Publications by authors named "Jens Duebel"

27 Publications

  • Page 1 of 1

Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy.

Front Cell Neurosci 2021 18;15:648210. Epub 2021 Mar 18.

Institut de la Vision, Sorbonne Université, Paris, France.

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.
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http://dx.doi.org/10.3389/fncel.2021.648210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012682PMC
March 2021

Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates.

Commun Biol 2021 Jan 27;4(1):125. Epub 2021 Jan 27.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012, Paris, France.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 10 photons.cm.s. Vector doses of 5 × 10 and 5 × 10 vg/eye transfected up to 7000 RGCs/mm in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.
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http://dx.doi.org/10.1038/s42003-020-01594-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840970PMC
January 2021

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells.

Front Cell Dev Biol 2020 27;8:585675. Epub 2020 Oct 27.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.
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http://dx.doi.org/10.3389/fcell.2020.585675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652757PMC
October 2020

cAMP-Dependent Co-stabilization of Axonal Arbors from Adjacent Developing Neurons.

Cell Rep 2020 10;33(1):108220

Sorbonne Université, Inserm, CNRS, Institut de la Vision, 17 rue Moreau, Paris 75012, France. Electronic address:

Axonal arbors in many neuronal networks are exuberant early during development and become refined by activity-dependent competitive mechanisms. Theoretical work proposed non-competitive interactions between co-active axons to co-stabilize their connections, but the demonstration of such interactions is lacking. Here, we provide experimental evidence that reducing cyclic AMP (cAMP) signaling in a subset of retinal ganglion cells favors the elimination of thalamic projections from neighboring neurons, pointing to a cAMP-dependent interaction that promotes axon stabilization.
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http://dx.doi.org/10.1016/j.celrep.2020.108220DOI Listing
October 2020

Towards optogenetic vision restoration with high resolution.

PLoS Comput Biol 2020 07 15;16(7):e1007857. Epub 2020 Jul 15.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France.

In many cases of inherited retinal degenerations, ganglion cells are spared despite photoreceptor cell death, making it possible to stimulate them to restore visual function. Several studies have shown that it is possible to express an optogenetic protein in ganglion cells and make them light sensitive, a promising strategy to restore vision. However the spatial resolution of optogenetically-reactivated retinas has rarely been measured, especially in the primate. Since the optogenetic protein is also expressed in axons, it is unclear if these neurons will only be sensitive to the stimulation of a small region covering their somas and dendrites, or if they will also respond to any stimulation overlapping with their axon, dramatically impairing spatial resolution. Here we recorded responses of mouse and macaque retinas to random checkerboard patterns following an in vivo optogenetic therapy. We show that optogenetically activated ganglion cells are each sensitive to a small region of visual space. A simple model based on this small receptive field predicted accurately their responses to complex stimuli. From this model, we simulated how the entire population of light sensitive ganglion cells would respond to letters of different sizes. We then estimated the maximal acuity expected by a patient, assuming it could make an optimal use of the information delivered by this reactivated retina. The obtained acuity is above the limit of legal blindness. Our model also makes interesting predictions on how acuity might vary upon changing the therapeutic strategy, assuming an optimal use of the information present in the retinal activity. Optogenetic therapy could thus potentially lead to high resolution vision, under conditions that our model helps to determinine.
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http://dx.doi.org/10.1371/journal.pcbi.1007857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416966PMC
July 2020

AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

Int J Mol Sci 2020 Feb 3;21(3). Epub 2020 Feb 3.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, 75012 Paris, France.

Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.
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http://dx.doi.org/10.3390/ijms21030994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036814PMC
February 2020

Opsins for vision restoration.

Biochem Biophys Res Commun 2020 06 23;527(2):325-330. Epub 2020 Jan 23.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012, Paris, France. Electronic address:

Optogenetics is a biological technique that combines the advantageous spatial-temporal resolution of optics and genetic cell targeting to control cellular activity with unprecedented precision. It has found vast applications both in neurosciences and therapy, particularly in view of its application to restore vision in blind patients. Optogenetics requires the ectopic expression of a so-called opsin to render neurons sensitive to light. There are two types of opsins for modulating membrane potential of neurons: (i) microbial opsins from unicellular organisms that respond to a light stimulus by mediating a flow of ions across the membrane (ii) animal opsins that are naturally present in mammalian retinas that initiate G protein coupled signaling in response to light. The former category has been extensively employed for vision restoration in the past decade with two ongoing clinical trials employing microbial opsins to restore light sensation in retinitis pigmentosa patients. The latter subtype of animal opsins is emerging more recently as strong candidates to restore vision with the promise of greater light sensitivity and tolerability. In this review we will discuss each approach in view of its utility for vision restoration in retinal blindness.
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http://dx.doi.org/10.1016/j.bbrc.2019.12.117DOI Listing
June 2020

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

Nat Commun 2019 10 4;10(1):4524. Epub 2019 Oct 4.

