help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seiler, M. P.
Right arrow Articles by Zabner, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seiler, M. P.
Right arrow Articles by Zabner, J.
American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 133-140, 2002
© 2002 American Thoracic Society

Thixotropic Solutions Enhance Viral-Mediated Gene Transfer to Airway Epithelia

Michael P. Seiler, Paul Luner, Thomas O. Moninger, Philip H. Karp, Shaf Keshavjee and Joseph Zabner

Department of Internal Medicine, Division of Pharmaceutics College of Pharmacy, and Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa; and Division of Thoracic Surgery, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada

Address correspondence to: Joseph Zabner, M.D., University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. E-mail: Joseph-Zabner{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adenovirus-mediated gene transfer to airway epithelia is inefficient in part because its receptor is absent on the apical surface of the airways. Targeting adenovirus to other receptors, increasing the viral concentration, and even prolonging the incubation time with adenovirus vectors can partially overcome the lack of receptors and facilitate gene transfer. Unfortunately, mucociliary clearance would prevent prolonged incubation time in vivo. Thixotropic solutions (TS) are gels that upon a vigorous shearing force reversibly become liquid. We hypothesized that formulating recombinant adenoviruses in TS would decrease virus clearance and thus enhance gene transfer to the airway epithelia. We found that clearance of virus-sized fluorescent beads by human airway epithelia in vitro and by monkey trachea in vivo were markedly decreased when the beads were formulated in TS compared with phosphate-buffered saline (PBS). Adenovirus formulated in TS significantly increased adenovirus-mediated gene transfer of a reporter gene in human airway epithelia in vitro and in murine airway epithelia in vivo. Furthermore, an adenovirus encoding the cystic fibrosis transmembrane regulator (CFTR) gene (AdCFTR) formulated in TS was more efficient in correcting the chloride transport defect in cystic fibrosis airway epithelia than AdCFTR formulated in PBS. These data indicate a novel strategy to augment the efficiency of gene transfer to the airways that may be applicable to a number of different gene transfer vectors and could be of value in gene transfer to cystic fibrosis (CF) airway epithelia in vivo.

Abbreviations: adenovirus encoding the CFTR gene, AdCFTR • coxsackievirus adenovirus receptor, CAR • cystic fibrosis, CF • CF transmembrane regulator, CFTR • phosphate-buffered saline, PBS • transepithelial resistance, Rt • thixotropic solutions, TS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Viral vectors have been extensively studied for gene transfer to airway epithelia. These studies have demonstrated the feasibility of using adenoviruses (1–9), adeno-associated viruses (10–13), and retroviruses (14, 15) for gene transfer to the airways. Adenoviral infection of HeLa cells is very efficient and the mechanism of infection has been extensively studied (16). In contrast to HeLa cells, the efficiency of adenovirus infection of ciliated airway epithelia is limited (1, 5, 6, 8, 17–20). The inefficiency of adenovirus to infect airway epithelia appears to be due to the lack of high-affinity fiber receptor, the coxsackievirus adenovirus receptor (CAR) (2), on the apical surface (21, 22). Thus the inability of adenovirus to bind the apical surface of airway epithelia seems to be the rate-limiting step for efficient adenovirus-mediated gene transfer.

Several strategies have been employed to increase adenoviral binding to airway epithelia. These include: targeting virus to other receptors expressed by epithelia, using serotypes that bind more efficiently to the apical surface of epithelia (23–25), utilizing phage display to identify other peptide sequences that bind the apical surface (26–28), employing cationic molecules and/or calcium phosphate coprecipitation to increase the local viral concentration (3, 29) and disrupting the epithelial tight junctions to allow virus access to the basolateral-localized receptor (22). It is of interest to note that the simple strategy of increasing the viral incubation time with airway epithelia has been shown to increase the efficiency of adenovirus-mediated gene transfer (18). Unfortunately, in vivo, normal mucociliary clearance would limit the incubation time of vector, and therefore limit gene transfer.

Cilia are found throughout the tracheobronchial tree, and though the percentage of ciliated cells is higher in the large airways than in the peripheral airways, the quantitative implications on clearance are not well understood. There are ~ 200 cilia per cell (30). Each cilium is roughly 6 µm long and 0.3 µm wide. The average ciliary beat frequency ranges from 12.5–15 beats/s (Hz) (31). Each cilium is anchored through the basal body and its interaction with the microtubules and dynein arms results in a ciliary beat that propels the mucus layer (32). This ciliary beat results in clearance of insoluble particles from the lungs at a half time of 30 min to 2 h (33) or at a speed of 0.5–1.5 cm/min. Though several diseases affect mucociliary clearance (34) and several interventions result in decreased ciliary beat (neuropeptide Y, dopamine, norepinephrine) (35) or destruction of the cilia (ozone, oxygen toxicity, tobacco smoke, sulfur dioxide) (36–39), these may not be safely translated into interventions that increase epithelia contact time by gene transfer vectors.

Mucociliary clearance has been studied as a limiting factor for drug delivery to the nasal mucosa and upper airway epithelia (40, 41). It has been shown that suspensions containing methylcellulose derivatives decrease mucociliary clearance in a frog palate model, thereby increasing the bioavailability of various pharmacologic agents (42), and in human nasal epithelia (41). The decrease in mucociliary clearance may be attributable to the rheologic properties of the solutions, specifically increased viscosity and surface tension. These solutions also have the advantageous property of thixotropy (43).

