Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer

Abstract

Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of alphavbeta3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.

FIG. 1

Interaction of AdV with human airway epithelial cell cultures. (A) Representative histological cross-sections of human airway cells after 5 days in culture showing PD epithelial cells and after >25 days in culture showing WD pseudostratified ciliated epithelial cells. Hematoxylin and eosin counterstain; magnification, ×215. (B to D) Comparative analyses of lacZ gene expression in PD and WD cultures 48 h after exposure to AdVlacZ (6 h at 37°C) (B), internalization of AdV into PD and WD cultures after exposure to ³⁵S-AdVlacZ (6 h at 37°C) (C), and attachment of AdV to PD and WD cultures after exposure to ³⁵S-AdVlacZ (6 h at 4°C) (D). Only the apical surfaces of cultures were exposed to AdV (10¹⁰ particles/ml). β-Gal activity and counts per minute (CPM) were measured per square centimeter of epithelial surface area. Values shown are mean ± standard error (SE) (n = 3). The results shown are representative of a total of three different experiments.

FIG. 2

Direct visualization of specific and nonspecific cellular uptake pathways with human cultures expressing both PD and WD cellular phenotypes. (A) Exposure of cellular islands to CyAdV (10¹⁰ particles/ml for 6 h at 37°C) (red) resulted in association of CyAdV with PD cells at the periphery of the islands (arrow) and only rarely with individual cells within the WD regions. (B) The cellular distribution of CyAdV is paralleled by the cellular distribution of lacZ expression (arrow) in the same cultures 24 h later. (C and E) To assess whether uptake into PD cultures was restricted to specific AdV uptake, the apical surfaces of confluent cultures were exposed to fluorescent microspheres (∼10¹⁰ spheres/ml for 6 h at 37°C), resulting in a large quantity of microspheres (red) associated with PD (C) but not WD (E) cultures viewed en face. (D and F) To determine the cellular localization of microspheres, confocal microscopy-generated XZ sections revealed that microspheres (red) were both attached to and internalized into PD cultures (D) but not WD cultures (F). For panels D and F, the cells were counterstained with calcein. Magnifications, ×48 (A and B), ×24 (C and E), and ×240 (D and F).

FIG. 3

Specificity of AdV interactions with human PD and WD airway cultures. Inhibition of AdV-mediated gene transfer (top) and AdV attachment (bottom) to cells preincubated with purified fiber-knob protein (10 μg/ml) (+knob). Fiber-knob protein produced only a partial inhibition of AdV-mediated gene transfer to PD cultures but inhibited the small amount of gene transfer to WD cultures. In contrast to HeLa cells, fiber-knob protein did not inhibit AdV attachment to either PD or WD cultures. AdV represents cultures exposed to AdV but not preincubated with fiber-knob protein. Values shown represent mean ± SE of n determinations, where n is shown in parentheses.

FIG. 4

Investigation of nonspecific attachment of AdV to human PD and WD airway cells. The apical surfaces of human cultures were exposed to AdV (10¹⁰ particles/ml for 6 h at 4°C), and tissues were processed for TEM to assess AdV attachment. (A) With human PD cultures, AdV (arrows) was associated with cellular glycocalyx-like structures on the apical membrane (inset). (B) With WD cultures, in agreement with the attachment studies, little AdV was associated with the apical surface. Magnifications, ×7,000 (A and B) and 20,000 (inset).

FIG. 5

Expression of hCAR mediates AdV attachment and gene expression. (A) Specific AdV attachment (i) and gene expression (ii) were measured for the HeLa, CHO, and CAR-CHO cell lines by incubating cells in the absence (solid bars) or presence (open bars) of purified fiber-knob protein (10 μg/ml) for 1 h at 4°C before adding ³⁵S-AdV (10¹⁰ particles/ml for 2 h at 4°C). Values shown represent the mean ± SE (n = 6 and 3 for solid and open bars, respectively). (B) Representative immunofluorescent detection of hCAR with HeLa (i), CHO (ii), and CAR-CHO (iii) cell lines exposed at 4°C to anti-hCAR MAb with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (green) and CyAdV (red). Cell nuclei are counterstained with DAPI (blue). Localization of hCAR and CyAdV was restricted to HeLa and CAR-CHO cells; there was no localization of hCAR and little CyAdV associated with CHO cells. Magnification, ×250.

