May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Development of HSV-Vectored Ribozymes for the Treatment of HSV Keratitis
Author Affiliations & Notes
  • J. Liu
    Molecular Genetics, University Florida, Gainesville, FL, United States
  • D.C. Bloom
    Molecular Genetics, University Florida, Gainesville, FL, United States
  • G.S. Schultz
    Ob/Gyn, University Florida, Gainesville, FL, United States
  • S.S. Tuli
    Ophthalmology, University Florida, Gainesville, FL, United States
  • A.S. Lewin
    Ophthalmology, University Florida, Gainesville, FL, United States
  • Footnotes
    Commercial Relationships  J. Liu, None; D.C. Bloom, None; G.S. Schultz, None; S.S. Tuli, None; A.S. Lewin, None.
  • Footnotes
    Support  AI48633, EY11596, EY05587
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4627. doi:
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      J. Liu, D.C. Bloom, G.S. Schultz, S.S. Tuli, A.S. Lewin; Development of HSV-Vectored Ribozymes for the Treatment of HSV Keratitis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4627.

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

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Abstract

Abstract: : Purpose: Herpes Simplex type I (HSV-1) is a neurotropic virus that causes HSV keratitis, which is one of the most frequent infectious causes of corneal blindness. Currently, prophylactic oral antiviral medications are effective in preventing recurrent infection in less than half of the patients. Our goal is to develop a gene therapy treatment for HSV keratitis using HSV-vectored ribozymes that target mRNAs of several HSV-1 genes that are essential for viral replication. Methods: Synthetic hammerhead ribozymes targeting four essential HSV genes (ICP4, ICP27, UL20, and UL30) were synthesized and the efficiency of their enzymatic cleavage of synthetic target RNAs were measured in vitro. The ribozyme targeting the ICP4 gene was cloned into an expression plasmid that was transfected into a cell line that stably expresses ICP4 gene, and the level of ICP4 mRNA in the transfected cells was assessed by RT-PCR. Results: The Kcat/Km values for the UL20 and ICP27 ribozymes were 15.9 and 11.7 µM-1 min-1 respectively, using 5 mM Mg2+ concentration. The Kcat/Km of the UL30 and ICP4 ribozymes were 2.7 and 0.3 µM-1 min-1, respectively, using 20 mM Mg2+ concentration. The ribozyme targeting ICP4 gene reduced ICP4 transcripts in transfected cells at least 50% after 48 hours using RT-PCR. Conclusions: The synthetic hammerhead ribozymes efficiently cleaved synthetic target RNAs in vitro for four HSV genes that are essential for viral replication. Also, the ribozyme targeted to the ICP4 gene dramatically reduces the level of ICP4 transcripts in transfected cells. These data suggest that HSV-vectored ribozymes targeting essential HSV-1 genes may be a new treatment strategy for reducing or preventing recurrent HSV keratitis.

Keywords: gene transfer/gene therapy • keratitis • herpes simplex virus 
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