Thixotropic solutions (TS) are gels that when subject to a shearing force, reversibly become liquid. This is an attractive characteristic for drug delivery because it allows even dispersal of an insoluble component by simply "shaking it." Once the insoluble agent is evenly distributed within the suspension and delivered to its target, the gel reassumes the viscoelastic properties that likely inhibit mucociliary clearance. We hypothesized that formulating recombinant adenovirus in a carboxymethyl cellulose solution exhibiting thixotropic properties would decrease virus clearance and thus enhance gene transfer to airway epithelia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human Airway Epithelia
Airway epithelial cells were obtained from trachea and bronchi of lungs removed for organ donation. Cells were isolated by enzyme digestion as previously described (44). Freshly isolated cells were seeded at a density of 5 x 105 cells/cm2 onto collagen-coated, 0.6 cm2 diameter millicell polycarbonate filters (Millipore Corp., Bedford, MA). The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and air. Twenty-four hours after plating, the mucosal media was removed and the cells were allowed to grow at the air–liquid interface (45, 46). The culture media consisted of a 1:1 mix of DMEM/Ham's F12, 2% Ultraser G (Biosepra SA, Cergy, Pontoise, France), 100 U/ml penicillin, and 100 µg/ ml streptomycin. Epithelia were tested for transepithelial resistance and morphology by Ussing chamber (Jim's Instruments, Iowa City, IA) and scanning electron microscopy, respectively. Transepithelial resistance (Rt) was measured with an ohmmeter (EVOM; World Precision Instruments, Inc., Sarasota, FL) by adding cell culture media to the apical surface and values were compared with untreated controls.

Formulation of Adenovirus Vector Delivery Vehicle
A vehicle was formulated using American Chemical Society (ACS) reagent grade and United States Pharmcopeia-National Formulary (USP-NF) excipients that facilitated uniform suspension of the adenoviral particles and resisted flow relative to phosphate-buffered saline (PBS) after application to epithelial surfaces. The vehicle consisted of an aqueous 0.6% (wt/vol) dextrose solution containing 0.25% (wt/vol) 2-phenylethyl alcohol and 0.005% (wt/vol) benzalkonium chloride, 1.0% (wt/vol) Avicel Type RC-591 (containing carboxymethylcellulose) (FMC, Philadelphia, PA) and 0.013% (wt/vol) polysorbate 80. The resultant solution was mixed thoroughly. The surface tension of TS was 36.14 ± 0.22 dynes/cm compared with the commercially available Flonase at 39.25 ± 0.28 dynes/cm, resulting in a gel that liquefied upon vigorous shaking (47, 48).

Recombinant Viruses
Recombinant adenoviral vectors expressing ß-galactosidase (Adßgal) and adenovirus encoding the CFTR cDNA (AdCFTR) were prepared as described previously (49) by the University of Iowa Gene Transfer Vector Core at titers of 1010 infectious units (I.U.)/ml. Recombinant adenovirus vectors expressing green fluorescent protein (GFP) (50, 51), AdGFP (1 x 1010 IU/ml) were made as previously described (52).

Viral Infection
Epithelia were allowed to reach confluence and develop an Rt indicating the development of tight junctions and an intact barrier. All epithelia had values of Rt > 500 {Omega} cm2. Fourteen days after seeding, 50 MOI of the recombinant adenoviruses in PBS were added to the apical surface diluted in different ratios of TS. Following the indicated incubation time, the viral suspension was removed and epithelia were rinsed twice with PBS. After infection, the monolayers were incubated at 37°C for an additional 30 to 72 h. Rt was measured with an ohmmeter (EVOM; World Precision Instruments) before infection. Transepithelial resistance was not altered by application of virus.

Measurement of Gene Transfer
We measured total ß-galactosidase activity using a commercially available method (Galacto-Light; Tropix, Inc., Bedford, MA). Briefly, after rinsing with PBS, cells were removed from filters by incubation with 120 µl lysis buffer (25 mM Tris-phosphate, pH 7.8; 2 mM DTT; 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; 10% glycerol; and 1% Triton X-100) for 15 min. Light emission was quantified in a luminometer (Analytical Luminescence Laboratory, San Diego, CA).

To analyze infectivity patterns in AdGFP-infected cells, epithelia were fixed with 4% paraformaldehyde and filter supports were removed and mounted on glass slides using vectashield (Vector Laboratories, Burlingame, CA) mounting media. Cells were imaged using a BioRad 600RC confocal microscope fitted with a 1x lens to show infection pattern on the entire epithelia.

Measurement of Mucociliary Clearance by Microscopy
To measure mucociliary clearance we suspended 100 nm FITC-labeled microspheres (Molecular Probes Inc., Eugene, OR) in PBS or TS, at 1:100 or 1:1,000,000 dilutions. One microliter of each preparation was added to either cultured airway epithelia grown on filter supports, or freshly excised rhesus monkey trachea. Excised tissue and cultured human airway epithelia were kept on a heated stage to keep specimens at 37°C for duration of the experiments. Using a Biorad 600RC confocal microscope, monkey trachea and human airway epithelia were scanned every 5 s for 2 min. Data were quantified using BioRad Lasersharp confocal software to calculate ciliary clearance as microns per second.

