Summary. The rapid advances associated with the
Human Genome Project combined with the development
of proteomics technology set the bases to face the
challenge of human gene therapy. Different strategies
must be evaluated based on the genetic defect to be
corrected. Therefore, the re-expression of the normal
counterpart should be sufficient to reverse phenotype in
single-gene inherited disorders. A growing number of
candidate diseases are being evaluated since the ADA
deficiency was selected for the first approved human
gene therapy trial (Blaese et al., 1995). To cite some of
them: sickle cell anemia, hemophilia, inherited immune
deficiencies, hyper-cholesterolemia and cystic fibrosis.
The approach does not seem to be so straightforward
when a polygenic disorder is going to be treated. Many
human traits like diabetes, hypertension, inflammatory
diseases and cancer, appear to be due to the combined
action of several genes and environment. For instance,
several wizard gene therapy strategies have recently
been proposed for cancer treatment, including the
stimulation of the immune system of the patient (Xue et
al., 2005), the targeting of particular signalling pathways
to selectively kill cancer cells (Westphal and Melchner,
2002) and the modulation of the interactions with the
stroma and the vasculature (Liotta, 2001; Liotta and
Key words: Skin, Gene therapy, Tissue engineeering
The skin constitutes a tempting target for gene
transfer as it is the most accessible tissue of the body and
it is possible to monitor the genetically modified area
and replace the tissue in case of adverse side effects.
Skin cells are easy to obtain and expand in vitro from a
small skin biopsy. Moreover, extraordinary advances
have recently been accomplished in the development of
tissue-engineered skin equivalents for the finest and
permanent skin regeneration in clinics (Pellegrini et al.,
1999; Ronfard et al., 2000; Llames et al., 2004). Finally,
the epidermal stem cell compartment, the required target
for any permanent gene therapy strategy in the skin, has
been shown to be efficiently modified using different
integrative vectors (Levy et al., 1998; Serrano et al.,
2003; Del Rio et al., 2004). These approaches provide
evidence for the rationale that corrective gene transfer is
feasible and constitutes a starting point for further
refinement and development of future in vivo and ex vivo
genetic therapies at preclinical and clinical levels (Fig.
1). Thus, recent major progress has been made to correct
genodermatoses. Cutaneous gene therapy is attractive
not only for the correction of skin diseases but also
because the epidermis can be used as a “bioreactor” to
deliver a therapeutic protein to the systemic circulation.
It has been proved that genetically modified human
keratinocytes grafted to immunodeficient mice are able
to act as a source of systemic proteins such as growth
hormone (Teumer et al., 1990), factor IX (Gerrard et al.,
1993) and leptin (Larcher et al., 2001). A major effort is
now being directed to optimise critical issues such as
long-term persistence of adequate therapeutic serum
levels of the modified keratinocyte-derived proteins
(Larcher unpublished data).
Two different gene delivery methods can be used to
achieve the genetic modification of skin: ex vivo and in
vivo. The ex vivo approach consists of the isolation and
in vitro propagation of skin cells (either keratinocytes or
fibroblasts or both). Then, cells are genetically modified
before grafting them back to the patient as part of a
tissue-engineered skin equivalent. In the in vivo setting,
direct gene transfer is accomplished either through the
delivery of plasmidic DNA (by direct injection, biolistic
“gene gun” or electroporation) or using viral vectors
(such as lentivirus, retrovirus and adenovirus).
Skin gene therapy for acquired and inherited disorders
M. Carretero1, M.J. Escámez1, F. Prada2, I. Mirones1, M. García1,
A. Holguín1, B. Duarte1, O. Podhajcer2, J.L. Jorcano1, F. Larcher1and M. Del Río1
1Regenerative Medicine Unit and Cutaneous Diseases Modeling Unit, Epithelial Biomedicine Division, Basic Research Department,
CIEMAT, Madrid, Spain and
2Leloir Institute, University of Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
Histol Histopathol (2006) 21: 1233-1247
Offprint requests to: Dr. Marcela del Río, Regenerative Medicine Unit,
Epithelial Biomedicine Division, Basic Research Department, CIEMAT,
Av. Complutense 22 edificio 7, 28040 Madrid, Spain. e-mail:
M.C. and M.J.E. have contributed equally to this manuscript.
Cellular and Molecular Biology
Some critical issues must be considered for the
adequate choice of the delivery system. Transient gene
expression would be desirable for applications such as
regenerative therapies. In this case, the use of non-
integrative vectors such as replication-deficient
recombinant adenoviruses appears to be the best option.
They have been successfully used in in vivo gene
transfer to skin by subcutaneous injection. The broad
tropism of adenoviral vectors allows for keratinocytes,
fibroblasts, smooth muscle cells, sebaceous cells and
adipocytes to be modified using this strategy (Setoguchi
et al., 1994). Adenovirus vectors are easy to produce in
high titers, transduce both proliferating and quiescent
cells and do not integrate in the genome of the
transduced cells. Nonviral vectors avoid potential safety
problems associated with viral vehicles, but they show
extremely low gene delivery efficacy. Electroporation
and gene-gun methodologies as well as synthetic vectors
such as liposomal formulations, polymers and
nanoparticles have been used to enhance the uptake of
naked DNA in tissue.
