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Viscoelastic Gel Formulations Enhance Airway Epithelial Gene Transfer with Viral Vectors Patrick L. Sinn, Ankur J. Shah, Maureen D. Donovan, and Paul B. McCray, Jr. Program in Gene Therapy, Department of Pediatrics, Carver College of Medicine, The University of Iowa; and Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa

Advances in gene transfer to the conducting airways for the treatment of pulmonary diseases such as cystic fibrosis have identified several vector classes that transduce airway epithelia in vitro and in animal models. One barrier to epithelial gene transfer is the rapid removal of materials from the airway surface via mucociliary clearance. This host defense mechanism limits gene transfer efficiency to airway epithelial cells. Here we show that formulation of gene transfer vectors with viscoelastic gels provides longer epithelial residence time and increases vector-mediated gene transfer efficiency. Gene transfer with adenoviral, adeno-associated, and lentiviral vectors all significantly improved after formulation with viscoelastic gels designed to slow mucociliary clearance. Importantly, viscoelastic gel formulations enhanced vector transduction to the conducting airways, the desired treatment target for diseases such as cystic fibrosis. Keywords: gene transfer; viral vectors; viscoelastic gel

Gene transfer vectors can be readily delivered to the airways by aerosol or instillation, but once at the mucosal surface they must overcome numerous barriers for efficient uptake and expression in epithelia. Cellular barriers to gene transfer include epithelial tight junctions that restrict paracellular uptake and access to some receptors, low endogenous rate of endocytosis at the apical membrane, extracellular glycocalyx, and rapid mucociliary clearance of foreign material entrapped in secreted mucins (reviewed in Ref. 1). Early attempts to bypass cellular barriers included the use of agents to transiently disrupt tight junctions, such as EGTA, to increase paracellular permeability (2–5), or the targeting of specific cellular receptors to enhance vector uptake (1, 3, 6–8). Because of safety considerations associated with modifying tight junctions in vivo, efforts to directly transduce cells from the luminal surface have increased. As a result, several different viral vectors are being investigated for improved gene transfer (1, 9). Progress has been reported with lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV) (9–13). Early success in the translation of genebased therapies with adenoviral viral vectors was seen in many cell culture and animal models. However, the location of the adenovirus receptor (CAR) on the basolateral surface of the airway epithelium diminished the transduction efficiency unless tight junctions were disrupted (7). In addition, adenovirus is a nonintegrating vector; thus chronic, repeated administration of the gene therapy would likely be required to sustain a population of normal respiratory epithelial cells. Other viral vectors, notably

AAV5 (13), AAV6 (11), AAV9 (14), and pseudotyped lentiviruses (9), can target receptors on the luminal airway surface. These vector systems are promising tools for gene therapy. Mucociliary clearance is a barrier to efficient gene transfer. The luminal surfaces of the large airways are populated with ciliated epithelia that act to remove inhaled particles trapped in mucus. Efficient mucociliary transport is accomplished by the coordinated beating of the cilia through the periciliary liquid layer with intermittent ciliary contact with the overlying mucus layer. Mucociliary clearance rates from the large airways of humans range between 0.25 and 0.5 cm/min (15). Consequently, the contact time of a gene transfer vector with the epithelial surface may be too short to achieve significant uptake of a genebased therapeutic agent, regardless of the uptake mechanism. Seiler and coworkers (16) previously demonstrated that formulating recombinant adenovirus in a thixotropic solution containing 1% Avicel RC-591, a mixture of microcrystalline cellulose and carboxymethylcellulose, decreased virus clearance and enhanced gene transfer to airway epithelia. Results in this study indicated that such gel formulated adenoviral vectors enhanced gene transfer in vitro and in vivo by inhibiting mucociliary clearance. We hypothesized that optimized viscoelastic gel formulations might increase the transduction efficacy for multiple classes of gene transfer vectors. We tested three viscoelastic gels, carboxymethylcellulose (CMC), methylcellulose (MC), and poloxamer 407 (P407), for their ability to enhance gene transfer of adenoviral, AAV5, and lentiviral vectors to the airways of mice.

MATERIALS AND METHODS Vector Production Recombinant adenoviral vectors expressing ␤-galactosidase (Ad-␤Gal) or luciferase (Ad-Luc) were prepared as previously described (17) by the University of Iowa Gene Transfer Vector Core at titers of ⵑ 1010 plaque-forming units (pfu) per ml. Recombinant AAV5 vector expressing eGFP (AAV-eGFP) at titer of 4 ⫻ 1012 viral genomes (vg) per ml was a kind gift from Jay Chiorini (13, 18). The FIV vector used in this study was reported previously (6, 19). The FIV vector construct expressed a nuclear targeted ␤-galactosidase cDNA directed by the CMV promoter (FIV-␤Gal) or a firefly luciferase cDNA directed by the RSV promoter (FIV-Luc). The Autographa californica GP64 envelope has efficiently pseudotyped an HIV-based vector (20), and will efficiently pseudotype the FIV-based vector (P.L.S. and P.B.M., unpublished observations).

