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

基本信息

批准号：
10354977
负责人：
Benjamin J Frankfort
金额：
$ 24万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10354977
关键词：
Adult Anatomy 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 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 Lead Lentivirus Ligands Mammalian Cell Mammals Measures Mediating Membrane 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 Subgroup System Techniques Testing Therapeutic Transgenic Animals Transgenic Mice Variant Virus Virus Receptors age related base 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

项目摘要

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残留神经节细胞（RGC）的等级变性。 RGC是高度的已经确定了小鼠视网膜中的异质性，大于40个RGC亚型。目前，我们的由于RGC的数量较小，因此对RGC亚型的理解受到了阻碍。因此，用于实验的可行成年RGC和RGC亚型种群的技术，例如免疫人员具有很大的价值。在这个新应用中，我们将开发一种体外/体内的小说基于免疫剂技术的修改，针对靶向RGC亚型操作的系统。我们的总体假设是RGC中禽肿瘤病毒受体A（TVA）的Cre依赖性生态表达 RGC亚型将允许高度纯化的RGC种群的隔离和培养。这将是通过TVA介导的免疫剂（TVAMI）实现，我们将开发一种新技术基于蛋白质与TVA结合的阳性免疫良好步骤。此外，通过与 andivirus用选择性TVA配体Enva进行了拟南芥，我们将尝试在该基因表达中诱导基因表达体外和体内表达TVA的RGC种群相同。有两个具体目标：1）建立和优化用于RGC隔离和文化的TVAMI系统； 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成年RGC。我们将在两者之后将几种TVAMI变体与标准免疫人员进行比较隔离和一周的细胞培养物，具有细胞染色和免疫细胞化学，以开发优化的协议。通过AIM 2，我们将研究在αRGC中表达TVA的转基因动物， RGC包括四个RGC亚型。首先，我们将将αRGC与TVAMI分离，并确认身份，验证纯度并测量分离后四个αRGC亚型的相对存活和一周细胞培养的细胞染色和免疫细胞化学。我们还将在这种富集的人群定义了αRGC的转录特征。其次，αRGC隔离后，我们将评估αRGC特异性病毒在体外转移的特异性和效率与Enva-Pseudotyped型转移慢病毒或AAV2载体，其中包含RGC特异性GFP表达盒。第三，我们将介绍通过玻璃体内注射和RGC相同的病毒，并比较体内αRGC转移的速率整个安装和切片视网膜的免疫组织化学以及随后的TVAMI之后。如果成功，这系统有可能解锁多种新方法，以在正常和疾病状态，并能够具有潜在的治疗价值的广泛应用。