Evaluation of AAV-based optogene transfer for the treatment of canine models of inherited retinal dystrophies

Abstract

Purpose : Recombinant Adeno-Associated Virus (AAV)-mediated optogene (coding for light-sensitive proteins) transfer into retinal ganglion cells has emerged as a promising strategy to restore photosensitivity in the context of inherited retinal dystrophies (IRD). Optogene transfer in rodent models of IRD demonstrated restoration of visual functions. Assessment of Opn4 or ChR2 optogene transfer in large animal models of IRD is a key step prior evaluation in clinical trials. In this study, we have tested optogenes expression and functionality in vitro. We have also evaluated the restoration of retinal functions after optogene transfer in the retinal ganglion cells of blind Rpe65-deficient dogs.

Methods : We produced plasmid vectors containing either the ChR2 or the human Opn4 sequence under the control of the CMV or the hSyn promoter. Optogenes expression and functionality were validated in vitro by immunocytochemistry and calcium sensor analysis. Moreover, we generated rAAV2/2 vectors carrying the ChR2 or hOpn4 cDNA under the control of the hSyn promoter. We performed an intravitreal injection of vectors in Rpe65^-/- dogs with advanced progression of the disease. Immunostaining of ChR2 injected retinas were performed to confirm the transduction of the canine retinal ganglion cells. We assessed the restoration of the retinal function by electroretinogram (ERG) based on standard and adapted procedures for optogene-based retinal ganglion cells responses, until 1 year post injection.

Results : The plasmid constructs containing each optogenes cDNA were able to drive expression of proteins in vitro resulting in an increase of intracellular calcium after light stimulation in HEK293 cells. We demonstrated wide spread transduction of injected retinas with expression of optogenes, as shown by immunostaining of the ChR2 protein on transduced flat mount retina. Nevertheless, we did not detect ERG responses after optogene transfer in a blind canine retina.

Conclusions : Although, the expression cassettes of optogenes have been validated in vitro and the surgical approach led to a large transduction of the retinal ganglion cell, we never detected ERG signal after optogene transfer in Rpe65^-/- retinas. Alternative approaches, such as Micro Electrode Array or Visual Evoked Potential experimentations, are still under investigation to evaluate the restoration of visual functions mediated by the optogenes.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.