An in vitro/in vivo system for targeted retinal ganglion cell subtype manipulation

项目总结 青光眼的特征是视网膜神经节细胞(RGC)逐渐变性。RGC是高度 异质性，在小鼠视网膜中有超过40种RGC亚型。目前，我们的 由于RGC数量少，一般无法接触到RGC，这阻碍了对RGC亚型的理解。 因此，丰富可存活的成体视网膜节细胞和视网膜节细胞亚型用于实验的技术， 例如免疫泛素，是非常有价值的。在这一新的应用中，我们将开发一种新的体外/体内 基于免疫泛素技术修改的靶向RGC亚型操作系统。我们的 总体假设是Cre依赖的禽类肿瘤病毒受体A(TVA)在RGCs中的异位表达 RGC亚型可用于分离和培养高纯度的RGC群体。这将是 通过TVA介导的免疫潘宁(TVAMI)实现的，我们将开发一种新技术，它集成了 基于蛋白质与TVA结合的阳性免疫泛素步骤。此外，通过转导与 慢病毒与ENVA，一个选择性的TVA配体，我们将尝试诱导基因表达在 在体外和体内，表达TVA的RGC群体相同。有两个具体目标：1)建立和 优化TVAMI系统分离培养RGC；2)调控靶向RGC的基因表达 亚群。在整个目标1中，我们将研究在细胞表面表达TVA的转基因小鼠 几乎所有的成年RGC。我们将比较几种TVAMI变种与标准免疫泛素 分离培养一周后进行细胞染色和免疫细胞化学，以建立优化的 协议。在整个目标2中，我们将研究在αRGC中表达TVA的转基因动物，RGC是一个 RGC包括四个RGC亚型。首先，我们将分离出患有TVAMI的αRGC并确认其身份， 分离后及1周后，验证4种αRGC亚型的纯度，并测定其相对存活率 用细胞染色和免疫细胞化学方法进行细胞培养。我们还将进行单细胞RNA测序 这个丰富的群体定义了αRGC的转录特征。第二，在α研资局分离后，我们将 用ENVA假型评价αRGC特异性病毒体外转导的特异性和有效性 慢病毒或AAV2载体，含有RGC特异性的GFP表达盒。第三，我们将介绍 玻璃体腔注射相同病毒感染视网膜节细胞并比较体内α视网膜节细胞转导效率 全视网膜、切片视网膜及TVAMI后的免疫组织化学染色。如果成功，这将是 该系统有可能解锁多种新的方法来研究正常和 疾病状态，并实现具有潜在治疗价值的广泛的新应用。

PROJECT SUMMARY Glaucoma is characterized by the gradual degeneration of retinal ganglion cells (RGCs). RGCs are highly heterogeneous, and greater than 40 RGC subtypes in the mouse retina have been identified. Currently, our understanding of RGC subtypes is hindered by the general inaccessibility of RGCs due to their small number. Therefore, techniques that enrich the population of viable adult RGCs and RGC subtypes for experimentation, such as immunopanning, are of great value. In this new application, we will develop a novel in vitro/in vivo system for targeted RGC subtype manipulation based on modifications of the immunopanning technique. Our overall hypothesis is that Cre-dependent ectopic expression of avian tumor virus receptor A (TVA) in RGCs and RGC subtypes will allow for the isolation and culture of highly purified RGC populations. This will be achieved through TVA-mediated immunopanning (TVAMI), a new technique we will develop which integrates a positive immunopanning step based on protein binding to TVA. Furthermore, through transduction with lentivirus pseudotyped with EnvA, a selective TVA ligand, we will attempt to induce gene expression in the same TVA-expressing RGC populations both in vitro and in vivo. There are two Specific Aims: 1) establish and optimize the TVAMI system for RGC isolation and culture; and 2) manipulate gene expression in targeted RGC subpopulations. Throughout Aim 1, we will study transgenic mice that express TVA on the cell surface of nearly all adult RGCs. We will compare several TVAMI variations against standard immunopanning both after isolation and one week of cell culture with cell staining and immunocytochemistry to develop an optimized protocol. Throughout Aim 2, we will study transgenic animals that express TVA in αRGCs, a population of RGCs which includes four RGC subtypes. First, we will isolate αRGCs with TVAMI and confirm the identity, validate the purity, and measure the relative survival of the four αRGC subtypes after isolation and one week of cell culture with cell staining and immunocytochemistry. We will also perform single cell RNA sequencing on this enriched population to define the transcriptional signature of αRGCs. Second, after αRGC isolation, we will assess the specificity and efficiency of αRGC-specific virus transduction in vitro with EnvA-pseudotyped lentivirus or AAV2 vector containing an RGC-specific GFP expression cassette. Third, we will introduce the same viruses to RGCs via intravitreal injection and compare rates of αRGC transduction in vivo with immunohistochemistry in whole mounted and sectioned retinas and after subsequent TVAMI. If successful, this system has the potential to unlock multiple new approaches to study RGCs and RGC subtypes in normal and disease states, and enable a broad range of novel applications with potential therapeutic value.