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

• 批准号: 10546445
• 负责人: Marc A Sommer
• 金额: $ 64.21万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 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/10546445
• 关键词: Anatomy; Animal Model; Animals; Area; Atlases; Basic Science; Behavior; Brain; Brain Diseases; Capsid; Cells; Chronic; Complex; Dedications; 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; Physiological; Population Projection; Primates; Proteins; Protocols documentation; Recombinant adeno-associated virus (rAAV); Regimen; Research; Rodent; Role; Sirolimus; Site; Specificity; System; T-Lymphocyte; Techniques; Technology; Topoisomerase; Topoisomerase Inhibitors; Transgenes; Transportation; V1 neuron; Validation; Viral; Viral Vector; Virus; Vision; Vision research; Visual; Visual System; Work; adaptive immune response; arm; brain disorder therapy; cell type; constitutive expression; effective therapy; efficacy evaluation; efficacy validation; frontal eye fields; gene therapy; immunoregulation; improved; in vivo; in vivo evaluation; inhibitor; insight; interest; neural; neural circuit; neuronal circuitry; neurophysiology; nonhuman primate; novel therapeutics; oculomotor; optogenetics; orbit muscle; pharmacologic; prevent; promoter; reagent testing; recruit; retrograde transport; success; superior colliculus Corpora quadrigemina; therapeutic gene; tool; transgene delivery; transgene expression; vector; virology; visual control; visual map; visual motor; visual neuroscience

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.

项目摘要 使用Opsins和其他执行器映射大脑的视觉和视觉电路电路，以靶向和控制 神经元是现代神经科学的核心目标。执行器已成为研究神经元电路的关键， 对脑疾病进行建模并开发新的疗法。神经执行器应用于啮齿动物的研究 其他小动物取得了巨大的成功。但是，在灵长类动物中，这些方法尚未 变革性。主要问题是需要病毒载体传递执行基因，但两者都病毒 在灵长类动物实验室，甚至内部的转导和基因表达一直不可靠。功效是 受到灵长类动物中的复杂先天和适应性报复的免疫反应的阻碍，即使 方法确实有效，缺乏细胞类型的特异性。该项目的总体目的是纳入最新 病毒学，基因疗法和免疫学的进步，以最大化病毒转导，维持慢性基因 表达，并通过在视觉回路中的病毒逆行转运来获得细胞类型的特异性 猕猴的大脑。在整个项目中，光遗传学是执行器介导的干预措施和视觉 眼动系统是测试床。我们专注于两种提供逆行运输的病毒：逆行腺 相关的病毒-2（RAAV2-RETRO）和融合糖蛋白-e假病毒载体（Neuret）。每个都会 传递编码可红色通道的基因（REACHR）。我们的团队，跨越杜克大学，纽约大学和 UNC-Chapel Hill在向量技术和猕猴神经生物学方面拥有广泛的专业知识。目标1是专用的 迈向将灵长类动物免疫系统的两个臂修改为的药理学方案的成熟 增强病毒转导并促进Opsin转基因的长期本构表达。 AIM 2意志 建立逆行转导神经元的综合表达图。该映射是关键的步骤 提供细胞和电路级特异性，并提供一种用于识别神经元的手段， 通过光吸收，基于它们的解剖连通性。 AIM 3将使用与投影配对的光吸收 靶向识别和神经生理学的特征，表征有助于特定电路的神经元 猕猴大脑的视觉和视觉运动电路。结合使用，这项工作将增强病毒的功效 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和 对开发协议的功能验证，并为功能作用提供新的见解 电路在视觉和视觉运动行为中起作用。最后，该项目将为 改善依赖于治疗基因的病毒递送的人类基因疗法。