Gene Therapy (2005) 12, 1752–1760
& 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00
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RESEARCH ARTICLE
Gene delivery to human sweat glands:
a model for cystic fibrosis gene therapy
H Lee1,2, DR Koehler1,3, CY Pang1,4,5, RH Levine5, P Ng6, DJ Palmer6, PM Quinton7 and J Hu1,2,3
1
Research Institute, The Hospital for Sick Children, Toronto, Canada; 2Institute of Medical Science, University of Toronto, Toronto,
Canada; 3Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, Canada; 4Department of Physiology,
University of Toronto, Toronto, Canada; 5Department of Surgery, University of Toronto, Toronto, Canada; 6Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX, USA; and 7Department of Pediatrics, UCSD School of Medicine University
of California, San Diego, La Jolla, CA, USA
Gene therapy vectors are mostly studied in cultured cells,
rodents, and sometimes in non-human primates, but it is
useful to test them in human tissue prior to clinical trials.
In this study, we investigated the possibility of using human
sweat glands as a model for testing cystic fibrosis (CF) gene
therapy vectors. Human sweat glands are relatively easy to
obtain from skin biopsy, and can be tested for CFTR function.
Using patients’ sweat glands could provide a safe model to
study the efficacy of CF gene therapy. As the first step to
explore using sweat glands as a model for CF gene therapy,
we examined various ex vivo gene delivery methods for a
helper-dependent adenovirus (HD-Ad) vector. Gene delivery
to sweat glands in skin organ culture was studied by topical
application, intradermal injection or submerged culture. We
found that transduction efficiency can be enhanced by
pretreating isolated sweat glands with dispase, which
suggests that the basement membrane is a critical barrier
to gene delivery by adenoviral vectors. Using this approach,
we showed that Cftr could be efficiently delivered to and
expressed by the epithelial cells of sweat glands with our
helper-dependent adenoviral vector containing cytokeratin
18 regulatory elements. Based on this study we propose that
sweat glands might be used as an alternative model to study
CF gene therapy in humans.
Gene Therapy (2005) 12, 1752–1760. doi:10.1038/
sj.gt.3302587; published online 21 July 2005
Keywords: gene delivery; Cftr; sweat glands; native tissue; helper-dependent adenovirus vector; basement membrane
Introduction
Cystic fibrosis (CF), the most common genetic disorder in
Caucasian populations, is caused by recessive mutations
in the gene encoding the cystic fibrosis transmembrane
conductance regulator (Cftr).1 CFTR is an ATP-binding
cassette (ABC) transporter that plays a critical role in salt
transport and fluid movement by controlling chloride
ion transport across epithelia.2 Dysfunction of CFTR
leads to disease in various organs including the
respiratory and digestive tracts. However, the complications in the lung caused by abnormal airway secretions,
chronic infection and inflammation pose the most serious
threat to CF patients.3 Currently, how the loss of CFTR
function causes the clinical manifestations and pathological features of CF lung disease is not fully understood,
so treatment is focused on management of respiratory
infection and inflammation in the lung.4
CF is considered one of the diseases that could be
treated by gene therapy because it is a single gene
disorder and the vector can be delivered directly to the
lung through the airway. Moreover, restoration of normal
Correspondence: Dr J Hu, Lung Biology Research Hospital for Sick
Children, 555 University Ave., Toronto, ON, Canada M5G 1X8
Received 20 March 2005; accepted 13 June 2005; published online
21 July 2005
Cftr mRNA to 5–10% could rescue the CF phenotype,5,6
although a recent review proposed that a higher level of
expression may be required at the protein level.7 In the
last decade, adenovirus, adeno-associated virus (AAV)
and nonviral vectors were developed and used in CF
gene therapy clinical trials.8 Gene therapy vectors are
mostly studied in cultured cells, rodents and other
animal models to verify their efficacy before clinical
trials. However, an appropriate animal model to study
CFTR functional correction in lung has not been achieved
because Cftr knockout mice do not show the pulmonary
pathologic features observed in human CF lung.9
Although the Cftr knockout mouse models have been
utilized to evaluate the therapeutic potential of CF gene
therapy,10–12 the lack of pathophysiology in the Cftr
knockout mouse lung limits the interpretation of experimental results from CF mouse models. Therefore, an
alternative model using a native human tissue is
critically needed to assess the efficacy of CF gene therapy.
