April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Cell SELEX- a Novel in vivo Evolutionary Approach for RNA Drug Discovery
Author Affiliations & Notes
  • M. C. Butler
    Ophthalmology, University at Buffalo- SUNY; VA Western NY Healthcare System, Buffalo, New York
  • J. N. Misasi
    Ophthalmology, Upstate Medical University, Syracuse, New York
    Infectious Disease, Children's Hospital, Boston, Massachusetts
  • J. M. Sullivan
    Ophthalmology, University at Buffalo- SUNY; VA Western NY Healthcare System, Buffalo, New York
    Ophthalmology, Upstate Medical University, Syracuse, New York
  • Footnotes
    Commercial Relationships  M.C. Butler, None; J.N. Misasi, None; J.M. Sullivan, None.
  • Footnotes
    Support  NIH Grant EY13433;Research to Prevent Blindness Challenge Grants
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3015. doi:
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      M. C. Butler, J. N. Misasi, J. M. Sullivan; Cell SELEX- a Novel in vivo Evolutionary Approach for RNA Drug Discovery. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3015.

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

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Purpose: : Develop a rapid in vivo evolutionary approach to identify highly active post-transcriptional gene silencing (PTGS) agents such as ribozymes.

Methods: : Selective amplification of ligands by exponential enrichment (SELEX) is a powerful, established in vitro evolution method used to isolate target ligands from combinatorial libraries. Selection is based upon relative binding of ligands to a purified target molecule fixed to surfaces. Since the performance of PTGS agents created in vitro does not predict their performance in vivo, additional labor intensive in vivo validation is required. The Cell SELEX strategy extends the SELEX to the intra-cellular milieu overcoming this limitation. First, a combinatorial library of episomal PTGS expression plasmids is stably expressed within a cell population, upon which evolutionary pressure will be applied. Second, selective pressure is achieved by transfecting these cells with an expression vector containing a target cDNA engineered to promote substantial cellular toxicity. Thus, cell viability is dependent upon the PTGS agent(s) to substantially and rapidly suppress target mRNA levels, resulting in a profoundly limited cell population harboring episomal expression plasmids of successful PTGS agents. Following clonal expansion, these episomes are harvested and further amplified in bacteria, completing the first round of Cell SELEX. Further rounds reestablish cell populations containing the episomes from the prior round. Increased stringency is produced using experimental control over PTGS expression level and/or the degree of cellular toxicity.

Results: : Human rod opsin (RHO) is a gene implicated in retinal degeneration. As an essential first step, we have engineered RHO cDNA to have two levels of cytotoxic expression-one under experimental control. Furthermore, two episomal PTGS expression systems containing ribozyme expression libraries were created with high levels of sequence diversity. Amplified nuclear episomes from a library of stably established cell populations have been successfully harvested. We have determined that our method can recover as few as 2 episomes per cell. These recovered plasmids were amplified in bacteria and reestablished in 293E cells. Hence, all three critical aspects of the Cell-SELEX strategy (diversity, selection, and amplification), were verified, allowing formal testing of the integrated platform.

Conclusions: : Cell-SELEX is expected to allow the rapid identification of highly potent PTGS agents for any target mRNA for possible therapeutics.

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

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