Translational vision science & technology · 2019
Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.
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Yau Edwin H, Taggart Robert T, Zuber Mohammed, Trujillo Alexandria J, Fayazi Zahra S, Butler Mark C, Sheflin Lowell G, Breen Jennifer B, Yu Dian, Sullivan Jack M
Аннотация
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.
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.