Also, novel strategies mimicking viral mechanisms
have been developed to overcome the major drawback of
these systems when aimed at permanent correction of a
given inherited disorder, which is their episomal nature
that precludes long-term expression of the transgene. To
achieve safe and stable integration, gene transfer based
on the φC31 integrase methodology (Ortiz-Urda et al.,
2002), on transposons (Ohlfest et al., 2005) and more
recently on human artificial chromosomes (Mecklenbeck
et al., 2002) are currently being explored at preclinical
As mentioned above, permanent gene expression in
the epidermis requires the efficient targeting of the stem
cell population. Ex vivo gene transfer using
oncoretroviral vectors (i.e. Moloney-based vectors) has
been efficiently used in pre-clinical studies for this
purpose (Del Rio et al., 2002). In this setting,
proliferation of the target cells is a mandatory requisite.
It has been hypothesized that under regular culture
conditions a proportion of epidermal stem cells may not
be induced to proliferate. Thus, a number of “dormant”
stem cells may not be transduced using oncoretroviral
vectors. Lentiviral vectors (HIV-based vectors) which
are able to infect both proliferating and quiescent human
keratinocytes may represent a promising strategy for
genodermatoses such as epidermolysis in which the
number of stem cells may be limited (Kuhn et al., 2002;
Serrano et al., 2003). Retroviral and lentiviral vectors
integrate randomly in the genome of their target cells, a
characteristic that has recently generated significant
safety concerns, particularly in applications involving
genetic modification of stem cells (Cavazzana-Calvo et
al., 2000). Major efforts are now being conducted to
clarify whether the important side effects observed in an
otherwise successful gene therapy trial using
hematopoietic stem cells (Hacein-Bey-Abina et al.,
2003) are specifically linked to that protocol or,
alternatively, we are in the presence of a general
phenomenon that may affect other tissue stem cells,
other gene products and other transduction and grafting
protocols. Last October, after a three year halt due to the
side effects reported in the above mentioned
hematopoietic gene therapy trial, a phase I-II trial of
Skin gene therapy
Fig. 1. Schematic diagram
showing the applicability of
human skin equivalents in
cutaneous gene therapy.
cutaneous gene therapy for laminin 5-deficiency (JEB)
based on autologous transplantation of epidermal stem
cells transduced by an oncoretroviral vector was re-
established in Italy (M De Luca and F Mavilio, personal
communication). The results of this study will be critical
to unravel some of these concerns.
Adeno-associated virus vectors (AAV) can
efficiently transduce nondividing cells and do not elicit a
significant inflammatory response. AAV vectors can
ensure long lasting expression of the therapeutic gene
with low risk of insertional oncogenesis. A new
technology based on large-capacity DNA vectors
capable of site-specific integration with lower risk for
insertional oncogenesis has recently been described
(Recchia et al., 2004). However, several technical
improvements have to be accomplished before these
vectors can be widely used in gene therapy protocols.
Vector design to achieve controlled transgene
expression in the skin through inducible promoters
points in that direction. For example, a growth factor
inducible element (FiRE) has been shown to target
transgene expression to cutaneous wound edge
keratinocytes (Jaakkola et al., 2000). Pharmacologically
regulated systems are of great interest for skin gene
therapy, as modulation could be accomplished by simple
topical application. Progress has been made in
transcription systems induced by tetracycline,
rapamycin, RU-486, steroid hormone and antibiotics
(reviewed in Toniatti et al., 2004).
Among the inherited skin diseases, monogenic
recessive disorders are the best candidate diseases to be
treated by reintroducing a single normal copy of the
gene into the keratinocyte. Some examples are recessive
disorders such as Xeroderma Pigmentosum, X-linked
Ichtyosis and Junctional and Dystrophic forms of
Epidermolysis bullosa (Uitto and Pulkkinen, 2000;
Spirito et al., 2001; Del Rio et al., 2004). An open
question is how to circumvent a potential immune
response against the foreign therapeutic protein in the
host, in particular in patients carrying null-mutations.
Large spontaneous immunocompetent animal models of
some genodermatoses are now available and will be
critical for accurate validation of cutaneous gene therapy
strategies (Capt et al., 2005; Magnol et al., 2005; Spirito
et al., 2006). Other skin disorders present a dominant
negative mutation resulting in an aberrant protein that
affects the function of its normal counterpart, such as
Epidermolysis Bullosa Simplex (EBS) and the recessive
form of Dystrophic Epidermolysis Bullosa (DEB) (Uitto
and Richard, 2005). In this case, treatment should aim at
suppressing the expression of the mutated gene or
ameliorate the disease by controlled overexpression of
the normal gene product. Most research efforts towards
cutaneous gene therapy have been conducted to treat
Epidermolysis bullosa (EB). The robust basic and
preclinical data gathered during the past years have
recently allowed the launching of a phase I-II clinical
Epidermolysis bullosa (EB) is a group of inherited
mechano-bullous skin diseases which results from
defects affecting either keratin cytoskeleton or proteins
involved in dermo-epidermal adhesion (Pulkkinen and
Skin gene therapy
Fig. 2. Molecular
defects in the
different forms of
Bullosa (JEB) is
such as integrins or
Bullosa (DEB) is
caused by genetic
mutations in the gene
Transmission electron microscopy images show the blistering defect in the epidermal-dermal adherence in each case. HD, hemidesmosome; LD,
lamina densa; LL, lamina lucida; AFl, anchoring filaments; AFb, anchoring fibrils; KIF, keratinocyte intermediate filaments. Electron microscopy pictures
were kindly provided by Dr. Y. Gache, Dr. F. Spirito and Dr. G. Meneguzzi from INSERM U634, Faculty of Medicine, Nice, France.