FIG. 6

Distribution of endogenous hCAR in human PD and WD cultures. (A) Human PD (i and iii) and WD (ii and iv) cultures after permeabilization were exposed to either RmcB MAb (i and ii) or an irrelevant isotyped MAb (iii and iv), and binding was detected with goat anti-mouse IgG-Texas Red. For PD cultures, hCAR was detected on all surfaces of epithelial cells, whereas WD cultures show a basolateral distribution of hCAR. Magnification, ×100. (B) Exposure of AdV to apical versus basolateral surfaces of human WD cultures. AdV (10¹⁰ particles/ml) was applied to the respective surfaces for 6 h at 37°C, and gene expression was measured 48 h later. The values shown represent mean ± SE (n = 3).

FIG. 7

Interaction of AdV with rat airway epithelial cell cultures. (A) Representative histological cross-sections of rat airway cells after 5 days in culture showing PD epithelial cells and after >19 days in culture showing WD pseudostratified mucociliary epithelial cells. Hematoxylin and eosin counterstain; magnification, ×220. (B to D) Comparative analyses of lacZ gene expression in PD and WD cultures 48 h after exposure to AdVlacZ (6 h at 37°C) (B), internalization of AdV into PD and WD cultures after exposure to ³⁵S-AdVlacZ (6 h at 37°C) (C), and attachment of AdV to PD and WD cultures after exposure to ³⁵S-AdVlacZ (6 h at 4°C) (D). Only the apical surfaces of cultures were exposed to AdV (10¹⁰ particles/ml). β-Gal activity and counts per minute (CPM) were measured per square centimeter of epithelial surface area. Values shown are mean ± SE (n > 8).

FIG. 8

α_vβ_3/5 integrin localization in human airway cultures and entry of AdV mediated by dynamin-associated uptake pathways. (A) Panel i shows immunoprecipitates of biotinylated human α_vβ_3/5 integrins with a rabbit anti-human α_vβ_3/5 integrin polyclonal antibody. Apical (Ap) or basolateral (Bl) membranes of either PD (lanes 1, 2, and 5) or WD (lanes 3, 4, and 6) cultures were biotinylated and, after immunoprecipitation, probed with streptavidin conjugated to horseradish peroxidase for detection. Lanes 5 and 6 show immunoprecipitation with a rabbit IgG control. The arrowhead shows approximately 120 kDa. The experiment shown is representative of four experiments. Panel ii shows lack of inhibition of gene transfer with α_vβ_3/5 integrin-interacting peptides. Neither cRGD peptide (0.4 mg/ml) with human cultures (HBE) nor RGD peptide (4 mg/ml) with rat cultures (RTE) significantly altered the level of transgene expression in the respective cultures after exposure to AdVlacZ (10¹⁰ particles/ml). Values shown represent mean ± SE (n = 4 and 3 for cultures in the absence [closed bars] and presence [open bars] of RGD peptides, respectively). (B) Comparison of AdV attachment (i) and AdV-mediated gene transfer (ii) to HeLa cells overexpressing either Wt or mutant (K44a) dynamin. Cells were exposed to AdVlacZ (10¹⁰ particles/ml) for 2 h at 4°C, and either attachment was measured immediately or expression was measured 24 h later. Values shown represent mean ± SE (n = 6 for each).

FIG. 9

Increased AdV attachment leads to increased gene transfer in cells with active nonspecific uptake pathways. (A) Effect on AdV attachment and gene transfer in human PD (i) and WD (ii) cultures with exposure to different numbers of AdV particles. Values shown represent mean ± SE (n > 3). (B) Panel i shows measurement of nonspecific fluid-phase uptake pathways in human PD and WD cells with [³H]inulin exposed to the luminal surface of the cultures at 37°C for 6 h. Values shown represent mean ± SE (n > 5). Panel ii shows gene expression in parallel cultures exposed to AdVlacZ (10¹⁰ particles/ml) for 6 h at 37°C, with enzyme activity measured 24 h later. Values shown represent mean ± SE (n > 8).