Measurement of Transepithelial Electrical Properties
The epithelial monolayers were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA) as previously described (53). Epithelia were bathed on the submucosal surface with a Ringer's solution containing, in mM: 135 NaCl, 2.4 KH2PO4, 0.6 KH2PO2, 1.2 CaCl2, 1.2 MgCl2, 10 Hepes (titrated to pH 7.4 with NaOH) and 10 dextrose. The mucosal solution was identical with the exception that NaCl was replaced with 135 Na gluconate. Amiloride (10 µM) was added to the mucosal solution to inhibit Na+ channels and thereby transepithelial Na+ transport. The cAMP agonists, forskolin 10 µM and IBMX 100 µM, were added to the mucosal and submucosal solutions to stimulate trans- epithelial Cl- current through CFTR Cl- channels. To assess total Cl- current, we then added 100 µM bumetanide to the submucosal solution and calculated the difference between current after forskolin/IBMX and current after bumetanide.

Transmission Electron Microscopy
Traditional aqueous fixatives (e.g., gluteraldehyde, osmium tetroxide) for electron microscopy are quite adequate for preserving the ultrastructure of airway epithelia. However, the application of aqueous buffers and fixatives to the lumen of an airway will result in the distortion or complete loss of the mucus blanket covering the epithelia. If the observation of this mucus layer or of other materials in contact with the epithelia is the goal, an alternative method of fixation must be pursued. To this end we employed 1% osmium tetroxide dissolved in perfluorocarbon (Fluorinert FC-72; 3M, St. Paul, MN) as the fixative to preserve the mucus layer, and TS-formulated adenovirus. Cell cultures were gently immersed in the perfluorocarbon (PFC) fixative for 1–2 h followed by one rinse in pure PFC. The samples were then dehydrated in three washes of 100% ethanol (3 x 6 min), transitioned to Eponate-12 resin (Ted Pella Inc., Redding, CA) (50:50 resin:100% ethanol) for 30 min at room temperature, three changes of 1 h in 100% Eponate-12 resin, and cured overnight at 65°C. Ultrathin sections were post-stained with uranyl acetate and lead citrate and imaged in a Hitachi H-7000 transmission electron microscope.

Studies in Mice
For in vivo analysis, 6- to 8-wk-old C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were studied. Mice were lightly anesthetized using a methoxyflurane chamber. 3 x 1010 particles of Adßgal were suspended in either PBS or TS and administered intranasally in two 62.5 µl instillations, delivered 5 min apart. The experiment was performed with four animals per group. Twenty-eight days after vector administration, animals were killed with CO2. PBS (10 ml) was instilled into the right ventricle and then the lungs and heart were removed intact. The trachea was intubated and instilled at 10 cm H2O of pressure with the following solutions in order: PBS, 4% paraformaldehyde, PBS, overnight with X-Gal stain, and finally rinsed with PBS. Lungs were cryosectioned and two independent reviewers that were unaware of the experimental identity of the samples analyzed sections. The reviewers counted the number of blue nuclei of ßgal-expressing cells from a 5-µm slice obtained every 50 µm (n = 20 fields/lung).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TS on Mucociliary Clearance of Virus-Sized Particles
When adenovirus is added to the surface of cultured ciliated human airway epithelia, the virus is moved around by the mucociliary clearance, leading to viral spread (54). Similarly, Matsui and coworkers (55) showed that cultures of human airway epithelia swirl the mucus layer in a counterclockwise fashion. Thus, we tested the effect on mucociliary clearance of 100-nm fluorescent beads formulated in either PBS or TS on human airway epithelia. Mucociliary clearance of the beads was recorded immediately by taking serial confocal images every 5 s for 2 min. Figure 1 shows the merged serial images of the beads formulated in PBS (Figure 1A) versus beads formulated in TS (Figure 1C). PBS-formulated beads generally moved in a uniform direction, appearing like "spaghetti" on the merged images. In marked contrast, merged serial images of TS-formulated beads resulted in an overlap of fluorescent signals, suggesting a reduction in movement. To better characterize bead movement, experiments were repeated using a PBS and TS formulation containing 104 fewer beads. Figures 1B and 1D show images that allow recognition of individual beads, and confirm the reduction in movement with the TS formulation. (To visualize ciliary clearance of the beads over time, please see website for a representative movie [http://genetherapy.genetics.uiowa.edu/TSfinal/movies.html].) Quantitative analysis of the clearance rates of five different experiments showed that the PBS-suspended particles were propelled at 30 to 40 µm/s, whereas the TS-suspended beads were moved at a significantly reduced rate (Figure 2A) . The alcohol 2-phenylethanol alone had no effect on ciliary function. These data suggest that particles at the surface of primary cultures of human airway epithelia are subjected to mucociliary movement. Furthermore, the TS solution appears to arrest or delay the clearance of fluorescent particles on the surface of epithelia.



View larger version (154K):
[in this window]
[in a new window]
  		Figure 1. Thixotropic solution inhibits mucociliary clearance of virus- sized particles. 100 nm FITC-labeled micro-spheres were suspended in PBS (A and B) or TS (C and D), at 1:100 or 1:1,000,000 (vol/vol) dilutions. One microliter of each preparation was then added to human airway epithelia. Epithelia were scanned every 5 s for 2 min using video-confocal microscopy. Confocal images are stacked time series, to illustrate uniform ciliary movement of virus-sized beads. Scale bar is 1 mm. Data from five different experiments.

 


View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. Quantification of the effect of a thixotropic solution on mucociliary clearance. The trajectory of fluorescent beads on airways was quantified using Laser-sharp 6.0 confocal software. The data represents ciliary movement of beads in microns per second. (A) Represents ciliary clearance of 100 nm fluorescent particles on an in vitro model of human airway epithelia. (B) Represents ciliary clearance of fluorescent particles on a monkey trachea model in vivo. *P < 0.05. Data from five different experiments.

