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M.-G. Spiga, T. Borras; Developing a Gene Therapy Vector for the Treatment of Steroid Glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1670.
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Glucocorticoid (GC) treatment leads to increased ECM deposition in the trabecular meshwork (TM) and to elevated IOP in 40% of patients. Our goal was to design a GC-inducible viral vector overexpressing matrix metalloproteinase protein 1 (MMP1) to counteract ECM deposition only in the presence of the steroid.
Human MMP1 cDNA from HTM cells was cloned downstream a GC response element (GRE) fused to a TATA-like promoter. The expression cassette was inserted into a pShuttle adenoviral vector to produce AdhGRE.MMP1. A negative functional control was generated using a MMP1 cDNA containing a His199 point mutation in the catalytic active site (AdhGRE.mutMMP1). Confluent primary HTM cells were infected with AdhGRE.MMP1 or AdhGRE.mutMMP1 in the presence or absence of 0.1µM DEX. MMP1 protein and mRNA were analyzed by western blotting and real time PCR. Activity of the secreted MMP1 enzymes was analyzed with a Fret assay, which uses a fluorogenic substrate mimicking collagen I, a major ECM component.
HTM cells treated with DEX show a decreased MMP1 expression compared to untreated controls. However, DEX-treated cells infected with AdGRE.MMP1 secreted high levels of MMP1, overriding the reduction and boosting its expression. MMP1 protein levels were ~ 5 and 20-fold the DEX-treated uninfected cells at 3 and 5 days post-infection, respectively; mRNA levels were >200-fold at 3 days. AdhGRE.MMP1 and AdhGRE.mutMMP1 infected cells treated with DEX had also higher MMP1 mRNA and secreted protein than those infected-untreated, confirming the functional activity of the GRE element. More important, the media of DEX-treated cells infected with AdhGRE.MMP1 showed a 90% higher fluorescent activity than untreated-infected controls while that of the cells infected with AdhGRE.mutMMP1 were similar to controls, which confirmed the functional state of the constructs. Collaborative in vivo experiments using a large animal steroid model are ongoing.
We have developed a novel glucocorticoid-inducible adenoviral vector system, which overexpresses MMP1 only in the presence of DEX. This tightly regulated gene transfer approach could be of great advantage for the potential gene therapy treatment of steroid responders glaucoma patients.
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