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Journal of Virology, October 2005, p. 12818-12827, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12818-12827.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

Patrick L. Sinn,1 Erin R. Burnight,1 Melissa A. Hickey,1 Gary W. Blissard,2 and Paul B. McCray Jr.1*

Program in Gene Therapy, Department of Pediatrics, Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242,1 Boyce Thompson Institute, Cornell University, Ithaca, New York 148532

Received 28 April 2005/ Accepted 22 July 2005


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ABSTRACT
 
Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 106 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (~109 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ~1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.


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INTRODUCTION
 
One aim of gene transfer vector development is to create vehicles to efficiently, safely, and persistently express therapeutic genes in the appropriate cell types (32, 51). For cystic fibrosis gene therapy, the goal is to deliver the cystic fibrosis transmembrane conductance regulator (CFTR) to surface epithelial cells of the conducting airways. The most common envelope currently used for retrovirus pseudotyping is the vesicular stomatitis virus glycoprotein (VSV-G). VSV-G pseudotyping has many advantages over other available glycoproteins, including extensive cell tropism and a virion-stabilizing effect, allowing higher attainable titers by centrifugation (4). However, VSV-G preferentially transduces airway epithelia at the basolateral surface (50). Furthermore, the toxicity of the VSV-G protein to recombinant retrovirus-packaging cells and vector inactivation by human sera limit clinical applications of VSV-G-pseudotyped vectors (7, 26). A new envelope pseudotype possessing the advantages of VSV-G, but lacking its shortcomings, could have wide utility for a number of target tissues, including airway epithelia.

Our laboratory and others have investigated several viral envelope glycoproteins for their ability to mediate gene transfer to airway epithelia (16, 18, 21, 38, 41, 50, 52). The prospect of increasing the specificity of lentiviral vector tropism for apical transduction of airway epithelia via pseudotyping is appealing. Filoviral glycoprotein (Ebola or Marburg)-pseudotyped lentiviruses were the first virus-based vectors that displayed preferential apical transducing properties (21, 41). However, the maximal titers were significantly lower than those obtained with VSV-G. Significant effort was devoted to improving the titers, as previously described (41); however, even under optimal conditions, the titers obtained were ~70-fold lower than VSV-G titers. While Jaagsiekte sheep retrovirus Env pseudotyped feline immunodeficiency virus (FIV) efficiently (40) and favored the apical surface for transduction, the transduction efficiency was very low (42).

The list of lentiviral envelope pseudotyping candidates for apical transduction of airway epithelial cells is daunting. Therefore, we focused our search on envelope proteins from viruses with known lung tropism. For example, paramyxoviruses (e.g., respiratory syncytial virus, parainfluenza virus, and measles virus) and orthomyxoviruses (e.g., influenza virus A and B) are well studied and known to enter and replicate in airway epithelia. However, some of these viruses use two envelope proteins to mediate viral binding and entry into host cells, complicating lentiviral pseudotyping (P. L. Sinn and P. B. McCray, Jr., unpublished observations). Retroviral envelope glycoproteins are typically type I transmembrane proteins that assemble into homotrimers. Within the orthomyxovirus family, a fourth genus (influenza virus D, also referred to as Thogoto virus) was defined by the composition and location of structural proteins and shared amino acid identity (31, 37). The influenza D genus has three members, Dhori, Thogoto, and Batken virus. These viruses have an envelope glycoprotein, GP75, that assembles into a homotrimer and mediates both binding and fusion (37).

Here we investigate the compatibility of influenza D and the related baculovirus family envelope glycoproteins for FIV pseudotyping and transduction of airway epithelia. Importantly, an FIV pseudotype using GP64 from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) transduces primary cultures of human airway epithelia from the apical surface and mediates persistent gene transfer to the respiratory epithelia of mice.


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MATERIALS AND METHODS
 
Vector production. The FIV vector system utilized in this study was reported previously (17, 52). The vector construct expressed either nuclear-targeted ß-galactosidase cDNA directed by the cytomegalovirus promoter or firefly luciferase directed by the Rous sarcoma virus promoter. Pseudotyped FIV vector particles were generated by transient transfection of plasmid DNA into 293T cells as described previously (17). Vector was concentrated 250-fold by centrifugation, and FIV vector preparations were titrated on HT1080 cells (ATCC no. 12012) at limiting dilutions.

