Letter to the Editor
Authors: Brian L. Mark, Don Mahuran
Journal: Molecular Therapy (2020)

We wish to bring to your attention inaccuracies in the commentary published by Brian W. Bigger in the recent October 2020 issue of Molecular Therapy.1Bigger B.W. Dual Purpose Vectors for Rare Neurological Diseases.Mol. Ther. 2020; 28: 2104-2105Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar The commentary discusses an article published in the same issue by Lahey et al.2Lahey H.G. Webber C.J. Golebiowski D. Izzo C.M. Horn E. Taghian T. Rodriguez P. Batista A.R. Ellis L.E. Hwang M. et al.Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.Mol. Ther. 2020; 28: 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar The first inaccuracy involves the role played by the human β-hexosaminidase isozymes HexA and HexB in GM2-ganglioside turnover. While it is true the α- and β subunits of human β-hexosaminidase combine to form three different isoforms—HexA (α/β), HexB (β/β), or HexS (α/α)—only HexA is able to hydrolyze GM2 ganglioside in humans. HexB is not involved in GM2 ganglioside turnover in humans since it lacks an α subunit and thus the α-active site needed to hydrolyze the ganglioside.3Mahuran D.J. Biochemical consequences of mutations causing the GM2 gangliosidoses.Biochim. Biophys. Acta. 1999; 1455: 105-138Crossref PubMed Scopus (223) Google Scholar,4Mark B.L. Mahuran D.J. Cherney M.M. Zhao D. Knapp S. James M.N. Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease.J. Mol. Biol. 2003; 327: 1093-1109Crossref PubMed Scopus (180) Google Scholar Further, elements of both the α- and β subunit (aside from the β subunit active site) are necessary for HexA to correctly interact with the GM2 Activator Protein.3Mahuran D.J. Biochemical consequences of mutations causing the GM2 gangliosidoses.Biochim. Biophys. Acta. 1999; 1455: 105-138Crossref PubMed Scopus (223) Google Scholar,5Lemieux M.J. Mark B.L. Cherney M.M. Withers S.G. Mahuran D.J. James M.N. Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.J. Mol. Biol. 2006; 359: 913-929Crossref PubMed Scopus (139) Google Scholar,6Tropak M.B. Yonekawa S. Karumuthil-Melethil S. Thompson P. Wakarchuk W. Gray S.J. Walia J.S. Mark B.L. Mahuran D. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.Mol. Ther. Methods Clin. Dev. 2016; 3: 15057Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar This latter protein correctly presents GM2 ganglioside to the α subunit active site of HexA and is required for ganglioside hydrolysis. It is a common misconception to assume HexB is needed for GM2 turnover due to the nomenclature that is used to describe the biochemistry of the hexosaminidase system and the associated diseases. Tay-Sachs disease occurs from loss of HexA activity due to mutations in HEXA (which codes for the α subunit), whereas, Sandhoff disease occurs from loss of HexA due to mutations in HEXB (which codes for the β subunit of HexA).3Mahuran D.J. Biochemical consequences of mutations causing the GM2 gangliosidoses.Biochim. Biophys. Acta. 1999; 1455: 105-138Crossref PubMed Scopus (223) Google Scholar Thus, mutations in the β subunit cause disease because of their negative effect on HexA formation, not because of a lack of the HexB isozyme. This misconception led to a second inaccurate comment about our own work on the development of HexM as a therapeutic strategy to treat both Tay-Sachs and Sandhoff disease. Since Dr. Bigger assumed HexB is needed for GM2 turnover in humans, he incorrectly stated that HexM could not be used to treat Sandhoff disease. This is not true. HexM is an engineered hybrid enzyme that combines the critical features of the α- and β subunits of human HexA into a single subunit (μ).6Tropak M.B. Yonekawa S. Karumuthil-Melethil S. Thompson P. Wakarchuk W. Gray S.J. Walia J.S. Mark B.L. Mahuran D. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.Mol. Ther. Methods Clin. Dev. 2016; 3: 15057Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar The resulting μ-subunit forms a stable homodimer (HexM) that degrades GM2 in a GM2 Activator Protein-dependent manner both in cellulo and in vivo when delivered to the CNS using an adeno-associated viral vector.6Tropak M.B. Yonekawa S. Karumuthil-Melethil S. Thompson P. Wakarchuk W. Gray S.J. Walia J.S. Mark B.L. Mahuran D. