TY - JOUR
T1 - Absence of metabolic cross-correction in Tay-Sachs cells
T2 - Implications for gene therapy
AU - Martino, Sabata
AU - Emiliani, Carla
AU - Tancini, Brunella
AU - Severini, Giovanni Maria
AU - Chigorno, Vanna
AU - Bordignon, Claudio
AU - Sonnino, Sandro
AU - Orlacchio, Aldo
PY - 2002/6/7
Y1 - 2002/6/7
N2 - 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 β-hexosaminidase A (HexA) (β-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 LαHexTN retroviral vector carrying the cDNA encoding for the human Hex α-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.
AB - 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 β-hexosaminidase A (HexA) (β-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 LαHexTN retroviral vector carrying the cDNA encoding for the human Hex α-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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U2 - 10.1074/jbc.M106164200
DO - 10.1074/jbc.M106164200
M3 - Article
C2 - 11923278
AN - SCOPUS:0037036461
VL - 277
SP - 20177
EP - 20184
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 23
ER -