Retrovirus-Mediated Transfer and Expression of β -Hexosaminidase α -Chain cDNA in Human Fibroblasts from G M2 -Gangliosidosis B1 Variant

Abstract

Mutations in the α-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 α-chains) with a retroviral vector encoding the human hexosaminidase α-chain leads to a complete correction of HexA (αβ dimer) activity with both synthetic and natural substrates. The α-subunit overexpression leads to a partial HexB (ββ 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 α-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.