Uitto, 1999; Fine et al., 2000; Uitto and Richard, 2004;
2005) (Fig. 2). The clinical phenotype is associated with
skin fragility, blisters, and erosion over trauma-prone
parts of the body that may be aggravated by infection,
usually during the first year of life. EB could also be
associated with a significant extracutaneous multiorgan
involvement leading to impaired growth and anaemia.
To date, medical care of EB patients is extremely limited
and major research efforts towards keratinocytes-based
gene therapy have been made for these disorders (Uitto
and Pulkkinen, 2000; Spirito et al., 2001; Del Rio et al.,
EB is classified into 3 major categories which differ
in the affected gene and therefore in the level at which
lesions split (Uitto and Richard, 2004, 2005).
Epidermolysis Bullosa Simplex (EBS), is an
autosomal dominant disorder produced by mutations in
the basal keratins K5 or K14 genes compromising the
keratin cytoskeleton which is no longer providing the
resistance stress needed to keep the mechanical stability
of the epidermis. The genetic correction of this disease is
complex due to the dominant-negative action of the
mutant protein. Gene therapy approaches should aim at
suppressing the expression of the mutated copy of the
gene. However, an alternative approach has been
proposed by Lane´s laboratory consisting of reinforcing
the weakened intermediate filament cytoskeleton of EBS
keratinocytes by providing an additional intermediate
filament network of human desmin, which plays a
similar role to the keratins in muscle cells (Magin et al.,
2000; D'Alessandro et al., 2004). By retroviral gene
transfer of human desmin into EBS keratinocytes, this
group reported the formation of a well defined desmin
filament network, independent of the endogenous keratin
cytoskeleton which is not susceptible to the negative
effect of the mutant keratin. These findings represent a
promising result for the treatment of EBS disorders.
Junctional Epidermolysis Bullosa (JEB) is an
autosomal recessive inherited disease produced by
mutations in 6 different genes encoding 3 components of
hemidesmosomes. The 3 chains that constitute the
trimeric laminin 5 (LAMA3, LAMB3 or LAMC2),
BPAG2/type XVII collagen (COL17A1) and α6/ß4
integrins (ITGA6 and ITGB4) could be affected. The
hemidesmosomes are multiprotein complexes linking the
epithelial intermediate filament network to the dermal
anchoring fibrils, thus maintaining the integrity of
integument in humans (McGrath et al., 1995; Vidal et
al., 1995; Fine et al., 2000).
The initial efforts at genetic correction of EB were
performed using keratinocytes of patients affected by the
severe form of JEB associated with lack of expression of
one of the 3 chains of laminin-5 heterotrimer. The
restoration of laminin 5 expression has been achieved in
primary JEB keratinocytes using either retroviral vectors
(Dellambra et al., 1998; Vailly et al., 1998; Robbins et
al., 2001) or non viral stable approaches mediated by the
φC31 integrase (Ortiz-Urda et al., 2003a) or the
transposon systems (Ortiz-Urda et al., 2003b). As
mentioned before, the first trial for JEB using an
oncoretroviral vector encoding laminin ß3 chain was
started in 2002 but was put on hold after the adverse
effect observed in the X-linkend SCID trial and was
restarted last October (DeLuca and Mavilio, personal
Similarly, re-expression of type XVII collagen as
well as integrin ß4 has been accomplished by retroviral
gene transfer to primary keratinocytes isolated from
collagen XVII-null (Seitz et al., 1999) and ß4-null
(Dellambra et al., 2001) patients, respectively.
The recovered expression of laminin 5 subunits, type
XVII collagen or integrin ß4, in keratinocytes from JEB
patients restored cell adhesion and colony forming
ability without affecting their polarity and differentiation
potential. JEB corrected cells were used to generate
either skin equivalents in vitro or human skin in vivo
after transplantation on immunodeficient mice.
Genetically modified keratinocytes successfully
assemble normal hemidesmosomal structures and correct
the major hallmarks of the disease.
Taken together these achievements have
demonstrated the feasibility of phenotypic reversion of
adhesion-defective disorders by keratinocyte-based gene
It is noteworthy, however, that although in vitro
reconstructed skin and specially regenerated skin on
immunodeficient mice are a valuable model to test gene
therapy approaches, they are unable to recapitulate the
host immune response. The identification of breeds of
dogs with an inherited form of JEB provides the unique
opportunity to verify the feasibility of the strategy in an
immunocompetent environment. The genetic defect of
dog keratinocytes has already been successfully
corrected by retroviral gene transfer (laminin α3)
(Spirito et al., 2006). Transplantation of autologous
genetically modified dog skin equivalents are expected
to provide information on the host immunoreaction and
on persistence of the therapeutic transgene.
Dystrophic Epidermolysis Bullosa (DEB) is an
autosomal dominant or recessive inherited disorder
caused by genetic mutations in the gene (COL7A1)
encoding collagen type VII, a large basement component
which plays a critical role in epidermal-dermal
adherence (Franzke et al., 2005). DEB is an untreatable
condition characterized by unremitting blistering of the
skin and mucosa, contractures and dystrophic scarring of
the lesions. In the most severe form of the disorder
(Hallopeau-Siemens recessive DEB), the lesions
progress with time to invasive squamous cell carcinoma.
Different groups have recently explored the feasibility of
a permanent correction of RDEB primary human
keratinocytes by ex vivo gene transfer. Promising results
in animal models have been reported.
