Fabry disease is a rare monogenic disease caused by mutations in the GLA gene, which encodes the enzyme α-galactosidase A (α-gal A). This enzyme catalyzes the lysosomal hydrolysis of glycosphingolipids such as globotriaosylceramide (Gb3). Because of α-gal A impairment, Gb3 accumulates throughout the body, leading to functional impairment in multiple organs, including the vascular system. The cardiac manifestation of Fabry disease is associated with cardiac remodeling, fibrosis and left ventricular hypertrophy, with the leading cause of death among patients being heart failure. However, the disease phenotype cannot be attributed solely to Gb3 accumulation, as evidence suggests that the activation of secondary metabolic processes contributes to cellular and organ damage. Approved therapies remain insufficient: chaperone therapy is available only to a subset of patients, while enzyme replacement only achieves incomplete substrate clearance, particularly in the heart. Since currently available in vivo and in vitro models fail to reproduce the complexity of the symptoms seen in patients, the establishment of a human-relevant cardiac 3D model is crucial for understanding processes underlying pathogenesis and identifying new therapeutic targets.
Building on a previously established protocol, we generated cardiac organoids composed of 50% iPSC-derived cardiomyocytes and 50% non-cardiomyocytes, including human cardiac fibroblasts, along with iPSC-derived endothelial cells and human adipose-derived stem cells in a 4:2:1 ratio, respectively. Cardiomyocytes and endothelial cells were differentiated from two iPSC lines: one generated from a Fabry disease patient and its isogenic control, corrected via CRISPR/Cas-based prime editing of the GLA mutation. As Fabry disease also heavily affects the vascular system, incorporating patient-derived endothelial cells enables a more comprehensive representation of the disease phenotype. The use of cells differentiated from these iPSC-lines further allows dissection of cell-cell interactions between mutant and non-mutant populations, as the other cell types included in the model are genetically unaffected.
Fabry organoids exhibited reduced enzyme activity compared to the isogenic control. In line with this observation, Gb3 accumulation was evident in diseased organoids, but was absent in healthy conditions. Strikingly, treatment of diseased cardiomyocytes with an AAV vector designed to overexpress wild-type α-gal A prior to organoid assembly effectively prevents this accumulation. Immunofluorescence-based analysis revealed altered endothelial organization in Fabry organoids, suggesting vascular structure abnormalities. Additionally, Fabry organoids presented increased oxidative DNA damage relative to its control. While no significant differences in key hypertrophic and fibrotic markers have been detected so far, ongoing work focuses on identifying Fabry-distinctive functional and metabolic differences, including different time points. Planned single-nuclei sequencing will further explore intercellular interactions between cardiomyocytes and endothelial cells within organoids, providing a more comprehensive understanding of Fabry disease pathogenesis.
