Specific Targeting of Gene Expression to a Subset of Human Trabecular Meshwork Cells Using the Chitinase 3-Like 1 Promoter

Glaucoma is a group of blinding disorders characterized by damage to the optic nerve. The most common form of the disease, primary open-angle glaucoma (POAG), is frequently associated with elevated intraocular pressure (IOP) that results from an abnormal resistance to the outflow of aqueous humor through the conventional outflow pathway.1,2 The conventional outflow pathway is the route by which most aqueous humor exits the anterior chamber of the eye, and it includes the trabecular meshwork (TM) and Schlemm’s canal (SC).3 The outflow pathway is a complex tissue composed of several different cell types that are morphologically and functionally different: (1) Schwalbe’s line (SL) cells are located in the anterior, nonfiltering portion of the TM and have been proposed to be the progenitor cells of the TM; (2) TM cells cover the surface of the connective tissue beams in both the uveal and corneoscleral filtering meshwork and appear to be involved in phagocytosis and tissue remodeling; (3) juxtacanalicular tissue (JCT) cells are randomly distributed within the extracellular matrix of the juxtacanalicular meshwork; and (4) the cells of the inner wall endothelium of SC constitute the only continuous cell layer in the outflow pathway.4,5 The specific functional differences of these cells and their role in the physiology of the aqueous humor process are not clear. Although the locus of both normal outflow resistance and the abnormal resistance in POAG is believed to be at the level of the JCT and/or the inner wall of SC,6–8 there is uncertainty as to the exact location. Although one school of thought postulates that the extracellular material within the JCT is responsible for normal resistance, the other school of thought emphasizes the role of the cells of the inner wall of SC as the major locus of the outflow resistance.7,9–13 A potential strategy for understanding these questions, which also has therapeutic implications, is directed gene delivery to specific cell types within the outflow pathway. We have recently demonstrated the feasibility of targeting gene expression to the outflow pathway with replication-deficient adenoviruses containing tissue-specific promoters that were identified from gene expression profile analyses of the TM.14 Analyses of the published TM libraries have provided important information about the genes preferentially expressed in the outflow pathway compared with other tissues18–20; however, the specimens used in these analyses also include cells from the inner wall of SC. Therefore, the identification of genes differentially expressed in the TM and SC is the next logical step in planning further experiments targeting specific cell types. One experimental approach for the identification of such differential markers is the comparison of gene expression profiles. However, because of the small size and complexity of the outflow pathway, it is impractical to dissect only the SC endothelia, making the construction of a direct SC cDNA library almost impossible. As an alternative approach, we present a comparative gene expression profile analyses between human primary cultured TM and SC cells. We identified genes with promoters potentially capable of targeting gene expression in specific cells in the outflow pathway. The expression of one of these promoters was tested in both TM cells and SC cells, and also in human perfused anterior segments.