Volume 19, Issue 7, Pages 1212-1219 (July 2011) LiGluR Restores Visual Responses in Rodent Models of Inherited Blindness  Natalia Caporale, Kathleen D Kolstad, Trevor Lee, Ivan Tochitsky, Deniz Dalkara, Dirk Trauner, Richard Kramer, Yang Dan, Ehud Y Isacoff, John G Flannery  Molecular Therapy  Volume 19, Issue 7, Pages 1212-1219 (July 2011) DOI: 10.1038/mt.2011.103 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Light-gated glutamate receptor (LiGluR) expression is selectively targeted to retinal ganglion cells in rd1 mouse retinas. (a) The LiGluR opens and closes upon the reversible photoisomerization of the tethered agonist maleimide-azobenzene-glutamate (MAG) between its trans (500 nm) and cis configuration (380 nm). The maleimide moiety of MAG binds covalently to the cysteine on the ligand-binding domain of the receptor and photoswitching occurs through reversible binding of the glutamate moiety of MAG, in and out of the ligand-binding pocket. MAG binding activates the receptor allowing cations to flow, resulting in membrane depolarization. TMD, transmembrane domain; C, C- terminus; N, N-terminus; glu, glutamate. (b) AAV-2 construct with the human Synapsin promoter (hSyn) driving iGLuR6 cDNA with a L439C mutation for MAG conjugation. A WPRE (woodchuck hepatitis post-transcriptional regulatory element) follows the LiGluR coding region for enhanced expression. (c–e, g–h) fluorescence images of rd1 mouse retinas 12 weeks post AAV2-hSyn-iGluR injection, stained with antibodies to iGluR6 (in green) and counterstained with NeuN antibodies (in red) in the cross-sections. Retinal ganglion cells (RGCs) were strongly labelled in both the rd1 (c–e) and triple knockout mice (TKO) retinas, as observed in top (c–e) and cross-sectional (g–h) views. Strong LiGluR expression was observed around the optic nerve head where RGCs cell bodies are dense (ONH, optic nerve head) (c), extending to the periphery (d) with clear membrane labeling of most RGCs (e). Cross-sections show LiGluR expression is restricted to RGCs (g–h). No detectable labeling was found in uninjected rd1 retinas (f). Blue, 4',6-diamidino-2-phenylindole-stained cell nuclei. Bars = 50 µm. INL, inner nuclear layer; IPL, inner plexiform layer. Molecular Therapy 2011 19, 1212-1219DOI: (10.1038/mt.2011.103) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Light-gated glutamate receptor (LiGluR) restores light responsiveness to rd1 retinas in the presence of the photoswitch maleimide-azobenzene-glutamate (MAG). (a) Example multiunit recording from a retina from an adult LiGluR-rd1 mouse in the absence of MAG. Top, light stimulation protocol (5 seconds 380 nm alternating with 5 seconds 500 nm). Middle, raster plot showing spikes for all recorded retinal ganglion cells (RGCs). Bottom, peri-stimulus time histogram (PSTH) for the population of recorded neurons. (b) Example of a typical multiunit recording from a retina of an adult LiGluR-rd1 mouse in the presence of MAG (same retina as a). Top, light stimulation protocol (5 seconds 380 nm alternating with 5 seconds 500 nm). Middle, raster plot showing spikes for all recorded RGCs. Bottom, PSTH for the population of recorded neurons. (c,d) Example raster plots of LiGluR-mediated light responses in both a transient (c) and a more sustained (d) ganglion cell for three different stimuli durations (380 nm). Top, 50 ms. Middle, 300 ms. Bottom, 5 s. Arrow indicates the onset of the 380 nm light pulse. (e) Comparison of the mean firing rates at 380 and 500 nm for the RGCs recorded in a. Gray lines, single units. Black lines, population mean. (f) Comparison of the mean firing rates at 380 and 500 nm for the RGCs recorded in b. Gray lines, single units. Black lines, population mean. (g,h) Scatter plot of the mean firing rates of all recorded RGCs (n = 6 retinas) in response to a 5 seconds pulse of 380 or 500 nm light in the absence (g) or presence (h) of MAG. Gray dots, individual cells. Red dot, population mean. Error bars, SEM. Molecular Therapy 2011 19, 1212-1219DOI: (10.1038/mt.2011.103) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Light-gated glutamate receptor (LiGluR) expression restores visually-evoked potentials (VEPs) in the primary visual cortex of rd1 mice. (a) Example VEPs recorded from the visual cortex in an rd1, LiGluR-rd1 (+ maleimide-azobenzene-glutamate (MAG)) and wild type mouse in response to a 300 ms pulse of 380 nm light (see Materials and Methods section). Bar shows the timing of the light stimulus. (b) Mean peak VEP amplitude for each experimental group (rd1, n = 6; LiGluR-rd1, n = 13; WT, n = 7). (c) Wavelength tuning of VEPs. Mean peak VEP amplitude is expressed as a percentage of the VEP at 380 nm (n = 4–6 for each wavelength tested). Error bars are SEM. WT, wild-type mice. Molecular Therapy 2011 19, 1212-1219DOI: (10.1038/mt.2011.103) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Restoration of pupillary reflex in triple knockout mice lacking phototransduction and melanopsin expression by transduction with light-gated glutamate receptor (LiGluR). (a) Representative infrared images of the pupil area taken in the dark (left) and during exposure to 380 nm (right) for WT, TKO, and TKO-LiGluR(+MAG) animals. (b) Mean maximal pupillary constriction across the population (WT, n = 5; TKO-LiGluR, n = 7; TKO, n = 5). Error bars are SEM. MAG, maleimide-azobenzene-glutamate; TKO, triple knockout mice; WT, wild-type mice. Molecular Therapy 2011 19, 1212-1219DOI: (10.1038/mt.2011.103) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Light-gated glutamate receptor (LiGluR) restores a water-maze based light-avoidance behavior to adult rd1 mice. (a) Diagram of the behavioral setup (see Material and Methods section). (b) Time to reach platform for the three experimental groups (rd1, LiGluR-rd1, WT, n = 10/group, mean ± SEM). (c) Mean number of retreats from the illuminated arm (which contained the platform) for the three experimental groups (rd1, LiGluR-rd1, WT, n = 10/group, mean ± SEM). Mice were run continuously for 7 days (one session/day). At day 8, animals were intravitreally injected with MAG. Testing continued for 7 more days. MAG, maleimide-azobenzene-glutamate. Molecular Therapy 2011 19, 1212-1219DOI: (10.1038/mt.2011.103) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions