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Jack Sullivan, Tiffany Kolniak; RNA Structure-Function Issues in Realizing Catalytically-Active Hammerhead Ribozymes in Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6118.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate enzymatic performance of a proven hammerhead ribozyme (hhRz) against human rod opsin (hRHO) in a smaller supportive RNA scaffold in a mutation-independent gene silencing therapeutic strategy for autosomal dominant retinitis pigmentosa. VAI RNA of chicken embryonic lethal orphan (CELO) virus was chosen as a smaller (91 nt) scaffold for the hhRz (725 HH16) (prior human VAI scaffolds >160 nt). Smaller chimeras allow 3D RNA algorithms for structural design and biophysical investigation of hhRz structure/function performance.
CELO-VAI scaffold, designed using RNAStructure, was PCR constructed with partially overlapping DNA primers, then directionally ligated into pNEB193-T7. hhRz cDNA or control sequence was cloned into an adapter placed at a unique CELO-VAI restriction site. Target hRHO fragment cDNA (510 nt RNA) containing the hhRz cleavage site was cloned into pBlueScript. Transcription with T7 RNA polymerase generated chimeric CELO-VAI-hhRzs and target RNA to test in vitro cleavage. In HEK293 cells full length hRHO cDNA was expressed from pCDNA3.1, and CELO-VAI-hhRzs or controls (in pNEB193-T7) were expressed from strong intragenic Pol-III promoters. hRHO mRNA was assayed by quantitative RT/PCR with controls. ANOVA and t-tests were used to statistically evaluate outcomes.
The hhRz that demonstrated cellular cleavage activity within a larger human VAI RNA scaffold had only pure antisense target suppression (64% knockdown, p<0.05) within the smaller CELO-VAI scaffold, and minimal in vitro target cleavage. A second CELO VAI scaffold with a stabilized stem/loop for the hhRz demonstrated no significant knockdown of cellular target and minimal cleavage in vitro. The stabilized stem loop with hhRz in isolation (H1 promoter) did not improve activity. Addition of a 5’ upstream tertiary accessory element (TAE) to drive formation of a catalytically active hhRz conformation state recovered both catalytic intracellular knockdown (41%, p<0.05) and robust in vitro cleavage.
RNA structure and expected dynamics of a small hhRz within an RNA scaffold strongly impacts enzymatic function. This suggests an RNA structure driven biophysical approach for enhanced hhRz-based gene therapeutics. TAE may be a generalized element to enhance intracellular hhRz catalytic performance. The CELO-VAI-hhRz-TAE is an agent for testing in our pre-clinical adRP trial pipeline.
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