original article© The American Society of Gene & Cell Therapy

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translat-ability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injec-tion into the eye’s easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell–specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intra-vitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.

Received 5 May 2014; accepted 29 July 2014; advance online publication 9 September 2014. doi:10.1038/mt.2014.154

INTRODUCTIONThe remarkable success in clinical trials for the childhood-onset blindness, Leber’s congenital amaurosis (LCA) established the proof-of-concept for AAV-mediated retinal gene therapy.1–4 Gene replacement approach is effective for treating diseases resulting from recessive null mutations but remains difficult to apply to dominantly inherited retinal dystrophies, affecting the major-ity of visually impaired patients. Furthermore, inherited retinal degenerations display wide variation in their mode of inheritance,

underlying genetic defects, age of onset, and phenotypic severity (https://sph.uth.edu/retnet/disease.htm). The genetic origin of the disease remains unknown in half of the patients. These pres-ent enormous obstacles for the development of broadly applica-ble gene therapy strategies for retinal degeneration. As one such example, over 80 gene loci are involved in retinal diseases that result in photoreceptor cell death, with the most common subtype being retinitis pigmentosa (RP). Given this constraint, mutation-independent gene therapeutic approaches have been widely devel-oped over the past 20 years.5–7 One such approach is optogenetics. Optogenetics aims at restoring vision in blind patients by express-ing microbial opsins,8–10 endogenous opsins,11 or engineered pho-tosensitive ion channels12 to reactivate residual retinal neurons in late-stage photoreceptor diseases.

The nonselective expression of optogenetic light switches in inner retinal cells does not restore the diversity of retinal output responses as they activate ON and OFF cells indistinctly.8,11,12 On the other hand, it has been shown that expressing halorhodop-sin in nonfunctional but surviving “dormant” cones preserves the processing of visual inputs by all layers of the retina.10 Clinical data shows that dormant cones appear in a restricted area of the macula, but it remains unclear what percentage of the patient pop-ulation displays this phenotype. Histopathologic studies of post-mortem retinas from patients with RP show that 78–88% of the inner nuclear layer cells are preserved in patients with severe and moderate RP.13 An attractive cell target for optogenetic therapies is thus the ON-bipolar cell. A pioneering study used electroporation to insert channelrhodopsin cDNA under the control of the ON bipolar cell promoter into bipolar cells of the rd1 mouse retina. This lead to the recovery of visually evoked potentials (VEP) and visually guided behaviors after the intervention.9 Electroporation, however, is not a viable delivery method for clinical application as it leads to transient and low expression levels (~7% of the targeted ON bipolar cells). To use a microbial opsin in vision restoration, the transgene expression must be stable and robust in specific cel-lular targets, and this can be best achieved using AAVs.

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The first three authors contributed equally to this work.Correspondence: Deniz Dalkara, Institut de la Vision, Sorbonne Universités, UPMC University of Paris 06, UMR_S 968, 17 rue Moreau, Paris, France. E-mail: deniz.dalkara@gmail.com or Jens Duebel, Institut de la Vision, Sorbonne Universités, UPMC University of Paris 06, UMR_S 968, 17 rue Moreau, Paris, France. E-mail: jens.duebel@gmail.com

Targeting Channelrhodopsin-2 to ON-bipolar Cells With Vitreally Administered AAV Restores ON and OFF Visual Responses in Blind MiceEmilie Macé1,2,3, Romain Caplette1,2,3, Olivier Marre1,2,3, Abhishek Sengupta1,2,3, Antoine Chaffiol1,2,3, Peggy Barbe1,2,3, Mélissa Desrosiers1,2,3, Ernst Bamberg4, Jose-Alain Sahel1,2,3,5,6, Serge Picaud1,2,3, Jens Duebel1,2,3 and Deniz Dalkara1,2,3

1INSERM, U968, Paris, France; 2Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France; 3CNRS, UMR_7210, Paris, France; 4Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt am Main, Germany; 5Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France; 6Fondation Ophtalmologique Adolphe de Rothschild, Paris, France

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Natural AAVs have been shown to effectively transduce retinal ganglion cells following intravitreal injection14,15 and photorecep-tors using subretinal injection in normal retinas.16 However, bipo-lar cells were more difficult to target, and they require engineered vectors. After degeneration of photoreceptors in the rd1 retina, a tyrosine capsid-mutated serotype, AAV8-Y733F, was effective at transducing bipolar cells via subretinal administration.17 In this study, hChR2-green fluorescent protein (GFP) was expressed in the ON-bipolar cells, and it was shown that continuous chan-nelrhodopsin expression after AAV delivery is safe from immu-nological standpoint. However, recently published results from clinical trials show that subretinal injections are associated with procedural risks in the foveal region.18 The progression of the dis-ease likely affects retinal structure, making it prone to damage by surgical detachment. The risk of compromising residual central vision in late-stage RP patients may represent a roadblock for this therapeutic option. Furthermore, subretinal injections only treat a fraction of the retina. To overcome these hurdles, new AAV vari-ants with the ability to deliver genes deep into the retina via intra-vitreal route have been engineered.19–21 We, for instance, recently developed a new AAV vector, 7m8, a genetic variant of AAV2, which has a peptide on its heparin-binding site.20 This variant was isolated from a large library of AAV mutants injected intra-ocularly into transgenic mice expressing a rhodopsin-GFP fusion protein in rod photoreceptors.20 Rods were subsequently isolated using flow cytometry, and AAV variants were recovered from these cells. This process enabled us to iteratively enrich for AAV variants capable of reaching the outer retina from the vitreous. All of the variants isolated through this screen had 7mer insertions in their heparin-binding site pointing to the relevance of hepa-rin binding. The increased retinal transduction efficiency of 7m8 compared to its parental serotype AAV2 may arise in part from this variant’s reduced heparin affinity,22 which may both decrease capsid sequestration in the inner limiting membrane and enable enhanced penetration through retinal layers.

