Generation and Characterization of an Osteoclastic Cell Model of Gaucher’s Bone Disease

Abstract Gaucher disease (GD) is caused by recessive mutations in the GBA1 gene (Glucosylceramidase beta 1) generating lysosomal accumulation of glucosylceramides. Bone phenotypes related to GD include Erlenmeyer flask deformity, osteosclerosis, osteonecrosis, osteopenia, and bone pain. Lysosomes modulate osteoclast differentiation, playing a fundamental role in osteoclastogenesis and bone resorption. However, the role of GBA1 mutations in the osteoclast function remains poorly understood. We aimed to generate and characterize an in vitro osteoclast cell model of GD. The Gba1 gene was knocked out with CRISPR/Cas9 in the murine macrophage cell line RAW264.7. One RAW-Gba1KO clone displaying no GBA1 enzymatic activity was selected. Non-edited RAW264.7 cells were used as controls (wt). Expression of osteoclast biomarkers, Rank , Trap , Nfatc1 , and pro-cathepsin K/cathepsin K as well as activity of TRAP protein and bone resorption were assessed. RAW-Gba1KO cells showed reduced expression of the osteoclast differentiation markers Rank and Nfatc1, suggesting that osteoclast differentiation is delayed or impaired compared with wt cells. Despite this, KO cells exhibited higher expression of Trap and cathepsin K, indicating enhanced osteoclast activity. Functional assays confirmed this finding, showing significantly increased TRAP activity and bone resorption in KO cells during differentiation. Additionally, KO cells displayed elevated levels of cathepsin K and pro-cathepsin K, particularly before differentiation, which may contribute to the increased TRAP activation. In conclusion, lack of Gba1 activity induced changes in our osteoclast model which enhanced its bone resorption potential, demonstrating a major intrinsic osteoclastic defect in Gaucher disease of bone.