Generation and functional characterization of a new recombinant molecule of human β-glycosylceramidase carrying synonymous mutations

Author(s)

Ana Carolina Coelho

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2018-09-25

Examining board members

Aparecida Maria Fontes; Jose Simon Camelo Junior; Vania D'Almeida; Paula Rahal

• Advisor: Aparecida Maria Fontes

Abstract

Gaucher disease (DG), caused by mutations in the GBA1 gene, is the first lysosomal storage disease described and has become the model for the clinical and phenotypic description of the diseases in that group. Enzyme replacement therapy is the main treatment available for patients with DG, but high cost. Thus, in order to obtain a human cell line with stable expression and high levels of recombinant human GBA lysosomal enzyme, the hypothesis of this study is that the aggregation of the use of synonymous codons, genetic engineering and lentiviral system will allow the generation of a new biologically active recombinant molecule in vitro. To achieve this goal, transient transfection was first performed to assess whether the lentiviral vectors were functional, which was demonstrated by the increase of GBA activity in the supernatant of the lineages by the biological activity assay of GCase. From these results lentiviral particles were produced relative to the GBA cDNA sequences with the original and synonymous codons, both constructs under the control of the HeF1α promoter. The lentiviral titers were about 5.24x108 and 7.88x108 PV / mL, referring to plasmids LV_HeF1α_GBA_WT and LV_HeF1α_GBA_SYN, respectively. Six cycles of MOI transduction were performed between 30-60, and after transduction, the lines were treated with puromycin. Following cloning of the mixed populations, clones with higher levels of the recombinant enzyme were selected. Analyzes of biological activity in the supernatant of the L18_293FT_GBA_WT_PM line was 2.72 U / ml, whereas for L17_293FT_GBA_SYN_PM it was 104.8 U / ml. In relation to the clones that presented higher levels of GBA activity, we obtained clone 4 (19.73 U / mL) and clone 9 (17.08 U / mL) for L18, and clones 15 (585.46 U / mL) and 16 (683.95 U / mL) for L17. Intracellular activity analyzes of GBA for both strains and their respective clones showed enzyme levels in the order of 108.4 U / mg for the mixed population of L18, 146.5 U / mg for clone 4 and 159.8 U / mg for clone 9. Intracellular activity for the mixed population of L17 was 307.5 U / mg and for clones 15 and 16 were about 586.3 and 752.3 U / mg. The mass spectrometry assay showed quantification of the recombinant lysosomal enzyme in the supernatant and intracellular produced by the cell line L18_293FT_GBA_WT_PM and the results showed levels similar to GBA produced by the virgin cell line. On the other hand, the lineage L17_293FT_GBA_SYN_PM showed high levels of GBA recombinant lysosomal enzyme in the supernatant on the order of 38.39 µg GBA and clones 15 and 16 about 60.15 and 53.23 µg GBA, respectively. The quantification of recombinant intracellular GBA enzyme was about 14.08 µg for the mixed population and for clones 15 and 16, it was 6.17 and 5.34 µg GBA, respectively. It was also carried out the scheduling of the mixed population L17 and the results showed that it is possible to expand the production of GBA on a large scale. In this way, we conclude that the lentiviral system was effective and this set of information leads to the interpretation that the use of synonymous codons may consist of a promising strategy to obtain a human cell line with high levels of the recombinant lysosomal enzyme. (AU)

• FAPESP's process: 14/15846-5 - Platform for the development of a human cell line with a high number copies of the synthetic b-glucocerebrosidase cDNA with optimized codons for Gaucher disease
• Grantee: Ana Carolina Coelho
• Support Opportunities: Scholarships in Brazil - Doctorate