In this study, we combined this potent AAV capsid variant designed for gene delivery into deep layers of the retina20 with the previously described 200-base pair enhancer sequence of the mouse Grm6 gene, fused to the SV40 eukaryotic promoter.9,17 Large numbers of ON-bipolar cells were targeted across the retina using this capsid-promoter combination. Unlike preceding stud-ies, treated rd1 retinas showed both ON and OFF responses indi-cating that these pathways are reactivated jointly by our approach. Furthermore, we show that these ON and OFF responses are transmitted to the visual cortex and lead to light-induced loco-motory behavior. Restoring vision at the bipolar cell level with a surgically less complex intravitreal injection of engineered AAV-channelrhodopsin is thus a promising strategy for RP patients with no remaining cones and fragile retinal architecture.

RESULTSGene delivery to ON bipolar cellsFor all experiments, we used a humanized version of channel-rhodopsin-2 with H134R mutation (ChR2/H134R). The H134R mutation provides a reduction in desensitization and increased light sensitivity.23 It was fused to GFP to facilitate cellular local-ization. Gene expression, however, was restricted to the ON

bipolar cells by the use of a 200-base pair enhancer sequence of the mouse Grm6 gene, which encodes the ON bipolar cell–spe-cific metabotropic glutamate receptor, mGluR6 (ref. 24). We used 7m8, a genetic variant of AAV2, to deliver ChR2 across the retinal layers. ChR2 was delivered to the retinas of C57BL/6J wild-type and C3HeN rd1 mice. Rd1 mice were used for this study as their photoreceptors rapidly degenerate and their electroretinogram is undetectable by 8 weeks of age.25,26 Four to 8-weeks-old rd1 mice were injected intraocularly. Four to six weeks after intravit-real delivery of AAV2-7m8 vector encoding hChR2/H134R-GFP lead to strong pan-retinal expression as seen by in vivo fundus imaging (Figure 1a). Retinas were then harvested and directly visualized using two-photon microscopy to characterize the cell types expressing hChR2/H134R-GFP (Figure 1b). Observation of retinal slices revealed strongly labeled axon terminals in the inner half of the inner plexiform layer (ON-sublamina), consistent with localization of axons of ON bipolar cells.27 These cells were imaged in a region of the retina where bipolar cells were sparsely labeled with GFP fluorescence to ensure that their axon terminals are not masked by processes of neighboring bipolar cells. Based on their branching pattern, dendritic morphology, and stratification of their axon terminals, we were able to identify both rod and cone bipolar cell types28 as shown in Figure 1b: the narrowly branching bipolar cell with compact dendritic field and short axon terminals was identified as type 7 (ref. 28). Another ON cone bipolar cell type with sparse, long meandering dendrites was identified as type 9. The rod bipolar cell type was identified by its typical bulbous shaped axon terminal.

Some eyes from the same group were enucleated, fixed, and embedded in optical coherence tomography for histology and confocal microscopy 120–140 days postinjection. Fifteen-micrometer-thick retinal cryosections were incubated with anti-protein kinase C (PKC) α antibodies (in red), and GFP signal was amplified with GFP antibodies (Figure 1c). There was strong label-ing of the ON strata of the inner plexiform layer across the retina (Supplementary Figure S1). GFP expression was localized to the plasma membrane, where it strongly labeled cell somas, dendritic arbors, axons, and axon terminals. Another series of animals were sacrificed 204–254 days postinjection. These eyes were used for cross-sections as shown in Supplementary Figure S1. In some areas, we observed sparse labeling of amacrine cells. No off-target expression was observed in OFF bipolar cells (Supplementary Figure S1). To examine the distribution of GFP across treated rd1 retinas, low-magnification images were acquired after anti-GFP staining (Figure 1d). Retinal flat mounts costained with ON bipolar cell antibodies (PKC-α and Go-α), the percentage of ON bipolar cell transduction, was studied using confocal microscopy. To evaluate the percentage of ON bipolar cells labeled across the retina, low-magnification confocal images were acquired at eight different locations, at two different eccentricities from the optic nerve head. Confocal image stacks were acquired across the bipo-lar cell layer, and cell bodies that stain positive for the ON bipolar cell markers and GFP were counted (Supplementary Table S1). We found that 6–10,000 ON bipolar cells were labeled per square millimeter across the retinas, which represents 52–74% of total ON bipolar cells counted (Figure 1e). Expression was stable for at least 12 months postinjection in both wild-type and rd1 retinas

