To assess the engraftment of hESC-derived photoreceptors and their ability to restore vision in early and advanced stages of Retinitis Pigmentosa.

The retina is an extension of the central nervous system that lines the back of the eye, transmitting information from our visual world to the brain via the optic nerve. One of the important roles of the retina is to convert light into electrical signals, a process called phototransduction. These electrical signals are subsequently transmitted across retinal networks, eventually generating impulses in the optic nerve that connects the eye to the brain. Once these impulses reach central visual brain areas, they lead to visual perception. The cells responsible for phototransduction are the photoreceptors, the rods (responsible for vision in dim light conditions) and the cones (responsible for colour vision in bright light conditions and for our ability to see sharp details).

One of the main causes of blindness is rod/cone malfunction, often due to genetic mutations (hereditary photoreceptor dystrophies). When these cells do not function properly, they gradually degenerate, leading to partial or total and irreversible blindness. There are currently no available preventative treatments or new therapeutic interventions that can successfully hinder disease progression or offer long-term promising outlook for patients suffering from these devastating conditions.

Retinitis pigmentosa (RP) is a common form of hereditary photoreceptor dystrophy associated with progressive rod degeneration of the mid-peripheral retina, leading to night blindness and loss of visual acuity. At later stages of the disease, cones degenerate as well, resulting in complete blindness at final disease stages. Therefore, there is a pressing need to develop novel approaches either for photoreceptor replacement or for reactivation of dysfunctional surviving photoreceptor by gene therapy (if performed at early stages of the disease).

In our group, we develop artificial retinas (organoids) derived from human pluripotent stem cells (hPSCs). We isolate photoreceptors from these organoids and inject them in retinas with photoreceptor dystrophies, with the goal to achieve integration of these new healthy photoreceptors into the host retina, eventually restoring visual function. We have successfully achieved these goals using a cone-enriched population of photoreceptor precursors in a mouse model of RP, resulting in partial restoration of visual function assessed by behavioural and electrophysiological testing. However, given the prevalence of rod degeneration in RP, our hypothesis is that transplantation of hPSCs-derived rods at early stages may lead to improved rod integration and function, while also protecting cones from degenerating at later stages. In addition, we suggest that a combination of rod and cone transplantation may achieve optimal results at more advanced stages of retinal degeneration.

Here we propose to test this hypothesis in a mouse model of RP. We will develop new hPSC lines, which will enable enrichment of cone and rod precursors, each one carrying a genetically encoded fluorescent marker of a different colour for easier identification once engrafted in the host retina. In one set of experiments, we will inject rods alone at early degeneration stages, with the goal of improving their integration into the host retina and protecting cones from later degeneration. In another set of experiments, we will inject a mixed population of rod and cone precursors at later stages of degeneration, to see whether this approach protects cones from ensuing degeneration.

Using all the tools we have developed to generate homogenous populations of cone and rod precursors from hPSCs, perform successful cell transplantation and assess vision restoration using behavioural and electrophysiological approaches, this project will provide fundamental knowledge to establish the optimal conditions necessary for successful large-scale engraftment of stem cell-derived healthy photoreceptors to restore sight in devastating photoreceptor dystrophies.

Retinitis pigmentosa (RP) is characterized by degeneration of the mid-peripheral retina, leading to night blindness and loss of visual acuity. The final impact is the loss of all photoreceptors. With the advances made in differentiation of human pluripotent stem cells (hPSCs), it is now feasible to generate with ease retinal organoids containing rod and cone photoreceptors. Given the prevalence of rod degeneration in RP, transplantation of hPSCs-derived rods into patient retinas at the early stages of RP could be a valid therapeutic option, which may lead to improved rod integration and function, while also protecting the remaining foveal cones. However, during the disease progression, the loss of rods leads to cone death; hence, a combined rod and cone transplantation approach may be needed at the advanced stages of RP. In this proposal, we will test this hypothesis by assessing the engraftment of hPSCs-derived rods in an animal model of RP alone and in combination with cones at the early and advanced stages of retinal degeneration respectively.

These goals will be achieved by modifying two new hPSCs cell lines, which enable the enrichment of both cone and rod precursors, each under a different reporter (CRX-GFP and OPN1LW/MW for cones and NRL-mCherry for rods) for easier identification following subretinal injection and engraftment in the host retina. Immunofluorescence will be used to identify how the engrafted precursors evolve into mature rods and cones and establish connections with other retinal neurones. Restoration of visual function will be tested using behavioural approaches, in vivo recordings from the visual cortex and ex vivo recording of light responses from the ganglion cell layer using a large-scale, high-density multielectrode array, allowing us to characterise receptive field properties in the ganglion cell layer at pan-retinal level.