Développement de vecteurs AAV sûrs et efficaces pour la thérapie génique rétinienne

Vision is our most cherished sense and its loss is a feared handicap. A highly diverse and complex array of inherited retinal degenerations leads to irreversible vision loss. Today, there is no cure for such disorders. However, in the last decade, many gene therapies entered clinical trials offering hope for the treatment of inherited retinal degenerations. In this thesis, we explored the contribution of viral vectors within the general context of retinal gene therapy. We focused on optimization of viral vectors for mutation-independent gene therapies broadly applicable across rod-cone dystrophies. We carefully designed vectors for targeting cones and studied their translational potential for optogenetic activation of cones in several relevant model systems.La vision est notre sens le plus cher et sa perte est un handicap redouté. Or, il existe un ensemble très hétérogène et complexe de dégénérescences rétiniennes héréditaires entraînant une perte de vision irréversible. Aujourd'hui, il n'y a pas de traitement pour ces maladies. Cependant, au cours de la dernière décennie, de nombreuses thérapies géniques ont été testées dans des essais cliniques, donnant de l'espoir pour le traitement des dégénérescences rétiniennes héréditaires. Dans cette thèse, nous avons exploré l'apport des vecteurs viraux dans le contexte général de la thérapie génique rétinienne. Nous avons plus particulièrement optimisé des vecteurs viraux pour des thérapies géniques indépendantes des mutations, largement applicables à toutes les dystrophies rétiniennes avec dégénérescence de bâtonnets puis cônes. Nous avons conçu des vecteurs pour cibler les cônes et étudié leur potentiel de translation pour l'activation optogénétique des cônes dans plusieurs systèmes modèles pertinents

La conception de vecteurs adaptés à la thérapie génique oculaire

L’utilisation d’un gène comme médicament est l’un des concepts les plus passionnants de la médecine moderne. Les vecteurs viraux ont été largement utilisés à cette fin et ont montré une efficacité thérapeutique dans une variété de modèles animaux de dégénérescence rétinienne. L’extension de ce succès à une application clinique a été initialement lente, mais une expression à long terme de gènes thérapeutiques a récemment été obtenue chez des patients atteints de déficits immunitaires, d’hémophilie B ou de troubles héréditaires de la rétine. Ces résultats ont suscité des espoirs pour le traitement de nombreuses autres maladies, et ont ouvert la voie au développement de nouveaux outils de transfert de gènes. Comme nous le verrons ici, les perspectives et les défis de la thérapie génique sont dans une large mesure dépendants du tissu ciblé, de la maladie concernée et, surtout, de l’efficacité du transfert génique. L’acheminement du gène vers les cellules cibles dépend de vecteurs, qui doivent assurer une expression durable du gène sans engendrer ni toxicité ni réaction immunitaire de la part de l’hôte. La conception de tels vecteurs, proposée pour la première fois dans les années 1970, s’est avérée être plus compliquée que prévu, limitant pendant de nombreuses années le succès de la thérapie génique. Les vecteurs doivent donc être adaptés à chaque scénario ; nous discuterons ici la conception de tels vecteurs pour un transfert de gènes vers la rétine

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion

International audienceTissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system

Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates

International audienceOver 90 years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs) based on their morphology and location within the spinal cord. In more than 200 vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF). Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus), macaque (Macaca fascicularis) and zebrafish (Danio rerio). In these species, the somata of spinal PKD2L1 + CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1 + CSF-cNs originate. pkd2l1 + CSF-cNs were all GABAergic and organized in two rows—one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer–Agduhr cells. Accordingly, pkd2l1 + CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species

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

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

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

International audienceHuman-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

Rescue of Defective Electroretinographic Responses in Dp71-Null Mice With AAV-Mediated Reexpression of Dp71

International audiencePurpose: To study the potential effect of a gene therapy, designed to rescue the expression of dystrophin Dp71 in the retinas of Dp71-null mice, on retinal physiology.Methods: We recorded electroretinograms (ERGs) in Dp71-null and wild-type littermate mice. In dark-adapted eyes, responses to flashes of several strengths were measured. In addition, flash responses on a 25-candela/square meters background were measured. On- and Off-mediated responses to sawtooth stimuli and responses to photopic sine-wave modulation (3-30 Hz) were also recorded. After establishing the ERG phenotype, the ShH10-GFP adeno-associated virus (AAV), which has been previously shown to target specifically Müller glial cells (MGCs), was delivered intravitreously with or without (sham therapy) the Dp71 coding sequence under control of a CBA promoter. ERG recordings were repeated three months after treatment. Real-time quantitative PCR and Western blotting analyses were performed in order to quantify Dp71 expression in the retinas.Results: Dp71-null mice displayed reduced b-waves in dark- and light-adapted flash ERGs and smaller response amplitudes to photopic rapid-on sawtooth modulation and to sine-wave stimuli. Three months after intravitreal injections of the ShH10-GFP-2A-Dp71 AAV vector, ERG responses were completely recovered in treated eyes of Dp71-null mice. The functional rescue was associated with an overexpression of Dp71 in treated retinas.Conclusions: The present results show successful functional recovery accompanying the reexpression of Dp71. In addition, this experimental model sheds light on MGCs influencing ERG components, since previous reports showed that aquaporin 4 and Kir4.1 channels were mislocated in MGCs of Dp71-null mice, while their distribution could be normalized following intravitreal delivery of the same ShH10-GFP-2A-Dp71 vector

Noninvasive gene delivery to foveal cones for vision restoration

International audienceIntraocular 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