Shaimardanova Alisa A, Chulpanova Daria S, Solovyeva Valeriya V, Aimaletdinov Aleksandr M, Rizvanov Albert A
Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia.
Neural Regen Res. 2022 Jan;17(1):122-129. doi: 10.4103/1673-5374.314310.
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.
泰-萨克斯病和桑德霍夫病是严重的遗传性神经退行性疾病，由β-己糖胺酶A（HexA）缺乏引起，导致GM2神经节苷脂在神经细胞中蓄积。在本研究中，我们使用了一种双顺反子慢病毒载体，该载体编码由P2A肽核苷酸序列分隔的HexAα和β亚基基因的cDNA，分析了细胞介导的基因治疗对桑德霍夫病和泰-萨克斯病的疗效和安全性。在HEK293T细胞和人脐血单个核细胞（hUCBMCs）培养物中分析了包含HEXA-HEXB基因盒的双顺反子构建体的功能。我们的结果表明，与天然细胞的条件培养基相比，从基因改造的HEK293T-HEXA-HEXB和hUCBMCs-HEXA-HEXB收获的条件培养基中HexA的酶活性分别提高了23倍和8倍。蛋白质印迹分析表明，hUCBMCs-HEXA-HEXB分泌完全分离的HEXA和HEXB蛋白，以及一种由P2A肽连接的包含HEXA+HEXB的未切割蛋白。对实验用Wistar大鼠静脉注射基因改造的hUCBMCs-HEXA-HEXB，并测定实验动物血浆中的HexA酶活性以及免疫系统器官（脾脏、胸腺、骨髓、淋巴结）的活细胞数量。结果显示，在注射hUCBMCs-HEXA-HEXB后的第6天和第9天，实验大鼠血浆中HexA的酶活性显著增加（分别增加2.5倍和3倍）。同时，所研究器官中的活细胞数量保持不变。因此，在体外和体内均显示了由P2A肽核苷酸序列分隔的编码HEXA和HEXB基因cDNA的双顺反子基因构建体的功能。我们推测，由于hUCBMCs具有克服生物屏障的天然能力，这种策略可以恢复GM2神经节苷脂沉积症患者中枢神经系统中缺失酶的活性。根据获得的数据，可以得出结论，静脉注射过表达HexA的hUCBMCs是治疗GM2神经节苷脂沉积症的一种有前景的方法。该动物实验方案于2020年6月30日获得喀山联邦大学动物伦理委员会批准（第23号）。
