Retrovirus-mediated transfer and expression of beta-hexosaminidase alpha-chain cDNA in human fibroblasts from G(M2)-gangliosidosis B1 variant.

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.