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Shaun Wood, Michelle McClements, Robert E MacLaren; Intracellular localisation of human bestrophin in neuronal cells after transduction with an AAV2.CBA.hBEST1.WPRE vector. Invest. Ophthalmol. Vis. Sci. 2014;55(13):384.
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Mutations in the human BEST1 gene are responsible for a wide-range of retinal disorders broadly classified as Bestrophinopathies. The BEST1 protein is known to be expressed in the retinal pigment epithelium (RPE) where it localises to the membrane and acts as a Cl- channel. Delivery of hBEST1 to the RPE cells of the retina is a potential treatment option for patients with bestrophinopathy. Potential expression of BEST1 in other cell types of the retina however could be problematic, particularly in neurons where a channel protein could influence function or even trigger apoptosis. We therefore sought to identify the membrane localisation patern of hBEST1 when expressed ectopically from a powerful expression cassette used in a current clinical trial - AAV2 CAG.hBEST1.WPRE.pA in HEK293T cells in vitro.
Human BEST1, driven by the ubiquitous CAG promoter and with a downstream WPRE sequence, was packaged into AAV2 capsids and used for in vitro assessment in HEK 293T cells. Commercially available anti-BEST1 antibodies were tested for their ability to detect the human BEST1 protein via western blot. Immunocytochemical (ICC) staining was also performed to determine localisation of BEST1 within the HEK293T cells. Histological sections of wild-type mouse eyes were prepared and immunostained to determine native BEST1 expression throughout the different layers of the retina using the human-specific antibody.
Western blots using transfected and transduced HEK293T lysates detected large amounts of hBEST1 protein at the predicted size of 67 kDa, indicating successful overexpression from the plasmid and AAV2 vectors respectively. ICC on the HEK293T cells however showed BEST1 expression in the cytosol with little localisation at the cell membranes. Staining of the wild type mouse eyes indicated some expression of BEST1 in the RPE confirming broad species specificity of the antibodies.
An AAV2 vector carrying CAG.hBEST1 has been generated and successfully used to express hBEST1 in vitro. Although initial tests suggest that the localisation of the protein would render it non-functional in this cell line, further functional tests will be required to confirm this.
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