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Figure 1 ChR2 delivery to ON bipolar cells using AAV2-7m8. (a) Fundus image of a representative rd1 mouse retina expressing ChR2(H134R)-GFP under the control of 200-base pair enhancer sequence of the mouse Grm6 gene, fused to the SV40 eukaryotic promoter. (b) Live two-photon images of ON-bipolar cells types (z-projections of image stacks recorded in retinal slice). Examples of different ON-bipolar cell types expressing ChR2(H134R)-GFP fusion: (left) ON cone bipolar cell type 7, (middle) ON cone bipolar cell type 9, and (right) rod bipolar cell. (c) A variety of ON bipolar cell types were labeled in the rd1 retina on the basis of cell morphology, colocalization with ON bipolar cell markers and stratification of axon terminals in the IPL. Red, PKC-α; blue, DAPI-stained cell nuclei; green, endogenous GFP. Scale bars = 10 μm in b and c. (d) Retinal whole-mount showing the spread of gene expression across the rd1 retina with the insets showing confocal stack projections across the inner nuclear layer showing ON bipolar cell bodies labeled with PKC-α, Go-α, and GFP. (e) ON-bipolar cell counts per square millimeter across retinal flat mounts (n = 4) stained with PKC-α, Go-α, and GFP antibodies. DAPI, 4',6-diamidino-2-phenylindole.

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(Supplementary Figure S1). Immunoreactivity against the rod bipolar cell marker PKC-α was observed in about half of the GFP-expressing cells in the AAV-injected retinas (Figure 1d). Our data show that both rod and cone ON bipolar cells expressed the hChR2/H134R-GFP fusion protein.

ChR2-evoked spike activity in retinal ganglion cellsTo demonstrate that selective ON-bipolar cell targeting of hChR2/H134R-GFP can restore visual function in blind rd1 retinas (age: >18 weeks), we recorded spiking activity from retinal ganglion cells using a multielectrode array (MEA). Mice were injected 4–8 weeks after birth, and MEA recordings were performed at 18–36 weeks of age. Light-evoked spiking activity was observed when stimulating treated rd1 retinas with full-field flashes (Figure 2b),

whereas control rd1 retinas did not show any increase in spik-ing in response to light (data not shown). We measured the light spectrum of the responses at 1017 photons/cm2/second using dif-ferent wavelengths. The spectrum recorded matches the excitation spectrum of channelrhodopsin-2 (ref. 29,30). Firing frequency was highest in response to 480 nm light, which corresponds to the excitation peak of ChR2 (ref. 31; Figure 2a). Firing rate frequency was intensity dependent, with a threshold of >1014 photons/cm2/second (Figure 2d). The ratio of responding cells increased linearly with increasing light intensities (Figure 2c). Importantly, treated rd1 retinas showed both ON and OFF responses to light stimuli. To investigate if these responses were produced by residual cone photoreceptors, we blocked the cone to ON bipolar synapse with a metabotropic glutamate receptor agonist. ON responses remained

Figure 2 Restoration of ON and OFF light responses at the retinal ganglion cell level. (a) Peristimulus time histogram: average response to a full field flash at 450nm, with the addition of LAP4 in the bath (middle), and with the addition of strychnine (below). Dashed vertical lines indicate the onset and offset of the stimulus. (b) Spectral tuning of ChR2(H134R)-GFP expressing rd1 retina. (c) Ratio of cells responding as a function of the luminance of the flash. (d) Ratio of cells responding to the onset (white) and the offset (gray) of the stimulus under the control condition (upper panel), with the addition of LAP4 (middle panel), or strychnine (lower panel). LAP4, l-(+)-2-amino-4-phosphonobutyric acid.

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after application of metabotropic glutamate receptor agonist l-(+)-2-amino-4-phosphonobutyric acid at 50µmol/l in ChR2-treated rd1 retinas (Figure 2c,d). Conversely, l-(+)-2-amino-4-phosphonobutyric acid blocked all ON responses in wild-type retinas by blocking the transmission between photoreceptors and ON bipolar cells (data not shown). These results demonstrate that all responses in treated rd1 retinas originated from ChR2/H134R expressing bipolar cells, bypassing the endogenous phototrans-duction step. Furthermore, OFF responses (92%) were blocked by the glycine receptor antagonist, strychnine, at 10µmol/l (Figure 2b,d). This observation is consistent with an indirect activation of the OFF pathway, likely through rod-bipolar cells to AII amacrine cells, in the treated rd1 retina.

