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Journal of Virology, December 1998, p. 9818-9826, Vol. 72, No. 12
Departments of
Pediatrics1 and
Internal
Medicine,2 University of Iowa College of
Medicine, Iowa City, Iowa 52242;
IntroGene, Leiden, The
Netherlands3; and
Chiron
Technologies-Center for Gene Therapy, San Diego, California
921214
Received 6 May 1998/Accepted 17 August 1998
Gene transfer with recombinant murine leukemia viruses (MuLV)
provides the potential to permanently correct inherited lung diseases,
such as cystic fibrosis (CF). Several problems prevent the application
of MuLV-based recombinant retroviruses to lung gene therapy: (i) the
lack of cell proliferation in mature pulmonary epithelia, (ii)
inefficient gene transfer with a vector applied to the apical surface,
and (iii) low titers of many retroviral preparations. We found that
keratinocyte growth factor (KGF) stimulated proliferation of
differentiated human tracheal and bronchial epithelia. Approximately
50% of epithelia divided in response to KGF as assessed by
bromodeoxyuridine histochemistry. In airway epithelia stimulated to
divide with KGF, high-titer ampho- and xenotropic enveloped vectors
preferentially infected cells from the basal side. However, treatment
with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the
mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells
expressed the transgene after gene transfer. By using this approach,
cells throughout the epithelial sheet, including basal cells, were
targeted. Moreover, the Cl Several vector systems for the
treatment of cystic fibrosis (CF) are under investigation. One
shortcoming of current nonviral vectors and recombinant adenovirus is
the short duration of transgene expression (41, 52). Since
CF is a chronic, progressive disease, it may be advantageous to develop
strategies that result in transgene integration and persistent
expression. Moloney murine leukemia virus (MuLV)-based retroviruses
have received extensive use in gene transfer studies and are approved
for human trials. However, there are limitations which prevent the
practical application of retroviral gene transfer to airway epithelia.
The mature lung presents several potential barriers for efficient gene
transfer with retroviral vectors. In vivo studies suggest that gene
transfer efficiency is low in the absence of cell proliferation (13, 16). With the exception of disease states such as CF, epithelial injury, or tumors, pulmonary epithelia are mitotically quiescent (<1% of cells dividing) (3, 9, 24, 37). Also, the secreted products of airway epithelia may cause vector entrapment in mucus or vector inactivation by a number of antimicrobial factors. For example, phagocytes resident in the lung, including alveolar macrophages, inhibit gene transfer (28, 48). Finally,
adequate titers of recombinant retrovirus preparations are required to achieve a useful multiplicity of infection (MOI) to impact the estimated 6 to 10% of epithelia required for correction of the Cl Recent studies have identified specific growth factors that stimulate
proliferation of pulmonary epithelia, including keratinocyte growth
factor (KGF) (18, 36, 44) and hepatocyte growth factor (25, 33, 36, 42, 51), suggesting a means to increase cell
proliferation. Further advances in retroviral-vector design and
concentration methods allow production of ampho- and xenotropic viruses
with titers of 108 to 109 CFU/ml (8, 19,
21, 22). We investigated the feasibility of using growth factor
stimulation and high-titer retrovirus to attain gene transfer to
differentiated human airway epithelia in vitro. We also tested the
ability of retrovirus to infect airway epithelia from the apical or
basolateral surface.
Primary culture of human airway epithelia.
Primary cultures
of human airway epithelia were prepared from trachea and bronchi by
enzymatic dispersion as previously described (23, 50, 54).
Briefly, epithelial cells were dissociated and seeded onto
collagen-coated, semipermeable membranes with a 0.4-µm pore size
(Millicell-HA; surface area, 0.6 cm2; Millipore Corp.,
Bedford, Mass.). Twenty-four hours after seeding, the mucosal medium
was removed and the cells were allowed to grow at the air-liquid
interface as reported previously (50). The culture medium
consisted of a 1:1 mixture of Dulbecco modified Eagle medium and Ham's
F-12 with 2% Ultroser G (Sepracor Inc., Marlborough, Mass.), 100 U of
penicillin per ml, and 100 µg of streptomycin per ml. Representative
preparations from all cultures were scanned by electron microscopy, and
the presence of tight junctions was confirmed by transepithelial
resistance measurements (resistance, >1,000
Reagents. (i) Growth factors.
