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