Optogenetics for visual restoration: From proof of principle to translational challenges

Introduction

Retinal degenerative disorders are a common cause of blindness

Degenerative retinal disorders are among the commonest cause of vision loss in industrial countries and represent a major socioeconomic burden [39]; [61]; [158]. These include the rather common multi-factorial Age-related Macular Degeneration (AMD) and the Inherited Retinal Degenerations (IRDs) like retinitis pigmentosa. While AMD leads to a predominantly central vision loss in late stages, usually after retirement age [107]; [115]; [120], IRDs may lead to full peripheral and central vision loss in working age [61]; [80]. Current estimates suggest that IRDs affect 1:3000 people worldwide [80], and to date disease causing mutations have been identified within >100 genes and loci [150]; [154]; [179], comprising >1000 individual variants [150]. Thus, degenerative retinal disorders are highly heterogeneous in terms of their underlying genetics, pathophysiology and their clinical course [15]; [42]; [60], yet they typically share a final common pathology and ultimately lead to irreversible photoreceptor death and loss of vision (Figure 1).

Gene therapy has become a clinical reality

The emergence of gene replacement therapies has begun to offer therapeutic options for some inherited retinal disease, providing a mechanism to correct underlying genetic defects that ultimately lead to photoreceptor loss. Clinical trials have begun for a number of conditions [187] and have now received regulatory approval in some jurisdictions. The first retinal gene therapy to be approved was Voretigene neparvovec (Luxturna®), an Adeno-Associated Virus serotype 2 (AAV2) vector containing the human RPE65 gene designed for

the treatment of recessive RPE65-linked Leber’s congenital amaurosis ([127]; [162]). Luxturna®, like other gene replacement therapies under development, are primarily intended to be administered early during retinal disease to correct the genetic cause of on-going pathology and arrest further visual loss. Recent years have seen an explosion in the molecular diagnosis of retinal disease – which enables a genetic diagnosis in up to three quarters of cases ([54]). Hence it would appear that gene replacement therapies may in future offer viable treatments for IRDs. However, the large number of disease-causing mutations identified to date (in >100 genes), the relatively large number of cases for which a genetic cause is yet unknown (around 30%; [20]; [21]; [150]; [179]), and the high cost of developing such tailored treatments (costs for Luxturna® are $400,000 per eye, with some differences between markets/jurisdictions) potentially limits this approach for widespread treatment of IRDs. Furthermore, and potentially most importantly, it is clear that such gene replacement approaches are not an effective treatment for end stage retinal degeneration, where a near complete loss of post-mitotic photoreceptors has already occurred. There is therefore a clinical need to identify regenerative medicine approaches to restore visual function for the large number of patients who are already in the later stages of retinal degeneration, lacking any significant number of photoreceptors but retaining the remainder of their visual pathways.

To this end, one approach being investigated is the use of prosthetic ‘bionic eye’ type devices. These devices comprise an external camera which records and then processes and transmits visual signals to a small electrode array-based device surgically implanted onto or beneath the retina ([214]). Some of these devices are FDA approved and have shown early promise, restoring basic visual perception and improving performance in a number of visual tasks ([56]; [147]; [203]). At present, technical limitations regarding the total number of electrodes, and the density of electrodes that can be incorporated into such devices, the inability to cover large areas of the retina, and issues of long-term biocompatibility remain the major hurdles for these devices. In 2019 both SecondSight Medical Products who manufacture the Argus II device, and Retina Implant AG who manufacture the Alpha IMS device ceased manufacturing and implantation of these artificial retina devices ([7]; [182]). In addition to prosthetic devices, various types of cell transplantation therapy are also currently under investigation: Pre-clinical studies aimed at directly replacing lost photoreceptors using either immature photoreceptor precursor cells, human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) ([173]; [217]). Although clinical trials have begun in a number of cases, in general there has been only limited success with photoreceptor stem cell therapy ([173]), not least due to problems of donor cell survival and functional integration, but also disease related changes in the retinal pigment epithelium that is essential for normal photoreceptor function. The current state of cell transplantation therapies for visual restoration have been recently covered in a number of excellent reviews ([30]; [66]; [96]; [173]; [217]) and will not be covered in further detail here.

Ectopic expression of opsins renders inner retinal neurons light sensitive

A novel strategy, termed therapeutic optogenetics now offers a viable treatment for late-stage retinal degeneration. Optogenetics involves the expression of transgenes encoding light sensitive proteins in surviving retinal cells to make them directly light sensitive ([18]; [19]; [32]; [67]; [112]; [121]; [170]; [199]). Such an optogenetic gene therapy could work independent of the underlying genetic cause of degeneration and does not depend on the integrity of the retinal pigment epithelium-photoreceptor complex. Thereby, it offers the potential to restore vision even in late-stage retinal degenerations, potentially using a universal approach for the treatment of many different degenerative retinal disorders. Work by several groups has shown that light responses can be restored in rodents with degenerate retina using this method ([19]; [26]). There is moreover data indicating that restored light responses can drive visually guided behaviours (functional vision) in treated animals. Consequently, early-stage clinical trials of optogenetic vision restoration have been initiated (for examples see the Phase I/II trials RST-001 and PIONEER including patients with advanced retinitis pigmentosa, NCT02556736 and NCT03326336 on clinicaltrials.gov, respectively).

Different classes of opsins are being studied as tools for vision restoration

To-date a broad spectrum of light sensitive proteins (opsins) have been studied for the purpose of vision restoration, but most commonly include microbial light gated ion channels, or mammalian light sensitive G-protein coupled receptors. There are several review papers describing their features in much detail (e.g.: [106]; [172]; [189]). Most of these reviews put a focus on one particular class of opsins. Here we will briefly introduce each of the relevant classes, highlighting their key features as well as the potential advantage and disadvantages of each in the context of vision restoration. A summary of preclinical studies in optogenetic vision restoration is given in Table 1.

Opsin class	Key features	Studies using these tools
Microbial Opsins
Channel Opsins	Fast poorly light sensitive Independent of external retinal recycling Concerns about immunogenicity No amplification/second messenger coupling	([19]; [26]; [33]; [41]; [46]; [62]; [64]; [77]; [90]; [91]; [112]; [125]; [126]; [149]; [166]; [170]; [192], [190], [191]; [202])
Light-driven pumps	Hyperpolarizing Independent of external retinal recycling No amplification/second messenger coupling	([26]; [38]; [64]; [77]; [103]; [216])
Mammalian Opsins
Melanopsin	Native human Independent of external retinal recycling Couples to ubiquitous amplifying second-messenger cascade Works at ambient light conditions	([44]; [116]; [122], [123])
Rhodopsin	Native human Couples to inhibitory amplifying cascades Works at ambient light conditions	([32]; [49]; [67])
Cone Opsin	Native human Couples to inhibitory amplifying cascades Works at ambient light conditions	[18]
Engineered mammalian opsins	Currently tested based on melanopsin Improved kinetics	([111]; [199])
Other
Vertebrate switchable opsins	Relatively fast On and off switchable	[160]
Chemically sensitized mammalian ion-channels	Independent of external retinal recycling No amplification/second messenger coupling	([29]; [68])
Chemically sensitized mammalian receptors	Independent of external retinal recycling	[17]
Multi-characteristic opsins	Broad spectrum Reported to work at ambient light conditions	[14]

Microbial opsins are fast but insensitive

Depolarizing opsins. The first opsin to be used in optogenetic vision restoration was Channelrhodopsin-2 (ChR2), a microbial ion-channel type opsin ([19]) (fig:2A). Upon light stimulation the chromophore bond in ChR2 photoisomerases resulting in the opening of an ion permeable pore. Under physiological conditions this leads to a depolarizing influx of cations. After a short time, the chromophore relaxes back into its resting state resulting in pore closure. The chromophore is not released from its pocket and hence Channelrhodopsins can be readily re-activated by the next incident photon ([106]; [215]). As chromophore is recycled, the presence of any intact retinal pigment epithelium/Muller cell chromophore recycling machinery is not a necessity for using ChR2-type optogenetic tools. Moreover, Channelrhodopsins operate with high temporal fidelity, showing rapid onset and offset kinetics – a feature that is critical for delivering useful visual information. Both these aspects make ChR2-type opsins highly attractive as tools for vision restoration. However, these advantages come at a price, which is a low level of light sensitivity (in terms of macroscopic conductance), the absence of any intracellular signal amplification cascade, and levels of desensitization ([117]). Channelrhodopsins have received major attention as tools in research as well as therapeutics ([209]), leading to the discovery or engineering of several variants with favorable properties. In particular, variants with a long wavelength-shifted excitation spectrum and larger macroscopic currents (at the cost of switching kinetics) like ReaChR or ChrimsonR have become available and have been employed in optogenetic vision restoration studies ([105]; [118]; [121]; [163]; [170]). These modifications have enabled stimulation of retinal neurons in vivo using light intensities below the safety thresholds as defined by regulatory authorities ([170]), and in the case of ChrimsonR their successful translation into clinical trials ([163]). Light intensities required to stimulate either of these Channelrhodopsins remain significantly above ambient light levels, and current clinical studies typically include the use of electronic ‘signal amplifying’ headwear (i.e. glasses, see also Section 9) to first detect, process and then amplify the intensity of light signals before they are transmitted to the retina ([163]). Ultimately, there has been one report detailing the generation of a ChR2 type variant that operates at ambient light levels and can be used to restore some degree of visual function ([202]).

