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. 2013 Mar 19;21(5):947–953. doi: 10.1038/mt.2013.49

Adenoviral Gene Transfer Corrects the Ion Transport Defect in the Sinus Epithelia of a Porcine CF Model

Andrea E Potash 1, Tanner J Wallen 1, Philip H Karp 2, Sarah Ernst 2, Thomas O Moninger 2, Nicholas D Gansemer 2, David A Stoltz 2, Joseph Zabner 2, Eugene H Chang 1,*
PMCID: PMC3666638  PMID: 23511247

Abstract

Cystic fibrosis (CF) pigs spontaneously develop sinus and lung disease resembling human CF. The CF pig presents a unique opportunity to use gene transfer to test hypotheses to further understand the pathogenesis of CF sinus disease. In this study, we investigated the ion transport defect in the CF sinus and found that CF porcine sinus epithelia lack cyclic AMP (cAMP)-stimulated anion transport. We asked whether we could restore CF transmembrane conductance regulator gene (CFTR) current in the porcine CF sinus epithelia by gene transfer. We quantified CFTR transduction using an adenovirus expressing CFTR and green fluorescent protein (GFP). We found that as little as 7% of transduced cells restored 6% of CFTR current with 17–28% of transduced cells increasing CFTR current to 50% of non-CF levels. We also found that we could overcorrect cAMP-mediated current in non-CF epithelia. Our findings indicate that CF porcine sinus epithelia lack anion transport, and a relatively small number of cells expressing CFTR are required to rescue the ion transport phenotype. These studies support the use of the CF pig as a preclinical model for future gene therapy trials in CF sinusitis.

Introduction

Topical gene therapy directed toward the paranasal sinus offers the possibility for treatment of cystic fibrosis (CF) related sinus disease. Nearly, all people with CF will have sinus hypoplasia and develop CF sinusitis, yet there is no cure for CF sinus disease.1 CF is due to mutations in the CF transmembrane conductance regulator gene (CFTR), a cyclic AMP (cAMP) and nucleotide activated anion channel expressed on the apical surface of epithelia.2,3 Although mutations in CFTR cause CF, the pathogenesis of CF sinus disease is unknown. The paranasal sinuses offer advantages to topical gene therapy approaches due to their proximal location, relative symmetry, and similar histology to lower airway epithelia.4 Past attempts at correcting the CFTR defect in the paranasal sinuses of people with CF have been unsuccessful, due to the advanced state of disease and lack of change in CF phenotype.5,6

The CF pig model provides an opportunity to define the ion transport defect in the CF porcine sinus and use gene transfer to test hypotheses to further understand the pathogenesis of CF sinus disease. Our group recently reported that newborn CF pigs had sinus hypoplasia and spontaneously developed CF sinusitis similar to humans.7 Our primary objective in this article is to characterize ion transport in the CF porcine sinus, as defective ion transport may be the initiating factor for the CF host defense defect.3,8,9,10,11,12,13 Our secondary objective is to use CFTR gene transfer to an in vitro model of CF porcine sinus epithelia to test what percentage of cells are necessary to correct the ion transport phenotype. This work will form a foundation for future studies to determine whether rescuing CFTR in the sinus will correct the CF sinus phenotype.

Results

Ion transport studies of CF and non-CF porcine sinus epithelia cultures

We cultured CF porcine sinus epithelial cells at the air–liquid interface as previously described.14 After 2 weeks, CF sinus epithelia were well differentiated and highly ciliated (Figure 1a,b). The ciliary cytoskeletal structures of CF sinus epithelia were composed of a normal 9+2 microtubule arrangement (Figure 1c). We then investigated the electrophysiology of CF and non-CF porcine sinus epithelia. We measured transepithelial voltage (Vt), short-circuit current (Isc), and transepithelial conductance (Gt). We altered transport with: (i) amiloride, which inhibits the apical epithelial sodium channel; (ii) 4,4′-diisothiocyanotostilbene-2,2′-disulfonicacid, which inhibits non-CFTR Cl− channels in the apical membrane; (iii) forskolin and 3-isobutyl-2-methylxanthine (IBMX),which increase cAMP levels leading to phosphorylation of CFTR by cAMP-dependent protein kinase; and (iv) GlyH-101, which inhibits the CFTR channel.15 We discuss our findings below.

