Fabry disease is a rare and inherited monogenetic disease caused by mutations in the alpha-galactosidase A gene GLA. The encoded protein, alpha-galactosidase A (α-Gal A), is a lysosomal enzyme of the glycosphingolipid pathway. Mutations lead to the accumulation of its substrate, globotriaosylceramide (Gb-3), in lysosomes of different organs, thereby leading to organ damage, including the heart with heart failure being the most prevalent cause for death in Fabry disease. Current treatment strategies (enzyme replacement therapy and chaperone therapy) are either not effective on cardiomyocytes or only available for certain mutations. Therefore, novel treatment strategies are highly needed. In order to identify such, a disease model that reliably reflects the patient’s cellular phenotype needs to be established.
We had previously shown that living myocardial slices (LMS) can be infected with AAV viral particles demonstrating GFP transgene expression in a dose-dependent manner. We further proved that shRNAs can efficiently knockdown Gla gene expression in cardiomyocytes. This led us to the hypothesis that a shRNA-mediated knockdown of Gla gene expression in LMS would allow us to establish a novel ex vivo Fabry disease model.

We first designed different shRNAs directed against the rat Gla gene and cloned these into AAV viral vectors. For a functional validation, neonatal rat cardiomyocytes (NRCMs) were infected with AAV6-shRNA virus. We identified one shRNA that mediated a very efficient knockdown (up to 95%) of Gla gene expression accompanied with a strong reduction (more than 60%) of α-Gal A enzyme activity. Aiming for a translation of this knockdown approach to an ex vivo model, rat LMS were infected with AAV6-shRNA and –scramble viral particles. After the maximum cultivation period of 48 hours, RNA was isolated and used for gene expression analyses. However, after transduction with an MOI of at least 5x10⁴ viral particles per slice, there was no knockdown of Gla gene expression detectable compared to the scramble control, which suggests that a stable knockdown may only be achieved after a prolonged cultivation period.
In parallel to optimizing LMS cultivation and genetic manipulation conditions, an alternative Fabry disease model was used to investigate a cellular disease phenotype in vitro. We generated induced pluripotent stem cells (iPSCs) from a male Fabry disease patient and differentiated these into cardiomyocytes (CMs). Fabry iPSC-CMs presented a loss of α-Gal A enzyme activity, an accumulation of Gb-3 in lysosomes, a compromised response to cellular stress stimuli leading to an increase in ROS production and apoptosis as well as mitochondrial dysfunction. Thereby, Fabry iPSC-CMs demonstrated hallmarks of the cellular disease phenotype and present a valuable disease model.

In conclusion, we successfully identified a shRNA that mediates a highly efficient knockdown of Gla gene expression in rat cardiomyocytes in vitro on mRNA as well as on functional level. Translation to an ex vivo disease model, however, will need further optimization, especially aiming for a prolonged cultivation of LMS. By using an alternative in vitro Fabry disease model based on iPSC-CMs, we identified a number of cellular dysfunctions that may underlie the patients’ disease phenotype. The identification of such will allow us to stringently analyze respective cellular functions in an optimized 3D ex vivo disease model.