Hyperpolarizing opsins. The other important family of microbial opsins in the context of vision restoration are the light-activated ion pumps, such as halorhodopsin. Upon light stimulation these pumps typically transport either chloride into or protons out of the cell [106] (Figure 2 B). Like channel opsins, these light gated pumps are capable of internal chromophore re-isomerisation making them attractive tools for visual restoration. Furthermore, the hyperpolarising effects of these pumps can be an attractive feature when targeting surviving/dormant cone photoreceptors or “OFF-pathway” cells, that naturally hyperpolarise in response to light (thus allowing for a conservation of signal polarity). However, light activated pumps show temporal kinetics that are about tenfold slower than channel opsins [55], [106]. Still, this has been shown to be fast enough to recover key features of retinal signal processing when expressed in residual cone photoreceptors of blind, retinally degenerate, mice [26].

Mammalian opsins show higher sensitivity and a broader dynamic range

In contrast to microbial opsins, mammalian opsins (including rhodopsin, cone opsin and melanopsin) are type A G-protein coupled receptors (Figure 2 C&D). Upon stimulation with light, they initiate an intracellular second-messenger cascade, which has a signal amplifying effect and thereby allows these opsins to evoke cellular responses following exposure to light stimuli that are considerably dimmer than those required for microbial opsins [67], [73]. This apparent sensitivity is one of the key arguments for the use of mammalian opsins for vision restoration. Given that ambient light levels are sufficient to activate these tools, when expressed in vitro and in vivo [32], [44], it is expected that normal daytime vision and perhaps even some night vision can be restored without the need for signal amplifying goggles and light safety thresholds would not be of a concern.

While the overall light-sensitivity of optogenetic tools is of clear relevance in the context of vision restoration, at least as important is the dynamic range of signal encoding that can be achieved. In their native cellular environment (i.e. rods, cones and intrinsically photosensitive retinal ganglion cells; ipRGCs) mammalian opsins enable encoding of a broad range of signal intensities, spanning at least 5 log-units of intensity [43], while microbial opsins operate over a range of only about 2 log-units. It is understood that at least in rods and cones intracellular modulatory mechanisms contribute to this property; still, a similarly broader dynamic range has been observed when comparing rhodopsin to ChR2 [67] and also when comparing melanopsin to ReaChR [73] outside their native environments as tools for vision restoration.

Melanopsin. The first mammalian opsin tested in the context of visual restoration was melanopsin [116], [137], the photopigment of the intrinsically photosensitive retinal ganglion cells [85], [104]. This choice had been straightforward as melanopsin had already been shown to be functional when expressed outside its native environment and confer photosensitivity to a number of cell types [116], [137]. Melanopsin can initiate both, ubiquitous G$_{q/11}$- and G$_i$-protein pathways [95], [174], [175] making it likely that melanopsin could also evoke cellular responses when expressed in other types of retinal neurons. Moreover, melanopsin putatively uses an intrinsic chromophore recycling mechanism and hence, similar to channelrhodopsins, does not depend on external retinal pigment epithelium/Müller cell mediated retinal supply [44], [110]. We have previously used this approach to restore vision in the rd1 mouse model, leading to restoration of retinal and cortical light responsiveness that persisted for at least one year after treatment [44] (Figure 6 C).

Rhodopsin and iodopsins. The main downside of melanopsin are its physiologically slow signalling kinetics, which has motivated researchers to test alternative opsins, like rhodopsin and cone-opsins [18], [32], [67] (Figure 2 D). Both these photopigments physiologically activate G$_t$ (Transducin, member of the G$_i/o$ family) to initiate a cGMP dependent signal cascade specific for rods and cones [52]. They are also capable of activating ubiquitous G$_i$ [10], [11], [114], though in the case of the cone-opsins they do so with low efficiency [83]. With regard to signalling speed achieved by the different optogenetic tools it is important to remember that for mammalian opsins, not just the opsin activation and inactivation kinetics are rate limiting. Rather, the availability of downstream effectors and their kinetic properties are important and can be equally rate limiting. Thus, the kinetic behaviour of mammalian opsins will differ depending on the cell type they are expressed in and the signalling cascade they couple to in those cells, and they can’t be extrapolated immediately from their kinetic behaviour in rods, cones and intrinsically photosensitive retinal ganglion cells, respectively. Interestingly, in heterologous expression systems, rhodopsin or cone opsin induced signalling show similar temporal kinetics (if not slower) as melanopsin signalling [11, 82]. In any case, detailed data on response kinetics of rhodopsin and middle-wave sensitive (MWS) cone opsin treated retinal neurons are available and show times to half-activation in the range of few 100 ms [18] and all three opsins seem to be able to mediate some level of image-vision guided behaviour [18, 32, 44, 67].

Opsin Chimeras. Given the above-mentioned limitations of mammalian opsins, attempts have been made to identify or engineer opsins with properties that more optimally suit the requirements of vision restoration. One such attempt is the melanopsin – glutamate receptor chimera Opto-mGluR6 [199] . Herein, the light sensitivity conferring transmembrane and extracellular segments of class A G-protein coupled receptor melanopsin were fused with the intracellular segments of class C G-protein coupled receptor mGluR6, in essence to produce a light sensitive mGluR6 receptor [188, 199]. Opto-mGluR6 is one of very few examples of a functional inter-class G-protein coupled receptor-chimera and was designed to utilize the native signal transduction cascade of ON-bipolar cells – that is initiated by changes in mGluR6 activation and ultimately results in TRPM-1 channel gating [167]. Indeed, when delivered into ON-bipolar cells, Opto-mGluR6 was able to drive retinal light responses with high temporal precision as well as image-vision guided behaviour [111, 199]. Such tools optimized to interact with native ON-bipolar signalling pathways are attractive in the long term as they could enable high-speed, high sensitivity light perception. Other reports of chimeric opsins include the generation of chimeras containing the intracellular domains of melanopsin and extracellular and transmembrane domains of rhodopsin or long-wave sensitive (LWS) cone opsin, designed to confer activation of the ubiquitous $G_q$ signalling pathway natively observed with melanopsin, to the faster switching and more sensitive rhodopsin and LWS-cone opsins, respectively [83]. Using this approach, $G_q$-activity was successfully conferred to rhodopsin (but not LWS-cone opsin) chimeras, albeit with a lower efficiency and slower rate of activation compared to wild-type melanopsin [83] Figure 3.

Further approaches

Multi-characteristic opsins. While microbial and mammalian opsins are receiving most attention, a number of further innovative strategies are also being pursued. One example is the so-called multi-characteristic opsin (MCO, Patent No PCT/US2017/059922), developed by Nanoscope Therapeutics and which has now progressed into clinical trials (NCT04919473, NCT04945772). Delivery of MCO into ON-bipolar cells of the rd10 degenerate mouse retina has been shown to restore visually guided behaviors in mice at ambient light levels [14], yet there is little information published on the nature of the MCO construct [206] making it difficult to fully interpret these results in the broader context of therapeutic visual restoration.

Light-gated mammalian ion-channels. An alternative to a purely optogenetic approach is to photosensitize normally non-light sensitive mammalian ion channel proteins using synthetic compounds. Such an approach could provide faster signalling kinetics compared to mammalian opsins without necessarily coming with the disadvantages of microbial opsins. However, the significant disadvantage of this approach is that the chemical photosensitising agents need to be regularly supplied to the site of the ion channel. In this regard, maleimide-azobenzene-glutamate tethered mutant GluK2 ionotropic glutamate receptors are most advanced, and have been shown to accurately restore time-resolved electrophysiological and behavioral responses when expressed in either ON-bipolar cells or RGCs [29, 68]. Perceptively, the burden of constant/repeated photosensitiser supply could be overcome by deployment of drug delivery systems similar to those employed in the treatment of neovascular retinal diseases [159].

Switchable opsins. Most recently, the spectrum of candidate tools for vision restoration has been expanded to include the class vertebrate opsins from non-mammalian origin. While also being G-protein coupled receptors, certain non-mammalian opsins may possess desirable features not found in their mammalian counterparts. Lamprey parapinopsin, in particular, is a fish opsin coupling to $G_i$ that can be switched on and off by different wavelengths of light with sub-second precision [48] and has been shown to restore retinal light responsiveness when expressed ON-bipolar cells of degenerate mouse retina [160].

Restoring cellular responsiveness is not restoring functional vision

For most of the opsins proposed for vision restoration it has been clearly shown that when ectopically expressed in retinal neurons they

render these cells light responsive. Moreover, it has been studied how transduced cells integrate with their neural environment inside the retina and to what extent physiological response patterns of healthy retina can be replicated (e.g. ([121]) Figure 4). These are essential prerequisites that critically determine the upper limits of any restored functional vision, but high-fidelity responses on retinal level alone are not necessarily sufficient to restore high fidelity functional vision. Signals leaving the retina need to encounter a central neural circuitry that can make sense of these signals in order to create a perceived visual scene that can be made sense of. Several techniques have been employed to demonstrate that exposing optogenetically treated eyes of laboratory animals to light stimuli evokes neural responses also in central brain areas. Examples therefore include optical imaging of changes blood flow or oxygenation in the murine visual cortex ([44]; [199]) (Figure 2A–C), recording of visually evoked potentials ([123]; [166]; [185]) or microelectrode array recordings from the dorsal lateral geniculate nucleus (dLGN) ([32]). Collectively, these data show without doubt that visual stimulation of optogenetically treated retinal neurons results in changes in activity within retino-recipient brain areas. Such non-behavioral data are highly important, as much of the early phase vision restoration studies are performed in mice, and mice are not regarded as highly visual animals. They naturally rely more on scent, and indeed visual performance on a behavioral level is not easily measured.

behavioral level is not easily measured. Still, behavioral level tests are necessary to prove that the entire axis from the “photoreceptorised” retinal neuron to functional image vision has been restored.

