Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect

Abstract

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.

Figure 1

FIV vectors transduce nondividing airway epithelia in vitro. (a) Application of FIV-βgal (moi 10) to the apical surface of the epithelial sheet resulted in no gene transfer. Representative en face view of X-gal stained epithelia 3 days after vector application. (b) FIV-βgal vector applied to airway epithelia from the basolateral surface in vitro (moi ∼10) resulted in gene transfer. Representative en face view is shown. (c) Gene transfer from the apical surface with VSV-G FIV is enhanced by physical disruption of epithelia. The epithelial sheet was scratched with a pipette tip prior to apical vector application. En face view shows gene transfer (gray cells) only along the area of epithelial disruption. (d) FIV transduces aphidicolin growth-arrested cells (en face view). Vector was applied to apical surface in the presence of 6 mM EGTA in hypotonic buffer. Approximately 17% of epithelia were transgene positive (range 12–22%; n = 5 epithelia from 2 different preparations). For cells in control media without aphidicolin, approximately 20% of cells expressed the transgene (data not shown; range 8–30%; n = 5 epithelia from 3 different preparations). (e) Quantification of gene transfer results under conditions shown in d (mean ± SEM; n = 5 epithelia; 3 different preparations).

Figure 2

FIV-CFTR corrects the CF Cl^– transport defect. CF epithelia were transduced from the apical surface in the presence of EGTA with ∼10 moi of FIV vector expressing either β-galactosidase or human CFTR. For comparison, another group of CF cells received Ad2/CFTR (moi 50, basolateral application). At time points of 3, 13, 30, 60, 90, and 180 days later, epithelia were mounted in Ussing chambers, and the change in short circuit current was measured in response to cAMP agonists (ΔIsc_(IBMX/Forsk)). (a) Comparison of ΔIsc_(IBMX/Forsk) in response to cAMP agonists in CF epithelia transduced with adenovirus or FIV vectors, 3 days after gene transfer. Both Ad- and FIV-transduced epithelia express cAMP-activated Cl^– currents similar to normal cells (n = 5 CF epithelia; n = 5 normal epithelia, for each time point). (b) CFTR expression persists in FIV-transduced epithelia. CF epithelia were transduced with FIV-βgal, FIV-CFTR, or Ad2/CFTR and ΔIsc_(IBMX/Forsk) measured at the indicated time points after gene transfer (n = 5 CF epithelia; n = 5 normal epithelia, for each time point). Data from each experiment were normalized to the mean Isc_(IBMX/Forsk) seen 3 days after infection. One CF preparation was viable 6 months after gene transfer.

Figure 3

Gene transfer to rabbit tracheal epithelia in vivo using FIV-βgal vector. Panels show results 5 days after gene transfer. Low magnification en face view of X-gal–stained trachea from control (a) or FIV vector–treated trachea (b). Blue cells were only seen in the trachea transduced with the FIV vector (b). (c) Low-magnification view of X-gal–stained tracheal section. β-galactosidase–expressing cells are noted at both the surface and basal cell levels of the transduced epithelium. (d–f) Higher-magnification views of tracheal epithelium showing cell types expressing β-galactosidase. No inflammatory cells were noted in control or transduced specimens (n = 4 animals). Scale bar in d also applies to e and f.

Figure 4

FIV-βgal transduction of lower airway epithelia 5 days after gene transfer. (a) En face view of pleural surface of lung after fixation and X-gal staining showing βgalactosidase–expressing cells. All treated animals had similar segments of β-galactosidase–expressing cells extending to the pleural surface. (b–f) Higher magnification views of tissue sections showing lower airway and parenchymal cells transduced. (b) Low-magnification view of a large bronchus (>750 μm diameter) demonstrating patches of β-galactosidase–expressing cells extending around the circumference of the epithelium. (c) High-magnification view of expression in a large bronchus (>750 μm diameter) showing expression in ciliated cells and basal cells. (d) High-magnification view of expression in a medium sized airway (500–750 μm diameter) demonstrating expression in nonciliated surface cells (Clara cells). (e) β-galactosidase expression in a small bronchus (250–500 μm diameter) showing expression in nonciliated surface cells (Clara cells). (f) Distal lung sample (airways 0–250 μm diameter) showing expression in cuboidal cells consistent with alveolar type II cells. (g) Gene transfer expressed as a function of airway size. Numbers above each bar represent the number of animals with transduced cells in the corresponding region. Tissues from 12 animals were studied. Scale bar in d also applies to e and f.

Figure 5

β-galactosidase expression persists in vivo 6 weeks after gene transfer. (a) En face view demonstrating β-galactosidase–positive cells in the trachea. Larger clusters of blue cells (arrows) were noted at 6 weeks than at the earlier time point, suggesting clonal expansion of transduced cells. (b–d) Cross sections of tissue shown in a. Multiples cell types were targeted as indicated by the arrows. Clusters of β-galactosidase–positive cells were noted, suggesting clonal expansion of targeted cells (n = 4 animals). Epithelial morphology appeared normal as determined by examination of hematoxylin and eosin stained sections. Scale bar in d also applies to b and c. SMG = submucosal gland.