Controlled and Site–selective Gene Delivery Into Mouse Keratocytes by Adeno–associated Viral, Lentiviral and Plasmid Vectors

Abstract

Abstract: : Purpose:Controlled and site–selective gene delivery techniques provide an alternative approach to study gene’s functions and pathological conditions in the cornea and prevent undesired side effects of surgery. The goals of this study were to: i) identify optimal viral and non–viral vectors for gene delivery in keratocyte; ii) determine changes in the quantity and/or location of transgene delivery mediated by vector–application methods; and iii) characterize the first point in time and duration of expression, and localization of transgene with different vectors in cornea. Methods: The efficacy of three vectors, adeno–associated virus serotype 5 (AAV5), lentiviral and plasmid, and two vector–delivery techniques, were investigated. Two microliters of a) AAV5–EGFP (9.6x10¹⁰ particles/µl), b) Lentiviral–EGFP (1.2x10¹⁰ particles/µl), c) pTR–ß–gal plasmid (50µg DNA mixed with 200nM of DDAB and DOPE) or d) BSS were injected into central stroma using a Hamilton microinjection system or applied topically on the exposed and dried stromal surface after removal of epithelium in the mouse cornea. Virus–treated eyes were collected at 2d, 3d, 1w, 2w, 4w, and 6w, and plasmid–treated eyes were collected at 2hrs, 4hrs, 24hrs, 36hrs, 3d and 7d. Transgene expression was analyzed with immunostaining, ELISA assay, and Confocal microscopy. Velocity software was used for generating 3–D reconstructions. Results: All three vectors delivered reporter genes selectively into keratocytes. Viral vectors delivered with higher efficiency and for longer duration compared to the plasmid vector. In virus–treated tissues, reporter gene expression was first detected at day 3. However, in plasmid treated tissues expression was first detected at 2 hrs. AAV– or lentivirus–exposed tissues expressed transgene up to 6 weeks (last tested time point), whereas no expression in plasmid–treated tissues was observed after 3 days. Vector–delivery techniques also influenced efficiency and the area of gene delivery. Topical application delivered reporter genes to the stromal surface exposed to the vector, in contrast to microinjection where transgene delivery was localized to the site of injection. Conclusions: AAV5, lentiviral, and pTR–plasmid vectors can be used for selective gene delivery into mouse keratocytes and may be useful for gene therapy in humans. Appropriate vectors and delivery techniques can be selected to achieve desired levels and duration of gene expression.

Keywords: gene transfer/gene therapy • cornea: stroma and keratocytes • wound healing