The probably best-established assay to assess mouse vision at the behavioral level is the optomotor reflex ([27]). This is attractive as it is comparably robust and widely validated to measure grating acuity and contrast sensitivity with reasonably good resolution. However, the optomotor reflex is mediated at the subcortical level ([27]) and thereby does not truly assess what is considered as functional vision. Another widely used assay, the light/dark box, on the contrary does require cortical activity, however this behaviour requires only detection of irradiance – as opposed to space-resolved pattern vision – and is in fact a behaviour that can be driven solely by melanopsin expressing intrinsically photosensitive retina ganglion cells (ipRGCs) in the absence of rods and cones ([169]). Several attempts have been made to overcome such limitations. These include a modification of the light/dark box into an open field arena where optomotor-drum type gratings are presented on one side of a dividing wall and equilluminsecent grey on the other ([32]). Also modified “hidden” platform ([199]), fear avoidance ([18]; [67]) and observance of behaviour upon exposure to naturalistic scenes ([199]) based approaches have been employed. Our group has successfully expanded the novel object recognition paradigm by adding

a modifiable visual context (Figure 6 D, E) to measure functional vision ([44]; [186]). All in all, such tests have provided good evidence that all four major candidate mammalian opsins can restore functional vision. However, this approach is limited, and fails to provide a graded measure for restored vision making comparison between different optogenetic tools difficult. Moreover, most of these tests are dependent on some period of learning, which may be impaired in blind mice. And finally, the high light intensities required for some tools – particularly microbial opsins – are likely to induce fear responses in nocturnal, species such as mice via the native light-responsivity of melanopsin-expressing ipRGCs ([169]). Such fear or light aversion responses would potentially mask changes in functional vision.

The discussion in this review thus far has mainly focused on preclinical animal models but similar considerations need to be taken into account when it comes to clinical trials, and how best to assess restoration of vision in human patients: It is clear that restoring electroretinogram responses in a patients eye is not predictive of a patients ability to identify optotypes or gratings in a visual acuity test. But even if an optogenetic treatment has enabled a patient to successfully complete a visual acuity test, this does not by itself confirm that they are able to use this visual information to perform tasks (relevant in every day’s life). This distinction is made by the terms “visual function” vs. “functional vision” ([8]). While the first one, “visual function”, is relatively straight forward to assess and of high importance in the proof-of-principle stage (see also Section 9), “functional vision” is what ultimately matters to the patients. To-date, there is a limited number of validated tools available and usually rather coarsely graded. Examples for such tests are mobility parcourse or standardized street or room-environments ([37]; [164]). At the current stage of optogenetic therapy development it is important to include and report validated tests of functional vision to enable inter-trial comparability. Importantly, there is yet no consensus on which assays of functional vision are best suited for this purpose, and it is likely that established assays alone may not be sufficient to ensure signs of therapeutic efficacy are detected.

Choice of target cells and subcellular targeting strategies

By targeting upstream neurons some intraretinal signal processing can be retained

A common hallmark of most degenerative retinal disorders is that early cell loss is largely restricted to the outer retina photoreceptors, with the bipolar cells and retinal ganglion cells, which are involved in the integration and transmission of visual signals and their associated interneurons (horizontal cells and amacrine cells), remaining largely intact. This provides a number of options regarding which cell type to target with optogenetic tools, and two main approaches are being investigated. The first approach is to target RGCs – located in the innermost layers of the retina, and the cells that are ultimately responsible for transmitting visual information to the brain. This approach offers a method to directly control the information transmitted from the retina to central brain regions. It is moreover relatively easy to target RGCs with current viral delivery strategies making this approach relatively easy to translate into clinical settings (Figure 2 E).

The alternative approach to targeting RGCs is to target the outermost surviving cells of the degenerate retina – degenerate cones or bipolar cells (Figure 2 F). Restoring photosensitivity at that level has several possible advantages compared to delivery to RGCs, not least the potential to make use of existing retinal circuitry to aid (more natural) processing of visual signals. This could potentially allow a more faithfully recreation of visual code – a feature that will likely be lacking when targeting RGCs, where light detection is simply converted into a widely uniform output to the brain.

Intraretinal signal processing. Visual information is not provided to the brain as simple space and time resolved brightness encoding.

Instead of just relaying visual information to the brain, the retina performs substantial signal processing. In the healthy retina, visual information is processed by as many as 40 parallel pathways and dedicated RGC subtypes ([9]; [193]), each responsible for encoding specific aspects of the visual scene, like contrast or direction of movement (Figure 7). To date the ability of optogenetics to restore this diversity of functional responses and truly replicate natural visual code has not been examined. The more of these functional channels can be recovered by an optogenetic gene therapy, the more natural will be the information transmitted to the brain, increasing its potential to extract useful information from the received data.

Tuning RGCs into photoreceptors. At the current stage, clinical trials employ intravitreal injections of AAV to target optogenetic tools to as many RGCs as possible. This current “non-selective” approach will usually result in the simultaneous delivery of optogenetic tools to multiple distinct classes of RGCs, regardless of which particular visual feature they naturally deliver to the brain. Such widespread delivery to RGCs is unlikely to replicate normal visual code. In this scenario, instead of encoding specific aspects of visual information (and performing distinct roles in visual processing) different RGC channels will now respond in a uniform manner, collectively now providing the brain with a jumbled or aberrant version of the normal visual code – potentially even sending conflicting signals, via ON and OFF channels for example (now presumably both showing ON responses). This “non-selective” approach also poses the risk of delivering optogenetic tools to RGCs naturally involved in non-visual tasks and may therefore disrupt other biological processes. For example, this may lead to transduction of melanopsin expressing ipRGCs – a class of endogenously photosensitive RGC classically involved in non-image forming pathways such as circadian entrainment ([58]; [85]; [104]; [148]). Such a situation could conceivably result in the generation of aberrant light responses in ipRGCs, leading to a disruption of circadian pathways and sleep wake cycles (and increased likelihood of conditions associated with circadian disruption) ([1]; [57]; [59]).

Notably, to date it is not understood how cortical circuits may handle or adapt to an aberrant visual code received from the retina. While there might be capacity for adaptation at the cortical level, at least efferences from those RGCs projecting into non-visual brain areas (like endogenously photosensitive RGCs) will remain problematic. Speculatively, it may ultimately be preferable to selectively target one or a few major classes of RGCs in order to restore a specific (and useful) feature of vision, rather than target all RGCs non-specifically. While more specific targeting of RGCs is possible using cell-type specific promoters (as suggested for bipolar targeting approaches) it is not currently clear which of the 40 plus RGC types ([9]; [193]) should be targeted to encode the most important and most useful features of vision, or indeed which cell type specific promoters can be used to specifically target such populations.

Reviving intraretinal signal processing by re-sensitizing cones. The most straightforward way to recover as many of these output channels as possible is by targeting optogenetic tools into the lowest order of retinal neurons surviving in the degenerate retina, thereby retaining the natural path the visual signal takes through the retina. The potential thereof has been demonstrated by Busskamp et al. in 2010 who targeted a hyperpolarizing microbial opsin into surviving cone photoreceptors that had no remaining native light responsivity. In fact, by this strategy they were able to restore key features of intraretinal signal processing, including direction sensitivity and center-surround inhibition ([26]). These experiments provide the important confirmation that intraretinal signal processing can be revived. Yet, cones are typically only available as target cells for optogenetic vision restoration for a narrow time interval: At the point of intervention, native vision should be mostly absent so that the optogenetic vision would not interfere with it and a sufficient number of cones should still survive. While in humans for the individual cone, this may be a time span of several years, retinal degeneration progresses over space and time and thus many cones might have already died in one area while they are just beginning to lose light sensitivity in another ([28]). This is a valuable proof of principle, though, on the path towards optogenetic vision restoration as a clinical reality it needs to be taken into account that the first patients participating in clinical trials are those with end-stage retinal degeneration and thus likely to have no relevant number of cones remaining. Moreover, cones seem to be unable to survive in a rod-less retina for yet not completely understood reasons ([28]) and it is unlikely that optogenetic treatment could halt or slow down cone death. Finally, as retinal degeneration progresses, extensive circuitry remodelling takes place (discussed in further detail in Section 6). Besides the pure availability of cones as a target cell, this remodelling will likely affect the way in which the retina is capable of computing visual features.

Bipolar cells are the lowest order of retinal neurons that are primarily retained throughout the course of retinal degeneration. Targeting these should therefore yield the highest potential to restore lifelike retinal output channels. Indeed, several groups have successfully expressed optogenetic tools in retinal ON bipolar cells of rd1 mice and have assessed features of intraretinal signal processing in some detail ([32, 38, 41, 46, 68, 67, 112, 121, 126, 199, 198]). An overt feature of retinal signal processing is the segregation of stimuli into “ON” and “OFF”-type responses, i.e. into responses that positively correlate with incident light intensity and responses that inversely correlate with incident light intensity. In the absence of native rod and cone input to bipolar cells, this would be mainly a consequence of the synaptic link from ON-bipolar cells via AII amacrine cells to OFF-bipolar cells ([23]). Interestingly, while ON responses could be observed in all studies, ON and OFF responses were only reported by some authors ([38, 41, 68, 67, 126, 199]), while others explicitly report that this feature of intraretinal signal processing had not been observable ([46, 68, 112, 121]). While all these studies were conducted in mice, thereby excluding inter-species effects, one possible explanation for this discrepancy are the strength (and specificity) of the different promoters and/or the transduction efficacy of the different delivery viral vectors used ([109]). This seems logical but presently alternative explanations cannot be ruled out: It is particularly noteworthy that in studies finding ON and OFF responses, mice were typically younger when they underwent behavioral or electrophysiological testing as compared to those in studies where only ON responses were detected (exception: ([126])). For these mice had a shorter course of retinal degeneration and thus probably a lower degree of retinal remodelling. It is therefore possible that active retinal remodelling – while not impeding signal propagation to the brain per se – abolishes the AII-OFF circuit and with it, possibly other relevant aspects of intraretinal signal processing. This would be consistent with the recently observed formation of novel gap-junctions from ON-bipolar cells to AII cells in the degenerating retina ([151]). Finally, it also needs to be considered that obtaining cell type-specific delivery within the retina is challenging (see Section 7), and the different AAV2 serotypes and promoters chosen in the various studies might have had slightly different (off-target) transduction patterns that could potentially explain the OFF-responses observed in some studies.

