TY  - BOOK
AU  - Yu, Dian
TI  - Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics
SN  - 2164-2591
PY  - 2019///
KW  - IN PROCESS -- NOT YET INDEXED
KW  - MedStar Washington Hospital Center
KW  - Ophthalmology
KW  - Journal Article
N2  - 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; 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; 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; 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; Translational Relevance: This work establishes a platform approach to develop RNA biologicals for the treatment of human disease. Copyright 2019 The Authors
UR  - https://dx.doi.org/10.1167/tvst.8.6.28
ER  -