Development of novel viral vectors to study and treat neuroinflammation

开发新型病毒载体来研究和治疗神经炎症

• 批准号: 9805884
• 负责人: Fredric Manfredsson
• 金额: $ 24.42万
• 依托单位: ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9805884
• 关键词: Address; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Animal Disease Models; Animals; Bioinformatics; Biological; Brain; Brain Diseases; Capsid; Cells; Characteristics; Computer Simulation; Coupled; Data; Databases; Dependovirus; Development; Directed Molecular Evolution; Disease; Disease model; Dose; Engineering; Etiology; Evolution; Foundations; Gene Transfer Techniques; Generations; Genetic; Genetic Transcription; Genomics; Glare; Goals; Histology; Human; Immune; Immune system; In Situ Hybridization; Individual; Infection; Inflammation; Inflammatory; Libraries; Ligands; Link; Methods; Microglia; Modality; Modeling; Molecular Evolution; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurologic; Neurons; Neurophysiology - biologic function; Neurosciences; Onset of illness; Parkinson Disease; Participant; Pathology; Pattern; Peptide Library; Play; Population; Process; Production; Refractory; Reporting; Research; Research Personnel; Resolution; Role; Series; Signal Transduction; Structure; TLR4 gene; Techniques; Testing; Time; Toxic effect; Tracer; Transgenic Animals; Translating; Variant; Viral; Viral Vector; aged; base; brain cell; cell type; cellular transduction; cytokine; design; experimental study; gene therapy; genetic manipulation; improved; indexing; innovation; mouse model; neuroinflammation; neuron loss; neuronal survival; novel; novel strategies; optogenetics; prevent; single-cell RNA sequencing; spatiotemporal; temporal measurement; tool; transcriptome; transcriptome sequencing; vector

Project Summary/Abstract Neuroinflammation is increasingly thought of as playing a causative role in neurodegenerative disease, with evidence of inflammatory processes preceding neuronal death. For instance, microglia are now thought of as crucial participants in the onset of diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). This shift in focus from neurons to microglia has made evident a glaring need for tools which allows for precise manipulation of microglia. In the last decades there has been great progress in the generation of tools aimed at studying brain form and function. However, despite these advances, barriers remain. Despite the crucial role of microglia in disease, tools to study their function and to manipulate their expression (in disease) are lacking. Certainly the use of inducible transgenic animals has represented a significant step forward. Still, such approaches have limits. For instance, CRE-based models do not facilitate the precise modulation of multiple cellular populations at the same time. To that end, we contend that viral vectors will represent an integral part in studying the role of microglia in disease, and that the use of vectors that can target non-neuronal cells with efficacy and fidelity will be a requisite and a significant advance in translational neurodegenerative research. Thus far, viral vectors have proven to be remarkably refractory to microglial transduction. Thus, herein we propose a novel approach whereby we will engineer novel vectors directed towards microglia with no off-target activity. Our preliminary data using AAV show that there is no biological reason for this apparent impediment in microglial infection. In a single Aim we will utilize an innovative workflow where we will insert a library of known microglial ligands into the capsid of AAV, and where each variant will be unequivocally linked to a unique genetic barcode. This library will be injected in to the brain of mice, and single cell RNAseq (scRNAseq; also identifying the barcode) will be performed. Using genomics we will thus be able to associate each unique barcode/capsid variant with one, or multiple, cellular transcriptional profiles. Importantly, this will allow us to perform in silico negative selection to screen for capsids that only transduce microglia. We will validate the novel viral vectors in aged and inflamed animals, and also demonstrate the utility of this microglial-specific vector in a the 5XFAD mouse model of AD, where we will test the hypothesis that inhibiting Toll-like receptor 4 in microglia per se is sufficient to prevent neuroinflammation and neurodegeneration. At the completion of this proposal we will have generated a novel tool to help us manipulate microglial function with precision, and under conditions that is reflective of the degenerating brain. Thus providing for a new and crucial foundation to move translational efforts within neurodegeneration forward. Moreover, we will also have generated a unique database of capsid variants and their associated transduction transcriptome pattern. This database can be queried by any researcher in order to identify vectors that transduce other cells, perhaps even subsets of neuronal cells. These novel tools will have far-reaching impact on the field of neurodegeneration.

项目总结/摘要 神经炎症越来越多地被认为在神经退行性疾病中起致病作用， 神经元死亡前炎症过程的证据。例如，小胶质细胞现在被认为是 阿尔茨海默病（AD）和帕金森病（PD）等疾病发病的关键参与者。 这种从神经元到小胶质细胞的焦点转移显然需要能够精确定位的工具。 操纵小胶质细胞在过去的几十年里，在开发工具方面取得了很大进展， 研究大脑的形态和功能然而，尽管取得了这些进展，障碍依然存在。尽管关键作用 尽管小胶质细胞在疾病中的作用越来越大，但缺乏研究其功能和操纵其表达（在疾病中）的工具。 当然，诱导型转基因动物的使用代表了向前迈出的重要一步。但这种 方法有局限性。例如，基于CRE的模型不便于多个信号的精确调制。 细胞生物与此同时为此，我们主张病毒载体将是 在研究小胶质细胞在疾病中的作用，以及使用载体，可以靶向非神经元细胞， 有效性和保真度将是转化性神经变性研究中必要的和显著的进步。 到目前为止，病毒载体已被证明是显着的小胶质细胞转导难治性。在此，我们 我提出了一种新的方法，我们将工程新的载体直接对小胶质细胞没有脱靶 活动我们使用AAV的初步数据表明，在细胞中这种明显的障碍没有生物学原因。 小胶质细胞感染在一个单一的目标，我们将利用创新的工作流程，我们将插入一个库的已知 在一些实施方案中，所述变体将小胶质细胞配体插入AAV的衣壳中，并且其中每个变体将明确地连接到一个独特的配体。 基因条码该文库将被注射到小鼠的大脑中，并进行单细胞RNA测序（scRNA测序;也 识别条形码）。使用基因组学，我们将能够将每个独特的 在一些实施方案中，所述抗体可以是具有一个或多个细胞转录谱的条形码/衣壳变体。重要的是，这将使我们能够 进行计算机模拟阴性选择以筛选仅覆盖小胶质细胞的衣壳。我们将验证 新的病毒载体在老年和发炎的动物，也证明了这种小胶质细胞特异性的效用， 在5XFAD小鼠AD模型中，我们将测试抑制Toll样受体4 小胶质细胞本身足以防止神经炎症和神经变性。在完成这一 我们将产生一种新的工具来帮助我们精确地操纵小胶质细胞功能， 在这种情况下，大脑会退化从而提供了一个新的和关键的基础， 在神经变性中推动翻译工作向前发展。此外，我们还将产生一个独特的 衣壳变体及其相关转导转录组模式的数据库。此数据库可 任何研究人员询问，以确定载体，使其他细胞，甚至可能的子集， 神经元细胞这些新工具将对神经退行性变领域产生深远影响。