The enzyme replacement therapy (ERT) has been clinically applied for genetic lysosomal enzyme deficiencies (lysosomal storage diseases, LSDs) by utilizing functional N-glycans carrying terminal mannose 6-phosphate (M6P) or mannose (Man) residues attached to recombinant human lysosomal enzymes produced by mammalian cell lines. However, the high costs of enzyme drugs and immunogenicity leading to the production of the neutralizing antibodies due to continuous administration and decrease in QOL of the patients have been problematic1). On the other hand, a transgenic technology was developed in 2000 to produce a transgenic (Tg) silkworm Bombyx mori by gene transfer utilizing a piggyBac vector2), and several Tg silkworm strains were established to produce glycoproteins in cocoons (ca 1~5 mg protein/cocoon), including human monoclonal antibodies and cytokines for industrial and medical use3,4). Furthermore, a novel glycotechnology was developed first in Japan to transglycosylate the N-glycans attached to N-glycoproteins to other N-glycan-types using microbial endo-β-N-acetylglucosaminidase (ENGase) mutants, including Endo-M N175Q from Mucor hiemalis5), and Endo-CC N180H from Coprinopsis cinerea6), and N-glycan oxazoline derivatives. Therefore, in recent years we have established Tg silkworms overexpressing the human lysosomal enzyme genes in the silk glands to produce the recombinant enzyme drugs at low cost7). We also applied the transglycosylation technology to produce “neoglycoenzymes” using functional N-glycans carrying terminal M6P residues as donors, and recombinant human lysosomal enzymes derived from TG silkworms as N-glycan acceptors for sustainable production and increase the effectiveness of neoglycoenzymes for the ERT at a less single dose.NeoglycoIDUA production and therapeutic effects on MPS1 disease modelsMucopolysaccharidosis type1 (MPS1) is an autosomal recessive LSD caused by the human lysosomal α-iduronidase (IDUA) gene mutations and IDUA deficiency. MPS1 is associated with excessive accumulation of natural substrates of IDUA, including heparan sulfate (HS) and dermatan sulfate (DS), in the patients’ Figure 1. N-Glycan structure comparison among mammal, insect and Tg silkworm cocoon Cocoon-derived human IDUA did not carry the insect-specific α1,3-linked fucose residues immunogenic for man.The basic N-glycan structures were similar to those of mammals and included but had no mannose 6-phosphate (M6P)-type glycans.87Development of neoglycobiologics using transglycosylation technologyKohji ItohDepartment of Medicinal Biotechnology, Institute for Medicinal Research,Graduate School of Pharmaceutical Sciences, Tokushima University
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