Development of human enzyme therapeutics and the tools for their engineering




Bonem, Matthew Wesley

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Enzyme therapeutics are a growing class of pharmaceuticals for the treatment of a plethora of diseases, including the treatment of cancer and metabolic disorders. In cancer, amino acid depletion therapy has seen success with the family of l-asparaginase enzymes utilized for the treatment of acute lymphoblastic leukemia (ALL). For metabolic disorders, a new treatment modality termed enzyme replacement therapy (ERT), has seen much success, in particular with lysosomal storage disorders. With the evolution of this field has come the need for improved tools and techniques for the engineering and characterization of potential drug candidates. This work highlights 1) the development of a tool for therapeutic enzyme development, 2) the validation of a therapeutic enzyme for treating homocystinuria, 3) the development of a tool for peptide synthesis and purification, 4) the identification of a scaffold enzyme to replace existing amino acid depletion treatments. Homocystinuria is a metabolic disorder that disrupts the transsulfuration pathway, causing a series of symptoms that typically result in cardiac arrest. This work showcases a novel enzyme therapeutic based on human cystathionine-[gamma]-lyase (hCGL) for the treatment of homocystinuria and demonstrates its efficacy in multiple mouse models, completely preventing the neonatal lethality associated with homozygous deletion of the cbs gene. To further improve the homocystinuria enzyme therapeutic, we explored a number of genetic selection strategies for the directed evolution of enzyme variants with superior catalytic and biophysical properties. Specifically, we designed a genetic selection based on an auxotrophy for α-ketobutyrate and, correspondingly, isoleucine. Also as part of this dissertation, beyond the homocystinuria enzyme therapeutic, we designed and developed an novel, orthogonal technology for the expression and purification of peptides based on human asparaginase-like protein 1 (hASRGL1). Lastly, we explored various human scaffold proteins in an effort to develop a successor to the bacterial asparaginase currently used for the treatment of acute lymphoblastic leukemia.


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