Figure 1.

Figure 1

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Primary porcine sinus epithelia cultures. Porcine sinus epithelia grown at an air–liquid interface 14 days after seeding. (a) Transmission electron microscopy of sinus epithelia were highly differentiated and pseudostratified. (b) Scale bar = 2.5 μm. Scanning electron microscopy of highly ciliated cultures. Scale bar = 500 nm. (c) Transmission electron microscope of cilia exhibiting a normal 9+2 microtubule ciliary cytostructure. Scale bar = 250 nm. Representative of three cultures.

CF porcine sinus epithelia lack anion transport and do not have increased sodium transport

We first measured voltage (Vt) and found that VtBasal and ΔVtAmiloride were higher in CF than non-CF porcine sinus epithelia (Figure 2a,b). In addition, CF cultures did not respond to the cAMP-stimulant Forskolin and IBMX (F&I) or CFTR inhibitor (GlyH) (Figure 2c,d). These findings are consistent with in vivo nasal measurements performed in CF pigs.16 Moreover, these results are similar to nasal transepithelial potential differences found in people with CF.17,18 Next, we measured current (Isc) and found IscBasal and ΔIscAmiloride to be lower in CF than non-CF porcine sinus epithelia (Figure 3a, b). There was no response to the cAMP-stimulant F&I (ΔIscF&I) or CFTR inhibitor (ΔIscGlyH) (Figure 3c,d). We then investigated conductance (Gt) to see whether changes in voltage and current after amiloride were secondary to increased Na+ absorption or due to loss of Cl− channel activity in CF. We found that basal conductance in CF was decreased (GtBasal), associating the absence of CFTR channels relative to non-CF sinus epithelia (Figure 4a). Moreover, non-CF epithelia had a greater change after amiloride (ΔGtAmiloride) than CF epithelia (Figure 4b). These findings suggest that changes in VtAmiloride and IscAmiloride in CF epithelia are secondary to lack of apical Cl− transport and not due to increased Na+ transport. In addition, there was no response to the cAMP-stimulant (ΔGtF&I) or CFTR inhibitor (ΔGtGlyH) (Figure 4c,d).

Figure 2.

Figure 2

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CF porcine sinus epithelia have elevated basal voltage (VtBasal), increased change after amiloride (ΔVtAmiloride), and no response to cAMP agonists. Data are means ± SE from newborn CF (closed bars) and non-CF (open bars). Studies performed on two different cultures from nine different pigs (n = 9) *P < 0.05. (a,b) Change in voltage after adding 100 μmol/l of amiloride (Amil) apically. (c) Change in voltage after adding cAMP agonists 10 mmol/l forskolin and 100 mmol/l 3-isobutyl-2-methylxanthine (F&I). (d) Change in voltage after adding 100 μmol/l of GlyH-101 (GlyH).

Figure 3.

Figure 3

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CF porcine sinus epithelia have decreased basal current (IscBasal), decreased change after amiloride (ΔIscAmil), and no response to cAMP agonists. Data are means ± SE from newborn CF (closed bars) and non-CF (open bars). Studies performed on two different cultures from nine different pigs (n = 9) *P < 0.05. (a,b) Change in current after adding 100 μmol/l of amiloride (Amil) apically. (c) Change in current after adding cAMP agonists 10 mmol/l forskolin and 100 mmol/l 3-isobutyl-2-methylxanthine (F&I). (d) Change in current after adding 100 μmol/l of GlyH-101 (GlyH).

Figure 4.

Figure 4

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CF porcine sinus epithelia have decreased basal conductance (GtBasal), decreased change after amiloride (ΔGtAmil), and no response to cAMP agonists. Data are means ± SE from newborn CF (closed bars) and non-CF (open bars). Studies performed on two different cultures from nine different pigs (n = 9) *P < 0.05. (a,b) Change in conductance after adding 100 μmol/l of amiloride (Amil) apically. (c) Change in conductance after adding cAMP agonists 10 mmol/l forskolin and 100 mmol/l 3-isobutyl-2-methylxanthine (F&I). (d) Change in conductance after adding 100 μmol/l of GlyH-101 (GlyH).