Vehicle Preparation

(Received in original and final form December 22, 2004) This work was supported by the Cystic Fibrosis Foundation SINN04G0 (P.L.S.), NIH RO1 HL-61460 (P.B.M), and PPG HL-51670 (P.B.M.). Correspondence and requests for reprints should be addressed to Paul B. McCray, Jr., Department of Pediatrics, 240G EMRB, The University of Iowa, Iowa City, IA 52242. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 32. pp 404–410, 2005 Originally Published in Press as DOI: 10.1165/rcmb.2004-0410OC on February 4, 2005 Internet address: www.atsjournals.org

Six-millimolar EGTA (pH 7.4) formulation was used for in vitro testing and 400 mM EGTA (pH 7.4) was used for in vivo testing as previously described (4–6). Three viscoelastic gel formulations were prepared as follows. MC (Methocel A4C; Dow Chemical Co., Midland, MI) was added to hot (90 ⬚C) serum-free Dulbecco’s modified Eagle’s medium (DMEM) and mixed thoroughly. DMEM was added to the desired volume and the gel was gently stirred until the polymer was in solution and a gel of consistent viscosity formed. Carboxymethlycellulose sodium (Aqualon 7MF; Hercules, Inc., Wilmington, DE) was dispersed in room temperature DMEM and mixed for ⵑ 30 min to form a homogeneous gel. Poloxamer 407 (Pluronic F127; BASF Wyandotte Co.,

Sinn, Shah, Donovan, et al.: Gene Transfer with Viscoelastic Vehicle

Parsipanny, NJ) polymer was added to an aliquot of cold DMEM and the mixture was placed in the refrigerator overnight for complete polymer dissolution. The solution was brought to room temperature and sufficient DMEM water was added to bring the system to the desired volume.

In Vitro Viral Vector Administration For this study we used a transformed human airway epithelial cell line (NuLi-1) grown at an air–liquid interface as described previously (21). This cell line forms a polarized epithelial sheet that exhibits a transepithelial resistance and vectoral ion transport. All preparations had initial resistances ⬎ 800 ⍀ ⫻ cm2 confirmed by transepithelial resistance measurements using a volt-ohm meter (World Precision Instruments, Sarasota, FL). The volt-ohm meter was also used to measure transepithelial resistance at indicated time points after addition of viscoelastic gels to NuLi cells. The NuLi cells were transduced by diluting vector preparations in serum-free media to achieve an MOI of 10, and 100 ␮l of the solution was applied to the apical cell surface for a 2-h period at 37 ⬚C. The cells were rinsed with media and cultured for 4 d. To transduce airway epithelia from the basolateral side, the Millicell culture insert containing the epithelium was turned over and virus applied to the bottom surface of the filter for 2 h in 100 ␮l of media. After the 2-h incubation, the vector was removed, cells were rinsed in media, and the culture insert was turned upright and cultured at 37 ⬚C, 5% CO2, for 4 d. After the 4-d incubation, cells were harvested and ␤-galactosidase activity quantified. The Galacto-light chemiluminescent reporter assay (Tropix, Bedford, MA) was used to quantify ␤-galactosidase activity according to the manufacturer’s protocol. The relative light units (RLUs) were quantified using a luminometer (Monolight 3010; Pharmingen, San Jose, CA) and standardized to total protein as determined by modified Lowry assay (23240; Pierce Biotechnology, Rockford, IL) using the manufacturer’s protocol.

In Vivo Viral Vector Pulmonary Administration This study was approved by the University of Iowa Institutional Animal Care and Use Committee. Approximately 2.5 ⫻ 108 pfu of Ad vector or 1.0 ⫻ 1011 viral genomes of AAV vector in a 50-␮l volume was delivered via direct orotracheal intubation with a 24-G Teflon catheter to the airways of 3-mo-old male Balb/c mice under ketamine anesthesia. Four days after Ad-␤Gal delivery, the mice were killed and the lungs removed, fixed in 2% formaldehyde/0.2% gluteraldehyde, and X-gal stained for 4 h at 37 ⬚C. After X-gal staining, lungs were paraffinembedded, sectioned, and counterstained with nuclear fast red. Four days after Ad-Luc delivery, the mice were imaged using a CCD camera as described below. Three weeks after AAV-eGFP delivery, the mice were killed, the lungs were removed, fixed in 4% paraformaldehyde, protected with 15% sucrose followed by 30% sucrose, and sectioned. Images of eGFP-expressing lungs were captured with a Zeiss 510 Confocal Microscope (Zeiss, Thornwood, NY).