 
To test the effect of TS on mucociliary clearance on the tracheal mucociliary escalator, we obtained freshly excised rhesus monkey tracheal specimens, and measured mucociliary clearance by fluorescent microscopy. In the PBS control (Figure 2A), the fluorescent beads were rapidly cleared toward the vocal cords at a 15 to 20 µm/s (for a representative movie please check http://genetherapy. genetics.uiowa.edu/TSfinal/movies.html). Figure 2B shows the calculated rate of clearance of the fluorescent beads was reduced 5-fold when formulated in TS compared with PBS. The effect of TS was completely and immediately reversible after rinsing the trachea with PBS. These data suggest that virus-size particles are rapidly cleared by mucociliary clearance in the trachea and that TS can decrease the clearance.

Effect of TS on Cilia
To evaluate the mechanism by which TS arrests mucociliary clearance of virus-sized particles, recombinant adenovirus formulated in PBS or TS was added to the apical surface of human airway epithelia, the cells were subsequently fixed using a perfluorochemical emulsion containing osmium tetroxide fixative. This technique allows fixation without dehydration steps that usually disrupt the mucous sol and gel layer of airway surface liquid. Figures 3A and 3B show characteristic transmission electron micrographs of human airway epithelia exposed to PBS-formulated adenovirus. The figures show the normal airway surface liquid compartment, as well as cilia at various stages of movement. Some cilia perpendicular to the surface with mucus on top of the sol layer can be seen on Figure 3B. Adenovirus capsids are visible in the sol layer (solid arrows). In contrast to what was seen in the PBS conditions, the cilia of epithelia exposed to adenovirus in TS appeared disoriented (Figures 3C and 3D). First, in the presence of TS the cilia appear flattened against the surface of the epithelia. Second, abnormal bends or kinks on the cilia of ~ 100° were observed close to the cell surface. Third, adenovirus can be seen adjacent to the cell membrane. These data suggest that the altered mucoliliary function induced by TS may be explained by changes in cilia morphology; likely the cilia are compressed by TS material.



View larger version (184K):
[in this window]
[in a new window]
  		Figure 3. Thixotropic solution affects morphology of human airway epithelia cilia. Transmission electron micrograph of human airway epithelia cells treated with either PBS (A and B) or TS (C and D). Samples were fixed for transmission electron microscopy using a perfluorocarbon emulsion procedure, to preserve the airway surface liquid and mucous layers. Arrows indicate adenovirus virions. Scale bar is 5 µm.

 
TS Increases Adenovirus-Mediated Gene Transfer to Airway Epithelia In Vitro and In Vivo
We investigated the ability of adenovirus to infect human airway epithelia through the apical side with a relatively low MOI of 50 and a short incubation time of 1 h. Because adenovirus-calcium coprecipitates (Ca:Pi) were previously shown to augment gene transfer (3), we compared the Adßgal-mediated gene transfer of TS-formulated adenovirus, PBS-formulated adenovirus, and adenovirus precipitated in Ca:Pi. We found that TS-formulated adenovirus resulted in a 1- to 2-log increase in gene transfer over PBS-formulated Adßgal and it was also more efficient than Ca:Pi-formulated adenovirus (Figure 4A) . Moreover, adenovirus-mediated CFTR gene transfer to cystic fibrosis airway epithelia resulted in only a partial correction of the chloride transport defect when AdCFTR was formulated in PBS and a correction similar to values seen in non-CF airway epithelia, when the virus was formulated in TS (Figure 4B). These data suggest that adenovirus formulated in TS results in enhanced gene transfer to both non-cystic fibrosis and cystic fibrosis airway epithelia in vitro.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 4. Thixotropic solution increases gene transfer to airway epithelia both in vitro and in vivo. (A) Human airway epithelia were treated with 50 MOI Adßgal suspended in PBS, CaPi, or TS for 1 h, or left untreated in the control condition. After 48 h, cells were assayed for ßgal activity. Data from n = 12; three different experiments. (B) CF epithelia were treated with 50 MOI of AdCFTR suspended in PBS or TS for 1 h. After 48 h, cells were studied for sensitive chloride current in an Ussing chamber. Data from n = 4. (C) C57 black mice were instilled with 3 x 109 particles of Adßgal suspended in either PBS or TS. Data from n = 8; two different experiments. Three days later mice were killed, their lungs extracted, fixed, and stained for ß-galactosidase activity. Data are represented as blue cells per 10 x field. *P < 0.05.

 
To compare the transduction efficiency of TS-formulated and PBS-formulated adenovirus in vivo, we administered either preparation with 3 x 1010 particles of Adßgal to 6- to 8-wk-old C57BL/6 mice, in a total volume of 125 µl via nasal instillation. After 3 d the mice were killed, and the lungs were fixed and stained with X-Gal. We observed a low-level transduction of airway epithelia cells in mice treated with PBS-formulated Adßgal (Figure 4C). In contrast, we found a significant increase in the number of transduced airway epithelia cells in the lungs of mice treated with TS-formulated Adßgal. These data support the in vitro observation that TS formulation enhances gene transfer to the luminal surface of airway epithelia in vivo.

