A Robust Optomotor Assay for Assessing the Efficacy of Optogenetic Tools for Vision Restoration

Abstract

PurposeTo develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies.MethodsA triple-knockout (TKO) mouse line, Gnat1−/−Cnga3−/−Opn4−/−, and a double-knockout mouse line, Gnat1−/−Cnga3−/−, were employed. The expression of channelrhodopsin-2 (ChR2) and its three more light-sensitive mutants, ChR2-L132C, ChR2-L132C/T159C, and ChR2-132C/T159S, in inner retinal neurons was achieved using rAAV2 vectors via intravitreal delivery. Pupillary constriction was assessed by measuring the pupil diameter. The optomotor response (OMR) was examined using a homemade optomotor system equipped with light-emitting diodes as light stimulation.ResultsA robust OMR was restored in the ChR2-mutant-expressing TKO mice; however, significant pupillary constriction was observed only for the ChR2-L132C/T159S mutant. The ability to evoke an OMR was dependent on both the light intensity and grating frequency. The most light-sensitive frequency for the three ChR2 mutants was approximately 0.042 cycles per degree. Among the three ChR2 mutants, ChR2-L132C/T159S was the most light sensitive, followed by ChR2-L132C/T159C and ChR2-L132C. Melanopsin-mediated pupillary constriction resulted in a substantial reduction in the light sensitivity of the ChR2-mediated OMR.ConclusionsThe OMR assay using TKO mice enabled the quantitative assessment of the efficacy of different optogenetic tools and the properties of optogenetically restored vision. Thus, the assay can serve as a valuable tool for developing effective optogenetic therapies.

Optogenetic restoration of retinal ganglion cell activity in the living primate

“…ptogenetic therapies aim to restore light sensitivity in postreceptoral retinal cells of patients with irreversible sight loss caused by photoreceptor degeneration. In vivo preclinical testing is typically performed in mice [1][2][3] , and while these animals provide access to genetic models of retinal disease, they have different immune function and retinal physiology to humans. Only the primate has a human-like fovea, the retinal structure which mediates high acuity central vision and dominates our visual experience.…”

The functional characteristics of optogenetic gene therapy for vision restoration

“…Side by-side comparisons of candidate opsins are scarce and either only provide comparison of very similar opsins or only assess very basic functional parameters [ 9 , 14 , 18 , 20 ]. The methodological strategy utilized here, employing MEAs to record neural responses from the output level of explanted retinae together with a system that allows for delivery of complex patterned light stimuli can serve to compare and benchmark the spectrum of candidate optogenetic constructs.…”

“…These are usually primarily intended to arrest visual loss, however, some degree of functional improvement, presumably by re-activating degenerating photoreceptors, has been reported [3,4]. A novel strategy involves the expression of transgenes encoding photosensitive proteins (translational optogenetics) in surviving retinal cells such as ganglion cells [5][6][7], bipolar cells [8][9][10][11][12] or outer segments degenerate photoreceptors [13], to make them directly light sensitive [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Such an optogenetic gene therapy is particularly attractive as it does not depend on the integrity of the retinal pigment epithelium photoreceptor complex.…”

“…To date, several groups have successfully shown that delivery of different optogenetic constructs into remaining retinal neurons can restore light responsiveness of retina degenerate rodents [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] and these promising results have fostered the initiation of early stage clinical trials (NCT02556736, NCT03326336 at clinicaltrials.gov). Despite these pivotal advances, the amount of information available regarding the extent to which complex image forming vision can be restored at the retinal level, and also what are the likely functional limits remains scarce.…”

Using Looming Visual Stimuli to Evaluate Mouse Vision

“…However, OKR requires surgery to attach a fixation device to the mouse skull 24 . OMR requires neither surgery nor mouse training; however, it requires training to allow an experimenter to subjectively detect subtle mouse head movements in response to a moving grating in an optic drum 25,26 . Pupil light reflex measures pupil constriction in response to light stimuli, which does not require anesthesia and exhibits objective and robust responses 19 .…”