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Jack M. Sullivan, Edwin H. Yau, Robert T. Taggart, Mark C. Butler, Tiffany A. Kolniak; RNA Drug Discovery- An Efficient Pipeline for Post-Transcriptional Gene Silencing Agent Development. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5128.
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Develop and utilize a technological pipeline for efficient RNA Drug Discovery (RDD) of candidate therapeutic post-transcriptional gene silencing (PTGS) agents (ribozyme, shRNA, miRNA).
A bioinformatics model operates on output of three algorithms (MFold, SFold, OligoWalk) to map accessible regions in target mRNAs at the single nt level. A novel cDNA mapping of accessible RNA sites (CMARS) uses a library of partially randomized oligodeoxynucleotides to map target RNA regions that support reverse transcription initiation. Large sets of PTGS agents are screened for knockdown efficacy in a human cell expression system (HEK293) co-transfected with expression plasmids for a discrete PTGS agent and the target cDNA fused to a reporter enzyme (SEAP) in a bicistronic mRNA. Knockdown efficacy is measured by a HTS enzyme fluorescence assay for SEAP and rank-ordered to identify a lead candidate. Lead optimization occurs by variation of antisense flank length, ribozyme structure, and nature of supportive RNA expression chimeras, and is facilitated by a HTS quantitative immunocytochemistry platform. Toxicity is evaluated in a HTS apoptosis assay. Subretinal vector delivery in mice occurs with a novel surgical stereo microscope optimized for the small rodent eye to achieve detailed real time in vivo imaging. Ultrahigh resolution OCT imaging identifies successful subretinal vector delivery.
Four target mRNAs (human RHO, mouse RHO, SEAP, RPE65) were mapped for accessible regions by bioinformatics and CMARS with correlative outcomes and large single stranded regions are rare in all targets evaluated. For hRHO and SEAP sets of PTGS agents were tested against accessible and inaccessible regions and lead candidate were identified and optimized for potent intracellular performance. PTGS expression vectors are not toxic in human cells. Vector delivery into the subretinal space is definitive to initiate preclinical evaluation.
A rational RDD technological platform can realize potent PTGS agents to validated disease target mRNAs in the outer retina.
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