March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Toward Cellular Directed Evolution of Therapeutic Ribozymes
Author Affiliations & Notes
  • Mark C. Butler
    Research Service, VA Western NY Healthcare System, Amherst, New York
    Ophthalmology-Ross Eye Institute,
    University at Buffalo/SUNY and SUNY Eye Institute, Buffalo, New York
  • Aishwarya Bhaskar
    Biotechnical Clinical Laboratory Sciences, University at Buffalo/SUNY, Buffalo, New York
  • John N. Misasi
    Ophthalmology, Upstate Medical University/SUNY, Syracuse, New York
  • Jack M. Sullivan
    Research Service, VA Western NY Healthcare System, Amherst, New York
    Ophthalmology-Ross Eye Institute, Pharmacology/Toxicology, Physiology/Biophysics,
    University at Buffalo/SUNY and SUNY Eye Institute, Buffalo, New York
  • Footnotes
    Commercial Relationships  Mark C. Butler, None; Aishwarya Bhaskar, None; John N. Misasi, None; Jack M. Sullivan, None
  • Footnotes
    Support  NIH Grant: R01 EY013433 (JMS), R24 EY016662, RPB Unrestricted Grant
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5127. doi:
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      Mark C. Butler, Aishwarya Bhaskar, John N. Misasi, Jack M. Sullivan; Toward Cellular Directed Evolution of Therapeutic Ribozymes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5127.

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

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Purpose: : Conduct a limited proof-of-principle test of a strategy (CELL-SELEX) for cellular directed evolution of knockdown efficacy by hammerhead ribozyme (hhRz) gene-based therapeutics.

Methods: : The hhRz target cDNA (human RHO) is engineered with the 3’UT converted to protein coding sequence, followed by an in-frame suicide gene cDNA to generate a toxic target gene (TTG). Expressed in human cells, the TTG promotes efficient cell death. A gene-specific hhRz library was made to all GUC cleavage sites within hRHO mRNA. HhRzs were expressed within a chimeric RNA (VAI) from plasmids containing EBV oriP episomal maintenance elements in HEK293E cells that express EBNA1. Cell numbers are measured with a SYTOX Green HTS assay. HhRz episomes are isolated from selected 293E cells by a modified Hirt procedure and amplified in bacteria. HhRz episomal evolution was assayed by restriction fragment gel analysis.

Results: : A RHO-TK TTG has a HSV thymidine kinase as the suicide gene that operates on added gancyclovir (GCV) (prodrug) to create toxic DNA metabolites. GCV concentrations (25-50 μg/ml) induced efficient cell death in 293E cells expressing RHO-TK. 293E cells co-expressing stabilized TTG with the hhRz library, or with the library vector without hhRzs (control), or with discrete hhRz expression plasmids targeting RHOmRNA were exposed to GCV and cell viability measured over time. Only the hhRz library and discrete positive control hhRzs capable of RHOtarget knockdown provided viability protection. HhRz library was polluted with empty vector (no hhRzs) (1:10 ratio) and selection in GCV of co-transfected cells for two months resulted in progressive selection-based enrichment of episomal plasmids containing hhRzs. Sequences of cell/bacteria amplified episomal plasmids were enriched for two hhRzs against RHO GUC sites not identified in rational RNA drug discovery.

Conclusions: : Cellular directed evolution involving a randomized population of episomal hhRz expression vectors, strong cell selection, and amplification of successful winners has potential for development of gene-based hhRz therapeutics.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • age-related macular degeneration 

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