Envelope constructs. Viral envelope gene sequences were PCR amplified and TA cloned into the mammalian expression plasmid pcDNA3.1-TOPO (K4800-01; Invitrogen) or pTARGET (A1410; Promega) by use of the manufacturer's protocol and the indicated amplification primers (Table 1). The plasmid backbone conferring the better titer (n ≥ 2; data not shown) was chosen for these studies.


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  		TABLE 1. Characteristics of envelope expression constructs

Culture of human airway epithelia. Airway epithelia were isolated from trachea or bronchi and grown at the air-liquid interface as described previously (19). Briefly, the culturing process is as follows: tissues undergo enzymatic dispersion, and epithelial cells are dissociated and are finally seeded onto collagen-coated semipermeable membranes (Millicell-PCF, Millipore) (0.6 cm2) (19). Twenty-four hours after seeding, the cells are allowed to grow and differentiate at the air-liquid interface. All preparations used were well differentiated (>2 weeks old; resistance > 1,000 {Omega} x cm2). This study was approved by the Institutional Review Board at the University of Iowa. A549 (ATCC CCL-185) cell lines are derived from human lung carcinoma. A549 cells were maintained in Dulbecco's minimal Eagle's medium-F12 (Gibco)-10% fetal bovine serum and supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml).

In vitro viral vector administration. Primary cultures of human airway epithelial cells were transduced with pseudotyped FIV by diluting vector preparations in serum-free medium to achieve the desired multiplicity of infection (MOI) and applying 100 µl of the solution to the apical cell surface. After incubation for 4 h at 37°C, the vector was removed and cells were further incubated at 37°C for 4 days. To infect epithelia with pseudotyped FIV from the basolateral side, the Millicell culture insert containing the epithelium was turned over and vector applied to the basolateral surface for 4 h in 100 µl of media. Following the 4-h infection, the vector was removed and the culture insert was turned upright and allowed to incubate at 37°C in 5% CO2 for 4 days.

Western blotting. Western blot analysis for AcGP64 envelope, VSV-G envelope, and p24 expression levels was performed using 10 µg of total protein, as determined by protein concentration assay (catalog no. 500-0111; Bio-Rad). Membranes were probed with mouse {alpha} p24 (IDEXX, Westbrook, Maine) (1:5,000 dilution), mouse {alpha} AcMNPV GP64 (AcV5 tissue culture supernatant) (1:1,000 dilution) (14, 30), or mouse {alpha} VSV-G conjugated to horseradish peroxidase (HRP) (catalog no. 1814133) (Roche) (1:10,000 dilution). Primary antibodies were detected with a secondary goat {alpha} mouse antibody conjugated to HRP (catalog no. 115-035-075; Jackson ImmunoResearch Laboratories Inc.) (1:5,000 dilution). HRP levels were detected with West Pico chemiluminescent substrate (catalog no. 34080; Pierce) and visualized with Kodak Biomax MR film (catalog no. 870-1302).

Electron microscopy. Formvar carbon-coated grids were prepared and glow discharged just before use to increase their hydrophilicity. Ten microliters of wild-type AcMNPV and AcGP64-FIV was applied to the grids. After 1 min, the excess liquid was removed by blotting with filter paper. One drop of 1% (wt/vol) uranyl acetate was added, and the excess was removed after 1 min. Samples were air dried and examined with a Hitachi H-7000 transmission electron microscope.

Endosomal acidification inhibition. A549 cells were treated with the carboxylic ionophore monensin (8 µM) (3, 8). Monensin was applied 1 h pretransduction at 4°C. The medium was changed, and AcGP64-FIV, VSV-G-FIV, and Ampho-FIV vectors were applied at an MOI of 10. Cells were incubated at 37°C for 30 min, and the medium was changed again. Control epithelia received vehicle treatment only. Four days later, gene transfer efficiency was assessed by ß-galactosidase enzyme assays and normalized to total protein by Lowry assay.

ß-Galactosidase quantification. The Galacto-light chemiluminescent reporter assay (Tropix; Bedford, MA) was used to quantify ß-galactosidase activity following the manufacturer's protocol. The relative light units (RLUs) were quantified using a luminometer (Monolight 3010; Pharmingen) and standardized to total protein as determined by a modified Lowry assay (catalog no. 23240; Pierce Biotechnology) using the manufacturer's protocol.

