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E. H. Yau, T. A. Kolniak, R. T. Taggart, J. M. Sullivan; Optimization of Ribozyme Agents for Human Rod Opsin Therapeutics. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5344.
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© ARVO (1962-2015); The Authors (2016-present)
To develop hammerhead ribozyme (hhRz) therapeutic agents that effectively suppress human rod opsin mRNA (Rho mRNA) in human cells. The major limiting factors in the success of hhRz agents are the difficulty in predicting target mRNA regions that are susceptible to hhRz cleavage in a cellular environment and the expression, trafficking, and structure of hhRz agents in human cells. We used rational design and experimental approaches to identify the best target region in Rho mRNA for hhRz cleavage in live human cells, and optimized hhRz agents targeted to this region.
Regions of accessibility in Rho mRNA were determined by a combination of in silico predictions of mRNA secondary structure and a PCR-based hybridization approach. A bicistronic vector consisting of the complete Rho cDNA followed by an internal ribosome entry site (IRES) and then the reporter SEAP cDNA sequence was engineered and stably expressed in human embryonic kidney (HEK) cells. hhRz cDNA sequences targeting regions of determined accessibility were cloned into a Pol-III vector that drives high level cytoplasmic expression of adenoviral VA1-hhRz RNA chimeras. VA1-hhRz plasmids and controls were transfected into HEK-Rho-IRES-SEAP cells and reporter expression assayed in our high throughput screening (HTS) system. hhRz knockdown against full-length native Rho mRNA target was validated using qRT-PCR.
Ten regions of Rho mRNA were experimentally found to be accessible, encompassing thirty potential hhRz cleavage sites. An additional four hhRz sites were chosen, two because of efficacy in previous studies and two known to reside in inaccessible regions. These 34 sites were then evaluated for efficacy using the SEAP bicistronic reporter system. Eighteen of the 34 sites showed significant knockdown of SEAP expression (p<0.01). We designed different optimizations of our hhRz agents against the most accessible target region. Tertiary structure elements that are highly efficacious in improving cleavage activity in cis-cleaving hhRz designs did not prove as efficacious in enhancing the cleavage activity of our trans-cleaving hhRz agents.
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