TS Enhances Gene Transfer by Immobilizing the Virus on the Airway Epithelia
To further investigate the enhancement of adenovirus-mediated gene transfer to airway epithelia by TS, we considered the possibility that TS disrupts the tight junctions, allowing viral access to the basolateral-localized receptor. We measured transepithelial resistance of well differentiated human airway epithelia exposed to PBS or TS via the apical side after 1 h and found no significant difference in Rt between the two groups (Figure 5) . We also measured the Rt every 5 min for 1 h, and found no changes after the addition of 10 µl TS. These data suggest the effect of TS does not depend on disruption of the epithelial barrier.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 5. Thixotropic solutions do not alter trans- epithelial resistance. Cells were exposed via the apical side to either 50 µl TS or PBS, and incubated at 37°C for 1 h. After this intervention, transepithelial resistances were measured with an ohmmeter and expressed as m{Omega}.cm2. Data from n = 6; two different experiments.

 
We reasoned that if TS-formulated adenovirus works by altering mucociliary clearance, then applying a small drop of TS-formulated adenovirus on ciliated human airway epithelia would result in gene transfer only where the drop is applied. To test this hypothesis we added 1 µl of Ad2GFP formulated either in PBS or TS for 1 h to the apical surface of ciliated human airway epithelia. Three days later, the few cells expressing GFP in the PBS-formulated AdGFP condition were seen throughout the entire 0.6 cm2 area of the epithelia; whereas the TS-formulated AdGFP infection resulted in a concentration of GFP-positive cells localized in a small area of the filter where the drop was applied (Figures 6A and 6B) .



View larger version (55K):
[in this window]
[in a new window]
  		Figure 6. Effect of volume on adenovirus-mediated gene transfer to human airway epithelia. Human airway epithelia were exposed to 5 MOI of AdGFP in a very small volume (1 µl) of either PBS (A) or TS (B) for 1 h. Two days later epithelia were fixed and imaged by fluorescent microscopy using a 1x objective lens. The figure shows a low-level infection with PBS-formulated virus seen throughout the epithelia, whereas the TS-formulated virus infects only on the site of application. The bright spots represent GFP expressing cells. (C) Shows the quantitative ß-galactosidase expression by epithelia infected with increasing volumes of PBS and TS-formulated adenovirus. As the volume of TS-formulated virus covers the entire epithelia, the level of gene transfer increases. Scale bar is 0.25 cm.

 
These data suggested the volume of TS-formulated adenovirus would have to cover the entire epithelia to result in maximum and uniform gene transfer. To test the effect of increased surface area coverage by TS-formulated adenovirus, we increased the delivery volume while keeping the number of I.U. constant. Three days after virus exposure, ßgal activity was measured as an indicator of gene transfer. As predicted, there was an increase in the total amount of ßgal activity when the volume increased from 1 to 50 µl, which correlated with the ability of the volume to cover the entire area of the epithelium. Not surprisingly, as volume increased over 50 µl the amount of gene transfer decreased. In the in vitro model of airway epithelia, volumes over 50 µl increase the thickness of the TS-virus solution above the epithelia, likely decreasing the local concentration to which the cells are exposed. These data further confirm that TS-formulated adenovirus results in increased gene transfer to ciliated airway epithelia by decreasing mucociliary spread and clearance.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because human ciliated airway epithelia lack high-affinity fiber receptor activity on the apical surface (17, 18), they are relatively resistant to adenovirus infection and adenovirus-mediated gene transfer. Increasing the time adenovirus is exposed to airway epithelia increases infectivity to a moderate level (18); however, mucociliary clearance in vivo is likely to prevent prolonged contact time. Here, we test a microcrystalline cellulose suspension that exhibits thixotropic properties as a vehicle for adenovirus-mediated gene transfer to the airways. Thixotropic properties describe gels that, upon application of a shearing force, reversibly become liquid. We tested whether formulating adenovirus in TS could inhibit ciliary clearance to increase viral contact time, and thereby increase adenovirus-mediated gene transfer.

We show that virus-sized particles suspended in TS are cleared at a slower rate, compared with particles in PBS in an in vitro model of human airway epithelia and in an in vivo monkey trachea. Electron microscopy data suggest that the ability of TS to inhibit mucociliary function correlates with altered morphology of cilia on the airway. This effect appears to be reversible when tested in monkey trachea; we found that cilia clearance was restored after 6 h. Moreover, similar methylcellulose formulations of pharmaceuticals targeted to nasal airway epithelia have been extensively used and they appear to be safe. Accordingly, we conclude that TS is effective at inhibiting mucociliary clearance and results in increased efficiency of adenovirus-mediated gene transfer to airway epithelia. This increase in efficiency allowed us to complement the CF defect with a relatively low multiplicity of infection. Other attempts to increase vector residence time on airway epithelia have employed intratracheal instillation of perfluorochemical liquids containing various viral vectors (56). Although preliminary data for this approach is very promising for diseases that require mechanical ventilation, our present strategy has the advantage that it can potentially be delivered by aerosolization, and obviates the need for mechanical ventilation.

Would this be relevant for CF airway epithelia? Recently, Matsui and coworkers showed in an in vitro model that lack of CFTR results in decreased airway surface liquid volume and impaired mucociliary clearance (57). In vivo studies have yielded conflicting results (58, 59). Nevertheless, we found a significant improvement with TS in adenovirus-mediated gene transfer in our in vitro model of CF airway epithelia, suggesting that mucociliary movement in CF epithelia was also reduced by TS.

