April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Optimization Of Promoter Sequences In Order To Mimic Physiological Pde6b Expression
Author Affiliations & Notes
  • JongEun Ihm
    Unit of gene Therapy & Stem Cell Biology, Jule-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland
  • Alberto Auricchio
    Telethon Institute of Genetics and Medicine, University of Napoli, Napoli, Italy
  • Yvan Arsenijevic
    Unit of gene Therapy & Stem Cell Biology, Jule-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland
  • Footnotes
    Commercial Relationships  JongEun Ihm, None; Alberto Auricchio, None; Yvan Arsenijevic, None
  • Footnotes
    Support  FP7 AAVEYE 223445
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 23. doi:
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      JongEun Ihm, Alberto Auricchio, Yvan Arsenijevic; Optimization Of Promoter Sequences In Order To Mimic Physiological Pde6b Expression. Invest. Ophthalmol. Vis. Sci. 2011;52(14):23.

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

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Abstract

Purpose: : Many retinal degenerations result from defective retina-specific gene expressions. Thus, it is important to understand how the expression of a photoreceptor-specific gene is regulated in vivo in order to achieve successful gene therapy. The present study aims to design an AAV2/8 vector that can regulate the transcript level in a physiological manner to replace missing PDE6b in Rd1 and Rd10 mice. In previous studies (Ogieta, et al., 2000), the short 5’ flanking sequence of the human PDE6b gene (350 bp) was shown to be photoreceptor-specific in transgenic mice. However, the efficiency and specificity of the 5’ flanking region of the human PDE6b was not investigated in the context of gene therapy during retinal degeneration. In this study, two different sequences of the 5’ flanking region of the human PDE6b gene were studied as promoter elements and their expression will be tested in wild type and diseased retinas (Rd 10 mice).

Methods: : Two 5’ flanking fragments of the human PDE6b gene: (-93 to +53 (150 bp) and -297 to +53 (350 bp)) were cloned in different plasmids in order to check their expression in vitro and in vivo by constructing an AAV2/8 vector. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP. jetPEI transfection in Y 79 cells was used to evaluate gene expression through luciferase activity. Constructs encoding EGFP under the control of the two promoters were performed in AAV2.1-93 (or 297)-EGFP plasmids to produce AAV2/8 vectors.

Results: : When pGL3-93 (150 bp) or pGL3-297 (350 bp) were transfected in the Y-79 cells, the smaller fragment (150 bp) showed higher gene expression compared to the 350 bp element and to the SV40 control, as previously reported. The 350 bp drove similar levels of expression when compared to the SV40 promoter. In view of these results, the fragments (150 bp or 350 bp) were integrated into the AAV2.1-EGFP plasmid to produce AAV2/8 vector, and we are currently evaluating the efficiency and specificity of the produced constructs in vivo in normal and diseased retinas.

Conclusions: : Comparisons of these vectors with vectors bearing ubiquitous promoters should reveal which construct is the most suitable to drive efficient and specific gene expression in diseased retinas in order to restore a normal function on the long term.

Keywords: retinal degenerations: cell biology • adenovirus • gene/expression 
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