Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

Viruses and recombinant viral vectors.

rAAV vectors were prepared in human HEK 293 cells as previously described (26, 31, 34). Vectors were purified by double CsCl gradient ultracentrifugation, dialyzed into 10 mM Tris-HCl–150 mM NaCl–10% glycerol, and stored at −20°C until used for infection of airway epithelial cells. Vector titers, depicted as transducing units (TU) per milliliter, were determined on Ad-infected HeLa cells by staining for β-galactosidase activity (32). This procedure typically produced vector lots with titers of 10⁸ to 10⁹ TU/ml. The ratio of TU to the genome-based particle number was approximately 1:100. The AAV vector lots were not heat inactivated, since this AAV vector preparation method produces AAV free of wild-type Ad.

The Ad vector AdCMVβgal was used in this study. This vector encodes a cytoplasmic β-galactosidase gene driven by the cytomegalovirus promoter-enhancer (9, 37). Ad vectors were propagated in HEK 293 cells, purified by CsCl gradient ultracentrifugation, and stored at −20°C until use. Vectors titers were determined on HeLa cells by staining for β-galactosidase activity. These titers ranged from 2 × 10¹¹ to 2 × 10¹² TU/ml. The ratio of TU to the number of viral particles (as determined by measuring the optical density at 260 nm) was approximately 1:20.

Wild-type Ad dl309 (23) was used to enhance AAV transduction. The Ad mutants dl366* (19) (designated Ad E4⁻) and E4dl ORF1–4 (21) (designated Ad E4⁻ ORF6⁺ 6/7⁺) have been described previously. These mutants were used in the studies of the helper function in AAV-mediated gene transfer in human airway cells.

Ad was inactivated with long-wave UV irradiation in the presence of the DNA intercalator psolaren (5). In brief, samples of Ad were added to a freshly prepared 8-methoxypsoralen solution (0.33 μg/μl; Sigma) and, while on ice, exposed to a 366-nm UV light source (model UVL-56; UVP Inc., Upland, Calif.) for 30 min. Following UV irradiation, the virus was purified by gel filtration (G-50 Sephadex; Boehringer Mannheim Corp., Indianapolis, Ind.) into phosphate-buffered saline (PBS) containing 3% (vol/vol) glycerol.

Cell culture.

Human nasal epithelial cells were isolated from nasal polyps of CF patients and from turbinates of normal subjects and cultured as described in detail elsewhere (35). All procedures were approved by the University of North Carolina committee for the rights of human subjects. The cells were fed on alternate days with serum-free, hormone-supplemented F-12 medium (supplements included insulin [5 μg/ml], endothelial cell growth supplement [3.7 μg/ml], epidermal growth factor [25 ng/ml], triiodothyroxine [3 × 10⁻⁸ M], hydrocortisone [10⁻⁶ M], transferrin [5 μg/ml], and cholera toxin [10 ng/ml], plus penicillin and streptomycin) (35).

Two immortalized airway epithelial cell lines from CF patients were also used in this study. CF/T43 cells were developed from the nasal epithelium of a homozygous ΔF508 CF patient and transformed with the simian virus 40 (SV40) T antigen (22). CFT1 cells were derived from the tracheal epithelium of a homozygous ΔF508 CF patient and transformed with the E6 and E7 genes of human papillomavirus (38). All experiments using CF/T43 cells or CFT1 cells were performed on a single clone, between passages 20 and 30. The cells were fed on alternate days with serum-free, hormone-supplemented F-12 medium.

Transduction of human airway epithelial cells.

Primary human nasal epithelial cells or immortalized airway epithelial cell lines from CF patients were plated at a density of 3 × 10⁴ cells per well in 24-well dishes (Costar, Cambridge, Mass.) and allowed to attach for 16 h, and then nonadherent cells were removed by gentle washing with PBS. Two days after plating, the total number of cells per dish was approximately 1 × 10⁴ to 3 × 10⁴. AAV vector (AAVβgal) or Ad vector (AdCMVβgal) was added to the dishes at various multiplicities of infection (MOIs) in 0.3 ml of medium. After incubation for various periods of time at 37°C in air plus 5% CO₂, the cells were washed with PBS and then fed with fresh, hormone-supplemented F-12 medium. Control groups were exposed to 0.3 ml of vehicle (F-12 medium) alone for comparable time periods and washed as described above.

Two days after infection, the transduction efficiency was calculated by determining the percentage of β-galactosidase-expressing cells by histochemical staining (32). To assess the time course of transgene expression, staining was performed at 1, 2, 3, 5, 7, and 10 days after vector exposure. All experiments were performed at least in triplicate, and a minimum of 500 cells/well were counted to determine the percentage of transduced cells.