The eccrine sweat gland (referred to as ‘sweat gland’
below) is one of the organs affected by CF. The sweat
gland is one of the smallest organs in the human body,
uniquely developed in humans to control body temperature by evaporation. It has a simple tubular structure
with only 2–3 layers of cells. Isotonic primary sweat is
produced in the lower part of the sweat gland called the
secretory coil, and, as the primary sweat moves to the
Gene delivery to human sweat glands
H Lee et al
skin surface through the sweat duct, electrolytes are
reabsorbed by the epithelial cells from the apical
membrane. CFTR plays an important role in sweat
secretion and salt reabsorption in sweat glands,13 and
its dysfunction in CF patients results in salty sweat. The
abnormal sweat composition in CF patients can be
detected by a ‘sweat test’, a common diagnostic method
used in CF clinics. Sweat glands do not manifest the
anatomical alterations or pathology observed in other
organs affected in the disease, and have been used as a
model to study CFTR function and electrolyte transport
pathophysiology.14,15
There are a number of advantages in using human
sweat glands as a model to study CF. They are relatively
easy to access from a skin punch biopsy, and the function
of CFTR can be measured electrophysiologically in the
apical membrane of the sweat duct ex vivo.16 With these
advantages, sweat glands could be a useful model to
assess efficacy of CF gene therapy. Delivery and
expression of normal Cftr to CF sweat glands is expected
to restore the normal chloride transport function. Thus, it
should be possible to evaluate the effectiveness of gene
transfer and expression by the secretory response,
biochemical analyses of the sweat, or electrophysiological properties according to well-established methods.16
Furthermore, using sweat glands in studying CF gene
therapy has significant implications for CF clinical trials.
It is inherently safer and more practical to administer
gene therapy vectors to a small punch biopsy or to a
small region of skin, rather than to CF affected organs
such as pancreas or lung. However, whether genes can
be effectively delivered to human sweat glands remains
to be shown. Although sweat glands may provide an
important model to study the effect of CF gene therapy
in human native tissues, differences in morphology
and physiology exist between the sweat gland and
the lung.
As a first step to explore the possibility of using sweat
glands as a model for CF gene therapy, we investigated
effective gene delivery methods using normal human
sweat glands. We showed that transgenes can be
efficiently delivered to the sweat gland cells by adenoviral gene therapy vectors. Furthermore, Cftr was
delivered to, and expressed by, the target cells in the
lumen of sweat glands using a helper-dependent
adenovirus (HD-Ad) vector containing an epithelial
cell-specific promoter. We also found that the basement
membrane is an important barrier to adenoviral vector
transduction.
culture). After 2 days of exposure to the vector, the skin
tissue was assayed for b-galactosidase activity determined by reaction with X-gal. The transduction efficiency was very low and only a few cells in a few sweat
glands stained with X-gal (Supplementary data, Figure
8). Those few transduced sweat glands were observed
only in the intradermally injected or submersion organ
cultures. No sweat gland transduction was observed by
topical application of virus to skin in air–liquid interface
culture.
Most transduced sweat glands were found at sites
where they had been directly exposed to virus. For
example, transduced sweat glands were found at the
immediate site of the injection or at the excised periphery
of the skin tissue in the submerged culture. This
observation indicated that the adenovirus vector has
limited ability to penetrate and transduce skin tissue
in the organ culture. In order to increase the number of
exposed sweat glands from the dermis, we treated the
skin tissue with collagenase. Although collagenase
partially digested the collagen-rich dermal matrix, it left
the tissue sticky and did not noticeably increase the
transduction efficiency. Nonetheless, we found that a
few sweat glands at the periphery of the tissue, which
were completely freed from the dermal matrix by the
collagenase treatment, were transduced by the adenoviral vector (Figure 1). These results using skin organ
culture suggested that the epidermis and dermal matrix
are significant barriers to adenoviral vectors.
