Duchenne muscular dystrophy (DMD) is a devastating incurable disease, affecting thousands with heavy burden on health systems. This project combines the development of a safe, “immune-privileged cell” with genetic engineering to correct many dystrophin gene mutations for an efficacious and cost affordable therapy. The applicant pioneered systemic intra-arterial transplantation of mesoangioblasts (blood vessel-derived progenitors) that proved safe in DMD patients and is being implemented for efficacy. However, this personalised approach would prove prohibitively expensive for healthcare systems, as pricing of successful gene therapies is showing. We made the striking observation that human mesoangioblasts can be indefinitely expanded with a novel culture medium, even after genetic manipulation and cloning. Cells will be first genome edited to delete endogenous HLA (β2-microglubin and class II CTA) while inserting tolerogenic HLA-E, fused to β2-microglubin and, as safety device, the Herpes Simplex Thymidine Kinase suicide gene with truncated NGF receptor for selection. Edited clones will be checked for genome integrity. Selected clones will be engineered to express a small nuclear RNA (snRNA) that causes skipping of a given exon of the dystrophin gene. Due to the syncytial nature of muscle fibres, the snRNA also enters and corrects the genetic defect in neighbouring, dystrophic nuclei, thus amplifying of one log the therapeutic effect. Five different cell lines would correct the mutation in 60% of DMD patients. The cell lines will be transplanted in humanized DMD mice and assessed for the ability to escape immune surveillance and to differentiate in dystrophin expressing myofibers, establishing pre-clinical safety and efficacy for an off the shelf, affordable product. The applicant has unique expertise to successfully complete this project, whose strategy may be expanded to other recessive monogenic diseases, for a ground breaking impact in regenerative medicine.

Current knowledge of neurodegenerative diseases is limited by poor understanding of how they progress through the central nervous system (CNS). It has recently been hypothesized that clinical progression in these conditions involves the systematic spreading of protein misfolding along neuronal pathways. Protein aggregates would trigger misfolding of adjacent homologue proteins in newly-affected regions, and this would propagate in a “prion-like” fashion across anatomical connections. This proposal seeks to decipher the mechanisms of network-based neurodegeneration by understanding how the complex architecture of brain networks (the connectome) shapes the evolving pathology of neurodegenerative diseases, and to develop tools for monitoring disease progression from presymptomatic to later stages of the disease. NeuroTRACK will apply emerging network science tools to longitudinal, structural and functional brain connectivity 3T magnetic resonance imaging data from patients with frontotemporal lobar degeneration (FTLD) – a devastating, relentlessly progressive, young onset, neurodegenerative disorder. The study will involve both sporadic and familial cases, including presymptomatic gene mutation carriers. The proposal addresses the following fundamental questions: i) How and where does pathological protein propagation occur in the FTLD phenotypes? ii) Can pathological spreading be predicted from brain connectome fingerprinting? iii) How do different protein abnormalities translate into large-scale network degeneration? iv) How early are brain network changes detectable in the (even presymptomatic) course of the disease? The ground-breaking nature of the experiments planned in this proposal will pave the way to the development of novel tools for understanding the biological underpinnings of other CNS proteinopathies such as Alzheimer’s disease and Parkinson’s disease, and to identifying individualized, early interventions to modify disease progression.

Excess of plasma glucagon is frequently reported in diabetic patients, a misregulation that exacerbates hyperglycemia, a major complication of diabetes. This has spurred research into alpha-cell function and glucagon regulation, to find potential treatments, independent from insulin, for diabetes. To normalize glucagon secretion as a therapeutic strategy, it is crucial to have a better understanding of its regulatory mechanisms and the expression of specific genes regulating those mechanisms. Unfortunately, there is no consensus model explaining the regulation of glucagon secretion from pancreatic alpha-cells. However, calcium is known to be required for glucagon secretion, but its role is not completely understood. We propose to evaluate the role of free calcium activity across islet α-cells in the regulation of glucagon secretion from mice, using a custom built light-sheet fluorescence microscope, allowing fast three-dimensional imaging for an extended amount of time. The interest will be focused on the heterogeneity of the response, which forms subpopulations of α-cells. After, the attention will be addressed in discovering cellular expression patterns of transcription factors known to be relevant in alpha-cells through immunofluorescence, and to determine how these regulate their target genes using single molecule Fluorescence In Situ Hybridization (smFISH). Particular attention will be focused on correlating the heterogeneities of calcium activity in subpopulations of alpha-cells with their differential gene expression. By finding this differential expression, it will be possible to target specific genes in order to trigger various single-cells behaviors and discover novel therapeutic targets.

Regulatory T cells (Tregs) are important for maintaining tolerance to self and are known to contribute to appropriate priming in the case of foreign challenges, however, their role in liver homeostasis and disease remains to be determined. Liver disease represents a significant global health issue with Hepatitis B Virus (HBV), Metabolic dysfunction-associated Steatotic Liver Disease (MASLD), and Hepatocellular Carcinoma (HCC) resulting in considerable morbidity and mortality worldwide and limited treatments are available to address these conditions. This project aims to identify novel mechanisms of targeting Tregs in liver disease to suggest methods to manipulate these cells for therapeutic benefit. Cutting-edge mouse models of HBV, MASLD, and HCC will be used to determine the phenotypes, kinetics, and localisation of Tregs in the liver under homeostasis and during disease. High parameter flow cytometry, spatial transcriptomics, and state-of-the-art intravital imaging will allow us to investigate high-resolution spatiotemporal dynamics of liver Tregs to identify novel molecules, interactions, and functions of these cells. Tissue samples from human patient HBV and HCC cohorts are available to the host lab and can be used to screen and refine the molecules of interest identified in mouse liver Tregs to focus further investigation on clinically relevant molecules. The role of Tregs in T cell priming in HBV, and effects on MASLD progression, and HCC development will be determined using mouse models to specifically deplete Tregs, or delete molecules of interest in Tregs, at defined disease stages. Overall, this project will significantly advance our understanding of Tregs in the liver and has the potential to reveal promising clinical targets. The training in experimental techniques, research communication, and transfer of knowledge my career will be developed towards my goal of becoming an independent researcher.