Sorbonne Université, Institut de la Vision, INSERM, CNRS, 75012, Paris, France.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.
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http://dx.doi.org/10.1038/s41467-019-12330-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778196PMC
October 2019

Optogenetic Light Sensors in Human Retinal Organoids.

Front Neurosci 2018 2;12:789. Epub 2018 Nov 2.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.
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http://dx.doi.org/10.3389/fnins.2018.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224345PMC
November 2018

Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids.

Stem Cell Reports 2018 09 9;11(3):665-680. Epub 2018 Aug 9.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, 17, Rue Moreau, Paris 75012, France. Electronic address:

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.
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http://dx.doi.org/10.1016/j.stemcr.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135113PMC
September 2018

Mini-Review: Cell Type-Specific Optogenetic Vision Restoration Approaches.

Adv Exp Med Biol 2018 ;1074:69-73

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, Paris, France.

The expression of light-sensitive microbial opsins is a promising mutation-independent approach to restore vision in retinal degenerative diseases. Using viral vectors, optogenetic tools can be genetically expressed in various subpopulations of retinal neurons. The choice of cell type depends on the availability of surviving retinal cells. If cones are still alive but they lack outer segments, they can be targeted with optogenetic inhibitors, such as halorhodopsin. Alternatively, it is possible to bypass the photoreceptors and to target bipolar cells. In late-stage degeneration, when bipolar cells degenerate, "artificial photoreceptors" can be made from retinal ganglion cells, but with this approach, upstream retinal processing cannot be utilized. However, when ganglion cells are stimulated directly, higher brain regions might be able to compensate for some loss of retinal processing, which is indicated by clinical studies with epiretinal implants, where patients can perform simple visual tasks. Finally, optogenetics in combination with neuroprotective approaches could serve as a valuable strategy to restore the function of remaining cells, as well as to rescue retinal neurons from progressive degeneration.
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http://dx.doi.org/10.1007/978-3-319-75402-4_9DOI Listing
May 2019

A spike sorting toolbox for up to thousands of electrodes validated with ground truth recordings in vitro and in vivo.

Elife 2018 03 20;7. Epub 2018 Mar 20.

Institut de la Vision, INSERM UMRS 968, UPMC UM 80, Paris, France.

In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously between hundreds and thousands of electrodes packed with a high density. However, they require novel methods to extract the spiking activity of large ensembles of neurons. Here, we developed a new toolbox to sort spikes from these large-scale extracellular data. To validate our method, we performed simultaneous extracellular and loose patch recordings in rodents to obtain 'ground truth' data, where the solution to this sorting problem is known for one cell. The performance of our algorithm was always close to the best expected performance, over a broad range of signal-to-noise ratios, in vitro and in vivo. The algorithm is entirely parallelized and has been successfully tested on recordings with up to 4225 electrodes. Our toolbox thus offers a generic solution to sort accurately spikes for up to thousands of electrodes.
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http://dx.doi.org/10.7554/eLife.34518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897014PMC
March 2018

Noninvasive gene delivery to foveal cones for vision restoration.

JCI Insight 2018 01 25;3(2). Epub 2018 Jan 25.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France.

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.
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http://dx.doi.org/10.1172/jci.insight.96029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821199PMC
January 2018

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina.

Mol Ther 2017 11 20;25(11):2546-2560. Epub 2017 Jul 20.

INSERM U968, Institut de la Vision, 75012 Paris, France; UMRS968, Institut de la Vision, Sorbonne Universités, Pierre et Marie Curie University (UPMC) University Paris 06, 75012 Paris, France; Centre National de la Recherche Scientifique (CNRS) UMR7210, Institut de la Vision, 75012 Paris, France. Electronic address:

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-Ca-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.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675708PMC
November 2017

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.

Stem Cells 2017 05 20;35(5):1176-1188. Epub 2017 Feb 20.

Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73 photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73 photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.
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http://dx.doi.org/10.1002/stem.2586DOI Listing
May 2017

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

EMBO Mol Med 2016 11 2;8(11):1248-1264. Epub 2016 Nov 2.

INSERM U968, Paris, France

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.
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http://dx.doi.org/10.15252/emmm.201505699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090658PMC
November 2016

The Thioredoxin Encoded by the Rod-Derived Cone Viability Factor Gene Protects Cone Photoreceptors Against Oxidative Stress.

Antioxid Redox Signal 2016 06 12;24(16):909-23. Epub 2016 May 12.