Results
Enhancement of gene delivery to isolated sweat
glands
Since sweat gland tubules are only 2–3 cell layers thick,
we suspected that the poor penetration of adenoviral
vector was due to some of the remnant dermal
component surrounding the sweat glands. We then
treated the isolated sweat glands with collagenase
(0.1 U/ml) and dispase (50 U/ml) overnight at 41C,
before inoculating with the HD-Ad vector (the unit
definition of the enzymes are defined by the manufacturers and do not correlate). These treatments not only
dramatically increased the overall transduction efficiency
but also resulted in transgene expression from the cells in
the lumen (Figure 3a and c). Transduction efficiency was
Transduction of human sweat glands in skin organ
culture
Considering the small size of sweat glands and their
ubiquitous distribution in human skin, we first attempted to transduce sweat glands embedded in the
dermis of full-thickness skin organ culture. A small block
of excised full-thickness skin (1 1 cm) was maintained
either at the air–liquid interface or submerged in culture
medium, and exposed to a HD-Ad vector expressing
b-galactosidase (HDD28E4LacZ)17 by topical application
or intradermal injection (air–liquid interface culture), or
by incubation in virus-containing media (submersion
1753
Gene delivery to isolated sweat glands
In an attempt to circumvent the barriers in skin tissue,
we isolated sweat glands from the dermis by mechanically mincing the skin.18 The isolated sweat glands were
then incubated with HD-Ad vectors added to the media.
Inoculating HDD28E4LacZ17 (5 1010 viral particles) into
100 ml media containing isolated sweat glands appeared
to increase the transduction and expression of the
transgene in the sweat glands, highlighted by the large
area of X-gal staining (Figure 2a). However, when the
histologic sections were examined most of the lacZ
positive cells were found at the outer layer of the
transduced sweat glands (Figure 2b). In fact, most of the
X-gal staining was in the remnant interstitial tissue,
indicating that the HD-Ad did not penetrate to the inner
layer of cells of the sweat gland when applied from the
basal side. This limitation is a problem for using sweat
gland as a CF gene therapy model because CFTR must be
expressed and function at the luminal membrane.19,20
Gene Therapy
Gene delivery to human sweat glands
H Lee et al
1754
Figure 1 Transduction of sweat glands in skin organ culture. Human skin
tissue blocks were pretreated with collagenase and transduced by culturing
in media containing HDD28E4LacZ. After 2 days the tissue blocks were
assayed for b-galactosidase activity by reaction with X-gal (blue) and the
transduced sweat glands were isolated and photographed. A part of a sweat
duct (a) and a whole sweat gland (b) were partially transduced by the
vector (original magnification 90).
correlated with the concentration of the proteases used.
The relationship between the degree of enzymatic
digestion and the transduction efficiency was further
confirmed in other experiments, where the sweat glands
were treated with dispase for various lengths of time as
well as at different temperatures (Supplementary data,
Figure 9).
Even though both collagenase and dispase significantly enhanced transduction efficiency at the higher
concentrations, they showed different effects on gene
delivery at lower concentrations. When sweat glands
were treated with collagenase at 0.004 U/ml, the transduction was limited to the outer cell layer of the glands
(Figure 3b). On the other hand, the dispase treatment
with the same factor of dilution (2 U/ml) resulted in
fewer transduced glands, but the adenoviral vector
delivered the transgene to the cells in the lumen of those
glands (Figure 3d). These results suggested that pretreatment with dispase might be a more effective and
specific method to improve gene delivery to the lumen of
the sweat glands.
Gene Therapy
Figure 2 Transduction of isolated sweat glands. (a) Sweat glands were
isolated from human skin tissue and transduced with HDD28E4LacZ.
After 2 days of exposure to the vector, the sweat glands were assayed for
lacZ expression (original magnification 90). (b) The X-gal stained sweat
glands were cryo-sectioned and counter stained with neutral red for
histologic analysis (original magnification 400).