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era. After imaging, animals recovered in a heated cage. Imaging data were analyzed and signal intensity quantified using Xenogen Living Image software.

Mucociliary Transport Rates Full-thickness segments of bovine trachea obtained from local meatpackers were depleted of endogenous mucus and the mucus layer was replaced with a thin layer of a reconstituted mucin solution (22). The explants were placed into Petri dishes containing sufficient Locke Ringers buffer to bathe the cartilaginous region of the tissue and were placed into a humidified chamber. The clearance rate across the tissue was measured by following the movement of the formulation across the surface using a stereomicroscope. To assist with visualization, 50–200 mesh (75–300 ␮m) charcoal particles (#05–690A; Fisher Scientific, Pittsburgh, PA) were incorporated into the formulation. The transit rate of individual charcoal particles was measured using a stereomicroscope with a calibrated eyepiece. Each explant was used for multiple measurements, and the explant was calibrated with control charcoal-containing mucus before each measurement.

Statistics All numerical data are presented as the mean ⫾ SD. Statistical analysis was performed using a two-tailed Student’s t test using Microsoft Excel software.

RESULTS Formulating Adenoviral Vectors in Viscoelastic Gels Markedly Enhances Gene Transfer to the Conducting Airways

Gene transfer to mouse airway epithelia with Ad-␤Gal formulated with saline vehicle (neat) is inefficient (Figures 1A and 1B). Consistent with previous observations (4, 5), the efficiency was enhanced by pretreating the airways with 400 mM EGTA (Figures 1C and 1D). We hypothesized that transient inhibition of mucociliary clearance by viscoelastic gel formulations would improve gene transfer efficiency. To test this hypothesis, we formulated Ad-␤Gal with final concentrations of 1% MC, 1% CMC, or 7.5% P407 and delivered each preparation to the lungs of mice via orotracheal intubation. Four days after vector delivery, the lungs were excised and X-gal stained. The appearance of ␤-galactosidase–positive cells was readily apparent to the naked eye in lungs receiving gel formulated vector (Figures 2A–

In Vivo Viral Vector Nasal Administration Approximately 1.25 ⫻ 107 transducing units of FIV vector was formulated with the indicated vehicle in a 50-␮l volume and delivered to the nasal epithelium of anesthetized mice via direct instillation. FIV vector preparations expressing luciferase were titered using real-time PCR as previously described (6). Three weeks after FIV-␤Gal delivery, mice were killed and ␤-galactosidase expression in the upper respiratory tract confirmed. Heads were removed, fixed in 2% formaldehyde/0.2% gluteraldehyde, X-gal stained, decalcified, paraffin-embedded, sectioned, and counterstained with nuclear fast red per standard techniques. After FIV-Luc delivery, animals were imaged at the indicated time points using a CCD camera as described below.

Bioluminescence Imaging In vivo luciferase expression was visualized using bioluminescent imaging. At the time-points indicated, animals were injected intraperitoneally with 100 ml/10 g body weight of D-luciferin (15 mg/ml in PBS; Xenogen, Alameda, CA) using a 25-G needle and anesthetized by isoflurane inhalation. Approximately 10 min after luciferin injection, the animals were placed in the Xenogen IVIS imaging cabinet, anesthetized with 1–3% isoflurane, and imaged using the Xenogen IVIS CCD cam-

Figure 1. In vivo gene transfer with Ad-␤Gal after EGTA pretreatment. Ad-␤Gal (2.5 ⫻ 108 pfu) was delivered to mouse airways via orotracheal intubation without pretreatment (Neat) (A and B) or 15 min after 400 mM EGTA nasal instillation (C and D). Mouse lungs were examined by X-gal staining 4 d after vector delivery. Tissue sections were counterstained with nuclear fast red. Scale bars ⫽ 500 ␮M; n ⫽ 4 animals/condition.

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Figure 2. In vivo gene transfer with Ad-␤Gal using viscoelastic vehicles. Ad-␤Gal (2.5 ⫻ 108 pfu) formulated with 1% MC (A, E, and I ), 1% CMC (B, F, and J ), or 7.5% P407 (C, G, and K ) was delivered to mouse airways via orotracheal intubation and compared with naı¨ve mouse controls (D, H, and L ). Mouse lungs were examined by X-gal staining 4 d after vector delivery. Tissue sections were counterstained with nuclear fast red. Scale bars ⫽ 250 ␮M; n ⫽ 4 animals/condition.

