Specific targeting of gene expression to a subset of human trabecular meshwork cells using the Chitinase–3–like–1 promoter in adenoviral–mediated gene transfer experiments.

Abstract

Abstract: : Purpose: To identify promoters for targeting gene expression to specific cells in the trabecular meshwork (TM) and Schlemm’s Canal (SC). Methods:Total RNA from 4 human TM and from 3 SC primary cultures were hybridized to Affymetrix U95Av2 Microarrays and the differential gene expression profile of HTM and SC cells was analyzed. Results from the arrays were confirmed by quantitative real time PCR. Based on the results, we constructed a recombinant adenovirus in which the expression of the reporter gene LacZ was driven by the 5’ promoter region of the chitinase–3–like 1 gene (AdCh3L1–LacZ). HTM and SC cells were infected with 100pfu/cell of the AdCh3L1–LacZ, and beta–galactosidase activity was analyzed 48 hours post infection. Expression of the chitinase–3–like 1 (Ch3L1) promoter was also analyzed in human perfused anterior segments infected with 10⁷pfu AdCh3L1–LacZ. Results:The genes showing higher levels of differential expression in TM versus SC cells were: Ch3L1, myocilin, and vascular cell adhesion molecule; while the genes more highly differentially expressed in SC versus TM cells were: gamma–sarcoglycan, fibulin–2, and collagen XV. Analysis of the expression of the best potential marker for TM cells (Ch3L1) showed targeted specific expression to a subset of the TM cells both in cell culture and in perfused anterior segments. Conclusions:Comparative analysis of gene expression between primary cultured SC and TM cells identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Our results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. Cells expressing high levels of this extracellular matrix protein have been reported in several disease models including atherosclerosis and rheumatoid arthritis. Targeting gene expression in these TM cell subtypes might provide important insight into normal outflow physiology and have future applications in glaucoma gene therapy.

Keywords: gene microarray • gene transfer/gene therapy • trabecular meshwork