Search for Promoters to Target Gene Expression in Selected Cells of the Outflow Pathway

Abstract

Abstract: : Purpose: The purpose of this study is to identify promoters capable of targeting gene expression in different cell types of the outflow pathway. Methods: Perfused anterior segments of human cadaver eyes were infected with 10⁷ plaque-forming units of replication-deficient recombinant adenoviruses expressing the beta-galactosidase reporter gene driven by either the CMV, the VE-Cadherin, or the Matrix GLA promoter. 48 Hours after infection the anterior segments were fixed by perfusion with 1% paraformaldehyde, 0.2% glutaraldehyde, 0.02 % NP40, 0.01 % Na DOC, at 15 mmHg, and stained for beta-galactosidase activity. Paraffin sections of the tissue were analyzed for beta-galactosidase expression. Results: The matrix GLA promoter targets gene expression much more specifically in the TM than the CMV promoter. Expression of this promoter is particularly high in the juxtacanalicular area. The VE-cadherin promoter showed high levels of expression in the aqueous venous plexi and episcleral veins, but did not show any expression in either TM or SC cells. Conclusions: The matrix GLA gene promoter targets HTM cells more specifically than the CMV promoter, while providing high levels of expression. The expression of the VE-cadherin promoter in the aqueous venous plexi and episcleral veins indicates that, at least some viral particles can pass through the HTM and across Schlemm’s canal endothelium, and reach the blood stream. Promoters from genes expressed in the cells of the outflow pathway might provide the means for a more specific targeting of gene expression in outflow pathway cells.

Keywords: gene/expression • molecular biology • trabecular meshwork