Ortiz-Urda and co-workers have used a φC31
integrase-based gene transfer system that stably
integrated the COL7A1 cDNA into RDEB keratinocytes
and fibroblasts isolated from patients (Ortiz-Urda et al.,
2002). Chen and co-workers have achieved efficient
Skin gene therapy
gene transfer using a self-inactivating lentiviral vector in
immortalized RDEB keratinocytes (Chen et al., 2002).
However, the exceedingly low transfer efficiency of non-
viral vectors and the absence of suitable packaging cell
lines for lentiviral vectors have hampered their use in
clinical trials. Alternatively, the use of Murine Leukemia
Virus (MuLV) based retroviral vectors are, at present, the
most convenient and widespread tool for ex-vivo gene
transfer of therapeutic genes in the context of clinical
trials. Recently, Meneguzzi and co-workers have
demonstrated that human primary RDEB keratinocytes,
transduced with a retroviral vector expressing the human
type VII collagen cDNA, were able to generate
transplantable tissue-engineering skin equivalents
(Gache et al., 2004).
When RDEB-null keratinocytes were grafted onto
the back of immunodeficient mice, the regenerated
human skin exhibited fragile epidermal-dermal
adherence and blistering, absence of type VII collagen
protein, and no detectable anchoring fibrils. In contrast,
and regardless of the viral vector used, all these
hallmarks were recovered after grafting genetically
corrected REDB cells. Two facts are noteworthy in these
studies. First, durable normalized col VII expression was
achieved (Chen et al., 2002). Second, ex vivo correction
of DEB was accomplished in a clinical setting by the use
of a new skin substitute successfully applied in burn
patients (Gache et al., 2004; Llames et al., 2004).
Finally, retroviral vectors have also been demonstrated
to mediate efficient transfer of dog collagen Tipe VII
cDNA into primary RDEB keratinocytes (Baldeschi et
al., 2003). The transduced cells fully reverted the RDEB
phenotype in vitro, which set the basis for preclinical
studies of RDEB gene therapy using a large
immunocompetent animal model.
Keratinocytes account for the majority of the
deposited collagen at the DEJ in human skin. However,
it has been proved that type VII collagen secreted solely
by normal (Ortiz-Urda et al., 2003c; Woodley et al.,
2003) or ex vivo corrected dermal fibroblasts (Ortiz-
Urda et al., 2003c) is enough to restore the DEJ after
being grafted to immunodeficient mice. Moreover, the
effect of direct intradermal injection of normal human
fibroblasts or gene corrected RDEB fibroblasts
(lentiviral vectors) into a RDEB human skin substitute
on nude mice has also been tested. Remarkably, the type
VII collagen synthesized and secreted by these
exogenously injected cells (for up to three months), is
precisely localized at the dermo-epidermal junction
forming anchoring fibrils probably due to the great
affinity of laminin-5 for this molecule.
In vivo gene transfer approaches have also been
tested. Woodley et al. have been able to correct RDEB
by a single intradermal injection of a lentiviral vector,
encoding the type VII collagen transgene, into a human
RDEB skin regenerated on immunodeficient mice by
providing stable type VII collagen at the basal
membrane for at least 3 months (Woodley et al., 2004a).
Based on the stability of the type VII collagen, this
group have proposed an even more simple strategy based
on protein therapy, proving that intra-dermal injection of
recombinant type VII collagen into a human RDEB skin
regenerated on immunodeficient mice can correct the
defect (Woodley et al., 2004b).
Xeroderma pigmentosum is an autosomal recessive
disease characterized by increased sensitivity to
ultraviolet light that leads to cutaneous and ocular
abnormalities. Patients are prone to develop cutaneous
basal and squamous cell carcinomas and melanomas.
About 30% of individuals also present neurological
disorders although these may become apparent later than
the cutaneous symptoms, which usually appear at one to
two years of age.
Mutations in different genes (XPA, XPB/ERCC3,
XPC, XPD/ERCC2, XPE/UV-DDB/DDB2, XPF/
ERCC4, XPG/ERCC5 and XPV/POLH) define the
seven complementation groups (XP group A to G) and
the variant group XP-V to which patients are assigned. A
defect in the nucleotide excision repair (NER)
machinery is observed in cells from patients belonging
to complementation groups A through G. Differently, the
XP variant cells show normal excision repair but instead
suffer from error-prone post-replication translesion
synthesis. In addition, there is a range of symptom
manifestations among individuals within any group.
Management of the disease is limited to strict
avoidance of sunlight exposure. One innovative
treatment for these patients is the topical application of
active enzymes in liposome formulations (bacteriophage
T4 endonuclease T4N5 and photolyase) (Kraemer and
DiGiovanna, 2002). The activity of these enzymes
results in the removal of lesions in the DNA. Treatment
of 30 patients (T4 endonuclease V) for 1 year resulted in
significant decrease in the onset of pre-cancerous lesions
without apparent immune reactions (Yarosh et al., 2001).
