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M. Eibschitz–Tsimhoni, R. Ambasudhan, N.A. Mandal, J.M. Bayliss, R. Ayyagari, M.A. Del Monte; A Molecular Approach to the Management of Graves’ Ophthalmopathy . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3586.
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Purpose:To develop a novel therapeutic strategy for the treatment of Graves’ Ophthalmopathy (GO), through siRNA mediated targeted silencing of orbital TSH receptor (TSHr) expression. GO is characterized by an increase in the volume of orbital tissues within the bony orbits leading to exophthalmos, extraocular muscle dysfunction, periorbital edema, compressive optic neuropathy, and vision loss. Few treatment options beyond palliative care are available. Present palliative treatments, including high dose corticosteroids, radiation therapy and surgery, are either of questionable effectiveness or could result in disabling side effects. TSHr is proposed to be an antigen responsible for the development of the autoimmune response resulting in GO. Through our new strategy, we hope to prevent the development of GO by eliminating the auto–antigen responsible, rather than merely treating the orbital damage caused by the autoimmune process. Methods:CHO cells characterized for stable expression of human TSHr were grown in Ham’s F12K medium. The presence of human TSHr was verified using RT–PCR and Western blotting. Using Oligofectamine the cells were transfected with several double stranded siRNA oligonucleotides targeted against different regions of TSHr mRNA. Cells were harvested at 0, 2, 4, 8, 12, 24, and 38 hours post transfection. TSHr RNA and protein levels were analyzed by using quantitative RT–PCR and Western blotting. Cellular GAPDH levels were measured as a control. Results:Among several siRNAs tested, one of the oligonucleotides yielded substantial inhibition of TSHr synthesis in the transfected cells, as compared to the controls and other siRNAs tested. This was confirmed at the RNA and protein levels. Conclusions:We have demonstrated that, through RNA interference, TSHr expression can be successfully depleted in an in vitro cultured cell line. Our results also indicate that certain regions of the TSHr mRNA are more suitable for effecting siRNA mediated silencing. This model sets the stage for the investigation of TSHr inhibition for the prevention or reversal of Graves’ Ophthalmopathy.
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