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Directed Evolution of L-Asparaginase for Improving In Vivo Stability

Grant number: 19/09354-6
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): September 16, 2019
Effective date (End): March 01, 2020
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Gisele Monteiro
Grantee:Guilherme Meira Lima
Supervisor abroad: Ratmir Derda
Home Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Local de pesquisa : University of Alberta, Canada  
Associated to the scholarship:18/15041-8 - Expression of L-asparaginase from Erwinia chrysanthemi in cell-free protein synthesis technology, BP.MS

Abstract

L-Asparaginase (L-ASNase) is a key component in the treatment of acute lymphoblastic leukemia (ALL). The introduction of this biopharmaceutical product in pediatric medical protocols improved ALL survival rates to approximately 90%. However, most L-ASNases in the market, especially the L-ASNase isolated from Erwinia chrysanthemi, display a relatively high immunogenicity and a short half-life, making ALL treatment difficult, particularly to adult patients. Drug clearance mechanisms such as antibody formation and protein degradation by lysosomal proteases are one of the main reasons responsible for the mentioned drawbacks. A L-ASNase with enhanced in vivo stability is therefore needed. Directed evolution could be used as an engineering strategy to design improved versions of L-ASNase. In this context, we propose to create novel L-ASNase mutants from E. chrysanthemi expressed in cell-free protein synthesis platform with improved biological stability through iterative rounds of mutations followed by in vitro and in vivo artificial selections. A library of L-ASNase gene variants created previously by our lab will be used and each expressed variant will be selected accordingly to their enzymatic activity, level of expression and stability on human serum. Finally, each viable L-ASNase mutant will be chemically modified and ligated to M13 phages. The resulting products will be combined and injected into mice, where the most stable variant will be selected and used as a starting point for the next round of mutations through site-directed mutagenesis. The strategy could elucidate important protein structure properties of L-ASNase and create variants better suited for medical applications in the future.