Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease
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 patien...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer International Publishing
2024
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| Online Access: | https://hdl.handle.net/1721.1/157396 |
| _version_ | 1824458290160992256 |
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| author | Figueiredo, Lílian L. S. Junior, Wilson L. da Silva Goncalves, Victor W. Ramos, Ester S. D’Almeida, Vania de Souza, Lucas E. B. Orellana, Maristela D. Abraham, Kuruvilla J. Lichtenstein, Flávio Bleicher, Lucas |
| author2 | Massachusetts Institute of Technology. Synthetic Biology Center |
| author_facet | Massachusetts Institute of Technology. Synthetic Biology Center Figueiredo, Lílian L. S. Junior, Wilson L. da Silva Goncalves, Victor W. Ramos, Ester S. D’Almeida, Vania de Souza, Lucas E. B. Orellana, Maristela D. Abraham, Kuruvilla J. Lichtenstein, Flávio Bleicher, Lucas |
| author_sort | Figueiredo, Lílian L. S. |
| collection | MIT |
| description | 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. |
| first_indexed | 2025-02-19T04:23:32Z |
| format | Article |
| id | mit-1721.1/157396 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2025-02-19T04:23:32Z |
| publishDate | 2024 |
| publisher | Springer International Publishing |
| record_format | dspace |
| spelling | mit-1721.1/1573962025-01-05T04:34:30Z Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease Figueiredo, Lílian L. S. Junior, Wilson L. da Silva Goncalves, Victor W. Ramos, Ester S. D’Almeida, Vania de Souza, Lucas E. B. Orellana, Maristela D. Abraham, Kuruvilla J. Lichtenstein, Flávio Bleicher, Lucas Massachusetts Institute of Technology. Synthetic Biology Center 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. 2024-10-21T20:18:59Z 2024-10-21T20:18:59Z 2024-10-04 2024-10-06T03:14:25Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/157396 Figueiredo, L.L.S., Junior, W.L., da Silva Goncalves, V.W. et al. Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease. Discov Appl Sci 6, 527 (2024). PUBLISHER_CC en https://doi.org/10.1007/s42452-024-06227-z Discover Applied Sciences Creative Commons Attribution-NonCommercial-NoDerivs License https://creativecommons.org/licenses/by-nc-nd/4.0/ The Author(s) application/pdf Springer International Publishing Springer International Publishing |
| spellingShingle | Figueiredo, Lílian L. S. Junior, Wilson L. da Silva Goncalves, Victor W. Ramos, Ester S. D’Almeida, Vania de Souza, Lucas E. B. Orellana, Maristela D. Abraham, Kuruvilla J. Lichtenstein, Flávio Bleicher, Lucas Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease |
| title | Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease |
| title_full | Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease |
| title_fullStr | Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease |
| title_full_unstemmed | Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease |
| title_short | Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease |
| title_sort | engineering synthetic and recombinant human lysosomal β glucocerebrosidase for enzyme replacement therapy for gaucher disease |
| url | https://hdl.handle.net/1721.1/157396 |
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