Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics
| Autor: | Edwin H. Yau, Robert T. Taggart, Mohammed Zuber, Dian Yu, Jack M. Sullivan, Mark C. Butler, Jennifer B. Breen, Zahra Fayazi, Alexandria J Trujillo, Lowell G. Sheflin |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Hammerhead ribozyme macular degeneration DNAzyme Biomedical Engineering Computational biology Small hairpin RNA ribozyme 03 medical and health sciences 0302 clinical medicine shRNA RNA interference Gene knockdown biology Drug discovery Ribozyme RNA Articles biology.organism_classification gene therapy Ophthalmology RNA silencing 030104 developmental biology siRNA retinal degeneration 030221 ophthalmology & optometry biology.protein high throughput screening |
| Zdroj: | Translational Vision Science & Technology |
| ISSN: | 2164-2591 |
| Popis: | Purpose To systematically evaluate human rod opsin (hRHO) mRNA for potential target sites sensitive to posttranscriptional gene silencing (PTGS) by hammerhead ribozyme (hhRz) or RNA interference (RNAi) in human cells. To develop a comprehensive strategy to identify and optimize lead candidate agents for PTGS gene therapeutics. Methods In multidisciplinary RNA drug discovery, computational mRNA accessibility and in vitro experimental methods using reverse transcription-polymerase chain reaction (RT-PCR) were used to map accessibility in full-length hRHO transcripts. HhRzs targeted predicted accessible and inaccessible sites and were screened for cellular knockdown using a bicistronic reporter construct. Lead hhRz and RNAi PTGS agents were rationally optimized for target knockdown in human cells. Results Systematic screening of hRHO mRNA targeting agents resulted in lead candidate identification of a novel hhRz embedded in an RNA scaffold. Rational optimization strategies identified a minimal 725 hhRz as the most active agent. Recently identified tertiary accessory elements did not enhance activity. A 725-short-hairpin RNA (shRNA) agent exerts log-order knockdown. Silent modulation of the 725-hhRz target site in hRHO mRNA resulted in resistance to knockdown. Conclusions Combining rational RNA drug design with cell-based screening allowed rapid identification of lead agents targeting hRHO. Optimization strategies identified the agent with highest intracellular activity. These agents have therapeutic potential in a mutation-independent strategy for adRP, or other degenerations where hRHO is a target. This approach can be broadly applied to any validated target mRNA, regardless of the disease. Translational relevance This work establishes a platform approach to develop RNA biologicals for the treatment of human disease. |
| Databáze: | OpenAIRE |
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