Basement membrane is the major barrier to adenoviral
vector delivery
To further analyze how the dispase treatment increased
transduction efficiency of sweat glands, we examined the
integrity of the basement membrane after dispase
treatment by immunostaining for type IV collagen. Type
IV collagen is the most abundant constituent of basement
membranes and assembles a sheet-like network with
laminin to form the scaffold for the basement membrane.21 When compared to intact sweat glands (Figure
4a), treatment with 50 U/ml dispase overnight at 41C
significantly reduced type IV collagen in the basement
membrane (Figure 4b). Digestion for 8 h at 371C
eliminated most of the type IV collagen at the basement
membrane (Figure 4c). From these results we conclude
that the basement membrane is one of the major barriers
to gene delivery to sweat glands using adenoviral
vectors.
Cftr gene delivery to sweat glands with HD-AdK18CFTR
Our laboratory has developed HD-Ad vectors with
control elements from the human cytokeratin 18 gene
Gene delivery to human sweat glands
H Lee et al
1755
Figure 3 Enhanced transduction of sweat glands by enzyme pretreatment. The sweat glands were isolated from human skin tissue and treated with (a)
collagenase, 0.1 U/ml; (b) collagenase, 0.004 U/ml; (c) dispase, 50 U/ml; or (d) dispase, 2 U/ml, prior to HDD28E4LacZ exposure. After overnight
incubation at 41C in enzymes containing KGM media, sweat glands were transduced with HDD28E4LacZ. The unit definitions of the two enzymes are
defined by the manufacturers and do not correlate (gross view magnification 90, histology magnification 200).
(KRT18 or K18) for CF gene therapy.10,22 These vectors
may be well suited for CF gene therapy because the K18
promoter can drive cell-specific expression in a pattern
similar to that of Cftr.23 K18 has been shown to be
expressed in epithelial cells of various organs including
sweat glands,24,25 and we confirmed its expression in
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Gene delivery to human sweat glands
H Lee et al
1756
Figure 4 Digestion of the basement membrane by dispase. Sweat glands
were isolated and treated with (a) no enzyme, (b) dispase overnight at 41C,
or (c) dispase for 8 h at 371C. Immunohistochemistry was carried out with
frozen sections of the sweat glands using anti-type IV collagen. Type IV
collagen was detected by DAB (brown) staining against methyl-green
counter staining (green) (original magnification 100).
human sweat glands by immunostaining (Supplementary data, Figure 10).
We tested whether the K18 control elements could
drive an expression pattern similar to that of native Cftr
in sweat glands. Isolated sweat glands treated with
dispase were exposed to HD-Ad-K18LacZ vector, which
contained the b-galactosidase gene with a nuclear
localization signal under the control of the K18 promoter.22 As shown in Figure 5 transgene expression by HDAd-K18LacZ in sweat glands was distinctive from that of
the human cytomegalovirus (CMV) promoter-driven
vector HDD28E4LacZ (Figure 3a and c). In general, the
Gene Therapy
Figure 5 Transduction of sweat glands by HD-Ad-K18LacZ. Isolated
sweat glands pretreated with dispase were transduced by HD-AdK18LacZ. The lacZ expression in the transduced sweat glands was
detected by X-gal staining (a, original magnification 90) and analyzed
by histologic section (b, original magnification 200). In addition, the
X-gal stained tissue sections were subjected to immunostaining with
anti-cytokeratin 18 antibody to identify epithelial cells (c, original
magnification 1000). Since the lacZ transgene in HD-Ad-K18LacZ
contains a nuclear localization signal, X-gal staining (blue) was observed
in the nuclei, whereas K18 was detected (brown) in the cytoplasm.
