Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells

Abstract

Transduction of endothelial cells (EC) with a vector that expresses apolipoprotein A-I (APOAI) reduces atherosclerosis in arteries of fat-fed rabbits. However, the effects on atherosclerosis are partial and might be enhanced if APOAI expression could be increased. We tested 4 strategies—primarily in vitro—to increase APOAI expression from our current highest-expressing vector: addition of 2 types of enhancers, addition of computationally identified EC-specific cis-regulatory modules (CRM), and insertion of the rabbit APOAI gene at the transcription start site (TSS) of genomic sequences cloned from genes that are highly expressed in cultured EC. Addition of a shear stress-responsive enhancer did not increase APOAI expression. Addition of 2 copies of a Mef2c enhancer increased APOAI expression from a moderately active promoter/enhancer, but decreased APOAI expression from our most highly active promoter/enhancer. Of 11 computationally identified CRM, 3 increased APOAI expression from the moderately active promoter (2–7-fold; P < 0.05); none increased expression from the highly active promoter/enhancer. Insertion of the APOAI gene into the TSS of highly expressed EC genes did not increase expression above levels obtained with moderately active promoter/enhancer. High performance of our current highest-expressing vector was confirmed; new strategies are needed to further increase APOAI transgene expression in EC.