Development of stem cell therapy to restore photopic vision

遗传性视网膜疾病和老年性黄斑变性(AMD)是欧洲不可逆性失明的主要原因，涉及视网膜中光敏视锥和视杆感光细胞的丧失。遗传性视网膜营养不良影响3000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。这些疾病缺乏有效的治疗方法，这意味着需要开发新的治疗方法。通过细胞移植替换丢失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连接。我们最近已经证明，将发育中的视网膜的未成熟光感受器细胞(前体)移植到视网膜疾病的小鼠模型中，可以整合新的杆状光感受器，恢复非进行性遗传性失明小鼠的弱光视觉。由于人类的视觉依赖于视锥感受器和视杆感受器，视锥感受器提供高敏锐度的日光和色觉，而视杆感受器提供弱光视觉，因此视锥细胞移植在临床应用中可能是必不可少的。晚期视网膜色素性视网膜炎，尽管失去了大部分视杆细胞，但直到最后残留的视锥细胞退化，患者仍能获得有用的视力。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，因为这种情况只涉及视网膜富含视锥的黄斑区的退化。由于视网膜只有5%的视锥细胞，我们也许能够通过更换相对较少的视锥细胞来恢复显着的功能。在某些情况下，黄斑变性患者可以在训练后注视并创造新的假性中心凹，如果通过移植，可以增加退化斑点外的视锥密度，这种情况可能会得到加强。在这个项目中，我们将确定在退行性视网膜疾病的动物模型中有效移植视锥感光细胞和恢复日光视力的条件，以期为开发类似的治疗人类疾病的方法提供框架。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，以改善移植到退化视网膜的情况。我们将研究视杆细胞移植，以开发改进的视锥细胞移植方案(II)我们将识别和操作可能限制视锥细胞整合的遗传控制，并利用我们所获得的如何有效分离和移植视锥细胞前体的知识，以实现新功能视锥细胞的最佳整合。(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视网膜内细胞(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)我们将移植干细胞系(胚胎干细胞系)产生的光感受器前体细胞，并检查这些细胞是否与我们在证明原理实验中使用的从发育中的视网膜分离的细胞一样好。这是至关重要的，因为要将我们在老鼠身上的发现转化为人类，我们需要证明来自可再生来源的光感受器前体，如胚胎干细胞，可以有效地恢复视力。我们将从涉及移植发育中的小鼠视网膜的光感受器前体的研究，到涉及移植人类干细胞衍生的视锥前体的研究，这些研究可能对临床应用有用。每个方案组成部分都将为下一步提供信息，为临床应用制定成功的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.