Approaches to Enhance Expression After Adenovirus-Mediated Gene Transfer to the Carotid Artery

Abstract

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 cytomegalovirus (CMV) promoter/enhancer. The CMV promoter drives constitutive expression, and response elements within the enhancer allow inducible expression through binding of active transcription factors, such as cAMP response element binding protein (CREB) and nuclear factor kappa B (NFkappaB). Rings of rabbit carotid artery were incubated ex vivo with a replication-deficient adenovirus that expresses beta-galactosidase (AdCMV-betagal). Virus was removed from the medium, and forskolin or phorbol-12-myristate-13-acetate (PMA), which can induce activation of CREB or NFkappaB, respectively, were added to the medium. Pyrrolidine dithiocarbamate (PDTC) was used to inhibit activation of NFkappaB. Following incubation for 24 hours, beta-galactosidase activity was assessed by chemiluminescent reporter assay. Forskolin and PMA enhanced transgene expression in the carotid artery. Activity increased from 56+/-13 mU/mg protein (mean+/-SE) in rings of carotid treated 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 treated with PMA. Phorbol didecanoate, an inactive phorbol, did not affect expression of beta-galactosidase. After pre-incubation with PDTC prior to PMA, expression of beta-galactosidase was less than in rings incubated with PMA alone (29+/-11, P<0.05). Histochemical staining of carotid artery for beta-galactosidase demonstrated enhanced endothelial expression following administration of PMA. These findings suggest that expression after gene transfer to the carotid artery using an adenoviral vector with the CMV promoter/enhancer may be enhanced by PMA and forskolin, perhaps by activation of transcription factors.

N‐acetylcysteine augments adenovirus‐mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry

“…Also, transgene expression from widely used viral promoters/enhancers including the CMV‐IEP and the Rous sarcoma virus promoter (RSV‐LTR) has been shown to be inhibited by the cytokines interferon‐gamma and tumor necrosis factor due to promoter attenuation and/or extinction4. On the other hand, transgene expression driven by the CMV‐IEP may be reactivated/enhanced by forskolin, phorbol ester, and lipopolysaccharides, probably by activation of the transcription factors NFκB and CREB, which bind to the CMV promoter5–7. Furthermore, activation of stress‐activated MAP protein kinases up‐regulates expression of transgenes driven by the CMV‐IEP8.…”

Gene therapy of hypertensive vascular injury

“…In addition, long-term expression of the transgene requires that the promoter driving expression of the transgene is active in a constant or regulated manner. Because current studies use viral promoters that can be inactivated or transiently activated by immune cytokines [47,48], future improvements may include the use of a cellular promoter to drive expression of the transgene. Furthermore, long-term expression of the transgene requires that the transduced cells have a slow turnover, and, perhaps preferably, progenitor capacity.…”