Melanopsin: targeted ectopic expression for optogenetic visual restoration

Abstract

Purpose : Ectopic expression of the light sensitive opsin protein Melanopsin renders neural cells sensitive to light and is emerging as a prime candidate in optogenetic approaches to vision restoration in the inherited retinal degenerations (IRDs).However, it is unclear if targeting specific neuronal populations within the retina is advantageous over non-specific delivery. In mouse models of IRD, a genuine comparison is confounded by the persistence of native intrinsically photosensitive retinal ganglion cell (ipRGC) responses. Here we describe a novel mouse model lacking all native retinal light responses (both canonical and ipRGC) allowing us to test the hypothesis that targeting expression of melanopsin to ON-bipolar cells is advantageous over a non-specific delivery.

Methods : Retina-degenerate mice lacking native melanopsin and expressing Cre recombinase in retinal ON-bipolar cells (L7.Cre,Opn4^-/-,Pde6b^rd1/rd1) of both sexes were used. At P45, intravitreal injections of adeno-associated virus containing the humanMelanopsin gene (OPN4) driven by either a “floxed” Ef1a (n=8) or a “non-floxed” Cba (n=8) promotor were administered alongside a saline (n=8) control group. Eight weeks later, behavioural visual assays were performed (pupillometery, optokinetic drum and novel object-visual-context tasks). Ex-vivo multiple electrode array recordings of retinal light responses were also undertaken. Comparison of means was performed by t-test where samples were normally distributed.

Results : No significant difference was seen in behavioural responses, sensitivity of electrophysiological responses nor onset kinetics between the two treated groups. However, expression in bipolar cells specifically led to significantly shorter duration (mean±SEM 38.82±2.68s n=83 vs. 60.56±4.28s n=54; p<0.0001) and faster recovery(t_1/2 5.01±0.65s n=84 s vs. 11.65±1.37s n=55s; p<0.00001) of electrophysiological responses.

Conclusions : This represents the first report in the literature of restoration of light responses in a retina devoid of all native photoreception. While there was no apparent advantage in sensitivity between targeted and non-specific delivery, when Melanopsin was specifically expressed in bipolar cells offset kinetics of electrophysiological responses were faster. These results demonstrate thatbipolar cells may represent favourabletargets for clinical optogenetic gene using melanopsin.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.