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

60多年来，蜡虫蛾（Galleria mellonella）的幼虫阶段一直用于研究微生物感染。昆虫免疫系统与人类免疫系统之间的相似性，以及昆虫在人体温度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。这些相同的优点也使其能够用作体内模型来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项实验程序。此外，21，495只小鼠和大鼠被用于创建新的遗传品系，更多的小鼠和大鼠被用于维持现有的转基因动物。因此，理论上，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。这些蛾幼虫也可用于进一步研究微生物致病机制或在开始哺乳动物研究之前缩小潜在的候选药物范围。然而，目前限制Galleria在研究界广泛使用和吸收的是缺乏可用于小鼠和大鼠的分子和遗传工具。这项奖学金提案将通过为Galleria开发先进的基因编辑方法来改变这种情况，并利用它们来创造新的菌株，以增加感染和药物筛选研究的实验能力。第一部分将采用已成功用于其他生物的现有CRISPR（基因编辑）技术，并将其修改用于Galleria。新的飞蛾品系将被创造出来，允许非常精确地将新的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，将有可能直接标记蛾自身基因的末端。然后，我们可以很容易地和非侵入性地在真实的时间告诉是什么导致这些基因打开，并收集证据，以了解它们的功能是什么。这项技术的进步将彻底改变科学家们使用Galleria进行研究的方式，大大加强了它们作为传统上使用小鼠和大鼠进行的实验的直接替代品的理由。第二部分将使用这些方法来荧光标记两个基因，我们知道这两个基因与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。通过对携带这些编辑基因的蛾幼虫进行免疫挑战，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过程中的确切变化。这些信息可以快速和非侵入性地收集。它不仅可以用于在实验前检测蛾的基线健康状况，而且还可以使用这些蛾幼虫来大规模筛选潜在的新抗菌治疗药物。该项目产生的技术，转基因菌株和数据将有助于推进Galleria用作啮齿动物的替代模型。除了增加其在微生物感染和药物筛选中的用途外，它还将向对其潜在用途感兴趣的各种领域开放其用途。

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.