Retrovirus-Mediated Transfer and Expression ofβ-Hexosaminidaseα-Chain cDNA in Human Fibroblasts from GM2-Gangliosidosis B1 Variant
| Autor: | Miguel Sena-Esteves, M.C. Sá Miranda, Carla Teixeira, Lurdes Lopes, M. G. Ribeiro |
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| Rok vydání: | 2001 |
| Předmět: |
DNA
Complementary Time Factors Genetic Vectors G(M2) Ganglioside Sandhoff disease Gangliosidosis Biology Cell Line Viral vector Mice Hexosaminidase A Hexosaminidase B Gangliosidoses GM2 Transduction Genetic Genetics medicine Animals Humans Hexosaminidase Transgenes Molecular Biology chemistry.chemical_classification Genetic transfer Gene Transfer Techniques Temperature 3T3 Cells Fibroblasts medicine.disease HEXA Precipitin Tests Molecular biology Recombinant Proteins beta-N-Acetylhexosaminidases HEXB Phenotype Retroviridae Enzyme Immunoglobulin M Microscopy Fluorescence chemistry Mutation Molecular Medicine Electrophoresis Polyacrylamide Gel Lysosomes Dimerization |
| Zdroj: | Human Gene Therapy. 12:1771-1783 |
| ISSN: | 1557-7422 1043-0342 |
| DOI: | 10.1089/104303401750476267 |
| Popis: | Mutations in the alpha-chain of lysosomal hexosaminidase (EC 3.2.1.52) underlie two distinct biochemical phenotypes known as variant B and variant B1 of G(M2) gangliosidosis. This paper shows that the transduction of human B1-type fibroblasts (producing catalytically inactive alpha-chains) with a retroviral vector encoding the human hexosaminidase alpha-chain leads to a complete correction of HexA (alpha beta dimer) activity with both synthetic and natural substrates. The alpha-subunit overexpression leads to a partial HexB (beta beta dimer) depletion corresponding to about 10% of control HexB activity. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. The high levels of recombinant enzyme correctly produced the metabolic defect, enabling the cells efficiently to degrade the accumulated storage product present in lysosomes. The transduced fibroblasts are also able to secrete HexA efficiently into the culture medium. Moreover, transfer of the human transgene product to B1-type deficient fibroblasts lead to an increase of activity against 4MUGS, the alpha-chain specific synthetic substrate, up to 30% of the control mean activity level. This level of activity might be sufficient to restore the normal ganglioside G(M2) metabolism in recipient cells. The data obtained demonstrate that B1-type phenotype can be efficiently corrected by retrovirus-mediated gene transfer. |
| Databáze: | OpenAIRE |
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