Adenovirus transduces in vitro sinus epithelia in a dose-dependent manner

We then asked whether we could transfer CFTR to the sinus epithelia of CF pigs via topical gene replacement. We used an adenoviral vector that expressed GFP-labeled CFTR (Ad5/GFP-CFTR) to transduce porcine sinus epithelial cultures.19 Cells that expressed CFTR were labeled green on the apical membrane and quantified by confocal microscopy. Increasing concentrations of Ad5/GFP-CFTR (5, 50, and 500 multiplicity of infection (MOI)) were applied to the apical surface of CF sinus epithelia after disruption of the tight junctions with ethylenediaminetetraacetic acid for 1 hour and then rinsed and further incubated with medium. At 48 hours after infection, the epithelial cultures were fixed and GFP fluorescence quantified. We found no GFP-positive cells in control epithelia (Figure 5a), although epithelial cells transduced with Ad5/GFP-CFTR expressed GFP-positive cells at 7% (MOI of 5), 17.5% (MOI of 50), and 28% (MOI of 500) of cells respectively (Figure 5b–e). These data suggest that Ad5/GFP-CFTR transduces sinus epithelia in a dose-dependent manner.

Figure 5.

Figure 5

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In vitro transduction of primary cultures. (a) Empty adenoviral vector and (b–d) various concentrations of Ad5/GFP-CFTR were applied to the apical surface of porcine sinus epithelia for 1 hour. Scale bar = 20 μmol/l. Cells were counterstained with ZO-1 (tight junctions, red) and DAPI (nuclei, blue). The epithelial cultures were then fixed and GFP fluorescence quantified. (e) We found no GFP-positive cells in control epithelia, although epithelial cells transduced with Ad5/GFP-CFTR expressed GFP-positive cells at 7% (MOI of 5), 17.5% (MOI of 50), and 28% (MOI of 500) of cells respectively (n = 6).

Ad5/GFP-CFTR restores cAMP-stimulated anion transport

We further investigated whether Ad5/GFP-CFTR vector could transduce CF and non-CF porcine sinus epithelia by measuring cAMP-mediated anion transport. CF epithelia were transduced after ethylene glycol tetraacetic acid with AdGFP-CFTR at MOIs of 5, 50, and 500 and non-CF epithelia with Ad5/GFP-CFTR at an MOI of 500. At the lowest MOI of 5, the response to Forskolin/IBMX was 6% of the non-CF response. At an MOI of 50, CFTR anion transport was 44% and at an MOI of 500, CFTR anion transport was 59% of non-CF levels. We found that CF epithelia transduced with 5, 50, or 500 MOI of AdGFP-CFTR demonstrated a dose-dependent increase in ΔIscF&I and ΔGtF&I (Figure 6a–c).

Figure 6.

Figure 6

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Ad5/GFP-CFTR restores cAMP-stimulated anion transport. (a) Representative Ussing chamber tracing of short-circuit current (Isc) after apical application of 100 μmol/l of amiloride (Amil), 100 μmol/l of 4,4′-diisothiocyanotostilbene-2,2′-disulfonicacid (DIDS), 10 mmol/l forskolin and 100 mmol/l 3-isobutyl-2-methylxanthine (F&I), and 100 μmol/l of GlyH-101 (GlyH) to CF epithelia transduced with 5, 50, and 500 MOI Ad5/GFP-CFTR, non-CF epithelia, and non-CF epithelia transduced with 500 MOI Ad5/GFP-CFTR. (b,c) Change in current and conductance after adding cAMP agonists 10 mmol/l forskolin and 100 mmol/l 3-isobutyl-2-methylxanthine (F&I). Studies performed on four different cultures from nine different pigs (n = 9) *P < 0.05. Different colors used to separate virus levels.

