ZEBRAFISH AS AN INNOVATIVE IN VIVO MODEL FOR REGENERATIVE MEDICINE AND RARE GENETIC DISEASES

This thesis investigates regeneration and early disease mechanisms using zebrafish as a unified in vivo platform. The work is organised into two complementary sections. The first explores the regenerative and immunomodulatory properties of microvesicles (MVs) derived from human immortalised amniotic epithelial cells (iAECs), with particular focus on their mitochondrial cargo. The second establishes and phenotypically characterises a severe early-developmental founder generation (F0) zebrafish model of Friedreich’s ataxia (FRDA) generated via CRISPR/Cas9, by targeting the zebrafish frataxin orthologue (fxn). This model serves as a tool to investigate the developmental impact of frataxin deficiency and provides a basis for exploring the therapeutic potential of iAEC-derived MVs in severe mitochondrial diseases. In the regenerative section, iAEC secretomes were fractionated to isolate an MV-enriched component, whose mitochondrial content was tracked in vivo using Turbo Red Fluorescent Protein (TurboRFP)-labelled iAEC mitochondria and qPCR for human mitochondrial DNA (hmtDNA). MV-treated larvae displayed enhanced caudal fin regrowth and altered patterns of proliferation and apoptosis following amputation. In parallel, analyses of neutrophils, macrophages and key cytokines (Tnfα, Il-1β, Tgfβ1) indicated that MVs modulate early inflammatory dynamics in a manner compatible with a pro-regenerative environment. These results suggest that iAEC-derived MVs support tissue repair through a combination of mitochondrial transfer and immune regulation. The identification of active, functional mitochondria within iAEC-derived MVs prompted us to investigate their potential as a treatment for severe mitochondrial diseases. Building on this premise, the disease-modelling section evaluates the efficiency of three single guide RNAs (sgRNAs) targeting exons 1, 3 and 4 of the fxn gene. Targeting fxn gene resulted in efficient F0 editing and produced a range of phenotypes consistent with FRDA, including developmental delay, craniofacial and cardiac defects, and reduced locomotor responsiveness. Analysis in transgenic neuronal, glial and cardiac reporter lines revealed impaired development of high-energy-demanding tissues, while cardio physiological assessment indicated mild bradycardia and altered ventricular activity. Although mosaic and early-onset in nature, this model captures developmental vulnerabilities associated with frataxin loss and provides a basis for future generation of stable mutant lines. Overall, this work highlights the value of zebrafish both as a regenerative model responsive to cell-free therapies and as a vertebrate system for dissecting early consequences of mitochondrial dysfunction. Together, the two complementary approaches offer a platform for future studies on microvesicle-based regenerative medicine and for the development of mitochondrial-targeted therapeutic strategies.