Recombinant human KGF was a
gift from Amgen, Inc. (Thousand Oaks, Calif.). Except when stated
otherwise, KGF was applied to the basal side of differentiated airway
cells at a concentration of 50 ng/ml. KGF-containing medium was changed
every 24 h.
(ii) Recombinant retrovirus and vector formulation.
High-titer recombinant amphotropic and xenotropic retroviruses were
prepared at Chiron Technologies-Center for Gene Therapy, Inc. (San
Diego, Calif.) as described previously (7, 28). Reporter
viruses used included DA-
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influence of Cell Polarity on Retrovirus-Mediated
Gene Transfer to Differentiated Human Airway Epithelia
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
transport defect in
differentiated CF airway epithelia was corrected. These findings
suggest that barriers to apical infection with MuLV can be overcome.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
transport defect associated with CF (20).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
· cm2). All preparations used in the study were
well differentiated, and only well-differentiated cultures >2 weeks
old were used in these studies. Previous studies showed that
differentiated epithelia in this model are multilayered and consist of
ciliated cells (cytokeratin 18 positive), secretory cells containing
granules that are reactive to goblet cell- and mucous-cell-specific
antibodies, and basal cells positive for cytokeratin 14 (50,
54). This study was approved by the Institutional Review Board of
the University of Iowa.
gal (-galactosidase [-Gal] reporter, amphotropic envelope) and DX-gal (-Gal reporter,
xenotropic envelope) (19, 21). The -Gal reporter gene was
driven by retroviral long terminal repeat. The vector formulation
buffer included 19.5 mM trimethamine at pH 7.4, 37.5 mM NaCl, and 40 mg
of lactose per ml. The osmolality of the viral buffer was 105 mmol/kg,
as measured with a vapor pressure osmometer (model 5500; Wescor, Inc.,
Logan, Utah). Polybrene was included in all infection mixtures at a
concentration of 8 µg/ml.
transport to undifferentiated CF epithelia in vitro,
and a stable producer cell line was selected (21, 27). For
gene transfer to differentiated CF airway epithelia, crude producer
cell supernatants were concentrated by centrifugation and applied to
epithelia with or without KGF stimulation. Epithelia were tested for
the presence of CFTR Cl currents in Ussing chambers 3 to
10 days after gene transfer as previously described (27).
Assessment of cell proliferation by BrdU immunohistochemistry. Bromodeoxyuridine (BrdU) labeling and immunostaining were performed with a kit from Zymed Laboratories Inc. (South San Francisco, Calif.). Cells were treated with 50 ng of KGF per ml for 36 h. A 1:100 dilution of the BrdU labeling reagent was added to the culture medium, and cells were labeled for 4 h followed by fixation in 10% neutralized Formalin. BrdU histochemistry was performed by following the methods of the Zymed BrdU kit. Labeled nuclei stained brown under these conditions. Epithelial cell preparations were examined microscopically en face or in cross sections of paraffin-embedded membranes, and the percentage of brown-staining nuclei was determined. Hematoxylin or 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Molecular Probes, Eugene, Oreg.) was used for counterstaining.
-Gal expression.
Epithelial cells were fixed with 2%
paraformaldehyde-phosphate-buffered saline (PBS) solution for 20 min
and rinsed with PBS twice for 5 min each.
5-Bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) staining solution was added to the cells at 37°C for
4 h or overnight as previously described (26). Cell
membranes were examined microscopically en face or in cross sections
for -Gal expression. The percentage of -Gal-positive cells was
determined by counting a minimum of 1,000 cells from cross sections of
each treated cell culture insert.
Identification of amphotropic retroviral receptor (GLVR-2) in cultured human airway epithelia. (i) GLVR-2 antibody. Affinity-purified polyclonal GLVR-2 antisera were prepared by immunizing rabbits with a synthetic peptide (GLVR-2A), an extracellular domain sequence that is conserved in rat Ram-1 and human GLVR-2 (31, 32, 44a). The peptide was coupled to keyhole limpet hemocyanin, and then the rabbits were immunized. Different postimmunization bleeds were tested by using enzyme-linked immunosorbent assay and immobilized free peptide. The resulting antipeptide antisera were pooled and affinity purified on columns of immobilized GLVR-2A peptides. These affinity-purified antibodies were then used for all GLVR-2 expression analyses.
