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Gene Therapy (2005) 12, 1275–1282
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RESEARCH ARTICLE
Mechanism of efficient transfection of the nasal airway
epithelium by hypotonic shock
JL Lemoine1, R Farley2 and L Huang1
1
Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, PA, USA; and 2Department of Gene Therapy, Imperial
College, National Heart and Lung Institute, London, UK
The main barrier to gene transfer in the airway epithelium is
the low rate of apical endocytosis limiting naked DNA uptake.
Deionized water is known to stimulate the exocytosis of
numerous intracellular vesicles during hypotonic cell swelling, in order to expand plasma membrane and prevent cell
lysis. This is followed by the phase of regulatory volume
decrease (RVD), during which the excess plasma membrane
is retrieved by intensive endocytosis. Here we show that the
more hypotonic the DNA solution, the higher the transfection
of the nasal tissue. P2 receptors are known to be involved in
RVD and we demonstrate that some P2 agonists and a P2
antagonist impair transfection in a time-dependent manner.
Our study strongly suggests that the nasal airway epithelial
cells take up plasmid DNA in deionized water during RVD,
within approximately half an hour. Our simple gene delivery
system may constitute a promising method for respiratory
tract gene therapy.
Gene Therapy (2005) 12, 1275–1282. doi:10.1038/
sj.gt.3302548; published online 12 May 2005
Keywords: airway; gene transfer; naked DNA; hypotonic shock; mechanism
Introduction
To date, transfection efficiencies in clinical trials of
cystic fibrosis (CF) are too low to result in a notable
correction of the chloride conductance defect.1,2 Two
main extracellular barriers impede DNA uptake by
airway epithelium. The first barrier is the mucus-gel
layer involved in the mucociliary clearance of foreign
bodies. The second is the low rate of endocytosis taking
place on the apical side of airway epithelial cells.3
Moreover, the basolateral access is prevented by the
presence of tight junctions. The respiratory epithelium
contains these mechanisms of defense to limit viral and
bacterial infections.
We hypothesized that a hypotonic shock would force
the apical membrane to undergo an intensive endocytosis, allowing plasmid DNA to access the intracellular
compartments of the airway epithelial cells. Indeed, it is
well known that a hypotonic shock comprises two
phases: cell swelling and cell shrinkage or regulatory
volume decrease (RVD). In the course of the first step,
water molecules penetrate into the cells by osmosis. This
initial cell swelling stimulates the exocytosis of numerous intracellular vesicles.4,5 By fusing with plasma
membrane, they allow the membrane to expand and
even to form some protrusions or blebs in fibroblasts and
Ehrlich ascites tumor cells.6,7 Thus, cell swelling does not
Correspondence: Professor L Huang, Center for Pharmacogenetics, School
of Pharmacy, University of Pittsburgh, 633 Salk Hall, 3501 Terrace Street,
Pittsburg, PA 15213, USA
Received 18 February 2005; accepted 26 March 2005; published
online 12 May 2005
lead to cell lysis. Indeed, cultured cells were kept in a
hypotonic medium for up to an hour without any loss of
viability.8 The second step of the hypotonic stress is the
internalization of the excess plasma membrane from both
the apical and the basolateral sides to reform the lost
intracellular vesicles.
It has been extensively shown that P2 receptors are
involved in this process. They are integral membrane
proteins present on the apical and the basolateral surfaces
and are divided into three subfamilies: X, Y and Z. The
most important receptors, P2Y, are coupled to G proteins.
P2Y1, P2Y2 and P2Y4 have been found on the apical side
of the airway epithelial cells and P2Y6 on the basolateral
side.9 Their natural agonist adenosine 50 -triphosphate
(ATP) is present at high concentrations in secretory
granules that fuse with the plasma membrane during
cell swelling.10 Thus, ATP released in the extracellular
medium can activate P2 receptors. A pathway transduced
by P2Y receptors leads to the opening of potassium
channels via a rise of intracellular calcium11 and another
pathway results to the opening of chloride channels,12
including the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), via the activation of the protein
kinase A.13 The release of potassium chloride reduces the
ionic strength inside the cells but increases it in the
extracellular environment, dragging water molecules out
of the cells by osmosis. Those events progressively evict
the excess intracellular water, allowing the retrieval of the
excess plasma membrane by endocytosis.