Effect of Ad on AAV transduction.

Two days after being plated, airway cells were incubated with AAV or Ad vector and then exposed to Ad dl309 for 1 h at 37°C. Forty-eight hours later, the transduction efficiency was determined as described above.

To elucidate the role of Ad in AAVβgal transduction of airway cells from CF patients, these cells were exposed to AAV vector at different MOIs for 1 h in the presence of either Ad dl309, UV- and psoralen-inactivated Ad dl309, Ad dl366* (Ad E4⁻), or E4dl ORF1–4 (Ad E4⁻ ORF6⁺ 6/7⁺) (100 particles/cell). The cells were then washed with PBS and fed with fresh, hormone-supplemented F-12 medium. Two days later, transduction efficiencies were determined by staining for β-galactosidase activity as described above.

Transduction of human tracheal explants with AAV.

Freshly excised human tracheal tissue (n = 3 [CF patients] or 4 [normal subjects]) was obtained from lung transplant CF patients and normal donors. Tracheal tissue was cut into 0.25-cm² samples and exposed to 20 μl of a solution containing 10⁸ TU (10¹⁰ genome-based particles) of AAVβgal vector/ml (MOI, ca. 1) for 30 min in the presence or absence of Ad dl309 (100 particles/cell). Tracheal specimens were then rinsed with PBS, incubated for 48 h at 37°C, fixed with 0.5% glutaraldehyde in PBS, and stained for β-galactosidase activity. The tissue was postfixed with OmniFixII (Aaron Medical Industries, St. Petersburg, Fla.), embedded in paraffin, sectioned, and counterstained with hematoxylin plus eosin.

AAV transduction in airway cells of CF patients and normal subjects.

We first tested the transduction efficiency of rAAV by infecting immortalized airway-specific cell lines and scoring for vector transduction by measuring lacZ expression. The transduction efficiency of the AAV vector increased in a dose-dependent manner in all cell types (Fig. 1). There was no difference in the transduction efficiency in primary nasal epithelial cells of CF patients versus those of normal subjects throughout the range of vector doses used (Fig. 1). The transduction efficiencies in the CF/T43 and CFT1 cell lines, however, differed dramatically. At the same MOI of AAV vector, the transduction efficiency in CF/T43 cells was approximately 4- to 10-fold higher than that in the CFT1 cell line (Fig. 1).

Ad and AAV vectors transduce airway epithelial cells with similar efficiencies.

We next compared the transduction efficiencies of AAV, lacZ, and Ad lacZ vectors in various airway-specific cell lines. The dose-effect relationships of vector MOI and transduction efficiency of airway cells from CF patients were remarkably similar for the AAV and Ad vectors (Fig. 2). The Ad vector was slightly more efficient in transduction of primary cells than was the AAV vector at the highest MOI (Fig. 2A). However, the efficiency of Ad vector transduction of the CF/T43 cell line was somewhat less than that of the AAV vector (Fig. 2B). In CFT1 cells there was no difference in the transduction efficiencies of the AAV and Ad vectors (Fig. 2C). These results suggest that transduction efficiencies can vary, depending on the specific cell line and vector being tested.

Enhancement of rAAV but not rAd transduction by subsequent wild-type Ad infection.

Previous work has demonstrated a rate-limiting step in rAAV-mediated transduction that can be alleviated by helper Ad coinfection (12, 13). To test this premise in airway epithelial cells, coinfections with rAAV and wild-type Ad were performed. AAV-mediated gene transfer to airway epithelial cells was enhanced in the presence of Ad dl309, whereas Ad-mediated gene transfer was unaffected by Ad dl309 (Fig. 3). In the presence of Ad, the AAV vector was considerably more efficient at transducing airway cells than was the Ad vector (Fig. 3A). We next compared the enhancement seen in airway cells after Ad coinfection to the reported observations in HeLa and 293 cells (12, 13). At 100 IU of Ad dl309 per cell, AAV transduction increased only two- to sevenfold, whereas at 2 IU of Ad dl309 per cell, AAV transduction of HEK 293 and HeLa cells increased by several orders of magnitude (Fig. 3B). The effect of Ad on AAV transduction was dose dependent for each CF cell type analyzed, although the magnitude of the effect differed among the cell lines (Fig. 4).