As for other genodermatoses, long-term effective
correction of XP skin can be approached through
cutaneous gene therapy. Early experiments, using XP
fibroblasts and integrative vectors encoding appropriate
wild-type genes, have shown stable complementation of
the cells and recovery of DNA repair (Carreau et al.,
1995; Zeng et al., 1997). However, gene delivery efforts
should be directed to the significant players of skin
cancer in these patients, such as keratinocytes, the cells
from which basal and squamous cell carcinoma
originate. The first steps towards XP gene therapy have
recently been achieved. Genetic correction of DNA
repair-deficient XPC keratinocytes has been
accomplished using a retroviral transduction strategy. In
this setting, reexpression of the wild-type XPC protein
resulted in restoration of normal DNA repair following
UVB irradiation (Arnaudeau-Begard et al., 2003). In
addition, an adenoviral vector carrying the XPA gene
used for in vivo gene delivery to the skin of XPA-
knockout mice led to the prevention of deleterious
Skin gene therapy
effects in the skin, including development of squamous
cell carcinoma (Marchetto et al., 2004). However, as for
other inherited skin disorders, permanent gene delivery
to the epidermal stem cell compartment through ex-vivo
gene transfer strategies appears as the most realistic and
clinically relevant approach. The latest in vitro
development of human XP skin equivalents (Bernerd et
al., 2001; Arnaudeau-Begard et al., 2003) is the basis for
imminent preclinical trials. In vivo regeneration of XP
skin onto immunodeficient mice is envisioned as a
unique humanized model suitable to validate an
adequate gene therapy approach to treat this disorder, as
previously performed for other genodermatosis.
Gene therapy to improve wound healing
Non-healing acute and chronic wounds are a diverse
group of diseases of different aetiology and
manifestation. The most common cause of acute wounds
is thermal injury. Chronic wounds include arterial,
diabetic, pressure and venous ulcers and cause
significant morbidity and impaired quality of life. The
comprehension of the tissue repair process and its failure
has been essential for the development of new
therapeutic approaches. The analysis of genetically
modified mouse models has been particularly important
to understand the role of individual genes (Scheid et al.,
2000). The first event in the normal wound repair
process is the immediate activation of the coagulation
cascade followed by an acute inflammatory response.
Leucocytes clear the wound and release growth factors
that induce the formation of a temporary granulation
tissue. The provisional matrix provides a scaffold that
allows extracellular matrix remodelling, angiogenesis
and re-epithelialization (Clark, 1996; Martin, 1997;
Singer and Clark, 1999). Growth factors and cytokines
orchestrate all these events to perform tissue repair
(Werner and Grose, 2003).
Clinicians realize the difficult task involved in the
management of impaired wound healing associated with
chronic and acute wounds. A conventional treatment
consists of transplantation of epidermal sheets (Gallico
et al., 1984; De Luca et al., 1989). An improved strategy
is based on the grafting of skin substitutes, resembling
native human skin as closely as possible, and whose
clinical efficacy has already been reported by different
groups, including ours (Hansbrough et al., 1989;
Falanga, 1998; Chang et al., 2000; Llames et al., 2004,
2006). Different growth factors and their receptors such
as PDGF (Reuterdahl et al., 1993), VEGF (Frank et al.,
1995; Nissen et al., 1998), FGF (Vogt et al., 1998), and
KGF (Marchese et al., 1995) appear to play a critical
role in wound repair. Since defects in their expression
may impair wound healing, many groups have been
interested in testing them as candidate genes for gene
therapy. The use of growth factors such as recombinant
proteins in clinical assays to improve wound healing has
been discouraging, probably due to their rapid
degradation in the wound environment (Falanga, 1993;
Meyer-Ingold, 1993; Lauer et al., 2000; Werner and
Grose, 2003). After a first report showing that the
expression of human epidermal growth factor mediated
by gene gun transfer accelerated wound repair in porcine
and mouse models (Andree et al., 1994), the idea that
gene therapy may also contribute to the therapeutics has
Two applications can be easily distinguished for
cutaneous gene therapy aimed at wound handling. One is
the ex-vivo gene transfer of healing promoting genes to
keratinocytes combined with either autologous or
allogenic skin equivalent transplantation. The other is
the in vivo gene transfer of these factors directly to the
wound site using vectors capable of achieving high
levels of protein expression and transducing both
proliferating and quiescent cells (Sylvester et al., 2000;
Gruss et al., 2003).
Platelet derived growth factor (PDGF) is produced
locally by most of the cells at the site of injury
promoting the granulation process (Heldin and
Westermark, 1999; Werner and Grose, 2003). Reduced
expression of PDGF and its receptors has been
associated with impaired cutaneous wound healing in
mice (Beer et al., 1997; Gao et al., 2005) and in vivo
gene transfer of both PDGF A and B cDNA by direct
particle bombardment of wounds promotes wound repair
in rats (Eming et al., 1999). PDGF-A overexpression
mediated by retroviral gene transfer improves graft
performance during the first critical week after
transplantation due to the acceleration of graft invasion
by fibrovascular cells and the deposition of collagen
(Eming et al., 1995, 1998). In addition, the effect of
PDGF-B is dramatically enhanced when it is produced
from an adenoviral vector (PDGF-B/Ad5) in three
different models: diabetic mice, ischemic rabbit ear
(Liechty et al., 1999a, 1999b) and human skin substitute
regenerated on immunodeficient mice (Sylvester et al.,
2000). In addition, topical administration of recombinant
PDGF–BB protein, which was the first growth factor
approved by FDA (becaplermin gel; Regranex®,
Janssen-Cilag Ltd), has already been shown to be safe
and effective in treating human chronic wounds
(Margolis et al., 2005). These encouraging results have
promoted the first gene therapy clinical trial for the
treatment of chronic ulcers using an adenoviral vector
encoding human PDGF (PDGF/Ad5) (NIAMS-044; N01
AR-9-2238; Margolis et al., 2000, 2004)
Vascular endothelial growth factor (VEGF) has been
explored as a candidate to induce angiogenesis since its
expression is enhanced in keratinocytes at the wound site
and induces potent proliferation and migration of
endothelial cells (Nissen et al., 1998, 2003). The
impaired wound healing in the diabetic mouse model has
been related to a reduction of VEGF expression (Frank
et al., 1995; Lauer et al., 2000; Altavilla et al., 2001). We
and others have also found that VEGF overexpression in
Skin gene therapy
the skin of transgenic mice increases vascularization
(Detmar et al., 1998; Larcher et al., 1998). Thus, our lab
undertook grafting of ex vivo gene transferred pig
primary keratinocytes overexpressing VEGF (through
lipid mediated cDNA transfection) to nude mice as part
of a transplantable skin substitute. The result of this
strategy was a dramatic increase in the number of blood
vessels in the host stroma (Del Rio et al., 1999).