2C), but not in naı¨ve animals (Figure 2D) or control animals receiving the adenovirus without gel formulation (data not shown). Upon microscopic examination, the transduction of the conducting airway epithelium with Ad-␤Gal formulated with 1% MC or 1% CMC was striking (Figures 2E, 2F, 2I, and 2J), whereas the conducting airway cell transduction achieved with the P407 was considerably less (Figures 2G and 2K). No ␤-galactosidase–positive cells were observed in the naı¨ve control (Figures 2H and 2L). The overall survival rates after orotracheal delivery of 50 ␮l of formulated vector was as follows: neat 31/35 (89%), MC 29/32 (90%), CMC 36/40 (90%), and P407 12/28 (43%). Mice tolerated vector formulated with MC and CMC well; however, less than half of the mice receiving vector formulated with P407 survived. The 1 in 10 deaths that occurred after delivery of MC or CMC formulated vector were not significantly different than unformulated vector and were likely a combined result of anesthesia and fluid volume. Due to the low survival rate and the lower gene transfer efficiency, P407 was not included as a tested vehicle in subsequent in vivo experiments. To quantify relative gene transfer efficiencies, serotype 5 adenoviral vector expressing firefly luciferase (Ad-Luc) was delivered to mouse airways via orotracheal intubation. Bioluminescent signal was assessed using a CCD camera 5 min after intraperitoneal luciferin delivery (Figure 3). As shown, the maximum gene transfer was observed with Ad-Luc formulated with 1% MC. Neat and EGTA-pretreated animals displayed approximately equal levels of gene transfer in the lung (Figure 3). However, EGTA-pretreated animals had much greater expression in the nasal epithelia, presumably due to enhanced vector access to CAR.

Effects of Viscoelastic Gels on Mucociliary Clearance and Transepithelial Resistance

The effect of gel formulation on mucociliary transport rate (MTR) was measured using a bovine tracheal explant model (23) (Table 1). The transport rate of 1% CMC, 1% MC, or 7.5% P407 was measured and compared with the transport rate of a control charcoal:mucin suspension. CMC was most effective at decreasing MTR (40% decrease). MC and P407 also decreased MTR (14% and 10% decrease, respectively). These data suggest that one method of action for polymeric gels is the slowing of mucociliary clearance. The resulting increase in mucosal contact time likely facilitates significantly improved uptake of the Ad␤gal vector via receptor-independent mechanisms (24). Increasing vector residence times on cells will lead to increased receptor-independent transduction with adenovirus applied to the apical surface (24). To test the effectiveness of our viscoelastic gel formulations at increasing receptor-independent cellular transduction, we used NuLi cells grown at an air–liquid interface with previously demonstrated basolateral CAR expression (21). Ad vector was formulated with gels or EGTA and applied to the apical surface of NuLi cells (Figure 4A). Control cells received vector formulated in serum-free media (MOI ⫽ 10) applied at the apical or basolateral surface. As expected, in the absence of formulation (neat), the Ad vector transduced the basolateral surface with greater efficacy than the apical surface. Consistent with previous observations, EGTA was effective at increasing transduction at the apical surface (7). In addition, each gel formulation enhanced transduction from the apical surface. EGTA is known to disrupt tight junctions and allow vector access to basolateral localized receptors (25, 26). To determine if the viscoelastic gels also disrupt tight junctions, formulations

Sinn, Shah, Donovan, et al.: Gene Transfer with Viscoelastic Vehicle

407

Figure 3. Bioluminescent quantification of in vivo gene transfer with Ad-Luc using viscoelastic vehicles. Ad-Luc (2.5 ⫻ 108 pfu) was delivered to mouse airways via orotracheal intubation. Vector was delivered with 1% MC, after 400 mM EGTA pretreatment, or with no formulation (Neat) and compared with naı¨ve mouse controls. Four days after instillation, mice were imaged and light emission (photons/s/cm2) from a region of interest drawn around the lungs quantified. *P ⬍ 0.01; n ⫽ 3 animals/condition.

identical to those used in the in vivo experiment were applied to the apical surface of NuLi cells (Figure 4B). The viscoelastic gels did not significantly affect the transepithelial resistance. In contrast, vector formulated with EGTA transiently lowered the resistance. Taken together, these data suggest that the gel formulations enhance adenoviral gene transfer by increasing the vector residence time at the cell surface leading to receptor-independent uptake. Formulating an AAV in Viscoelastic Gels Enhances Gene Transfer to the Conducting Airways