(ii) Western blotting. Airway epithelial cells were lysed in 10 mM Tris-HCl buffer (pH 7.4) containing 0.5% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were collected and protein concentrations were determined by the Lowry method. Thirty-five micrograms of protein in loading buffer was denatured at room temperature (not boiled) for 40 min and run on a 10% polyacrylamide-sodium dodecyl sulfate gel. Following electrophoresis, the proteins were transferred to a Nytran membrane (Schleicher & Schuell, Inc., Keene, N.H.) by electroblotting and blocked with 10% skim milk powder. Immunoblotting was performed with the polyclonal antisera at a 1:10,000 dilution. Goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase was used for the secondary antibody (Bio-Rad, Hercules, Calif.), and the proteins were identified by autoradiography by using the ECL system (Amersham, Arlington Heights, Ill.). The specificity of the antibody was confirmed by preincubating the antibody with 20 µM free synthetic peptide for 30 min in PBS-1% bovine serum albumin at room temperature prior to incubation with the blots.
Measurement of transepithelial resistance. Differentiated epithelial cells were treated with 50 ng of KGF per ml for 24 h. Solutions of water or 1.5 mM EGTA in water were used to rinse the apical side for 20 min. The solution was then replaced with viral formulation buffer alone, or a 1:1 mixture of viral formulation buffer plus 3 mM EGTA water solution, and incubated for 4 h. Control cells received KGF treatment and PBS washes instead of water or EGTA washes. Transepithelial resistance was monitored with an ohmmeter (EVOM; World Precision Instruments, Inc., Sarasota, Fla.) over 16 to 18 h until resistance returned to baseline.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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KGF induces proliferation of differentiated human airway epithelia. We first tested the hypothesis that KGF stimulates proliferation of human airway epithelia grown at the air-liquid interface (50). To determine the percentage of cells dividing and identify the cell types responsive to KGF, human airway epithelia were treated with 50 ng of KGF per ml in the basal medium for 36 h, followed by BrdU labeling and immunostaining. As shown in Fig. 1, 52% ± 8% (mean ± standard error [SE]) of the nuclei of KGF-treated airway epithelia were labeled, compared to only 7% ± 3% of control cells. Cross sections of the cell membranes demonstrated that the height of the epithelial sheets increased in KGF-treated samples and that basal cells, ciliated surface cells, and cells between the basal cell layer and the surface of the membrane divided in response to the growth factor. Apical application of KGF had the same effects as basolateral treatment (data not shown). These findings show conclusively that KGF stimulates a marked proliferative response in differentiated human airway epithelia.
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Retroviral gene transfer via the apical surfaces of proliferating
airway epithelia.
While KGF can stimulate epithelial cell
proliferation, it is not clear whether cell division is sufficient to
enhance retrovirus-mediated gene transfer to differentiated human
airway epithelia. To address this question, airway epithelia were
stimulated with 50 ng of KGF per ml for 24 h followed by
application of DA-gal amphotropic vector (MOI, ~20) to the apical
side of the membrane for 4 h. Three days after infection, X-Gal
staining was performed to evaluate transgene expression. As shown in
Fig. 2, when the vector was applied to
the apical membranes of quiescent or KGF-stimulated cells, there was no
gene transfer. When the same approach was used with xenotropic vector,
we again saw no gene transfer with the vector applied to the mucosal
surface (not shown). These results were unexpected and suggested that
either the receptors for amphotropic and xenotropic viruses were not
present or they were inaccessible to virus applied to the apical
surface.