Similarly, Supp and co-workers using human
keratinocytes overexpressing VEGF through retroviral
gene transfer, have also found an improvement in graft
performance (Supp et al., 2000a,b; Supp and Boyce,
2002). On the other hand, in vivo VEGF delivered either
by means of an adenoviral vector or an adeno-associated
viral vector has shown improved angiogenic response
and has subsequently enhanced the overall wound
healing process in different animal models of impaired
wound repair (Deodato et al., 2002; Romano Di Peppe et
al., 2002; Galeano et al., 2003a,b). Preliminary results
from our lab indicate that the use of VEGF-producing
autologous keratinocytes, as a part of a transplantable
skin substitute, improves stable engraftment and tissue
regeneration quality in a porcine immunocompetent
model (Garcia et al., 2003). Together, these results
warrant additional studies to evaluate the clinical
usefulness of VEGF gene transfer to improve skin
The potential value in the acceleration of re-
epithelialization of keratinocyte growth factor (KGF), a
well known growth factor playing an important role
during cutaneous injury, has been explored (Werner,
1998; Werner and Grose, 2003). Expression of KGF by
fibroblasts is stimulated during normal wound healing,
and its expression is significantly reduced and retarded
in diabetic and glucocorticoid treated mice (Werner et
al., 1992, 1994a; Brauchle et al., 1995). In addition,
dominant negative KGF receptor transgenic mice are
characterized by a severe delay in wound re-
epithelialization (Werner et al., 1994b). Preclinical data
from animal models demonstrated that KGF stimulates
both epithelialization and granulation tissue (Staiano-
Coico et al., 1993; Pierce et al., 1994; Andreadis et al.,
2001). Interestingly, KGF also stimulates capillary
endothelial cells together with endothelial barrier
stabilization (Gillis et al., 1999). Additionally, KGF gene
transfer demonstrated a beneficial effect on wound
healing in different animal models. In a pre-clinical
wound healing model based on the transplantation of ex
vivo modified skin equivalents to analyse graft take, as a
wound healing end point (7-15 days post-
transplantation), KGF overexpressing human
keratinocytes were able to demonstrate beneficial effects
on early graft performance in immunodeficient mice
(Erdag et al., 2004). On the other hand, enhanced wound
healing has also been reported after in vivo gene transfer
of KGF to excisional wounds of diabetic mouse (naked
DNA injection with subsequent electroporation) (Marti
et al., 2004) and to thermally injured rats (liposomal
injection) (Jeschke et al., 2002). Finally, protein therapy
using recombinant KGF was found to reduce the
duration and severity of oral mucositis in a phase III
clinical trial (Spielberger et al., 2004). Collectively, these
results support KGF as a candidate growth factor for
Hepatocyte growth factor (HGF) is a potent mitogen
for hepatocytes but it has also been revealed as a factor
with interesting activities in cutaneous tissue repair,
having an effect on epithelial cell proliferation,
migration and morphogenesis (Brinkmann et al., 1995).
Recently, the overexpression of HGF gene by human
keratinocytes (retroviral-mediated gene transfer) after
transplantation on nude mice regenerated an epidermis
showing a transitory hyperproliferation that subsides by
2 weeks (Hamoen and Morgan, 2002). Therefore,
activated skin substitutes containing cells that express
and locally deliver HGF whose time frame action
coincides with the graft take process may be useful in
cutaneous tissue repair and wound healing. Recently, a
phase I clinical trial using HGF has demonstrated that
intramuscular injection of naked HGF plasmid achieves
clinically significant improvement of ischemic ulcers
(Morishita et al., 2004). These findings are preliminary
and do not establish the long-term safety of HGF but
encourage further studies.
Recently, we have described a novel wound healing
model based on the injury of skin-humanized mice
(Escamez et al., 2004). The main advantage of our
system is that full thickness wounds are performed in a
mature, “quiescent” regenerated human skin (9-12
weeks after grafting). This model offers the possibility to
test both in vivo and ex vivo gene transfer approaches by
using normal-control skin or stable genetically modified
human skin, respectively (Fig. 3). So far, we have
applied exogenously recombinant human KGF protein
by intradermal injection to wounds and it recapitulated
the accelerated wound closure previously reported in
other animal models. This skin-humanized mouse model
represents a useful platform to study candidate genes to
improve wound healing.
Infection is the major cause of skin graft failure in
burn patients and sepsis increases mortality rates.
Immediately after injury, gram-positive organisms
usually invade the burn wound surface. Subsequently,
more virulent gram-negative organisms may replace
them, proliferate and disseminate to underlying viable
tissues and reach the circulation. The increasing problem
of multi-drug resistant bacteria limits the use of topical
and systemic antibiotics to treat infection and results in
secondary opportunistic infections with fungal
pathogens. The most common cause of nosocomial
infection in severely burned patients is Methicillin-
resistant S. aureus (MRSA), multiple resistant P.
aeruginosa and vancomycin-resistant Enterococci
infection (Steinstraesser et al., 2004). Recent studies
focus on the use of novel compounds to treat infection
Skin gene therapy
such as naturally occurring antimicrobial peptides. They
are major components of the innate defence system from
plants to vertebrates and display a broad spectrum of
microbicidal activity. Antimicrobial peptides are
attractive candidates for clinical development because
bacteria may not easily develop resistance to them.
