Distil the benefit of bone marrow transplants into safer microglia-targeting treatments for childhood leukodystrophies..

Immune system replacement therapy, also called bone marrow transplant (BMT), has been used for decades to treat inherited white matter disorders (leukodystrophies, LDs). LDs are devastating neurological diseases, affecting mainly children, and associated with loss of white matter, neuroinflammation and decreased survival. BMT is offered to young patients, preferably pre-symptomatically, and has been successful at increasing lifespan and decreasing symptoms. However, BMT is a high-risk treatment associated with 20% mortality rate. The challenge here is to define which aspects of BMT are effective to develop safer treatments. Which immune cell type has a therapeutic role during BMT is hard to dissect since many cell types are involved, ranging from innate to adaptive immunity. My research has identified the microglia as the immune cell driving the benefit of BMT (Hamilton et al, Glia, 2020; Rutherford at al, BioRxiv DOI:10.1101/2023.12.04.569924, 2023). I therefore hypothesise that by targeting microglia and understanding their role in the disease, we can develop safer treatment by keeping the benefit of BMT without the associated mortality. I pioneered LD modelling using the zebrafish as small vertebrate animal model in one form of LD, RNASET2-deficient LD (Haud -Hamilton maiden name- et al, PNAS 2011). We have now developed multiple models of LDs using zebrafish (Green* and Hamilton* et al, ResearchSquare DOI:10.21203/rs.3.rs-4356120/v1 2024- *co first author), which we will be using in this project, alongside new translational models combining human microglia in zebrafish larvae. We have 3 objectives: Identify microglial effectors driving brain-wide neuroinflammation. Preliminary RNA sequencing experiments from our archetype LD model rnaset2-deficient zebrafish identified chemokine-related genes over-expressed specifically in microglia. We will target those genes to assess their role in driving the pathology using our validated and published assays: locomotion, microglia behaviour and morphology, survival and white matter integrity. Define common pathological pathways across multiple LD models. We found common dysregulated pathways across different leukodystrophies: microglial engorgement resulting in activation of the antiviral immune (AV) response. We will use multiple models of LDs ranging from metabolic (Krabbe disease) to auto-inflammatory disorders (SUPV3L1-deficiency), alongside our archetype RNASET2-decifient leukodystrophy model combining both metabolic and auto-inflammatory components. Develop new translational human-zebrafish chimeric models to validate our findings and test new therapies. Validating our zebrafish microglial results in human has been a challenge due to the lack of models allowing in vivo studies of human microglia in a brain environment. Here we will use the transparent zebrafish larvae as a host for human microglia. I have published that the zebrafish embryos can successfully host human stem cells and zebrafish macrophages. Our preliminary experiment show that human iPSC-derived macrophages can be transplanted into fish using our published transplantation protocol. This will allow for the first time the visualisation of healthy and sick human microglia (using RNASET2-deficient iPSCs) in vivo in real time, while performing crucial physiological actions, such as populating the embryonic brain and neurodevelopment apoptosis clearing. This work will identify key common microglial pathways and effectors that are controlling the start of the pathology using different LD models to ensure a broader impact of our work on larger patient numbers. We will develop a new model to visualise in vivo human microglia in zebrafish for the first time, which will be used to validate our zebrafish results and identify human therapeutic targets for developing better and safer therapies for leukodystrophy patients.