title: Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.
abstract: Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.
authors.affiliation: Genetics and Genome Biology, SickKids , Toronto, Ontario, Canada.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Tropak
authors.affiliation: Genetics and Genome Biology, SickKids , Toronto, Ontario, Canada.
authors.forename: Sayuri
authors.initials: S
authors.lastname: Yonekawa
authors.affiliation: Department of Ophthalmology and Gene Therapy Center, University of North Carolina , Chapel Hill, North Carolina, USA.
authors.forename: Subha
authors.initials: S
authors.lastname: Karumuthil-Melethil
authors.affiliation: Medical Genetics/Department of Pediatrics, Queen's University , Kingston, Ontario, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Thompson
authors.affiliation: Department of Chemistry and Biology, Ryerson University , Toronto, Ontario, Canada.
authors.forename: Warren
authors.initials: W
authors.lastname: Wakarchuk
authors.affiliation: Department of Ophthalmology and Gene Therapy Center, University of North Carolina , Chapel Hill, North Carolina, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Gray
authors.affiliation: Medical Genetics/Department of Pediatrics, Queen's University , Kingston, Ontario, Canada.
authors.forename: Jagdeep S
authors.initials: JS
authors.lastname: Walia
authors.affiliation: Department of Microbiology, University of Manitoba , Winnipeg, Manitoba, Canada.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
authors.affiliation: Genetics and Genome Biology, SickKids, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Don
authors.initials: D
authors.lastname: Mahuran