The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells

Lysosomalreplacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycanreceptors, and glycanstructures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycanfeatures. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cellsthat enables production of lysosomalenzymes with N- glycanscustom designed to affect key glycanfeatures guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry diseasemouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphateand mannose receptorsexhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrixand engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics.