Overcoming Barriers to retinal ganglion cell replacement in experimental glaucoma

项目摘要/摘要 视网膜神经节细胞(RGC)是视网膜的输出神经元，负责将视觉世界的信息从眼睛传递到大脑。因此，RGC损伤和丢失是许多视觉系统疾病的特征，直接导致视力损害，或者当RGC丢失更严重时导致失明。我们的翻译启用方法建立在一个非常成熟、充分表征和验证的实验性青光眼(EG)模型基础上。这为我们的研究提供了独特的优势，即在一个可靠的RGC变性模型的框架内进行每个拟议的假设驱动的实验，该模型紧密概括了在人类青光眼中观察到的解剖变化和病理生理过程。此外，我们的初步结果确立了我们方法的可行性，表明我们已经成功地将人类诱导多能干细胞(IPSC)来源的RGCs移植到EG视网膜，同时也描述了需要有针对性的解决方案的主要障碍。因此，我们建议对供体RGC和受体EG视网膜进行一系列操作，以克服RGC替换的现有障碍，从而实现恢复青光眼或其他视神经疾病盲人视力的大胆目标。我们的每个目标都是基于相关生物学的现有知识，并将导致有意义的提高供体RGC在青光眼EG视网膜中的存活、整合和功能。我们将利用严格的定量电生理学和解剖学评估来检验每个目标核心的假设。目标1将针对神经炎症，以提高移植的视网膜节细胞的长期存活率。我们将创造低免疫原性的IPSCs，并使用全身免疫抑制剂或抑制小胶质细胞激活来操纵宿主视网膜环境。AIM 2将通过调节细胞年龄来增加供体细胞的存活和整合，宿主视网膜神经胶质细胞通过细胞再生实验诱导到未成熟状态。AIM 3将增强供体视网膜神经节细胞的连通性和轴突生长。供体RGCs将被编辑为表达用于化学发生刺激的hM3Dq DREADD受体和mTOR激活剂。凝血酶反应蛋白将在宿主视网膜星形胶质细胞和供体视网膜节细胞中过度表达，利用星形胶质细胞衍生的促进轴突生长和突触形成的因子。总而言之，这些目标将为科学界创造丰富的知识和资源，并使我们更接近通过取代研资局恢复视力的现实。

Project Summary / Abstract Retinal ganglion cells (RGCs) are the output neurons of the retina responsible for transmitting information about the visual world from the eye to the brain. Thus, RGC damage and loss, a characteristic of many disorders of the visual system, has the direct consequence of vision impairment, or blindness when RGC loss is more severe. Our translation-enabling approach builds on a very well-established, thoroughly characterized and validated experimental glaucoma (EG) model. This affords our study the distinct advantage of conducting each of the proposed hypothesis-driven experiments within the framework of a reliable model of RGC degeneration that closely recapitulates the anatomical changes and pathophysiological processes observed in human glaucoma. Moreover, our preliminary results establish the feasibility of our approach, demonstrating that we have already achieved successful transplantation of human induced pluripotent stem cell (iPSC)-derived RGCs into the EG retina, while also characterizing major barriers that require targeted solutions. Hence, we propose to employ a series of manipulations to both donor RGCs and the recipient EG retina in order to overcome the existing barriers to RGC replacement and thus make a giant leap forward toward realization of the audacious goal to restore vision in persons blinded by glaucoma or other optic neuropathies. Each of our Aims is soundly based on existing knowledge of the relevant biology and will lead to meaningful enhancement of donor RGC survival, integration, and function in the glaucomatous EG retina. We will utilize rigorous quantitative electrophysiological and anatomical assessments for testing the hypothesis at the core of each Aim. Aim 1 will target neuroinflammation to improve the long-term survival of transplanted RGCs. We will create hypoimmunogenic iPSCs and manipulate the host retinal environment using systemic immunosuppressive agents or inhibition of microglial activation. Aim 2 will augment donor cell survival and integration through modulation of cellular age, with host retinal glia experimentally induced to an immature state through cellular rejuvenation. Aim 3 will enhance the connectivity and axon outgrowth of donor RGCs in the retina. Donor RGCs will be edited to express hM3Dq DREADD receptors for chemogenetic stimulation and mTOR activators. Thrombospondin will be overexpressed in host retinal astrocytes and donor RGCs, leveraging astrocyte-derived factors that promote axonal outgrowth and synaptogenesis. Together, these Aims will generate a wealth of knowledge and resources for the scientific community and bring us significantly closer to the reality of vision restoration through RGC replacement.