RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. AbbreviationsAMD, age-related macular degeneration; Apaf-1, apoptotic protease-activating factor-1; CNV, choroidal neovascularization; CTGF, connective tissue growth factor; DR, diabetic retinopathy; dsRNA, double-stranded RNA; ECM, extracellular matrix; HIF-1α, hypoxia-inducible factor-1α; IOP, intraocular pressure; ONC, optic nerve crush; POAG, open-angle glaucoma; RGC, retinal ganglion cell; RISC, RNA-induced silencing complex; RNAi, RNA interference; RP, retinitis pigmentosa; Smad, signalling mathers against decapentaplegic; SPARC, secreted protein acidic and rich in cysteine; TLR3, toll-like receptor 3; TM, trabecular meshwork; TXNIP, thioredoxin interacting protein IntroductionRNA interference (RNAi) technology has been used to elucidate gene function, to generate model systems and to identify new molecular targets. In addition, RNAi is currently progressing from basic research to potential therapeutic applications. Because any gene that causes or contributes to a disease is susceptible to suppression by RNAi, RNAi therapy represents a promising biomedical strategy for treating a diverse range of diseases including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory conditions, viral infections and ocular diseases (Guo et al., 2010). In particular, the accessibility of the eye facilitates small-interfering RNA (siRNA) delivery, and naked siRNA has been efficiently applied by topical administration to the anterior segment or by intravitreal injection to the posterior segment (de Fougerolles, 2008). As the localized delivery of siRNA to the eye is less challenging than for other tissues, there has been significant progress made towards its use as a therapeutic procedure for eye diseases. In fact, several siRNA-based therapeutic agents for o...

BACKGROUND AND PURPOSEHere, we have studied the effects of the dinucleotide P 1 , P 4 -Di (adenosine-5′) tetraphosphate (Ap4A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency. EXPERIMENTAL APPROACHExperiments in vitro were performed using human corneal epithelial cells (HCLEs) treated with Ap4A (100 μM) for 5 min. Western blot analysis and transepithelial electrical resistance (TEER) were performed to study the TJ protein levels and barrier function respectively. Intracellular pathways involved were determined using an ERK inhibitor and P2Y2 receptor siRNAs. In in vivo assays with New Zealand rabbits, TJ integrity was examined by zonula occludens-1 (ZO-1) staining. The hypotensive compound 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) was used to assess improved delivery, measuring its levels by HPLC and measuring intraocular pressure using 5-MCA-NAT, P2Y receptor antagonists and P2Y2 siRNAs. KEY RESULTSTwo hours after Ap4A pretreatment, TJ protein levels in HCLE cells were reduced around 40% compared with control. TEER values were significantly reduced at 2 and 4 h (68 and 52% respectively). TJ reduction and ERK activation were blocked by the ERK inhibitor U012 and P2Y2 siRNAs. In vivo, topical application of Ap4A disrupted ZO-1 membrane distribution. 5-MCA-NAT levels in the aqueous humour were higher when Ap4A was previously instilled and its hypotensive effect was also increased. This action was reversed by P2Y receptor antagonists and P2Y2 siRNA. CONCLUSIONS AND IMPLICATIONSAp4A increased corneal epithelial barrier permeability. Its application could improve ocular drug delivery and consequently therapeutic efficiency. -Di (adenosine-5′) tetraphosphate; HCLE, human corneal epithelial cells; IOP, intraocular pressure; NGS, normal goat serum; PPADS, pyridoxal phosphate-6-azo (benzene-2′,4′-disulfonic acid); RB2, reactive blue 2; TEER, transepithelial electrical resistance; TJ, tight junctions; U0126, 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene ethanolate; ZO-1, zonula occludens-1

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