Novel strategies for gene transfer to lung epithelia cells have recently emerged. Creation of new or modified viral vectors can result in recombinant viruses with a specific tropism for airway epithelia. Once these vectors have been rigorously tested for the ability to specifically bind and infect airway cells in culture, they will meet the limitation of mucociliary clearance, potentially reducing their ability to infect. By developing an alternative delivery vehicle for transiently inhibiting mucociliary clearance, we would predict that the efficiency of gene transfer would improve regardless of the vector used. We show that reducing mucociliary clearance increases receptor-independent infection of airway epithelia with adenovirus. Finally, advancements in vehicle formulation specifically tailored for delivery to the airway may provide insight into other methods to increase the therapeutic potency and viability of vector-mediated gene therapy.


   Acknowledgments
 
The authors thank Deana Van DerKamp, Janice Launspach, Pary Weber, Phil Karp, Theresa Mayhew, and Rosanna Smith for excellent assistance. They especially appreciate the help of Michael Welsh and Dwight Look for stimulating discussions. They appreciate the support of the Gene Transfer Vector Core, the Cell Culture Core and the Morphology Core of the Gene Therapy Center at the University of Iowa. This work was supported by the National Heart Lung and Blood Institute PPG.

Received in original form December 6, 2001

Received in final form February 25, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Arcasoy, S. M., J. Latoche, M. Gondor, S. C. Watkins, R. A. Henderson, R. Hughey, O. J. Finn, and J. M. Pilewski. 1997. MUC1 and other sialoglycoconjugates inhibit adenovirus-mediated gene transfer to epithelial cells. Am. J. Respir. Cell Mol. Biol. 17:422–435.[Abstract/Free Full Text]
  2. Bergelson, J. M., J. A. Cunningham, G. Droguett, E. A. Kurt-Jones, A. Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320–1323.[Abstract/Free Full Text]
  3. Fasbender, A., J. H. Lee, R. W. Walters, T. O. Moninger, J. Zabner, and M. J. Welsh. 1998. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo. J. Clin. Invest. 102:184–193.[Medline]
  4. Ginsberg, H. S. 1984. The Adenoviruses. Plenum Press, New York.
  5. Goldman, M., Q. Su, and J. M. Wilson. 1996. Gradient of RGD-dependent entry of adenoviral vector in nasal and intrapulmonary epithelia: implications for gene therapy of cystic fibrosis. Gene Ther. 3:811–818.[Medline]
  6. Grubb, B. R., R. J. Pickles, H. Ye, J. R. Yankaskas, R. N. Vick, J. F. Engelhardt, J. M. Wilson, L. G. Johnson, and R. C. J. Boucher. 1994. Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans. Nature 371:802–806.[Medline]
  7. Joseph, P. M., B. P. O'Sullivan, A. Lapey, H. Dorkin, J. Oren, R. Balfour, M. A. Perricone, M. Rosenberg, S. C. Wadsworth, A. E. Smith, J. A. St George, and D. P. Meeker. 2001. Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis: I. methods, safety, and clinical implications. Hum. Gene Ther. 12:1369–1382.[Medline]
  8. Knowles, M. R., K. W. Hohneker, Z. Zhou, J. C. Olsen, T. L. Noah, P. C. Hu, M. W. Leigh, J. F. Engelhardt, L. J. Edwards, K. R. Jones, M. Grossman, J. M. Wilson, L. G. Johnson, and R. C. Boucher. 1995. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N. Engl. J. Med. 333: 823–831.[Abstract/Free Full Text]
  9. Perricone, M. A., J. E. Morris, K. Pavelka, M. S. Plog, B. P. O'Sullivan, P. M. Joseph, H. Dorkin, A. Lapey, R. Balfour, D. P. Meeker, A. E. Smith, S. C. Wadsworth, and J. A. St George. 2001. Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis: II. transfection efficiency in airway epithelium. Hum. Gene Ther. 12:1383–1394.[Medline]
  10. Duan, D., Y. Yue, Z. Yan, P. B. McCray, and J. F. Engelhardt. 1998. Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia. Hum. Gene Ther. 9:2761–2776.[Medline]
  11. Ferrari, F. K., T. Samulski, T. Shenk, and R. J. Samulski. 1996. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J. Virol. 70:3227–3234.[Abstract]
  12. Flotte, T. R., and B. J. Carter. 1995. Adeno-associated virus vectors for gene therapy. Gene Ther. 2:357–362.[Medline]
  13. Zabner, J., M. Seiler, R. Walters, R. M. Kotin, W. Fulgeras, B. L. Davidson, and J. A. Chiorini. 2000. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J. Virol. 74:3852–3858.[Abstract/Free Full Text]
  14. Johnson, L. G., J. P. Mewshaw, H. Ni, T. Friedmann, R. C. Boucher, and J. C. Olsen. 1998. Effect of host modification and age on airway epithelial gene transfer mediated by a murine leukemia virus-derived vector. J. Virol. 72:8861–8872.[Abstract/Free Full Text]
  15. Wang, G., P. L. Sinn, and P. B. McCray, Jr. 2000. Development of retroviral vectors for gene transfer to airway epithelia. Curr. Opin. Mol. Ther. 2:497–506.[Medline]