Here we demonstrate for the first time that in our
in vivo model of transfection of the murine airway, the
nasal airway epithelial cells take up plasmid DNA
delivered in the deionized water during the step of cell
Mechanism of transfection of the airway epithelium with naked DNA in water
JL Lemoine et al
1276
shrinkage of hypotonic shock, leading to its efficient
transfection without damaging the cells.
Results
Effect of tonicity of the DNA solution on transfection
of the nasal tissue
We evaluated gene transfer in the mouse nose, a
commonly used model of the airway epithelium because
of the easy access,14 with plasmid DNA (pDNA) in the
deionized water, sodium chloride and phosphatebuffered saline (PBS). Luciferase activity was reduced
in a dose-dependent manner by sodium chloride and
maximal transfection efficiency was produced in the
deionized water, with values 92- and 102-fold higher
than those obtained in 150 mM NaCl (saline) and PBS,
respectively (Figure 1). It was recently shown that PBS
and saline are more potent vehicle for pDNA than
deionized water in the skin15 and the muscle.16 This
suggests that a different mechanism takes place in the
airway than in the skin or the muscle. Either the tonicity
or the ionic strength of the pDNA solution prevents the
transfection of the nasal epithelium.
To determine whether transfection efficiency depends
on the tonicity of the pDNA solution, we perfused the
nasal epithelium with plasmid DNA in sucrose solutions
of increasing tonicity, which also decreased transfection
in a dose-dependent manner. The most hypotonic pDNA
solution is therefore the most potent condition for
transfecting the nasal tissue.
Localization of transgene expression in the nasal
tissue by b-galactosidase immunostaining
To localize transgene expression within the nasal tissue,
100 mg of b-galactosidase expression plasmid pSVb was
delivered in deionized water or in saline. No specific
staining was found in the nasal tissue of untreated mice
(Figure 2a), the nasal tissue perfused with pNGVL3-luc
in deionized water (Figure 2b) or with pSVb in saline
(Figure 2c). On the other hand, a positive and uniform
staining was observed in the airway epithelial cells
following their perfusion with pSVb in deionized water
(Figure 2d). In all areas examined, more than 90% of the
airway epithelial cells were significantly transfected
(arrowheads).
Compared to the untreated tissue (Figure 2a) and the
tissue perfused with pSVb in saline (Figure 2c), the nasal
tissues that were perfused with pNGVL3-luc in deionized water (Figure 2b) or with pSVb in deionized water
(Figure 2d) were not significantly changed. This result
indicates that hypotonic shock treatment did not damage
the nasal epithelial tissue.
Effect of the preperfusion of deionized water on
transfection of the nasal airway epithelium with pDNA
We then sequentially perfused mice with deionized
water for different periods of time, followed by pDNA
in deionized water. There was a time-dependent
decrease of the luciferase activity. The administration
of deionized water for 20 min followed by the perfusion
of the nose with pDNA resulted in a maximal reduction
of the luciferase expression (seven-fold).
Transfection efficiency returned to the level achieved
with the simple perfusion of mice with pDNA, when
deionized water was predelivered for as long as 30 min.
Neither the perfusion of the nasal tissue with deionized
water after pDNA, nor the delivery of saline prior to the
one of pDNA affected transfection of the nasal epithelium (Figure 3).
These data suggest that the simple perfusion of
deionized water improves gene transfer within a 30min period, after which the system has recovered to its
original state and could be restimulated. We hypothesized that the perfusion of the nose for a period shorter
than 30 min with deionized water prior to the delivery of
pDNA would start a hypotonic shock before the nasal
tissue sees the pDNA, thus the endocytosis of DNA
could not take the full benefit of the hypotonic shock.
