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Matthew Hartzell, Maria Parker, Andrew Stempel, Trevor McFarland, Binoy Appukuttan, John T. Stout; Evaluation Of AAV-DJ Vector As A Therapeutic Delivery System For Ocular Cells And Tissue. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1895.
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Gene therapy for the treatment of Leber Congenital Amerosis (LCA) using an AAV vector is currently under a Phase I trial. The development of adeno-associated viral (AAV) vectors that can deliver to multiple ocular cell types with high efficiency via subretinal or intravitreal delivery methods is essential for effective therapy. The AAV-DJ (type2/type8/type9 chimera) was engineered from shuffling 8 different wild type native viruses (Kay et al 2008). The goal of this study is to investigate whether the chimeric AAV-DJ-GFP vector is capable of transducing a variety of ocular cell types in vitro and in vivo.
The chimeric AAV- DJ vector serotype (AAV-DJ) encoding enhanced green fluorescent protein (GFP) was produced (Vollum Viral Core). One microliter of virus with a titer of 4x10e12 GC/ml was added to the following cell lines: HEK 293T, mouse Muller glia (C57MIO), human Muller glia (MIO-M7), primary human and primary pig trabecular meshwork, human retinal pigment epithelial (ARPE 19), rhesus retinal/choroidal endothelial (RF/6A), human primary iris endothelial /fibroblast, retinoblastoma cells (Y79), human choroidal endothelial and ocular melanoma (Mel 202). Cells were cultured and GFP expression was measured by fluorescent microscopy at 18 and 42 hours.1.5ul of AAV-DJ was injected intravitreally in each eye of a C57BL/6 mouse. Mice were euthanized on post-injection day 5. Retinal flat mounts were prepared. GFP -positive cells were viewed using fluorescent microscopy.
With the exception of Y79 retinoblastoma cells, all cell types cultured were transduced with high efficiency and GFP expression was observed at all time points. Multiple GFP positive cells were observed within the neural retina after intravitreal injection.
The 2-8-9 chimera AAV-DJ vector can transduce multiple ocular derived cells in culture. The AAV-DJ vector has the potential to be useful for ocular cell studies as well as gene therapy experiments.
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