  16. Greber, U. F., M. Willetts, P. Webster, and A. Helenius. 1993. Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75:477–486.[Medline]
  17. Zabner, J., P. Freimuth, A. Puga, A. Fabrega, and M. J. Welsh. 1997. Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J. Clin. Invest. 100:1144–1149.[Medline]
  18. Zabner, J., B. G. Zeiher, E. Friedman, and M. J. Welsh. 1996. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J. Virol. 70:6994–7003.[Abstract/Free Full Text]
  19. Pickles, R. J., P. M. Barker, H. Ye, and R. C. Boucher. 1996. Efficient adenovirus-mediated gene transfer to basal but not columnar cells of cartilaginous airway epithilia. Hum. Gene Ther. 7:921–931.[Medline]
  20. Goldman, M. J., and J. M. Wilson. 1995. Expression of {alpha}vB5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J. Virol. 69:5951–5958.[Abstract]
  21. Pickles, R. J., D. McCarty, H. Matsui, P. J. Hart, S. H. Randell, and R. C. Boucher. 1998. Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer. J. Virol. 72:6014–6023.[Abstract/Free Full Text]
  22. Walters, R. W., T. Grunst, J. M. Bergelson, R. W. Finberg, M. J. Welsh, and J. Zabner. 1999. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J. Biol. Chem. 274:10219–10226.[Abstract/Free Full Text]
  23. Drapkin, P. T., C. R. O'Riordan, S. M. Yi, J. A. Chiorini, J. Cardella, J. Zabner, and M. J. Welsh. 2000. Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia. J. Clin. Invest. 105:589–596.[Medline]
  24. Haisma, H. J., J. Grill, D. T. Curiel, S. Hoogeland, V. W. van Beusechem, H. M. Pinedo, and W. R. Gerritsen. 2000. Targeting of adenoviral vectors through a bispecific single-chain antibody. Cancer Gene Ther. 7:901–904.[Medline]
  25. Wickham, T. J. 2000. Targeting adenovirus. Gene Ther. 7:110–114.[Medline]
  26. Romanczuk, H., C. E. Galer, J. Zabner, G. Barsomian, S. C. Wadsworth, and C. R. O'Riordan. 1999. Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice. Hum. Gene Ther. 10:2615–2626.[Medline]
  27. Romanczuk, H., C. E. Galer, J. Zabner, G. Barsomian, S. C. Wadsworth, and C. R. O'Riordan. 1999. Dressing up adenoviruses to modify their tropism: interview by Florence Paillard. Hum. Gene Ther. 10:2575–2576.[Medline]
  28. Zabner, J., M. Chillon, T. Grunst, T. O. Moninger, B. L. Davidson, R. Greg- ory, and D. Armentano. 1999. A chimeric type 2 adenovirus vector with a type 17 fiber enhances gene transfer to human airway epithelia. J. Virol. 73:8689–8695.[Abstract/Free Full Text]
  29. Walters, R., and M. Welsh. 1999. Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer. Gene Ther. 6:1845–1850.[Medline]
  30. Satir, P., and M. A. Sleigh. 1990. The physiology of cilia and mucociliary interactions. Annu. Rev. Physiol. 52:137–155.[Medline]
  31. Sleigh, M. A., J. R. Blake, and N. Liron. 1988. The propulsion of mucus by cilia. Am. Rev. Respir. Dis. 137:726–741.[Medline]
  32. Puchelle, E., J. M. Zahm, C. Duvivier, J. Didelon, J. Jacquot, and D. Quemada. 1985. Elasto-thixotropic properties of bronchial mucus and polymer analogs: I. Experimental results. Biorheology 22:415–423.[Medline]
  33. Wanner, A., M. Salathe, and T. G. O'Riordan. 1996. Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154(6, Pt. 1):1868–1902.[Medline]
  34. Brody, S. L., X. H. Yan, M. K. Wuerffel, S. K. Song, and S. D. Shapiro. 2000. Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am. J. Respir. Cell Mol. Biol. 23:45–51.[Abstract/Free Full Text]
  35. Berggren, L. 1970. Further studies on the effect of autonomous drugs on in vitro secretory activity of the rabbit eye ciliary processes. A. Inhibition of the pilocarpine effect by isopilocarpine, arecoline, and atropine. B. Influence of isoproterenol and norepinephrine. Acta Ophthalmol. (Copenh.) A8:293–302.
  36. Fraser, D. A., M. C. Battigelli, and H. M. Cole. 1968. Ciliary activity and pulmonary retention of inhaled dust in rats exposed to sulfur dioxide. J. Air Pollut. Control Assoc. 18:821–823.[Medline]
  37. Laurenzi, G. A., S. Yin, and J. J. Guarneri. 1968. Adverse effect of oxygen on tracheal mucus flow. N. Engl. J. Med. 279:333–339.
  38. Albert, R. E., M. Lippmann, and W. Briscoe. 1969. The characteristics of bronchial clearance in humans and the effects of cigarette smoking. Arch. Environ. Health 18:738–755.[Medline]
  39. Carnow, B. W., R. M. Senior, R. Karsh, S. Wessler, and L. V. Avioli. 1970. The role of air pollution in chronic obstructive pulmonary disease. JAMA 214:894–899.[Medline]
  40. Merkus, F. W., J. C. Verhoef, N. G. Schipper, and E. Marttin. 1998. Nasal mucociliary clearance as a factor in nasal drug delivery. Adv. Drug Deliv. Rev. 29:13–38.[Medline]
  41. Zhou, M., and M. D. Donovan. 1996. Intranasal mucociliary clearance of putative bioadhesive polymer gels. Int. J. Pharm. 135:115–125.
