June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Evaluation of the efficiency and specificity of short variants of the human PDE6b promoter in photoreceptor-like cells in vitro and ex vivo.
Author Affiliations & Notes
  • Caroline F Peddle
    NDCN, University of Oxford, Oxford, United Kingdom
  • Harry O Orlans
    NDCN, University of Oxford, Oxford, United Kingdom
    Oxford Eye Hospital, Oxford, United Kingdom
  • Michelle E. McClements
    NDCN, University of Oxford, Oxford, United Kingdom
  • Robert E MacLaren
    NDCN, University of Oxford, Oxford, United Kingdom
    Oxford Eye Hospital, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships   Caroline Peddle, None; Harry Orlans, None; Michelle McClements, None; Robert MacLaren, None
  • Footnotes
    Support  Mabel Churn Scholarship at St Cross College, Oxford University. Oxford NIHR Biomedical Research Centre. Royal College of Surgeons of Edinburgh
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 629. doi:
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      Caroline F Peddle, Harry O Orlans, Michelle E. McClements, Robert E MacLaren; Evaluation of the efficiency and specificity of short variants of the human PDE6b promoter in photoreceptor-like cells in vitro and ex vivo.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):629.

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

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Abstract

Purpose : Retinitis pigmentosa (RP) describes a group of genetic diseases characterised by progressive loss of rod photoreceptor cells with accompanying visual impairment. The identification of a short rod-specific promoter has application in RP research as many pre-clinical treatment strategies use adeno-associated viral (AAV) vectors which have a carrying capacity limited to around 4.8kb DNA. The PDE6B promoter has been shown to be rod cell specific and active in both human and mouse tissue. A short 146 bp region of PDE6B promoter has been identified which drives expression of a reporter gene in both Y-79 (human retinoblastoma) cells and Xenopus embryo heads. This study aims to evaluate the efficacy and specificity of two short regions of the PDE6B promoter through a direct comparison with both rod-specific and ubiquitous promoters in three cell types.

Methods : A 216 bp (-163 to +53) and a 158 bp (-108 to +53) region of the PDE6B promoter were amplified from human DNA and cloned into separate plasmids to drive expression of DsRed. Both plasmids were individually transfected into two human cell lines, retinoblastoma-derived WERI-Rb-1 cells and human embryonic kidney 293 cells, as well as dissociated retinal cells from Nrl-GFP mice (which express GFP in rod photoreceptors). DsRed expression (and GFP expression in the Nrl-GFP cells) was monitored over two weeks using fluorescence microscopy. RNA was extracted from all cell lines and DsRed expression was quantified using reverse transcription quantitative PCR. For all three cell types, DsRed expression under the PDE6B promoters was compared to that driven by the rod-cell specific human rhodopsin promoter (RHO), the ubiquitous CAG promoter, or no promoter, as well as that measured from untransfected samples.

Results : Both short variants of the PDE6B promoter were successfully transfected into all three cell types and resulted in DsRed expression both in vitro and ex vivo. In all cases, cells transfected with the CAG.DsRed plasmid demonstrated red fluorescence. Both rod-specific promoters showed reduced activity in the non-retinal HEK293 cells.

Conclusions : The PDE6B promoter is capable of driving expression in photoreceptor-like cells when utilising either a 158 bp or 216 bp fragment. These sequences therefore represent attractive potential candidates as promoters for use in AAV constructs for targeting rod cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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