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

用于靶向视网膜神经节细胞亚型操作的体外/体内系统

• 批准号: 10546443
• 负责人: Benjamin J Frankfort
• 金额: $ 20万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10546443
• 关键词: Adult; Animal Model; Binding; Binding Proteins; Birds; Blindness; Cell Culture System; Cell Culture Techniques; Cell Death; Cell Separation; Cell Survival; Cell physiology; Cell surface; Cells; Cellular Membrane; Cellular biology; Cre lox recombination system; DNA cassette; Data; Disease; Disease model; Dissociation; Ectopic Expression; Effectiveness; Gene Expression; Gene Expression Profile; Genetic Transcription; Genus Alpharetrovirus; Glaucoma; Hour; Immunoglobulin Constant Region; Immunoglobulin G; Immunohistochemistry; In Vitro; Injury; Lentivirus; Ligands; Mammalian Cell; Mammals; Measures; Mediating; Membrane Proteins; Methods; Modification; Morphology; Mus; Nerve Crush; Neurons; Newborn Animals; Oncogenic Viruses; Optic Nerve; Optic Nerve Injuries; Oryctolagus cuniculus; Partner in relationship; Pattern; Physiological; Population; Predisposition; Preparation; Procedures; Protocols documentation; Regulatory Element; Research; Resistance; Retina; Retinal Ganglion Cells; Specificity; Stains; Subcellular Anatomy; Subgroup; System; Techniques; Testing; Therapeutic; Transgenic Animals; Transgenic Mice; Variant; Virus; Virus Receptors; age related; cell injury; extracellular; fetal; genetic manipulation; human pluripotent stem cell; immunocytochemistry; improved; in vivo; injured; interest; intravitreal injection; novel; novel strategies; prevent; protein protein interaction; response; response to injury; restoration; retinal ganglion cell degeneration; single-cell RNA sequencing; tool; vector

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.

项目摘要 青光眼的特征在于视网膜神经节细胞（RGC）的逐渐变性。RGC高度 在小鼠视网膜中，已经鉴定了超过40种RGC亚型。目前我们的 RGC亚型的理解受到RGC由于其数量少而通常难以接近的阻碍。 因此，富集存活的成年RGC和RGC亚型的群体用于实验的技术， 例如免疫淘选，具有很大价值。在这个新的应用中，我们将开发一种新的体外/体内 基于免疫淘选技术的改进的靶向RGC亚型操作系统。我们 总体假设是，RGCs中禽肿瘤病毒受体A（TVA）的Cre依赖性异位表达 和RGC亚型将允许高度纯化的RGC群体的分离和培养。这将是 通过TVA介导的免疫淘选（TVAMI）实现，这是我们将开发的一种新技术， 基于蛋白质与TVA结合的阳性免疫淘选步骤。此外，通过与 用EnvA（一种选择性TVA配体）假型化的慢病毒，我们将尝试诱导基因表达。 在体外和体内均具有相同的TVA表达RGC群体。有两个具体目标：1）建立和 优化用于RGC分离和培养的TVAMI系统;和2）操纵靶向RGC中的基因表达 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。我们将比较几个TVAMI变化对标准免疫淘选后， 分离和一周的细胞培养，细胞染色和免疫细胞化学，以开发优化的 议定书在整个目标2中，我们将研究在α RGC中表达TVA的转基因动物， RGC包括四种RGC亚型。首先，我们将用TVAMI分离α RGC并确认身份， 验证纯度，并测量分离后和一周后四种αRGC亚型的相对存活率。 用细胞染色和免疫细胞化学方法检测细胞培养。我们还将进行单细胞RNA测序， 这个富集的群体来定义α RGC的转录特征。第二，在αRGC分离之后，我们将 评估用EnvA假型化的α RGC特异性病毒体外转导的特异性和效率 慢病毒或含有RGC特异性GFP表达盒的AAV 2载体。第三，我们会介绍 通过玻璃体内注射相同的病毒至RGC，并将体内αRGC转导率与 在整个封片和切片的视网膜中以及在随后的TVAMI之后的免疫组织化学。如果成功，这 该系统有可能解锁多种新方法来研究正常和非正常人中的RGC和RGC亚型， 疾病状态，并使具有潜在治疗价值的广泛的新应用成为可能。