March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Gene Transfer of Self Complimentary Adeno-associated Virus into Rabbit Corneas After Photorefractive Keratectomy
Author Affiliations & Notes
  • Paulette M. Robinson
    OB-GYN,
    University of Florida, Gainesville, Florida
  • Sriniwas Sriram
    OB-GYN,
    University of Florida, Gainesville, Florida
  • William Hauswirth
    Molecular Genetics and Microbiology,
    University of Florida, Gainesville, Florida
  • Alfred S. Lewin
    Molecular Genetics & Microbio,
    University of Florida, Gainesville, Florida
  • Gregory S. Schultz
    Dept of OBGYN and Ophthalmology,
    University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  Paulette M. Robinson, None; Sriniwas Sriram, None; William Hauswirth, None; Alfred S. Lewin, None; Gregory S. Schultz, None
  • Footnotes
    Support  NIH Grant EY05587 and NIH Grant EY08571
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1914. doi:https://doi.org/
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      Paulette M. Robinson, Sriniwas Sriram, William Hauswirth, Alfred S. Lewin, Gregory S. Schultz; Gene Transfer of Self Complimentary Adeno-associated Virus into Rabbit Corneas After Photorefractive Keratectomy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1914. doi: https://doi.org/.

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

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Abstract

Purpose: : Self complementary (double stranded) adeno-associated viruses (scAAVs) have been shown to have a faster onset of gene expression because the scAAV DNA is transcribed rapidly. In addition, scAAV generally have higher transduction efficiency than conventional rAAV vectors. In this study, we investigated scAAV-mediated gene transfer in rabbit cornea after photorefractive keratectomy (PRK). Our goal was to characterize the time of first detectable expression, determine the duration of transgene expression, and identify what cell types were transfected.

Methods: : Self complementary AAV expressing green fluorescent protein (scAAV-GFP) was applied following PRK for two minutes. Corneas were removed at 0, 1, 2, 3, 4 , 7, 30 and 180 days after vector application and fixed in 2% formaldehyde then cryosectioned. Slides were analyzed for direct fluorescence using confocal microscopy. Images were analyzed using Optimas Imaging software and were analyzed for statistical significance by student t-test using GraphPad prism.

Results: : The GFP fluorescence was first detected 24 hours after application and the peak fluorescence detected occurred at 7 days. Day 7 fluorescence was 22 times greater than day 0. Levels of the fluorescence from day 180 were statistically the same as day 0 levels of fluorescence. The transgene was expressed in all cell types of the cornea; epithelium, keratocytes and endothelium.

Conclusions: : These data indicate that topically applied scAAV vectors rapidly express transgenes in all cell types in the cornea. Transduction of corneal cells with scAAV vector expressing ribozymes or shRNA targeting profibrotic genes may provide an optimal therapy to reduce scar formation in the cornea.

Keywords: gene transfer/gene therapy • cornea: basic science • microscopy: light/fluorescence/immunohistochemistry 
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