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Sriniwas Sriram, Paulette Robinson, Alfred Lewin, Gregory Schultz; Nanoparticle Vectored siRNAs Reduce Profibrotic Gene Expression in Wounded Rabbit Corneas. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3878.
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Corneal haze remains a major problem following injury or refractive surgery. We tested the efficiency of a commercially available histidine-lysine peptide nanoparticle formulation to deliver siRNAs to a laser ablated cornea, to reduce pro-fibrotic mRNA, and to reduce corneal haze in a rabbit model.
To test the efficacy of siRNA delivery, excimer laser ablated rabbit corneas were topically dosed with fluorescently labeled scrambled siRNA sequences complexed with polymeric nanocarriers (22.5 uM). Excised corneas were maintained in organ culture for 8 hours then fixed, sectioned, and analyzed via fluorescent microscopy. To test the efficacy of an anti-fibrotic formulation of siRNAs delivered with these nanoparticles, corneas of nine rabbits were ablated with an excimer laser. One eye was treated with nanoparticles complexed with a triple combination of siRNA that targeted major scarring genes (TGFβ1, TGFβR2 and CTGF), while the other eye received empty nanoparticles as a control. To test the molecular efficacy of the siRNAs, corneas of 3 rabbits were excised 24 hours post-wounding, RNA was extracted and quantified by RT-qPCR. Knockdown percentages of the target genes were compared to the paired control eyes. To determine therapeutic effect, corneas of 6 rabbits were assessed for levels of haze on days 14 and 15 using digital photographs, in vivo confocal microscopy, and were graded for haze using the standard 0-4 semiquantitative scale.
Tissue sections from organ cultured corneas analyzed by confocal microscopy showed high levels of fluorescence in all layers of the treated cornea when compared to untreated controls. In anti-fibrotic treated rabbit corneas, the siRNA triple combination produced an average knockdown of 57% for TGFB1, 25 % for TGFBR2 and 24% for CTGF. One rabbit had a maximum knockdown of 80% for TGFB1, 57% for TGFBR2 and 46% for CTGF, indicating the siRNA triple combination was effectively delivered to the cornea in this animal. Results from haze grading on day 14 showed a decrease in haze formation in three out of the six treated rabbits.
These results indicate histidine-lysine peptide nanoparticles were effective in delivering siRNAs to all cell layers of excimer ablated corneas. With optimization of the dose and delivery of the triple siRNA combination, this gene targeted therapy may be an innovative treatment to reduce scar formation.
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