To date, most studies using specific targeting to bipolar cells have focused on selective transduction of ON bipolar cells. However, a significant advantage of targeting bipolar cells is the ability to also specifically target the OFF pathway via selective delivery to OFF bipolar cells (see also ([184])). Restoration of the ON pathways is considered to be more functionally important than restoration of the OFF pathway, and thus while cell specific promoters have been identified for OFF bipolar cells this line of study has received far less attention. Ultimately it may be possible to target both ON and OFF pathways with simultaneous delivery of different optogenetic tools to ON (depolarizing tool) and OFF bipolar cells (hyperpolarising tool) to more fully replicate normal visual processing in the retina.

Evidence for a benefit of targeting upstream neurons is indirect. By simply analyzing the polarity of light-responses in electrophysiological assays, the presence of such ON/OFF circuitry is relatively easy to assess and observations from the aforementioned studies show us that under the right conditions some intraretinal computation can be restored when targeting ON-bipolar cells. But overall, it is unclear whether this is in any way an informative surrogate for the integrity of the retinal circuitry. It is also unclear how it relates to function at a behavioral level. Behavioral assays employed in reported studies are too diverse to allow comparison across study borders and so far, only a few studies employing sensitive behavioral tests directly compare intraretinal computation retaining ON-bipolar cell delivery and non-intraretinal computation retaining RGC delivery: using rhodopsin as an optogenetic tool Cehajic-Kapetanovic and colleagues observed that rd1 mice that expressed rhodopsin in bipolar cells performed better in light/dark box tests as compared to rd1 mice expressing rhodopsin within RGCs. Moreover, they observed that only ON-bipolar cell delivery enabled mice to respond to complex naturalistic scenes ([32]).

Pertinent in this regard are the findings of [68]. In their study, a light-activatable ion-channel was delivered into either RGCs or ON-bipolar cells. When targeting RGCs, responses to a single 1s flash stimulus recorded from the retinal output level appeared highly similar, and – consequently – all strongly correlated. In contrast, when delivering the light-activatable ion-channel onto ON-bipolar cells, responses to an identical stimulus were much more diverse, and correlated with each other only in clusters, indicating distinct functional RGC groups ([68]). Indeed, we can confirm this observation re-analyzing our own data ([121]) in an analogous fashion: When delivering red-shifted Channelrhodopsin (ReaChR) into (predominantly) RGCs, obtained peri-stimulus histograms correlated with 0.58 [IQR: 0.18–0.81] while correlation was significantly weaker, when targeting ON-bipolar cells (0.51 [IQR: 0.14–0.73], data from 76 to 57 responsive units observed on microelectrode array recordings from RGC level, $p < 0.05$).

Subcellular targeting strategies are explored to mimic ON and OFF responses or improve kinetics

From a translational perspective, delivering optogenetic tools into specific cell types – in particular bipolar cells – bears certain challenges. By contrast, widespread RGC delivery can be obtained relatively easy using intravitreal delivery strategies and ubiquitous promoters (see Section 7). Therefore, efforts are being made to optimize the way optogenetic tools operate inside RGCs.

Physiologically RGCs receive input originating from a number of rod and cone photoreceptors. In many types of RGCs, via differential usage of ON and OFF pathways and lateral connectivity, a center-surround antagonism is obtained which supports contrast and initiates edge detection ([52]). It has been proposed that by differential subcellular targeting of excitatory and inhibitory optogenetic tools to the soma and dendrites of an RGC, such center-surround inhibition could be re-enacted ([77]). Indeed at the level of individual RGCs, this strategy has proven successful to create center-surround inhibition and “edge detection” ([77]).

A challenge with this strategy is that until now it is not possible to ensure that both excitatory and inhibitory opsins are expressed in each cell transduced. While smaller targeting motifs could be identified and tested ([207]), the size of two opsins alone exceeds the packaging capacity of a single AAV. Thus, a mix of different AAVs would need to be injected, resulting in the formation of pure ON, OFF RGCs besides the intended center-surround RGCs. Such functional diversity might seem attractive at first, but as it only depends on the random event of which cell becomes transduced by which virus, it still needs to be determined if and how this would carry additional information to the brain, and how this information might be interpreted. Once the challenge of uneven delivery is solved it will be interesting to see in what way such subcellular targeting strategies are beneficial for system-level responses. In any case, reflecting the size of the dendritic field of an RGC as compared to a rod or cone photoreceptor, sharpening the receptive field of optogenetically treated RGCs by subcellular targeting of a single opsin might be beneficial even without aiming for center-surround inhibition.

Interaction of optogenetic tool with the target cell environment

Another consideration to make when choosing the optimal target for optogenetic gene therapy is how the selected optogenetic tool operates inside its host cell environment. This is most obvious for mammalian opsins, which need to be able to activate an intracellular signalling cascade which is available in the host cell, as discussed in Section 3. But also, microbial opsins may differentially shape retinal light responsiveness depending on the cell type they are expressed in. For example, in a recent study we observed that when delivered into RGCs, ReaChR-mediated light responses decay relatively quickly upon continuous stimulation. While this is considerably less evident when ReaChR is delivered into (predominantly) ON-bipolar cells (Figure 5). This observation is not yet well understood but it appears likely that spike-generating RGCs might be less well equipped to handle the large, continuous depolarizing ion-flux mediated by channel opsins. On the other hand, for non-spiking ON-bipolar cells such continuous depolarizing flux might more closely resemble the physiological mode of operation enabling these cells to sustain light responsiveness also upon long-term stimulation.

Inner retinal remodelling in retinal degeneration

The nature of retinal and central remodelling

While it has been known for many years that the post-photoreceptor visual pathway is functional in very late-stage retinal degeneration, it is only more recently that attention has been given to the impact of such remodelling of visual pathways on the potential success of optogenetic visual restoration. Following the loss of rods and cones, the remaining cells of the retina are initially spared from further widespread degeneration. However, the loss of photoreceptor input is accompanied by extensive retinal network rewiring over time (Figure 8) [130], [152]. Significant levels of retinal and central remodelling are apparent in both mouse models [101]; [181] and human subjects [99], [100], and furthermore it appears this remodelling is not confined to the retina, but also occurs in the retino-recipient visual pathways in the brain [34]; [212].

Robert Marc’s laboratory originally examined the late stages of retinal degenerations in rd1 mice, a widely used model of human retina degeneration [31]; [53] containing a mutation in the β subunit of phosphodiesterase 6b (PDE6b) that is also known to cause autosomal recessive retinitis pigmentosa in humans McLaughlin et al., 1993. Using computational molecular phenotyping Marc et al. revealed a sustained period of remodelling in the degenerate retina [130], initially showing relatively subtle changes in neuronal structure in the inner retina, progressing to large-scale reorganization of cell to cell contacts and retinal circuitry, and including cell loss [99]; [130] (Figure 8). Three distinct phases of degeneration were described by Marc et al. [130]. Phase I and phase II refer to the different stages of rod (phase I) and then cone (phase II) photoreceptor degeneration. Additionally, more subtle changes are also known to occur during these periods including sprouting of neurites from various cell types, changes in bipolar cell glutamate and NMDA receptor expression [98]; [129], retraction of bipolar and horizontal cell dendrites, cell movements, and initial glia cell activation [152]. Some particularly interesting changes during these early phases have been described in detail recently: Rod bipolar cells form unphysiological gap junctions with AII-amacrine cells and GABAergic amacrine cells begin to form synapses with horizontal cells [151]. Phase III represents a more sustained period of retinal remodelling, with significant sprouting of neurites, formation of new synaptic sites and re-wiring of cell-to-cell contacts, with increasing levels of cell migration, disruption of inner retinal layers and cell death. Phase III is subdivided into early, mid and late phase III reflecting distinct periods of retina remodelling [130]. Early phase III involves significant neurite remodelling, with increased sprouting from amacrine and horizontal cells (Fariss

et al., 2000; [130], accompanied by Müller cell hypertrophy and initial neuronal cell death. By mid-phase III global remodelling is evident, with progressive cell death, formation of multicellular fascicles, significant microneuroma growth, and migration of amacrine and bipolar cells into the inner plexiform and ganglion cell layers. Late phase III shows a plateau in levels of remodelling with continued cell death

and a regression of microneuromas, increased levels of Müller cell hypertrophy and completion of the glial seal, invasion of blood vessels and migration of retinal pigmented epithelium cells to the inner retina layers and progressive cell death.

In the context of optogenetic vision restoration the fate of bipolar cells as one proposed target for optogenetic tools is of particular interest.