expression level was moderate compared to that
achieved by HDD28E4LacZ, and showed a punctate
transduction pattern (Figure 5a). The histologic examination of HD-Ad-K18LacZ transduced sweat glands
showed cell type-specific expression of the transgene
(Figure 5b). Unlike sweat glands transduced with
HDD28E4LacZ, where all of the cells expressed the
transgene, HD-Ad-K18LacZ induced lacZ expression
specifically in the luminal cells. The condition of
histologic sections of isolated sweat glands after dispase
treatment and ex vivo culture prevented clear morpholo-
Gene delivery to human sweat glands
H Lee et al
1757
1
2
337
305
178
127
M
3
4
HD-Ad-K18-CFTR
K18 promoter K18 intron 1
K18 exon 1
Cftr
Pvu II
transgene Cftr mRNA
endogenous Cftr mRNA
5’ UTR
Figure 6 Expression of Cftr by sweat glands transduced with HD-AdK18-CFTR. A total of 50 sweat glands were treated with dispase overnight
and transduced with HD-Ad-K18-CFTR for 2 days. The total RNA was
isolated and cDNA was generated using a common reverse primer (black
arrow head). Forward primers designed specifically at the K18 exon 1 (gray
arrow head) or 50 UTR of Cftr (unfilled arrow head) were used in PCR (35
cycles) to detect the transgene Cftr (Lane 2, 305 bp) or endogenous Cftr
(Lane 3, 337 bp) expression, respectively. The transgene expression was
confirmed by restriction enzyme (PvuII) digestion (Lane 1). Lane 4 is RTPCR product (25 cycles) of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
gical identification of the cell types on sections. However,
the epithelial cell-specific expression of the transgene
was confirmed by double staining to detect K18 (by
immunostaining) in the cytoplasm and LacZ (by X-gal
staining) in the nucleus (Figure 5c). The expression of
lacZ transgene in luminal cells in this study was similar
to the Cftr expression pattern detected in normal human
sweat glands by immunohistochemistry in other studies.19,26 These results show that the HD-Ad vector with
K18 control elements can drive an epithelial cell specific
transgene expression in sweat glands.
Finally, we transduced sweat glands with HD-AdK18CFTR, our HD-Ad containing the human Cftr gene
tagged with a VSV-G epitope under control of the K18
promoter.10 Cftr expression was detected by RT-PCR
using transgene-specific primers designed across K18
intron 1, which allowed us to distinguish the PCR
product of the transgene Cftr mRNA from that of the
endogenous Cftr mRNA or the vector DNA (Figure 6).
Furthermore, we confirmed Cftr gene expression in the
epithelial cells of the lumen by immunostaining the
sweat gland sections with anti-VSV-G antibody 3 days
after transduction (Figure 7b). CFTR-VSV-G was detected
in the perinuclear region and at the plasma membrane
of transduced cells in the sweat glands (Figure 7c),
consistent with our immunohistochemistry results for
the endogenous CFTR of the human sweat gland
(Supplementary data, Figure 11) as well as previous
reports of the intracellular localization of CFTR.27
Discussion
This study investigated gene delivery to human sweat
glands to explore the possibility of using sweat glands as
a model for CF gene therapy. Prior to this study there
have been no reports of gene delivery directed to human
sweat glands. One study reported partial transduction of
sweat glands when an AAV vector was intradermally
injected into pig skin.28 Using various methods we
attempted to deliver genes using an HD-Ad vector to
human skin in organ culture. We found that gene
delivery to sweat glands in intact human skin was very
inefficient ex vivo. In general, topical gene delivery has
been considered to be difficult for cutaneous gene
Figure 7 Gene delivery and expression of Cftr. Sweat glands were
transduced with (a) no virus, or (b) HD-Ad-K18CFTR, and Cftr
expression was detected by immunostaining with anti-VSV-G antibody
(brown) against methyl-green counter staining (original magnification
200). (c) is at a higher magnification of (b) showing intracellular
localization of transduced Cftr gene product (original magnification
1000).
therapy.29 A number of gene delivery studies with
adenoviral vectors showed that it is possible to achieve
a significant transduction of mouse epidermis in vivo,
and skin appendages such as hair follicles and sebaceous
glands can be transduced in mouse skin.30–32 The
discrepancy between the transduction we observed with
our human skin organ culture and what others have
observed in vivo may reflect inherent differences between
eccrine sweat glands and other skin appendages, mouse
versus human species differences, and/or differences
between skin in organ culture versus skin in vivo.
