Developing gene editing technologies in the non-mammalian infectious disease model organism, Galleria mellonella

蜡蛾的幼虫阶段已经被用来研究微生物感染超过60年。昆虫免疫系统与人类免疫系统的相似之处，以及昆虫在人体体温下长时间存活的不寻常能力，使其成为研究医学相关细菌和真菌感染的方便系统。这些相同的优势也使其成为一种体内模型，用于筛选可以对抗这些感染的新疗法。目前，大多数研究微生物感染机制和潜在新药安全性和有效性的体内实验程序都是在小鼠和大鼠身上进行的。2020年，在免疫、感染和毒理学领域，对这些动物进行了326,495次实验程序。此外，21495只小鼠和大鼠被用来创造新的遗传品系，更多的被用来维持现有的转基因动物。因此，从理论上讲，使用Galleria作为中间模式生物可以大大减少推动药物发现所需的啮齿动物数量。这些蛾子幼虫也可以用于进一步研究微生物发病机制或在开始哺乳动物研究之前缩小潜在候选药物的范围。然而，目前限制Galleria在研究界广泛使用和吸收的是缺乏小鼠和大鼠均可获得的分子和遗传工具。这项奖学金提案将通过开发先进的Galleria基因编辑方法，并利用它们来创建新的菌株，从而增加感染和药物筛选研究的实验能力，从而改变这种情况。第一部分将采用现有的CRISPR（基因编辑）技术，这些技术已经成功地应用于其他生物体，并对其进行修改，以用于Galleria。新的飞蛾品种将被创造出来，它们可以非常精确地将新的DNA片段插入生物体自身的基因组中，而不会影响基因本身。通过插入编码天然荧光蛋白的DNA片段，将有可能直接标记飞蛾自身基因的末端。然后，我们可以很容易地、无创地实时了解是什么原因导致这些基因开启，并收集证据，证明它们的功能是什么。这一技术进步将彻底改变科学家利用Galleria进行研究的方式，极大地加强了使用Galleria直接替代传统上用小鼠和大鼠进行实验的理由。第二部分将使用这些方法荧光标记两个基因，我们知道这两个基因与飞蛾对细菌和真菌微生物感染的免疫反应有关。这两种蛋白质参与识别潜在的微生物入侵者并帮助杀死它们。通过向携带这些编辑过的基因的蛾幼虫引入免疫挑战，并测量幼虫对免疫挑战的总荧光，我将确定这些蛋白质水平在感染的时间过程中究竟是如何变化的。这些信息可以快速、无创地收集。它不仅可以在实验前用于检测蛾子的基线健康状况，而且还可以使用这些蛾子幼虫大规模筛选潜在的新型抗菌疗法。该项目产生的技术、转基因菌株和数据将有助于推进Galleria作为啮齿动物的替代模型的使用。除了增加其在微生物感染和药物筛选方面的应用外，它还将向对其潜在用途感兴趣的各种领域开放其使用。

The larval stage of the waxworm moth (Galleria mellonella) has been used to study microbial infection for over 60 years. The similarities between the insect immune system and that of humans, and its unusual ability for an insect to survive for prolonged periods at human body temperature, make it a convenient system for investigating medically relevant bacterial and fungal infections. These same advantages also enable its use as an in vivo model to screen for new therapeutics that can fight these infections. Currently, most in vivo experimental procedures to investigate both the mechanisms of microbial infection and the safety and efficacy of potential new drugs are performed upon mice and rats. In 2020 across the fields of immunity, infection, and toxicology, 326,495 experimental procedures were performed on these animals. Additionally, 21,495 mice and rats were used to create new genetic strains with many more used just to maintain existing transgenic animals. The use of Galleria as an intermediate model organism could therefore, theoretically, vastly reduce the number of rodents required to drive forward drug discovery. These moth larvae could also be used to further investigate the mechanisms of microbial pathogenesis or narrow down potential drug candidates before commencing studies in mammals.What is currently limiting the widespread use and uptake of Galleria within research communities, however, is the lack of molecular and genetic tools that are readily available for both mice and rats. This Fellowship proposal will change that by developing advanced gene editing methodologies for Galleria, and by using them to create new strains that increase the experimental power of both infection and drug screening studies. The first part will take existing CRISPR (gene editing) technologies that have been successfully used in other organisms and modify them for use in Galleria. New strains of moth will be created that allow the very precise insertion of new sections of DNA, into the organism's own genome, without affecting the genes themselves. By inserting sections of DNA that encode naturally fluorescent proteins, it will be possible to directly tag the end of the moth's own genes. We can then easily and non-invasively tell in real time what causes these genes to switch on, and gather evidence as to what their function is. This technological advance will revolutionise the way scientists are able to use Galleria for research, dramatically strengthening the rationale for their use as a direct replacement for experiments traditionally carried out using mice and rats.The second part will be to use these methods to fluorescently tag two genes that we know are involved in the moth's immune response to microbial infection from bacteria and fungi. The two proteins are involved in recognising potential microbial invaders and in helping to kill them. By introducing an immune challenge to moth larvae carrying these edited genes and measuring the total larval fluorescence in response to an immune challenge, I will determine how exactly these proteins levels change over the time course of an infection. This information can be collected quickly and non-invasively. Not only can it then be used to detect the baseline health of a moth before experiments, but it is also a step forward to being able to use these moth larvae to mass screen potential new antimicrobial therapeutics.The techniques, transgenic strains and data generated by this project will be instrumental in advancing Galleria use as a replacement model for rodents. As well as increasing its use for microbial infection and drug screening, it will also open up its use to a wide variety of fields interested in its potential use.