Ex vivo cell-mediated gene therapy for metachromatic leukodystrophy using neurospheres
Ex vivo cell-mediated gene therapy for metachromatic leukodystrophy using neurospheres
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DOI：
10.1016/j.brainres.2006.03.116
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发表时间：
2006-06-13
期刊：
影响因子：
2.9
通讯作者：
Shimada, Takashi
中科院分区：
文献类型：
--
作者：
Kawabata, Ken;Migita, Makoto;Shimada, Takashi
异染性脑白质营养不良（MLD）是一种常染色体隐性遗传疾病，由编码溶酶体酶芳基硫酸酯酶A（阿萨）的基因突变引起。在MLD中，底物硫酸化糖蛋白在中枢和外周神经系统中的蓄积导致进行性运动和精神恶化。神经前体细胞被认为可用于神经退行性疾病的细胞替代疗法和细胞介导的基因疗法。在本研究中，我们研究了使用神经祖细胞进行MLD离体基因治疗的可行性。从E14胚胎MLD敲除小鼠或GFP转基因小鼠的纹状体制备神经祖细胞（神经球），并用携带阿萨基因的VSV假型HIV载体（HIV-ASA）转导。对于体内研究，将来自GFP小鼠的神经球用HIV-ASA转导并接种到成年MLD小鼠的脑实质中。HIV载体转导的祖细胞保留了体外分化为神经元、星形胶质细胞和少突胶质细胞的潜力。用分光光度酶分析和蛋白质印迹法证实了阿萨在HIV-ASA转导的神经球中的表达。在体内，注射后1个月可检测到GFP阳性细胞。这些细胞包括GFAP-和MAP 2-阳性细胞。使用抗ASA抗体的免疫组织化学显示阿萨在GFP阳性和阴性细胞中的定位。在MLD敲除小鼠体内证实了累积的硫苷脂的部分清除。目前的研究结果表明，阿萨酶从迁移的神经球释放，并能够消化周围细胞中的硫苷脂。我们的研究结果表明，基因工程神经祖细胞（神经球）的MLD离体治疗的潜力。(c)2006 Elsevier B. V.保留所有权利。
Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. in the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striaturn of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophoto metric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD. (c) 2006 Elsevier B.V. All rights reserved.