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Journal of Virology, July 1999, p. 6085-6088, Vol. 73, No. 7
Departments of Medicine and Molecular and
Cellular Engineering,
Received 30 November 1998/Accepted 30 March 1999
The limitations of adeno-associated virus (AAV)-mediated vectors
for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells
and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with
glycosidases. These results indicate that transduction of airway
epithelia by AAV vectors is limited by entry and reinforce the
importance of a physical barrier on the airway surface.
Cystic fibrosis is a common, lethal
genetic disease caused by inherited defects of the cystic fibrosis
transmembrane conductance regulator (CFTR) and is characterized by
chronic lung infection and inflammation (2).
Adeno-associated virus (AAV) is currently being considered as a vector
for gene transfer into airway epithelium (7). Recent phase I
clinical trials with AAV vectors have also shown efficient and safe
delivery of the CFTR gene into epithelial cells with low levels of gene
transfer (18). AAV vectors offer several potential
advantages over adenoviral vectors, including more stable gene
expression and diminished cellular immunity to transgene products
(10, 21). However, the transduction efficiency of AAV in
vivo is rather low in the lung (15, 18). In other organs or
tissues, such as muscle, brain, and liver, infection with AAV vectors
resulted in efficient and stable gene expression (6, 11, 20,
21). The reason for the low performance of this vector system in
lungs is not fully understood.
It is the goal of this study to investigate barriers to efficient
transduction with AAV into airway epithelium. Our hypothesis is that a
physical barrier of negatively charged molecules, such as mucins or
glycosaminoglycans, or limited virus receptors at the apical surfaces
of epithelial cells limit viral entry, resulting in inefficient
transduction. Studies were performed with AAV vectors containing
enhanced green fluorescent protein (eGFP) as a reporter gene driven
from the 5' flanking region of the immediate-early gene of
cytomegalovirus (CMV) and an air-liquid interface culture system of
well-differentiated human bronchial epithelium. Recombinant AAV was
created by a transfection approach that does not require coinfection
with adenovirus (20). Cultures of human tracheobronchial epithelial cells in transwells were established and maintained as
described by Wang et al. (19) by using a medium consisting of a 50:50 mixture of Ham's F-12 medium and Dulbecco modified Eagle
medium as the base, supplemented with 2% Ultroser G (BioSepra, Villeneuve-la-Garenne Cedex, France). Differentiated cultures were
selected for experiments by measuring the transepithelial resistance
(Rt) with an ohmmeter (EVOM; World Precision
Instruments, Inc., Sarasota, Fla.). Cultures were considered confluent
and differentiated if the Rt was greater than
1,300 Initial experiments evaluated the impact of cell differentiation
on transduction. At different time points after seeding onto transwells, cultivated cells were infected with 1011
genomic particles of AAV-CMV-eGFP in 200 µl of medium applied to the upper or lower buffer compartment of the transwell. After 2 h the cultures were washed and assayed over time for gene expression. Numbers of positive cells were determined by counting ten randomly selected fields with a Nikon Diaphot inverted microscope under UV
illumination. A microscopic field usually contained 800 to 900 cells as determined by counting of nuclei under phase contrast. Cultures used had an Rt of >1,000
Transduction was much more efficient in undifferentiated cells, as
determined by GFP expression (Fig. 1A).
Total cellular DNA analyzed by DNA hybridization demonstrated
significant levels of both single- and double-stranded monomers of the
viral genome under most conditions (Fig.
2). This analysis does not specifically evaluate randomly integrated proviral DNA that would migrate with high-molecular-weight DNA as a smear. Cells infected in an
undifferentiated state (Fig. 2, lane 1) contained amounts of vector DNA
equal to those in cells that were infected in a differentiated state
(lane 2). The number of transgene-positive cells, however, was much higher in the cells infected in an undifferentiated state (Fig. 1A),
indicating that postentry processes may contribute to differences in transduction efficiency. This suggests that cellular factors present in nondifferentiated cells or absent in mature cells are necessary for efficient gene expression. The relationship between transduction and the state of differentiation has been described for
adenoviral and retroviral vectors (8, 13, 19) and proposed for AAV vectors (17); for integrating viruses, postentry
processes related to vector genome replication have been proposed
(4, 5).
We then investigated whether entry limitations contribute to the low
transduction efficiency of differentiated, polarized epithelium.
Application of AAV vector to the basolateral compartment resulted in
substantially enhanced transduction compared to transduction from
apically applied vector (Fig. 1B and 3).
Disruption of tight junctions by glycerol increased the numbers of
positive cells when vector was applied to the apical surface (Fig. 1C
and D and Fig. 3). Southern blots of total cellular DNA at day 40 of
the culture revealed increased vector DNA in cells where virus was applied from the basolateral side (Fig. 2, lane 4) or in cultures treated with glycerol (Fig. 2, lane 3) compared to cultures infected from the apical side without any further treatment (Fig. 2, lane 2).
The increased transduction efficiency following basolateral administration of vector may be due to the enhanced permissivity of
basally located cells, higher concentrations of viral receptors, or the
absence of a physical barrier. Putative AAV receptor structures, such
as heparan sulfate (12, 16) and fibroblast growth factor receptor 1 (14), have been localized to the basolateral
compartment of the respiratory epithelium (1, 3, 9).