Some of them are induced in human keratinocytes in
inflammatory conditions, such as for example ß-
defensins 2 and 3 (Harder et al., 2001; Nomura et al.,
2003) and the cathelicidin LL-37 (Frohm et al., 1997). A
defect in HBD-2 expression in full-thickness burn
wounds and in burn blister fluid has been previously
demonstrated. This fact might influence microbial
growth (Ortega et al., 2000).
Several biotechnological companies have been
interested in the therapeutic application of recombinant
peptides to treat infection. Some examples of the
developed products are: MBI-226 for the treatment of
central venous catheter related infections (Migenix Inc.,
Vancouver), Immucept for the treatment of nosocomial
pneumonia (Inimex Pharmaceutical Inc., Vancouver) and
P-113D for cystic fibrosis (Demegen Inc., Pittsburgh).
The high cost of scaling up a human quality recombinant
protein makes the antimicrobial peptide gene therapy
approach a promising therapeutic strategy for the
treatment of infected wounds. An adenoviral vector for
the delivery of LL-37 was previously employed to
reverse the bacterial killing defect observed in cystic
fibrosis by using a human bronchial xenograft model
(Bals et al., 1999). We have also demonstrated the
efficacy of adenoviral-mediated overexpression of
different antimicrobial peptides in preventing bacterial
growth in a human skin equivalent setting (Carretero et
al., 2004). It has also been reported that transient
adenoviral delivery of LL-37 is effective in reducing
bacterial growth in an infected rat burn model (Jacobsen
et al., 2005).
In addition to the activity of antimicrobial peptides
as natural antibiotics, other actions have been described
on eukaryotic cells that could be beneficial for wound
repair. LL-37 is upregulated in the re-epithelializing
front of human skin wounds. However, no protein
expression is detected in non-healing chronic ulcers. In
addition, inhibition studies using antibodies against LL-
37 showed a dose-dependent impaired re-
epithelialization in ex vivo wounds (Heilborn et al.,
2003). LL-37 has also been shown to induce
proliferation and migration of airway epithelial cells
(Shaykhiev et al., 2005). Growth factors, such as IGF-I
and TGF-α, as well as proinflammatory cytokines may
act as inducers of antimicrobial peptide expression in
wounded tissue (Sorensen et al., 2003). Interestingly,
mice deficient for the murine homologue CRAMP
presented a decreased vascularization during wound
Skin gene therapy
Fig. 3. Wound healing studies
in skin-humanized mice. The
experiments are performed in
regenerated skin (12 weeks
post-grafting). At day 0
excisional wounds were
performed by using 2mm
biopsy punches. The healing
response is analyzed at day 3
post-wounding (pw). A. Two
different protocols are used
for gene transfer to human
skin. Upper panel: The ex
vivo approach consists of the
in vitro genetic modification of
human keratinocytes using
retroviral vectors before
grafting them as part of a skin
immunodeficient mice. Stable
GFP expression is observed
in the entire graft. Lower
panel: The in vivo approach
consists in the direct
subcutaneous injection of
adenoviral vectors. Using this
approach only local
expression of GFP is
observed around the wound
consistent with the injection
site. B. Immunohistochemical
analyses of the reepithelialization process at different days post-wounding (pw) using an antibody directed against human involucrin. The involucrin
positive labelling indicates that the closure of the wound is performed by human keratinocytes.
repair, suggesting an important role of this peptide in
cutaneous wound neovascularization. LL-37 has been
shown to induce angiogenesis through formyl peptide
receptor-like 1 expressed on endothelial cells (Koczulla
et al., 2003). Other activities of this peptide are related to
its anti-endotoxic properties (Scott et al., 2002) and the
involvement in the enhancement of adaptive immunity
either by chemoattracting different immune cells (De et
al., 2000) or modulating dendritic cell differentiation
(Davidson et al., 2004). Altogether, these data support
the use of an antimicrobial peptide gene therapy for
wound repair, as it might control the spread of infection
as well as contribute positively to tissue regeneration by
acting at different stages of the wound healing process.
Antitumoral gene therapy to treat melanoma
Cancer is commonly defined as an illness where
multiple genes and environmental factors are interacting
in a complex way to give rise to the disease phenotype.
Moreover, tumor generation is a multistep process, in
which successive genetic changes, each conferring a
growth advantage, leads to the progressive
transformation of normal cells into highly malignant
derivatives. These changes range from deregulated
proliferation and limitless replicative potential to tissue
invasion and metastasis (Hanahan and Weinberg, 2000).
Melanoma is a very severe form of skin cancer that
begins in melanocytes. Although melanoma accounts for
only about 4% of all skin cancer cases, it causes most
skin cancer-related deaths (Hanahan and Weinberg,
2000). Current treatment of malignant melanoma using
chemotherapy or high-dose of interferon and IL-2 is
ineffective and associated with a high cost and toxicity
(Meric et al., 2003).
The recent advances in our understanding of the
functional genomics and molecular abnormalities
underlying the progression of malignant melanoma, the
identification of melanoma specific tumor antigens and
the easy accessibility to tumor lesions have brought to
the clinical trial arena the use of gene therapy as a
promising strategy against this disease (Rosenberg,
1999; Hengge, 2001).