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Expression of the amphotropic retroviral receptor GLVR-2 in human airway epithelia. Retroviral transduction begins with the interaction and binding of viral envelope glycoproteins and cell surface receptors. In the case of amphotropic enveloped vectors, the receptor (Ram-1 or GLVR-2) has been cloned and identified as a sodium-dependent phosphate transporter (31, 45), a 656-amino-acid transmembrane protein. We used a rabbit polyclonal antibody raised against a synthetic peptide sequence from the extracellular domain of GLVR-2 to learn if the receptor is expressed and whether KGF influences receptor abundance. As shown in Fig. 3, treatment of human airway epithelia with KGF for 24 h increased the abundance of the GLVR-2 protein as determined by Western blot analysis in comparison to that in untreated controls. Thus, human airway epithelia express GLVR-2, and its expression is increased by KGF. We also performed immunohistochemistry on human airway epithelia with or without KGF stimulation by using the same polyclonal antisera used for the Western blot and an additional GLVR-2 antibody (10). However, the results were inconclusive because it was not possible to localize the protein by using these reagents. Together, these data suggested that the receptor was inaccessible to virus applied to the apical surface.
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) or presence (+) of KGF. The left panels
show results in the absence of preincubation of antibody with GLVR-2
peptide. A band of ~70 kDa (indicated by arrowhead) was present in
KGF-treated cells but barely perceptible in untreated cells. The
experiment was repeated three times, with identical results.
Polarity of retroviral gene transfer to proliferating airway
epithelia.
In marked contrast to the results with apically applied
virus, vector application to the basal sides of KGF-treated epithelia resulted in efficient gene transfer, with 17% ± 1.5% (mean ± SE for six membranes from two preparations) of cells expressing the transgene. Cross sections of epithelia showed that most -Gal expressing cells were located basally in the epithelial sheet (Fig.
4). Occasional -Gal-positive cells
were noted in the cell layer above the basal cells. Occasional
-Gal-positive cells were also noted among cells that received no KGF
when the vector was applied to the basal surface, in agreement with the
lower mitotic indices of cells grown under these conditions (Fig. 1).
Similar results were obtained with xenotropic enveloped virus (data not shown). These data demonstrate that KGF-induced proliferation enhances
retroviral gene transfer when the vector is applied to the basal cell
surface.
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Increasing transepithelial permeability facilitates gene transfer from the apical side. The polarity of gene transfer to proliferating cells suggested that receptors may be accessible only from the basal side. However, the most direct means for in vivo application would be to deliver the vector to the apical surface. To address this issue, experiments were designed to test if increasing epithelial permeability would facilitate gene transfer to dividing cells with an apically applied vector. We reasoned that treatments expected to increase transepithelial permeability might allow access of virus to all epithelial cells, even when applied apically.
Two methods were tested to increase transepithelial permeability. The first was the application of water to the mucosal surface. Widdicombe and colleagues have shown that hypotonic solutions rapidly increase transepithelial permeability in cultured airway epithelia (47). The second method was the application of EGTA, a calcium chelator, under isotonic or hypotonic conditions. Since the tight junction complex is calcium dependent, EGTA treatment causes a reversible increase in paracellular permeability (1, 5, 15). Differentiated airway epithelia were first treated with 50 ng of KGF per ml for 24 h to induce proliferation. Then the apical surfaces of cells were exposed to 50 µl of water or 1.5 mM EGTA in water for 20 min. During this period, the basolateral surfaces of the cells remained in contact with the normal nutrient medium containing calcium. After the specified 20-min wash, DA-gal vector was applied to the
apical membrane at an MOI of ~20 for 4 h in formulation buffer
or buffer spiked with EGTA to a final concentration of 1.5 mM. -Gal
expression was detected by histochemical staining 3 days later. In
contrast to the previous findings of inefficient gene transfer with the
apically applied vector (Fig. 2), gene transfer was greatly enhanced
for all treatment groups. As shown in Fig. 5A and
B, 3% ± 0.5% of epithelia from
preparations pretreated with water expressed -Gal (mean ± SE
for 13 membranes from three preparations). Treatment with water and
then EGTA in water for 10 min each, followed by addition of vector,
resulted in 8% ± 1.3% (mean ± SE) positive cells (Fig. 5C and
D) (nine membranes from three preparations). A further incremental
increase in expression was seen in cells pretreated with a combination of water and EGTA for 20 min followed by addition of vector (20.3% ± 2.5% cells positive [mean ± SE for nine membranes from two
preparations] (Fig. 5E and F). Finally, cells pretreated with water
and EGTA for 20 min followed by the addition of vector containing EGTA showed 34.3% ± 5.4% -Gal-positive cells 3 days following vector delivery (mean ± SE for nine membranes from two preparations) (Fig. 5G and H). Application of vector solution, spiked 1:1 with 6 mM
EGTA to obtain a final concentration of 3 mM EGTA in the formulation
buffer, with no wash steps also resulted in ~35% transduction (data
not shown). Cells treated with the water or EGTA solution without
vector showed no evidence of increased proliferation or endogenous
-Gal activity (not shown). Importantly, cross sections of epithelia
demonstrated that these maneuvers facilitated gene transfer to cells at
all levels of the epithelial sheet, including surface cells and basally
located cells (Fig. 5). Gene transfer with apically applied xenotropic
enveloped MuLV was also enhanced in a fashion similar to that seen with
the amphotropic vector when epithelial permeability was increased by
treatment with water or EGTA (not shown).
