Adeno-associated virus serotype-9 efficiently transduces the retinal outer plexiform layer

PURPOSE: Adeno-associated virus serotype-9 (AAV-9) is a promising gene delivery vector. In this study, we evaluated AAV-9 transduction in the mouse retina. METHODS: Three different AAV vectors were used in our study: AAV-9.RSV.AP, AAV-9.CMV.eGFP, and AAV-9.CMV.∆R4–23/∆C. In these vectors, two different promoters (the cytomegalovirus promoter-CMV promoter and the Rous sarcoma virus-RSV promoter) were used to express three different transgenes including the alkaline phosphatase (AP) gene, the enhanced green fluorescent protein (eGFP) gene, and a therapeutic microdystrophin gene (the ∆R4–23/∆C). Specifically, 1 µl AAV-9 reporter gene vectors (1×10(9) viral genome particles of AAV-9.RSV.AP or 1×10(10) viral genome particles of AAV-9.CMV.eGFP) were administered subretinally to young (2–3-week-old), adult (3-month-old), and old (12-month-old) C57BL/6J mice. To evaluate AAV-9 transduction in a diseased retina, we injected subretinally 1×10(9) viral genome particles of AAV-9.CMV.∆R4–23/∆C to mdx(3cv) mice, which we used as a model for Duchenne muscular dystrophy (DMD). Transgene expression was examined by histochemical as well as immunofluorescence staining at three and five weeks after injection. Electroretinograms were recorded five weeks after subretinal AAV-9.RSV.AP injection. RESULTS: Subretinal injection yielded widespread transduction throughout the retina in all age groups. Robust expression was seen in the retinal pigment epithelium, outer nuclear layer, and in Müller cells. Interestingly a synaptic layer, the outer plexiform layer (OPL), also showed intensive expression. Transduction of the synaptic layer was further confirmed by immunostaining for C-terminal binding protein 2 (CtBP2), a marker for the photoreceptor synaptic ribbon. Dystrophin is normally expressed in the OPL photoreceptor terminals. This expression is lost in DMD patients and mdx(3cv) mice. Consistent with our findings in normal mice, we observed efficient microdystrophin expression in the OPL after AAV-9.CMV.∆R4–23/∆C infection. At five weeks after subretinal delivery of AAV-9.RSV.AP, no morphology or ERG abnormalities were observed. CONCLUSIONS: We demonstrated that AAV-9 is a potent vector for retinal gene delivery. Furthermore, subretinal AAV-9 administration did not cause appreciable acute retinal damages. In summary, AAV-9-mediated OPL transduction holds promise for treating diseases that primarily affect this layer.