This interpretation is in agreement with the report that
cells incubated in a hypotonic medium swell and reach
their maximal size in 2–3 min and shrink back to their
initial volume in approximately 30 min.17 Our data
suggest that DNA uptake by the nasal respiratory tissue
occurs during the step of cell shrinkage (RVD). When a
Figure 1 Effect of tonicity of the pDNA solution on transfection of the nasal tissue. Comparison of luciferase expression following the delivery of plasmid
DNA in deionized water, sodium chloride, phosphate-buffered saline (PBS 1 ) and sucrose (n ¼ 3–4 mice per group); **Po0.005 and ***Po0.0005 versus
deionized water.
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Mechanism of transfection of the airway epithelium with naked DNA in water
JL Lemoine et al
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Figure 2 b-Galactosidase immunostaining of the nasal airway epithelium. Nasal tissues harvested from mice that received 100 mg of plasmid DNA were
fixed, sectioned and immunohistochemically stained for the b-galactosidase 24 h following administration. Representative sections of the airway epithelium
of untreated animals (a), animals that received the luciferase expression plasmid pNGVL3-luc in deionized water (b), animals that received pSVb in saline
(c) and animals that received pSVb in deionized water (d) are shown. Hematoxylin stain (blue) marks nuclei and AEC stain (red) marks b-galactosidase
expression. The arrowheads point to some of the b-galactosidase-expressing nasal epithelial cells. 40, original magnification.
Figure 3 Sequential perfusion of deionized water and plasmid DNA in water. Deionized water was delivered for 10–30 min shortly followed by pDNA in
deionized water for 15 min. pDNA in deionized water was delivered for 15 min shortly followed by deionized water for 20 min (DNA-20 min water) and
saline (150 mM NaCl) was delivered for 20 min shortly followed by plasmid DNA in deionized water for 15 min (20 min saline-DNA). **Po0.05,
++
Po0.01 and ***Po0.0005 versus pDNA alone (n ¼ 3–4 mice per group).
cycle of cell swelling and cell shrinkage is over (in about
30 min), a second cycle can begin and luciferase
expression returns to the level achieved with a simple
perfusion of pDNA (Figure 4).
Effect of ATP on transfection of the nasal epithelium
The crucial role played by P2 receptors in RVD has been
abundantly illustrated.18,19 Their overstimulation by
exogenous ATP accelerates RVD,20 whereas their blockade by the antagonist suramin slows it down.21 To assess
whether P2 receptors are involved in deionized watermediated transfection, we delivered pDNA along with
ATP in the deionized water. There was a dose-dependent
decrease in luciferase expression in the nasal tissue, with
values 10-fold lower at 400 nmol of ATP (Figure 5). We
hypothesized that exogenous ATP accelerates the phase
of cell shrinkage, during which nasal cells take up
pDNA. Since the DNA solution was delivered within
15 min, in the presence of exogenous ATP, RVD would
nearly be over while most of the pDNA were not yet
delivered (Figure 4).
We tested this hypothesis by shortening the duration
of delivery of the DNA/ATP solution. The faster the
perfusion of mice with the plasmid DNA/ATP solution,
the higher the luciferase activity, and the perfusion of
the nose lasting as short as 7 min gave rise to gene
expressions as high as pDNA alone delivered in
deionized water in 7 min (Figure 5).
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Mechanism of transfection of the airway epithelium with naked DNA in water
JL Lemoine et al
1278
Effect of suramin on transfection of the nasal
respiratory tissue
Similarly, we perfused the nasal epithelium with pDNA
and a P2 antagonist, suramin. There was also a dosedependent reduction of the luciferase activity, as high as
15-fold at 50 nmol of suramin. Interestingly, the delivery
of pDNA along with 400 nmol of ATP and 50 nmol of
suramin resulted in a similar transfection efficiency as
that of the pDNA alone (Figure 6). This demonstrates
that neither exogenous ATP nor suramin are toxic to the
nasal tissue. In the presence of suramin, RVD would last
longer and after the 15-min perfusion most of pDNA
would have been degraded by extracellular DNases
prior being taken up by cells22 (Figure 4).
