AAV-mediated efficient gene transfer in retinal ganglion cells of dogs and non-human primates

Abstract

Purpose: In the context of retinal diseases, AAV vectors carrying optogenes (light-sensitive protein) into retinal ganglion cells (RGCs) have emerged as a promising strategy to restore photosensitivity in degenerated retinas. To promote the translation of AAV-mediated optogenes transfer in RGCs of blind patients, dogs and non-human primates (NHPs) are clinically relevant models. Indeed, the eyes of both animal models share great anatomical homologies with the human eye. Furthermore, the surgical procedure and the amount of vector delivered are similar for the future human retinal clinical trials. Therefore, the present study was designed in the effort to optimize and characterize AAV transduction in RGCs of dog and NHP retinas following intravitreal injection.

Methods: We generated AAV2/2 vectors carrying the enhanced green fluorescence protein (eGFP) cDNA under the control of the cytomegalovirus promoter. We performed intravitreal injection of the vector (500µl, 10¹²vg/ml) in the dog and NHP eyes. Native eGFP expression was monitored by funduscopic imaging. Using confocal microscopy, eGFP expressing cells were analyzed in retinal flat mounts and sections. Finally, to label RGCs, immunocytochemistry analyzes were applied using neurochemical markers: NeuN and TUJ1.

Results: In dogs, funduscopic imaging displayed a strong native eGFP expression across the retina. In NHPs, a gradient of eGFP expression was observed, with the highest in the peripheral retina and the lowest in the central retina, except for the annulus transduction around the fovea. In both animal models, native eGFP expression was localized along the axons and in the optic nerve head which indicates that RGCs were transduced by AAV2/2. Using confocal microscopy, we observed in retinal flat mounts of dogs and NHPs, that eGFP expression was localized in numerous cells bodies of the ganglion cells layer. Our analysis also demonstrated that native eGFP-expressing RGCs specifically co-localized with NeuN and TUJ1.

Conclusions: Here, we describe that AAV2/2 is able to mediate a strong and efficient transduction of RGCs in dog and NHP retinas following intravitreal injection. These findings in both animal models, especially in dog, are primordial steps to set the stage to AAV-mediated optogenes transfer in RGCs of blind patients because a variety of human genetic diseases have their equivalent in the canine population.