The canonical ways to targeted gene therapy aimed
at melanoma cells themselves were the introduction of
suicide genes, the expression of tumor suppressor genes,
the inactivation of oncogenic signalling pathways and
the introduction of genes encoding immunologically
relevant molecules. So far, limited positive results have
been reported in previous clinical trials. However, these
studies have shown the feasibility and safety of this
approach (Sotomayor et al., 2002).
Although these strategies directed to kill cancer cells
themselves or inhibit their proliferation have been the
focus of oncological treatments, recent advances have
proved that the tumor environment is actively involved
in the development of cancer. Tumor progression results
from an imbalanced interaction of tumor cells with host
cells and the extracellular matrix (ECM), in which
neoplastic cells recruit vasculature and stroma through
production and secretion of stimulatory growth factors
and cytokines, while host-activated cells and ECM
modify the proliferative and invasive behavior of tumor
cells (Liotta, 2001; Liotta and Kohn, 2001).
Pleiotropic molecules capable of affecting more than
one component of the tumor-stroma system will be
potential targets of gene therapy for the treatment of
An interesting example of this is the glycoprotein
SPARC (Secreted Protein Acidic and Rich in Cysteine).
This secreted molecule is expressed during
embryogenesis and tissues undergoing remodelling
(Lane and Sage, 1994). SPARC interacts with several
ECM components, binds and modulates the activity of
specific growth factors, and regulates matrix
metalloproteinase expression and activity (Bradshaw and
Sage, 2001). Moreover, SPARC expression is associated
with tissue remodelling processes, like wound healing
and angiogenesis, both of which include physiological
steps of invasive phenotypes activity (Bradshaw and
Sage, 2001). Several reports associated SPARC
expression with the invasive and metastasic capacity of
different human cancers (Ledda et al., 1997a,b; Jacob et
al., 1999; Briggs et al., 2002; Schultz et al., 2002; Rich
et al., 2003). In addition, we have demonstrated that
suppression of SPARC expression in human melanoma
cells abrogated their tumorigenic capacity (Ledda et al.,
1997b). SPARC is expressed not only by malignant cells
but also by surrounding fibroblasts, endothelial cells and
certain inflammatory cells. Interestingly, its production
by stroma cells has also been associated with the
neoplastic progression of tumors in which SPARC is
hardly detected in the malignant cells themselves
(Podhajcer et al., 1996; Brown et al., 1999; Lussier et al.,
2001; Koukourakis et al., 2003). Suppression of SPARC,
using adenoviral vectors carrying the antisense-RNA
technology, in three different human melanoma cell
lines, promoted the in vivo PMN recruitment and
rejection of tumor cells in nude mice. Suppression of
SPARC expression also promoted the rejection of
bystander non-engineered melanoma cells in vivo and
triggered the in vitro anti-tumor cytotoxic capacity of
human PMN. Overall, these results suggest that SPARC
produced by malignant cells might be involved in the
escape of tumor cells from primary anti-tumor immune
surveillance (Alvarez et al., 2005).
Another attractive case of a pleiotropic protein that
may be used for improving melanoma treatment is the
molecule called pigment epithelium-derived factor
(PEDF). Initially identified as a neuronal differentiation
factor produced by culture human retinal pigment
epithelial cells (Tombran-Tink and Barnstable, 2003),
PEDF has recently proved to be a potent inhibitor of
angiogenesis in the eye by inducing apoptosis in actively
dividing endothelial cells (Stellmach et al., 2001). The
potential role of PEDF as an antiangiogenic agent in the
context of solid tumors has recently begun to be
explored, pointing to a more widespread angioinhibitory
Skin gene therapy
role for this multifunctional factor (Crawford et al.,
2001; Abramson et al., 2003; Wang et al., 2003). Studies
in PEDF-knockout mice showed increased vessel density
in several organs, combined with marked hyperplasia of
the pancreas and prostate epithelium. Interestingly,
highly tumorigenic prostate cell lines showed reduced
PEDF expression compared with less tumorigenic ones.
Thus, it is likely that PEDF has an inhibitory effect on
prostate tumor development (Doll et al., 2003). We have
recently reported that retroviral mediated overexpression
of PEDF produced a dramatic direct growth inhibition of
primary melanoma and metastases, which contributes to
the canonical antiangiogenic effect of PEDF (Garcia et
al., 2004). Our results emphasize that PEDF is targeting
both the tumor cells and vasculature, leading to a more
efficient blockade of tumor growth than that achieved by
using purely antiangiogenic compounds.
Thus, the search for multifunctional molecules as
new putative targets that may affect several tumorigenic
pathways could be one of the main foci of scientific
investment in oncotherapy.
Aknowledgements. Our work is supported by grants SAF-2004-07717
from Ministerio de Ciencia y Tecnología (Spain) and LSHG-512073 from
UE to M. Del Rio, LSHG-503447 from UE to J.L. Jorcano and LSHG-
512102 from UE to F. Larcher. We express our gratitude to Dr. Y.
Gache, Dr. F. Spirito and Dr. G. Meneguzzi for providing EM pictures to
illustrate this work.
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Zeng L., Quilliet X., Chevallier-Lagente O., Eveno E., Sarasin A. and
Mezzina M. (1997). Retrovirus-mediated gene transfer corrects DNA
repair defect of xeroderma pigmentosum cells of complementation
groups A, B and C. Gene Ther. 4, 1077-1084.
Accepted May 22, 2006
Skin gene therapy