1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, 901 19th Street South, BMR2-502, Birmingham, AL 35294, USA
2 Departments of Pathology, Cell Biology and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-2172, USA
Correspondence
David T. Curiel
curiel{at}uab.edu
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ABSTRACT |
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INTRODUCTION |
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Vector systems based on other, less pathogenic human adenovirus serotypes and various xenotypes have been proposed to overcome these concerns (Both, 2004; Gao et al., 2003
; Rasmussen et al., 1999
; Reddy et al., 1999
). Among the diverse approaches, a canine adenovirus type 2 (CAV2) vector demonstrated promising features, including high titres, undetectable replication-competent revertants, inability to replicate in various human cell lines tested, transduction efficiency comparable to that of human adenovirus serotype 5 (Ad5) vectors, minimal neutralization by human sera (Kremer et al., 2000
) and neurotropism (Peltékian et al., 2002
). CAV2 vectors have also gained importance in the field of adenoviral virotherapy for the treatment of cancer. Its disease presentation and prevalence in the dog population parallel similar properties of Ad5 found in the human population. These considerations establish the rationale for studying the effects of the immune system on a syngeneic CAV2 oncolytic adenovirus in the setting of a spontaneous osteosarcoma dog model (Hemminki et al., 2003
).
The interest in CAV2 as a gene-delivery vector and as a model oncolytic adenovirus for immunological evaluation motivated us to consider genetic labelling of CAV2 via the fusion protein IXenhanced green fluorescent protein (pIXEGFP) as a means for vector detection. By using this system, we and others have previously generated fluorescent Ad5 particles with minimal effect on viral function, whilst demonstrating utility for vector detection in tracking, binding and biodistribution assays (Le et al., 2004; Meulenbroek et al., 2004
). Likewise, another group was able to incorporate enhanced yellow fluorescent protein into a bovine adenovirus type 3 vector via pIX (Zakhartchouk et al., 2004
). Based on studies involving human adenovirus serotypes 2 and 5, pIX is a small polypeptide of 140 aa (14·7 kDa) that acts as a cement protein to stabilize hexonhexon interaction and therefore the capsid structure itself (Parks, 2005
); however, pIX is structurally dispensable during virion formation (Colby & Shenk, 1981
). Data suggest that four trimers of pIX interact with a group of nine hexons in each facet of the icosahedron (Stewart et al., 1991
), resulting in 240 copies of the protein per virion (Lehmberg et al., 1999
; van Oostrum & Burnett, 1985
). During infection, pIX reorganizes the structure of the nucleus by sequestering the promyelocytic leukaemia protein (Rosa-Calatrava et al., 2003
). In addition, pIX also serves as a transcriptional activator of several viral and cellular TATA-containing promoters, including adenoviral E1A, E4 and major late promoters (Lutz et al., 1997
), although its transcriptional role during normal infection is not significant (Sargent et al., 2004
). Taking advantage of its surface localization (Akalu et al., 1999
), pIX was initially exploited as a locale for incorporation of a heterologous polylysine peptide onto its carboxy terminus for transduction purposes (Dmitriev et al., 2002
). More recently, the fusion of spacers (Vellinga et al., 2004
) and a biotin-acceptor peptide (Campos et al., 2004
) onto Ad5 pIX have further expanded its utility for targeting.
Published reports suggest that various human adenoviruses possess the IX gene (Chroboczek et al., 1992; Dijkema et al., 1981
; Engler, 1981
; Mei et al., 2003
). Although ovine and avian adenoviruses do not appear to express pIX (Hess et al., 1997
; Ojkic & Nagy, 2000
; Vrati et al., 1996
), analyses of some canine, bovine and porcine adenovirus genomes have shown the presence of the IX gene (Aggarwal & Mittal, 2000
; Salmon & Haj-Ahmad, 1994
; Shibata et al., 1989
; Zheng et al., 1999
). Of note, our CAV2 vector of interest does express pIX. We accomplished genetic labelling of CAV2 by generating a recombinant virus that expresses the fusion pIXEGFP. The results indicate that pIX labelling was achieved, resulting in fluorescent viral particles that could be detected in vitro during viral binding and nuclear localization, and in situ after systemic administration. Furthermore, pIXEGFP expression could be exploited to visualize virus replication and spread. Genetic labelling of CAV2 via pIXEGFP provides a means for vector detection in gene-therapy applications and has potential utility for studying virotherapy in a canine osteosarcoma model.
