Figure 1.

Generation and confirmation of the glioma-specific adenovirus. (a) Schematic diagram of a glioma-specific chimera fiber of Ad5GliomaFF. The knob and the shaft domain of HAd5 fiber were replaced with the fiber fibritin trimerization domain. The glioma-targeted chimeric fiber named GliomaFF was generated by incorporating the phage-panned glioma-specific peptides (VTW) on the C terminus of the chimeric fiber. (b) Molecular validation of a glioma-specific chimeric fiber of Ad5GliomaFF: detection of fiber trimerization and incorporation of viral particles by Western blot analysis with an HAdV-5 tail antibody (4D2). 1×10¹⁰ VP of Ad5CMV-GFP (lanes 1 and 2) or Ad5GliomFF-CMV-GFP (lanes 3 and 4) in Laemmli buffer was analyzed. Samples, identified as B (boiled, incubation at 95°C for 10 min) in lanes 2 and 4 and marked as U (unboiled, incubation at room temperature for 10 min) in lanes 1 and 3, show the denatured monomeric fiber composition and the nondenatured trimeric fiber composition, respectively. Makers indicate kilodaltons (kDa). (c) Fluorescence micrograph and (d) flow cytometric analysis of CAR-independent infection with Ad5GliomaFF-CMV-GFP. Cells were infected with Ad5GliomaFF-CMV-GFP or Ad5CMV-GFP at an MOI of 300 VP/cell for 2 hr on CAR-negative CHO cells and CAR-positive CHO-hCAR cells, and the virus-containing medium was removed and replaced with fresh medium containing 2% FBS. Fluorescence micrographs were taken 72 hr postinfection, followed by flow cytometric analysis. All images are representative of three different experiments. Scale bar, 100 μm. Each column is the average of three independent replicates. Means±SEM are plotted. Color images available online at www.liebertpub.com/hum