Switching on the Lights: The Use of Optogenetics to Advance Retinal Gene Therapy
Authors: Therese Cronin, Jean Bennett
Journal: Molecular Therapy (2011)

Photoreceptors carry a high number of mitochondria necessary to maintain their high metabolic rate. This allows them to respond rapidly to small changes in illumination. However, the metabolically demanding role of light reception appears to render photoreceptors particularly vulnerable to mutations; this is manifested in the many clinical forms of hereditary retinal degeneration (RD).1Punzo C Kornacker K Cepko CL Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa.Nat Neurosci. 2009; 12: 44-52Crossref PubMed Scopus (379) Google Scholar Remarkably, in many cases the remainder of the retinal circuitry remains capable of firing despite the death of the photoreceptor cells.2Mazzoni F Novelli E Strettoi E Retinal ganglion cells survive and maintain normal dendritic morphology in a mouse model of inherited photoreceptor degeneration.J Neurosci. 2008; 28: 14282-14292Crossref PubMed Scopus (184) Google Scholar,3Lagali PS Balya D Awatramani GB Münch TA Kim DS Busskamp V et al.Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.Nat Neurosci. 2008; 11: 667-675Crossref PubMed Scopus (435) Google Scholar In this issue of Molecular Therapy, Caporale et al. exploit this residual capacity for signal transmission so as to endow retinal ganglion cells with the ability to take over the task of light detection from the photoreceptors in conditions of RD.4Caporale N Kolstad KD Lee T Tochitsky I Dalkara D Trauner D et al.LiGluR restores visual responses in rodent models of inherited blindness.Mol Ther. 2011; 19: 1212-1219Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar The retina is a thin sheet of photosensitive neural tissue that lines the inside of the eyeball. It comprises three distinct layers of cells that vertically transmit the information carried by photons from the light-sensitive rod and cone photoreceptors via bipolar cells to the inner ganglion cells. Individuals with RD are progressively losing their rods and cones and face the prognosis of blindness. Many of these patients cannot hope to benefit from the ongoing clinical trials for gene replacement or pharmaceutical therapies, and a restoration of light sensitivity to the retina in its degenerated state may be the only possible treatment. The work by Caporale et al. was enabled by the growing field of optogenetics, which involves the heterologous expression of light-sensitive channels or receptors in a neuron, allowing control of neuronal firing by an external light impulse.5Nagel G Szellas T Huhn W Kateriya S Adeishvili N Berthold P et al.Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.Proc Natl Acad Sci USA. 2003; 100: 13940-13945Crossref PubMed Scopus (1937) Google Scholar When applied to retinal gene therapy, these tools allow non-light-sensitive retinal cells to fire directly in response to light without the need to be triggered by the photoreceptor cells.3Lagali PS Balya D Awatramani GB Münch TA Kim DS Busskamp V et al.Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.Nat Neurosci. 2008; 11: 667-675Crossref PubMed Scopus (435) Google Scholar These latest results from the Flannery laboratory4Caporale N Kolstad KD Lee T Tochitsky I Dalkara D Trauner D et al.LiGluR restores visual responses in rodent models of inherited blindness.Mol Ther. 2011; 19: 1212-1219Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar support the potential of optogenetics to address the ambitious goal of vision restoration. Several groups in retinal research have used optogenetic tools from nature, such as channelrhodopsin2 (ChR2; ref. 3Lagali PS Balya D Awatramani GB Münch TA Kim DS Busskamp V et al.Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.Nat Neurosci. 2008; 11: 667-675Crossref PubMed Scopus (435) Google Scholar) and halorhodopsin (ref. 6Busskamp V Duebel J Balya D Fradot M Viney TJ Siegert S et al.Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa.Science. 2010; 329: 413-417Crossref PubMed Scopus (478) Google Scholar). By contrast, Caporale et al.4Caporale N Kolstad KD Lee T Tochitsky I Dalkara D Trauner D et al.LiGluR restores visual responses in rodent models of inherited blindness.Mol Ther. 2011; 19: 1212-1219Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar made use of an artificial optical switch that was engineered to generate large currents in neuronal cells.7Szobota S Gorostiza P Del Bene F Wyart C Fortin DL Kolstad KD et al.Remote control of neuronal activity with a light-gated glutamate receptor.Neuron. 