Fabry disease is a monogenetic, inherited lysosomal storage disorder with a worldwide incidence of 1:40,000 – 1:117,000, even though recent newborn screenings have suggested a higher prevalence (1:3200). The underlying mutations in the α-Galactosidase A gene GLA lead to misfolding and/or degradation of α-galactosidase A (α-Gal A) enzyme and thereby to the accumulation of its substrate, the glycosphingolipid globotriaosylceramide (Gb-3), in the cells’ lysosomes. Subsequently, the impaired glycosphingolipid metabolism results in a severe clinical phenotype, which is characterized by multiorgan symptoms like vascular dysfunction, left ventricular hypertrophy, cardiac fibrosis, proteinuria, neuropathies, and renal failure. Current treatment options include enzyme replacement therapy, substrate reduction therapy, chaperone therapy (e.g. Migalastat) or gene therapy. Nonetheless, all mentioned therapies are cost-intensive; require a high patient-compliance and only insufficiently target/cure already developed phenotypes like cardiac fibrosis or proteinuria. Therefore, new treatment options are of high need. Unfortunately, an important limitation to screen and test new Fabry therapeutics is the lack of a suitable animal model, which faithfully recapitulates the human clinical phenotype, especially in the heart. Therefore, we successfully generated patient-specific induced pluripotent stem cells (iPSC) and differentiated them into iPSC-derived cardiomyocytes (CMs) as an in vitro platform to gain further insights into the disease phenotype as well as for the screening for new therapeutic options for Fabry disease.

We generated iPSCs from a male Fabry patient carrying a 959A>T [N320I] mutation by isolating and reprogramming of patient-derived CD34⁺ hematopoietic stem cells using a lentiviral OKSM-vector.

The reprogrammed iPSC clones were characterized for their pluripotency and genetic integrity. They displayed expression of pluripotency markers, chromosomal stability, and preservation of the patient-specific mutation in the GLA gene. Furthermore, the iPSC line showed reduced α-Gal A protein levels and almost no α-Gal A enzyme activity when compared to a wild-type iPSC line. These characteristics (lower protein levels, absent enzyme activity) were retained in the Fabry CMs after successful cardiomyocytes differentiation. Moreover, the Fabry CMs showed a nearly 20-fold increase in Gb-3 accumulation compared to the control CMs after 60 days of differentiation. The Fabry CMs (d60) additionally displayed an impaired autophagic flux, a significantly higher ROS production after H₂O₂ stimulation as well as a significantly increased number of pro-apoptotic cells after doxorubicin treatment.

In summary, we have established a functional Fabry disease model, which reflects the cellular disease phenotype. Utilizing this model, we are now able to further investigate the cardiac specific disease mechanism via metabolomics and proteomics and screen for novel therapeutic options.