May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Hammerhead Ribozyme Mediated Knockdown of TGF-β1 in Cell Culture Using an scAAV Vector
Author Affiliations & Notes
  • P. M. Kuznia
    University of Florida, Gainesville, Florida
    OB/GYN,
  • L. Glickman
    University of Florida, Gainesville, Florida
    OB/GYN,
  • R. Yuan
    University of Florida, Gainesville, Florida
    OB/GYN,
  • T. D. Blalock
    University of Florida, Gainesville, Florida
    OB/GYN,
  • W. W. Hauswirth
    University of Florida, Gainesville, Florida
    Molecular Genetics and Microbiology,
  • A. S. Lewin
    University of Florida, Gainesville, Florida
    Molecular Genetics and Microbiology,
  • G. S. Schultz
    University of Florida, Gainesville, Florida
    OB/GYN,
  • Footnotes
    Commercial Relationships  P.M. Kuznia, None; L. Glickman, None; R. Yuan, None; T.D. Blalock, None; W.W. Hauswirth, None; A.S. Lewin, None; G.S. Schultz, None.
  • Footnotes
    Support  NEI Grant EY05587
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3385. doi:
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      P. M. Kuznia, L. Glickman, R. Yuan, T. D. Blalock, W. W. Hauswirth, A. S. Lewin, G. S. Schultz; Hammerhead Ribozyme Mediated Knockdown of TGF-β1 in Cell Culture Using an scAAV Vector. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3385.

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

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Abstract

Purpose: : Corneal trauma, surgery or infection initiate a complex cascade of growth factors, cytokines, chemokines and proteases that may result in stromal scarring that impairs vision. Transforming growth factor- beta 1 (TGF- β1), a secreted growth factor, has been implicated in multiple roles in the corneal scaring pathway. Currently, there is no clinically available agent to reduce the activity of TGF- β1. We investigated whether a hammerhead ribozyme that targets TGF-β1 mRNA.can reduce the amount of TGF- β1 protein synthesized in tissue culture cells.

Methods: : A ribozyme cleavage site in human TGF- β1 mRNA was identified, and the coding sequence for a 33 nucleotide hammerhead ribozyme was chemically constructed. The ribozyme gene was cloned into a self complementary Adeno associated virus (scAAV) expression plasmid and transfected into HEK293 cells. An inactive hammerhead ribozyme cloned into the same scAAV plasmid was used as a negative control. The effects of the ribozyme on expression of TGF-β1 protein levels in media were measured using an ELISA specific for TGF-β1.

Results: : Stable transfection of the TGF- β1 ribozyme into HEK293 cells showed a statistically significant (p<0.05) reduction of 77% of TGF- β1 protein in the media as compared to control cell media. The TGF- β1 ribozyme knocked down 46% more protein in the media than the inactive ribozyme. The difference between TGF-β1 levels in media from untransfected cells and cells transfected with inactive ribozyme plasmid was not statistically significant.

Keywords: wound healing • growth factors/growth factor receptors • gene transfer/gene therapy 
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