Optogenetic Light Sensors in Human Retinal Organoids

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

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

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