Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

• Main Authors: Michael B Tropak, Sayuri Yonekawa, Subha Karumuthil-Melethil, Patrick Thompson, Warren Wakarchuk, Steven J Gray, Jagdeep S Walia, Brian L Mark, Don Mahuran
• Format: Article
• Language: English
• Published: Elsevier 2016-01-01
• Series: Molecular Therapy: Methods & Clinical Development
• Online Access: http://www.sciencedirect.com/science/article/pii/S2329050116301474

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.