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N. Comes, T. Borrás; Functional Delivery of Naked siRNA to the Human Trabecular Meshwork (TM) in Perfused Organ Cultures. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2051.
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© ARVO (1962-2015); The Authors (2016-present)
RNA interference (RNAi) is a powerful technology for silencing the expression of a specific gene. Our goal here was to investigate a) whether siRNA molecules could be directly delivered to the intact human TM through the anterior chamber, b) whether this naked siRNA could silence a TM specific gene and most important, c) whether direct siRNA treatment could counteract the downstream effect of a deleterious agent (DEX) by silencing its receptor.
Anterior segments from post-mortem normal human donors were perfused at either 2-5 µl/min-constant flow (C=0.18±0.04) (n=11) or 15 mmHg-constant pressure (C=0.19±0.04) (n=2) for 24 h to baseline. Commercial siRNAs were diluted directly in DMEM perfusion medium and used without coupling to transfection reagents ("naked"). Perfusion of labeled siRNA (Darmacon siGLO RISC-free Cy3) was performed at 100 nM for 48 h followed by 24 h with DMEM medium (OD); OS was injected twice with 20 µl of 200 nM (2 pairs). Perfusions of siRNA (100 nM) targeting the Matrix GLA (MGP) gene (OD) and scrambled-siRNA (control) (OS) were performed for 48 h (Ambion IDs#122163 & #1) (2 pairs). Two other eye pairs were pre-treated with perfused glucocorticoid receptor (GR)-siRNA (ID#3909, OD) and scrambled-control (OS) for 48 h and continued by adding 0.1 µM DEX to the perfusion media for an additional 24 h. Frozen sections of labeled anterior segments were analyzed by fluorescence microscopy. Relative quantification of GR, MGP, MYOC and CDT6 transcripts was determined by RT-TaqMan PCR on RNA extracted from dissected TMs. Levels of secreted MYOC in the effluents were analyzed by western blot.
Histological evaluation of anterior segments perfused with labeled siRNA showed intense Cy3 fluorescence in the TM region. MGP gene expression was silenced in the TM perfused with naked MGP siRNA; MGP transcripts were reduced -16.95-fold from those present in the contralateral TM perfused with scrambled-control. In the presence of GR silencing, DEX treatment caused a reduction of the MYOC and CDT6 expressions (fold changes -142.9 and -41.7 respectively) obtained with scrambled-control/DEX eyes. Western blots revealed a significant decrease of secreted MYOC protein in effluents from GR silenced cultures.
Readily available siRNA can be delivered to the intact human trabecular meshwork by intracameral perfusion. The delivered naked siRNA is functional, inhibiting not only the targeted gene but their downstream effectors. This functional intracameral delivery might be used to protect the TM from unwanted insults and might have important therapeutical applications.
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