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

视网膜是中枢神经系统的延伸，位于眼睛的后部，通过视神经将信息从我们的视觉世界传递到大脑。视网膜的重要作用之一是将光转换为电信号，这一过程称为光转导。这些电信号随后通过视网膜网络传输，最终在连接眼睛和大脑的视神经中产生脉冲。一旦这些冲动到达中央视觉脑区，它们就会导致视觉感知。负责光传导的细胞是光感受器、视杆细胞（负责在昏暗光线条件下的视觉）和视锥细胞（负责在明亮光线条件下的色觉和我们看到清晰细节的能力）。失明的主要原因之一是视杆/视锥功能障碍，通常是由于基因突变（遗传性感光细胞营养不良）。当这些细胞不能正常工作时，它们会逐渐退化，导致部分或全部和不可逆转的失明。目前没有可用的预防性治疗或新的治疗干预措施可以成功阻止疾病进展或为患有这些破坏性疾病的患者提供长期有希望的前景。视网膜色素变性（RP）是一种常见的遗传性感光细胞营养不良，与中周边视网膜的进行性杆变性相关，导致夜盲和视力丧失。在疾病的后期，视锥细胞也会退化，导致最终疾病阶段的完全失明。因此，迫切需要开发新的方法，通过基因治疗（如果在疾病的早期阶段进行）来替代感光细胞或重新激活功能障碍的存活感光细胞。在我们的小组中，我们开发了来自人类多能干细胞（hPSC）的人工视网膜（类器官）。我们从这些类器官中分离出光感受器，并将其注射到患有光感受器营养不良的视网膜中，目的是将这些新的健康光感受器整合到宿主视网膜中，最终恢复视觉功能。我们已经成功地实现了这些目标，在RP的小鼠模型中使用的光感受器前体的锥体富集的人口，导致视觉功能的部分恢复评估的行为和电生理测试。然而，鉴于RP中视杆变性的普遍性，我们的假设是在早期阶段移植hPSC衍生的视杆可能导致视杆整合和功能的改善，同时还保护视锥细胞在后期阶段免于变性。此外，我们认为，杆和锥移植的组合可能会达到最佳的结果，在更先进的阶段retinal degeneration.Here，我们建议测试这一假设在小鼠模型RP。我们将开发新的hPSC系，这将使锥和杆前体的富集，每一个携带不同颜色的遗传编码的荧光标记，以便在植入宿主视网膜后更容易识别。在一组实验中，我们将在早期变性阶段单独注射视杆细胞，目的是改善它们与宿主视网膜的整合，并保护视锥细胞免受后期变性的影响。在另一组实验中，我们将在退化的后期注射混合的视杆细胞和视锥细胞前体，以观察这种方法是否能保护视锥细胞免受随后的退化。使用我们开发的所有工具，从hPSC中产生同质的锥和杆前体群体，进行成功的细胞移植，并使用行为和电生理方法评估视力恢复，该项目将提供基础知识，以建立成功大规模移植干细胞衍生的健康光感受器所需的最佳条件，以恢复破坏性光感受器营养不良的视力。

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.