In vivo viral vector administration. This study was approved by the University of Iowa Institutional Animal Care and Use Committee. Female 6- to 10-week-old 18- to 22-g BALB/c mice (Harlan, Indianapolis, IN) were used in this study. Approximately 1.25 x 107 transducing units (TU) of FIV vector in a 50-µl volume were delivered to the nasal epithelia in anesthetized mice via direct instillation. This volume was chosen as a maximal tolerated dose. The vector was typically delivered to the right nostril; however, within an individual animal, no effort was made to ensure that only the right nostril received vector. Reporter gene expression was consistently bilateral. Vector was formulated with saline (neat) or with 1% methylcellulose (MC) as previously described (42). Instillation of neat vector required 5 to 10 s, whereas instillation of MC-formulated vector required 45 to 90 s. Mice received either a single dose or seven doses over seven consecutive days. Three weeks following the initial FIV-ß-galactosidase delivery, mice were sacrificed and the expression of the ß-galactosidase reporter gene in the upper respiratory tract was confirmed. Following FIV-Luc delivery, the mice were imaged at the indicated time points using a charge-coupled device (CCD) camera as described below.

Histochemical analysis. Heads were removed, fixed, X-Gal (5-bromo-4-chloro-3-indolyl-ß-galactopyranoside) stained, decalcified (27), paraffin embedded, sectioned, and counterstained with nuclear fast red per standard techniques. Sections were 8 µm thick and collected at 200-µm intervals. Stereology was performed using basic methods as previously described (6). Respiratory and olfactory epithelial areas were examined by capturing all images at x20 with an Olympus DP70 digital camera and analyzed using Image-Pro Plus version 4.1 computer software (Media Cybernetics, Inc., Silver Spring, MD). Epithelial areas of cells whose apical membrane reached the airway lumen were traced, and the number of ß-galactosidase-positive cells were calculated compared to the total number of cells within the traced area. Calculations are based on images taken from four mice, and four epithelial areas from each animal were examined. The epithelial areas were collected ~3.0 to 3.4 mm caudal from the nose. Images were coded and counted by an observer blinded to this code. Measurements were counted twice independently with reproducible results.

Bioluminescence imaging. In vivo luciferase expression was visualized using bioluminescence imaging. At the time points indicated, animals were injected intraperitoneally with 100 µl/10 g of body weight of D-luciferin (Xenogen, Alemeda, CA) (15 mg/ml in PBS) using a 25-gauge needle and anesthetized by isoflurane inhalation. Approximately 5 min after luciferin injection the animals were placed in the Xenogen IVIS imaging cabinet, anesthetized with 1 to 3% isoflurane, and imaged using a Xenogen IVIS CCD camera. After imaging, animals recovered in a heated cage. Imaging data were analyzed and signal intensity was quantified using Xenogen Living Image software.

Statistics. All numerical data are presented as means ± standard deviations. Statistical analyses were performed under consultation with the University of Iowa College of Public Health Department of Biostatistics by use of SAS (SAS Institute, Cary, NC) and R (www.r-project.org/) software packages.


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RESULTS
 
Transduction of human airway epithelia with influenza D GP75-pseudotyped FIV. Our efforts to pseudotype FIV with the hemagglutinin (HA) protein from influenza A (A/Japan/305/57) (H2N2) have not been successful. Visual titers on HT1080 cells were below the limit of detection (<102 TU/ml) (Fig. 1A). Unlike influenza A, which utilizes the HA protein for entry, influenza D requires the envelope protein GP75 for binding and entry. We evaluated envelopes from the influenza D family for their compatibility with FIV. Dhori (10) and Thogoto (37) virus GP75-pseudotyped FIV resulted in titers of 4.2 x 103 TU/ml and 5.0 x 106 TU/ml, respectively (Fig. 1A). To test the polarity of transduction on well-differentiated human airway epithelial cells, Thogoto virus GP75-pseudotyped FIV (Thogoto-FIV) expressing nuclear targeted ß-galactosidase was applied to the apical or basolateral surface of the epithelial sheet for 4 h at an MOI of ~0.5. Four days later, gene transfer was quantified by ß-galactosidase activity assay as described previously (41). As shown, transduction of polarized human airway epithelial cells by Thogoto-FIV occurred preferentially at the apical surface (Fig. 1B). These data indicate that influenza D envelope glycoproteins confer apical transducing properties to a lentiviral vector.



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  		FIG. 1. (A) The indicated envelope proteins from influenza A or D were used to pseudotype FIV. The maximum titer from at least two independent vector preparations is indicated in TU per milliliter. The phylogenetic tree diagrammatically shows the relative relationships between the envelopes tested and is not inclusive of all species within the family. (B) Primary cultures were transduced with Thogoto virus GP64-pseudotyped FIV vector applied to the apical (gray bars) or basolateral (black bars) surface. Four days after initial vector incubation, cells were harvested and the ß-galactosidase activity was quantified and normalized to total protein. Each bar represents one culture each from three independent human specimens. *, P < 0.01 as determined by a paired t test (MOI = 0.5). The y axis begins at the background level of the assay (102 RLU per microgram of protein).

