Enhancement, mapping, and validation of viral vectors for primate optogenetics

用于灵长类光遗传学的病毒载体的增强、绘图和验证

基本信息

批准号：
10391957
负责人：
Marc A Sommer
金额：
$ 68.87万
依托单位：
DUKE UNIVERSITY
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：
2022-01-15 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10391957
关键词：
Anatomy Animal Model Animals Area Atlases Basic Science Behavior Brain Brain Diseases Capsid Cells Chronic Complex Dependovirus Disease Electrophysiology (science)Enzymes FDA approved Gene Expression Genes Genetic Glycoproteins Goals Histologic Human Immune response Immune system Immunology Injections Innate Immune Response Intervention Lentivirus Lentivirus Vector Light Macaca Maps Mediating Methods Modeling Modernization Molecular Monkeys Neurobiology Neurons Neurosciences Opsin Outcome Output Pattern Pharmaceutical Preparations Pharmacology Physiological Population Projection Primates Proteins Protocols documentation Regimen Research Rodent Role Sirolimus Site Specificity System T-Lymphocyte Techniques Technology Topoisomerase Topoisomerase Inhibitors Transduction Gene Transgenes Transportation V1 neuron Validation Viral Viral Vector Virus Vision Vision research Visual Visual system structure Work adaptive immune response arm base brain disorder therapy cell type effective therapy efficacy evaluation efficacy validation frontal eye fields gene therapy improved in vivo in vivo evaluation inhibitor insight interest neural circuit neuronal circuitry neurophysiology nonhuman primate novel therapeutics oculomotor optogenetics orbit muscle prevent promoter reagent testing recruit relating to nervous system retrograde transport success superior colliculus Corpora quadrigemina therapeutic gene tool transgene delivery transgene expression vector virology visual control visual motor visual neuroscience

项目摘要

PROJECT SUMMARY Mapping the visual and visuomotor circuits of the brain using opsins and other actuators to target and control neurons is a central goal of modern neuroscience. Actuators have become key to studying neuronal circuits, modeling brain disorders, and developing new therapies. Neural actuator applications to research in rodents and other small animals have achieved great success. In primates, however, these approaches have yet to be transformative. The main problem is that viral vectors are required to deliver actuator genes, but both viral transduction and gene expression have been unreliable across, and even within, primate labs. Efficacy is hindered by the complex innate and adaptive retaliatory immune response in primates, and even when the approach does work, cell-type specificity is lacking. The overall purpose of this project is to incorporate recent advances in virology, gene therapy, and immunology to maximize viral transduction, maintain chronic gene expression, and gain cell-type specificity through retrograde transportation of viruses in visual circuits of the macaque brain. Throughout the project, optogenetics is the actuator-mediated intervention and the visual- oculomotor system is the testbed. We focus on two viruses that provide retrograde transport: retrograde adeno- associated virus-2 (rAAV2-retro) and fusion glycoprotein-E pseudotyped lentiviral vector (NeuRet). Each will deliver genes encoding the Red-activatable Channelrhodopsin (ReaChR). Our team, spanning Duke, NYU, and UNC-Chapel Hill, has extensive expertise in vector technology and macaque neurobiology. Aim 1 is dedicated towards the maturation of pharmacological regimens that modify both arms of the primate's immune system to enhance viral transduction and promote long-term constitutive expression of opsin transgenes. Aim 2 will establish comprehensive expression maps of retrogradely transduced neurons. This mapping is a critical step toward providing cell- and circuit-level specificity and supplies a means for physiologically identifying neurons, through phototagging, based on their anatomical connectivity. Aim 3 will use phototagging paired with projection targeting to identify and neurophysiologically characterize neurons contributing to specific circuits within the visual and visuomotor circuitry of the macaque brain. In combination, this work will enhance the efficacy of viral vectors for neuroscientific research of the macaque visual and visuomotor system, provide both anatomical and functional validation of the developed protocols, and provide new insights into the functional role these specific circuits serve in vision and visuomotor behaviors. Finally, this project will provide fundamental insights for improving human gene therapies that depend on viral delivery of therapeutic genes.

项目概要使用视蛋白和其他执行器来定位和控制大脑的视觉和视觉运动回路神经元是现代神经科学的中心目标。执行器已成为研究神经元回路的关键，模拟大脑疾病并开发新疗法。神经执行器在啮齿类动物研究中的应用其他小动物也取得了巨大的成功。然而，在灵长类动物中，这些方法尚未得到证实。变革性的。主要问题是病毒载体需要传递致动基因，但两种病毒载体都需要传递致动基因。在灵长类动物实验室中，甚至在灵长类动物实验室内部，转导和基因表达都是不可靠的。功效为受到灵长类动物复杂的先天性和适应性报复性免疫反应的阻碍，甚至当该方法确实有效，但缺乏细胞类型特异性。该项目的总体目的是整合最近病毒学、基因治疗和免疫学的进展，以最大限度地提高病毒转导，维持慢性基因表达，并通过视觉回路中病毒的逆行运输获得细胞类型特异性猕猴的大脑。在整个项目中，光遗传学是执行器介导的干预和视觉- 动眼系统是试验台。我们关注两种提供逆行运输的病毒：逆行腺病毒相关病毒-2 (rAAV2-retro) 和融合糖蛋白-E 假型慢病毒载体 (NeuRet)。每个都会传递编码红色激活通道视紫红质 (ReaChR) 的基因。我们的团队涵盖杜克大学、纽约大学和北卡罗来纳大学教堂山分校在载体技术和猕猴神经生物学方面拥有丰富的专业知识。目标1是专注朝着改变灵长类动物免疫系统双臂的药物治疗方案的成熟方向发展增强病毒转导并促进视蛋白转基因的长期组成型表达。目标2将建立逆行转导神经元的综合表达图谱。这个映射是关键的一步提供细胞和电路水平的特异性，并提供一种生理学识别神经元的方法，通过基于其解剖连接性的照片标记。目标 3 将使用照片标签与投影相结合旨在识别和神经生理学表征对特定神经回路有贡献的神经元猕猴大脑的视觉和视觉运动回路。结合起来，这项工作将增强病毒的功效用于猕猴视觉和视觉运动系统神经科学研究的载体，提供了解剖学和对所开发的协议进行功能验证，并为这些特定的功能作用提供新的见解神经回路负责视觉和视觉运动行为。最后，该项目将为以下方面提供基本见解：改善依赖病毒传递治疗基因的人类基因疗法。