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

项目总结 使用视蛋白和其他致动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。致动器已经成为研究神经元电路的关键， 建立大脑紊乱的模型，并开发新的治疗方法。神经致动器在啮齿动物和动物研究中的应用 其他小动物也取得了巨大的成功。然而，在灵长类动物中，这些方法尚未得到应用。 具有变革性。主要的问题是需要病毒载体来传递致动器基因，但这两种病毒 在整个灵长类实验室，甚至在灵长类实验室内部，转导和基因表达都是不可靠的。功效是 受到灵长类复杂的先天和适应性报复性免疫反应的阻碍，甚至当 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将最近的 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，并通过病毒在视觉回路中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- 动眼系统为实验床。我们专注于两种提供逆行运输的病毒：逆行腺病毒- 联合病毒2(rAAV2-retro)和融合糖蛋白E伪型慢病毒载体(NeuRet)。每个人的遗嘱 传递编码红色可激活通道视紫红质(ReaChR)的基因。我们的团队，横跨杜克大学、纽约大学和 北卡罗来纳大学教堂山分校在媒介技术和猕猴神经生物学方面拥有广泛的专业知识。Aim 1致力于 走向成熟的药物疗法，改变灵长类免疫系统的双臂，以 加强病毒转导，促进视蛋白转基因的长期结构性表达。目标2将 建立逆行转导神经元的全面表达图谱。这一映射是关键的一步 为了提供细胞和电路级的特异性并提供用于生理识别神经元的手段， 通过光标记，基于它们的解剖连接性。Aim 3将使用与投影配对的照片标记 靶向识别和神经生理学特征神经元在脑内的特定回路 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疗效 为猕猴视觉和视觉运动系统的神经科学研究提供了解剖学和 对开发的协议进行功能验证，并为这些特定的功能角色提供新的见解 回路服务于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 改进依赖于治疗性基因的病毒传递的人类基因治疗。

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.