Our observations indicated that the basement membrane is a critical barrier to the delivery of adenoviral
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Gene delivery to human sweat glands
H Lee et al
1758
vectors from the basal side of sweat glands. The
basement membrane has been suggested as a physical
barrier to herpes simplex virus (HSV) vectors,33 and
Weeks et al34 showed that the intradermal injection of
HSV below the epidermal basement membrane did not
cause pathogenesis. In Duchenne muscular dystrophy
gene therapy, the ineffectiveness of viral vectors to
transduce mature muscle has been a major impediment.35 Huard et al33 reported that the more mature
basement membrane of the muscle fiber in adult mice,
compared to newborn mice, acted as barrier for HSV
vector transduction. Van Deutekom et al35 also showed
that treating myofibers with streptokinase, a fibrinolytic
drug, enhanced transduction efficiency of the HSV vector
in mature skeletal muscle, suggesting basement membrane and surrounding extracellular matrix as a physical
barrier. Interestingly, Pruchnic et al36 reported that AAV
could transduce mature as well as immature muscle
fibers, and proposed that the small size of AAV,
compared to other viral vectors, might have contributed
its ability to penetrate the basement membrane.
We were able to significantly enhance adenoviral gene
delivery to sweat glands by dispase pretreatment. Saito et
al37 used collagenase to enhance the gene transfer to hair
follicle from mouse and suggested that partial digestion
of the dermis might have increased the transduction
efficiency. We also achieved comparable transduction
efficiencies with sufficient collagenase treatment instead
of dispase. However, dispase treatments were more
effective than the collagenase at higher dilutions (Figure
3b and d). Commercially available collagenase and
dispase are extracts from bacterial cultures and exhibit
various proteinase activities, but dispase is considered an
effective reagent for digestion of basement components
such as fibronectin and type IV collagen.38 Our immunostaining for type IV collagen demonstrated that
degradation of the basement membrane was correlated
with enhanced transduction efficiency. Although we
used dispase treatment to enhance gene delivery from
the basal side of the sweat gland in our ex vivo study,
an alternative method could be used to deliver genes to
sweat glands without the enzyme pretreatment, to
preserve tissue integrity. One of the plausible methodologies is microperfusion. Microperfusion of gland tubules
has been used to study electrophysiology and CFTR
functions in sweat ducts.14,15 This technique would allow
delivery of the vector directly to the lumen of sweat
glands instead of from the basal side, without enzyme
pretreatment. Once the vector is delivered to the sweat
tubule, the chloride conductance could be quantitatively
measured to evaluate CFTR function in the sweat
gland.16
In this study, we have demonstrated that it is possible
to deliver Cftr to sweat glands using an HD-Ad, and to
express CFTR in the epithelial cells in the lumen. Our
results provide the basis to study the functional efficacy
of CF gene therapy using human sweat glands, and
we propose that sweat glands could be used as an
alternative model. Delivery of Cftr to sweat glands from
a CF patient should restore normal CFTR function of
b-adrenergically stimulated secretion, lower sweat salt
concentrations, and enhance Cl conductance in sweat
ducts. These changes can be quantitatively evaluated by
biochemical and electrophysiological methods.14–16
Future studies should be directed toward detecting these
Gene Therapy
changes in sweat secretion function using sweat glands
from CF patients.
The study of functional efficacy is an important step
toward a successful CF gene therapy. In addition to
molecular end points, such as mRNA and protein
expression, clinical trials often relied on nasal electrical
potential difference to detect correction of the chloride
transport function.8 However, measuring nasal potential
difference is known to be a variable technique39 and
there have been conflicting results questioning the
sensitivity of the method for measuring CFTR function.40,41 Although the nasal epithelium is more closely
related to the lung epithelium in its electrophysiology,
the sweat gland could provide an alternative model to
assess the efficacy of the gene therapy vector in an organ
without disease pathology. One difficulty would be the
currently less widespread use of sweat gland electophysiology techniques14–16 compared to nasal potential
difference measurement.
In addition to quantitative assays for the functional
evaluation of gene therapy, the sweat gland model could
provide advantages in clinical trials. Gene delivery to
sweat glands from the skin surface or to sweat glands
isolated from a punch biopsy would be more convenient
than delivery to the nasal cavity or the lung. More
importantly, ex vivo transduction of sweat glands, or
transduction of sweat glands in a small region of skin,
would not be a major safety and ethics concern
compared to applying viral vectors to pathologically
compromised organs such as the CF lung. The sweat
gland model can also provide a way to predict
compliance to the gene therapy for each individual
patient, by testing his own sweat glands prior to
treatment of the CF affected organ. With these potential
clinical implications, the sweat gland could be considered as an alternative model to study efficacy of CF gene
therapy in humans.
