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；大脑中的神经细胞和脊髓的交流性产生，长时间的小腔室在Toir Enviration dry且热烈的策略中生存下来。 OPED的目的是开发“病毒载体”，这些媒介的非感染病毒可以传递到并操纵基因。发现的病毒可以用其他鸟类感染地狱，并可能揭示出有关感染另一种宿主物种的新想法，例如从蝙蝠跳跃到人类。 。 。这项研究表征了这两个物种的血液屏障，或者是在加州型血清型的NSF支持的三叶草中心的表现。这两个物种。 - 基于XENBASE的网站（例如，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。该法定任务值得支持该基金会的知识分子和影响埃里亚。