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

两栖动物（青蛙和蝾螈）是环境变化的关键指示物种；许多动物受到栖息地丧失、海平面上升和气温变化的威胁，因为它们是“冷血动物”，不能调节体温。 然而，一些物种在生理学（例如温度调节）、发育要求以及大脑和脊髓神经细胞活动产生的行为变化方面都能够应对气候变化。 非洲爪蛙（非洲爪蟾）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时间。非洲爪蟾利用这些弹性策略在全球灭绝事件中生存下来。 西班牙肋蝾螈（Pleurodeles）可以再生整个神经系统，即使是成年后也是如此。 为了理解为什么这些特殊的两栖动物如此顽强，我们需要找出它们身体的特定部分在压力条件下如何工作。 该项目旨在开发“病毒载体”，即可以传递到并操纵身体不同部位基因的非传染性病毒。 这些载体可以帮助测试一些想法，例如，青蛙大脑的哪些部分与恢复能力有关，以及蝾螈和蝾螈在受伤时如何再生身体的整个部分。 此外，寻找可以感染两栖动物的病毒的过程将有助于研究人员使用鸟类等其他物种，并可能揭示病毒感染不同宿主物种的能力如何进化的新想法，例如从蝙蝠跳跃到人类。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，并在公共数据库上共享方案和非传染性病毒，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——进化到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）的腺相关病毒 (AAV) 工程是该 EDGE 项目的重点。重组 AAV 的生产能够采用定向进化方法，在两种两栖动物（无尾爪蟾和蝾螈）中进行高通量选择和筛选。 这项研究描述了两个物种的血脑屏障的特征，以确定它是否或在哪个发育阶段形成。 利用 NSF 支持的加州理工学院 CLOVER 中心，研究人员通过静脉注射 AAV 血清型来转染这两个物种；然后，他们收获动物的中枢神经系统，通过两轮筛选对产生的变异进行生产、测序和生物信息分析。 由于 AAV 的承载能力有限，该项目正在开发在这两个物种的神经元中特异性表达的转基因 cre 系。 使用携带 floxed-CRISPR 构建体和经过验证的 gRNA 的 AAV，研究人员通过眼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。 使用经过验证的抗体通过免疫组织化学方法验证敲除。 AAV 按成本与合作者共享并存放在 Addgene 中。 结果通过美国虚拟会议、美国实践研讨会和国际会议分享。 通过基于有机体的网站（例如 Xenbase）向更广泛的研究界提供协议和验证结果。 所有数据和协议均存放在公开可用的数据库中，并在哥伦比亚大学存档。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.