Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency of β-hexosaminidase A (HexA) enzyme, which results in the accumulation of GM2 gangliosides in the nervous system cells. In this work, we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexA α- and β-subunit genes separated by the nucleotide sequence of a P2A peptide (HEXA-HEXB). The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells (hUCBMCs). Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times, respectively, compared with the conditioned medium of native cells. Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins, and an uncleaved protein containing HEXA + HEXB linked by the P2A peptide. Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out, and the HexA enzymatic activity in the blood plasma of experimental animals, as well as the number of live cells of immune system organs (spleen, thymus, bone marrow, lymph nodes) were determined. A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9 (by 2.5 and 3 times, respectively) after the administration of hUCBMCs-HEXA-HEXB was shown. At the same time, the number of live cells in the studied organs remained unchanged. Thus, the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo. We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers, such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses. Based on the obtained data, it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses. The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University (No. 23) on June 30, 2020.