Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia

Abstract

The choice of adeno-associated virus serotypes for clinical applications is influenced by the animal model and model system used to evaluate various serotypes. In the present study, we sought to compare the biologic properties of rAAV2/1, rAAV2/2, and rAAV2/5 transduction in polarized human airway epithelia using viruses purified by a newly developed common column chromatography method. Results demonstrated that apical transduction of human airway epithelia with rAAV2/1 was 100-fold more efficient than rAAV2/2 and rAAV2/5. This transduction profile in human airway epithelia (rAAV2/1 >> rAAV2/2 = rAAV2/5) was significantly different from that seen following nasal administration of these vectors to mouse lung (rAAV2/5 > rAAV2/1 >> rAAV2/2), emphasizing differences in transduction of these serotypes between these two species. In stark contrast to rAAV2/2 and rAAV2/5, rAAV2/1 transduced both the apical and basolateral membrane of human airway epithelia with similar efficiency. However, the overall level of transduction across serotypes did not correlate with vector internalization. We hypothesized that differences in post-entry processing of these serotypes might influence the efficiency of apical transduction. To this end, we tested the effectiveness of proteasome inhibitors to augment nuclear translocation and gene expression from the three serotypes. Augmentation of rAAV2/1 apical transduction of human polarized airway epithelia was 10-fold lower than that for rAAV2/2 and rAAV2/5. Cellular fractionation studies demonstrated that proteasome inhibitors more significantly enhanced rAAV2/2 and rAAV2/5 translocation to the nucleus than rAAV2/1. These results demonstrate that AAV1 transduction biology in human airway epithelia differs from that of AAV2 and AAV5 by virtue of altered ubiquitin/proteasome sensitivities that influence nuclear translocation.

FIGURE 1

HPLC purification of rAAV type 1, 2, and 5 vectors by ion-change HPLC. A, HPLC chromatographic profiles for rAAV2/1, rAAV2/2, and rAAV2/5 on a 4.6 × 100-mm Poros PI column at a flow rate of 4 ml/min. The chromatograph was monitored by absorption at 280 nm on the left y axis. The buffer set consisted of low salt loading buffer (A buffer: 0.02m NaCl, 20 mm Tris-HCl, pH 8.0) and high salt buffer (B buffer: 1m NaCl, 20 mm Tris-HCl, pH 8.0). The same buffer set was used for isolation of all three AAV serotypes. A linear 0.02-1m NaCl gradient (from 0 to 100% B buffer) with 15 column volumes was adopted in the elution. The right y axis denotes percent Buffer B in the effluent. rAAV2/1, rAAV2/2, and rAAV2/5 were eluted from the column at the gradient at 0.51, 0.46, and 0.41m NaCl, respectively. B,5 × 10¹⁰ particles of each HPLC-purified virus were resolved on a SDS-PAGE, and the capsid proteins (VP1, VP2, and VP3) were visualized with Coomassie Brilliant Blue staining. No obvious impurities were detected under these conditions. The infectivity of purified rAAV serotypes was compared on 293 cells (C ) and CHO-K1 cells (D). Cells were cultured in 24-well plates to 70% confluence and infected with AV2/1.Luc, AV2/2.Luc, or AV2/5.Luc at a multiplicity of infection of 100 particles/cell. Transduction efficiencies, as assessed by luciferase expression, were evaluated at 24 h post-infection. Data represents the mean (±S.E.) relative luciferase activity (per well) for four independent experiments.

FIGURE 2

Species-specific differences in airway transduction with AAV1, -2, and -5 serotypes. A, comparison of rAAV vector serotypes in mouse lung. C57/BL6 male mice (4-week-old) were infected with 6 × 10¹⁰ particles of AV2/1.Luc, AV2/2.Luc, or AV2/5.Luc by nasal inhalation. At 2 weeks post-infection, relative luciferase activity of whole lung homogenates was assessed and normalized per mg of total protein. The value represents the mean (±S.E.) relative luciferase activity per mg protein of lung homogenate (n = 3). B, a similar serotype comparison of transduction was performed on differentiated human airway epithelia grown at an air-liquid interface in Millicell inserts. 2 × 10⁹ particles of AV2/1.Luc, AV2/2.Luc, or AV2/5.Luc were applied to the apical surface of polarized human airway epithelial cultures for 24 h. Luciferase expression was assayed at 3 days post-infection. Data represents the mean (±S.E.) relative luciferase activity (per well) from 10 independent infections (n = 10).

FIGURE 3

Comparisons of transduction efficiencies for different rAAV vector serotypes following infection of the basolateral and apical membrane of human polarized airway epithelia. 2 × 10⁹ particles of AV2/1.Luc, AV2/2.Luc, or AV2/5.Luc were applied to either the apical surface or the basolateral side of human polarized airway epithelia for 24 h. At 3 days post-infection, relative luciferase activities in each well were assessed. Data represent the mean (±S.E.) relative luciferase activity (per well) in airway epithelia from three independent tissue donor samples: donor 1 (n = 3) (A), donor 2 (n = 4) (B), and donor 3 (n = 3) (C ).

FIGURE 4

Serotype differences in rAAV internalization from the apical and basolateral membrane. Polarized human airway epithelia were infected from the apical or basolateral membrane with 10¹⁰ particles of each rAAV serotype, and the abundance of vector genomes in Hirt DNA was assessed at 2 and 24 h post-infection. Viral infections were performed for 2h by applying virus to the apical or basolateral membrane followed by extensive washing to remove unbound virus. Hirt DNA was extracted from infected cells immediately after washing at 2 h post-infection (A) or after 22 h of an additional incubation period (24 h post-infection) (B). TaqMan PCR was then performed to quantify viral genomes in the Hirt DNA. Data represent the mean (±S.E.) viral genome copies from five independent infections (n = 5).

FIGURE 5

Co-administration of proteasome-modulating agents LLnL and doxorubicin induced rAAV2 and rAAV5 transduction from the apical membrane more effectively than rAAV1. 2 × 10⁹ particles of AV2/1.Luc, AV2/2.Luc, or AV2/5.Luc were applied to the apical membrane of polarized human airway epithelial cultures for 24 h in the absence or presence of LLnL (40 μm) and doxorubicin (5 μm). A, luciferase expression was assayed at 3 days post-infection. Data represent the mean (±S.E.) relative luciferase activity (per well) for n = 10 independent infections from three independent tissue samples. B, the fold induction in luciferase reporter expression shown in A following the addition of proteasome inhibitors for each serotype (mean ± S.E.). C, cellular fractionation was performed to evaluate the cellular distribution of viral genomes in the absence or presence of proteasome inhibitors. Cells were lysed, and the cytoplasmic and nuclear fractions were isolated at 3 days post-infection for quantification viral genome copy number using Taq-Man PCR. Data represent the mean (±S.E., n = 4) percentage of viral genomes in the cytoplasmic (solid bars) and nuclear (open bars) fractions for each sero-type in the presence or absence of proteasome inhibitors (P.I.).