To determine if the viscoelastic gels were effective at restricting gene transfer to the conducting airways with other classes of encapsidated viral vectors, AAV5-eGFP was tested in a similar manner as described in Figure 2. AAV5-eGFP (1.0 ⫻ 1011 vg) was gel formulated as described for adenovirus and delivered to the lungs of mice via orotracheal intubation. Three weeks after vector delivery, the presence of eGFP expression was determined by examining tissue sections under confocal microscopy. As shown in Figure 5, GFP-positive cells were evident in each sample with the exception of the naı¨ve control. As with the Ad vector, CMC and MC markedly enhanced gene transfer to the conducting airways as compared with the neat or EGTA controls. Pretreating animals with EGTA before delivering AAV5eGFP formulated with 1% MC did not improve gene transfer over 1% MC alone.

TABLE 1. VISCOELASTIC GEL EFFECTS ON MUCOCILIARY TRANSPORT RATE

Carboxymethylcellulose (1%) Methylcellulose (1%) Poloxamer 407 (7.5%)

Basal MTR (cm/min)

Formulation MTR (cm/min)

% Decrease in MTR

n

0.90 0.67 0.67

0.44 0.57 0.60

51% 14% 10%

3 1 1

Definition of abbreviation: MTR, mucociliary transport rate.

Formulation of Lentiviral Vectors with Viscoelastic Gels Enhances Gene Transfer to Nasal Epithelia

Based on our in vivo and in vitro data, we conclude that viscoelastic gel formulations have no significant deleterious effect on the titer of encapsidated vectors such as adenovirus and AAV. However, stability of enveloped viruses in such formulations has not been assessed. To test if such gels inactivated enveloped viruses, we first incubated each gel with feline immunodefiency virus (FIV)-based vector for 30 min at 37 ⬚C, then titered the vector by limiting dilution on HT1080 cells. Importantly, there was no significant drop in vector titer after incubation with any of the gels evaluated (Table 2). This result suggests that these, and perhaps other, gel formulations might be developed to enhance delivery of enveloped viral vectors to airway epithelia. FIV-based vector expressing luciferase (FIV-Luc) or an Ad5Luc control were formulated as described in Materials and Methods and delivered directly to the nasal epithelia of mice (Figure 6A). The nasal epithelium was selected as a relevant target because cystic fibrosis (CF) mouse models are available that manifest measurable ion transport and other defects at this site (27). Gene transfer efficacy was evaluated 19 d after vector delivery by quantifying bioluminescence as described in Materials and Methods. As shown in Figure 6, the MC formulation greatly enhanced FIV gene transfer to the nose of mice in contrast to the neat or EGTA controls. Unexpectedly, pretreating the nasal epithelia with EGTA before delivering FIV-Luc formulated with MC resulted in less transduction than the MC formulation alone. The reasons for this discrepancy are currently unclear and are under further investigation. A disadvantage of luciferase as a reporter is the difficulty in determining cell types expressing the transgene. It is necessary to determine the transduced cell types in nasal airways (28); therefore, in parallel experiments, FIV expressing nuclear targeted ␤-galactosidase (FIV-␤Gal) was formulated with MC and delivered to the nasal epithelia of mice (Figure 6B). Three weeks after vector delivery, paraffin-embedded sections of nasal tissues were prepared as described in Materials and Methods. Morphologic analysis confirmed ␤-galactosidase expression in both respiratory and olfactory epithelia (Figure 6B).

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Figure 4. In vitro gene transfer with Ad-␤Gal using viscoelastic vehicles. Polarized human airway epithelial cells were transduced with Ad-␤Gal (A ). Vector was formulated with 1% CMC, 1% MC, 6 mM EGTA, 7.5% P407, and applied to the apical surface in a 100-␮l volume. Control cells received unformulated (neat) Ad-␤Gal applied to the apical or basolateral surface. Four days after initial vector incubation, cells were harvested and the ␤-galactosidase activity was quantified and normalized to total protein. RLU, relative light units, n ⫽ 4; MOI ⫽ 10. The transepithelial resistance was quantified using a volt-ohm meter at the indicated time points (B ) after addition of viscoelastic gels, 6 mM EGTA, or serum-free media in a 100-␮l volume (n ⫽ 4 epithelial sheets/condition).

