Improving therapy for patients with metabolic diseases

Problem Title

Improving therapy for patients with metabolic diseases

Scientific Title

Expression and characterization of monomeric human alpha-galactosidase; a proposition for second generation enzyme replacement therapy for Fabry Disease patients

Seamus Hughes
Biochemistry and Molecular Biology
iCons Concentration: 
iCons Class Year: 
Class of 2014
Executive Summary 

Fabry's Disease is a lysosomal storage disease caused by a paucity of the alpha-galactosidase (α-Gal) activity. This diminished activity causes the enzyme's substrates, primarily globotriaosylceramide (Gb3), to accumulate in the cell lysosomes of various organs and tissues, impeding their function. Currently, the only treatment for this disease is enzyme replacement therapy (ERT), in which exogenously produced recombinant enzyme is administered to patients intravenously.

However, this approach has demonstrated diminished efficacy in clearing Gb3 from the critical podocyte cells of the kidney's glomerular filtration barrier. This observation is likely due to the inability of the α-Gal homodimer to translocate the glomerular membrane, which inhibits the crossing of large molecules.

We propose that a monomeric version of human α-Gal would possess an enhanced capacity to traverse this size-selective barrier, thereby making ERT more efficient. In order to produce a human monomeric α-Gal, we removed two critical hydrophobic residues on the homodimer interface through site-directed mutagenesis and expressed the protein in insect cells. Size-exclusion chromatography and analytic ultracentrifugation will be used to verify the monomeric state of the protein. Additionally, circular dichroism and kinetic assays will be used to further characterize the enzymology of the monomeric protein. Preliminary data from SEC and kinetic assays have implicated that the mutant α-Gal is in a monomeric state and has retained activity.

Problem Keywords: 
Fabry's Diease
metabolic disease
Scientific Keywords: 
enzyme replacement therapy
human alpha-galactosidase activity
lysosomal storage
podocyte cells