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T. Kolniak, E. Yau, R. Taggart, J. M. Sullivan; Identification of Lead Candidate Ribozymes for Human Rod Opsin Therapeutics. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1682.
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© ARVO (1962-2015); The Authors (2016-present)
To functionally evaluate the conformational landscape of human rod opsin mRNA (Rho mRNA) in order to locate the most efficacious site for hammerhead ribozyme (hhRz) cleavage in a cellular environment. Post transcriptional gene silencing (PTGS) methods hold unrealized clinical potential for gene therapy in inherited retinal dystrophies. A major limitation is the difficulty in predicting target mRNA regions that are susceptible to PTGS attack in a cellular environment. We used rational design and experimental approaches to identify the best target region in Rho mRNA for hhRz cleavage in live human cells.
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 in Rho mRNA 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 SEAP expression assayed in our high throughput screening (HTS) system.
Computational analysis of Rho mRNA revealed several areas of potential hhRz accessibility. These regions were ranked and used to generate probes for an in vitro PCR-based hybridization scheme (gene specific mRNA Accessibility Site Tagging, gsMAST). Eight regions found to be experimentally accessible encompassed 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) with five of these sites providing robust knockdown of SEAP measures. We identified these five ribozyme constructs as lead candidates for further optimization prior to animal studies.
Combining mRNA accessibility determination methods and a cell based HTS approach; we have identified five lead candidate hhRzs against Rho mRNA. As Rho mRNA harbors many mutations responsible for retinal degenerations, a robust hhRz targeted against Rho mRNA is of therapeutic interest. This combined approach is also extendable to development of therapies against any arbitrary mRNA target.
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