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H.E. Abdel-maksoud, J.M. Sullivan; Knockdown Ribozyme Development for adRP Gene Therapy . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2340.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop efficacious hammerhead ribozymes (hhRz) that significantly knockdown target gene expression in a human cell expression system. Methods: A rational computational approach is used to identify local accessible regions in human rod opsin mRNA secondary structure that could support rapid hhRz annealing and efficient cleavage. Energy minimization algorithms determine an ensemble of secondary structures in a moveable computational window and the probabilities of recurrence of stable local RNA structures are convolved with the length of embraced single stranded annealing platforms to obtain accessibility scores. These scores are rank ordered to select regions of secondary structures with NUH↓ motifs for hhRz targeting. Testing in human cells presents a additional range of target variables likely to be important for ribozyme function such as tertiary structures, RNA binding proteins and RNA compartmentalization. Symmetrical (7 nt in stem I and III) hhRz genes (catalytic active or inactive) are cloned into a pol-III expression vector that presents the hhRz as a chimeric adenoviral VA1-hhRz RNA for high level expression and cytoplasmic colocalization with opsin mRNA. HEK293 cells are cotransfected with opsin, VA1-hhRz (active, inactive), or VA1-hhRz-null (control, irrelevant sequence replaces hhRz) expression vectors. hhRz-mediated knockdown of opsin expression is measured by Western and slot blot analyses and statistically evaluated (ANOVA, t-tests) relative to controls. Results: To functionally test predicted accessibility at six target sites, hhRzs were designed against NUH↓ motifs at CUU 262, CUC 266, CUC 272, UUC 1197, CUC 1411, AUC 1414. When measured against the VA1-hRz-null control, hhRzs targeting CUC 272 and CUU 262 demonstrate substantial opsin knockdown (greater than 50%), while others (CUC 266, UUC 1197, CUC 1411) show partial knockdown and one (AUC 1414) appears to be ineffective. Conclusions: Knockdown hhRzs with substantial efficacy to suppress expression of most known human rod opsin mutants are identified. This moderate throughput target screening approach has substantial potential to rapidly identify candidate hhRz therapeutics that are expected to perform equivalently well in retinal neurons. This approach could be used to develop hhRzs targeting arbitrary disease-related mRNAs or in functional genomics applications. A major advantage is that the target is the same human mRNA that would exist in human gene therapy clinical trials.
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