Generation and characterization of cellular models of Fabry disease exploiting CRISPR/Cas9 technology for the study of disease patophysiology

Fabry disease (FD) is an X-linked genetic lysosomal storage disorder caused by pathogenic muta-tions in GLA gene, resulting in deficient alfa galactosidase A (α-Gal A) activity. This deficiency leads to globotriaosylceramide (Gb3) deposits, and its derivative globotriaosylsphingosine (lyso-GL-3) in cells, plasma, and urine, causing progressive tissue damage in affected organs, resulting in multisystemic disease, life-threatening complications, and a reduced life expectancy in both males and females. The main affected organs are the kidney and heart. The early symptoms of FD, including neuropathic pain in the extremities, hypohidrosis, and gastrointestinal symptoms such as abdominal pain, diarrhea, and food intolerance, typically begin during childhood. Later in life, many patients develop life-threatening disease manifestations, including chronic renal disease, cardiovascular disease and stroke.
Gb3/lysoGb3 accumuation, triggers a pathogenic cascade that includes an inflammatory response, which continues over time, leading to a chronic state characterized by fibrosis and loss of function of target organs.
Still, much research is needed to pursue a better understanding of the bases of the disease in target organs and the effect of available therapies. However, the analysis of the molecular pathways leading to cellular damage in the kidney and heart has been challenging due to the lack of appro-priate human cellular models
Recent advances in site-specific genome editing techniques have opened new possibilities for the in vitro generation of specific disease cellular models. The use of engineering nucleases to target specific DNA sequences, enables the introduction of mutations in specific genes generating cells that represent excellent disease models. Indeed, they present several advantages in comparisons with other sources of cellular models: (i) could be generated from commercial cell lines without the need to collect cells from patients, (ii) being obtained from continuous cell lines they provide more reproducible results than primary cultured cells, (iii) they grow fast in culture, are easy to transfect and therefore they are suitable for highthroughput screening experiments, (iv) being isogenic cell lines they represent an ideal system to study mutation specific phenotypes.
The general aim of this proposal is to develop and characterize cellular models of FD, exploiting editing technology using human cell lines of kidney and heart organs, the main targets of the dis-ease. In addition, the possible role of lysosomal storage in triggering inflammation and fibrosis will be explored.