Materials and methods
Helper-dependent adenoviral vectors
Construction of the HDD28E4LacZ, HD-Ad-K18LacZ
and HD-Ad-K18CFTR HD-Ad vectors has been described previously. HDD28E4LacZ contains bacterial lacZ
under control of the CMV immediate-early promoter.17
HD-Ad-K18LacZ and HD-Ad-K18CFTR have 2.5 kb
of genomic sequence upstream from the cytokeratin 18
(K18) gene containing the promoter plus first intron of
K18 followed by a translation enhancer.10,22 HD-AdK18LacZ contains a nuclear localization signal from SV40
antigen.22 HD-Ad-K18CFTR has the vesticular stomatitis
virus G glycoprotein (VSV-G) epitope tag at the Cterminus of CFTR.10 The HD-Ads were prepared as
previously described.17 The final concentration of virus
was 5–8 1012 viral particles/ml.
Preparation of full thickness skin and gene delivery
using skin organ culture
Human skin tissue was obtained from abdominal
pannus excised from otherwise healthy subjects undergoing dermolipectomy. A clinical protocol (HSC protocol
# 001960042) was approved for ex vivo experiments on
the skin tissue. The hypodermal fat was removed from
the skin and the full-thickness skin was briefly washed
Gene delivery to human sweat glands
H Lee et al
with PBS. For skin organ culture study the full-thickness
skin was cut into 1 1 cm pieces and either submerged
in media or placed on the Transwell culture system
(Costar, Corning, NY, USA), such that the dermis
contacted the medium while the epidermis remained
exposed to the air. RPMI 1640 containing 10% FBS was
used as the culture media. The skin was transduced
by topical application or injection of adenovirus, or
by addition of adenovirus to the submersion culture
medium. For topical application, 10 ml of virus solution,
containing 2.5 1010 virus particles, was carefully laid on
the epidermis in the center of the skin sample. For
injection, the same number of virus particles (in 5 ml) was
intradermally injected using a 30G needle. For submersion culture, the same number of virus particles was
added to 1 ml media, which contained the skin sample.
The tissue was incubated for 2 days in a 5% CO2
incubator at 371C and then stained with X-gal. In order to
digest the dermal matrix, full-thickness skin was placed
in 2 mg/ml of collagenase (274 U/mg) (Worthington
Biochemicals, Lakewood, NJ, USA) in aMEM with 1%
FBS for 2 h at 371C.
Isolation of sweat glands and transduction
Sweat glands were isolated under a dissection microscope after mincing 1 0.5 cm full-thickness skin with
fine surgical scissors.18 The isolated sweat glands were
maintained in KGM media (Clonetics, Walkersville, MD,
USA) during isolation and the transduction experiment.
The sweat glands were digested in 100 ml of KGM
containing 10% dispase (5000 U/ml) (Collaborative
Research, Bedford, MA, USA) overnight at 41C, unless
specified otherwise. Alternatively, collagenase H
(0.2 U/ml) (Roche, Mannheim, Germany), diluted in
KGM, was used to compare transduction efficiencies.
The unit definitions of dispase and collagenase were
defined by the manufacturers and do not correlate. Five
to ten isolated sweat glands in each well of a 96-well dish
were washed twice in PBS and cultured in 100 ml of
KGM. The HD-Ad (2.5–5 1010 particles) was added
directly to the media and the culture maintained for
2 days, except when specified otherwise.
