Developing Cone-Dominant Retinal Disease Models as a Resource for Translational Vision Research

项目摘要/摘要。 NEI的大胆目标倡议(2012年启动)提出了挑战，即在 人类通过再生眼睛和视觉系统中的神经元和神经连接。虽然有一个明显的 对新疗法的亲和力、当前的资源和技术差距排除了许多疗法的翻译 接近了。其中一个差距与具有人类视网膜关键特征的动物模型的可用性有关 解剖学，以及忠实地模拟患者的机制和过程的疾病模型 视网膜退行性变(可能适用于再生疗法的致盲疾病)。不存在 容易获得的锥体占优势的哺乳动物模型是阻碍努力的主要技术差距 开发和评估视网膜再生治疗策略。我们建议提前两个月 前景看好的模型系统比更广泛使用的更接近人类视觉解剖和功能的模型系统 小鼠和大鼠模型。第一种是13行地松鼠(13-LGS)：一种白天活动的锥体优势啮齿动物(~85% 视锥细胞)有大片大脑区域专门用于处理视觉信息。第二种是树精：一种非 啮齿动物，类似灵长类的哺乳动物，也是锥体占优势的(~95%的锥体)。这些模型已被用于研究 冬眠期间的视觉传导(13-LGS)、外节形态发生、脱落和重构(13-LGS) LGS)、视锥双极细胞电路(13-LGS)、近视(树鼠)和中央视觉处理(树鼠)。 然而，它们被用作评估再生的生存和整合的翻译使能模型 锥体感光细胞一直受到限制；主要是因为缺乏允许对这些细胞进行遗传操作的工具 动物(因此缺乏疾病模型)。我们建议将这些物种作为与疾病相关的物种 通过以下具体目标建立模型：(1)开发、优化和验证成像方法和功能 13-LGS和树鼠的检测；(2)IPSCs产生13-LGS和树鼠视锥感受器 在体外；(3)在体内建立rAAV介导的13-LGS和树鼠视网膜变性模型；(4)启用 胚系转基因13-LGS人类疾病模型的建立；(5)移植13-LGS整合的检测与优化 在正常和退化的13-LGS和树鼠视网膜中，LGS、树鼠和人类IPSC衍生的视锥细胞。一个 这一建议的主要特点是通过比较这些模型的细胞分辨率表型来验证它们 与具有类似条件/突变的患者的情况相同。在整个项目中，我们将分享和传播 我们的协议、方法和数据为更广泛的视力研究社区提供资源；这 将使用现有的和新创建的在线工具来完成。此应用程序的一个主要优点是 已经组建的多学科团队来承担这一具有挑战性的项目。这支球队带来了 模型开发、干细胞治疗和细胞评估所需的必要补充专业知识 生存、整合和功能。这项工作将产生重大的积极影响，不仅提供经过验证的 不仅是疾病模型，也是在这些物种和其他物种中创建额外模型的通用工具。

PROJECT SUMMARY/ABSTRACT . The NEI’s Audacious Goal Initiative (launched in 2012) put forward the challenge of “restoring usable vision in humans by regenerating neurons and neural connections in the eye and visual system.” While there is an obvious affinity towards novel therapies, current resource and technology gaps preclude translation of many therapeutic approaches. One such gap pertains to the availability of animal models that share key features of human retinal anatomy, as well as disease models that faithfully emulate the mechanisms and processes seen in patients with retinal degenerations (blinding diseases that might be amenable to regenerative therapies). The absence of readily available cone-dominant mammalian models represents a major technology gap impeding efforts to develop and evaluate regenerative treatment strategies in the retina. We propose to advance two promising model systems that are closer to human visual anatomy and function than the more widely used mouse and rat models. The first is the 13-lined ground squirrel (13-LGS): a diurnal, cone-dominant rodent (~85% cones) with large brain regions dedicated to processing visual information. The second is the tree shrew: a non- rodent, primate-like mammal that is also cone dominant (~95% cones). These models have been used to study visual transduction (13-LGS), outer segment morphogenesis, shedding, and remodeling during hibernation (13- LGS), cone-bipolar cell circuitry (13-LGS), myopia (tree shrew) and central visual processing (tree shrew). However, their use as translation-enabling models for evaluating both survival and integration of regenerated cone photoreceptors has been limited; mainly due to a lack of tools that allow for genetic manipulation of these animals (and thus a dearth of disease models). We propose to advance these species as disease-relevant models through the following Specific Aims: (1) Develop, optimize, and validate imaging methods and functional assays for the 13-LGS and tree shrew; (2) Generate 13-LGS and tree shrew cone photoreceptors from iPSCs in vitro; (3) Create rAAV-mediated retinal degeneration models for the 13-LGS and tree shrew in vivo; (4) Enable germline transgenic 13-LGS models of human disease; (5) Test and optimize integration of transplanted 13- LGS, tree shrew, and human iPSC-derived cones in normal and degenerated 13-LGS and tree shrew retinas. A key feature of this proposal is the validation of these models by comparing their cellular-resolution phenotype with that seen in patients with similar conditions/mutations. Throughout the project, we will share and disseminate our protocols, methods, and data to provide resources for use by the broader vision research community; this will be done using existing and newly-created online tools. A major strength of this application is the multidisciplinary team that has been assembled to take on this challenging project. The team brings the necessary complementary expertise required for model development, stem cell treatment, and evaluation of cell survival, integration, & function. This work will have a significant positive impact by providing not only validated disease models but also generalizable tools with which to create additional models in these and other species.