May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Microarray Analysis of Retinal Responses to AAV-Vectored Ribozymes Against the Rod PDE in Wild Type Mice
Author Affiliations & Notes
  • J. Liu
    Department of Ophthalmology and Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
  • A.M. Timmers
    Department of Ophthalmology and Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
  • A.S. Lewin
    Department of Ophthalmology and Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
  • W.W. Hauswirth
    Department of Ophthalmology and Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
  • Footnotes
    Commercial Relationships  J. Liu, None; A.M. Timmers, None; A.S. Lewin, None; W.W. Hauswirth, AGTC, Inc. P.
  • Footnotes
    Support  EY07864, EY11123, EY11596, NS3602, FFB, MVRF, RPB. CR: P
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4511. doi:
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      J. Liu, A.M. Timmers, A.S. Lewin, W.W. Hauswirth; Microarray Analysis of Retinal Responses to AAV-Vectored Ribozymes Against the Rod PDE in Wild Type Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4511.

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

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Abstract

Abstract: : Purpose: To create an animal model of retinal degeneration by applying ribozyme-mediated in vivo somatic knockdown of the γ-subunit of the rod cGMP phosphodiesterase (PDEγ) in wild type mice and to profile the resulting expression response globally using microarray analysis. Methods: Four hammerhead ribozymes with or without CTEs (constitutive transport elements) were designed to target the PDEγ gene in C57BL/6 mice and were packaged into serotype 2 AAV vectors containing a proximal 472bp murine rod opsin promoter. For subretinally injected and partner control retinas, retinal responses to light were measured by electroretinograms (ERG) at 4 or 6 weeks post-treatment. To identify sets of retinal genes that are up- or down-regulated in response to ribozyme treatment, retinal RNA from treated and control eyes were analyzed by Affymetrics mouse genome microarrays. GeneChips algorithms were applied to the data. Results: Hammerhead ribozymes with the CTE suppressed target gene efficiently. After subretinal injection of the AAV-ribozyme vector, ERG amplitudes were 30~90% lower than in partner control retinas 4~6 weeks post-injection. Microarray analysis showed that of the approximately 12,000 genes scored, 14 were significantly up-regulated and 7 were significantly down-regulated. Hierarchical cluster analysis showed that relative to control, wild type retinas, retinal gene changes in ribozyme-treated eyes grouped with rd mouse retinal gene changes and were from the control retina group. Gene expression profiling confirms that somatic down-regulation of the PDEγ gene by AAV-ribozymes in wild type mice creates an retinal expression pattern mimicking that of the rd retina, i.e. parallel patterns of genes encoding proteins that interact with or are part of phototransduction. Relative quantitative RT-PCR will be used to confirm these results. Conclusions: Ribozyme-mediated in vivo somatic knockdown of the wild type PDEγ gene can efficiently create an animal model of retinal degeneration resembling human RP. Microarray analysis of such ribozyme-treated retinas may be a useful tool for identifying co-regulated gene expression changes related to retinal degenerations.

Keywords: gene microarray • animal model • retinal degenerations: hereditary 
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