Optogenetic photoresponses in the visual cortexTo assess whether the ChR2-mediated retinal photoresponses were transmitted to higher visual centers of the brain, visually evoked responses were recorded from the visual cortex of AAV2-7m8-ChR2/H134R-treated rd1 mice and compared with responses obtained from untreated rd1 and wild-type mice. The treated eye (contralateral to the recording hemisphere) was stimulated with 200 ms pulses of blue light (light intensity: 1.7 1017 photons/cm2/second) repeated 200 times at 1 Hz. Both local field poten-tials (LFP) and multiunit spiking activity were recorded. As the amplitude and shape of a visually evoked LFP response, also called VEP, depends on cortical depth, we used linear multisite silicon microprobes (16 electrodes at 50 μm intervals) to perform VEP recordings. For each acquisition, after averaging over the 200 tri-als, the electrode showing the VEP with maximal peak amplitude was selected for quantification. No VEPs were visible (flat traces) on recordings from untreated rd1 mice. For comparison, we mea-sured the maximal amplitude of the LFP trace due to the noise level in these recordings from untreated rd1 mice. The maximal VEP amplitude was significantly larger in treated animals (54 ± 6 µV; n = 5) than maximal LFP amplitudes recorded in untreated rd1 mice (17 ± 2 µV; n = 5; P = 2.3 10−4, P < 0.001, unpaired one-tailed t-test; Figure 3a). The amplitude of the VEP in treated rd1 mice was consistently lower than wild-type VEPs (464 ± 37 µV; n = 7; P = 1.8 10−6, P < 0.001, unpaired one-tailed t-test). Interestingly, the shape of the VEPs was similar between wild-type and treated mice, with clearly visible ON and OFF responses (Figure 3). The age of the five treated rd1 animals analyzed were 245, 323, 304, 337, and 346 days at the time of recordings, and all animals in this group were injected 8 weeks after birth.

VEP responses are dominated by synaptic currents because the synaptic currents are slow events that can overlap locally to build a macroscopic current response and a large fluctuation in the field potential.32 The presence of VEPs does not guarantee that visual stimulation induces spiking responses in neurons in the vicinity of the electrode. Therefore, VEPs and spikes in the primary visual cor-tex convey independent information.33 We thus studied, for the first time, the multiunit activity recorded by the silicon probe, extracted from the same data set. Light stimuli produced strong ON and OFF spike discharges in treated rd1 mice similar to those observed in sighted animals (Figure 3b). No modulation of spike discharges was observed in untreated rd1 mice. We calculated the latency of both ON and OFF responses in the two groups after building the

peristimulus time histogram (Figure 3c). The latency of the ON response was consistently shorter in treated rd1 mice (20.3 ± 0.6 ms) than in wild-type animals (50.9 ± 3.3 ms) (Figure 3d). On the contrary, OFF responses exhibit similar latencies in both treated (55.2 ± 1.5 ms) and wild-type mice (53.0 ± 4.5 ms). Altogether, our data demonstrate that cortical neurons generate light-induced spike discharges at ON and OFF light onsets in blind mice whose retinas have been reactivated in a circuit-specific manner.

Light-induced locomotory BehaviorWe tested whether a light-induced locomotory behavior was vis-ible in rd1 blind mice lacking photoreceptors treated with bilateral injection of AAV2-7m8-ChR2/H134R. The mouse was placed into a cylindrical transparent tube with a bright blue (470 nm) LED light source at one end (Figure 4a). The LED light source was controlled by a function generator generating light flashes at 2 Hz. Initially, the light source was kept off, and the mice were allowed to acclimate in the chamber for 2 minutes. Mice were filmed by an infrared camera during the whole experiment. The LED light source was switched on (at an intensity of 3,000 lux) when the mice approached the light source, facing the LED. We observed that wild-type mice quickly turned their whole body away from the light (n = 8; mean latency to first turn = 4.62 ± 1.25 seconds) once the light was switched on. Untreated rd1 mice took significantly longer than the wild-type mice to perform a first body turn after the light was switched on (n = 15; mean latency to first turn = 15.8 ± 3.04 seconds, significance wild-type versus rd1: P < 0.05 by one-way ANOVA followed by Bonferoni’s post hoc test). However, the rd1 mice treated with bilat-eral injection of AAV2-7m8-ChR2/H134R showed significantly faster body movement in response to the light being switched on (n = 12; mean latency to first turn = 6.67 ± 1.12 seconds; significance rd1 versus treated rd1: P < 0.05 by one-way ANOVA followed by Bonferoni’s post hoc test) than the untreated rd1 mice (Figure 4b). The difference between the wild-type mice and the treated rd1 mice was not statistically significant for the body movement response.

DISCUSSIONIn this study, we report a broadly applicable optogenetic gene therapy approach for restoring visual function in patients suffer-ing from photoreceptor degeneration. The landmark clinical trials for LCA2 gene therapy support the safety and efficacy of retinal gene transfer via AAV vectors when the retinal structure is intact. Gene replacement therapy requires intervention at an early stage of the disease and knowledge of the mutated gene. However, disease-causing mutations remain currently unknown for a large population of affected patients, and their retina has undergone full photoreceptor degeneration by the time an intervention is underway. For gene therapy in these patients, a surgically safe delivery route is crucial for maximal therapeutic benefit with min-imal damage. It has been shown that subretinal injections pres-ent risks of retinal damage in the macular region,18 which might be accentuated in late stages of retinal degenerative disease. On the other hand, intravitreal injections are performed routinely in ophthalmic practice. A major challenge of vision restoration by optogenetics is thus to preserve the retinal processing of visual information by targeting the most upstream surviving neurons, in the retina, while avoiding procedural effects.

