Dataset Information
Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.
Ontology highlight
ABSTRACT: 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.
SUBMITTER: Yau EH
PROVIDER: S-EPMC6908138 | biostudies-literature | 2019 Nov
REPOSITORIES: biostudies-literature
Publications
Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.
Translational vision science & technology 20191101 6
<h4>Purpose</h4>To systematically evaluate human rod opsin (<i>hRHO</i>) 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.<h4>Methods</h4>In multidisciplinary RNA drug discovery, computational mRNA accessibility and in vitro experimental methods using reverse transcription-polym ...[more]