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METHODS |
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CAV2-wt-IX-EGFP construction.
Similar to our previous report of pIXEGFP-labelled Ad5 (Le et al., 2004), we generated replication-competent CAV2-wt-IX-EGFP with a carboxy-terminal fusion of EGFP onto pIX. Construction involved the cloning of an E1 shuttle vector produced by self-ligation of the 7·6 kb NdeI fragment liberated from pTG5412 (Transgene SA), which contains the CAV2 genome (strain Toronto A 26/61). Briefly, the EGFP gene from pEGFP-N1 (Clontech) was inserted into the Klenow large fragment-blunted AseI site between the last codon of the IX gene and the stop codon. The bovine growth hormone polyadenylation signal was placed after the EGFP gene. Finally, we inserted a kanamycin-resistance gene after the polyadenylation signal to facilitate double-selection recombination with the rescue backbone, which contains an ampicillin-resistance gene. The final shuttle plasmid was linearized with NdeI and NotI enzymes and recombined homologously with pTG5412 in BJ5183 cells. Colonies were selected on ampicillin/kanamycin double-selection plates to allow identification of recombinants containing the IX-EGFP modification with the kanamycin gene located in the CAV2 genome plasmid, which itself expresses ampicillin. This double-selection system was designed so that non-recombined original shuttle and genome-rescue plasmids would be eliminated, whilst preferentially selecting for recombinant plasmids that contain the IX-EGFP modification in the CAV2 genome. A correct clone was linearized with ClaI (which flanked the kanamycin gene) to remove the kanamycin gene and self-ligated to generate the final recombinant genome. The NotI-cut genome was transiently transfected into DK cells for virus generation and then amplification.
Virus propagation and purification.
CAV2-wt-IX-EGFP was propagated in DK cells and purified by double caesium chloride (CsCl) ultracentrifugation and dialysis against PBS with 0·9 M Mg2+, 0·5 M Ca2+ and 10 % (v/v) glycerol. Final aliquots of virus were analysed for viral-particle titre (A260) and cytopathic effect unit titre (CPEU). CPEU was determined by infecting DK cells in 96-well plates with 1/10 serial dilutions of the virus and assayed on day 10 post-infection with an MTS viability assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega] to determine the viral dilution that causes 50 % cytopathic effect (n=6). Based on the cell number seeded (15 000), CPEU was calculated where one CPEU unit is defined as the amount of virus that causes cytopathic effect in one DK cell in 10 days. A similar protocol has been reported previously (O'Carroll et al., 2000). The viral particle (vp) : CPEU ratio of CAV2-wt-IX-EGFP was 49, whilst that of a control CAV2-wt was 21. All viruses were stored at 80 °C until use.
Fluorescence microscopy.
Fluorescence microscopy was performed with an inverted IX-70 microscope (Olympus) equipped with a Magnifire digital CCD camera (Optronics). Glass slides and coverslips were used to mount samples (Fisher Scientific). Images were acquired with a 100x objective by using oil immersion and deconvoluted digitally with IRIS version 4.15a (http://www.astrosurf.com/buil/) by applying the RichardsonLucy algorithm with 15 iterations. An image of a single, fluorescent virus particle with strong signal-to-noise ratio was used to estimate the point-spread function as suggested by the software documentation. Green fluorescence, red autofluorescence and blue Hoechst-stain images were finally merged by using Adobe Photoshop 7.0. For pIXEGFP expression and localization analysis, DK and MDCK cells seeded on glass coverslips were infected with CAV2-wt-IX-EGFP (100 vp per cell) and processed for imaging at 24 and 48 h post-infection.
