Development of stem cell therapy to restore photopic vision

遗传性视网膜疾病和年龄相关性黄斑变性（AMD）是欧洲不可逆失明的主要原因，并且涉及视网膜中的光敏感锥和视杆感光细胞的损失。遗传性视网膜营养不良影响3,000人中的1人，年龄相关性黄斑变性（AMD）影响60岁以上的10人中的1人。缺乏对这些疾病的有效治疗意味着需要开发新的疗法。通过细胞移植来替代失去的光感受器是一种可能的方法，但移植的细胞需要与宿主视网膜建立功能性连接。我们最近已经证明，从发育中的视网膜移植未成熟的感光细胞（前体细胞）到视网膜疾病的小鼠模型中，导致新的视杆细胞的整合，从而恢复具有非进行性遗传性失明的小鼠的弱光视力。由于人类视觉依赖于提供高敏锐度日光和颜色视觉的视锥光感受器以及提供低光视觉的视杆光感受器，因此视锥移植对于临床应用可能是必不可少的。在晚期视网膜色素变性中，尽管大多数视杆细胞丧失，但患者仍具有有用的视力，直到最后剩余的视锥细胞退化。此外，视锥细胞移植对于AMD患者的最佳视网膜修复至关重要，因为这种病症仅涉及视网膜的富含视锥细胞的黄斑区域的变性。由于视网膜仅由5%的视锥细胞组成，我们可能能够通过相对较少的视锥细胞的替换来恢复重要的功能。在某些情况下，患有黄斑变性的患者可以在训练后固定并产生新的假中央凹，并且如果通过移植，可以增加退化黄斑外部的视锥密度，则这可能会增强。在这个项目中，我们将确定有效移植视锥光感受器的条件，并在退行性视网膜疾病的动物模型中恢复日光视觉，以便为开发类似的方法来治疗人类疾病提供框架。为了实现这一目标，我们将进行以下调查。(i)我们将进行实验，以确定可能限制移植后连接的新光感受器数量的重要相互作用，并将修改条件以改善移植到退化的视网膜中。我们将研究杆移植，以开发改进的协议移植锥（ii）我们将确定和操纵的遗传控制，可能会限制锥整合，并使用我们所获得的知识，如何有效地分离和移植杆前体，以实现新的功能锥细胞的最佳整合。(iii)我们将确定新的视锥细胞和视杆细胞的突触连接，因为为了恢复视力，移植的光感受器必须与内层视网膜的细胞（双极细胞）形成连接，我们需要知道这是否发生在退化的环境中。(iv)我们将移植从干细胞系（胚胎干细胞系）产生的感光细胞前体，并检查这些细胞是否与从发育中的视网膜分离的细胞一样好，我们在原理实验中使用这些细胞。这是至关重要的，因为要将我们在小鼠中的发现转化为人类，我们需要证明来自可再生来源的感光细胞前体，如胚胎干细胞，可以有效地恢复视力。我们将从涉及移植来自发育中的小鼠视网膜的光感受器前体的研究进展到涉及移植可能对临床应用有用的人类干细胞衍生的视锥前体的研究。每个计划组成部分将为下一步提供信息，为制定成功的临床应用长期战略提供最佳机会。

Hereditary retinal disease and age related macular degeneration (AMD) are major causes of irreversible blindness in Europe and involve the loss of the light sensitive cone and rod photoreceptor cells in the retina. Inherited retinal dystrophies affect 1 in 3,000, and age related macular degeneration (AMD) affects 1 in 10 people over the age of 60. The lack of effective treatments for these conditions means there is a requirement to develop new therapies. The replacement of lost photoreceptors by cell transplantation is one possible approach, but transplanted cells need to make functional connections with the host retina. We have recently demonstrated that transplantation of immature photoreceptor cells (precursors) from the developing retina into mouse models of retinal disease results in the integration of new rod photoreceptors that restore low light vision in mice with a non progressive form of inherited blindness. As human vision is dependent upon cone photoreceptors, which provide high acuity daylight and colour vision, as well as rod photoreceptors, which provide low light vision, cone transplantation is likely to be essential for clinical application. In late stage retinitis pigmentosa, despite loss of most rods, patients have useful vision until the last remaining cones degenerate. Furthermore, cone transplantation is critical for optimal retinal repair in patients with AMD as this condition involves degeneration only of the cone-rich macula region of the retina. Since the retina consists of only 5% cones we might be able to restore significant function with the replacement of relatively few cones. In certain instances patients with macular degeneration can fixate and create new pseudo-fovea following training and this might be enhanced if, by transplantation, cone density could be increased outside the degenerate macula. In this project we will determine the conditions for effective transplantation of cone photoreceptors and the restoration of daylight vision in animal models of degenerative retinal disease, in order to provide the framework for developing similar approaches to treat human disease. We will conduct the following investigations to achieve this goal. (i) We will perform experiments to identify important interactions that may limit the number of new photoreceptors connecting after transplantation and will modify conditions to improve transplantation into the degenerating retina. We will study rod transplantation in order to develop improved protocols for transplanting cones (ii) We will identify and manipulate the genetic controls that may limit cone integration and use our acquired knowledge of how to effectively isolate and transplant rod precursors in order to achieve optimal integration of new functional cone cells. (iii) We will determine the synaptic connectivity of new cone and rod cells as, in order to restore vision, it is necessary for transplanted photoreceptors to form connections with cells of the inner retina (bipolar cells) and we need to know whether this occurs in the degenerating environment. (iv) We will transplant photoreceptor precursors generated from stem cell lines (embryonic stem cell lines) and examine whether these are as good as the cells isolated from the developing retina, which we use in our proof of principle experiments. This is essential as to translate our findings in mice to humans we need to show that photoreceptor precursors from a renewable source, such as embryonic stem cells can effectively restore vision. We will progress from studies involving transplantation of photoreceptor precursors derived from developing mouse retinae, to studies involving transplantation of human stem cell-derived cone precursors that might be useful for clinical application. Each programme component will inform the next step, providing the best opportunity to develop a successful long-term strategy for clinical application.