Abstract 21136: The Regulation of Elastin expression by Engineered Zinc Finger Proteins
As a major matrix protein in the vasculature, elastin provides strength and elasticity. The haploinsufficient expression of the elastin gene resulting from heterozygous deletion is responsible for most of the vascular abnormalities in Williams-Beuren Syndrome (WBS) patients. It is generally thought that elastin is synthesized and assembled during development and early post-natal growth, and that the elastin gene is quiescent in adult cells. We hypothesized that transcriptional activation of the elastin gene could be accomplished in adult cells with an engineered transcription factor approach, and that this could in turn to be used to rescue the haploinsufficiency of WBS patients. Using bacteria one hybrid selection, we screened and selected out three zinc finger protein (ZFP) clones that were able to bind 9-nucleotide sequence in human elastin promoter region with high affinity. The best of these ZFP clones, after being fused with transaction domain VP16, was able to upregulate elastin mRNA 68.2 ± 15.1 folds in HEK293 cells which normally do not express elastin, while a proven VEGF-ZFP as well as a VP16 alone plasmid had very minimum effect on elastin mRNA induction (3.3 ± 0.1 and 1.2 ± 0.2 folds respectively). All elastin-ZFPs had no effect on VEGF and HIF-1α mRNAs. Elastin-ZFPs increased elastin mRNA dramatically (13.5 ± 1.3 fold for elastin-ZFP1 and 35.9 ± 3.9 fold for elastin-ZFP3 vs 1.2 ± 0.3 fold for control VEGF-ZFP) in human dermal fibroblast cells. In addition, both elastin-ZFPs resulted in more than 10 fold increase in elastin protein secreted into media from those cells. Therefore, engineered elastin-ZFPs can drive elastin expression from the normal allele in the adult cells with specificity and have the potentials to improve vessel and smooth muscle cell functions in engineered tissue vessels and in WBS cells.
- © 2010 by American Heart Association, Inc.