Transduction of human airway epithelia with baculovirus GP64-pseudotyped FIV. The suggestion has been made that horizontal transfer of the GP64/GP75 envelope protein gene occurred during the evolution of influenza D and baculovirus (31). Given this sequence homology and prior reported success of pseudotyping human immunodeficiency virus (HIV) with AcMNPV GP64 (22), we hypothesized that baculovirus GP64 might efficiently pseudotype and confer apical transducing properties to FIV. We screened the envelope glycoproteins from several species of baculovirus for their FIV-pseudotyping properties (Fig. 2A). Efficient FIV pseudotyping (ranging from 107 to 109 TU/ml) resulted with baculovirus GP64 envelope glycoproteins from AcMNPV, Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV), and Anticarsia gemmatalis multicapsid nuclear polyhedrosis virus (AgMNPV) (Fig. 2A). Conversely, the pseudotyping efficiency of the F proteins from Spodoptera exigua multicapsid nuclear polyhedrosis virus (SeMNPV), Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) (23), and Plutella xylostella granulovirus (PxGV) was much lower (ranging from 102 to 103 TU/ml) (Fig. 2A). In addition, a metavirus (TED) F protein with high sequence homology to baculovirus F protein (33) pseudotyped FIV with low efficiency (data not shown). These data demonstrate that baculovirus GP64 envelope glycoproteins efficiently pseudotype FIV, whereas baculovirus F envelope proteins do not.



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  		FIG. 2. (A) The indicated baculovirus envelope proteins were used to pseudotype FIV. The maximum titer from at least two independent vector preparations is indicated. The phylogenetic tree diagrammatically shows the relative relationships between the envelope proteins tested and is not inclusive of all species within the family. (B) Primary cultures were transduced with AcGP64 (MOI = 30)-, VSV-G (MOI = 30)-, or OpGP64 (MOI = 5)-pseudotyped FIV vector applied to the apical (gray bars) or basolateral (black bars) surface. Four days after initial vector incubation, cells were harvested and the ß-galactosidase activity was quantified and normalized to total protein. Each bar represents one culture each from seven (AcGP64-FIV and VSV-G-FIV) or three (OpGP64-FIV) independent human specimens. *, P < 0.01; **, P < 0.025 as determined by a paired t test. The y axes begin at the background level of the assay (102 RLU per microgram of protein).

FIV pseudotyped with envelopes from AcMNPV (AcGP64-FIV), VSV-G (VSV-G-FIV), or OpMNPV (OpGP64-FIV) and expressing ß-galactosidase was evaluated for the ability to transduce primary cultures of well-differentiated human airway epithelia. The vector was applied to the apical or basolateral surface of the epithelial sheet for 4 h. As before, 4 days following vector delivery, gene transfer was quantified by measuring ß-galactosidase activity. As shown (Fig. 2B), AcGP64-FIV (MOI = 30) and OpGP64-FIV (MOI = 5) transduced the cells from either surface; however, the apical efficiency was significantly greater than the basolateral. This finding was in sharp contrast to our results with VSV-G-FIV (MOI = 30), where entry was predominantly basolateral. Importantly, at the same MOIs the ß-galactosidase enzyme activity levels for the AcGP64 were equal to or greater than those achieved with the VSV-G envelope, the most efficient pseudotype we have identified to date. Gene transfer was inhibited by the reverse transcriptase inhibitor AZT or the transcriptional inhibitor actinomycin D, indicating that these results do not simply represent protein transfer or "pseudotransduction" (data not shown).

Vector characteristics and cell entry route of baculovirus GP64-pseudotyped FIV. Because the titers and transduction efficiency were greatest for the AcGP64-FIV, we focused the remainder of our studies on this glycoprotein. Consistent with previous studies (48), we observed under electron microscopy that wild-type AcMNPV is cigar shaped and ~250 to 300 nM in length (Fig. 3A), whereas AcGP64-FIV is spherical and within a typical lentiviral particle diameter of ~50 to 100 nM (Fig. 3B). Ratios of FIV p24 to AcGP64 and FIV p24 to VSV-G were similar by Western blotting (Fig. 3C), which is consistent with efficient packaging into virions.



