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（青蛙和蝾螈）是环境变化的关键指示物种;许多两栖动物受到栖息地丧失、海平面上升和温度变化的威胁，因为它们是“冷血动物”，不能调节体温。 然而，有些物种在面对气候变化时在生理上都具有弹性（例如，温度调节），发育要求，以及大脑和脊髓中神经细胞活动产生的行为变化。 非洲爪蟾（Xenopus）虽然一生都生活在淡水中，但当环境变得干燥和炎热时，它们可以在地下的小房间里隔离很长一段时间。非洲爪蟾利用这些恢复策略在全球灭绝事件中幸存下来。 西班牙肋蝾螈（Pleurodeles）可以再生他们的整个神经系统，即使是成年人。 为了理解为什么这些特殊的两栖动物如此哈代，我们需要找出它们身体的特定部位在压力条件下是如何工作的。 该项目旨在开发“病毒载体”，即非传染性病毒，可输送到身体不同部位并操纵基因。 这些载体可以帮助测试一些想法，例如，大脑的哪些部分与青蛙的恢复力有关，以及蝾螈和蝾螈在受伤时如何再生身体的整个部分。 此外，发现可以感染两栖动物的病毒的过程将有助于研究人员使用其他物种，如鸟类，并可能揭示病毒感染不同宿主物种的能力如何演变的新想法，例如从蝙蝠到人类。该项目还包括对本科生和研究生进行培训，让他们接触国际团队科学，以及会议和研讨会，并在公共数据库中分享协议和非传染性病毒，以便其他研究人员进行类似的研究。病毒-天然的多基因表达和传递载体-进化为针对不同的物种和组织。 针对冷血脊椎动物（半水生或水生两栖动物）的工程化腺相关病毒（AAV）是EDGE项目的重点。重组腺相关病毒的生产，使定向进化方法的高通量选择和筛选两种两栖动物：无尾类非洲爪蟾和蝾螈Pleurodeles。 这项研究描述了这两个物种的血脑屏障，以确定它是否或在哪个发育阶段形成。 利用加州理工学院NSF支持的三叶草中心，研究人员通过静脉注射提供了一种能够抑制两种物种的AAV血清型;然后他们收获了动物的中枢神经系统，通过两轮筛选来产生，测序和生物信息学分析所产生的变体。 由于AAV携带能力的限制，该项目正在开发在两个物种的神经元中特异性表达的转基因cre系。 使用携带双链CRISPR构建体和经验证的gRNA的AAV，研究人员通过眶内递送敲除眼睛中的两种天然基因-视紫红质和酪氨酸酶。 使用经验证的抗体以化学方法验证敲除。AAV与合作者按成本共享并存放在Addgene中。 通过美国的虚拟会议，动手美国研讨会和国际会议分享结果。 通过基于生物体的网站（例如，Xenbase）。 所有数据和协议都存放在一个公开的数据库中，并在哥伦比亚大学存档。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估。