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Until recently, AAV vectors were unable to access primary and secondary neurons situated in deeper retinal layers via intravit-real injection route. We combined an AAV capsid specially engi-neered to penetrate deeper in the retina20 with a highly specific ON-bipolar cell promoter.9 Our results demonstrate that the bipo-lar cell layer of the retina can be targeted efficiently via an intravit-real injection for the delivery of a therapeutic gene.

Targeting dormant cones for optogenetic vision restoration offers the advantage of preserving intrinsic neural image–pro-cessing capabilities of the inner retina.7 A fundamental aspect of this retinal processing is the splitting between ON and OFF pathways.34 ON cells are activated by light increments, whereas OFF cells are activated by light decrements, and these two path-ways remain spatially segregated in the lateral geniculate nucleus before being combined in the visual cortex.35,36 While previous studies reactivating ON-bipolar cells only showed restoration

of ON responses,9,17 here we also observed OFF responses when light was turned off. As ChR2(H134R) expression was absent in OFF bipolar cells, these OFF responses can be attributed to the activation of rod ON bipolar cells, which activate AII amacrine cells who have a glycinergic synapse with OFF bipolar cells37 (Figure 5). This hypothesis was supported by the suppression of the OFF responses by blocking glycinergic synaptic transmission with strychnine. Alternatively, AII amacrine cell activation can be induced through electrical synapses with ON cone bipolar cells. In both cases, at light decrements, the release of the glycinergic inhi-bition induces a rebound of excitatory responses in OFF bipolar cells (Figure 5). Our ability to restore the OFF response might be related to the higher number of ON bipolar cells targeted with our intravitreal approach.

ON and OFF responses observed by MEA recordings from the ganglion cells were also observed in the visual cortex. For the first

Figure 3 Restoration of ON and OFF light responses in the visual cortex of ChR2-treated blind mice. (a) Examples of local field potential (LFP) traces in response to a 200 ms blue light stimulus (1.7e17 photons/cm2/second) for rd1, treated rd1, and wt mice. The signal is averaged on 200 repetitions presented at 1 Hz. (b) Multiunit spike rasters obtained from the same raw data as in a. (c) Peristimulus time histograms (PSTHs) built from the previous rasters. The bin size is 5 ms. (d) Latencies of ON and OFF responses for treated rd1 and wt mice, showing that the ON latency is significantly shorter in treated rd1. ON and OFF latencies are calculated from PSTHs as the bin with maximal spike rate during and after the stimulus respectively. Mean and SEM are shown for each group: treated rd1 (n = 5) and wt (n = 7). wt, wild type.

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time, spiking activity was recorded in treated mice and showed reliable ON and OFF responses generated by cortical neurons. Measuring this spiking activity was important given that VEPs do not reflect spiking but rather account for synaptic activity. It proves that the visual inputs are processed by the visual cortex and transmit-ted to downstream brain centers. ON latency is shortened by 30 ms which is consistent with the notion that in treated rd1 mice, the ON signal bypasses the phototransduction cascade as well as the synap-tic transmission from cones to ON bipolar cells through the slow metabotropic glutamate receptors.30,38 Instead, the signal is directly generated in ON bipolar cells. Conversely, the OFF responses were not faster than those from wild-type mice, which can be explained by the indirect synaptic pathway, through AII amacrine cells, likely responsible for the generation of OFF responses. The restoration of OFF responses is very promising in the perspective of recover-ing a visual perception as close to natural vision as possible as it is necessary to activate both channels for an efficient computation of visual inputs by the higher visual centers.34 We believe preserving both ON and OFF channels is likely to improve patients’ perfor-mance at visual tasks compared to strategies that activate only one pathway, or that abolish ON and OFF parallel processing. From a functional standpoint, all neurons of the visual cortex display an ON and OFF component in their receptive fields as they combine

both pathways via the lateral geniculate nucleus inputs. Therefore, restoring both the ON and OFF responses preserves normal inputs to the cortex. From a perceptual standpoint, several studies have demonstrated the importance of ON pathway in visual perception. Pharmacological block of the ON responses in primates profoundly impairs key aspects of their perception, the most pronounced change being the large decrease in contrast sensitivity.34 Unlike the ON pathway, no pharmacological agent can specifically block OFF responses. As a result, it is less clear to what extent the loss of the OFF pathway would affect visual perception, but we anticipate that at least symmetric effects to loss of ON pathway would occur.39 Patients without OFF responses are likely to perform poorly at tasks involving contrast such as discriminating objects from the back-ground, in particular dark objects from light background.

Finally, light-induced locomotory behavior was observed in the rd1 mice treated with the AAV encoding ChR2 (H134R). Normal mice avoid light by orienting themselves away from sources of illumination. This response is much slower in rd1 mice. We found that the treatment of the rd1 mice with ChR2 encoding AAV significantly reduces this response time and brings it close to the wild-type levels. Some of the light-induced locomotory behavior might have been mediated by melanopsin expressed by intrinsically photosensitive ganglion cells. However, in our assay, the difference of response time between wild-type and rd1 mouse was highly significant. Hence, it is unlikely that endogenous mela-nopsin alone could account for the restoration of visual behavior in our AAV-treated rd1 mice.