1 INSERM , U968, Paris, France .

Aims: Rod-derived cone viability factor long (RdCVFL) is an enzymatically active thioredoxin encoded by the nucleoredoxin-like-1 (Nxnl1) gene. The second product of the gene, RdCVF, made by alternative splicing is a novel trophic factor secreted by rods that protects cones in rodent models of retinitis pigmentosa, the most prevalent inherited retinal disease. It acts on cones by stimulating aerobic glycolysis through its interaction with a complex containing basigin-1 and the glucose transporter GLUT1. We studied the role of Nxnl1 in cones after its homologous recombination using a transgenic line expressing Cre recombinase under the control of a cone opsin promoter.

Results: We show that the cones of these mice are dysfunctional and degenerate by 8 months of age. The age-related deficit in cones is exacerbated in young animals by exposure to high level of oxygen. In agreement with this phenotype, we found that the cones express only one of the two Nxnl1 gene products, the thioredoxin RdCVFL. Administration of RdCVFL to the mouse carrying a deletion of the Nxnl1 gene in cones reduces the damage produced by oxidative stress. Silencing the expression of RdCVFL in cone-enriched culture reduces cell viability, showing that RdCVFL is a cell-autonomous mechanism of protection.

Innovation: This novel mode of action is certainly relevant for the therapy of retinitis pigmentosa since the delivery into cones of the rd10 mouse, a recessive model of the disease, rescues cones.

Conclusion: Our work highlights the duality of the Nxnl1 gene, which protects the cones by two distinct mechanisms. Antioxid. Redox Signal. 24, 909-923.
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http://dx.doi.org/10.1089/ars.2015.6509DOI Listing
June 2016

Optogenetics.

Curr Opin Ophthalmol 2015 May;26(3):226-32

aInstitut de la Vision, Sorbonne Universités, UPMC Univ Paris, UMR_S 968 bINSERM, U_968 cCNRS, UMR_7210 dCentre Hospitalier National d'Ophtalmologie des Quinze-Vingts, DHU ViewMaintain, INSERM-DHOS CIC 1423 eFondation Ophtalmologique Rothschild, Paris, France fInstitute of Ophthalmology-University College London, London, UK.

Purpose Of Review: In this review, we will discuss the recent developments in optogenetics and their potential applications in ophthalmology to restore vision in retinal degenerative diseases.

Recent Findings: In recent years, we have seen major advances in the field of optogenetics, providing us with novel opsins for potential applications in the retina. Microbial opsins with improved light sensitivity and red-shifted action spectra allow optogenetic stimulation at light levels well below the safety threshold in the human eye. In parallel, remarkable success in the development of highly efficient viral vectors for ocular gene therapy led to new strategies of using these novel optogenetic tools for vision restoration.

Summary: These recent findings show that novel optogenetic tools and viral vectors for ocular gene delivery are now available providing many opportunities to develop potential optogenetic strategies for vision restoration.
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http://dx.doi.org/10.1097/ICU.0000000000000140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395664PMC
May 2015

Gene therapy for the eye focus on mutation-independent approaches.

Curr Opin Neurol 2015 Feb;28(1):51-60

aINSERM, U968 bSorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision cCNRS, UMR_7210 dCentre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503 eFondation Ophtalmologique Adolphe de Rothschild, Paris, France.

Purpose Of Review: This review will discuss retinal gene therapy strategies with a focus on mutation-independent approaches to treat a large number of patients without knowledge of the mutant gene. These approaches rely on the secretion of neurotrophic factors to slow down retinal degeneration and the use of optogenetics to restore vision in late-stage disease.

Recent Findings: Success in clinical application of adeno-associated virus (AAV)-mediated gene therapy for Leber's congenital amaurosis established the feasibility of retinal gene therapy. More clinical trials are currently on their way for recessive diseases with known mutations. However, the genetic and mechanistic diversity of the retinal diseases presents an enormous obstacle for the development of gene therapies tailored to each patient-specific mutation. To extend gene therapy's promise to a large number of patients, evidence suggests retina-specific trophic factors, such as rod-derived cone viability factor, can be used to slow down loss of cone cells responsible for our high acuity vision. In parallel, it has been shown that microbial opsins are able to restore light sensitivity when expressed in blind retinas.

Summary: Recent findings imply that using the viral technology that has been demonstrated as well tolerated in patients, there are opportunities to develop widely applicable gene therapeutic interventions in clinical ophthalmology.
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http://dx.doi.org/10.1097/WCO.0000000000000168DOI Listing
February 2015

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice.

Mol Ther 2015 Jan 6;23(1):7-16. Epub 2014 Aug 6.

1] INSERM, U968, Paris, France [2] Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France [3] CNRS, UMR_7210, Paris, France.