X-gal staining
Sweat glands transduced with HD-Ad vectors containing
lacZ were assayed for b-galactosidase activity by reaction
with X-gal. The sweat glands were briefly washed in PBS
and fixed in fixative solution (1% formaldehyde, 0.1%
glutaraldehyde, 2 mM MgCl2, 5 mM EGTA in 0.1 M
sodium phosphate buffer, pH 7.8) by incubating for
30 min at 41C with gentle mixing. After washing twice
for 15 min in washing solution (2 mM MgCl2, 0.02% NP40, 0.01% deoxycholate in 0.1 M sodium phosphate
buffer, pH 7.8) at 41C, the tissue was stained with X-gal
solution (wash solution plus 5 mM K4Fe(CN)6:3H2O,
5 mM K3Fe(CN)6, 1 mg/ml 5-bromo-4-choloro-3-indolyl
b-D-galactopyranoside) for 2 h at 371C. Samples were
then rinsed in 70% ethanol, and further fixed in 4%
paraformaldehyde for 1 h at 41C. We used same methods
for skin tissue but allowed longer incubation times. The
stained tissue samples were sectioned after embedded
in paraffin or OCT (Tissue-Tek, Torrance, CA, USA) and
counter stained with neutral red.
1759
Immunohistochemisty
The transduced sweat glands were treated in fixative
solution (as above) for 30 min at 41C and embedded in
OCT for cryosection, or in paraffin after a series of short
dehydration steps in 70% (20 min), 90% (20 min), 100%
ethanol (2 20 min), and xylene (2 30 min). Both frozen
and paraffin sections were cut 5 mm in thickness and
paraffin sections were deparaffinized prior to immunostaining. Tissue sections were rehydrated in PBS, treated
with 0.2% Triton X-100 in PBS for 4 min, quenched with
0.3% H2O2 for 10 min, and blocked with blocking buffer
(2% BSA, 5% goat serum) for 30 min. Then, monoclonal
antibody against VSV-G (1:2000 dilution, Boehringer
Mannheim, Indianapolis, IN, USA) or human type IV
collagen (1:2500 dilution, Chemicon, Boronia, Australia)
diluted in blocking buffer was applied to the section. The
primary antibodies were detected using the Vectastain
Kit and DAB Substrate Kit for Peroxidase (Vector
Laboratories, Burlingame, CA, USA), according to the
manufacturer’s recommendations. Finally, the sections
were counter stained with either neutral red or methyl
green. For K18/LacZ double staining, the X-gal stained
sweat glands were embedded in paraffin and subjected
to immunostaining with anti-cytokeratin 18 antibody
(1:20 dilution, RDI, Flanders, NJ, USA).
RT-PCR
A total of 50 isolated sweat glands were transduced with
1.24 1011 particles of HD-Ad-K18-CFTR in 100 ml of
KGM for 2 days as described above. The total RNA was
isolated in 500 ml of TRIzol (Invitrogen, Carlsbad, CA,
USA), according to the manufacturer’s recommendations
following tissue disruption by a cycle of freezing and
thawing and vigorous pippetting. cDNA was generated
using a common reverse primer (50 -ccgaagggcattaatgagtttagg-30 ) in the coding region of Cftr, and the forward
primers designed specifically at the K18 exon 1 (50 GTCCGCAAAGCCTGAGTCCTGTCC-30 ) or 50 UTR of
Cftr (50 -ATCACAGCAGGTCAGAGAAAAAG-30 ) were
used in PCR to detect the transgene or endogenous Cftr
expression, respectively. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also
detected by RT-PCR using a reverse primer (50 -GATG
ATGACCCGTTTGGCTCC-30 ) and a forward primer
(50 -CTGGTAAAGTGGATATTGTTGCC-30 ). The PCR reactions were performed for 35 cycles, except for GAPDH,
whose PCR reaction was performed for 25 cycles.
Acknowledgements
We thank Bernard G Martin for preparation of the
helper-dependent adenoviral vectors, and Dr Jeffery
Wang and Mr Kirk Tayler for technical assistance. We
also thank Dr Hugh O’Bradovich and Dr Johanna
Rommens for critical comments during this study. This
work was supported by an Operating Grants from the
Canadian Cystic Fibrosis Foundation, an Operating
Grant from the Canadian Institutes of Health Research
to JH and to CYP (MOP 8048), the Sellers Fund to JH and
CYP, and an Operating Grant from the National
Institutes of Health (P50 HL59314) to PN. JH is a CCFF
Scholar and holds a Premier’s Research Excellence
Award of Ontario, Canada.
Gene Therapy
Gene delivery to human sweat glands
H Lee et al
1760
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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt).
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