Characterization of virus-gradient fractions.
CAV2-wt-IX-EGFP was propagated in ten 150 mm dishes and then purified by CsCl ultracentrifugation where the top and bottom viral bands were retained after two centrifugation steps, yielding one gradient from the ten dishes. After the second spin, fractions (approx. 100 µl) were collected dropwise through a perforation at the bottom of the tube into a 96-well white opaque plate. Plates with the viral fractions were measured with a microplate fluorometer (Fluostar Optima; BMG Labtechnologies) by using 490/10 nm excitation and 510/10 nm emission filters. To determine viral DNA content, a sample of each fraction (10 µl) was diluted in 90 µl 0·5 % SDS/PBS and incubated at room temperature for 10 min to release the viral genomes. A260 was then measured for each sample (MBA 2000; Perkin Elmer).
Western blot.
Fractionated samples (4 µl) were ethanol-precipitated, pelleted and resuspended in 20 µl RIPA buffer. Samples (5 µl) were resolved with SDS-PAGE and then transferred to a PVDF membrane (Bio-Rad). Blotting was performed with a primary monoclonal GFP antibody (1 : 1000 dilution; BD Biosciences Clontech) followed by a secondary horseradish peroxidase-linked anti-mouse antibody (1 : 5000 dilution; Amersham Biosciences). Bands were detected with a chemiluminescent ECL kit (Amersham Biosciences).
Tracking of CAV2-wt-IX-EGFP infection.
The day prior to infection, DK and MDCK cells (2·5x105) were seeded on glass coverslips that were placed in six-well plates with growth medium lacking phenol red (5 % fetal calf serum/Dulbecco's modified Eagle's medium). The cells were incubated for 1 h at 4 °C with CAV2-wt-IX-EGFP (10 000 vp per cell) in 1 ml phenol red-free growth medium containing 25 mM HEPES buffer. The viruses were allowed to bind to the cells at 4 °C for 1 h (cell binding), after which the cells were washed three times with PBS and then fixed to the coverslip with 3 % formalin (Tousimis) for 10 min. After another three washes, Hoescht (33342) staining was performed for 5 min, followed by three final washes with PBS before mounting and sealing. Another set of coverslips with the cells and virus were transferred to 37 °C for 1·5 h after incubation at 4 °C for 1 h (nuclear localization). Likewise, these samples were prepared for fluorescence imaging.
In situ detection of CAV2-wt-IX-EGFP.
All methods were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham, AL, USA, and performed according to their guidelines. C57/BL6 mice (Charles River Laboratories) were anaesthetized with 2 % isoflurane at about 0·5 l min1 for an open laparotomy procedure and injected via the inferior vena cava with 1011 vp CAV2-wt-IX-EGFP in 200 µl PBS. Twenty minutes after virus injection, the mice were sacrificed and the liver was frozen until sectioning (Minotome PLUS; Triangle Biomedical Sciences). Frozen sections (5 µm thick) of the liver were fixed onto glass slides and stained with Hoechst 33342 (Molecular Probes) for nuclear DNA. Glass coverslips were mounted on the slides with mounting medium (Biomeda) prior to fluorescence microscopy as described above.
In vitro visualization of CAV2-wt-IX-EGFP replication and spread.
Dog osteosarcoma D22 and human lung adenocarcinoma A549 cells were seeded on glass-bottomed 3 cm diameter dishes (200 000 cells per dish; Willco Wells) using phenol red-free medium. The next day, the cells were infected with 100 vp CAV2-wt-IX-EGFP per cell. Fluorescence microscopy was performed to detect pIXEGFP expression in the same field of view over the course of 8 days.
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RESULTS AND DISCUSSION |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 February 2005;
accepted 9 September 2005.
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