Restoration of visual function by transplantation of optogenetically engineered photoreceptors

Abstract

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.

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

“…Cryosections were prepared and confocal images were acquired after labeling with antibodies against KIAA0319L (red) and nuclear staining with DAPI (blue). We observed that AAVR is most strongly expressed in the center of the rosettes at day 44 ( Figure S1) and in the outer layers of our cone-enriched retinal organoids at day 70, were photoreceptor cells are found at each timepoint of differentiation [12,13]. This finding is consistent with the expression pattern in mature human and mouse retinas ( Figure 2B-G).…”

“…At this stage, the organoids presented a neural-retina like structure, giving rise to all the different retinal neuronal cell types. rAAVs were applied on day 44, corresponding to the onset of cone differentiation, as we have shown by upregulation of human cone arrestin by a qPCR time course analysis [13]. Cell cycle arrest due to Notch signaling inhibition was achieved by addition of DAPT, a selective gamma secretase inhibitor added for a week, 2 days prior to infection.…”

“…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.…”

Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective

“…Transplanted photoreceptors may not facilitate their physiological roles fully due to lack of interaction with the host RPE. One way to enhance the photosensitivity of transplanted photoreceptors from human stem cell-derived retinal organoids is to use optogenetically transformed photoreceptors [222]. Material transfer is found as well from conjugates formed from transplanted NTPDase2-positive CellTracker Green labelled Müller Glial Cells [223,224].…”

The primate model for understanding and restoring vision

“…Cone replacement has suffered from challenges in the production of cone photoreceptors in vitro from human iPSC-derived retinal organoids, although some strategies have been reported to bias cells to adopt this fate (126,127). There is now renewed interest in investigating human cell transplantation to the retina (128), as advances in the past decade using mouse models appear to be in part secondary to material transfer between transplanted cells and endogenous cells (129,130).…”