Volume 5, Issue 6, Pages 770-779 (June 2002) In Vivo Hepatic Adenoviral Gene Delivery Occurs Independently of the Coxsackievirus– Adenovirus Receptor  Theodore Smith, Neeraja Idamakanti, Helen Kylefjord, Michele Rollence, Laura King, Michele Kaloss, Michael Kaleko, Susan C. Stevenson  Molecular Therapy  Volume 5, Issue 6, Pages 770-779 (June 2002) DOI: 10.1006/mthe.2002.0613 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Western immunoblot analysis of capsid proteins in adenoviral vectors containing mutated fiber proteins and a targeting peptide. The following adenoviral vectors were subjected to SDS–PAGE and western immunoblot analysis under denaturing conditions: lane 1, Av3nBg; lane 2, Av3nBgFKO1; lane 3, Av1nBgHIRGD; lane 4, Av1nBgFKO1RGD. A total of 5 × 109 viral particles was applied per lane. (A) Levels of fiber were evaluated with a rabbit anti-Ad5 fiber polyclonal antiserum. (B) To control for loading differences, levels of penton were also analyzed with a rabbit anti-Ad5 penton polyclonal antiserum. The bound antibodies were then detected by chemiluminescence. The positions of fiber and penton monomer are indicated. Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Adenoviral-mediated transduction of A549 and HDF cells. A549 (A) or HDF cells (B) were infected with each adenoviral vector with 0–12,500 particles per cell (PPC) of Av3nBg (open circle), Av3nBgFKO1 (filled circle), Av1nBgHIRGD (open square), or Av1nBgFKO1RGD (filled square) for 1 hour at 37°C. After 24 hours, the cells were fixed and stained with X-gal. The percentage of transduced cells was determined per high-power field for each vector dose. The data represent the mean percentage of transduction ± SD (n = 3 wells) from one representative experiment. Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Competition analysis of adenovirus binding to A549 cells. A549 cell monolayers were transduced with 200 PPC of Av3nBg (wt), Av3nBgFKO1 (KOI), Av1nBgHIRGD (wt+RGD), or Av1nBgFKO1RGD (KO1+RGD) in the presence or absence of fiber knob (16 μg/ml), penton peptide (500 nM), heparin (3 mg/ml), or with a combination of all competitors (mix) for 1 hour at 37°C. After 24 hours, the cells were fixed and stained with X-gal. The number of transduced cells per high-power field was determined for each condition and expressed as a percentage of the total transduction in the absence of competitor. The level of transduction for each vector in the absence of competitor was defined as 100%. The data show the average percentage ± SD from a representative experiment. Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Liver adenoviral-mediated gene delivery in vivo. Adenoviral vectors were administered to C57BL/6 mice via tail vein injection at a dose of 1 × 1013 particles/kg in cohorts of five mice per treatment group. We killed the mice 3 days after vector delivery and analyzed livers for β-galactosidase gene expression (A) and adenoviral DNA content (B). (A) The average β-galactosidase activity level (relative light units, RLU/µg protein) ± SD is shown for each treatment group (n = 5 per group). (B) Liver DNA was isolated and analyzed by real-time PCR for adenoviral hexon DNA content. The average hepatic adenoviral DNA content (adenoviral genome copies per cell) ± SD is shown for each treatment group. Asterisk indicates significant difference from Av3nBg virus control according to an unpaired, two-tailed t-test analysis (P < 0.01). Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Immunohistochemical localization of β-galactosidase in mouse livers. The indicated adenoviral vectors were administered to C57BL/6 mice via tail vein injection at a dose of 1 × 1013 particles/kg. We killed the mice 3 days after vector delivery and analyzed livers for β-galactosidase expression by immunohistochemistry of liver sections. Brown nuclear staining indicates the presence of β-galactosidase. (A) Mock-treated, (B) Av3nBg, (C) Av3nBgFKO1, (D) Av1nBgFKO1RGD, (E) Av1nBgHIRGD. Representative photomicrographs are shown. Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Adenoviral-mediated liver transduction of fiber-modified viral particles after systemic delivery. C57BL/6, Balb/C, or CD-1 mice were injected intravenously via the tail vein with HBSS (–), Av3nBg (wt), or Av3nBgFKO1 (KO1) at a dose of 1 × 1013 particles/kg. The mice were killed 3 days after vector delivery and livers were analyzed for β-galactosidase gene expression (A) and adenoviral DNA content (B). (A) The average β-galactosidase activity level (relative light units, RLU/µg protein) ± SD is shown for each treatment group (n = 5 per group). (B) Liver DNA was isolated and analyzed by real-time PCR for adenoviral hexon DNA content. The average hepatic adenoviral DNA content (adenoviral genome copies per cell) ± SD is shown for each treatment group. Asterisk indicates significant difference from Av3nBg virus control according to an unpaired, two-tailed t-test analysis (P < 0.01). Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 7 In vitro adenoviral gene delivery to human and mouse hepatocytes. Human hepatocarcinoma cells, HepG2 (A), the mouse hepatocyte cell line, FL83B (B), or primary CD-1 mouse hepatocytes (C) were infected with each adenoviral vector from 0 up to 12,500 particles per cell (PPC) of Av3nBg (open circle) or Av3nBgFKO1 (filled circle) for 1 hour at 37°C. After 24 hours the cells were fixed and stained with X-gal. The percentage of transduced cells was determined per high-power field for each vector dose. The data represent the mean percentage of transduction ± SD (n = 3 wells) from one representative experiment. Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 8 Competition analysis of adenoviral transduction of primary murine hepatocytes. CD-1 murine primary hepatocytes were transduced with Av3nBg (wt) at 2500 PPC or Av3nBgFKO1 (KO1) at 12,500 PPC in the presence or absence of fiber knob (10 μg/ml), an RGD peptide (200 μg/ml), or heparin (3 mg/ml) for 1 hour at 37°C. After 24 hours, the cells were fixed and stained with X-gal. The number of transduced cells was determined per high-power field for each condition and was expressed as a percentage of the total transduction in the absence of competitor. The level of transduction for each vector in the absence of competitor was defined as 100%. The data show the average percentage control ± SD from a representative experiment. Molecular Therapy 2002 5, 770-779DOI: (10.1006/mthe.2002.0613) Copyright © 2002 American Society for Gene Therapy Terms and Conditions