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  		FIG. 3. Characterization of AcGP64-FIV by transmission electron microscopy, Western blotting, and monensin treatment. Wild-type AcMNPV (A) and FIV pseudotyped with AcMNPV GP64 (B) were subjected to negative staining and transmission electron microscopy. Scale bars = 50 nm. (C) FIV pseudotyped with AcGP64 and VSV-G was subjected to Western blot analysis with envelope and p24 antibodies. (D) A549 cells received no pretreatment (black bars) or were pretreated with 8 µM monensin (gray bars). Following monensin pretreatment, cells received GP64-FIV, VSV-G-FIV, or Ampho-FIV at an MOI of 10. Four days following vector delivery, cells were lysed and ß-galactosidase was quantified and normalized to total protein (n = 3). *, P < 0.01 as determined by a paired t test.

As an initial characterization of the cellular transduction process, we asked whether AcGP64-FIV fuses at the cell surface or within a low pH endosome. Monensin is an inhibitor of intracellular protein transport and a carboxylic ionophore that prevents endosomal acidification. Using monensin pretreatment of A549 cells, we found that AcGP64-FIV transduction was monensin sensitive, in similarity to VSV-G-FIV (Fig. 3D). In contrast, amphotropic envelope-pseudotyped FIV (Ampho-FIV) fuses at the cell surface in a pH-independent fashion and was unaffected by monensin (Fig. 3D). Thus, pretreating A549 cells with monensin disrupted transduction by AcGP64-FIV and VSV-G-FIV but not Ampho-FIV. These data suggest that, like VSV-G-FIV and wild-type baculovirus (56), AcGP64-FIV transduces cells via a low-pH endosome route.

AcGP64 pseudotyped FIV persistently transduces respiratory epithelia in vivo. We utilized the firefly luciferase reporter gene to test the efficiency of FIV transduction in vivo. Pseudotyped FIV vector expressing luciferase was formulated with 1% methylcellulose and delivered to mice via nasal instillation. Mice received FIV pseudotyped with VSV-G (VSV-G-Luc; 2.5 x 107 TU), Ebola envelope GP (previously optimized [15, 41]) (EBO{Delta}O-Luc; 3 x 106 TU), or AcMNPV GP64 (AcGP64-Luc; 2.5 x 107 TU) via nasal instillation (Fig. 4). Transgene expression was quantified 42 days later by bioluminescent imaging. Equal transducing units of VSV-G-Luc and AcGP64-Luc were delivered; however, the transduction efficacy of AcGP64-Luc was significantly higher than that of VSV-G-Luc (Fig. 4). AcGP64-Luc transduction was also significantly higher than that of EBO{Delta}O-Luc, although the lower transduction efficiency of EBO{Delta}O-Luc was made more difficult to evaluate by the lower MOI delivered. The transduction efficiencies of both EBO{Delta}O-Luc and VSV-G-Luc were significantly greater than that of the naïve control (P < 0.01, as determined by analysis of variance [ANOVA]).



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  		FIG. 4. In vivo gene transfer with FIV-Luc to mouse nasal epithelia. FIV pseudotyped with AcGP64, EBO{Delta}O, or VSV-G and formulated with 1% methylcellulose was delivered to mice via nasal instillation. At 42 days following instillation, mice were imaged and photons per second per square centimeter were quantified as described in Materials and Methods. Red squares indicate individual animals. *, P < 0.0001 as determined by ANOVA using the Holm correction for multiple comparisons.

Encouraged by these initial results, we used longitudinal bioluminescence imaging to evaluate reporter gene persistence for the AcGP64-Luc and VSV-G-Luc constructs following nasal application (Fig. 5). Prior to instillation, the vector was formulated with MC or with PBS (neat) as described in Materials and Methods. MC-formulated adenoviral vector-expressing luciferase (Ad-Luc) was delivered in parallel as a positive control. Three weeks following vector delivery luciferase expression was quantified by bioluminescence imaging with a CCD camera (Fig. 5A). The greatest level of expression was observed in mice that received MC-formulated AcGP64-Luc. Bioluminescence from mice that received formulated or neat VSV-G-Luc was not significantly different than naïve control results (Fig. 5B). Gene transfer efficacy was further evaluated 3, 9, 19, 25, 35, and 50 weeks following vector delivery. Luciferase expression from AcGP64-Luc (with or without MC) remained constant over the tested time period. Conversely, Ad-Luc expression dropped sharply between the 3-week and 9-week time points.