Indeed, there is evidence for some bipolar cell death occurring over the late phases (Phase III) of degeneration. A human post-mortem study found that loss of cells in the inner nuclear layer is moderate (20% lower than age matched controls in a population aged 77 years in median), and only evident in eyes from individuals that had a visual acuity of “light perception only” or worse before their death ([141]; [165]). Accordingly, optical coherence tomography (OCT) studies indicate a 12% thinning of the inner nuclear and inner plexiform layer together only in patients with no remaining light perception but not individuals with better visual function ([197]). OCT data also indicate that this effect might be genotype-dependent, with patients with RPGR mutants even showing an increase in inner nuclear layer thickness ([63]). In comparison, data for bipolar cell loss reported for mouse models are variable and range from none ([178]), over 10% and 20% ([63]; [180]) to 60% ([35]). But not only does the survival of bipolar cells matter in the context of using these cells as a target structure for optogenetic gene therapy. Especially when using mammalian GPCR-type opsins it is important to consider to what extent native signal transduction cascades are preserved. Evidence has accumulated over the last fifteen years that bipolar cell glutamate signalling is altered following loss of rod and cone input. In particular, mGluR6 mediated TRPM1 modulation is found to be lost by some authors ([12]; [98]; [129]). Notably, this does not essentially mean components of this signalling cascade are no longer expressed. Reduction of mGluR6 mRNA expression is reported to be transient over the course of degeneration ([3]) and we could recently show, that in P90 rd1 mice (resembling stage III remodelling) mRNA expression of both mGluR6 and TRPM1 was back to normal ([72]). Rather, there is evidence for a mis-localization of its individual components, in particular mGluR6 ([2]; [47]; [156]) and TRPM1 ([70]) that likely impedes functional coupling but not necessarily utilization in the context of mammalian opsin-based vision restoration approaches.

Oscillating retinal networks and their role in vision restoration

It is clear from numerous different animal models of retina degeneration (including rd1 and rd10 mice, and P23H-1 and RCS rats) that loss of photoreceptors leads to the emergence of rhythmic activity in the retina, with the generation of oscillatory local field potentials and spontaneous bouts of RGC spike firing ([24]; [36]; [75]; [133]; [140]; [155]; [176]; [177]). Indeed, studies have now shown that two distinct oscillatory networks are evident in the degenerate retina ([51]), including an outer retina network driven by interactions between remaining cones, horizontal cell and rod bipolar cells – where retrograde release of GABA from horizontal cells onto cones produces oscillating local field potentials at a frequency of around 3 Hz ([78]). A second oscillatory network is located in the inner retina, and produces oscillatory field potentials in the range of 8–10Hz ([24]; [195]). Multiple mechanisms have been proposed to explain this oscillatory activity. It has been proposed that remodelling and rewiring of the retina following degeneration may be responsible ([97]; [130], [129]; [153]; [181]), yet current evidence suggests that such oscillations are an intrinsic property of electrically-coupled AII amacrine cells and ON bipolar cells ([24]; [36]; [132]; [194]; [195]), and that these events are normally suppressed by photoreceptor input. Support for this theory is supplied by observations that similar ∼10 Hz oscillations are induced in healthy wildtype retina following pharmacological blockade of photoreceptor input to bipolar cells ([140]; [194]; [195]), or photoreceptor bleaching ([139]). At the molecular level, it has been shown that gap junctions assembled from Connexin36, physiologically functioning to electrically couple AII amacrine cells are critical for the development of retinal oscillations ([92]) and that not their expression but rather their phosphorylation state (possibly being the result of an imbalance of dopaminergic and glutamatergic signalling) changes during retinal degeneration ([93]). Thus, no major rewiring of the retina seems to be required to generate these rhythmic bouts of oscillatory activity – just a loss of photoreceptor input.

This increase in spontaneous activity, and – at an output level - the increased spontaneous firing of RGCs has implications for visual restoration and may impact the success of optogenetic treatments. Increased spontaneous firing from RGCs likely adds significant noise to transmitted signals, reducing signal to noise ratio, and ultimately reducing the clarity of information sent to brain ([49]). Furthermore, the efficiency by which external electrical stimulation induces changes in RGC spike firing has been shown to be reduced in multiple mouse models of retina degeneration compared to healthy wildtype retina ([75, 81, 131, 144, 210, 211]), and there appears to be a strong correlation between levels of oscillatory activity and efficiency of RGC stimulation ([81]). Furthermore, pharmacological blockade of oscillatory activity results in improved RGC responses to electrical stimulation in rd10 mice ([71]). Thus it seems possible that such oscillatory signals may directly act in some way to suppress or reduce the efficacy of exogenous stimulation of RGCs. Therefore, combined such oscillatory potentials may act to both reduce the potency of optogenetic based stimulation, whilst also reducing the signal to noise ratio of restored responses. Indeed, several studies have now investigated the potential consequences of these oscillatory signals for optogenetic vision restoration. Pharmacological blockade of oscillations with gap junction blockers have been shown to increase the signal-to-noise ratio of optogenetic restored light responses, following viral delivery of channelrhodopsin to RGCs ([13]) and also following delivery of human rhodopsin to ON bipolar cells of degenerate retina ([49]). Interestingly, recent studies have shown that expression of the optogenetic tool Opto-mGluR6 selectively in ON bipolar cells can by itself reduce levels of oscillatory activity and spontaneous RGC firing to levels similar to that seen in wildtype retina ([111]). Notably, it has been shown that benzodiazepines can be used to abolish retinal oscillations and improve RGC stimulation efficiency ([71]), and similar pharmacological approaches may in future be combined with optogenetic therapies to improve functional outcomes.

How does remodelling impact upon optogenetic restoration therapy?

In the majority of human retinal diseases degeneration is slow and not uniform across the retina. As such, patients can experience prolonged periods of only partial visual loss, where the retina still retains some level of basic physiological function. Current clinical trials for restorative therapies typically only target patients with end stage disease (see Section 9). However, it is currently unknown how Phase III remodelling would affect therapeutic outcome.

What is the impact of remodelling on the functional capacity of surviving visual circuits? The profound nature of retinal remodelling raises some important issues regarding optogenetic therapies for visual restoration. It is strongly predicted that the anatomical changes associated at least with Phase III, but possibly also with earlier phases of degeneration, will lead to changes in retinal pathways and connectivity and potentially impact the quality of restored vision following regenerative therapy. Whether photoreception is restored at the level of bipolar cells or retinal ganglion cells, the visual signal produced must be transferred through remaining circuits in the retina and brain to support vision. In visually intact individuals these circuits efficiently transmit the visual signal and perform appropriate computations. As elements of this circuitry degenerate and connections between remaining neurones become disordered, it would be most surprising if the functional properties of the circuit were not impacted. In this regard, Pfeiffer et al. recently speculated how pathologic gap junctions formed between rod bipolar cells and AII amacrine cells, as early as in Phase I remodelling could impact basic intercellular interactions such as lateral inhibition, a key perquisite for complex vision ([151]). In fact, while there is a wealth of information on the anatomy of remodelling, the impact on circuit function remains largely untested. There has not been a systematic assessment of the impact that remodelling has on the spatial and temporal resolution of the visual code, or on its ability to perform neuronal computations. This is of fundamental importance for restorative therapies, as circuit performance could realistically place an upper limit on the quality of vision supported, and it is possible that late intervention may limit the efficacy and success of regenerative therapy in the retina.

Is intervention at early stages of degeneration advantageous to functional outcome? While there are obvious advantages to intervening later in disease progression, when the possibility of disrupting residual rod and cone driven vision is avoided, it is possible that remodelling at later stages of disease could potentially degrade the fidelity of retinal circuitry to such an extent as to fundamentally limit the quality of restored vision. While studies in animal models at late stage of degeneration ([44, 111]) and early results from clinical trials ([163]) (see Section 9) suggests that optogenetic therapies can function in a highly remodelled state, it does not exclude the possibility that intervening earlier could deliver better therapeutic outcomes. If end stage remodelling does indeed significantly impede higher levels of restored visual function, this could lead to poor results in ongoing late-stage remodelling trials and an overall underestimation of the potential benefits of optogenetics for visual restoration. Intervening in earlier stages of retinal remodelling would be associated with additional risks for the trail participants. Thus, animal studies that are able to evaluate if and what the functional consequences of advanced remodelling for optogenetic vision restoration are, are urgently needed.

Late-stage remodelling may affect the choice of retinal target. The profound changes in connectivity or rewiring associated with late-stage Phase III remodelling can ultimately affect the choice of retinal survivor cells to target with optogenetic treatments. In the later stages of disease, it may be more effective to target the retinal output neurons – the RGCs, rather than bipolar cells. Whilst you may lose benefits associated with increased retinal processing you may retain some fidelity through the preservation of retinotopic mapping. It needs to be mentioned that, to date, this is still a theoretical concept. Worth considering, RGCs are also affected by aberrant connectivity in the late stages of degeneration. Though possibly to a lesser extent than upstream neurons, it has not been investigated what this exactly means in functional terms.

Expression patterns may change during degeneration. Besides circuit rewiring – an additional aspect needs to be taken into account: (how) does the protein machinery in the different retinal neurons change during degeneration and how does this affect the suitability of that cell type as a target for optogenetic gene therapy? This question is particularly pressing in the context of G-protein coupled receptor-type opsins, as these need an intracellular cascade to couple to. In fact, data from our group and others indicate that this seems not to be a major issue: even after long-term degeneration melanopsin is able to induce light-evoked responses following expression in RGCs ([45]) and similarly Opto-mGluR6 (see Section 3) can do so when expressed in ON bipolar cells ([111]). There is still relatively little understanding of which intracellular signalling cascades are utilised. With regard to ON bipolar cells we recently showed that key components of the native Go-signalling pathway remain transcriptionally unchanged over the course of retinal degeneration, suggesting that expression of a compatible opsin will likely simply revive the native cascade ([72]).

Retinal remodelling in response to regenerative therapy – potential for neuroprotection and compensatory remodelling of visual circuits? The final critical unknown regarding the significance of remodelling for visual restoration is whether early intervention (prior to large scale remodelling) could either prevent disruptive remodelling or ensure that it progresses in such a way as to maximize the impact of restored photoreception. Either or both outcomes are possible. If delivered during early stages of retina disease optogenetic therapy may by itself impact on the rate of further retinal degeneration, remodelling of visual circuits and pathological cell loss. Delivery of therapy could prove neuroprotective, not just for the visual circuits, but also for the primary photoreceptor degeneration. There are reports that reactivation of rod signalling circuity can lead to a reversal of retinal remodelling and recovery of visual signalling circuitry ([201]). Secondly, it is possible that therapy may lead to compensatory remodelling. Increased external input could constitute a form of environmental enrichment, which is protective for degenerative conditions in general, and retinal degeneration specifically. Optimistically, this could lead to gradual improvements in therapeutic outcome over time as circuits reorganise to optimally process and interpret the newly restored visual signals.