DISCUSSION Progress in gene transfer to the airways has resulted from focused studies of both epithelial biology and vector development. Combining improved vectors with modified formulations appears to be a promising strategy for enhancing the efficiency of airway epithelial gene transfer for CF and other airway diseases (2, 3, 5, 6, 8, 16). These studies demonstrate that two simple gel formulations (MC and CMC) dramatically enhance the efficiency

of gene transfer to the airways. Importantly, by formulating Ad5 and AAV5 vectors with viscoelastic gels, gene transfer is concentrated in the conducting airways, with limited gene transfer to the alveoli. This is a novel result with important implications for future CF applications, as the cells of the conducting airway epithelium are a key target population for correction. In contrast, it is believed that gene transfer to the alveolar epithelium may not be required for prevention or treatment of CF lung disease. By restricting gene transfer to the airway epithelium and avoiding delivery to the large alveolar surface area, we also effectively increase the vector MOI delivered to the target cells compared with simple water-based vehicles. Rat lungs have been reported to have ⵑ 5 ⫻ 107 total epithelial cells from trachea to respiratory bronchioles and ⵑ 9 ⫻ 108 alveolar epithelial cells (29). Estimating 109 total cells/gram (30), 3.57% of a 1.4-g rat lung is composed of airway epithelial cells and 64.3% is alveolar epithelia. Assuming such ratios hold true for mice, a 230-mg mouse lung contains ⵑ 8.2 ⫻ 106 epithelial cells from the trachea to the respiratory bronchioles and ⵑ 1.5 ⫻ 108 alveolar epithelial cells. We delivered 2.5 ⫻ 108 pfu of Ad5 vector to the lungs. Assuming an even distribution of vector across the entire airway and alveolar surface area, we achieved an MOI of 1.6. Restriction of the vector to the conducting airways with viscoelastic gel increases the effective MOI to 30.5. This ⵑ 20-fold increase in MOI is consistent with the significant increase in gene transfer of the conducing airways observed with viscoelastic gel formulated vector. We presumed that gel formulations of adenoviral vectors enhanced gene transfer via CAR-independent mechanisms (24). The receptor for AAV5, PDGFR (31), appears to be functionally present on the apical surface of airway epithelia (13). Likewise, GP64 pseudotyped FIV preferentially transduces airway epithelia from the apical surface (P.L.S. and P.B.M., unpublished observations). The finding that CMC and MC gel formulations substantially enhanced AAV5 and GP64 pseudotyped FIV gene transfer is consistent with the idea that improving the vector residence time by inhibiting mucociliary clearance increases the probability for receptor binding and uptake.

Figure 5. In vivo gene transfer with AAV-eGFP using viscoelastic vehicles. AAV-eGFP (1.0 ⫻ 1011 vg) formulated with 1% CMC or 1% MC and delivered to mouse airways via orotracheal intubation. Additional mice were treated with 400 mM EGTA via nasal instillation; 15 min later mice received AAV-eGFP formulated with 1% MC (MC⫹EGTA) or unformulated AAV-eGFP (EGTA). AAV-treated mice were compared with naı¨ve mouse controls. Mouse lungs were examined by confocal microscopy 3 wk after vector delivery. All panels are the same scale. Scale bar ⫽ 200 ␮m. v, blood vessels; asterisks indicate large airways. Representative images from three mice/ condition.

Sinn, Shah, Donovan, et al.: Gene Transfer with Viscoelastic Vehicle TABLE 2. VISCOELASTIC GEL EFFECTS ON LENTIVIRAL VECTOR TITER Titer (TU/ml) Carboxymethylcellulose (1%) Methylcellulose (1%) Poloxamer 407 (7.5%) Culture media control

5.1 4.7 2.7 6.0

⫻ ⫻ ⫻ ⫻

107 107 107 107

⫾ ⫾ ⫾ ⫾

36% 6% 16% 23%

Data are presented as averages ⫾ percent SD (n ⫽ 3). TU ⫽ transducing units.

Based on the cow tracheal explant model, the concentrations of viscoelastic gels used in this study transiently decreased mucociliary transport rate by ⬍ 50% (Table 1). Higher gel concentrations may be more effective at enhancing gene transfer to conducting airways. Indeed, 2% methylcellulose gels can decrease mucociliary transport rate by ⵑ 85% (A.S. and M.D., unpublished observations). For this study, the maximum “work-

