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Combined replacement effects of human modified ?-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

ABSTRACT: GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal ?-hexosaminidase (Hex) ?- and ?-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered ?-subunits containing a partial amino acid sequence of the ?-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (?? heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB ?-subunit to that in the native HexA ?-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.

SUBMITTER: Kitakaze K

PROVIDER: S-EPMC5613250 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

Kitakaze Keisuke K Tasaki Chikako C Tajima Youichi Y Hirokawa Takatsugu T Tsuji Daisuke D Sakuraba Hitoshi H Itoh Kohji K

Biochemistry and biophysics reports 20160608

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A ...[more]

PMID: 28955902

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Project description: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.

Dataset Information

Combined replacement effects of human modified ?-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

Publications

Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

Similar Datasets

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