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Scott D. Lawrence, Lakisha K. Buie, Terete Borras; Direct Delivery of Synthetic Naked siRNA to Trabecular Meshwork in Living Rats. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6616.
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Small interference RNA (siRNA) has the potential to silence unwanted, disease-relevant genes. Delivery of siRNA to trabecular meshwork could potentially provide an important therapeutic means of modifying glaucoma risk factors. Having previously demonstrated successful transfer of siRNA to trabecular meshwork (TM) of post-mortem human eyes, we sought to investigate whether naked siRNA molecules could be delivered in vivo to the TM of living rats.
We performed intracameral injections of 5 µl (5 nmol) of fluorescently labeled siRNA (Dharmacon siGLO RISC-free DY-547) in 5 eyes of 4 Wistar rats. The siRNA (20 nmol) was diluted in phosphate buffered saline (PBS) without coupling to transfection reagents ("naked"). Transfer of siRNA was achieved via a 30-gauge needle inserted anterior to the corneal limbus and parallel to the iris plane with delayed needle retraction to minimize reflux of the vehicle. As controls, 5 µl of PBS was injected in the fellow eye of 3 rats. The rats were euthanized, and the ocular tissues harvested at 24 hours post-injection. An additional 8 eyes of 4 rats received 5 µl injections (5 siRNA, 3 PBS) and were harvested at 48 hours post-injection. One uninjected rat was euthanized and eyes harvested for autofluorescence control. Harvested anterior segments were fixed in 4% paraformaldehyde for 1 hour and cryoprotected in 30% sucrose overnight. Tissue was then embedded and frozen in OCT medium. Ten µm cryosections were analyzed by fluorescence imaging for detection of DY-547 (rhodamine filter) siRNA in all eyes.
Histological evaluation at 24 hours demonstrated intense DY-547 hyperfluorescence in the trabecular meshwork region of all 5 eyes injected with siRNA. The 3 eyes injected with PBS showed mild to moderate hyperfluorescence - in each case, the degree of fluorescence was less than the fellow (siRNA) eye. Of the 8 eyes harvested at 48 hours, only 2 (both injected with siRNA) showed very low levels of DY-547 hyperfluorescence in the region of the TM while the remaining specimens exhibited no fluorescence. Control, uninjected eyes showed minimal autofluorescence at the same exposure times.
Synthetic naked siRNA can be successfully delivered to the trabecular meshwork in a living animal by intracameral injection. That hyperfluorescence was also detected in fellow eyes receiving PBS suggests systemic absorption of siRNA. Decreased DY-547 hyperfluorescence at 48 hours may indicate rapid degradation of siRNA or the DY-547 label.
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