溶酶体α-甘露糖苷贮积症基因治疗的初步研究

目的探讨溶酶体α－甘露糖苷贮积症基因治疗的可行性。方法以逆转录病毒为载体，将编码人溶酶体α－甘露糖苷酶ｃＤＮＡ导入病猫皮肤成纤维细胞。结果人源ｃＤＮＡ在病猫细胞内表达高活性α－甘露糖苷酶，重组酶的ｐＨ活性范围与正常酶相同，定位于细胞的溶酶体，分泌到细胞外后能通过甘露糖－６－磷酸受体介导的胞饮作用被其他细胞吸收。在体内，由重组病毒感染细胞组成的类器官表达有活性酶可达６周。结论此表达系统是研究溶酶体α－甘露糖苷贮积症基因治疗的有用模型。

Preliminary studies on gene therapy for lysosomal alphamannosidosis

bjective Exploration of the feasibility of treating human lysosomal mannosidosis by gene therapy. MethodsRetroviral vectormediated transfer of human lysosomal mannosidase cDNA into the diseased cat skin fibroblasts. Detection of the enzymatic activity in the cells and their culture media at different pH conditions. Localization of the recombinant enzyme in the cells by chemical staining. Crosscorrection of untransduced cat cells by incubation in medium containing the secreted enzyme in the absence and presence of mannose6phosphate or fetal calf serum. In vivo expression of the recombinant enzyme in organoids containing the vectortransduced cells. Results Among the two human cDNAs tested, only one encoded high levels of mannosidase activity in the cat cells, which shared the same pH profile and lysosomal localization with the normal enzyme. The recombinant enzyme was proportionally secreted into the medium of the cell culture and taken up by untransduced cells via mannose6phosphate receptormediated endocytosis. The uptake was partially inhibited in the presence of fetal calf serum, which was dosedependent. In vivo, organoids containing the vectortransduced cells expressed detectable mannosidase activity for up to 6 weeks. Conclusion The human cDNA was functional in the cat cells and the expression system could be used for development of gene therapy for lysosomal mannosidosis.