EDGE FGT: NSF-BSF: Development of Viral Vectors for Amphibian Gene Delivery and Manipulation

EDGE FGT：NSF-BSF：用于两栖动物基因传递和操作的病毒载体的开发

• 批准号: 2110086
• 负责人: Darcy Kelley
• 金额: $ 100万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2110086&HistoricalAwards=false
• 关键词: EDGE; FGT; NSF; BSF; Development

Amphibians (frogs and salamanders) are key indicator species for environmental change; many are threatened by habitat loss, rising sea levels and changing temperatures as they are “cold-blooded” and do not regulate body temperature. Some species, however, are resilient in the face of climate change both in physiology (e.g., temperature regulation), developmental requirements, and changes in behavior produced by the activity of nerve cells in the brain and spinal cord. African clawed frogs (Xenopus), though they live in fresh-water throughout life, can sequester in small chambers underground for very long periods when their environment becomes dry and hot. Xenopus used these resilience strategies to survive global extinction events. Spanish ribbed newts (Pleurodeles) can regenerate their entire nervous system, even as adults. To understand why these particular amphibians are so hardy, we need to find out how particular parts of their bodies work under stressful conditions. This project aims to develop “viral vectors”, non-infectious viruses that can be delivered to, and manipulate, genes in different parts of the body. These vectors can help test ideas about, for example, which parts of the brain are involved in resilience in frogs and how newts and salamanders regenerate whole parts of the body when they are injured. Also, the process of finding viruses that can infect amphibians will help investigators using other species such as birds and may reveal new ideas about how the ability of a virus to infect a different host species evolves, leaping from bats, for example, to humans. The project also includes training of undergraduate and graduate students, exposing them to international team science, as well as conferences and workshops, and sharing of protocols and non-infectious viruses on public databases to enable similar research by other investigators.Viruses - natural multigene expression and delivery vehicles - evolved to target different species and tissues. Engineering Adeno-Associated Viruses (AAVs) for cold-blooded vertebrates (semi-aquatic or aquatic amphibians) is the focus of this EDGE project. Recombinant AAVs production enables a directed evolution approach for high-throughput selection and screening in two amphibians: the anuran Xenopus and the newt Pleurodeles. This research characterizes the blood brain barrier in both species to identify whether – or at what developmental stage – it forms. Leveraging the NSF-supported CLOVER Center at CalTech, researchers intravenously deliver an AAV serotype that transfects both species; they then harvest the animals’ central nervous system to produce, sequence, and bioinformatically analyze the resulting variants through two rounds of screening. Because of limits in the carrying capacity of AAVs, the project is developing transgenic cre lines that express specifically in neurons for both species. Using AAVs carrying floxed-CRISPR constructs and validated gRNAs, investigators knock out two native genes – rhodopsin and tyrosinase – in the eye via intraorbital delivery. Knocks outs are verified immunohistochemically using validated antibodies. AAVs are shared at cost with collaborators and deposited in Addgene. Results are shared via a US-based virtual conference, a hands-on US workshop, and an international conference. Protocols and validated results are rendered available to the broader research community via organism-based websites (e.g., Xenbase). All data and protocols are deposited in a publicly available data base and archived at Columbia University.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

两栖动物（青蛙和sal）是环境变化的关键指标。许多人受到栖息地丧失，海平面上升和温度不断变化的威胁，因为它们“冷血”并且不调节体温。然而，在生理学（例如温度调节），发育要求以及神经细胞在大脑和脊髓中的活性产生的行为变化的气候变化时，有些物种在气候变化方面具有抗药性。非洲爪蛙（Xenopus）尽管它们一生都生活在淡水中，但当它们的环境变得干燥和热时，可以在地下小室中隔离。 Xenopus使用这些弹性策略来幸免于全球扩展事件。即使是成年人，西班牙肋骨的纽特（Pleurodeles）也可以再生其整个神经系统。要了解为什么这些特殊的两栖动物如此强大，我们需要找出他们身体的特定部位在压力条件下如何工作。该项目旨在开发“病毒载体”，即可以在身体不同部位传递并操纵基因的非感染病毒。这些向量可以帮助测试有关大脑在青蛙中涉及的韧性的想法，以及在受伤时纽特和萨拉曼德人如何再生身体的整个部位。同样，寻找可以感染两栖动物的病毒的过程将帮助研究人员使用其他物种，例如鸟类，并可能揭示有关病毒感染不同宿主物种的能力如何从蝙蝠跳到人类的新想法。该项目还包括对本科和研究生的培训，将其暴露于国际团队科学以及会议和研讨会，以及在公共数据库上的协议和非感染病毒的共享，以启用其他研究人员的类似研究。病毒 - 自然多基因表达和递送工具 - 进化为靶向不同的物种和组织。该边缘项目的重点是与冷血脊椎动物（半水生或水生两栖动物）的工程腺相关病毒（AAVS）。重组AAVS生产可以采用两种两栖动物的高通量选择和筛查的定向进化方法：Anuran Xenopus和Newt Pleurodeles。这项研究表征了这两个物种的血脑屏障，以确定它是在哪个发育阶段形成的。利用加州理工学院的NSF支持的三叶草中心，研究人员静脉注射了一种AAV血清型，可翻译这两种物种。然后，他们通过两轮筛选来收集动物的中枢神经系统，以产生，序列和生物信息分析所得的变体。由于AAV的承载能力的限制，该项目正在开发转基因CRE系，这些CRE系在这两种物种的神经元中都表达。研究人员使用携带Floxed-Crispr构建体并经过验证的GRNA的AAV敲除了两个本机基因 - 视紫红质和酪氨酸酶 - 在眼内递送中。使用经过验证的抗体对敲门爆炸进行了免疫组织化学验证。 AAV与合作者的成本分享，并存入Addgene。结果通过美国的虚拟会议，美国动手研讨会和国际会议共享。协议和经过验证的结果将通过基于组织的网站（例如Xenbase）提供给更广泛的研究社区。所有数据和协议都存放在公共可用的数据库中，并在哥伦比亚大学存档。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，被视为通过评估而被视为珍贵的支持。