2007; 54: 535-545Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar The switch, developed by Volgraf et al., is based on the ionotropic glutamate receptor (iGluR) family.8Volgraf M Gorostiza P Numano R Kramer RH Isacoff EY Trauner D Allosteric control of an ionotropic glutamate receptor with an optical switch.Nat Chem Biol. 2006; 2: 47-52Crossref PubMed Scopus (483) Google Scholar In addition to mediating the principal excitatory currents of the central nervous system, the iGluR family carries a structurally ubiquitous ligand-binding domain that serves as a suitable template to achieve generic cell signaling. To the iGluR ligand-binding domain is incorporated a chemical called azobenzene which also serves as the carrier for the ligand glutamate; the entire moiety is termed maleimide-azobenzene-glutamate (MAG; see Figure 1). The light-dependent mechanics of the switch is achieved by the light-sensitive azobenzene, which undergoes isomerization from a trans to cis configuration in response to short wavelengths of light (≈380 nm). This azobenzene isomerization induces the molecular motion that allows glutamate (or its analogs) to be drawn into the pore of the receptor and the cation channel to open. This is the trigger for membrane depolarization, the necessary step for the neuron to fire. The geometry is reversed and the channel closed under longer-wavelength light (≈500 nm) and in conditions of darkness. Caporale et al. applied this light-dependent iGluR6 prototype (LiGluR6) to the ganglion cell and demonstrated its potential as a platform for further engineering to meet the goal of reversal of blindness. To accomplish their results, the authors achieved a high level of ganglion cell-specific LiGluR6 expression in the retinas of mice with retinal degeneration (rd1 mice). Tested ex vivo, electrical recordings show the retinas to be responsive to 380-nm light only when exposed to MAG, demonstrating the tight control of the switch. In vivo, photoresponses transmitted to the cortex were correlated in the behavior of entrained rd1 mice in a water-maze test. However, it is the restoration of the pupillary light reflex that sets this report apart from earlier studies of retinal optogenetic therapies. The pupillary light reflex refers to the contraction of the pupil as an involuntary response to light. This truly objective measure of non-image-forming photoresponse is mediated not only by the photoreceptors but also by a small subset of melanopsin-expressing ganglion cells.9Lucas RJ Hattar S Takao M Berson DM Foster RG Yau K-W Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice.Science. 2003; 299: 245-247Crossref PubMed Scopus (666) Google Scholar,10Tsujimura S Ukai K Ohama D Nuruki A Yunokuchi K Contribution of human melanopsin retinal ganglion cells to steady-state pupil responses.Proc Biol Sci. 2010; 277: 2485-2492Crossref PubMed Scopus (74) Google Scholar The authors selected the ideal mouse model in which to test this response: triple-knockout mice that lack melanopsin in addition to essential components of the phototransduction machinery and therefore do not show a pupillary light reflex.11Hattar S Lucas RJ Mrosovsky N Thompson S Douglas RH Hankins MW et al.Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice.Nature. 2003; 424: 76-81Crossref PubMed Scopus (934) Google Scholar Caporale and colleagues show that the reflex is restored in the mutant mice if their ganglion cells express LiGluR6 and they have been treated with MAG. It is important to note that targeting the ganglion cells may not take full advantage of the signal-processing power of the inner retina, where a lateral as well as a vertical transmission of information occurs.12Lagali PS Balya D Awatramani GB Münch TA Kim DS Busskamp V et al.Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.Nat Neurosci. 