Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to deficient α-galactosidase A activity and the accumulation of its substrate globotriaosylceramide (Gb3) within lysosomes. FD predominantly affects the kidneys, cerebrovascular system, and the heart, with left ventricular hypertrophy, tachyarrhythmia, and myocardial fibrosis often progressing to heart failure - the leading cause of death in FD patients. While the pathogenesis remains poorly understood, secondary metabolic effects and elevated hypertrophy-signalling pathways are described to be involved. Since current treatments only slow disease progression and are inefficient in advanced stages (enzyme replacement therapy) or are only available to a subset of patients (chaperone therapy), novel treatment approaches are needed. Consequently, this project aims to develop a miRNA-based therapy targeting the cardiac FD phenotype. This potential miRNA-based FD therapy could act by modulating dysregulated secondary metabolic processes, as miRNAs are effective post-transcriptional regulators of gene expression.
To identify miRNAs with therapeutic potential for FD, two in vitro disease models based on induced pluripotent stem cells (iPSCs) have been established at our institute: (1) a GLA knockout (KO) was introduced into an iPSC line derived from a healthy donor and (2) an iPSC line was generated from a FD patient, Both show clinically relevant phenotypes once differentiated into cardiomyocytes (CMs), which makes them a promising tool for identifying novel therapies.
First, miRNA sequencing of GLA-KO-CMs as well as their isogenic control has been performed to identify potentially dysregulated FD-associated miRNAs. Next, significantly regulated miRNA candidates were shortlisted in silico if previously associated with FD-related biological processes, e.g. lysosomal and mitochondrial dysfunction, autophagy and ROS handling capacity. We focussed on miRNAs that were upregulated in the FD model since blocking dysregulated miRNAs with antisense-oligonucleotides is an approach that is already in clinical testing for heart disease. For the selected miRNAs, transcript levels were validated via RT-qPCR using an independent set of iPSC-CM samples (GLA-KO & Fabry patient-derived). In a next step, experimentally validated mRNA targets of the shortlisted miRNAs were retrieved form miRTarBase and checked for overlap with mRNA sequencing data sets of both the GLA-KO and Fabry patient-derived disease model, generated by our institute. Strikingly, this combined approach led us to identify miRNA target LIMS1, which has been implicated before in the context of cardiac remodelling and heart failure. Therefore, an investigation into a putative role of LIMS1 in FD pathogenesis is promising.
In addition, we will further explore the role of our lead miRNA candidates and their mRNA targets in 2D cell culture and 3D cardiac organoid FD models, via gain- and loss-of-function experiments. This will be accompanied by in silico network analyses to elucidate pathophysiological secondary processes.
