Optogenetic Light Sensors in Human Retinal Organoids

Abstract

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

“…Based on a previously published protocol [27], we generated cone-enriched retinal organoids [12]. After 30 days of culture, self-forming organoids were mechanically isolated and placed in 3D conditions.…”

“…In the retina, the cell-type specificity also depends on the injection route with intravitreal injections, bringing the rAAVs in contact with inner retinal cells and subretinal injections and facilitating vector access to the outer retinal cells [8,11]. In our hiPSC-derived organoids [12,13], laminated neural retina obtained after several weeks of differentiation displays photoreceptor precursors on the outside and ganglion cell precursors in the center of the structures. In these organoids, the medium containing the rAAVs is first and foremost in contact with the photoreceptor precursors, mimicking subretinal injections.…”

Optogenetic therapy: High spatiotemporal resolution and pattern recognition compatible with vision restoration in non-human primates

“…A more likely explanation resides in differential protein trafficking with td-Tomato working as a trafficking helper, as well as possibly preventing protein aggregation 38 . The result would be an increased membrane targeting of the opsin construct 39 . We demonstrated the greater efficacy for both the mutated AAV capsid, AAV2.7m8, and the fused protein, ChR-tdT, in non-human primates.…”

The primate model for understanding and restoring vision

“…An engineered AAV2 variant (AAV2-7m8) delivers genes to foveal cones with a welltolerated dose when administered intravitreally. Although a healthy NHP is an excellent model to determine the adequacy of AAV capsids and doses in combination with appropriate injection routes (96,101,104,105), studies in mouse models, human iPSC-derived organoids (20,106), and postmortem human retinal explants (70,71,95) are required to complement findings in normal macaques. Although use of macaques can efficiently predict the distribution of AAV in a normal retina, viral transduction patterns change dramatically in retinal disease (107-109), making it necessary to add further model systems.…”