To test whether suramin affects transfection by
slowing down RVD, we slowed down the delivery of
the DNA/suramin solution. The slower the perfusion of
Figure 4 Schematic of the changes of the cell volume in the course of a
hypotonic shock. Cell volume in the absence of any drug (—), in the
presence of ATP (- - -) and in the presence of suramin (– – –).
mice with the pDNA/suramin solution, the higher the
gene expression, and the delivery lasting 45 min completely abolished the deleterious effect of this drug on
gene transfer (Figure 6).
Interestingly, it has been shown that RVD is also
slowed down in CF because of a limited release of
endogenous ATP during cell swelling.23,24 This last
experiment suggests that pDNA in deionized water
should preferentially be administered over a long period
of time in the airway of CF patients.
Impairment of transfection of the nasal tissue by other
P2 agonists
One may argue that ATP may interact with ATP-binding
proteins other than the P2 receptors. To determine
whether P2 receptors are the real target of ATP, pDNA
was delivered along with other well-known P2 agonists.
ADP, dATP and UTP are potent ligands of P2 receptors,
whereas adenosine and AMP are not.25 At the same dose
as ATP (400 nmol), ADP, dATP and UTP affected
luciferase activity as much as ATP and adenosine
had no effect (Figure 7). Gene expression achieved with
AMP is intermediate between adenosine and P2 agonists.
This result can be explained by the presence of ectoadenylate kinase on the apical surface of the airway.26
The enzyme converts one molecule each of AMP and
ATP into two molecules of ADP. We hypothesize that
ecto-adenylate kinase would convert a part of exogenous
AMP into ADP, which is an agonist. Since the P1 agonist
adenosine does not impair gene transfer, the data
demonstrate that P1 receptors are not involved in the
transfection and it is known that they are not involved in
RVD either.27
Similarly to ATP, the perfusions of the nasal epithelium with plasmid DNA along with 400 nmol of P2
agonists and 50 nmol of suramin resulted in luciferase
activities as high as the ones achieved with the plasmid
DNA alone. This strongly suggests that the target of the
nucleotides and suramin is the P2 receptor and not other
ATP-binding proteins.
Figure 5 Effect of ATP on transfection of the nasal epithelium. Luciferase activities achieved following a 15-min perfusion of the nose with plasmid DNA
and up to 400 nmol of ATP; +++Po0.001 and ***Po0.0005 versus pDNA alone. Transfection of the nose with pDNA and 400 nmol of ATP for as short as
7 min; *Po0.05 versus 400 nmol of ATP for 15 min (n ¼ 3–4 mice per group).
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Mechanism of transfection of the airway epithelium with naked DNA in water
JL Lemoine et al
1279
Figure 6 Effect of suramin on transfection of the nasal respiratory tissue. Transfection of the nasal tissue with pDNA and up to 50 nmol of suramin for 15
min. Luciferase expression following delivery of plasmid DNA along with 400 nmol of ATP and 50 nmol of suramin for 15 min (ATP+suramin-15 min);
++
Po0.01 and ***Po0.0005 versus pDNA alone. Perfusions of the nasal passages with plasmid DNA and 50 nmol of suramin for up to 45 min; *Po0.05
and **Po0.005 versus 50 nmol of suramin for 15 min (n ¼ 3–4 mice per group).
Figure 7 Effects of other P2 agonists on transfection of the nasal epithelium. Transfections efficiencies achieved with pDNA, 400 nmol of nucleotide(side)s
and 50 nmol of suramin; +++Po0.001 and ***Po0.0005 versus pDNA alone (n ¼ 3–4 mice per group).
Discussion
Our data are in agreement with the model of transfection
by hypotonic shock of the nasal tissue. Indeed, we have
shown that the more hypotonic the DNA solution was,
the more potent was the transfection of the nasal
respiratory epithelium. pDNA must be delivered within
a 30-min period to take the full benefit of the hypotonic
treatment. We have demonstrated that P2 agonists,
which are known to accelerate the phase of cell shrinkage
in the process of hypotonic shock, accelerated the
transfection as well. Similarly, we have shown that a P2
antagonist, suramin, which slows down the RVD, also
slowed down the transfection.
