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Sinéad Connolly, Qistina Pilson, Sally-Ann Cryan, Joan Ní Gabhann, Conor C Murphy; Optimizing microRNA Therapeutics in Sjögren's Syndrome Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5691.
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© ARVO (1962-2015); The Authors (2016-present)
Sjogren’s Syndrome (pSS) an inflammatory disease, primarily affecting mucous membrane epithelia leading to the clinical phenotype of dry eyes and dry mouth, but may manifest extraglandular involvement in up to 50% of patients. While the exact aetiology is unknown, dysregulated expression of Type 1 IFNs and proinflammatory cyctokines is strongly associated with pSS. MiR regulated genes may contribute to the pathogenesis of pSS by increasing cytokine production. Previous studies conducted by our group identified a panel of miRs with differential expression in primary conjunctival epithelial cells (CEC) from pSS compared to healthy controls.Validation of one such novel miR and its predicted gene target confirmed that miR-744-5p expression is significantly increased whilst its predicted gene, Pellino3, a known negative regulator of type I IFN, was significantly reduced in CECs from pSS patients.Manipulation of miR-744-5p expression using a mimic or antagomir resulted in reduced and increased expression of Pellino3 respectively.Given the potential of Pellino3 to reduce inflammation, we are investigating strategies to deliver miR modulating compounds to the ocular surface.
psiCHECK-2 luciferase constructs were designed such that one contained the potential miR-744 binding sites within the 3’ UTR of Pellino3, while the second contained an unrelated fragment with no binding sites as a control vector.Biodegradable cationic nanoparticles were loaded with varying concentrations of miR and antagomiR cargo or scrambled short-chain nucleotides as control. Particle size and charge were determined using Malvern Zetasizer Nano 3000.
Co-transfection of psiCHECK-2 construct studies containing both of the potential binding sites with a miR-744 mimic demonstrated a significant decrease in luciferase gene expression when compared to the unrelated fragment control.Nanoparticles incorporating miR mimic and antagomiR were fabricated in a range of sizes and concentration. The presentation will describe preparation and optimization of nanoparticles to date.
Overall our results confirming a direct interaction have identified miR-744-5p as a negative regulator of Pellino3. An optimised nanoparticle delivery system has therapeutic potential for modulating inflammation at the ocular surface.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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