May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Cell–Based High–Throughput Screening of Rationally Designed Ribozymes
Author Affiliations & Notes
  • E. Yau
    SUNY at Buffalo, Buffalo, NY
    Pharmacology & Toxicology,
  • J.M. Sullivan
    SUNY at Buffalo, Buffalo, NY
    VA Medical Center, Buffalo, NY
  • Footnotes
    Commercial Relationships  E. Yau, None; J.M. Sullivan, None.
  • Footnotes
    Support  NIH Grant EY13433
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 848. doi:
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      E. Yau, J.M. Sullivan; Cell–Based High–Throughput Screening of Rationally Designed Ribozymes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):848.

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

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Purpose: : To develop a high throughput screening (HTS) platform to rapidly test hammerhead ribozyme (hhRz) construct optimizations. The complexity of mRNA target structure within living cells also represents a major obstacle in the design of successful hhRz agents. Methods for rapid screening of hhRz constructs in live human cells are needed to realize the clinical potential of hhRz agents against disease targets.

Methods: : Human embryonic kidney (HEK293S) cells were engineered to stably express a secreted form of human placental alkaline phosphatase (SEAP). SEAP protein is secreted into culture medium in proportion to its RNA levels making it an ideal "model" target mRNA. A HTS assay for SEAP levels was developed that employs the substrate 4–methyl–umbelliferyl phosphate, which is converted by SEAP to a fluorescent product that is readily measured by a fluorescent plate reading HTS system. A series of candidate hhRz designs targeting SEAP mRNA were generated following rational design strategies based on identifying potential accessible regions of target mRNA. hhRz cDNA sequences were cloned into a Pol–III expression vector that drives high level expression of adenoviral VA1–hhRz RNA chimeras to the cytoplasm. VA1–hhRz plasmids and controls were transfected into HEK–SEAP cells and media from these cells assayed at 72 hours.

Results: : Computational analysis of SEAP mRNA revealed a highly ordered secondary structure with few accessible single–stranded regions. Three sites were chosen based on their predicted accessibility: AUA↓ 1654, CUA↓ 800 and CUC↓ 246. Statistically significant knockdown of SEAP (p<0.01) occurred with the 1654 (22%) and 246 (16%) hhRzs (n=14) relative to control expression vector. Further evaluation of the active 1654 hhRz construct vs. a catalytically inactivated core construct targeting the same site found no significant reversal of knockdown (both 18% knockdown, p<0.01, n=31).

Conclusions: : A HTS platform for SEAP was developed with operational sensitivity, dynamic range, and low variability that could reliably detect even moderate levels of SEAP knockdown by rationally designed hhRz constructs. The lead candidate (1654–hhRz) has equal efficacy in both catalytically active and inactive forms suggesting knockdown occurs by antisense or catalytic antisense effects. With substantial dynamic assay range remaining, current efforts are directed toward optimizing antisense flank sequences and VA1 chimera expression construct designs for 1654–hhRz and adding accessory elements that enhance activity. Optimized hhRz–VA1 chimera design and the HTS approach will be transposed to mRNA targets that promote retinal and macular degenerations.

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

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