Abnormal CFTR transport has been suggested as a factor in some people with chronic sinusitis. Therefore, we asked whether we could overcorrect CFTR activity in non-CF porcine sinus epithelia. Prior attempts at overexpression of CFTR in the lower airway have been unsuccessful, as CFTR was expressed on the basal surface of the epithelia, shunting chloride and reducing anion transport.20 We were surprised to find that transducing non-CF porcine sinus epithelia with Ad5/GFP-CFTR resulted in a further increase in ΔIscF&I and ΔGtF&I that exceeded non-CF cells treated with empty vector (Figure 6a–c). This finding highlights the differences in sinonasal and lower airway epithelia, and may be a possible therapy for non-CF sinusitis.

Discussion

Our data show that primary CF porcine sinus epithelia cultures are well differentiated, highly ciliated, and lack anion transport. We successfully transduced porcine sinus epithelia with an adenoviral vector expressing CFTR and achieved a dose-dependent restoration of cAMP-mediated anion transport. We found that GFP expression in approximately 7% of the cells resulted in a 6% increase in CFTR anion transport levels with 17.5–28% of GFP expression in transduced cells correcting CFTR transport to half the levels of non-CF epithelia. Very low levels of expression of CFTR can restore chloride current; in contrast, robust expression of GFP is required to catalogue a cell as GFP positive.21 Thus, these numbers are just an approximation and are likely to overestimate the number of cells required. Sinus epithelia were different than airway epithelia in that non-CF epithelia transduced with Ad5/GFP-CFTR expressed CFTR anion transport activity that was equal to or exceeded non-CF epithelia. These findings indicate that a relatively small percentage of cells may need to express CFTR to restore anion transport, and are the first step in gene therapy correction in an animal model of CF.

CFTR effects on sinus electrophysiology

Our data indicate that porcine CF sinus epithelia lack cAMP-stimulated anion transport. This finding is consistent with similar studies in porcine and human airway epithelia, which show defects in Cl− and bicarbonate (HCO3−) transport via CFTR.16 In vitro CF porcine sinus epithelia demonstrate elevated VtBasal similar to findings in CF porcine nasal epithelia and humans with CF. Although CF porcine sinus epithelia demonstrated elevated VtBasal, they had decreased IscBasal levels. Historically, Vt or potential difference and Isc have been used interchangeably. As shown in Chen et al., since Ohm's law: Isc = Vt × Gt; Vt does not have to be correlated necessarily with Isc.16 For example, in non-CF tissue, if Vt = 2 mV and Gt = 2 mS, then Isc = 4 μA. In CF tissue, if Vt = 4 mV and Gt = 1 mS, then we would have the same Isc = 4 μA even though the Vt is higher in CF.

CF porcine sinus epithelia also exhibit increased ΔVtAmiloride and decreased ΔIscAmiloride, leading some to hypothesize that CFTR mutations eliminate CFTR inhibition of epithelial sodium channel and cause Na+ hyperabsorption and mucus dehydration.8 If true, we would expect to see a greater change in conductance after the application of amiloride, an epithelial sodium channel inhibitor, in CF epithelial cells. Our findings of decreased change in conductance in CF versus non-CF epithelia suggest that CF epithelia do not have increased Na+ transport, but that changes in Vt and Isc reflect residual Cl− current in non-CF epithelia. Although we did not directly measure Na+ flux or volume absorption, studies in CF nasal porcine epithelia suggest that there is no hyperabsorption of Na+ in CF.16

One unexpected finding was that non-CF porcine sinus epithelia transduced with Ad5/GFP-CFTR expressed CFTR anion transport activity that exceeded non-CF epithelia. This finding differentiates sinus from the lower airway epithelia, where prior studies that overexpressed CFTR in non-CF lung epithelia generated CFTR channels on the basolateral surface, thereby reducing net Cl− transport.20 This result is exciting, as there is some evidence that decreased Cl− transport may be associated with the development of chronic sinusitis in people without CF, highlighting a possible therapy for non-CF sinusitis.22,23 Future studies in human in vitro sinus epithelia are necessary to distinguish if this finding is secondary to characteristics of sinus epithelia or due to the differences between porcine and human epithelia.

