Download Gene delivery to the epidermis

 1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 10 Review
1761–1767
Gene delivery to the epidermis
Alison H. Trainer* and 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
*To whom correspondence should be addressed: Tel: +44 141 201 0365; Fax: +44 141 357 4277; Email: [email protected]
+Present address: Department of Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, UK
1762 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review
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.
tissue, in contrast to the more specific epidermal expression seen
in both human and porcine skin.
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 epidermalspecific 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
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Figure 2. Histological analysis of transfected mouse skins. Mouse dorsal skins were transfected with 10 µg K5-driven β-galactosidase DNA, and the skins were stained
for β-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) β-galactosidase activity in the ear of a transgenic mouse, containing a K5-driven β-galactosidase gene, with a more restricted staining pattern in the hair follicle.
e, epidermis; f, dermal fibroblast; g, hair germ; h, hair follicle.
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 ∼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
1764 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review
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 β (TGF-β).
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-β
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 expression
when 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
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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 α and γ 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 costimulatory 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.
1766 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review
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