Central remodelling

Whilst the implications of photoreceptor loss on retinal anatomy have been studied, significantly less is known about the consequences of photoreceptor loss for central visual circuitry. It has been established that in the early stages of retinal degeneration in a rat model cortical V1 cells exhibited weaker orientation selectivity, lower optimal spatial and temporal frequency values, and smaller receptive fields compared to controls ([34]). It remains unclear to what extent this reflects a central reorganization of visual circuitry. And there are no functional studies of central visual circuit properties when all retinal input is lost. However, from clinical studies on retinitis pigmentosa patients it is understood that post-thalamic pathways remain structurally mostly intact ([145, 168]). Moreover, subjects treated with gene replacement therapy for Leber’s congenital amaurosis appear to show normal visual pathway anatomy and cortical responses measured by (functional) magnetic resonance imaging ([16]). Using established paradigms to test for cortical plasticity in ex-vivo electrophysiological recordings from rd10 mouse visual cortex a recent study could moreover show that cortical plasticity is retained, even in very late disease stages of retinal degeneration ([16]). Certainly, more research is required in this regard, but the current evidence suggests that cortical remodelling secondary to retina degeneration – if any – is not a significant limiting factor in optogenetic vision restoration.

Strategies for targeted optogenetic tool delivery

Cell type specific targeting of optogenetic tools has proven feasible in animal models, most notably the targeted expression of optogenetic tools in ON-bipolar cells. While potentially beneficial in terms of final outcomes and the nature of vision restored (see sec:5), there are a number of hurdles to overcome before these approaches can be translated into clinical therapies. In animal research models, many studies have used Cre-lox based approaches, typically employing a genetically engineered mouse in which Cre recombinase is expressed in a specific cell type (under control of a cell type specific promoter), and thus permits expression of AAV constructs containing floxed genes only within cell types of interest.

While successful in animal models it is clear that such Cre-lox based approaches are obviously not translatable into human studies. An alternative approach is the inclusion of cell type specific promoters within the AAV constructs themselves. Again, proof of concept has been demonstrated using the Grm6 promoter (and shortened variants) for targeting ON-bipolar cells ([41, 86]). However, there are some disadvantages of using such promoters. Cell type specific promoter constructs (such as grm6-sv40 ([41])) are often seen to produce lower levels of trans-protein expression compared to non-specific constructs using more ubiquitous promoters (such as CAG or EF1α) ([41]). This results in a practical trade-off between the potential advantages of targeted expression described above and achieving sufficient levels of optogenetic protein expression to induce functional light responses – as they might be limiting for some optogenetic tools.

The use of cell type specific promoters may be further compounded in the context of retina degeneration and remodelling, where changes in gene expression may reduce the functional specificity and efficacy of such approaches. However, transcriptomic studies from our own group ([74]) suggest only relatively minor changes in gene expression within the ON-bipolar cells during the progression of degeneration, and Grm6 has been specifically demonstrated to persist during degeneration (e.g.: [86]). However, studies at the protein level ([198]) show a markedly reduced ability of the grm6-sv40 construct to drive cell specific protein production in degenerate retina compared to wild type.

Equally important for translation to human studies is the specificity and efficacy of vector constructs across species. Evidence is accumulating that cell specific promoter constructs shown to work in mice are likely to have altered efficacy and specificity when expressed in other species ([84, 102, 124]) and thus careful consideration will be required before adopting this approach for human trials ([69]). Ultimately, due to reasons of technical complexity (and issues of remodelling), at the current stage, it may be necessary to settle on the simpler delivery of optogenetic therapies to RGCs, and forgo the potential benefits of specifically targeting bipolar cell populations.

Safety and toxicity of optogenetic therapies

In the human data thus far reported, there has been no evidence of severe toxicity or immune reaction following deployment of retinal optogenetic therapies, however, of necessity these examinations have been limited to assessments of gross anatomy on clinical examination (e.g: [163]). Whilst this precludes any cataclysmic systemic rejection of the tools so far tested, it does little to assess toxicity at lower, microscopic levels that nonetheless may have damaging effects, slowly over time, which may limit efficacy of the therapy. Such potential toxic effects can be thought of in four main categories: 1. Phototoxicity, 2. Immunotoxicity, 3. Direct toxic effects of optogenetic protein, or 4. Indirect toxic effects due to the function of the protein. Each of these should be considered and investigated while we are now more and more entering the stage of clinical translation.

Phototoxicity

Phototoxic effects, defined as effects on the retina related to light incident onto the retina are today well investigated and have been reviewed in detail elsewhere ([87]; [113]; [213]). In brief, photothermal, photomechanical and photochemical toxicity can be distinguished, with photochemical toxicity (light-induced changes to biological molecules) being one of the main relevance in the context of optogenetic vision restoration. The potential of a certain light stimulus to induce phototoxic damage mainly depends on the energy delivered, being a function of its intensity and wavelength. Consensus agreement has been achieved regarding which light intensities are acceptable for ocular exposure and which must be avoided ([88]). Such consensus has been widely implicated into national legislations. As discussed in sec:3, some optogenetic tools cannot be activated by ambient light would require stimulation by light intensifying goggles to function and such goggles must operate within the limits of light safety thresholds. While more recently a number of microbial opsin variants have emerged that can be operated within these safety thresholds, phototoxicity concerns had been a hurdle for opsins operated by (high energy) blue light. This point has been excellently made in [170], where details on the calculation of light safety thresholds are presented.

Immunotoxicity

Investigations of immunotoxicity have so far focused on the immune responses to AAV based vectors themselves [208] and this area has recently been elegantly reviewed in this journal [25]. Less has been reported on the immunogenic effects of the delivered tools to the retina independent of this. [103] found greater toxic effects on delivery of a non-mammalian protein (GFP) when compared to a native, retinal protein (retinoschisin) using the same AAV vector. Thus, giving some reason concerns regarding the immunogenic potential of exogenous expression of non-mammalian proteins (such as channelrhodopsins) in the retina and highlights an area of unmet need in research.

While the immunotoxic effects of channelrhodopsin expression have been examined at the retinal level in a small number of studies ([14, 46, 183, 205]), this has generally been limited to qualitative or semi-quantitative immunohistochemistry. Examining such local immune responses (e.g. glial activation) is further complicated by the process of retinal degeneration, which can in itself activate similar processes (see Section 6). While these, and other, qualitative studies have been helpful to exclude massive deleterious immune responses to optogenetic tools ([14, 46, 183]) they require expansion with a systematic quantitative and likewise sensitive analysis of reactions that may have subtle effects on efficacy or long-term toxic effects.

Indeed, in vivo primate data raises concerns that such low-level toxic effects might exist: Gauvain et al. report levels of ocular inflammation assessed by fundoscopy [69]. These data [69], Suppl Fig 1) shows that after treatment, vitreous cells were present in eight out of twelve examined treated macaque eyes and persisted over several examinations until the end of the study in six eyes. In the wider (non-ocular) literature, more in-depth studies on optogenetic tool immunotoxicity have been performed [171] including on central ([142]) and peripheral ([128]) neurons, demonstrating the existence of immune responses that limit long-term functionality [128].

Only microbial optogenetic tools have so far moved to clinical trial (see below) and while similarly there is no evidence of massive, deleterious immune reactions in one trial [134]; another trial reports one patient with vitritis and anterior chamber cells (NCT02556736) – however details have not yet been published. Further observations are therefore eagerly awaited from these early trials to determine the risk of a significant immune-type reaction in human eyes/patients.

Direct protein toxicity

Further observation both in disease models and humans will also be essential in forms of optogenetic induced toxicity beyond immune considerations. There is a considerable body of work regarding the direct toxic effects of proteins in retinal cells – especially in relation to dominant negative effects leading to autosomal dominant retinitis pigmentosa ([138]) secondary to mal- and over-expression of rhodopsin. These effects can lead to cell death by various routes ([6]) and are in themselves providing a fruitful target for novel gene therapies (e.g.[146]). While the mechanisms of direct protein toxicity resulting from opsin expression could be inferred from the example of rhodopsin, as yet direct toxic effects of exogenous (over)expression of mammalian opsins have not yet been systematically investigated.

While, similar to mammalian opsins, the direct toxic effects of microbial opsins have not been reported for the retina, observations have been made of such proteins when exogenously expressed in both central ([142]) and peripheral ([128]) neurons. Indeed, Miyashita et al. demonstrate morphological changes in cortical neurons following ChR2 expression. They speculate on possible mechanisms for this: either as a direct toxic effect of protein overexpression, or indirect, perhaps via disruption of intracellular calcium signalling due to the non-native conductance introduced by the channelrhodopsin protein [142]. Notably, observations regarding protein toxicity of microbial opsins are yet limited and observations made for one opsin can not necessarily be generalized to all opsins of that class. There are, for instance, engineered microbial opsins that are codon optimized ([76, 118]) and are designed for improved membrane targeting which may affect toxicity of such tools [117].

Functional toxicity

Exogenously expressed optogenetic tools could be predicted to have deleterious effects secondary to their functional activation, especially when we consider microbial opsins mediating directly light activated membrane conductances. This direct action is in contrast to the highly regulated receptor cascades activated by mammalian opsins with associated compensatory pathways to efficiently restore homeostasis and protect the cell. Such control is particularly important to bring intracellular calcium concentrations back into the physiological range to avoid excitotoxic processes [5]; [55, 108]. While activation of exogenously expressed microbial opsin does not appear to lead to loss of retinal ganglion cells [185, 205] our own data indicates that it may alter neuronal physiology to the extent that electrophysiological parameters - such as the rate of resting action potential firing could be attenuated [73]. Therefore, reinforcing that toxicity can have effects short of cell survival and further investigation is necessary to ensure the most efficient deployment of optogenetic approaches to visual restoration.

