594. Exosome-Associated AAV Enhances Retinal Transduction Following Intravitreal Injection

Introduction Adeno-associated virus (AAV) has been shown to be associated with cell derived exosomes (exo-AAV). Exo-AAV outperforms regular AAV in transduction efficacy and evades neutralizing anti-AAV antibodies. Here we investigate the retinal transduction profile of exo-AAV vectors when administered intravitreally in BL6 adult mice. Methods Exo-AAV vectors were isolated from the culture media of triple-transfected 293T cells (AAV2 rep/cap, GFP transgene and adenovirus helper plasmid) by differential centrifugation. Regular AAV vectors were isolated from the cell lysate by iodixanol density gradient ultracentrifugation. To assess the transduction ability of the regular AAV2 and exo-AAV2 , we performed intravitreal injections into mice (n= 12 eyes, 1×1012 VG/mL), using AAV2 as the control (n=14 eyes, 1×1012 VG/mL). Fundus imaging was performed at 2 and 4 weeks post-injection, at which point the eyes were collected for immunohistological processing. Results We found that the exo-AAV2 showed an early onset of robust GFP expression via fundus imaging at 2 weeks post injection, as compared to regular AAV2. This was further enhanced by larger spread and brightness of GFP expression at 4 weeks. Histological processing of retinal sections shows GFP expression after exo-AAV2 expression in the nerve fiber layer, retinal ganglion cell layer, inner nuclear layer, outer nuclear layer and photoreceptor inner and outer segments. Staining with ganglion cell and bipolar cell markers show co-localization of GFP in these cell types. AAV2 injected animals show GFP expression mostly restricted to the nerve fiber and ganglion cell layers. Conclusion Intravitreal injections are an ideal delivery route to the retina in humans as it is minimally invasive and performed routinely in the clinic. Repeated pre-clinical work by others and us using AAV2 shows that an intravitreal injection is limited to targeting the nerve fiber layer (consequently, optic nerve) and retinal ganglion cells. Here, we demonstrate that exo-AAV2 targets all cell layers of the retina at robust levels and successfully targets retinal bipolar cells. As a result, an intravitreal injection of exo-AAV with a cell specific promoter may be the vector of choice for future gene therapy.