Development of a Lentiviral Vector for High-Yield Production of Synthetic and Recombinant GCase for Gaucher Disease Therapy

Gaucher disease (GD) is an autosomal recessive disorder caused by the deficient activity of the lysosomal enzyme glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) remains the most effective treatment for GD patients, its high cost significantly limits accessibility. To enhance production efficiency, we developed a lentiviral system encoding a codon-optimized GCase gene driven by the human elongation factor 1a (hEF1) promoter for stable production in human cell lines. A functional lentiviral vector, LV_EF1_GBA_Opt, was generated at a titer of 7.88 x 108 LV particles/mL as determined by qPCR. Six transduction cycles were performed at a multiplicity of infection of 30-50. The transduced heterogeneous human cell population showed GCase-specific activity of 307.5 ± 53.49 nmol/ mg protein/ h, which was 3.21-fold increase compared to virgin 293FT cells (95.58 ± 16.5 nmol/ mg protein/ h). Following single-cell cloning, two clones showed specific activity of 763.8 ± 135.1 and 752.0 ± 152.1 nmol/mg/h (clones 15 and 16, respectively). These results show that codon optimization, a lentiviral delivery system, and clonal selection together enable the establishment of stable human cell lines capable of producing high levels of biologically active, synthetic recombinant GCase in vitro. Further studies are warranted for the functional validation in GD patient-derived fibroblasts and animal models.