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.

Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease

“…The HEXM gene, which encodes the modified Hex a-subunit variant, l, overcomes this challenge because the Hex l-subunit can form a stable homodimer, HexM, which functionally replaces the HexA heterodimer. 28 Overexpression of HexM has been shown to result in its enhanced secretion, and secreted HexM is taken up by other deficient cells through plasma membrane mannose 6-phosphate receptors. In addition, unlike HexA, both active sites in the HexM homodimer can potentially bind the G M2 AP-G M2 complex and hydrolyze G M2 .…”

“…Likely, the encouraging results reported by Matsuoka and colleagues are attributable to restoration of HexB-like activity that takes advantage of the alternative sialidase pathway in mice. As an alternative approach to develop a hybrid subunit, Tropak and colleagues 28 designed a modified asubunit incorporating both the unique b-subunit sequences required to form a stable, HexB-like, homodimer and those sequences needed for the homodimer to interact with the G M2 AP-G M2 complex. The a-active site was retained in order to facilitate the hydrolysis of negatively charged substrates, for example, G M2 .…”

Tay-Sachs disease: a case study

“…Jakkolwiek nie ma jeszcze zastosowania w TSD. Celem badań jest określenie drogi jaką enzym zastępczy mógłby pokonać barierę krew-mózg [14,15]. Terapia metodą ograniczenia substratu (SRT, substrate reduction therapy) ma na celu ograniczenie produkcji cząstek, których rozkład jest upośledzony.…”

Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2Gangliosidosis in Sandhoff Mice

“…As part of the initial characterization of HexM, Tropak et al 28 who used the same mice for short-term analysis of G M2 ganglioside, reported that these neonatal mice injected with scAAV9.47/ HEXM vector had a significant reduction in the storage of the G M2 ganglioside compared with the scAAV9.47/GFP and PBS control mice, but there was still a significant amount of G M2 ganglioside stored in the scAAV9.47/HEXM-injected mice compared with the heterozygous control mice. However, at the long-term terminal endpoint, the G M2 ganglioside levels in scAAV9.47/HEXMinjected mice at 36-49 weeks of age were not different from those in untreated mice at 12-16 weeks of age (Fig.…”

“…This HexM enzyme has been shown to be capable of catabolizing the G M2 ganglioside substrate in association with the human GM2AP. 28 The gene for this new l-subunit, HEXM, is *1.6 kb, and, combined with short regulatory sequences, is able to fit within the scAAV packaging constraints. 29 The current study evaluates the therapeutic effects of systemic delivery of scAAV9.47/HEXM vector in the neonatal SD mouse model.…”