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  		FIG. 5. Time course of in vivo gene transfer to mouse nasal epithelia with FIV-Luc by use of viscoelastic vehicle. (A) FIV-Luc was delivered to mouse airways via nasal instillation. Vector was delivered in saline formulation (Neat) or with a 1% MC formulation. FIV-Luc-treated mice were compared to mice treated with Ad-Luc formulated with 1% methylcellulose or naïve mouse controls. (B) At 3, 9, 19, 25, 35, and 50 weeks following instillation, mice were imaged and photons per second per square centimeter were quantified. n = 3 mice for each condition. *, P < 0.0001 compared to naïve control mouse results by ANOVA, using the Tukey-Kramer adjustment for multiple comparisons.

All bioluminescent imaging was initially performed with the animal in the supine position. The origin of luciferase expression was examined by rotating the mouse in 90° increments (Fig. 6A). Rotation of a luciferase-positive mouse localized the expression to a region consistent with the nasal passages. The greatest expression was observed over the most caudal portion of this region. Intense expression localized at the eye results, in part, from improved photon transmission through the optic canal and the thinner bone surrounding the orbit.



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  		FIG.6. In vivo localization of gene transfer with FIV in mouse nasal epithelia. (A) The origin of AcGP64-FIV-derived luciferase expression was examined by rotating a mouse in 90° increments. Scale for pseudocolor intensity is photons/s/cm2/sr. A single dose (B to F) or seven doses over seven consecutive days (G to K) of AcGP64-ßGal formulated with 1% methylcellulose were delivered to mouse airways via nasal instillation. High-power images reveal examples of olfactory (D, I) and respiratory (F, K) cell types. Single dose, n = 3 animals; seven consecutive doses, n = 1 animal. The computed tomatography scan, sagittal view inset of the mouse head displays the approximate location and orientation of the section. With nasal diagrams described by Mery et al. (29) used as a guide, panel B approximately corresponds to section level 9 and panel G approximately corresponds to section level 14. Scale bars = 500 µm (panels B and G; photo montage), 200 µM (C, E, H, and J), or 25 µM (D, F, I, and K). OE, olfactory epithelia; RE, respiratory epithelia; S, septum.

To determine the cell types transduced in the nasal airways, FIV-vector-expressing nuclear-targeted ß-galactosidase was pseudotyped with AcGP64 (AcGP64-ßGal). The vector was again formulated with 1% methylcellulose and delivered to mice via nasal instillation (Fig. 6B to K). Mice received either a single dose (Fig. 6B to F) or seven doses given once daily over seven consecutive days (Fig. 6G to K). At 10 days following vector delivery, paraffin-embedded sections of nasal tissues were prepared as described in Materials and Methods. Consistent with our previous observations (43), morphological analysis confirmed ß-galactosidase expression in both respiratory (Fig. 6E, F, J, and K) and olfactory (Fig. 6C, D, H, and I) epithelia in animals that received AcGP64-ßGal. As might be expected, a greater number of transduced cells was observed in nasal epithelia following seven consecutive doses of vector.

A greater number of ß-galactosidase-positive cells was observed caudally than rostrally (data not shown). This regional preference may be the result of the architecture of the nasal airways, gravity, and/or the delivery protocol. Furthermore, this observation is consistent with the luciferase expression from bioluminescence imaging (Fig. 6A). Rare ß-galactosidase-positive cells that were morphologically and regionally consistent with squamous or transitional epithelia could be found (data not shown), However, within the nasal epithelium, these cells are not the predominant cell types and are generally localized to rostral portions of the nasal airways. Therefore, the transduction efficiency of squamous and transitional epithelia was too low to quantify.

ß-Galactosidase-positive cells from two low-power (x20) fields each of respiratory cells and olfactory cells from four mice that received a single dose of AcGP64-ßGal were quantified as described in Materials and Methods. Within the respiratory epithelia, an average of 1.98% of the epithelial cells were ß-galactosidase positive (range, 0/1,056 to 29/629 cells). Within the surface (cells whose apical membrane reached the lumen) of the olfactory epithelia, 1.69% of the epithelial cells were ß-galactosidase positive (range, 5/1,865 to 32/580 cells).

