Development of a Lentiviral Vector for High-Yield Production of Synthetic and Recombinant GCase for Gaucher Disease Therapy
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ijms-26-07089.pdf
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Author(s)
Coelho, Ana Carolina
Wiezel, Claudia Emília Vieira
de Campos, Alline Cristina
Figueiredo, Lílian Louise Souza
Suardi, Gabriela Aparecida Marcondes
de Paula Bernardes, Juliana
da Cunha Tirapelli, Daniela Pretti
Faça, Vitor Marcel
Abraham, Kuruvilla Joseph
Carlotti-Júnior, Carlos Gilberto
Date Issued
July 23, 2025
Journal
International Journal of Molecular Sciences
Publisher
Multidisciplinary Digital Publishing Institute
Citation
Coelho, A.C.; Wiezel, C.E.V.; de Campos, A.C.; Figueiredo, L.L.S.; Suardi, G.A.M.; de Paula Bernardes, J.; da Cunha Tirapelli, D.P.; Faça, V.M.; Abraham, K.J.; Carlotti-Júnior, C.G.; et al. Development of a Lentiviral Vector for High-Yield Production of Synthetic and Recombinant GCase for Gaucher Disease Therapy. Int. J. Mol. Sci. 2025, 26, 7089.
Version
Final published version
Abstract
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 standard of care for non-neuropathic 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 (hEF1α) promoter for stable production in human cell lines. A functional lentiviral vector, LV_EF1α_GBA_Opt, was generated at a titer of 7.88 × 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 represents a 3.21-fold increase compared to wild-type 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.
MIT Department
Massachusetts Institute of Technology. Synthetic Biology Center
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DOI of Published Version
https://doi.org/10.3390/ijms26157089
