ArticleExperimental eye research2016
A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets.
Article in Experimental eye research, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 13 citations in OpenAlex.
- Enhanced hammerhead ribozyme turnover rates: Reevaluating therapeutic space for small catalytic RNAs.Molecular therapy. Nucleic acids · 2025Article
- Non-Viral Delivery Systems to Transport Nucleic Acids for Inherited Retinal Disorders.Pharmaceuticals (Basel, Switzerland) · 2025Review
- Retinitis Pigmentosa: Progress in Molecular Pathology and Biotherapeutical Strategies.International journal of molecular sciences · 2022Review
- A Dual-Functioning 5'-PPP-NS1shRNA that Activates a RIG-I Antiviral Pathway and Suppresses Influenza NS1.Molecular therapy. Nucleic acids · 2020Article
- Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.Translational vision science & technology · 2019Article
- Molecular Therapies for Inherited Retinal Diseases-Current Standing, Opportunities and Challenges.Genes · 2019Review
- Reduced expression of theMolecular vision · 2019Article
- Effects of Pathogenic Variations in the Human Rhodopsin Gene (hRHO) on the Predicted Accessibility for a Lead Candidate Ribozyme.Investigative ophthalmology & visual science · 2017Article
- Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents.Experimental eye research · 2011Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.