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transduction of Well-Differentiated Airway Epithelium by
Recombinant Adeno-Associated Virus Is Limited by Vector
Entry
ABSTRACT
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/cm2 (19).
/cm2.
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FIG. 1.
Levels of transgene expression after infection of
human airway cultures in transwell cultures. (A) The status of
differentiation is an important determinant of
transduction efficiency. Cells were infected from the apical side
at different time points after seeding onto the transwells, and numbers
of transgene-expressing cells were determined 7 days
after infection. (B) In polarized epithelium, transduction efficiency
is dependent on the site of vector application. AAV was applied to
fully differentiated epithelium (after 3 weeks of culture)
from the apical or basolateral side, and the expression of the
transgene was assayed over 40 days. Recombinant adenovirus (rAd) coding
for GFP (Ad5.010-CMV-eGFP) was applied to the apical or basolateral
side of the cultures (106 particles/200 µl). This
treatment did not result in a change in the Rt
(data not shown). (C) Treatment with glycerol (10%) or pretreatment
with 0.05 g of trypsin/liter-0.05 mM EDTA for 2 min, followed by
three washes, increased the transduction efficiency of AAV vectors.
Coinfection with wild-type adenovirus (serotype 5) (Ad) resulted in
increased numbers of positive cells compared to no treatment (normal).
(D) To determine whether the apical application of glycerol or trypsin
to differentiated cultures resulted in temporal disruption of the
apical tight junctions, Rt was measured up
to 24 h after the application of the agent. Trypsinization
did not decrease the Rt compared to the medium
control (normal); however, glycerol resulted in a significant
decrease in Rt.
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FIG. 2.
Southern blots of total cellular DNA extracted
after 40 days of culture (except for lane 1, where cells were harvested
14 days after seeding and infection) were hybridized to radiolabeled
eGFP cDNA as a probe. Shown are amounts of viral DNA in cells after
infection immediately after seeding of the cells into the transwells
(lane 1); after infection of differentiated cells by apical application
with no further treatment (lane 2), apical application together with
10% glycerol (lane 3), basolateral application (lane 4), or apical
application after predigestion with trypsin for 2 min (lane 5); and
after infection of differentiated cells that had been pretreated with
neuraminidase (lane 6) or endoglycosidase H (lane 7) compared to no
treatment (lane 8). DS, double-stranded viral DNA; SS, single-stranded
viral DNA.
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FIG. 3.
Micrographs showing representative areas of the
epithelial layer using UV illumination. Cells were infected with a
standard dose of 1011 genome particles of AAV vector coding
for eGFP. All cultures were fully differentiated at the time of
infection. Different treatments are indicated at the top; the times
after infection are indicated on the right. Bar, 100 µm.
To determine whether a physical barrier at the apical surface might hinder AAV-mediated gene transfer to the airways, we analyzed the impact of enzymatic modification of surface structures on transduction frequency. Pretreatment of the culture surface with trypsin (Fig. 1C and D and Fig. 3) and several glycosidases (Fig. 4) resulted in increased transduction, whereas application of the vector together with anionic macromolecules, such as heparin or mucin, resulted in decreased numbers of positive cells (Fig. 4). Exposure of cultures to trypsin or glycerol 14 days after infection of the cells with AAV vector did not impact on transgene expression, indicating that treatment did not induce changes in postentry steps (data not shown). Capsid proteins of AAV contain many basic amino acid residues (16), making entrapment of the viral particles by negatively charged macromolecules likely. Southern blots of total cellular DNA at day 40 of the culture showed that viral DNA was more abundant in cultures treated with trypsin (Fig. 2, lane 5) or certain glycosidases (Fig. 2, lanes 6 and 7) than in cultures that received the AAV vector apically without any further treatment (Fig. 2, lane 2 or 8). This indicates that apical structures or secretions interact with the virus and limit entry, contributing to reduced transduction efficiency.
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In conclusion, the results of the present study indicate that AAV-based gene transfer to respiratory epithelium is limited by entry restrictions and postentry events, which are a function of the biology of the target tissue. Differentiated cells are far less transducible than nondifferentiated cells for reasons that go beyond differences in entry. This indicates complex interactions of cellular components with vector DNA in postentry steps. Further characterization of differentiated cells confirmed that entry is a rate-limiting step in transduction. The physical barrier created by the mucous layer clearly contributes to this block. Strategies to improve the performance of AAV vectors for lung-directed gene transfer should consider impediments to transduction at each level.
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ACKNOWLEDGMENTS |
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R. Bals and W. Xiao contributed equally to this work.
This work was supported by the Cystic Fibrosis Foundation and the NIH (P30 DK 47757 and R01 HL 49040) as well as Genovo, Inc., a biotechnology company that J. M. Wilson founded and in which he has equity. R. Bals was a recipient of a fellowship of the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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* Corresponding author. Mailing address: 3601 Spruce St., 204 Wistar Institute, Philadelphia, PA 19104-4268. Phone: (215) 898-3000. Fax: (215) 898-6588. E-mail: wilsonjm{at}mail.med.upenn.edu.
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Journal of Virology, July 1999, p. 6085-6088, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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