CHARACTERIZATION OF A NEW CELLULAR MODELS OF GAUCHER DISEASE GENERATED USING CRISPR/CAS9 TECHNOLOGY.

ORMAZABAL, MAXIMILIANO; PAVAN, ELEONORA; VAENA, EMILIO; DARDIS, ANDREA; ROZENFELD, PAULA

Orlando

Congreso; 16TH ANNUAL WORLDSymposium; 2020

Gaucher disease (GD) is the most common lysosomal storage diseases (LSDs). It is caused by mutations in the GBA1 gene that encodes acid- β -glucocerebrosidase (GCase), the enzyme responsible for the degradation of glucosylceramide (GlcCer) that accumulates progressively in lysosomes, particularly in macrophages. Gaucher´s macrophage is the classic pathological hallmark of the disease. Moreover, GBA1 mutations constitute the main genetic risk factor for developing Parkinson´s disease (PD), a neurodegenerative disorder.To better understand the pathophysiological molecular mechanism, it is completely necessary to have a cellular model that represents the characteristics of the disease. The available cell lines that model Gaucher´s disease do not demonstrate the storage of lysosomal glycolipids, or a complete pathological phenotype. In addition, the development of therapeutic strategies has been hampered by the shortage of appropriate cellular models of Gaucher´s disease. In this sense, our study focused on the development and characterization of new cellular models of GD, exploiting the new technology for genome editing CRISPR/Cas9. It is a programmable tool for site-specific gene editing with low cost, easy application, and high efficiency. Specifically, monocytes (THP-1) and glial (U87) cells have been used for disease modelling by knocking out the GBA1 gene. After editing, these two continuous cells lines, THP-1 and U87 clones had altered GCase protein expression, displayed mutations in the GBA gene, reduced or absent GCase activity and accumulation of LysoGL1.In summary, we developed new GD cellular models using CRISPR/Cas technology in two different cell lines: THP-1 GBA KO model may result extremely useful to the purpose of pursuing a better understanding of GD pathophysiology and developing novel therapeutic approaches, since it can be differentiated in into macrophage-like cells; and U87 GBA KO clones were used to study neurological features of GD and a possible link to PD, studying the expression of α-synuclein.