2008; 11: 667-675Crossref PubMed Scopus (273) Google Scholar Much of this processing takes place in the interneuron retinal bipolar cell layer before the light signal reaches the ganglion cells. Skipping the bipolar cells sacrifices to some extent two of their important processing roles: (i) discrimination of an object darker than background as compared with an object lighter than background and (ii) detection of the edges of an object whereby the center of the field is contrasted to an inhibitory surround. The absence of such processing would in theory lead to loss of resolution and noise reduction. A more immediate obstacle to be overcome for clinical application of this strategy is the requirement that a sufficient level of the photoswitch MAG be delivered to meet the demands of newly turned-over iGluR6 channels. Encapsulated cell therapy may offer the best solution by allowing long-term administration of the glutamate analog in the eye.13Tao W Application of encapsulated cell technology for retinal degenerative diseases.Expert Opin Biol Ther. 2006; 6: 717-726Crossref PubMed Scopus (89) Google Scholar With this technology still under development, its incorporation may slow the progress of the LiGluR6 optical switch toward the clinic. Nonetheless, LiGluR6 has a significant advantage over naturally occurring optical switches such as ChR2. The main deficiency of ChR2 is its high level of desensitization, which reduces the overall current that can be achieved.5Nagel G Szellas T Huhn W Kateriya S Adeishvili N Berthold P et al.Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.Proc Natl Acad Sci USA. 2003; 100: 13940-13945Crossref PubMed Scopus (1937) Google Scholar,14Ishizuka T Kakuda M Araki R Yawo H Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.Neurosci Res. 2006; 54: 85-94Crossref PubMed Scopus (298) Google Scholar,15Lin JY Lin MZ Steinbach P Tsien RY Characterization of engineered channelrhodopsin variants with improved properties and kinetics.Biophys J. 2009; 96: 1803-1814Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar In the future, channel modifications may improve such limitations; in the meantime the LiGluR6-MAG channel offers a higher level of channel conductance and can therefore achieve more adequate photon capture. Mammalian opsin is a tough act to follow for those who endeavor to engineer photosensitive channels. For optogenetic retinal therapies to be translated to the clinic, specific hurdles must be overcome. Among these, the channels should be made receptive to more physiologically useful wavelengths, the efficiency of gene transfer to primate inner retinal cells needs to be improved, and the retinal cells that express the channel must be carefully selected so as to optimize visual processing while occupying regions of intact retinal circuitry. Alternative strategies aimed at restoring the visual function lost in RD are being investigated, and these too have their own hurdles. Retinal regeneration through the manipulation of stem cells combined with tissue engineering has been the focus of intense research efforts and yielded some recent successes.16Silva GA Silva NM Fortunato TM Stem cell and tissue engineering therapies for ocular regeneration.Curr Stem Cell Res Ther. 2011; (e-pub ahead of print 11 April 2011)PubMed Google Scholar However, the molecular mechanisms needed for appropriate stem cell differentiation remain largely unknown, as are the requirements for successful integration and survival of the derived cells following transplantation. The microelectronic retinal implant has also shown much promise since clinical trials commenced with the epiretinal chip (SecondSight) in February 2002 (ref. 17Chader GJ Weiland J Humayun MS Artificial vision: needs, functioning, and testing of a retinal electronic prosthesis.Prog Brain Res. 2009; 175: 317-332Crossref PubMed Scopus (195) Google Scholar). The progress of these chips to full clinical use remains limited until improved resolution and full biocompatibility can be achieved. As for the optogenetic approach, all these strategies require that layers of the inner retina and associated cell types maintain their architecture for a period of time following photoreceptor degeneration. Although this is broadly true in many instances of RD, it may nonetheless be of benefit to promote the parallel development of neurotrophic therapies that may optimally support the health of inner retinal cells. The study described here reminds us that if creative but methodical approaches are used to test strategies for vision restoration in advanced RD, the obstacles to the clinic will be overcome.