Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA

Virus-Host Coevolution in a Persistently Coxsackievirus B3-Infected Cardiomyocyte Cell Line

“…To test this assumption, we investigated the ability of CVB3-HL1 to infect and replicate in mouse myoblast C2C12 cells and CHO-K1 cells. CHO-K1 cells do not express CAR (57), whereas a low level of CAR expression in C2C12 cells was reported previously (32,44). Real-time RT-PCR analysis, however, demonstrated an absence of CAR expression in the C2C12 cells used in this study (results not shown).…”

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper‐dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

“…The dystrophin expression cassette was subsequently harvested from this plasmid. The LacZ expression cassette consisted of the Escherichia coli LacZ gene controlled by the Rous sarcoma virus long terminal repeat (RSV) promoter [20], and the CAR expression cassette consisted of the human CAR cDNA controlled by the RSV promoter [11]. The whole multiple cloning site of pBluescript was replaced by the sequence ApaI-PacI-NotI-PacI-BamHI by ligation of the synthetic oligonucleotide, restriction enzyme digestion and self-ligation; the generated plasmid was named pPN.…”

“…The secondary antibody was biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA, 1 : 200), and the stained sections were visualized by horseradish peroxidase. Serial frozen sections from muscle injected with HDAdLacZ-dys or HDAdCAR-dys were stained for detection of β-gal or CAR as described previously, respectively [11]. For detection of β-gal, frozen sections were fixed with 2% paraformaldehyde and 0.2% glutaraldehyde in phosphate-buffered saline (PBS), and stained with 0.1% X-gal in 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl 2 .…”

“…The injected muscles were removed and frozen in isopentane pre-cooled with liquid nitrogen. Frozen sections (10 µm in thickness) were stained with hematoxylin and eosin (H&E) or used for immunohistochemical detection of dystrophin as described previously [11]. Primary antibodies used for dystrophin detection were rabbit polyclonal anti-dystrophin antibody (anti 6-10 [23], 1 : 2000) or mouse monoclonal antidystrophin antibody (MANDRA1 (Sigma) labeled with the fluorescent dye Alexa 568 (Molecular Probes, Eugene, OR, USA) [24], 1 : 1200).…”

“…We previously reported that Ad-mediated high expression of coxsackievirus and adenovirus receptor (CAR) in mouse muscle improved the efficiency of subsequent Admediated marker gene transfer to the muscle [11]. In the present study, we investigated whether HDAd-mediated CAR expression contributed to the improvement of subsequent therapeutic gene expression, not marker gene expression, that is, whether repetitive injections of our new HDAd vector which contained the CAR and the dystrophin expression cassette could increase the efficiency of subsequent dystrophin gene transfer to mature muscle.…”