Sandhoff disease is an autosomal recessive neurodegenerative disease characterized by a GM2 ganglioside intralysosomal accumulation. It is due to mutations in the beta-hexosaminidases beta-chain gene, resulting in a beta-hexosaminidases A (alphabeta) and B (betabeta) deficiency. Mono and bicistronic lentiviral vectors containing the HEXA or/and HEXB cDNAs were constructed and tested on human Sandhoff fibroblasts. The bicistronic SIV.ASB vector enabled a massive restoration of beta-hexosaminidases activity on synthetic substrates and a 20% correction on the GM2 natural substrate. Metabolic labeling experiments showed a large reduction of ganglioside accumulation in SIV.ASB transduced cells, demonstrating a correct recombinant enzyme targeting to the lysosomes. Moreover, enzymes secreted by transduced Sandhoff fibroblasts were endocytosed in deficient cells via the mannose 6-phosphate pathway, allowing GM2 metabolism restoration in cross-corrected cells. Therefore, our bicistronic lentivector supplying both alpha- and beta-subunits of beta-hexosaminidases may provide a potential therapeutic tool for the treatment of Sandhoff disease.