September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Functional expression of Bestrophin-1 in vitro following transduction with viral vectors.
Author Affiliations & Notes
  • Shaun Roger Wood
    Nuffield Department of Clinical Neurosciences, The University of Oxford, Didcot, United Kingdom
  • Michelle McClements
    Nuffield Department of Clinical Neurosciences, The University of Oxford, Didcot, United Kingdom
  • Maria Ines Patricio
    Nuffield Department of Clinical Neurosciences, The University of Oxford, Didcot, United Kingdom
  • Robert E MacLaren
    Nuffield Department of Clinical Neurosciences, The University of Oxford, Didcot, United Kingdom
    Oxford University Hospitals NHS Foundation Trust, Oxford Eye Hospital, Oxford, United Kingdom
  • Carolina Uggenti
    The Institute of Human Development, The University of Manchester, Manchester, United Kingdom
  • Sumathi Sekaran
    Nuffield Department of Clinical Neurosciences, The University of Oxford, Didcot, United Kingdom
  • Forbes D Manson
    The Institute of Human Development, The University of Manchester, Manchester, United Kingdom
  • Footnotes
    Commercial Relationships   Shaun Wood, None; Michelle McClements, None; Maria Patricio, None; Robert MacLaren, Nightstarx (C), University of Oxford (E), Wellcome Trust (F); Carolina Uggenti, None; Sumathi Sekaran, None; Forbes Manson, None
  • Footnotes
    Support  Fight for Sight UK DPhil Studentship
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1174. doi:
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      Shaun Roger Wood, Michelle McClements, Maria Ines Patricio, Robert E MacLaren, Carolina Uggenti, Sumathi Sekaran, Forbes D Manson; Functional expression of Bestrophin-1 in vitro following transduction with viral vectors.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1174.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Mutations in the human BEST1 gene are responsible for a range of inherited macular dystrophies known collectively as the “bestrophinopathies”. The protein product of this gene, bestrophin-1, is a 67 kDa protein that localises to the basolateral membrane of the retinal pigment epithelium (RPE) where it is thought to act as a Ca2+-activated chloride ion channel. The purpose of this study was to compare the ability of two recombinant adeno-associated viral (AAV) vectors to express functional bestrophin-1 following transduction in Human Embryonic Kidney (HEK) 293 cells.

Methods : HEK293 cells were transduced with AAV2/2 vectors, expressing BEST1 under the control of a ubiquitous ‘CAG’ promoter, with or without a Woodchuck Post-translational Regulatory Element (WPRE). A construct expressing GFP under the same promoter was used as a control. Bestrophin-1 expression was assessed by western blot and immunocytochemistry. The effect of each vector on chloride ion conductance was measured using whole-cell patch clamp with extracellular chloride at 146 mM and intracellular chloride at 38.4 mM.

Results : Bestrophin-1 was detected in cells transduced with both vectors by western blot and higher expression was obtained from the AAV2/2.CAG.hBEST1.WPRE.pA vector (n=5 in each group, p=0.036). This result was confirmed with immunocytochemistry. Whole-cell patch-clamp showed an increase in chloride ion conductance in the presence of AAV2/2.CAG.hBEST1.pA and AAV2/2.CAG.hBEST1.WPRE.pA when compared to AAV2/2.CAG.GFP.WPRE.pA and non-transduced cells.

Conclusions : Both AAV2/2.CAG.hBEST1.pA and AAV2/2.CAG.hBEST1.WPRE.pA vectors can deliver functional bestrophin-1 to cells in vitro. Both vectors may, therefore, be useful in a gene therapy strategy to treat patients with mutations in BEST1.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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