Reversion of the biochemical defects in murine embryonic Sandhoff neurons using a bicistronic lentiviral vector encoding hexosaminidase a and b Audrey Arfi,* Rivka Zisling, Emmanuel Richard,* Lionel Batista,* Livia Poenaru,* Anthony H. Futerman and Catherine Caillaud* *Universite ´ Paris-Descartes, Faculte ´ de Me ´decine, INSERM, Centre National de la Recherche Scientifique, Institut Cochin (U567-UMR 8104), Paris, France  Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel Abstract Sandhoff disease, a neurodegenerative disorder characterized by the intracellular accumulation of GM2 ganglioside, is caused by mutations in the hexosaminidase b-chain gene resulting in a hexosaminidase A (ab) and B (bb) deficiency. A bicistronic lentiviral vector encoding both the hexosaminidase a and b chains (SIV.ASB) has previously been shown to cor- rect the b-hexosaminidase deficiency and to reduce GM2 levels both in transduced and cross-corrected human Sandhoff fibroblasts. Recent advances in determining the neuropatho- physiological mechanisms in Sandhoff disease have shown a mechanistic link between GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplasmic reticulum Ca 2+ - ATPase (SERCA) activity, and axonal outgrowth. To examine the ability of the SIV.ASB vector to reverse these pathophys- iological events, hippocampal neurons from embryonic Sandhoff mice were transduced with the lentivector. Normal axonal growth rates were restored, as was the rate of Ca 2+ uptake via the SERCA and the sensitivity of the neurons to thapsigargin-induced cell death, concomitant with a decrease in GM2 and GA2 levels. Thus, we have demonstrated that the bicistronic vector can reverse the biochemical defects and down-stream consequences in Sandhoff neurons, reinforcing its potential for Sandhoff disease in vivo gene therapy. Keywords: axonal morphology, calcium uptake, hexosami- nidases, hippocampal neurons, lentiviral vector, Sandhoff disease. J. Neurochem. (2006) 96, 1572–1579. Sandhoff disease is an autosomal recessive lysosomal storage disorder caused by mutations in HEXB, the gene encoding the b-subunit of b-hexosaminidases, resulting in hexosa- minidase A and B deficiency. Hexosaminidase A, an ab heterodimer, and hexosaminidase B, a bb homodimer, are the two major forms of b-hexosaminidase (Gravel et al. 2001). The aa homodimer, hexosaminidase S, is a minor form that appears to have negligible catalytic activity (Ikonne et al. 1975; Hepbildikler et al. 2002). Sandhoff disease is one of three GM2 gangliosidoses, characterized by lysosomal accumulation of GM2 ganglioside and related glycolipids such as GA2, especially in the CNS. In its most severe (infantile) form, Sandhoff disease presents with progressive neurodegeneration that generally leads to death by 3–5 years of age (Neufeld 1991; Mahuran 1999; Gravel et al. 2001). A mouse model of Sandhoff disease has been created by targeted disruption of the mouse Hexb gene. Hexb–/– mice display an acute, rapidly progressive neurodegenerative course very similar to the human disease (Sango et al. 1995; Phaneuf et al. 1996; Suzuki et al. 1997). This model therefore appears to be an excellent tool with which to delineate the mechanisms underlying the pathogenesis of this disease. Recent studies have revealed the presence of Received September 1, 2005; revised manuscript received October 20, 2005; accepted November 24, 2005. Address correspondence and reprint requests to A. H. Futerman, Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: tony.futerman@weizmann.ac.il Abbreviations used: BMT, bone marrow transplantation; CMV, cyto- megalovirus; cPPT, central polypurine tract; cs, coverslip; GFP, green fluorescent protein; Hex, hexosaminidase; IRES, internal ribosome entry site; LTR, long terminal repeat; MPS, mucopolysaccharidosis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; n.a., nu- merical aperture; NB-DNJ, N-butyl deoxynojirimycine; PBS, phos- phate-buffered saline; RRE, rev-responsive element; SERCA, sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase; SIV, Simian Im- munodeficiency Virus; SV40, Simian Virus 40. Journal of Neurochemistry , 2006, 96, 1572–1579 doi:10.1111/j.1471-4159.2006.03665.x Ó 2006 The Authors 1572 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1572–1579