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.Mol. Ther. Methods Clin. Dev. 2016; 3: 15057Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7Karumuthil-Melethil S. Nagabhushan Kalburgi S. Thompson P. Tropak M. Kaytor M.D. Keimel J.G. Mark B.L. Mahuran D. Walia J.S. Gray S.J. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.Hum. Gene Ther. 2016; 27: 509-521Crossref PubMed Scopus (21) Google Scholar, 8Osmon K.J. Woodley E. Thompson P. Ong K. Karumuthil-Melethil S. Keimel J.G. Mark B.L. Mahuran D. Gray S.J. Walia J.S. Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.Hum. Gene Ther. 2016; 27: 497-508Crossref PubMed Scopus (18) Google Scholar Indeed, we have shown HexM is able to reduce GM2 levels in both Tay-Sachs and Sandhoff disease mouse models.6Tropak M.B. Yonekawa S. Karumuthil-Melethil S. Thompson P. Wakarchuk W. Gray S.J. Walia J.S. Mark B.L. Mahuran D. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.Mol. Ther. Methods Clin. Dev. 2016; 3: 15057Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7Karumuthil-Melethil S. Nagabhushan Kalburgi S. Thompson P. Tropak M. Kaytor M.D. Keimel J.G. Mark B.L. Mahuran D. Walia J.S. Gray S.J. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.Hum. Gene Ther. 2016; 27: 509-521Crossref PubMed Scopus (21) Google Scholar, 8Osmon K.J. Woodley E. Thompson P. Ong K. Karumuthil-Melethil S. Keimel J.G. Mark B.L. Mahuran D. Gray S.J. Walia J.S. Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.Hum. Gene Ther. 2016; 27: 497-508Crossref PubMed Scopus (18) Google Scholar Unfortunately, while Lahey et al.2Lahey H.G. Webber C.J. Golebiowski D. Izzo C.M. Horn E. Taghian T. Rodriguez P. Batista A.R. Ellis L.E. Hwang M. et al.Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.Mol. Ther. 2020; 28: 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar mention our work on treating Sandhoff mice with HexM, they failed to cite our article.8Osmon K.J. Woodley E. Thompson P. Ong K. Karumuthil-Melethil S. Keimel J.G. Mark B.L. Mahuran D. Gray S.J. Walia J.S. Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.Hum. Gene Ther. 2016; 27: 497-508Crossref PubMed Scopus (18) Google Scholar Additionally, a statement by Lahey et al.2Lahey H.G. Webber C.J. Golebiowski D. Izzo C.M. Horn E. Taghian T. Rodriguez P. Batista A.R. Ellis L.E. Hwang M. et al.Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.Mol. Ther. 2020; 28: 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar that “the efficiency [of HexM in Sandhoff disease mice] was limited by an immune response” should have been tempered by a comment that, unlike their construct, which was made from mouse α- and β sequences, HexM is constructed using the human sequences,6Tropak M.B. Yonekawa S. Karumuthil-Melethil S. Thompson P. Wakarchuk W. Gray S.J. Walia J.S. Mark B.L. Mahuran D. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.Mol. Ther. Methods Clin. Dev. 2016; 3: 15057Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar which would likely cause an increase in antigenicity in treated mice. Finally, caution is needed when translating results obtained from mouse models of GM2 gangliosidosis to humans. Mice have a metabolic pathway that converts GM2 ganglioside to its asialo derivative, GA2, which can be catabolized by HexB. This “shunt” pathway bypasses HexA and can thus rescue the phenotype of HexA-deficient mice using HexB alone.9Igdoura S.A. Mertineit C. Trasler J.M. Gravel R.A. Sialidase-mediated depletion of GM2 ganglioside in Tay-Sachs neuroglia cells.Hum. Mol. Genet. 1999; 8: 1111-1116Crossref PubMed Scopus (18) Google Scholar Humans lack this shunt pathway. With this in mind, predicting the therapeutic benefit of a gene therapy in humans must discount the benefit observed in mouse models that is associated with HexB activity. We appreciate the efforts of Lahey et al.2Lahey H.G. Webber C.J. Golebiowski D. Izzo C.M. Horn E. Taghian T. Rodriguez P. Batista A.R. Ellis L.E. Hwang M. et al.Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.Mol. Ther. 2020; 28: 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar to advance a therapeutic approach to treating these devastating genetic diseases. However, we felt it necessary to correct the published record on the biochemistry of human hexosaminidase isozymes that are directing important efforts now underway to develop gene-based treatments for Tay-Sachs and Sandhoff disease.