Synthetic biology, use of humanized codons and microRNAs for the production of a biopharmaceutical for Gaucher disease

• Grant number: 13/50450-2
• Support Opportunities: Research Grants - Research Partnership for Technological Innovation - PITE
• Start date: October 01, 2013
• End date: September 30, 2017
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Agreement: Agilent
• Principal Investigator: Aparecida Maria Fontes
• Grantee: Aparecida Maria Fontes
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Company: Agilent Technologies Brasil Ltda
• City: Ribeirão Preto

Abstract

Gaucher disease is an autosomal recessive disease with a prevalence of 1:60,000 to 1:200,000 births. So far there is no cure, but there is treatment. Current treatments consist of replacement therapy with placenta-derived glucocerebrosidase (pdGBA) or recombinant glucocerebrosidase (rGBA). Despite the advantages of rGBA compared to pdGBA, such as reduced risks of viral contamination, the recombinant product is expensive, a fact that impairs adequate treatment. One innovation consists on the generation of a biopharmaceutical by synthetic biology. This experimental approach has contributed to the understanding of the fact that the use of preferential codons can increase gene expression by more than 1,000 times. Also, genomic sequences that generate a transcriptionally active environment (euchromatin and ubiquitous chromatin opening elements, UCOE) have been used to maximize the expression of the transgene. Finally, recent studies have identified microRNAs, such as miR-199a-5p or miR-132/212 which inhibit the endoplasmic reticulum chaperone or play an important role in N-glycosylation in the endoplasmic reticulum, respectively, and may play an important role in cellular bioprocesses. Taken as a whole, this information led us to raise the hypothesis that the association of synthetic biology and of the use of humanized codons, of specific genome sequences and microRNAs will permit the development of an optimized platform for obtaining a human cell line permanently producing synthetic GBA. In this respect, the proposal starts with the development of four constructs: vectors 1 and 2 - carrying the Beta-GBA gene with original codons, one in the presence and the other in the absence of the selected microRNA. Four human cell lines carrying the synthetic GBA gene (syntGBA) in their genome will then be generated and the level and biological activity of the synthetic biopharmaceutical will be evaluated in vitro and in vivo assays. The execution of this project will lead to the development of a fourth-generation synthetic biopharmaceutical for replacement therapy for patients with Gaucher disease and will open perspectives for the development of synthetic biopharmaceuticals for other monogenic diseases due to inborn errors of metabolism. (AU)