Although the delivery of pDNA in water was first
reported by others,28–30 we herein provided some
evidence that deionized water is not solely a vehicle for
pDNA, but actively participates in the transfection
process.
Naked pDNA in deionized water specifically transfected the airway epithelial cells, as demonstrated by
immunohistochemistry for b-galactosidase, whereas
pDNA in saline did not. The major concern about the
delivery of water in the airway is the potential damage
caused by the hypotonic DNA solution to the tissue.
However, the nasal tissues that received pDNA in
deionized water were not detectably different from the
untreated tissue (Figure 2), indicating that the hypotonic
shock procedure was well tolerated by the tissue.
One may argue that a hypotonic shock might increase
the paracellular permeability of the airway epithelium
to plasmid DNA, allowing this latter to gain access to the
basolateral side of the cells. The epithelial cells would
take pDNA up from a larger surface area of plasma
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Mechanism of transfection of the airway epithelium with naked DNA in water
JL Lemoine et al
1280
membrane. However, we were unable to detect any
specific staining of the mesenchymal cells underlying
the airway epithelium (Figure 2d). If pDNA was able to
cross the airway epithelium, those underlying mesenchymal cells would have been transfected. Moreover,
from Wang et al,31 upon a hypotonic shock, the transepithelial resistance of human airway epithelial cells
drops in a short period of time, plateaus for about 3 h
and slowly increases back to the base line in about 24 h.
A similar time course was obtained with human tracheal
cells by Widdicombe et al.32 The model of transfection
by hypotonic shock involving the opening of the tight
junctions fails to explain the data shown in Figure 3.
Indeed, the preperfusion of water for 10–25 min should
not significantly impair the transfection of the airway
epithelium. On the other hand, the model of transfection
involving an exacerbated rate of uptake of pDNA is
consistent with the results of Figure 3.
Widdicombe et al32 have shown that human tracheal
cells exposed to RITC-labeled dextran (2000 kDa) in
deionized water efficiently take up this fluorescent
macromolecule. Koberna et al33 have also demonstrated
that various fluorescent low-molecular weight compounds, such as nucleotides, peptides and dyes, are
rapidly taken up by cell lines, a human primary cell
culture, drosophila and xenopus cells, as well as slices of
the rat liver by hypotonic shock. In contrast, the same
molecules were not significantly internalized when the
cells were exposed to an isotonic medium. Finally, van
der Wijk et al34 have more recently shown that the
hypotonic swelling of intestine 407 cells induces the
endocytosis of TRITC-labeled dextran (10 kDa). Numerous fluorescent vesicles appeared in the hypo-osmotically stimulated cells but not in the cells exposed to an
isotonic solution of TRITC-labeled dextran. The rate of
endocytosis increased after a lag phase of 2–3 min and
lasted for about 15 min. It is highly likely that pDNA is
also endocytosed by the nasal epithelial cells in our
study, although the direct evidence will have to be
obtained in future studies.
So far, the shortcoming of nonviral vectors was their
insufficient potency to deliver genes in the normal
airway.35 The luciferase expression achieved by our
simple nonviral system approaches that of recombinant
Sendai virus in the nose36 and is comparable to that
achieved with pDNA/PEI and pDNA/CK30PEG10K.37
Unlike viral vectors, pDNA alone is not proinflammatory, especially when the CpG dinucleotides are eliminated.38 It could therefore be repeatedly administered in
the airway.
Furthermore, several currently used antiasthmatic
drug solutions, such as cromoglycate and salbutamol,
were found to be hypo-osmolar.39,40 Consequently, it is
clinically conceivable to deliver a hypotonic pDNA
solution in the airway of patients suffering from
inherited or acquired respiratory disorders.
The nebulization of a naked DNA solution results in a
nearly complete fragmentation of the DNA molecule,
which becomes therapeutically inactive.41 The patient
does not inhale most of the misted solution, which falls
back in the reservoir of the nebulizer and is misted again.
