May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
High Throughput Cellular Screening for Ribozyme Development Against Arbitrary mRNA Targets
Author Affiliations & Notes
  • E. Yau
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • J. M. Sullivan
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • Footnotes
    Commercial Relationships E. Yau, Research Foundation of SUNY, P; J.M. Sullivan, Research Foundation of SUNY, P.
  • Footnotes
    Support NEI R01 EY13433 and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1681. doi:
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      E. Yau, J. M. Sullivan; High Throughput Cellular Screening for Ribozyme Development Against Arbitrary mRNA Targets. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1681.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: To develop a high throughput screening (HTS) platform that can rapidly test efficacy of hammerhead ribozyme (hhRz) constructs in human cells. The complex determinants of mRNA target structure and hhRz expression within living cells represent major obstacles in the design of successful hhRz agents. Methods for rapid screening of efficacy in live human cells are needed to realize the clinical potential of hhRz or similar agents.

Methods:: Human embryonic kidney (HEK) cells were engineered to stably express secreted human placental alkaline phosphatase (SEAP). SEAP reporter protein is secreted into culture medium in proportion to its RNA levels making it an ideal "model" target mRNA for assaying gene expression. A series of candidate hhRz constructs targeting SEAP mRNA were designed using in silico predictions of mRNA accessibility. hhRz cDNA sequences were cloned into a Pol-III vector that drives high level cytoplasmic expression of adenoviral VA1-hhRz RNA chimeras. VA1-hhRz and controls were transfected into HEK-SEAP cells and SEAP protein assayed. This model system was then transposed to human rod opsin (Rho) mRNA by constructing a bicistronic expression vector (Rho-IRES-SEAP) consisting of the complete Rho cDNA followed by an internal ribosome entry site (IRES) and then SEAP cDNA, which was stably expressed in HEK cells. As an additional embodiment of this approach, a small region of target Rho mRNA was ligated into the early 3’UT of SEAP mRNA (SEAP-Rho).

Results:: Computational analysis of SEAP mRNA revealed a very compact secondary structure with few accessible single-stranded regions. Five sites were chosen based on their predicted accessibility or use in previous studies: GUU↓ 150, CUC↓ 246, CUA↓ 800, AUA↓ 965, AUA↓ 1654. Statistically significant knockdown of SEAP (p<0.01) occurred with the 800 (16%) and 965 (14%) hhRzs (n=24) relative to control. Enhanced versions of these two hhRz constructs containing tertiary stabilization elements promoted higher efficacy. An hhRz known to successfully target Rho mRNA was analyzed in the Rho-IRES-SEAP and SEAP-Rho paradigms and demonstrated significant efficacy relative to controls (27% and 37.7% knockdown respectively).

Conclusions:: A HTS platform to screen cellular hhRz efficacy was developed that exploits SEAP as a reporter of gene expression that is easily assayed in 96 or 384 well format. This approach allows rapid screening of hhRz efficacy in a cellular environment prior to preclinical studies. This approach can be extended to any arbitrary mRNA disease target. Other types of ribozymes, antisense, or RNAi could also be screened for knockdown efficacy using this screen.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • photoreceptors 

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