Enhancer Stimulation Unmasks Latent Gene Transfer After Adenovirus-Mediated Gene Delivery Into Human Vascular Smooth Muscle Cells

Abstract

Recombinant adenoviral vectors are being used increasingly for gene transfer studies in mammalian cells and gene therapy protocols in humans. High adenoviral titers are often required for successful transduction of vascular smooth muscle cells (VSMCs), defined as uptake and detectable expression of the foreign gene, but the relative contributions of efficiency of viral uptake and control of transcription are poorly understood. To explore the extent to which a lack of detectable gene expression may be due to inefficient transcription of a successfully transferred gene, we have used a replication-deficient adenovirus expressing β-galactosidase (RAd35β-Gal), under the control of the human cytomegalovirus major immediate-early promoter (CMV-IEP), which contains cAMP and nuclear factor-κB response elements, to investigate constitutive and inducible gene expression after gene transfer into human VSMCs. Histochemical staining with 5-bromo-4-chloro-3-indolyl β- d -galactopyranoside (X-gal), a quantitative spectrophotometric assay, SDS-PAGE, Western blotting, and Northern analysis were used to evaluate β−galactosidase expression in infected cells. After infection with RAd35β-Gal at 30, 100, and 1000 plaque-forming units per cell (pfu/cell), expression of β-galactosidase was augmented up to 17-, 19-, and 23-fold, respectively, in human VSMCs treated with forskolin and phorbol ester compared with unstimulated cells. After infection, the proportion of detectably transduced cells was increased by enhancer stimulation from 58% to 100% at 100 pfu/cell and from 9% to 62% at 10 pfu/cell, indicating quiescent viral DNA in unstimulated cells. At high adenoviral titers (1000 pfu/cell), the recombinant gene became the most abundant protein in cell extracts. These findings demonstrate that in human VSMCs, limited constitutive expression from the CMV-IEP, rather than failure of translocation of adenoviral DNA, may be responsible for the apparent failure of transduction at a low multiplicity of infection.

Temporally regulated expression patterns following in utero adenovirus-mediated gene transfer

“…25 In addition, a published study from our laboratory documented promoter-dependent variation in the tissue expression of a reporter transgene following injection of adenovirus into the early stage mouse embryo in culture (7.5-9 days); 26 CMV promoter control resulted in primarily endothelial expression while RSV control resulted in preferential expression within the myocardium. Similarly, Woo et al 16 found that the expression pattern of ␤-gal under RSV promoter control resulted primarily in myocardial cell expression when adenovirus was injected at a single developmental timepoint (12.5 d.p.c.)…”

High adenoviral loads stimulate NFκB-dependent gene expression in human vascular smooth muscle cells

“…9 Stimulation of NF B and CREB response elements in the CMV-IEP were required to unmask ␤-galactosidase expression after low multiplicity infection, however, high multiplicity infection led to effective transduction without enhancer stimulation. This raised the hypothesis that high multiplicity infection itself was able to modify the transcriptional environment to augment expression from the CMV-IEP.…”

A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer

“…However, pharmacological enhancement increased the proportion of SMC expressing transgene following gene transfer at MOI¼10 from 5 to 55%, suggesting, that in the absence of enhancer stimulation, 490% of copies of MIEhCMV entering SMC fail to express transgene. 4 Unfortunately, these methods of enhancement are not well suited to in vivo application. Furthermore, the ideal vector for vascular gene therapy should allow maximal transgene expression after a single dose without further physical or pharmacological manipulations.…”

“…After adenovirusmediated gene transfer at a multiplicity of infection (MOI) of 10, only a small proportion (E5%) of infected SMC expresses detectable levels of transgene when transcriptional regulation is governed by the MIEhCMV. 4 Other viral promoters such as the Rous Sarcoma Virus long terminal repeat promoter or Simian Virus 40 early promoter typically give rise to less transgene expression in SMC than MIEhCMV. 5 Transcriptional targeting employing SMC-specific promoters has successfully conferred cell-type specificity, 6 but the magnitude of transgene expression achieved using the SMC-specific SM22a promoter was less than that achieved using MIEhCMV.…”