The effect of Ad on AAV transduction was abolished by UV-psoralen inactivation of Ad gene expression (Fig. 5). In addition, an Ad mutant lacking the E4 region (E4⁻ Ad) was unable to enhance AAV transduction (Fig. 5). However, a partially deleted Ad mutant, lacking E4 ORF1–4 but containing ORF6 and ORF6/7 (Ad E4⁻ ORF6⁺ 6/7⁺), maintained the ability to increase AAV vector transduction similarly to that of Ad dl309 (Fig. 5), supporting previous published observations for the role of Ad gene expression in rAAV transduction (12, 13).

Prolonged incubation increases transduction of cultured human airway epithelial cells.

We tested another variable, involving extended vector exposure, for its effect on vector transduction. Both the AAV and Ad vectors exhibited a relationship between the length of exposure of airway epithelial cells and the efficiency of gene transfer (Fig. 6). All of the cell types showed similar requirements of prolonged exposure to achieve maximal transduction. No additional effect was detected when incubation times were extended longer than 24 h (data not shown). However, since transduction was determined 48 h after each of the different exposure periods (1, 6, 12, or 24 h), the cells from each experimental group were ultimately maintained in culture for different periods of time (2 to 3 days). Thus, we sought to determine if the duration of exposure or the duration of time in culture was responsible for the increase in transduction. Cells were infected for 1 h, washed free of unbound vector, and then monitored for different lengths of time in culture prior to staining for LacZ to access the amount of gene transfer. For AAV vectors, the percentage of LacZ-expressing cells increased with increased time in culture (Fig. 7). The highest levels of transduction were seen 3 to 5 days after infection of primary airway cells and CFT1 cells (Fig. 7A and C) and 3 days after infection of CF/T43 cells (Fig. 7B). These results are similar to those of the previous experiment, in which the highest level of transduction occurred following a 24-h incubation period and 48 h of growth in culture, i.e., 3 days after initial vector administration.

For the Ad vector, maximal transduction was observed at the earliest time points (Fig. 7). The percentage of transduced cells decreased over time in culture and was nearly zero at 10 days postinfection (Fig. 5). Because of these differences in the effect of time on expression from Ad and AAV vectors, there was a significant difference in the efficiencies of transduction of airway cells by AAV and Ad vectors at three or more days following vector administration. The maximal effect of time in culture on transduction was approximately three- to sevenfold. Interestingly, this was similar to the increase in transduction observed during coinfection with Ad.

To investigate the relationship between the effect of the presence of Ad and the duration of incubation on AAV transduction, airway epithelial cells were infected with AAV for a short period of time and then infected with Ad at various times following the initial vector infection. It was determined that either time or Ad coinfection could be used to increase AAV transduction, but the effects were not additive (Fig. 8). When exposure to the AAV vector was limited to 1 h, nearly the same level of transduction could be achieved with a 1-h incubation in the presence of Ad as could be achieved with a 24-h incubation in the absence of Ad (Fig. 8). Ad was able to increase AAV transduction at the early time points. However, given time, transduction increased to the same level on its own and Ad no longer had an effect. UV- and psoralen-inactivated Ad or E4⁻ Ad was unable to alleviate the need for a prolonged incubation time, whereas the E4⁻ ORF6⁺ 6/7⁺ virus functioned similarly to Ad dl309 (Fig. 8). Since the effects of time and the presence of Ad on AAV transduction of airway epithelial cells were not additive and could substitute for one another, it is assumed that they are acting in a similar manner; i.e., both result in the accumulation of functional genomes.

As mentioned above, a number of other agents have also been shown to increase AAV-mediated gene transduction. Both camptothesin and hydroxyurea were able to increase AAV transduction of airway epithelial cells (Fig. 9). However, by 3 days after vector delivery, these agents were no longer able to increase gene transduction (Fig. 9). These results support the above-mentioned interpretation of the conversion of singled-stranded AAV genomes to doubled-stranded templates capable of gene expression over time or enhanced by the addition of genetic (Ad coinfection) or chemical (hydroxyurea) stimuli.

Transduction of human tracheal explants with rAAV is limited to basal-like cells.

In the absence of Ad, three of eight freshly exercised human tracheal specimens infected with rAAV exhibited lacZ gene expression; however, the transduction efficiency, as assessed by determining the percentage of LacZ-expressing cells, was less than 0.1% (data not shown). Whereas basal-like cells were transduced in relatively ample quantities by the AAV vector (Fig. 10C and D), well-differentiated, ciliated columnar cells were not transduced by this vector (Fig. 10A and B). In the presence of Ad, LacZ-positive cells were found in one of three specimens. Because transduction efficiency, as assessed by determination of LacZ-positive cells, was less than 0.1%, there was no obvious enhancement of AAV transduction in the presence of Ad (data not shown). Further, features of cell tropism (columnar cells versus basal cells) of AAV vectors were not affected by coadministration of Ad.