To examine the persistence of expression, a single dose of methylcellulose-formulated AcGP64-ßGal and VSV-G-ßGal was delivered to mice via nasal instillation and the in vivo expression was followed for a longer period of time. Paraffin-embedded sections of nasal tissues were prepared 10 (Fig. 7A and D) and 90 (Fig. 7B, C, E, and F) days following vector delivery. At 90 days posttransduction, clusters of cells were evident in the olfactory epithelium. We also observed occasional ß-galactosidase-positive clusters of cells in the respiratory epithelium (data not shown). Such clusters suggest clonal expansion; however, further research is necessary to support such a speculation. No ß-galactosidase-positive cells were observed in animals that received VSV-G-ßGal (Fig. 7C and F).



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  		FIG. 7. A single dose of AcGP64-ßGal (A, B, D, and E) or VSV-G-ßGal (C and F) formulated with 1% methylcellulose was delivered to mouse airways via nasal instillation. Mouse nasal sections were examined by X-Gal staining 10 days (A and D) or 90 days (B, C, E, and F) following vector delivery. Tissue sections were collected ~2.8 mm caudal from the nose and counterstained with nuclear fast red. Scale bar = 500 µm (A, B, and C) or 200 µm (D, E, and F).


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DISCUSSION
 
We sought to adapt the respiratory tropism of glycoproteins from existing viruses and use them in a pseudotyped retrovirus. Others have reported successful retroviral pseudotyping with the HA envelope glycoprotein from influenza A fowl plague virus (9, 12). However, to date the only success at adapting respiratory tropism in retroviral pseudotyping has been with envelopes from the filovirus family. We, and others, have shown that HIV (21) or FIV (41) vectors pseudotyped with the Env from Marburg or Ebola virus transduce human or murine airway epithelia from the apical surface. The filovirus pseudotypes with the highest titers were generated by removing the mucin-like domain rich in O-glycosylation sites (EBO{Delta}O) (15, 41). We found that glycoproteins from members of the orthomyxovirus (influenza D virus) and baculovirus families were compatible with FIV. The best candidate, AcMNPV GP64, generated excellent FIV titers and conferred persistent gene transfer in vivo, consistent with progenitor cell transduction.

Others have suggested that, during the evolution of influenza D and baculovirus, horizontal transfer of an ancestral GP64/GP75 gene occurred (reviewed in reference 34). Based on the high ~30% amino acid identity between influenza D GP75 and baculovirus GP64, we further tested the ability of baculovirus envelope glycoproteins to pseudotype FIV and transduce airway epithelia at the apical surface. We observed that baculovirus F proteins pseudotyped FIV poorly, whereas pseudotyping FIV with baculovirus GP64 glycoproteins generated high vector titers ranging from 107 to 109 TU/ml. Of note, titers for AcGP64-FIV were, on average, 7.8 x 108 TU/ml, with maximal titers of 6.0 x 109 TU/ml. These titers are comparable to typical titers of VSV-G-FIV. AcGP64-FIV was consistently produced at titers exceeding those achieved from Marburg-, Ebola-, or EBO{Delta}O (15, 41)-pseudotyped FIV by use of identical procedures. We further tested the capacity of these pseudotyped vectors to transduce airway epithelia. Thogoto-FIV, OpGP64-FIV, and AcGP64-FIV each transduced well-differentiated human airway epithelia more efficiently when applied to the apical surface rather than the basolateral surface.

Wild-type baculovirus AcMNPV infects mammalian cells very efficiently (48), although it replicates only in insect cells. Baculovirus GP64-induced membrane fusion has been extensively studied (5, 20, 24, 35, 36, 46, 56). GP64 assembles into a homotrimer (28) and is responsible for receptor-mediated endocytosis as well as acid-induced endosomal escape (13). The kinetics of uptake and release from low-pH endosomes was similar to that of vesicular stomatitis virus (13). Expression of GP64 alone is sufficient to induce low-pH-triggered cell-cell fusion, thus defining it as a true fusion protein (3, 24). Recently, AcMNPV GP64 was demonstrated to efficiently pseudotype an HIV-based vector (22). Kumar and colleagues further demonstrated that this vector transduced cells in vitro in a manner similar to that VSV-G pseudotyped HIV. Schauber et al. recently characterized the transduction efficiencies of GP64-pseudotyped HIV type 1 (HIV-1)-based vector in other cell lines and demonstrated that hepatic transduction in mice was comparable to VSV-G-pseudotyped HIV-1 results following portal-vein injection (39).

