Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease

• Published in: Discover Applied Sciences 2024-10, Vol.6 (10), p.527-21, Article 527
• Main Authors: Figueiredo, Lílian L. Souza, Junior, Wilson Lau, da Silva Goncalves, Victor Wendel, Ramos, Ester Silveira, D’Almeida, Vania, de Souza, Lucas Eduardo Botelho, Orellana, Maristela Delgado, Abraham, Kuruvilla Joseph, Lichtenstein, Flávio, Bleicher, Lucas, Azevedo, Vasco, Chaves, Rigoberto Gadelha, Bonfá, Giuliano, Siciliano, Velia, Weiss, Ron, Gerson, Stanton, Fontes, Aparecida Maria
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Animal models; Catalytic activity; Coevolution; Conserved sequence; Cytomegalovirus; Dementia disorders; Disease; Endoplasmic reticulum; Enzymatic activity; Enzyme coevolution; Enzymes; FDA approval; Gaucher disease; Gaucher's disease; Gene expression; Gene regulation; Gene therapy; Glucocerebrosidase; Glucosylceramidase; Health risks; In silico molecular evolution; Lewy bodies; Lysosomal storage diseases; Mammals; Missense mutation; Molecular evolution; Mutants; Mutation; Nucleotide sequence; Peptides; Plasmids; Predictive control; Protein structure; Proteins; Signal peptide; Synthetic biology; Tertiary structure; Transfection; Vectors (Biology)
• ISSN: 3004-9261; 2523-3963; 3004-9261; 2523-3971
• DOI: 10.1007/s42452-024-06227-z

Gaucher Disease (GD) is an autosomal recessive, lysosomal storage disease caused by pathogenic variants in the glucocerebrosidase gene, leading to the loss of β-glucocerebrosidase (GCase) enzymatic activity. Enzyme replacement therapy (ERT) with recombinant GCase is the standard of care in GD patients. Our study investigates the combined use of in silico molecular evolution, synthetic biology and gene therapy approaches to develop a new synthetic recombinant enzyme. We engineered four GCases containing missense mutations in the signal peptide (SP) from four selected mammalian species, and compared them with human GCase without missense mutations in the SP. We investigated transcriptional regulation with CMV and hEF1a promoters alongside a GFP control construct in 293-FT human cells. One hEF1a-driven mutant GCase shows a 5.2-fold higher level of transcription than control GCase. In addition, this mutant exhibits up to a sixfold higher activity compared with the mock-control, and the predicted tertiary structure of this mutant GCase aligns with human GCase. We also evaluated conserved and coevolved residues mapped to functionally important positions. Further studies are needed to assess its functionality in a GD animal model. Altogether, our findings provide in vitro evidence of the potential of this engineered enzyme for improved therapeutic effects for GD.Article highlights. Conservation and coevolution analysis of amino acid frequencies in GBA1 homologs of pathogenic variants associated with Gaucher disease, Parkinson’ disease risk or Dementia with Lewy bodies risk.Graphic AbstractArticle HighlightsA novel synthetic recombinant GCase enzyme engineered with missense mutations in the signal peptide from selected mammalian speciesCorrelation between transfection efficiency and catalytic activity indicates possible intrinsic sequence features influencing DNA uptake and gene expression