Translation of optogenetic vision restoration into clinics is ongoing

Optogenetic studies in large animals

Targeted approaches to optogenetic therapies can be well modelled in small animal models – the mouse is particularly attractive both due to its ease of husbandry, the extensive experience in its genetic manipulation and available models of retinal degeneration. Modelling the higher register of visual processing (especially that related to foveal vision) will however be more difficult in mice and along with regulatory requirements for preclinical safety work there is a need for optogenetic approaches to be investigated in larger animal models.

Indeed, there is increasing evidence (Table 2) that some but not all [172] optogenetic constructs delivered by AAV can induce appropriate expression also in larger animal models. These can even restore electrophysiological responses to light at the level of the retina and cortex and a case report of optogenetics being used in a patient has been presented [163]. As primate models of retinal degeneration become increasingly available [143], this will become an increasingly attractive area of investigation, over and above its attraction for gathering regulatory pre-clinical safety data.

Study	Species	Approach	Construct	Results
[90]	Marmoset	In vivo intravitreal injection; ex vivo Immunohistochemistry (IHC) & Multiple Electrode Array Electrophysiology (MEA).	$ChR2$	Protein expression confirmed by IHC, Light dependent responses on MEA
[170]	Macaque, human	Ex vivo culture; with IHC and MEA	$ReaChR$	Protein expression & MEA light responses confirmed in cultured retinal explants of both species
[33]	Macaque	In vivo intravitreal injection, in vivo ocular imaging and cortical electrophysiology, ex vivo IHC and MEA	$CatCh$	Protein expression & MEA light responses confirmed, visually evoked cortical responses confirmed. In vivo ocular inflammation noted in small number of animals
Ameline et al., (2017)	RPE65-deficient Briard dog	In vivo intravitreal injection & OCT imagining. Ex vivo IHC & MEA	Melanopsin, $ChR2$	Protein expression confirmed by IHC, no change in retinal thickness over 21 months on OCT; restoration of light dependent responses on MEA.
[103]	Macaque, human	Ex vivo human retina explant; in vivo intravitreal and subretinal injection to macaque.	$Jaws$	Protein expression confirmed by IHC. Patch clamp electrophysiology demonstrates Jaws mediated light response
[123]	Macaque, Royal College of Surgeons Rat	In vivo intravitreal injection, examination electrophysiology & visual evoked potentials. Ex vivo patch clamp electrophysiology	Melanopsin	Protein expression confirmed by IHC. Reconstitution of electrophysiological light responses.
[65]	Human induced pluripotent stem cells	Ex vivo expression of optogenetic tool in ipRGC derived human cone photoreceptors which were transplanted into transplanted blind mice (Cpfl1/Rho−/−)	$Jaws$	Restoration of electrophysiological responses at photoreceptor and ganglion cell level
([135], [136])	Macaque	In vivo expression off optogenetic tool with adaptive optics ophthalmoscopy & in vivo calcium imaging	$Chrimson R$	Light induced optogenetic responses seen in vivo up to one year post injection.
[163]	Human	In vivo (“PIONEER” clinical trial)	$Chrimson R$	Light dependent tasks performed by reported patent

Also of note are the development of other large eye animal models of retina degeneration. These include the P23H miniature pig [161] and the P347L rabbit [98, 196] models of human RP. These animals have a visual streak which represents a better model of the human fovea than any available mouse model. They are also far more affordable and logistically simpler to work with than primate models, yet to date these models have not been studied in the context of optogenetic vision restoration. Future studies of optogenetic therapies in non-primate large eye animal models are likely an important step in translating optogenetic therapies to clinical use, and will likely be highly beneficial for optimization of AAV delivery to the human retina. Finally, it is worth mentioning that more recently primate models for retinitis pigmentosa ([89]) and a cone dystrophy ([143]) have been identified and may have their place in translational optogenetic vision restoration studies in future.

Clinical trials: challenges and directions

This increasing body of evidence illustrates the potential of optogenetic techniques to restore, at the very least, sensitivity to light in degenerate retina in multiple animal models as well as behavioral level light responses in some cases. The logical extension of this has been to translate these encouraging results into human patients. However, in common with the panoply of emerging therapies in IRDs, this presents a collection of challenges, not least in constructing a well-controlled clinical trial. The vast heterogeneity of retinal degenerations, especially in their natural history, emphasizes the importance of patient assessment and selection ([94]) to both ensure a fair comparison and to allow an appropriately phrased research question to be answered efficiently. Similarly, adopting useful, comparable, functional endpoints is a challenge in an area where therapeutic trials are still in their infancy. More practical realities of such trials are also important with a tension in trial design between demands for best-possible safety and the necessity to proof efficacy ([79]; [119]). For instance, while from a safety perspective it seems logical to include only patients with end stage retinal degeneration it could be argued that advanced retinal remodelling may impede optogenetic gene therapy to restore functional vision (see Section 6). Similarly, with several gene therapy vectors now being studied in humans (and some even approved for clinical use) it could be argued that optogenetic trials should only utilize these given their existing safety data. Conversely, with optogenetic approaches to visual restoration, transfecting a sufficient proportion of the target cell is critical and therefore utilizing the most efficient vectors is ever more important and could justify the selection of alternative vectors ([119]). Many of these challenges are articulated, and associated areas of unmet research need are presented in a recent manifesto by the Monaciano consortium ([187]).

Questions for clinical trials: selecting a tool and target cell population

Selecting the most appropriate optogenetic tool and target population has proven complex in animal models (Section 5), even before the documented interspecies variability discussed above (Section 7) is considered. In such a complex system, animal models are best employed to determine the “most likely” candidates to pursue in humans. While clinical trials may then provide the information to what extent a chosen optogenetic strategy can result in a restored functional visual perception, they will likely not be able to deliver the details necessary to fully understand why a chosen treatment has—or has not—worked and where optimization is needed. Herein, a steady flow of information between clinical trials and more flexible cell or animal models-based research that can resolve modes of operation of a certain treatment approach in organ and even cellular detail will be needed.

Multiple factors may influence candidate selection at different stages of clinical investigation: the selection of ChrimsonR in the PIONEER trial provides an apposite example ([163]). The overall light sensitivity of Chrimson R is below the level that would allow activation by ambient light ([105]) and is therefore not immediately an obvious choice for visual restoration. Its selection, and the incorporation of signal amplifying goggles, does however have several advantages. Firstly, in this phase I/II trial, any activity mediated toxicity or adverse effects (e.g. circadian disruption, unpleasant sensation of light, photophobia) could be avoided simply by keeping the patient in general room light. Secondly, the requirement for light amplifying goggles for activation allowed for an additional control: where light responses seen only with the goggles on, but not off, could more confidently be ascribed to the treatment. Secondly, although not activated by low levels of light, channelrhodopsin variants do provide virtually optimal kinetic performance under bright light conditions, with fast onset and offset kinetics and independently of the availability of second-messenger cascades – and therefore offer a near perfect tool to test the potential of optogenetics. Finally, the advantages of including computer assisted glasses are that they allow a level of signal processing and optimization to be performed prior to presentation of the light signal on the retina, including increasing levels of contrast and or edge detection, and image stabilisation. Combined, these features potentially offer the best opportunity to assess the feasibility of optogenetic visual restoration in humans at this stage. What needs to be kept in mind is that, if goggles are required, there is a need for long-term technical support – beyond the duration of the trial – and discontinuation of support of two main retinal implant devices has recently exemplified how this can be problematic ([182]). Finally, these goggles may come with a (possibly neglectable) wearing discomfort.

Current clinical trials have made use of high efficiency, non-specific capsid and promoter constructs to maximize the quantity of optogenetic protein expressed (and so the effect size seen). Again, this approach is useful in early phase clinical trials where questions relate to proof of principle, requiring transgene expression at sufficient levels in the maximum number of cells possible. However, there is now evidence that cis-regulatory elements evoking strong ubiquitous transgene expression are somehow linked to toxicity ([208]) and thus consideration should be given in clinical trials to population specific delivery possibly tolerating more moderate expression levels.

Efficacy measurement and design considerations in clinical trials

Selecting efficacy endpoints for clinical trials in optogenetic vision restoration gene therapy bears certain challenges that are substantially different from those faced in other gene therapy trails. In many ways, the obstacles are more akin the situation in retinal implant development ([22]) and experience gathered in this field can support efficient translation of optogenetic vision restoration into clinics. This is reflected in recent consensus paper on outcome measures for vision restoration approaches ([8]).

Controls. Comparison of a functional metric before and after treatment is the classical way of assessing treatment efficacy. Like retinal implants, stimulation of optogenetically treated retinas with tools that require light intensities above the ambient light levels (microbial opsins) requires utilization of signal enhancing goggles. Functional tests can be performed with goggles switched on and off thereby offering a valuable additional instance of control. This approach has been chosen in the PIONEER trial ([163]) and is recommended by the HOVER taskforce ([8]). Also, in instances where no signal enhancement is required, an additional instance of control can be obtained. For example, by using spectrally defined stimuli that do or do not match the spectral sensitivity of the optogenetic tool.

Measuring light responsiveness. In early optogenetic trials – from a researcher’s perspective – it will be critical to provide objective measures that evidence restoration of light responsiveness in a treated retina. This will not answer the question whether a patient is able to make use of this light responsiveness in any meaningful way (functional vision), but it will provide robust biological proof of principle. Electrophysiological approaches like electroretinogram recordings and visually evoked potentials can be employed and the (temporal) properties of the optogenetic tool under study can serve to discriminate tool-restored from residual native responses ([8]).

