Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated ...virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell–specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.
The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore ...retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca2+-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus CMV) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.
Vision restoration using microbial opsins has substantial clinical potential; however, it requires high-level expression of a foreign protein in the patient’s eyes. Our study shows the feasibility of obtaining safe and functional expression in primates using a cell-specific promoter and provides the basis for further clinical development of this optogenetic strategy.
The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation ...is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.
Targeting the photosensitive ion channel channelrhodopsin‐2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, ...the high intensity of blue light necessary to activate channelrhodopsin‐2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red‐shifted light is vastly lower than that of blue light. Here, we show that a red‐shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV‐ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV‐ and lentivirus‐mediated optogenetic spike responses in ganglion cells of the postmortem human retina.
Synopsis
A red‐shifted channelrhodopsin (ReaChR) was targeted to retinal ganglion cells using three models in parallel: mouse, macaque, and human. Safe orange illumination was able to trigger light responses in all three systems.
The red‐shifted channelrhodopsin ReaChR restored light responses at the retinal, cortical, and behavioral levels in blind rd1 mice, using light intensities below the safety limit for the human retina.
Optogenetic light responses were demonstrated in explanted postmortem macaque and human retina, infected ex vivo with viral vectors encoding ReaChR.
The study presents the first electrophysiological recordings of optogenetic light responses in ganglion cells obtained directly from the human fovea as well as the far peripheral human retina.
A red‐shifted channelrhodopsin (ReaChR) was targeted to retinal ganglion cells using three models in parallel: mouse, macaque, and human. Safe orange illumination was able to trigger light responses in all three systems.
Complete congenital stationary night blindness (cCSNB) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. GRM6 mutations are the third most prevalent cause ...of cCSNB. The Grm6-/- mouse model mimics the human phenotype, showing no b-wave in the electroretinogram (ERG) and a loss of mGluR6 and other proteins of the same cascade at the outer plexiform layer (OPL). Our aim was to restore protein localization and function in Grm6-/- adult mice targeting specifically ON-BCs or the whole retina.
Adeno-associated virus-encoding Grm6 under two different promoters (GRM6-Grm6 and CAG-Grm6) were injected intravitreally in P15 Grm6-/- mice. ERG recordings at 2 and 4 months were performed in Grm6+/+, untreated and treated Grm6-/- mice. Similarly, immunolocalization studies were performed on retinal slices before or after treatment using antibodies against mGluR6, TRPM1, GPR179, RGS7, RGS11, Gβ5, and dystrophin.
Following treatment, mGluR6 was localized to the dendritic tips of ON-BCs when expressed with either promoter. The relocalization efficiency in mGluR6-transduced retinas at the OPL was 2.5% versus 11% when the GRM6-Grm6 and CAG-Grm6 were used, respectively. Albeit no functional rescue was seen in ERGs, relocalization of TRPM1, GPR179, and Gβ5 was also noted using both constructs. The restoration of the localization of RGS7, RGS11, and dystrophin was more obvious in retinas treated with GRM6-Grm6 than in retinas treated with CAG-Grm6.
Our findings show the potential of treating cCSNB with GRM6 mutations; however, it appears that the transduction rate must be improved to restore visual function.
Abstract
Variations in
the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in ...children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.
Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns ...within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.
Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of inner retinal neurons is a promising avenue to ...restore vision in retinas having lost their photoreceptors. Expression of optogenetic proteins in surviving ganglion cells, the retinal output, allows them to take on the lost photoreceptive function. Nonetheless, this creates an exclusively ON retina by expression of depolarizing optogenetic proteins in all classes of ganglion cells, whereas a normal retina extracts several features from the visual scene, with different ganglion cells detecting light increase (ON) and light decrease (OFF). Refinement of this therapeutic strategy should thus aim at restoring these computations. Here we used a vector that targets gene expression to a specific interneuron of the retina called the AII amacrine cell. AII amacrine cells simultaneously activate the ON pathway and inhibit the OFF pathway. We show that the optogenetic stimulation of AII amacrine cells allows restoration of both ON and OFF responses in the retina, but also mediates other types of retinal processing such as sustained and transient responses. Targeting amacrine cells with optogenetics is thus a promising avenue to restore better retinal function and visual perception in patients suffering from retinal degeneration.
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Trapani and colleagues show that targeting specific cells called AII amacrine cells to express optogenetic proteins shows promise in restoring various visual responses, holding potential for better retinal function in those with degenerative retinal conditions.
Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection ...of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.
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