Our results on a murine model of RP show that optogenetic reactivation of ON-bipolar cells is a viable option for restoring a vision in patients with late-stage disease through a surgically safe delivery procedure. However, an important consideration with the use of intravitreal injections is the possibility of inflamma-tory response,40 namely, in nonhuman primates.20 While surgi-cally less invasive than a subretinal injection, there are important immunological issues to be considered when using intravitreal injections of high-dose AAV.20 Furthermore, there are interspe-cies differences in transduction patterns obtained by intravitreal

Figure 4 Restoration of a visual behavior in treated blind rd1 mice. (a) Experimental setup for the light avoidance behavior test. The mouse is placed in a transparent tube and filmed with an infrared camera. A bright 470 nm LED is placed on one end of the tube and flashes at 2 Hz. (b) Time to first body turn away from the light for treated, untreated blind rd1, and wild-type mice. Mean and SEM are shown for each group.

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AAV administration in primates.41 Ocular characteristics between mouse and primates affect the ability of AAV to transduce neu-ral retina, leading to limited gene delivery to the retina along the vasculature and perifoveal region where inner limiting mem-brane is thinnest. However, we have previously shown that retinal transduction by AAV increases in retinal disease.42,43 In view of these findings, in patients with late-stage retinal degeneration, we would expect an increased spread of transduction with our deliv-ery approach. These aspects need to be evaluated in further stud-ies to explore our technology’s translational potential.

Targeting bipolar cells in patients with late-stage RP is feasible even when degeneration goes beyond the loss of cone cell bodies, and it will restore the main processing capabilities of the retina, including the ON and OFF pathways. A current limitation in restor-ing visual function by optogenetic therapies is potential immune responses to the foreign transgene product, as high levels of expres-sion are required to get light responses from cells engineered to express microbial opsins. Another remaining issue in restoring physiological visual processes by optogenetic therapies is the high light intensity required to activate these opsins. The threshold light intensity to elicit spike responses in ganglion cells was 1014 photons/cm2/second, which is significantly higher than that of intrinsic rod and cone opsins (106 and 1010 photons/cm2/second, respectively). Therefore, external prosthetic devices (such as projecting systems or microLED arrays mounted on glasses) will be necessary to stimu-late ChR2(H134R) expressing bipolar cells with high light inten-sity (Degennar, Roska, Benosman et al., personal communication). However, recent efforts in molecular engineering of channelrhodop-sin-2 to modify its channel kinetics and ion selectivity lead to new mutants with higher light sensitivity, such as CatCh44 or bi-stable opsins45,46 We expect that these novel constructs have great potential to reduce the light level needed for optogenetic vision restoration. Moreover, novel opsins such as ReaChR47 and ChrimsonR48 have the potential to red shift the light used for stimulating the retinal neu-rons. This will be a major improvement since red light is less harm-ful for the retina. Our new AAV vector can be used to deliver these second-generation optogenetic tools in future studies.

MATERIALS AND METHODSAnimals. All experiments were done in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. The protocol was approved by the Local Animal Ethics Committee of Paris 5 (CEEA 34) and conducted in accordance with Directive 2010/63/EU of the European Parliament. All mice used in this study were C3H/HeN (rd1 mice) or C57Bl6J mice (wild type) from Janvier Laboratories (Le Genest Saint Isle, France).

AAV production. Recombinant AAVs were produced by the plasmid cotransfection method,49 and the resulting lysates were purified via iodixa-nol gradient ultracentrifugation as previously described. Briefly, 40% iodixanol fraction was concentrated and buffer exchanged using Amicon Ultra-15 Centrifugal Filter Units (Millipore, Molsheim, France). Vector stocks were then tittered for DNase-resistant vector genomes by real-time PCR relative to a standard.50

Injections. Mice were anesthetized with ketamine (50 mg/kg) and xyla-zine (10 mg/kg; Rompun]). Pupils were dilated, and an ultrafine 30-gauge disposable needle was passed through the sclera, at the equator and next to the limbus, into the vitreous cavity. Injection of 1 µl stock containing

1.8 × 10e11 particles of AAV was made with direct observation of the nee-dle in the center of the vitreous cavity.

Immunohistochemistry. Transduced retinas were dissected and fixed in 4% paraformaldehyde for 30 minutes at room temperature. For prepara-tion of retinal sections, fixed and washed retinas were embedded in optical coherence tomography, and 15 μm vertical sections were cut with a Leica cryostat (Leica Biosystems, Wetzlar, Germany). For immunohistochem-istry, retinal sections were incubated with blocking solution (2% normal goat or donkey serum, 1% bovine serum albumin, 0.5% Triton X-100 in phosphate-buffered saline (pH 7.4)) for 1 hour and subsequently immu-nostained with mouse anti-GFP (1:400) and anti-PKC antibodies (1:100 dilution; BD Biosciences, Grenoble, France). Alexa Fluor–conjugated sec-ondary antibodies (Molecular Probes, Invitrogen, Cergy Pontoise, France) were applied at a dilution of 1:200 for 2 hours. Following final washes in phosphate-buffered saline, tissue preparations were mounted on slides with Vectashield containing 4',6-diamidino-2-phenylindole.

