Enzyme replacement therapy for Pompe disease

Pompe Disease is a rare genetic lysosomal storage disease resulting from mutations in the gene for acid alpha- glucosidase. Mutations in this gene cause a buildup of glycogen within lysosomes, leading to lysosomal engorgement and a disruption of cellular processes. The heart and diaphragm are most often affected leading to death from cardiac or pulmonary failure. The only clinically available treatment methods are enzyme replacement therapy and nutritional and exercise therapy. Neither strategy completely resolves the syndrome, and enzyme replacement therapy is often too expensive for individuals. New treatment options for Pompe disease are needed. Guanidinylated neomycin (GNeo) is a novel molecular transporter which can target large bioactive molecules to the lysosome. If conjugated to acid alpha- glucosidase, GNeo may increase the efficacy of the currently available enzyme replacement therapy for Pompe disease by increasing the capacity of cells to take up the enzyme. In this thesis, the techniques required to demonstrate the efficacy of the GNeo molecular transporter are established. Methods for quantifying acid alpha- glucosidase presence and delivery to fibroblast lysosomes are demonstrated in vitro. A technique for inhibiting the mannose 6-phosphate (M6P) delivery of acid alpha- glucosidase to the endosomal pathway in vitro is described. Finally, a method for conjugating acid alpha- glucosidase to guanidinylated neomycin in order to increase the enzymes affinity for negatively charged glycosaminoglycans is developed