response. In the clinical setting, this would suggest that diseases due to null mutations are less likely to be amenable to supplementive gene therapy.

Subcutaneous cancer models. As most malignancies arise in immuno-competent patients, tumour cells must develop stratagems for evading the host immune system. As clinically effective immunotherapy is hindered by the toxic effects of systemic delivery, the production of a local anti-tumourigenic immune response is a plausible alternative.

Interleukin(IL)-8 is naturally chemotactic for neutrophils. Hengge et al. intra-dermally injected the gene encoding IL-8 into porcine skin and found a functional response within 4 h. IL-8 expressed in the epidermis produced a neutrophil chemotactic response in the underlying dermis equivalent to 30 ng of recombinant IL-8 (23). Extrapolating this strategy from functional assays to effective treatment modalities, Sun et al. expressed cytokines with known anti-tumour effects, including IL-2, IL-6 and tumour necrosis factors [alpha] and [gamma] in the epidermis, overlying early onset subcutaneously implanted tumours (27). Cytokine-specific tumour regression and increased mouse survival was demonstrated.

Alternatively, Rakmilevich et al. recently focused on well established primary and metatastic murine tumours using an expression construct encoding the gene for both the p35 and p40 subunits of IL-12, a stimulator of natural killer cells and promoter of cytotoxic T cell maturation (26). The epidermis overlying and surrounding five subcutaneously established tumour types was treated by particle bombardment. Immunohistological techniques indicated that IL-12 expression was limited to the epidermis and not within the tumour. Tumour-specific regression or transient growth suppression was observed. Subsequent re-challenge with tumour cells indicated that a tumour-specific immunological response had been elicited in the previously treated mice.

Squamous cell carcinoma

Squamous cell carcinoma, consisting of transformed keratinocytes, is a common skin malignancy. A recent study by O'Malley et al. showed that by adenoviral-mediated transfer of a sequence encoding the herpes simplex thymidine kinase `suicide' gene, followed by treatment with gancyclovir, regression of squamous head and neck tumours established in nude mice could be achieved (54). Similarly, adenoviral delivery of the wild-type p53 gene also caused regression of these tumours as a consequence of apoptosis (55). An in vivo liposome-mediated approach using the herpes simplex thymidine kinase/gancyclovir format has also shown a tumour growth suppressive, but not regressive, effect (34).

Williams et al. produced transgenic mice which constitutively expressed B7-1(CD80) on keratinocytes (56). CD80 is a co-stimulatory molecule with an anti-tumour effect through augmentation of cytotoxic T cell responses to tumour antigens (57). They showed that keratinocyte-directed expression of CD80 was not effective in preventing chemical induction of benign tumour formation or malignant transformation.

Novel tumour-specific adenoviral vectors are also being developed in which the adenoviral E1B protein is deleted. During infection of normal cells this protein binds and inactivates cellular p53, preventing cell death by apoptosis. Adenoviral vectors deleted for E1B can only survive in cells with no functioning p53 protein, as is the case in the majority of human cancer cells. This deletion, therefore, renders the adenovirus tumour-cell specific. These exciting new mutant adenoviral vectors are being used in clinical trials in patients with p53- squamous cell carcinomas of the head and neck (58).

Melanoma

Due to its accessibility in the skin, melanoma is commonly used as a model for tumour-directed gene therapeutic treatments. A whole spectrum of modalities has been attempted, including direct injection of vaccinia (59) and adenoviruses encoding cytokines (60), genetically modified fibroblasts (61) and tumour cells (62). In the future there may also be the possibility of melanocyte-specific gene expression by tyrosinase gene regulatory elements (63).

FUTURE DEVELOPMENTS

As this review indicates, the epidermis has great potential in a range of gene therapy applications, but there are many limitations to be overcome before its full potential can be realised.

With our present knowledge, and with the new generation of viral vectors being developed, it is evident that the most success to be gained in epidermal gene transfer lies in the local delivery of exogenous gene products for vaccination purposes and in the treatment of cancer. The future is promising in these fields as they require only transient localised gene expression eliciting an attenuated immunological response.

The biggest hurdle in the use of the epidermis as an organ for gene therapy is the lack of a highly efficient in vivo gene transfer method producing high level, prolonged gene expression. There is a need to develop vectors with high efficacy and safety, with low cost and ease of handling. Current transgenic and in vivo studies indicate that our knowledge of epidermal-specific gene regulatory sequences is insufficient to provide gene expression with a systemic therapeutic role. In the future, as research focuses on keratinocyte biology, epidermal-specific gene regulatory sequences and the molecular aspects of disease pathogenesis, it may be possible to attain higher levels of epidermal gene expression or to target expression to specific cell types.

In conclusion, it is evident that gene therapy via the epidermis will complement more conventional therapies and become an accepted clinical treatment modality.

ACKNOWLEDGEMENTS

We would like to thank Dr Rosemary Akhurst for her helpful comments on the manuscript and Dr M. Fallowfield for the human skin figure. The work carried out in the authors' laboratory was supported by grants from the Wellcome Trust and Medical Research Council.

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*To whom correspondence should be addressed: Tel: +44 141 201 0365; Fax: +44 141 357 4277; Email: gpva10@udcf.gla.ac.uk
+Present address: Department of Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, UK

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