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.
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http://dx.doi.org/10.1038/mt.2014.154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270733PMC
January 2015

Functional rescue of cone photoreceptors in retinitis pigmentosa.

Graefes Arch Clin Exp Ophthalmol 2013 Jul;251(7):1669-77

Institut de la Vision, UPMC Univ Paris 06, UMR_S 968, Paris, 75012, France.

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http://dx.doi.org/10.1007/s00417-013-2314-7DOI Listing
July 2013

Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa.

Science 2010 Jul 24;329(5990):413-7. Epub 2010 Jun 24.

Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Retinitis pigmentosa refers to a diverse group of hereditary diseases that lead to incurable blindness, affecting two million people worldwide. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. It is unknown if these cones are accessible for therapeutic intervention. Here, we show that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore light sensitivity in mouse models of retinitis pigmentosa. Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions (including directional selectivity), activate cortical circuits, and mediate visually guided behaviors. Using human ex vivo retinas, we show that halorhodopsin can reactivate light-insensitive human photoreceptors. Finally, we identified blind patients with persisting, light-insensitive cones for potential halorhodopsin-based therapy.
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http://dx.doi.org/10.1126/science.1190897DOI Listing
July 2010

Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs.

Cell 2010 May;141(4):618-31

Friedrich Miescher Institute for Biomedical Research, PO Box 2543, 4002 Basel, Switzerland.

Adaptation to different levels of illumination is central to the function of the retina. Here, we demonstrate that levels of the miR-183/96/182 cluster, miR-204, and miR-211 are regulated by different light levels in the mouse retina. Concentrations of these microRNAs were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes. We identified the voltage-dependent glutamate transporter Slc1a1 as one of the miR-183/96/182 targets in photoreceptor cells. We found that microRNAs in retinal neurons decay much faster than microRNAs in nonneuronal cells. The high turnover is also characteristic of microRNAs in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro. Blocking activity reduced turnover of microRNAs in neuronal cells while stimulation with glutamate accelerated it. Our results demonstrate that microRNA metabolism in neurons is higher than in most other cells types and linked to neuronal activity.
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http://dx.doi.org/10.1016/j.cell.2010.03.039DOI Listing
May 2010

Genetically timed, activity-sensor and rainbow transsynaptic viral tools.

Nat Methods 2009 Feb 4;6(2):127-30. Epub 2009 Jan 4.

Department of Medical Biology, Faculty of Medicine, University of Szeged, Somogyi B. u. 4., Szeged, H-6720, Hungary.

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.
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http://dx.doi.org/10.1038/nmeth.1292DOI Listing
February 2009

Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon.

Neuron 2006 Jan;49(1):81-94

Department of Biomedical Optics, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany.

A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.
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http://dx.doi.org/10.1016/j.neuron.2005.10.035DOI Listing
January 2006

The primordial, blue-cone color system of the mouse retina.

J Neurosci 2005 Jun;25(22):5438-45

Department of Neuroanatomy, Max-Planck-Institute for Brain Research, D-60528 Frankfurt/Main, Germany.

Humans and old world primates have trichromatic color vision based on three spectral types of cone [long-wavelength (L-), middle-wavelength (M-), and short-wavelength (S-) cones]. All other placental mammals are dichromats, and their color vision depends on the comparison of L- and S-cone signals; however, their cone-selective retinal circuitry is still unknown. Here, we identified the S-cone-selective (blue cone) bipolar cells of the mouse retina. They were labeled in a transgenic mouse expressing Clomeleon, a chloride-sensitive fluorescent protein, under the control of the thy1 promoter. Blue-cone bipolar cells comprise only 1-2% of the bipolar cell population, and their dendrites selectively contact S-opsin-expressing cones. In the dorsal half of the mouse retina, only 3-5% of the cones express S-opsin, and they are all contacted by blue-cone bipolar cells, whereas all L-opsin-expressing cones (approximately 95%) are avoided. In the ventral mouse retina, the great majority of cones express both S- and L-opsin. They are not contacted by blue-cone bipolar cells. A minority of ventral cones express S-opsin only, and they are selectively contacted by blue-cone bipolar cells. We suggest that these are genuine S-cones. In contrast to the other cones, their pedicles contain only low amounts of cone arrestin. The blue-cone bipolar cells of the mouse retina and their cone selectivity are closely similar to primate blue-cone bipolars, and we suggest that they both represent the phylogenetically ancient color system of the mammalian retina.
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http://dx.doi.org/10.1523/JNEUROSCI.1117-05.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6725002PMC
June 2005

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

PLoS Biol 2004 Jun 15;2(6):e163. Epub 2004 Jun 15.

Max Planck Institute for Medical Research, Heidelberg, Germany.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.
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http://dx.doi.org/10.1371/journal.pbio.0020163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC423138PMC
June 2004