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.
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 beta-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 beta-galactosidase expression 90-fold in SMC in vitro and approximately 40-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.
Appleby
C E
CE
Vascular Gene Therapy Unit, Manchester, UK.
Kingston
P A
PA
David
A
A
Gerdes
C A
CA
Umaña
P
P
Castro
M G
MG
Lowenstein
P R
PR
Heagerty
A M
AM
D000818:Animals; D003327:Coronary Disease / Q000628:therapy*; D003331:Coronary Vessels / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D007763:Lac Operon; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics; D011401:Promoter Regions, Genetic; D011817:Rabbits; D013552:Swine; D014161:Transduction, Genetic / Q000379:methods*
D018995:Myosin Heavy Chains