Presumably, AcGP64-FIV transduces airway epithelial cells at the apical surface in vitro because of an apically localized cellular receptor. Furthermore, airway epithelial cells were transduced in vivo in the absence of agents that disrupt tight junctions, most likely via the same apical localized cellular receptor. A receptor for baculovirus GP64 has not been identified. In prior studies, a soluble form of OpMNPV GP64 was shown to compete for AcMNPV viral binding, suggesting that OpMNPV and AcMNPV GP64 proteins use the same receptor (13). Competition experiments between influenza D GP75 and baculovirus GP64 glycoproteins have not been performed; however, such experiments would have interesting implications for the idea of lateral transfer of GP64 between the viral families and the possibility of common receptor recognition. We found that transduction by both VSV-G-FIV and AcGP64-FIV was inhibited by monensin, suggesting that fusion occurs in low-pH endosomes, in similarity to wild-type baculovirus results. These data suggest that in polarized cells, access to low-pH endosomal compartments from either the apical or basolateral surface results in effective gene transfer. We previously reported that gene transfer with Ampho-FIV from the apical surface was ineffective despite evidence of receptor expression (53). Therefore, targeting receptors that use low-pH endosome entry pathways may be more productive in polarized epithelia.

AcGP64-FIV expressing a firefly luciferase reporter was delivered to mice via nasal instillation and compared to VSV-G-FIV and EBO{Delta}O-FIV. The nasal epithelium was selected as a relevant target because cystic fibrosis mouse models are available that manifest measurable ion transport and other defects at this site (55). Many of the experimental conditions included vector formulated with 1% methylcellulose. Importantly, we previously demonstrated that viscoelastic gels such as methylcellulose significantly enhance vector transduction of airway epithelia without disrupting transepithelial resistance (43). Methylcellulose is a Food and Drug Administration-approved additive found in many common pharmaceuticals, including nasal sprays. With or without viscoelastic gel formulation, AcGP64-FIV transduced the nasal epithelia at greater efficacy than VSV-G-FIV. Furthermore, luciferase expression persisted for >11 months, suggesting successful targeting of a progenitor cell population. Histological analysis using a nuclear-targeted ß-galactosidase reporter revealed transduction of both respiratory and olfactory epithelial cells. As might be expected, the extent of transduction was greater in mice receiving repeated doses. Previous success at transducing murine nasal epithelia and persistence for ~3 months was reported using VSV-G-pseudotyped HIV-1 following pretreatment with lysophosphatidylcholine to disrupt tight junctions (25).

Because a large number of ß-galactosidase proteins/cell are required to stain a cell blue in the presence of X-Gal substrate, the number of blue cells detected microscopically likely underestimates transduction efficiency. Conversely, the number of CFTR mRNA transcripts expressed in surface epithelia has been estimated to be as little as 1 to 2 copies/cell (47). The groups of ß-galactosidase-positive cells at 90 days and the >11-month persistence of luciferase expression suggests that progenitor cells were targeted. We speculate that these groups of cells may represent clonal expansion of individually targeted progenitor cells. The turnover rates of respiratory and olfactory cells in the nasal epithelia have not been extensively studied in mice; however, the rates in rodents will differ as a result of developmental stage or injury (54).

We demonstrated that an AcGP64-pseudotyped lentiviral vector targets the apical surface of airway epithelia and persistently transduces mouse nasal epithelia in vivo. The successful development of a high-titer pseudotyped lentiviral vector capable of transducing respiratory epithelia in the absence of agents that disrupt tight junctions brings us closer to a clinical trial reagent for CFTR delivery with an integrating vector system.


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ACKNOWLEDGMENTS
 
We are grateful for the contributions of Beverly Davidson, Doug Dylla, Christine Rowley, Jennifer Springsteen, and Christine Wohlford-Lenane. We thank Micheal Henry for providing access to the Xenogen camera. We also thank Paul Friesen for providing the TED env and Fred Fuller for the Dhori construct.

We acknowledge the support of the DNA Sequencing Core, the Cell Morphology Core, and the Gene Transfer Vector Core partially supported by the Cystic Fibrosis Foundation, National Heart, Lung, and Blood Institute (PPG HL-51670), and the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-54759). This work was supported by National Institutes of Health grant RO1 HL-61460 (P.B.M.), PPG HL-51670 (P.B.M.), RO1 AI-33657 (G.W.B.), and the Cystic Fibrosis Foundation (grant SINN04G0 to P.L.S.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pediatrics, Carver College of Medicine, The University of Iowa, 240G EMRB, Iowa City, IA 52242. Phone: (319) 356-4866. Fax: (319) 335-6925. E-mail: paul-mccray{at}uiowa.edu. Back


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Journal of Virology, October 2005, p. 12818-12827, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12818-12827.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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