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

项目总结/摘要。 NEI的大胆目标倡议（2012年启动）提出了“恢复可用视力”的挑战。 通过再生眼睛和视觉系统中的神经元和神经连接来治疗人类。”虽然有一个明显的 由于对新疗法的亲和力，目前的资源和技术差距阻碍了许多治疗方法的转化， 接近。一个这样的差距涉及到动物模型的可用性，这些动物模型具有人类视网膜的关键特征， 解剖，以及疾病模型，忠实地模仿在患者中看到的机制和过程， 视网膜变性（可能适合再生疗法的致盲疾病）。没有 现有的锥优势哺乳动物模型是一个主要的技术差距，阻碍了努力 开发和评估视网膜再生治疗策略。我们建议提前两个月 有前途的模型系统，更接近人类的视觉解剖和功能，而不是更广泛地使用 小鼠和大鼠模型。第一种是13线地松鼠（13-LGS）：一种白天活动的、以锥体为主的啮齿动物（约85%）。 视锥细胞），具有专门处理视觉信息的大的大脑区域。第二种是树鼩：一种非- 啮齿类、灵长类哺乳动物，也是视锥占优势（约95%视锥）。这些模型被用来研究 视觉传导（13-LGS）、外节形态发生、脱落和冬眠期间的重塑（13-LGS） LGS）、视锥双极细胞回路（13-LGS）、近视（树鼩）和中枢视觉处理（树鼩）。 然而，将其用作评估再生植物的存活和整合的预防模型， 视锥光感受器是有限的;主要是由于缺乏允许对这些进行遗传操纵的工具。 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 模型通过以下具体目标：（1）开发，优化和验证成像方法和功能 （2）从iPSC产生13-LGS和树鼩视锥光感受器 （3）建立rAAV介导的13-LGS和树鼩视网膜变性的体内模型;（4） 人类疾病的生殖系转基因13-LGS模型;（5）测试和优化移植的13-LGS的整合; LGS，树和人类iPSC衍生的锥在正常和退化的13-LGS和树视网膜。一 该建议的一个关键特征是通过比较这些模型的细胞分辨率表型来验证它们 与在具有类似病症/突变的患者中观察到的相同。在整个项目中，我们将分享和传播 我们的协议，方法和数据，为更广泛的视觉研究社区提供资源;这 将使用现有的和新创建的在线工具完成。此应用程序的主要优点是 我们已经组建了一个多学科团队来承担这个具有挑战性的项目。团队带来了 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，不仅提供验证 疾病模型，但也可用于在这些和其他物种中创建其他模型的可推广工具。

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.