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J. Biol. Chem., Vol. 277, Issue 23, 20177-20184, June 7, 2002

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Absence of Metabolic Cross-correction in Tay-Sachs Cells
IMPLICATIONS FOR GENE THERAPY^*

Sabata Martino^§, Carla Emiliani, Brunella Tancini, Giovanni Maria Severini^¶, Vanna Chigorno, Claudio Bordignon^§, Sandro Sonnino, and Aldo Orlacchio^**

From the  Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, University of Perugia, 06126 Perugia, Italy, ^§ San Raffaele Telethon Institute for Gene Therapy, H. S. Raffaele, Milano, Italy, ^¶ Children Hospital "Burlo Garofolo," 34100 Trieste, Italy, and the  Department of Medical Chemistry and Biochemistry, Center of Excellence on Neurodegenerative Diseases, Study Center for the Biochemistry and Biotechnology of Glycolipids, University of Milan, 20090 Segrate, Italy

We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme -hexosaminidase A (HexA) (-N-acetylhexosaminidase, EC 3.2.1.52). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LHexTN retroviral vector carrying the cDNA encoding for the human Hex -subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells.

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