CF and history of prior gene therapy trials

CF is an attractive candidate for gene therapy, as the disease is secondary to defects in a single gene. Human trials of CFTR delivery to the airway have shown transient gene expression24,25,26,27,28,29,30 and changes in ion transport.27,28,29,31,32 But, there have been no reports of significant improvement in primary or secondary outcomes of CF airway disease.6,31,32,33,34,35 One possibility for the lack of change in clinical phenotype is the relative late stage of CF disease and the associated inflammation and infection of the airway. In addition, there have been only a few studies that have used more than one application dose, thus the clinical significance is unknown.25,34,35,36,37,38,39

The paranasal sinuses provide several advantages as a model for CF airway research. The sinus is lined with respiratory epithelia with ion transport and histology similar to that of the lower airways. The sinuses are symmetric and separate, enabling the use of a single subject as a case and control. The sinus is also prominent and accessible, allowing experimentation throughout the life of the subject.4 CFTR has been delivered to the sinus of CF humans via an adeno-associated virus vector.6,28 Topical delivery of adeno-associated virus vector carrying the CFTR gene showed dose-dependent gene transfer and ion transport changes in a Phase I/II study,28 but did not show a change in primary or secondary outcomes of the disease in a Phase II trial.6

Advantages of the CF pig sinus as a model for gene therapy

Clinical studies examining the use of CFTR gene therapy should include outcome measures that are pertinent to people with CF. The CF pig presents a unique opportunity to test gene therapy interventions due to the spontaneous development of both CF sinus and lung disease similar to humans. We also have the advantage of beginning therapy before clinical disease begins, thereby focusing on an early window of opportunity to prevent or slow progression of chronic sinus and lung disease.40 We found that only 7% of transfected CF porcine sinus cells were sufficient to restore 6% of CFTR current with 17–28% of transfected cells sufficient to correct the anion transport defect to 50% of non-CF levels. Previous studies that have looked at the percentage of non-CF cells needed to restore current range from 6–10% to 20–25% of cells.20,41,42 Future studies of gene transfer to the sinus of the CF pig will allow us to test whether CFTR correction can alter the CF phenotype of sinus hypoplasia and disease.

Disadvantages of adenovirus

There are several limitations with our study. Although adenovirus is an efficient viral gene transfer vector, transgene expression is transient and repeated administrations are inefficient due to the blocking immune response. Because of this, future therapies will need to consider alternative gene transfer vectors that can integrate into the DNA or avoid immune responses.43 Several recent publications highlight novel gene therapy vectors that can decrease the immune response (adeno-associated virus) or integrate into the genome (SIV, HIV-1, FIV).44,45,46,47,48 Another limitation is that the study was performed in sinus cultures, rather than entirely in an in vivo model.

In summary, relatively low levels of CFTR gene delivery are enough to restore a portion of the ion transport defect in CF porcine sinus epithelia. The CF pig allows us to use gene transfer to test hypotheses for the pathophysiology of CF sinus disease in an animal model with a clinical phenotype of CF sinusitis. Further studies using different gene therapy vectors and application to an in vivo model are necessary.

Materials and Methods

Animals. We previously reported generation of CFTR+/− pigs, and production of CFTR−/− and pigs.49,50 Animals were mated, and progeny was studied. After euthanasia, CF sinus epithelia were harvested from the ethmoid sinus of CFTR−/− newborn piglets, and non-CF sinus epithelia from the ethmoid sinus of CFTR+/− and CFTR+/+ newborn piglets. Standard procedures for animal husbandry and anesthesia were used The Institutional Animal Care and Use Committees of the Universities of Iowa and Missouri approved all animal experiments.

Electrophysiological measurements in cultured epithelia. Epithelial tissues were excised from the ethmoid sinus immediately after animals were euthanized. Cultured epithelia were studied in modified Ussing chambers. Epithelia were bathed on both surfaces with solution containing (mmol/l): 135 NaCl, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, 10 dextrose, 5 HEPES (pH = 7.4) at 37 °C and gassed with compressed air. Vt was maintained at 0 mV to measure short-circuit current (Isc). Transepithelial electrical conductance (Gt) was measured by intermittently clamping Vt to +5 and/or −5 mV.

