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

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.

Over the last 3.5 years, I have pioneered transgenic technology in Galleria, generating a microinjection pipeline that has resulted in not only the world's first transgenic Galleria but also the first strains carrying CRISPR mediated gene knockouts.
During this 24-month Fellowship, these methodologies will be employed and further built upon to increase the suite of genetic tools available for this organism. A strain expressing germline specific Cas9 will be generated and used to develop and optimise CRISPR based gene knock-in techniques via both homology and end-joining repair mechanisms.
These precise gene editing techniques will then be used to insert fluorescent reporters in frame with the endogenous coding sequences. Two proteins known to be involved in Galleria's humoral immune response to both bacterial and fungal infection will be tagged: hemolin and apolipophorin III. These proteins have previously been shown to be differentially expressed post infection and are known to both be involved in pattern recognition, opsonisation and phagocytosis of microbial pathogens.
Once these strains have been created, they will be used for in vivo infection assays by injecting larvae with variable doses of immunogenic substances. The qualitative and quantitative data on both localised and total larval fluorescence obtained over the time course of the experiments will provide insight not only as to the kinetics of these tagged proteins but also their suitability as reporters for larval health in automated high throughput screening systems.
The combination of both these new tools and strains will provide a lasting impact in not only Galleria's ability to directly replace rodents in infection studies and drug screens but also to its applications in a wide variety of fields.