**Generation of** *GLA***-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy**

**Hui-Yung Song 1,2, Chian-Shiu Chien 1,2, Aliaksandr A. Yarmishyn 1,2, Shih-Jie Chou 1,2, Yi-Ping Yang 2,3,4, Mong-Lien Wang 1,2,3,4, Chien-Ying Wang 3,5, Hsin-Bang Leu 2,3,6, Wen-Chung Yu 3,6, Yuh-Lih Chang 1,3,4,7,\* and Shih-Hwa Chiou 1,2,3,8,\***


Received: 25 February 2019; Accepted: 4 April 2019; Published: 8 April 2019

**Abstract:** Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of *GLA* gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.

**Keywords:** Fabry disease; human embryonic stem cells; CRISPR/Cas9 genomic editing; Mass spectrometry proteomic analysis; hypertrophic cardiomyopathy; disease model

#### **1. Introduction**

Approximately 3–5% of inherited hypertrophic cardiomyopathies result from lysosome storage disorders [1]. One of these cardiomyopathies is Fabry disease (FD), which is caused by alpha-galactosidase A (GLA) deficiency leading to accumulation of globotriaosylceramide (Gb3) in several tissues. FD is particularly manifested in renal and cardiovascular dysfunctions [2]. A number of in vivo and in vitro studies revealed that the loss of GLA results in left ventricular hypertrophy and develops into heart failure, myocardial infarction and life-threatening arrhythmias due to Gb3 deposition. The levels of Gb3 and its deacylated derivative, globotriaosylsphingosine (lyso-Gb3) in plasma and tissues are used as diagnostic biomarkers of FD, and are applied for the screening/monitoring of FD patients by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3–6]. However, the limited sample volume and variable levels of lyso-Gb3 in different matrices makes the quantitation very challenging [7]. Furthermore, the detailed mechanism of how Gb3 accumulation results in hypertrophic cardiomyopathy still needs to be elucidated. In order to develop new therapeutic strategies for FD-associated vasculopathy, it is essential to understand the underlying pathogenesis mechanisms, as well as to discover the potential prognostic biomarkers.

Previously, we generated CRISPR/Cas9-edited GLA-null human HEK293 cells; however, this cell line may not be an appropriate model with which to study pathological events occurring in cardiomyocytes [8]. The pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs), offer a great potential for modelling human diseases as they can be differentiated into the tissue affected by the pathology. Although several FD-specific iPSC lines have been obtained from patients carrying different *GLA* gene mutations, which claimed to be useful for FD cardiomyopathy research [9,10], one major limitation of such approach is the influence of variable genetic background, which can be significant even for monogenic, dominant and highly penetrant disease in FD [11,12]. Currently, CRISPR/Cas9 emerges as a powerful genome editing technique, providing the opportunity to efficiently delete genes to establish isogenic cells [13–15]. Therefore, our strategy in this study was to generate *GLA* knockout in human pluripotent stem cells by CRISPR/Cas9-mediated gene editing, and compare them with the parental cells of the same genetic background to study the mechanisms of FD-associated cardiomyopathy.

Recently, it became increasingly clear that lysosomal storage disorders have an impact on autophagic dysfunction [16]. Dysregulated ceramide metabolism can trigger cytotoxic signaling cascades, including apoptosis and necroptosis, missorting and accumulation of these sphingolipids in the membrane subdomains may destabilize lipid bilayer and cause their permeabilization [17]. Exosomes are small vesicles secreted upon fusion of multivesicular endolysosomal compartments with the plasma membrane and are derived from the intraluminal vesicles (ILVs) of those organelles. Exosomes may participate in the control of cellular homeostasis by promoting the release of intracellular harmful components, including proteins, lipids or nucleic acids. Emerging evidence from the studies of normal development, as well as multiple disease studies, is beginning to reveal a coordinated exosome–autophagy response that functions to maintain homeostasis through lysosomal degradation and release of cellular cargo [18–20]. However, little effort has been made to investigate the impact of autophagic dysfunction in FD on biogenesis and secretion of exosomes.

In the current study, we applied CRISPR/Cas9-mediated genomic editing to deplete *GLA* expression in ESC-derived cardiomyocytes to recapitulate FD cardiac hypertrophy in vitro and performed proteomic analysis by LC-MS/MS. We identified that Rab GTPase signaling-related vesicle secretion is the factor that may initiate or exacerbate the development of FD-associated cardiomyopathy. Such information will be extremely important for potential application in the prevention of and in interventions for the adverse effects of the cardiac hypertrophy in FD patients.
