In Vitro Adenovirus–Mediated Delivery of the Large ABCA4 Transgene Is Enhanced in a Simulated Ocular Environment

Abstract

Abstract: : Purpose: Mutations in the ABCA4 gene have been associated with Stargardt disease and adult–onset macular degeneration. The ABCA4 protein is expressed exclusively in photoreceptor outer segments. The first–generation chimeric adenoviral vector AdV5/F35 can efficiently deliver reporter transgenes to the retina including photoreceptors in vivo. The objective of this study is to evaluate the feasibility of using the AdV5/F35 vector to express the large ABCA4 protein in a simulated ocular environment. Methods: As an initial step towards gene therapy for diseases caused by ABCA4 mutations, adenoviral vectors containing an ABCA4 transgene were constructed and characterized. HEK293 cells and WERI retinoblastoma cells were transduced with a first–generation chimeric adenoviral vector AdV5/F35 containing a transgene encoding ABCA4 (AdV5/F35–ABCA4) under the control of a CMV promoter. To examine the in vitro transduction efficiency in a simulated ocular environment, expression of ABCA4 in the presence or absence of vitreous (5%) or hyaluronic acid (0.5 mg/ml) was analyzed by Western blot analysis using anti–ABCA4 monoclonal antibody Rim3F4 (R. Molday). Results: AdV5/F35 can efficiently deliver the ABCA4 gene to either HEK293 cells or WERI retinoblastoma cells. Quantitative analysis of membrane proteins isolated from transduced cells indicates that ABCA4 protein expressed in the presence of vitreous or hyaluronic acid was approximately 6–fold greater than in the absence of vitreous or hyaluronic acid. The amount of ABCA4 protein expressed in these cell lines was at least as great as that expressed on native photoreceptor outer segments. Conclusions:These results demonstrate that the AdV5/F35–ABCA4 vector is a suitable candidate for testing gene therapy in a murine abcr(–/–) knock–out model of rod/cone dysplasia.

Keywords: gene transfer/gene therapy • adenovirus • retinal degenerations: hereditary