The recirculation of the solution leads to the shear
damage of the DNA molecules. However, Trudell
Medical International (London, Ontario, Canada) has
recently developed the AeroProbet, which is an intra-
Gene Therapy
corporeal nebulizing catheter suitable for the delivery of
shear sensitive drugs (McLachlan, G et al, abstract # 496,
ASGT’s 7th annual meeting). The drug solution and a gas
flow through different capillaries composing the catheter,
which converge at the distal tip as minuscule orifices.
The pressurized gas mists the drug solution into fine
droplets. The drug solution is directly aerosolized in the
lung and does not recirculate in the catheter, which
might be used in conjunction with a bronchoscope. It is
expected that a single passage of the naked DNA
solution in this type of nebulizing catheter would not
extensively break the DNA molecules. The device may
be connected to a pump to slowly deliver a DNA
solution into the airway of CF patients, in order to take
into account of the slow rate of cell shrinkage of CF
epithelial cells after a hypotonic treatment (limited
release of ATP during the cell swelling).
Finally, this simple technique of transfection might
be used to deliver other nucleic acids in the airway, such
as siRNAs to treat viral infections (influenza), as well
as genes in other mucosa, such as the uro-genital and
the gastro-intestinal tracts.
Material and methods
Materials
All nucleotides, adenosine and suramin were purchased
from Sigma-Aldrich (St Louis, MO, USA).
Plasmid DNA
pNGVL3-luc carries the firefly luciferase gene driven by
the cytomegalovirus (CMV) promoter. It was custom
prepared as endotoxin-free DNA by Bayou Biolabs
(Harahan, LA, USA). The pSVb plasmid comprises the
b-galactosidase gene under the control of the SV40
enhancer/promoter.
Gene transfer to the murine nasal epithelium
All experiments were carried out in female CD1 mice
(6–8 weeks old, Harlan) anesthetized by intraperitoneal
injection of 2,2,2-tribromoethanol (Sigma-Aldrich). They
were handled according to the instructional guidelines
for animal care and use. As previously described by
Griesenbach et al,42 a catheter was placed 2.5 mm within
the left nostril and the diverse solutions of naked
plasmid DNA (100 mg/mouse in 75 ml of deionized
water) were perfused at a rate of 5 ml/min using a
peristaltic pump (pump P-1, Amersham Biosciences).
During the perfusion, the mice were kept in an inverted
position in order to maximize the time contact between
plasmid DNA and the nasal tissue. At 24 h after
perfusion, nasal turbinates were harvested, homogenized, frozen/thawed three times and assayed for
luciferase activity (luciferase assay, Promega, Madison,
WI, USA). The luciferase activity of untreated mice was
nullified.
b-Galactosidase immunostaining
CD1 mice were perfused with the b-galactosidase
expression plasmid pSVb, in deionized water or saline,
and with pNGVL3-luc in deionized water, as described
above. After 24 h, the nasal passages were harvested,
fixed in formalin, embedded in paraffin and sectioned.
The sections were incubated with a biotinylated anti-b-
Mechanism of transfection of the airway epithelium with naked DNA in water
JL Lemoine et al
galactosidase antibody (Sigma-Aldrich) and then a
complex avidin/biotinylated peroxidase (Vectastain,
Vector laboratories). Finally, the sections were incubated
with the peroxidase substrate AEC (3-amino-9-ethylcarbazol, Sigma-Aldrich) and counterstained with hematoxylin (Sigma-Aldrich). In the group of mice perfused
with pSVb in water, the determination of the percentage
of transfected airway epithelial cells was carried out
by counting the number of b-galactosidase-positive and
-negative cells in the airway epithelium in several
sections of the nasal tissue of three animals.
Acknowledgements
We thank Xinsheng Nan (University of Edinburgh, UK)
for the gift of the plasmid pSVb and Uta Griesenbach
(Imperial College, UK) for helpful discussions. This
study was supported by the Cystic Fibrosis Research
Trust (UK).
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