  42. Harris, A. S., E. Svensson, Z. G. Wagner, S. Lethagen, and I. M. Nilsson. 1988. Effect of viscosity on particle size, deposition, and clearance of nasal delivery systems containing desmopressin. J. Pharm. Sci. 77:405–408.[Medline]
  43. Dolz, M., J. Bugaj, J. Pellicer, M. J. Hernandez, and M. Gorecki. 1997. Thixotropy of highly viscous sodium (carboxymethyl) cellulose hydrogels. J. Pharm. Sci. 86:1283–1287.[Medline]
  44. Rich, D. P., L. A. Couture, L. M. Cardoza, V. M. Guiggio, D. Armentano, P. C. Espino, K. Hehir, M. J. Welsh, A. E. Smith, and R. J. Gregory. 1993. Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. Hum. Gene Ther. 4:461–476.[Medline]
  45. Kondo, M., W. E. Finkbeiner, and J. H. Widdicombe. 1991. Simple technique for culture of highly differentiated cells from dog tracheal epithelium. Am. J. Physiol. 261:L106–L117.[Abstract/Free Full Text]
  46. Yamaya, M., W. E. Finkbeiner, S. Y. Chun, and J. H. Widdicombe. 1992. Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. 262:L713–L724.[Abstract/Free Full Text]
  47. Dolz-Planas, M., F. Gonzalez-Rodriguez, R. Belda-Maximino, and J. V. Herraez-Dominguez. 1988. Thixotropic behavior of a microcrystalline cellulose-sodium carboxymethylcellulose gel. J. Pharm. Sci. 77:799–801.[Medline]
  48. Dolz-Planas, M., C. Roldan-Garcia, J. V. Herraez-Dominguez, and R. Belda-Maximino. 1991. Thixotropy of different concentrations of microcrystalline cellulose: sodium carboxymethyl cellulose gels. J. Pharm. Sci. 80:75–79.[Medline]
  49. Welsh, M. J., J. Zabner, S. M. Graham, A. E. Smith, R. Moscicki, and S. C. Wadsworth. 1995. Adenovirus-mediated gene transfer for cystic fibrosis: A. Safety of dose and repeat administration in the nasal epithelium. B. Clinical efficacy in the maxillary sinus. Hum. Gene Ther. 6:205–218.[Medline]
  50. Flotte, T. R., S. E. Beck, K. Chesnut, M. Potter, A. Poirier, and S. Zolotukhin. 1998. A fluorescence video-endoscopy technique for detection of gene transfer and expression. Gene Ther. 5:166–173.[Medline]
  51. Zolotukhin, S., M. Potter, W. W. Hauswirth, J. Guy, and N. Muzyczka. 1996. A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70:4646–4654.[Abstract]
  52. Bai, M., B. Harfe, and P. Freimuth. 1993. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J. Virol. 67:5198–5205.[Abstract/Free Full Text]
  53. Zabner, J., S. C. Wadsworth, A. E. Smith, and M. J. Welsh. 1996. Adenovirus-mediated generation of cAMP-stimulated Cl- transport in cystic fibrosis airway epithelia in vitro: effect of promoter and administration method. Gene Ther. 3:458–465.[Medline]
  54. Novoa, E., E. Langewisch, T. M. Grunst, and J. Zabner. 1999. Pathogenesis of Wild Type Adenovirus Infection on Human Airway Epithelia. ATS 1999, 95th International Conference. ATS Publications, San Diego, CA. B29.
  55. Matsui, H., S. H. Randell, S. W. Peretti, C. W. Davis, and R. C. Boucher. 1998. Coordinated clearance of periciliary liquid and mucus from airway surfaces. J. Clin. Invest. 102:1125–1131.[Medline]
  56. Weiss, D. J., T. P. Strandjord, D. Liggitt, and J. G. Clark. 1999. Perflubron enhances adenovirus-mediated gene expression in lungs of transgenic mice with chronic alveolar filling. Hum. Gene Ther. 10:2287–2293.[Medline]
  57. Matsui, H., B. R. Grubb, R. Tarran, S. H. Randell, J. T. Gatzy, C. W. Davis, and R. C. Boucher. 1998. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95:1005–1015.[Medline]
  58. Rutland, J., and P. J. Cole. 1981. Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis. Thorax 36:654–658.[Abstract]
  59. Regnis, J. A., M. Robinson, D. L. Bailey, P. Cook, P. Hooper, H. K. Chan, I. Gonda, G. Bautovich, and P. T. Bye. 1994. Mucociliary clearance in patients with cystic fibrosis and in normal subjects. Am. J. Respir. Crit. Care Med. 150:66–71.[Abstract]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. L. Farmen, P. H. Karp, P. Ng, D. J. Palmer, D. R. Koehler, J. Hu, A. L. Beaudet, J. Zabner, and M. J. Welsh
Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR
Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L1123 - L1130.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
P. L. Sinn, A. J. Shah, M. D. Donovan, and P. B. McCray Jr.
Viscoelastic Gel Formulations Enhance Airway Epithelial Gene Transfer with Viral Vectors
Am. J. Respir. Cell Mol. Biol., May 1, 2005; 32(5): 404 - 410.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
J. M. Pilewski
Gene Therapy for Airway Diseases: Continued Progress toward Identifying and Overcoming Barriers to Efficiency
Am. J. Respir. Cell Mol. Biol., August 1, 2002; 27(2): 117 - 121.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seiler, M. P.
Right arrow Articles by Zabner, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seiler, M. P.
Right arrow Articles by Zabner, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2002 American Thoracic Society.