In some cases, it may be possible to identify the area of the retina or even the individual cells that express an optogenetic tool after treatment. This may be the case after subretinal vector delivery or when fluorescent proteins are delivered together with the optogenetic tool ([163]). In those cases above-mentioned electrophysiological approaches may be paired with focal stimuli to provide a spatial correlation between the treated cells and obtained responses. Similarly, psychophysical techniques either using modified, validated microperimetry devices ([40]) or in a more experimental context single (photoreceptor) stimulating adaptive-optics devices ([157]) may be informative.

Particular to the field of optogenetic vision restoration is the great variability amongst the optogenetic tools that could be employed. This includes aspects like light sensitivity, desensitization or “slow” response kinetics. Stimulus paradigms therefore need to be carefully designed to meet the requirements of the optogenetic tool used in a particular trial.

On the other hand, some low-level stimuli (bright, broadband, sustained, long inter-stimulus-intervals) that are compatible across the spectrum of optogenetic tools currently under debate could enable basic inter-study comparability should be included into trial protocols.

Measuring vision in the ultra-low range. The first patients receiving a trial optogenetic therapy are those with little, or no, remaining perception of light. The improvement in visual function from current optogenetic therapies should be expected to bring patients beyond the range of ultra-low vision. Therefore, endpoints are needed that can robustly and sensitively detect functional gain within the range of ultra-low vision – and ideally beyond. As detailed in the HOVER consensus paper the availability of validated tests operating in this range is limited ([8]). Nevertheless, adherence to validated tests is highly important, not only to ensure best possibly comparability between studies ([8]). This situation becomes even more complex when aiming to measure visual ability (i.e. how well a patient is able to complete visual tasks of every day relevance), instead of pure visual function.

What if trials fail to meet their functional endpoint? We currently know hardly anything about how optogenetic “vision” would manifest in a treated individual. Moreover, there is a possible tension between regulators preferring primary functional endpoints that are both, validated and relevant to the patient and researchers prioritizing those best ensuring detection of any biological sign of efficacy. While we are still in the early days of optogenetic vision restoration it is important that functional data from trials failing to meet their predefined functional endpoint is not simply disregarded but equally reported. Where the reported tests or analysis strategies diverge from what was primarily planned, this obviously needs mentioning, but they may still contain valuable information for the design of future optogenetic treatment approaches.

Current clinical trials

Four groups have currently registered early phase clinical trials of optogenetic therapy in retinal degeneration patients: the ‘PIONEER’ trial (NCT NCT03326336), ‘RESTORE’ (NCT04945772), ‘RST-001 Phase I/II Trial’ (NCT NCT02556736) and the ‘BS01 Phase I/II Trial’ (NCT04278131). All four have involved delivery of microbial type opsins via AAV constructs under high efficiency, non-specific promoters with a primary endpoint of safety and tolerability and improvement in visual function as a secondary endpoint – details of their specific trial protocols are summarised in Table 3.

Phase	Patients (main inclusion criteria)	Intervention (tool, promoter, capsid, injection route)	Outcome/endpoints	Visual assessment (method used)	Inflammation assessment (method used)	Toxicity Assessment (method used)	Any results reported?
PIONEER (NCT03326336)	I/IIa	“Non-syndromic Retinitis Pigmentosa (confirmed on full field ERG)”	Chrimson R; AAV 2.7m8 intravitreal injection to worse seeing eye	Primary: safety & tolerability of escalating doses Secondary: Visual acuity, Visual function, mobility, quality of life measures, immune response	Freiburg Visual Acuity & Contrast Test, full field threshold stimulus test (FST), perimetry, Novel visual search, counting, mobility and detection tasks	Slit lamp biomicroscopy and grading according to Standardization of Uveitis Nomenclature Working Group	Slit lamp biomicroscopy, fundus autofluorescence, OCT	Results from one participant have been published ([163])
RESTORE (NCT04945772, NCT04919473)	I/IIa& IIb	“Advanced Retinitis Pigmentosa based on clinical examination, dilated fundus examination, and genetic testing”	Multi-characteristic opsin (MCO); delivered by AAV2 vector via intravitreal injection	Primary: Effect on ability to perform Y maze task; Adverse reactions Secondary: Effect of therapy on vision, light sensitivity, visual function. Secondary: Pharmacokinetics	Freiburg visual acuity, pupillometry, FST, perimetry, VQ-25, Shape recognition, optical low task	Not reported	Not reported	Press release only https://www.ophthalmologytimes.com/view/optogenetic-gene-therapy-restores-vision-in-11-rp-patients
RST-001 Phase I/II Trial’ (NCT NCT02556736). Allergan	I/IIa	“Advanced Retinitis Pigmentosa, vision no better than hand motion”	Intravitreal delivery of “RST-001 a gene therapeutic”	Primary: Number of Participants with Any Grade 3 or Greater Adverse Event (AE) considered Related to RST-001 (Grade 3 being: Severe or medically significant but not immediately life-threatening). Secondary: Change in Ambulation, Visual Acuity, Quality of life (VQ-25 score)	Not reported	Not reported	Not reported	Primary result summary on clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/results/NCT02556736)
NCT04278131	I/II	“Retinitis Pigmentosa, bare light perception in at least one eye”	AAV vector delivering channelrhodopsin-variant ChronosFP	Safety, change in threshold sensitivity	Electrophysiology	Not reported	Not reported

PIONEER in particular sparked much interest when individual results from a patient were reported ([163]). In this case report a patient with IRD and visual acuity of “light perception only“ was seen to recover the ability to perform low-level visually guided tasks and electroencephalographic light responses to return following injection with AAV encoding the microbial opsin ChrimonR. There were no apparent deleterious alterations in anatomy following treatment or evidence of intraocular inflammation. However, full results of this phase I/II dose escalation study yet to be reported.

The Multi-characteristic opsin (MCO) construct used in the RESTORE trial has been demonstrated to be safe and tolerable in a phase I/IIa trial (NCT04919473, press release: [50]). Little detail has been published with regard to the nature of MCO in the academic literature. It has however allowed a Phase IIb trial to commence with a saline injected control group with the hope of demonstrating efficacy (NCT04945772).

Recently several major pharmaceutical companies have entered the field to successfully translate further optogenetic vision restoration into clinics. Novartis has recently acquired Arctos Medical (aiming to commercialize Opto-mGluR6 ([199]; [204]) as well as Vedere Bio (cone-opsin ([18]; [200]; [204])) and Acucela has licensed a Rhodopsin based approach ([32]) and is preparing to enter clinical tails ([4]). It is therefore likely that we will see a significant

increase in the number of active clinical trials in the field. From a scientist’s perspective, this underscores the importance of efficacy and safety metrics that enable best possible inter-trail comparability.

Conclusion and future directions

Animal models have demonstrated the power and potential of optogenetic vision restoration and proof-of-principle clinical data has just recently been released. The next big challenge will be to move from restoration of a rudimentary form vision to a vision that offers the space and time resolution necessary for (basic) routine daily tasks. Achieving this goal will require a closer synergy between clinical and preclinical efforts: Clinical trials will provide the information to what extent a chosen optogenetic strategy can result in a restored functional visual perception. They may even provide cues on what is missing to deliver a better perception. However, they will be unlikely to deliver the level of detail for a systematic optimization of the approach. Flexible cell or animal models enable resolving modes of operation of a certain treatment approach in organ and even cellular detail. Fed with specific questions from clinical trials, these allow designing next, improved treatment approaches for re-translation.

Decisions are still to be made regarding the optimal tool and target cell type(s) for clinical treatments, ultimately timely progress is more likely to be constrained by our ability to develop protocols allowing safe and reliable delivery of appropriate levels of tools to the target cells of choice – and obviously the translation of these approaches to human subjects. In this respect there is an urgent need to develop reliable short-promoter constructs to allow optogenetic tools to be delivered to individual cell populations in human subjects.

It is expected that future developments in opsin engineering may yield tools with more favorable properties (improved combinations of light sensitivity and temporal kinetics for example). Moreover, the potential of specifically targeting the soma of survivor retinal ganglion cells with an optogenetic tool offers the potential to spatially sharpen light responses at the retinal output level.

Currently from the animal experiments we know that treatment with optogenetic therapy at the very late stage of the disease, when all photoreceptors have been lost and the retinal circuitry is remodeled, is still beneficial – including at behavioral level. However, as these optogenetic approaches translate into treatments, we are yet to really define the optimum time window to result in maximum treatment benefit. This may make the difference between restoring rudimentary vision allowing for coarse orientation and restoring complex functional vision.

Immediate questions are how safe such treatments are in long-term, and if delivery of such tools does not accelerate degeneration or cause damage to recipient cells. Equally, from what we know about retinal remodelling, early intervention could even result in slowing the rate of inner retinal remodelling as a form of neuroprotection. Many of these questions could and should be approached in the current preclinical model systems.

In the immediate future the promise of optogenetic treatments rests on the outcome of ongoing and commencing clinical trials aimed at showing the potential efficacy of such approaches. Though expectations are high, at this stage what is most needed are robust, reproducible signs of efficacy that justify and direct future studies.

Financial support

1. University Hospital Gießen and Marburg Research Funds (15/2020MR to ML)

2. German Research Foundation (LI 2846/6-1 to ML)

3. ProRetina Foundation (Pro-Re/Projekt/Gilholey-Whitehead-Lindner.04-2021 to ML and MJG)

4. Medical Research Council UK (MR/S026266/1 to MWH)

5. Wellcome Trust (205151/Z/16/Z to MJG)

6. NIHR Clinical Lectureship (CL-2019-18-004 to MJG)