Fabry disease is a rare lipid storage disorder, but is more frequent within selected patient populations, such as those suffering from chronic kidney disease or hypertrophic cardiomyopathy [1-3]. Fabry disease results from an X-linked mutation in the α-galactosidase A (GLA) gene, producing GLA enzyme deficiency or absence. This leads to progressive lysosomal globotriaosylceramide (Gb3) accumulation and thus multi-organ damage and dysfunction (including renal, cerebrovascular, and cardiac) [4]. The vast majority of Fabry disease patients experience kidney damage, resulting in end-stage renal disease and early mortality [5]. While enzyme replacement therapy is available [6], it is extremely expensive and cannot reverse damage which has already occurred (e.g., in advanced disease) [7]. To receive enzyme replacement therapy treatment, Fabry disease patients must visit the hospital once every 2 weeks and receive intravenous injections for several hours. Oral treatments are being developed, and patient convenience is expected. However, there are hurdles to conducting effective evaluation of therapeutics. Because it is difficult to recruit sufficient numbers of patients for clinical trials, because no available in vivo animal model species fully recapitulates the human phenotype, and because no satisfactory predictive toxicology assay model currently exists [8]. Therefore, there is a critical need for an appropriate model that predictive high-throughput screening for drug development.

Organoids derived from pluripotent or adult stem cells are increasingly employed by various studies [9,10]. Given that organoids exhibit organ-like architecture and may recapitulate in vivo disease mechanisms, they are attractive tools for disease modelling [11]. While Itier et al. [12] developed a human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte model of Fabry disease [13], no Fabry disease model based on nephron organoids differentiated from stem cells yet exists. The present study aimed to develop an organoid-based Fabry disease model derived from human embryonic stem cells (hESCs) featuring GLA mutations introduced via CRISPR/Cas9.

Ethics statement: The research described in this thesis utilized cell lines. As these cell lines were not directly obtained from human subjects, Institutional Review Board (IRB) approval was not sought. Research involving cell lines should consider the origin and ownership of the cell lines. The cell lines used in this study were provided by reputable suppliers, and their use was conducted in accordance with ethical guidelines and regulations.The authors of this thesis affirm that all research procedures involving the use of cell lines were conducted in compliance with ethical principles and guidelines, and efforts were made to ensure the accuracy and integrity of the research findings.

Disease modelling employing human-derived organoid cultures is beneficial for both disease mechanism elucidation and drug screening [16]. Furthermore, this approach is extremely valuable in the investigation of rare diseases such as Fabry disease, as the limited number of available patients hampers drug evaluation.

Recent advances in the generation of kidney organoids, together with gene editing systems such as CRISPR/Cas9, have extended this approach to renal disease [17]. One example is an in vitro polycystic kidney disease model derived via differentiation of hPSCs featuring targeted PKD1 and PKD2 gene mutations. Similarly, Freedman et al. [18] used the CRISPR/Cas9 system to introduce appropriate targeted mutations, followed by differentiation of mutated cells into kidney organoids which recapitulate the renal tubule cell cysts characteristic of renal cystic disease. Among the organs affected during Fabry disease, the kidney is particularly significant: renal failure is the major cause of death. Although many studies have attempted to generate in vitro and in vivo models of Fabry disease, none have developed kidney organoid-based models. For example, Pereira et al. [19] generated a Fabry disease model based on CRISPR/Cas9-modified immortalized podocytes, and Itier et al. [12] differentiated patient-derived iPSCs into cardiomyocytes recapitulating lysosomal Gb3 accumulation.

The present study differentiated GLA knockout hESCs to successfully develop a kidney organoid-based Fabry disease model, in which organoids form nephron-like structures expressing podocyte- and proximal tubule epithelium-specific markers and recapitulate absent GLA expression concomitant with lysosomal Gb3 accumulation and Fabry disease-pathognomonic zebra body formation. Because the Fabry disease organoid model created through this study mimics the characteristics of human diseases, it can be used for screening new drug candidates or assessing the effectiveness of therapeutic agents. To the best of our knowledge, this is the first study to report that GLA knockout hESC-derived kidney organoids recapitulate Gb3 accumulation. To further validate model robustness for drug screening, it would be necessary to investigate whether currently available interventions (e.g., GLA replacement therapy) decrease Gb3 accumulation.

In conclusion, we report the first kidney organoid-based Fabry disease model, which may provide a cornerstone for further disease mechanism elucidation and therapeutic drug discovery.