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able” concentration of each gel was chosen. For example, a 3% CMC gel is too viscous to accurately pipette and deliver to the airways. At room temperature the polymer concentrations of MC and CMC used in the present study produced freely flowing gels with viscosities qualitatively similar to normal mucus. In contrast, the reverse-thermally gelling poloxamer P407 system was too viscous to accurately pipette at room temperature and had to be formulated and delivered cold. We observed that orotracheal delivery of 7.5% P407 formulated vector resulted in low survival rates in mice; however, in a large animal model it is possible to deliver formulated vector to a localized lobe or segment of the lung. Perhaps under these conditions, higher viscosity gels would be better tolerated and even more effective at inhibiting MTR and promoting gene transfer. Importantly, all of the gel-forming polymers tested in these studies have been shown to be safe for use in humans (32–34). MC, CMC, and P407 are present in numerous FDA-approved commercial drug products that are administered topically for cutaneous, ophthalmic, oral, or nasal applications. Although further testing of the safety of these and other gels in the lungs is important, preliminary examinations (35) of the respiratory tissues treated with MC or CMC do not show any evidence of local toxicity, and the safety history of these gel-forming polymers suggests that these are promising formulations for clinical use. Interestingly, MC formulations increased the transduction efficiency for both enveloped and nonenveloped viral vectors. Although not a subject of our studies, gel formulations might also increase the transduction efficiency of nonviral vectors such as liposome/DNA complexes or naked DNA. These studies indicate that viscoelastic gel formulations are promising vehicles for several classes of gene transfer vectors. Conflict of Interest Statement : P.L.S has no declared conflicts of interest; A.J.S. has no declared conflicts of interest; M.D.D. has no declared conflicts of interest; and P.B.M. has no declared conflicts of interest. Acknowledgments : The authors are grateful for the contributions of Micheal Henry, Haley Sinn, Beverly Davidson, Melissa Hickey, and Christine Rowley. They acknowledge the assistance of the DNA Sequencing Core, Cell Morphology Core, Cell Culture Core, and the Gene Transfer Vector Core partially supported by the Cystic Fibrosis Foundation, NHLBI (PPG HL-51670), and the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-54759).

References

Figure 6. In vivo gene transfer with FIV-Luc and FIV-␤Gal using viscoelastic vehicles to mouse nasal epithelia. FIV-Luc was delivered to mouse airways via nasal instillation (A ). Vector was delivered in saline formulation (Neat) or with 1% MC formulation. Additional mice were pretreated with 400 mM EGTA via nasal instillation; 15 min later mice received FIV-Luc formulated with 1% methylcellulose (EGTA/MC) or unformulated FIV-Luc (EGTA). FIV-Luc–treated mice were compared with AdLuc formulated with 1% MC or naı¨ve mouse controls. Nineteen days after instillation, mice were imaged and light emission (photons/s/cm2) quantified. *P ⬍ 0.05; ns, not significant; n ⫽ 3. FIV-␤Gal formulated with 1% MC was delivered to mouse airways via nasal instillation (B ). Mouse lungs were examined by X-gal staining 21 d after vector delivery. Tissue sections were counterstained with nuclear fast red. Scale bars ⫽ 125 ␮m. oe, olfactory epithelia; re, respiratory epithelia; s, septum. n ⫽ 4 animals/condition.

1. Ferrari S, Geddes DM, Alton EW. Barriers to and new approaches for gene therapy and gene delivery in cystic fibrosis. Adv Drug Deliv Rev 2002;54:1373–1393. 2. Parsons DW, Grubb BR, Johnson LG, Boucher RC. Enhanced in vivo airway gene transfer via transient modification of host barrier properties with a surface-active agent. Hum Gene Ther 1998;9:2661–2672. 3. Coyne CB, Kelly MM, Boucher RC, Johnson LG. Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am J Respir Cell Mol Biol 2000;23:602–609. 4. Chu Q, St George JA, Lukason M, Cheng SH, Scheule RK, Eastman SJ. EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo. Hum Gene Ther 2001;12:455–467. 5. Wang G, Zabner J, Deering C, Launspach J, Shao J, Bodner M, Jolly DJ, Davidson BL, McCray PB Jr. Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo. Am J Respir Cell Mol Biol 2000;22:129–138. 6. Wang G, Slepushkin V, Zabner J, Keshavjee S, Johnston JC, Sauter SL, Jolly DJ, Dubensky TW Jr, Davidson BL, McCray PB Jr. Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect. J Clin Invest 1999;104:R55–R62. 7. Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ, Zabner J. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem 1999;274: 10219–10226. 8. Croyle MA, Cheng X, Sandhu A, Wilson JM. Development of novel formulations that enhance adenoviral-mediated gene expression in the lung in vitro and in vivo. Mol Ther 2001;4:22–28.