Confocal microscopy. Confocal microscopy was performed on an Olympus FV1000 laser-scanning confocal microscope. Images were acquired sequentially, line-by-line, in order to reduce excitation and emission cross talk, and step size was defined according to the Nyquist–Shannon sam-pling theorem. Exposure settings that minimized oversaturated pixels in the final images were used. Twelve bit images were then processed with FIJI, and Z-sections were projected on a single plane using maximum intensity under Z-project function and finally converted to 8-bit RGB color mode. For quantification of percentage of ON-bipolar cells transduced with channelrhodopsin-GFP, retinal flat mounts were incubated over 24 hours with primary antibodies against GFP, PKC-α, and Go-α applied in blocking buffer. After extensive rinsing with phosphate-buffered saline, secondary antibodies in green, red, and far red were applied over 6 hours. Retinas were rinsed and mounted between two coverslips using mounting medium. Confocal stacks through the bipolar cells were acquired using the 20× objective, in regions and two adjacent regions—above, below, to the right, and left—of the optic nerve (see Supplementary Figure S1).

MEA recordings. MEA recordings were obtained from ex-vivo isolated flat mounted retinae of rd1 mice aged from 132 to 324 days. Mice were sacri-ficed by quick cervical dislocation, and eyeballs were removed and placed in Ames medium (Sigma-Aldrich, St Louis, MO; A1420) bubbled with 95% O2 and 5 % CO2 at room temperature. Isolated retinas were placed on a cellu-lose membrane and gently pressed against a MEA (MEA256 100/30 iR-ITO; Multi-Channel Systems, Reutlingen, Germany), with the retinal ganglion cells facing the electrodes. GFP fluorescence was checked prior to recordings with a Nikon Eclipse Ti inverted microscope (Nikon, Dusseldorf, Germany) mounted under the MEA system, in order to ensure that the recording area expressed the transgene. The retina was continuously perfused with bubbled Ames medium at 34 °C at a rate of 1–2 ml/minute during experi-ments. Metabotropic glutamate receptor agonist l-(+)-2-amino-4-phospho-nobutyric acid (Tocris Bioscience, Bristol, UK; cat No. 0103) and Glycine receptor antagonist strychnine hydrochloride (Sigma-Aldrich; S8753) were freshly diluted to concentrations of 50 and 10µmol/l, respectively, and were bath applied through the perfusion system 10 minutes prior to recordings. Full-field light stimuli were applied with a Polychrome V monochromator (Olympus, Hamburg, Germany) driven by a STG2008 stimulus generator (MCS). Output light intensities were calibrated to range from 1.1014 to 1.1017 photons/cm2/second. Stimuli were applied for 2 seconds, with 10 seconds interval. Wavelength sensitivity of responses was determined by stimulat-ing 10 times, from 400 to 650 nm, with 10 nm steps. The order of the tested wavelength was randomized in order to prevent any adaptation of the retina.

Raw RGC activity was amplified and sampled at 20 kHz. Resulting data was stored and filtered with a 200 Hz high pass filter for offline subsequent analysis using Spike2 software v.7 (CED Co, Cambridge, UK). Single unit raster plots were obtained using a combination of template matching and cluster grouping based on principal component analysis

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of the waveforms. In our population analysis, significant responses were determined based on a z-score analysis. We estimated the mean and SD of the activity prior to stimulus and considered that a response was detected if the activity exceeded the mean by more than four times the SD in the 2 seconds after the onset or the offset of the stimulus (for a bin size of 50 ms). Error bars were calculated over the different experiments (n = 4). For the responses to light at different wavelengths, we measured the response to each flash in a 1 second window after the stimulus. We then normalized the response of each cell by its maximum firing rate response. For the responses to light at different intensities, we estimated the error bars by bootstrapping over the set of recorded cells.

Two-photon imaging. A custom-made two-photon microscope equipped with a 25× water immersion objective (XLPLN25xWMP/NA1.05, Olympus) with a pulsed femtosecond laser (InSight; DeepSee, Newport, Irvine, CA) was used for imaging ChR2(H134R)GFP-positive bipolar cells. AAV-treated retinas from rd1 mice were isolated in oxygenized (95% O2, 5% CO2) Ames medium (Sigma-Aldrich). For live two-photon imaging, retinal slices (300 μm) were cut with a razor blade tissue chopper (Stoelting, Wood Dale, IL), placed in the recording chamber of the microscope, and z-stacks were acquired using the excitation laser at a wavelength of 930 nm. Images were processed offline using ImageJ (NIH, Bethesda, MD). During imaging, the retina was superfused with oxygenized Ames medium.