Gene therapy delivery using adenoviral vectors

Primary porcine sinus epithelia cultures. The Ad5/GFP-CFTR vector was generated by homologous recombination using the pEGFP-CFTR with the adenovirus transfer plasmid pTG14682 as previously described.19 Porcine sinus epithelia were maintained at 37 °C and 5% CO2. On the day of the experiment, primary porcine sinus epithelia were treated apically with 5 mmol/l ethylene glycol tetraacetic acid in water for 5 minutes to transiently dissociate the tight junctions. ethylene glycol tetraacetic acid was then removed and various concentrations of Ad5/GFP-CFTR in 40 μl were placed on the apical surface for 1 hour and then rinsed. Cell monolayers and epithelia were processed and fixed for confocal microscopy after 48 hours.

Immunocytochemistry. Epithelia were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 2% bovine serum albumin in SuperBlock (Pierce, Perbio Science France, Brebières, France) for 1 hour at room temperature, cells were incubated with primary antibody (mouse antihuman ZO-1 antibody, 1:250; BD Biosciences, San Jose, CA) for 3 hours at 37 °C, rinsed in phosphate-buffered saline, then incubated with secondary antibody conjugated to fluorophore (1:1,000; Alexa Fluor 568-conjugated goat antimouse IgG; Invitrogen, Carlsbad, CA) for 90 minutes at 37 °C. After several rinses, cells were mounted on glass slides, a coverslip was placed with Vectashield 1 DAPI mounting solution (Vector Labs, Burlingame, CA), and samples were studied by confocal fluorescence microscopy. Samples were observed using a Leica TCS SP2 (Leica Microsystems, Wetzlar, Germany) or an Olympus FluoView V1000 (Olympus, Center Valley, PA) confocal microscope using a 360 oil immersion lens.

Calculation of transduced GFP cells. Percent GFP expression of treated cultured epithelial cells was quantified using ImageJ software. Single confocal images of each condition were divided into quadrants. Cells in each quadrant were counted separately to improve accuracy. Three populations of cells were counted in each quadrant: GFP-expressing cells, non GFP-expressing cells, and the total number of cells. The percent GFP expression was calculated in each quadrant as the number of GFP-expressing cells divided by the total cells in the respective quadrant. The percent GFP for each quadrant was averaged to determine the overall percent GFP expression for each confocal image. These measurements were performed in three different fields for each condition.

Electron microscopy. Filters for transmission electron microscope were processed using conventional methods. Briefly, samples were fixed in 2.5% gluteraldehyde in 0.1 mol/l Cacodylate buffer, post-fixed in 1% osmium tetroxide and en bloc stained 2.5% aqueous uranyl acetate. The filters were then dehydrated in a graded series of ethanol washes, transitioned to Eponate-12 resin (Ted Pella, Redding, CA) and cured overnight at 65 °C. Ultrathin sections were post stained with uranyl acetate and lead citrate, and imaged in a JEOL 1230 transmission electron microscope (JEOL, Peabody, MA) equipped with a Gatan 2 k × 2 k digital camera (Gatan, Pleasanton, CA). Filters destined for SEM were fixed in 2.5% gluteraldehyde in 0.1 mol/l Cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol washes, transitioned to hexamethyldisilizane and air-dried overnight. The filters were then removed from the support, mounted on aluminum stubs, sputter coated with gold/palladium and imaged in a Hitachi S-4800 SE (Hitachi High Technologies America, Dallas, TX.)

Statistical analysis. Data are presented as mean ± SE. Unpaired t-test was performed using GraphPad Prism version 5.00 for Mac, (GraphPad Software, San Diego, CA; www.graphpad.com). Differences were considered statistically significant at P < 0.05.

Acknowledgments

We thank Paul B McCray Jr, Michael Welsh and Peter Taft for their valuable assistance. This work was supported by the NIH (DE021413-01A1, HL51670, HL091842), the Cystic Fibrosis Foundation, the University of Iowa CTSA (KL2RR024980), and the Roy J Carver Charitable Trust (P.B.M.). D.A.S. is supported by the Gilead Sciences Research Scholars Program in CF. We also acknowledge the support of the In Vitro Models and Cell Culture Core and Cell Morphology Core, partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-54759). The authors declare no conflict of interest.

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