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9. Sinn PL, Hickey MA, Staber PD, Dylla DE, Jeffers SA, Davidson BL, Sanders DA, McCray PB Jr. Lentivirus pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independent of folate receptor-alpha. J Virol 2003;77:5902– 5910. 10. Kobinger GP, Weiner DJ, Yu QC, Wilson JM. Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 2001;19:225–230. 11. Halbert CL, Allen JM, Miller AD. Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors. J Virol 2001; 75:6615–6624. 12. Duan D, Yue Y, Yan Z, Yang J, Engelhardt JF. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J Clin Invest 2000;105:1573–1587. 13. Zabner J, Seiler M, Walters R, Kotin RM, Fulgeras W, Davidson BL, Chiorini JA. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol 2000;74:3852–3858. 14. Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X, Wilson JM. Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol 2004;78:6381–6388. 15. Suarez S, Hickey AJ. Drug properties affecting aerosol behavior. Respir Care 2000;45:652–666. 16. Seiler MP, Luner P, Moninger TO, Karp PH, Keshavjee S, Zabner J. Thixotropic solutions enhance viral-mediated gene transfer to airway epithelia. Am J Respir Cell Mol Biol 2002;27:133–140. 17. Anderson RD, Haskell RE, Xia H, Roessler BJ, Davidson BL. A simple method for the rapid generation of recombinant adenovirus vectors. Gene Ther 2000;7:1034–1038. 18. Davidson BL, Chiorini JA. Recombinant adeno-associated viral vector types 4 and 5: preparation and application for CNS gene transfer. Methods Mol Med 2003;76:269–285. 19. Johnston JC, Gasmi M, Lim LE, Elder JH, Yee J-K, Jolly DJ, Campbell KP, Davidson BL, Sauter SL. Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors. J Virol 1999;73:4991–5000. 20. Kumar M, Bradow BP, Zimmerberg J. Large-scale production of pseudotyped lentiviral vectors using baculovirus gp64. Hum Gene Ther 2003;14:67–77. 21. Zabner J, Karp P, Seiler M, Phillips SL, Mitchell CJ, Saavedra M, Welsh M, Klingelhutz AJ. Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am J Physiol Lung Cell Mol Physiol 2003;284: L844–L854.

22. Chen TM, Dulfano MJ. Mucus viscoelasticity and mucociliary transport rate. J Lab Clin Med 1978;91:423–431. 23. Wills P, Pritchard K, Cole P. Mucus transportability: the bovine trachea and frog palate models compared. Eur Respir J 1998;12:837–841. 24. Zabner J, Zeiher BG, Friedman E, Welsh MJ. Adenovirus-mediated gene transfer to cilitaed airway epithelia requires prolonged incubation time. J Virol 1996;70:6994–7003. 25. Wang G, Davidson BL, Melchert P, Slepushkin VA, Es HHGv, Bodner M, Jolly DJ, McCray PB Jr. Influence of cell polarity on retrovirusmediated gene transfer to differentiated human airway epithelia. J Virol 1998;72:9818–9826. 26. Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ, Zabner J. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem 1999;274: 10219–10226. 27. Zeiher BG, Eichwald E, Zabner J, Smith JJ, Puga AP, McCray PB Jr, Capecchi MR, Welsh MJ, Thomas KR. A mouse model for the delta F508 allele of cystic fibrosis. J Clin Invest 1995;96:2051–2064. 28. Parsons DW, Hopkins PJ, Bourne AJ, Boucher RC, Martin AJ. Airway gene transfer in mouse nasal-airways: importance of identification of epithelial type for assessment of gene transfer. Gene Ther 2000;7:1810– 1815. 29. Mercer RR, Russell ML, Roggli VL, Crapo JD. Cell number and distribution in human and rat airways. Am J Respir Cell Mol Biol 1994;10:613– 624. 30. Parent RH. Treatise on pulmonary toxicology: comparative biology of the normal lung. Boca Raton, FL: CRC Press, Inc.; 1999. 31. Di Pasquale G, Davidson BL, Stein CS, Martins I, Scudiero D, Monks A, Chiorini JA. Identification of PDGFR as a receptor for AAV5 transduction. Nat Med 2003;9:1306–1312. 32. Grindel JM, Jaworski T, Piraner O, Emanuele RM, Balasubramanian M. Distribution, metabolism, and excretion of a novel surface-active agent, purified poloxamer 188, in rats, dogs, and humans. J Pharm Sci 2002;91:1936–1947. 33. Ismail FA, Napaporn J, Hughes JA, Brazeau GA. In situ gel formulations for gene delivery: release and myotoxicity studies. Pharm Dev Technol 2000;5:391–397. 34. Johnston TP, Miller SC. Toxicological evaluation of poloxamer vehicles for intramuscular use. J Parenter Sci Technol 1985;39:83–89. 35. Ugwoke MI, Agu RU, Jorissen M, Augustijns P, Sciot R, Verbeke N, Kinget R. Toxicological investigations of the effects carboxymethylcellulose on ciliary beat frequency of human nasal epithelial cells in primary suspension culture and in vivo on rabbit nasal mucosa. Int J Pharm 2000;205:43–51.