In vivo extracellular recordings in the visual cortex. Mice (245 to 346 days old) were sedated with a low dose of ketamine–xylazine injection (ket-amine: 40 mg/kg and xylazine: 8 mg/kg) and then anesthetized with ure-thane (1.0 g/kg, 10% weight/volume in saline). The animal was placed in a stereotaxic holder. The temperature was maintained at 37 °C using a heat-ing pad and controlled with a rectal probe (ATC 1000, WPI, Sarasota, FL). Pupils were dilated using drops of tropicamide (Mydriaticum Dispersa). A small coverslip covered with vitamin A (Allergan, Irvine, CA) was placed on both eyes to prevent corneal dehydration throughout the procedure. The scalp was removed, and the skull was exposed. A small craniotomy (1 mm × 1 mm square) was drilled above V1 in the left hemisphere, centered 3 mm lateral and 0.5 mm rostral from the lambda point. The dura was removed using a sharp needle to facilitate electrode insertion. Gelfoam (Pfizer, New York, NY) soaked in cortex buffer was used to clean and hydrate the exposed area during the surgical procedures. Extracellular recordings were made with silicon microprobes from NeuroNexus Technologies (Ann Arbor, MI). We used a probe with 16 sites spaced at 50 μm intervals (model a1 × 16-3mm50-703), to span across multiple layers of cortex and with large sites for improved recording of LFP. The electrode was inserted into the cortex using a 3-axis micromanipulator (Sutter Instruments, Novato, CA) with a 30° angle to penetrate perpendicularly to the cortical surface. It was advanced 600 µm into the cortex. When the electrode was in place, the exposed surface was covered with agarose (1.2% in cortex buffer).

Visual stimuli were generated by a 470 nm collimated LED (model M470L3, Thorlabs, Dachau, Germany) placed 1 cm away from the eye. The light intensity at the level of the cornea was measured to be 1.7 × 1017 photons/cm2/second. An isolating cone ensured that the illumination was restricted to the stimulated eye. The stimulation consisted of 200 ms pulses of blue light repeated 200 times at 1 Hz triggered by a Digidata (Axon; Molecular Devices, Sunnydale, CA). Extracellular signals were amplified using a 16-channel amplifier from MultiChannel Systems (model ME16-FAI-μPA-System), sampled at 10 kHz and recorded using the software MC Rack (Multi-Channel Systems). Signals were then transferred to Matlab for further analysis using custom scripts. For LFP, signals were low pass filtered at 300 Hz and averaged over the 200 trials. For quantification, we selected for each experiment the electrode presenting the highest amplitude signal. For multiunit activity, signals were high pass filtered at 200Hz. Spiking events were detected by a threshold set at six times the median absolute deviation of the trace. Peristimulus time histograms were generated with bins of 5 ms. The latency was calculated as the start of the

bin with the highest spike rate after the onset/offset of the stimulus for ON/OFF responses, respectively. Three groups of animals were compared in this study: five treated rd1 mice, five untreated rd1 mice, and seven wild-type mice.

Light-induced locomotory behavior. A transparent tube with a bright blue (470 nm) LED light source (Lumitronix, Graefelfing, Germany) controlled by a function generator (Thandar Instruments, Cambridgeshire, UK) was used to assess light-induced locomotory behavior of mice in a setup adapted from Polosukhina et al.26. All mice were dark adapted for at least 15 minutes before being tested. The animal was introduced into the trans-parent tube and allowed to acclimate in the chamber for at least 2 min-utes. When mice approached the LED at the end of the tube with its head directly facing the LED, the illumination was turned on (flashing at 2 Hz, 3,000 Lux) for 2 minutes. We recorded the time to a whole body turn after light was switched on. Between each test subject, the position of the LED was randomly switched from one end of the tube to the other to minimize any effects of the mouse being located on one side of the tube. The test-ing chamber was thoroughly cleaned between each subject. All behavioral tests were conducted at the same time period each day (between 3 pm and 5 pm). Only female mice were tested. All data sets were confirmed by a blind observer. This behavior assay was tested on three groups of animals: blind rd1 mice (n = 15), blind treated rd1 mice (n = 12), and wild-type mice (n = 8). All animals were over 15 weeks old. The latency to first turn for mice was analyzed in GraphPad PRISM 6 (GraphPad Software, San Diego, CA) using one-way ANOVA and a Bonferoni’s multiple compari-sons test to compare the means of groups. Data are expressed as Mean ± SEM. Significance was set at P < 0.05 by Bonferoni’s post hoc test.

SUPPLEMENTARY MATERIALFigure S1. Retinal eye cups showing the spread of GFP expression across the retina.Table S1. Quantification of ON-bipolar cells transduced.

ACKNOWLEDGMENTSWe thank Constance Cepko and Botond Roska, for providing the AAV expression plasmid encoding ChR2 (H134R) under the control of 200–base pair enhancer sequence of the mouse Grm6 gene. We also thank Zoltan Raics for providing the 2-p Imaging Software. We are grateful to Stephan Fouquet and the imaging facility of Institut de la Vision. We are thankful to Marusa Lampic for blinded analysis of behavioral results and to Elisabeth Dubus for helpful advice on histology. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Pierre et Marie Curie University (UPMC), the Centre National de la Recherche Scientifique (CNRS), Agence Nationale pour la Recherche (ANR: OPTIMA), The Fondation Fighting Blindness (Wynn-Gund translational research award), the Fédération des Aveugles de France, the city of Paris, the Regional Council of Ile-de-France, and the French State program “Investissements d’Avenir” managed by the Agence Nationale de la Recherche [LIFESENSES: ANR-10-LABX-65] and ERC Starting Grant.D.D. is a consultant for Gensight Biologics. S.P. is a founder and consul-tant for Pixium Vision and GenSight Biologics. J.-A.S. is a founder and consultant for Pixium Vision and GenSight Biologics and a consultant for Sanofi-Fovea and Genesignal.

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