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

Abstract

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of b-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases b-galactosidase expression 90-fold in SMC in vitro and E40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.

Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses

“…We cannot conclusively state that the human CMV is stronger than the murine CMV in the context of HC-Ad-hTetON-␤-Gal and HC-Ad-mTetON-␤-Gal, respectively, because the orientation of the promoter constructs is different within each gutless vector genome. However, previous publications from our group and others have shown that the murine CMV promoter is stronger than the human promoter in first-generation adenoviruses (1,4,14,50). In vivo studies within the CNS showed that although both vectors exerted strong and regulatable transgene expression in the striatum, transgene expression levels from the HC-mTetON-␤-Gal vector were 2.5 times higher than those from HChTetON-␤-Gal.…”

Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

“…To do so, it is desirable to increase the levels of transgene expression per vector genome (vg). This can be achieved through the use of genetic sequences that either increase the number of transcript copies (e.g., stronger promoters) or stabilize the mRNA (e.g., the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) (3,16,21,22,26,29,30,32,40,48,52).…”

“…To do so, we engineered expression cassettes carrying the reporter transgene (lacZ) under the control of the powerful murine cytomegalovirus (mCMV) promoter with potential regulatory sequences linked to the 3Ј end of lacZ. The regulatory sequences tested were as follows: HSV1 TK gene (1, 131 bp); the ⌬TK gene, a truncated form of the HSV1 TK gene (1,072 bp) with a 60-bp deletion downstream of the first initiation codon; and WPRE (594 bp), used as a control posttranscriptional regulatory element (3,16,30,39,48). A control construct of lacZ without a posttranscriptional sequence was also engineered.…”

“…The expression levels of only two vectors (Ad-mCMV. ␤gal and Ad-mCMV.␤gal.WPRE) were described by our group in detail previously (3,15), and these vectors are included here only as controls. Thus, in this paper we report four completely novel vectors never described before and, for the first time, the biological activity for seven vectors.…”

Adenovirus-mediated gene transfer of fibromodulin inhibits neointimal hyperplasia in an organ culture model of human saphenous vein graft disease

“…20 Transgene expression from each virus was confirmed in a bioassay of TGF-b activity based on induction of luciferase expression by TGF-b in mink lung epithelial cells stably transfected with the luciferase cDNA under control of a TGF-b-responsive promoter (MLEC(PAI/ L)). 21 Conditioned medium (CM) from Ad5-Fmod-and Ad5-Dcn-infected SMC inhibited induction of luciferase expression (which correlates with TGF-b activity) at all concentrations of TGF-b1 (Figure 1a, all Po0.001).…”