|
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-Gal was substituted for DA-gal by using conditions
similar to those described for Fig. 5G and H. While the ecotropic
vector infected the control 3T3 mouse fibroblasts, no infection of
KGF-stimulated airway epithelia occurred (data not shown).
Retrovirus-mediated gene transfer to differentiated CF epithelia
corrects the Cl transport defect.
The feasibility of
gene transfer to correct the Cl transport defect in
differentiated CF airway epithelia was also tested. As described above
for non-CF epithelia, fully differentiated CF nasal-polyp epithelia
were pretreated for 24 h with KGF to induce proliferation and then
infected with the amphotropic retroviral vector expressing CFTR. The
DA-CFTR vector was applied apically with hypotonic buffer and EGTA as
described for Fig. 5G and H. Control cells received the DA-gal
vector. As shown in Fig. 7, when the
vector was applied apically to KGF-stimulated CF epithelia treated with
hypotonic shock and EGTA, cAMP-activated Cl current was
detectable 10 days following gene transfer. In control cells that
received the DA-gal vector, no correction of the CFTR transport
defect was detected.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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These novel findings demonstrate the feasibility of gene transfer to differentiated human airway epithelia by using MuLV-based retroviruses. First, KFG stimulated proliferation in more than 50% of cells. The use of this agent overcomes the previous limitation of low mitotic indices in differentiated airway epithelia. Second, we found that cell proliferation was required but not sufficient to support gene transfer to airway epithelia with the vector applied to the apical surface. Gene transfer was markedly polar, but the inefficiency of gene transfer from the apical surface was overcome by treatments which transiently decreased transepithelial resistance. Importantly, the percentage of transgene-expressing cells attained after increasing transepithelial permeability was within the range required for complementing defective ion and fluid transport in CF airway epithelia (20, 53).
Recombinant retroviruses were used in gene complementation studies
following the identification of the CF gene (2, 11) and have
been successfully applied in cell culture systems to transfer the CFTR
cDNA and generate cAMP-activated Cl secretion in a
variety of cell types, including human airway epithelia (2, 11,
20, 34). In vitro studies of basal and secretory cells isolated
from rabbit trachea showed that both cell populations were susceptible
to gene transfer with amphotropic vectors while actively proliferating
(16). Amphotropic MuLV vectors were also used to stably
transfer genes to human and rat pulmonary epithelia in xenografts
(12, 13). Studies of xenografts populated with human
bronchial epithelial cells demonstrated that the efficiency of gene
transfer to regenerating epithelia with 40% BrdU-positive cells was
good, while well-differentiated grafts (1% of cells BrdU positive)
showed poor infectivity with retrovirus (13). In vivo
studies of gene transfer with retrovirus to the lung are limited but
suggest that efficiency is low in the absence of injury to the
epithelium (16, 29, 38). The present study indicates that in
addition to overcoming the limitation of low mitotic indices, the low
transduction efficiency of the vector applied to the mucosal surfaces
of differentiated cells must be addressed.
