May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Testing RPGR Specific Ribozyme and siRNA in vitro: Tools for the Treatment of Dominant X-Linked Retinitis Pigmentosa
Author Affiliations & Notes
  • B. Chen
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida
    Center for Vision Research, Gainesville, Florida
  • M. B. O’Donoghue
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida
  • A. S. Lewin
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida
    Center for Vision Research, Gainesville, Florida
  • M. S. Gorbatyuk
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida
    Center for Vision Research, Gainesville, Florida
  • Footnotes
    Commercial Relationships  B. Chen, None; M.B. O’Donoghue, None; A.S. Lewin, None; M.S. Gorbatyuk, None.
  • Footnotes
    Support  NIH EY018335
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5345. doi:https://doi.org/
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    • Get Citation

      B. Chen, M. B. O’Donoghue, A. S. Lewin, M. S. Gorbatyuk; Testing RPGR Specific Ribozyme and siRNA in vitro: Tools for the Treatment of Dominant X-Linked Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5345. doi: https://doi.org/.

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

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Abstract

Purpose: : X-linked retinitis pigmentosa (XLRP) caused by RPGR ORF15 mutations cause severe, early onset retinal disease, and these mutations may also affect female carriers leading to adult onset RP. Such a dominant effect might be reduced by suppressing the expression of RPGR-ORF15. A canine model (XLPRA2) exists for dominant XLRP caused by an ORF15 frameshift. Therefore, we constructed a new RPGR plasmid based on the dog RPGR sequence and tested the knockdown effect of a ribozyme and a siRNA in HEK293 cells.

Methods: : We reconstructed the new RPGR plasmid pBL-sAP-RPGR from psiTEST vector (Invivogen) and the first 11 exons of the dog RPGR gene. We constructed plasmids containing a hammerhead ribozyme (Rz1273) expressed from the chicken β-actin promoter or a siRNA expressed as a small hairpin RNA from the H1 promoter. The ribozyme and the siRNA were designed to cleave at the same location in RPGR. These were co-transfected with pBL-sAP-RPGR in HEK293 cells and secreted alkaline phosphatase activity was measured in the culture supernatant in quadruplicate in order to estimate the knockdown effect.

Results: : The RPGR plasmid could effectively transfect HEK293 cells and lead to the secretion of alkaline phosphatase. HEK293 cells co-transfected with the sAP-RPGR plasmid and the ribozyme or siRNA at a ratio 1:5 showed a reduction of the RPGR expression by 89% and 57%, respectively.

Conclusions: : The RPGR plasmid (pBL-sAP-RPGR) can provide a rapid, simple, and convenient method to screen for functional siRNA and ribozyme sequences in XLRP. Knockdown mutant RPGR gene transcripts may be a good gene therapy for dominant XLRP.

Keywords: gene transfer/gene therapy • gene/expression • photoreceptors 
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