New insights in Gaucher cells models: characterization of a monocyte edited cells using CRISPR/Cas9 technology

PAVAN E; VAENA EMILIO; ROZENFELD PA; ORMAZABAL MAXIMILIANO; DARDIS A

Orlando

Simposio; Lysosomal Disease Network-15th Annual WORLD Symposium; 2020

Gaucher disease (GD), the most common lysosomal storage diseases, 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. 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 GD. 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. Specifically, monocytes (THP-1) cells have been used for disease modelling by knocking out the GBA1 gene. 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. Our previous results with this technology allow us to develop the protocol to obtain the edited cells in the Hek293 context. In addition, we obtained a glial model in the U87 cell line. In this opportunity, edited THP-1 clones had altered GCase protein expression, displayed mutations in the GBA gene, reduced or absent GCase activity and accumulation of LysoGL1.For a deeper characterization, we analyze different cell pathways, such as apoptosis, inflammation and more specifically to bone homeostasis, osteoclast differentiation. All these phenotypes reflect the typical characteristics described for different Gaucher cell models. In summary, we have a new cellular model of GD capable of reproducing several of the typical phenotypes of the disease, an increase in apoptosis associated with enzymatic deficiency, higher levels of the pro-inflammatory cytokine, and finally a greater generation of osteoclasts.