The tight junction, also known as zonula occludens, is the apical-most component of the epithelial junctional complex (1). A variety of agents transiently increase epithelial permeability by disrupting tight junctions. Bhat and coworkers reported that lowering intra- or extracellular calcium levels or disrupting the cytoskeleton reversibly increased permeability in rabbit tracheal epithelium (5). Widdicombe and colleagues found that hypotonic shock from the application of water to the apical surface transiently increased the permeability of cultured bovine and human tracheal epithelia (47). Hypotonic shock reversibly increased both transcellular and paracellular permeability (47).
The efficiency of infection with retroviruses is determined in part by the availability of specific cellular receptors that mediate virus entry (30, 46). In the case of amphotropic enveloped vectors, the receptor (Ram-1 or GLVR-2) is a cell surface protein that functions as a sodium-dependent phosphate transporter (31, 32). In hematopoietic cells (35) and hepatocytes (17), levels of Ram-1 mRNA expression correlate directly with infection efficiencies. In some cases receptor abundance and infectivity are regulated by nutritional or hormonal conditions. For example, there is evidence for the regulation of Ram-1 mRNA expression by insulin, dexamethasone (49), or hypophosphatemia (10, 32, 39). Our findings suggest that, in addition to stimulating cell proliferation, KGF also increases the expression of the GLVR-2 amphotropic receptor protein.
The observation that retroviral gene transfer to proliferating airway epithelia is polar is quite striking. This may be due to (i) a polarized distribution of retroviral receptors to the basolateral membrane, (ii) inaccessibility or impaired function of apical receptors, or (iii) inhibition or inactivation of apically applied virus by secreted products of epithelia. Other than the observations that amphotropic (34) or gibbon ape leukemia virus (4) enveloped vectors can infect airway epithelia, there has been little research to characterize the abundance, cellular location, or regulation of receptor expression in airway epithelia. In support of the first explanation, conditions known to transiently disrupt the integrity of epithelial tight junctions (i.e., hypotonic shock and low Ca2+ levels) enhanced gene transfer efficiency with an amphotropic or xenotropic vector applied to the mucosal surface. Disruption of tight junctions may have also caused a transient loss of cell polarity and shifting of receptors to the apical pole. However, there is considerable precedence in epithelia for viral infection to occur in a polarized fashion. Studies with high-resistance MDCK cells showed that vesicular stomatitis virus infected them at least 100 times more efficiently when applied to the basal side than when applied to the apical surface (14). In contrast, cytomegalovirus (43) and measles virus (6) infect more efficiently from the apical surface. While virus may bind when applied apically, perhaps cytoplasmic elements involved in the internalization and translocation of the nucleocapsid are inoperative at the apical surface, thus limiting gene transfer efficiency. We found that washing the cells with PBS to remove mucus or inhibitory factors from the apical surface did not enhance gene transfer. This suggests that factors elaborated by airway epithelia, such as mucus or antimicrobial peptides, are not a barrier to gene transfer in this model.
These studies provide novel approaches to stimulate differentiated human airway epithelia to divide and to facilitate gene transfer to proliferating cells from the apical surface. Further studies of MuLV-based gene transfer and virus-receptor interactions in airway epithelia may advance the development of this integrating vector system for the treatment of lung disease.
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ACKNOWLEDGMENTS |
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We thank Phil Karp and Pary Weber for culturing the human epithelial cells and Emily Appleton and David Lewis for technical assistance. We thank Phil Karp for assistance with electrophysiology studies. We especially thank Mike Welsh, Joe Zabner, John Engelhardt, and Mike Shasby for helpful discussions. We thank Tom Ulich at Amgen, Inc., for discussions and for providing recombinant human KGF.
This work was funded by Cystic Fibrosis Foundation PO96 (P.B.M. and B.L.D.) and the Children's Miracle Network Telethon. We acknowledge the support of the Cell Culture Core, partially supported by the Cystic Fibrosis Foundation and NHLBI (PPG HL51670-05). P.B.M. is a recipient of a Career Investigator Award from the American Lung Association. B.L.D. is a fellow of the Roy J. Carver Trust.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pediatrics, University of Iowa Hospitals and Clinics, 200 Hawkins Dr., Iowa City, IA 52242. Phone: (319) 356-4866. Fax: (319) 356-7171. E-mail: paul-mccray{at}uiowa.edu.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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