Approaches to enhance expression after adenovirus-mediated gene transfer to the carotid artery

The goal of this study was to enhance transgene expression after adenoviral -mediated gene transfer to the carotid artery. We used an adenoviral vector with a transgene that expresses beta-galactosidase, driven by the human cy tomegalovirus (CMV) promoter/enhancer. The CMV promoter drives constitutive expression, and response elements within the enhancer allow inducible expr ession through binding of active transcription factors, such as cAMP respon se element binding protein (CREB) and nuclear factor kappa B (NF kappa B). Rings of rabbit carotid artery were incubated ex vivo with a replication-de ficient adenovirus that expresses beta-galactosidase (AdCMV-beta gal). Viru s was removed from the medium, and forskolin or phorbol-12-myristate-13-ace tate (PMA), which can induce activation of CREB or NF kappa B, respectively , were added to the medium. Pyrrolidine dithiocarbamate (PDTC) was used to inhibit activation of NF kappa B. Following incubation for 24 hours, beta-g alactosidase activity was assessed by chemiluminescent reporter assay. Fors kolin and PMA enhanced transgene expression in the carotid artery. Activity increased from 56+/-13 mU/mg protein (mean+/-SE) in rings of carotid treat ed with virus alone (10(9) pfu) to 159+/-23 mU/mg protein (P<0.05) in rings treated with forskolin, and to 189+/-40 mU/mg protein (P<0.05) in rings tr eated with PMA. Phorbol didecanoate, an inactive phorbol, did not affect ex pression of beta-galactosidase. After pre-incubation with PDTC prior to PMA , expression of beta-galactosidase was less than in rings incubated with PM A alone (29+/-11, P<0.05). Histochemical staining of carotid artery for bet a-galactosidase demonstrated enhanced endothelial expression following admi nistration of PMA. These findings suggest that expression after gene transf er to the carotid artery using an adenoviral vector with the CMV promoter/e nhancer may be enhanced by PMA and forskolin, perhaps by activation of tran scription factors.