Gaucher disease (GD) is a rare lysosomal storage disease caused by pathogenic variants in the glucocerebrosidase gene (GBA1) resulting in a markedly decreased activity of the lysosomal enzyme b-glucocerebrosidase (GCase). Enzyme replacement therapy is the gold standard for treating GD patients.

Here, we investigate whether in silico molecular evolution can be combined with synthetic biology and gene therapy for the development of a new recombinant enzyme with higher activity.

We developed novel GBA variants by introducing nonsynonymous mutations into the GBA gene obtained using in silico molecular evolution. These GBA variants are denoted as GBA-7, GBA-8, GBA-9 and GBA-12 and were cloned into lentiviral plasmids using synthetic biology approaches. Additionally, we obtained the wild-type GBA sequence from Homo sapiens, which is labeled GBA-Opt. In addition, we also aim to explore the differences in the transcriptional regulation using two different promoters: the CMV and the human elongation factor 1A (hEF1A). As a control, we generated a construct that expresses the green fluorescent protein (GFP) gene under control of the same promoters. The characterization of these DNA constructs was performed using 293FT human cell line. We generated a total of 166 293FT cell lines with transient production of GBA (n = 100), GBA + GFP (n = 40), GFP (n = 8), and included virgin cells (n = 18). Out of the 100 GBA cell lines, 50 were utilized for real-time PCR analysis to investigate the transcription levels, while the remaining 50 were used to assess the enzyme-specific activity of GCase variants by fluorimetric assay. Furthermore, we determine mRNA secondary structures of GBA variants using the ViennaRNA and three-dimensional structures of the GCase-Opt and GCase-7 enzymes were elucidated using the AlphaFold2.

Quantitative real time PCR revealed that for 4 GBA transcripts (GBA-opt, GBA-7, GBA-9 and GBA-12) under control of hEF1A promoter showed higher expression in 293FT cells compared with CMV promoter (p < 0.05). Among the GBA variants, we observed that 293FT_GBA-7 cells express 5.2-fold higher transcript levels compared with 293FT_GBA-opt cells (p < 0.05). In cells lines co-transfected with GFP and GBA, we observed that comparing normalized activity values while considering transfection efficiencies can provide a more accurate assessment of the enzyme catalytic properties. We also screened the cell lysates for GCase specific activity. We observed that 293FT_GBA-7 cells and 293FT_GBA-Opt cells produced 507.6 ± 38.16 and 426.6 ± 25.25 nmol substrate hydrolyzed/mg protein/h, respectively. For the other transgenic 293FT cell lines these values were lower. Also, for 293FT transfected with mock plasmid this value was 82.92 ± 5.83 nmol/mg/h, respectively (p = 0.014). The 3D structures of GCase-Opt and GCase-7 revealed a high degree of alignment with the Homo sapiens enzyme (UniProt P04062).

These findings demonstrate the promise of using in silico molecular evolution and synthetic biology approaches for developing enhanced enzyme variants for Gaucher disease, and suggest that GBA-7 could be a viable candidate for further investigation for replacement therapy in GD. Additional studies are warranted to investigate the biodistribution and long-term effects of GBA-7 in animal models.