The lysosomal storage disorder Fabry disease is caused by numerous mutations (>1000) in the GLA gene. The gene encodes for the lysosomal hydrolase α galactosidase A (α-gal A), which, under physiological conditions, degrades glycosphingolipids, mainly globotriaosylceramide (Gb3). Mutations lead either to a reduction or to the loss of enzyme function, which impairs glycosphingolipid metabolism. As a result, the enzymes’ substrates accumulate in the lysosomes of the patient’s cells with symptoms manifesting essentially in every organ system. However, the range of symptoms varies greatly between individual patients. Nonetheless, heart and kidney failure are the most prevalent causes for mortality in Fabry disease. And while different treatment options such as chaperone therapy or enzyme replacement therapy are available, these only benefit patients with specific mutations or fail to effectively clear Gb3 deposits from cardiomyocytes (CM) and other severely affected cell types. This creates the urgent need for novel therapeutic approaches.
 

However, any efforts to broaden the therapeutic spectrum require reliable disease models for preclinical and translational research. Unfortunately, existing animal models only insufficiently replicate the disease phenotype developed by patients. Especially so when looking into the cardiac manifestation of Fabry disease. Patient-derived induced pluripotent stem cell (iPSC)-based models have proven to be one way to close that gap. However, two identical GLA genotypes do not necessarily correlate with the same disease phenotype. Additional and individual genetic variants might explain this observation. Therefore, we aimed to generate a more general disease model independent of the individual patients’ genetic background. We successfully generated a Fabry disease model using CRISPR Cas9-mediated GLA gene editing in a healthy iPSC line, which is now used in downstream applications such as compound screenings or validation of pre-identified therapeutic targets. 

Selected GLA KO iPSC clones showed the phenotypical characteristics of pluripotent cells, as they stained positive for the pluripotency markers SOX2, NANOG and OCT4. Furthermore, they had the potential to differentiate into the three germ layers underlining their pluripotent characteristics. Following cardiac differentiation, GLA KO iPSC-CM showed the Fabry disease specific phenotype with absent α gal A protein expression, undetectable α-gal A enzyme activity, extensive Gb3 deposits and an impaired reactive oxygen species handling capacity. 

Importantly, GLA KO iPSC-CM represent a potent model system for future drug screening approaches as we demonstrated the feasibility of functionally rescuing the cellular disease phenotype by treatment with a nucleoside-modified GLA mRNA (modRNA) and successfully measuring the treatment effect. Namely, the α-gal A enzyme activity in GLA KO iPSC-CM was restored and Gb3 deposits drastically reduced as measured by automated fluorescence microscopy. In the future, we will screen compound libraries for putative lead compounds with the potential to reduce Gb3 deposits.