Introduction of an N-Glycan Sequon Into HEXA Enhances Human β-Hexosaminidase Cellular Uptake in a Model of Sandhoff Disease

• Published in: Molecular therapy 2010-08, Vol.18 (8), p.1519-1526
• Main Authors: Matsuoka, Kazuhiko, Tsuji, Daisuke, Aikawa, Sei-ichi, Matsuzawa, Fumiko, Sakuraba, Hitoshi, Itoh, Kohji
• Format: Article
• Language: English
• Subjects: Animals; beta-Hexosaminidase alpha Chain - chemistry; beta-Hexosaminidase alpha Chain - genetics; beta-Hexosaminidase alpha Chain - metabolism; beta-Hexosaminidase alpha Chain - therapeutic use; beta-Hexosaminidase beta Chain - genetics; beta-Hexosaminidase beta Chain - metabolism; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - genetics; beta-N-Acetylhexosaminidases - metabolism; beta-N-Acetylhexosaminidases - therapeutic use; Cells, Cultured; CHO Cells; Chromatography, Thin Layer; Cricetinae; Cricetulus; G(M2) Ganglioside - metabolism; Glycosylation; Humans; Immunoblotting; Mice; Original; Polysaccharides - chemistry; Polysaccharides - metabolism; Sandhoff Disease - drug therapy; Sandhoff Disease - genetics; Sandhoff Disease - metabolism
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2010.113

Human lysosomal β-hexosaminidase A is a heterodimer composed of α- and β-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the β-subunit. The mutant HexA (NgHexA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHexA was more efficiently taken up than the wild-type HexA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHexA to SD model mice, NgHexA more efficiently restored the HexA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HexA. These findings indicate that i.c.v. administration of the modified human HexA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HexA deficiencies including Tay–Sachs disease and SD.