PMC

Proteasome-modulating agents augment rAAV transduction in airway cell lines. The effects of proteasome-modulating agents LLnL, aclarubicin (ACL), and doxorubicin (DOX) on AV2.Luc and AV2/5.Luc transduction of immortalized human airway cell lines IB3 (A) and A549 (B) were evaluated. Proteasome-modulating agents were coadministered with each rAAV vector (MOI of 500 particles/cell) at the time of infection, and transduction was evaluated 24 h later. Various concentrations of each chemical were evaluated, as indicated on each graph. Luciferase activity was measured with a luminometer at 47.7% sensitivity. Data represent the means (± standard errors of the means) of the relative luciferase activity (per well) results from four independent experiments.

Matrix dose-response assays of rAAV-2 transduction with combined application of LLnL and doxorubicin. IB3 (A) and A549 (B) cells, cultured in 96-well plates, were infected with 500 particles/cell of AV2.Luc. Different concentrations of each proteasome inhibitor alone or in combination (as indicated on each axis) were applied at the time of infection. At 24 h postinfection, luciferase activity was measured with a luminometer at 47.7% sensitivity with 1/10 of the total cell lysate. The level of luciferase expression in the diagrams represents the total from each well. The color signifies the range of the mean relative luciferase activity (measured in relative luciferase units [RLU]) per well for four independent experiments.

Proteasome-modulating agents augment rAAV transduction from the apical membrane of polarized human airway epithelia. Primary bronchial human airway epithelia were cultured and differentiated at an air-liquid interface on transwell filters 1 cm in diameter. Particles (10⁹) of AV2.Luc or AV2/5.Luc in a volume of 50 μl were applied to the apical surface of airway epithelia in the presence or absence of LLnL (40 μM), Z-LLL (4 μM), aclarubicin (ACL; 0.25 μM), or doxorubicin (DOX; 5 μM). Luciferase activity was measured at 3 days (A) and 17 days (B) postinfection with a luminometer setting of 80% sensitivity. Data represent the means (± standard errors of the means) of the relative luciferase activity (per well) results from three independent experiments.

LLnL and doxorubicin both facilitate translocation of rAAV to the nucleus. IB3 cells were infected with AV2eGFP (MOI of 1,000 particles/cell) in the presence or absence of 40 μM LLnL or 1 μM doxorubicin (DOX). At 24 h postinfection, cytoplasmic (Cyt.) and nuclear (Nuc.) fractions were isolated (n = 3 infections for each experimental point). (A) Viral DNA in each fraction was detected by slot blot hybridization against a ³²P-labeled eGFP probe and visualized with a Bio-Rad phosphorimager. (B) Purities of the cytoplasmic and nuclear fractions were confirmed by immunoblotting against the cytoplasmic marker Rab5 and nuclear antigen histone 3. (C) The percentage distributions of the viral genome signals in the nuclear and cytoplasmic fractions were calculated based on the mean (± standard error of the mean) signals for three experimental points. The ³²P signal was quantified with Bio-Rad software.

Combined administration of proteasome-modulating agents can synergistically induce rAAV transduction from the apical surface of polarized human airway epithelia. Particles (10⁹) of AV2.Luc (A and C) or AV2/5.Luc (B and D) were applied to the apical surface of polarized human airway epithelial cultures in the absence or presence of various combinations of LLnL (40 μM), Z-LLL (4 μM), and/or doxorubicin (DOX; 5 μM). Luciferase expression was assayed at 3 and at 17 days postinfection. Data represent the means (± standard errors of the means) of the relative luciferase activity (per well) results from three independent experiments. (E) Similar results were observed following apical infection with AV2.GFP under the above conditions. Representative fluorescent photomicrographs of GFP expression at 3 and 15 days postinfection are shown for the labeled conditions.

Doxorubicin induces rAAV transduction without directly enhancing the efficiency of second-strand synthesis. Polarized human airway epithelia grown at the air-liquid interface were infected with 5 × 10⁹ particles of full-length AV2.eGFP (A) or self-complementary scAV2.eGFP (B) from the apical surface at day 0. GFP expression was quantified at the time points indicated on the graphs by fluorescent microscopy and the following calculation: the mean area of GFP fluorescence multiplied by the mean intensity of fluorescence. Ten images were acquired randomly from each experimental point. The following experimental protocols were performed: (i) rAAV infection without doxorubicin (DOX), (ii) rAAV infection in the presence of 5 μM doxorubicin, and (iii) rAAV infection without doxorubicin and subsequent application of 5 μM doxorubicin for 24 h at 13 days postinfection. Results depict the means ± standard errors of the means for three independent epithelia for each experimental point.

Proteasome inhibitors augment rAAV transduction to mouse lungs and human bronchial xenografts in vivo. (A) Aerosol delivery of rAAV with Z-LLL or doxorubicin to the mouse airway. C57BL/6 mice were infected with a total of 6 × 10¹⁰ particles of AV2.Luc or AV2/5.Luc through nasal aspiration in the absence or presence of either 400 μM Z-LLL or 200 μM doxorubicin (DOX). Viruses and the proteasome inhibitor were mixed together (40 μl of inoculum/mouse) and delivered through the nose three times on sequential days. Two weeks after the final infection, the mouse lungs and trachea were harvested and homogenized in a Promega luciferase reporter lysis buffer. Samples were normalized for protein concentration, and luciferase activity was measured in a luminometer at 80% sensitivity. The values represent the mean (± standard error of the mean) relative luciferase activities in both the lungs and trachea (n = 3). (B) Human bronchial xenografts were infected with 10¹¹ particles (in a volume of 100 μl) of AV2.Luc. The administration of proteasome inhibitors was either through a local application mixed with virus and applied to the lumen of grafts at the time of infection (doxorubicin and Z-LLL) or through systemic application to the mouse by tail vein injection 1 and 2 days after rAAV infection of the airway lumen (Doxil). Total microgram doses and working concentrations of drugs are summarized below the chart (+, present; −, absent). The xenograft airways were harvested at 2 weeks postinfection, and luciferase assays were performed on the entire airway. Results depict the means ± standard errors of the means for three independent xenografts for each experimental point.