Dai Y F, Qiu X F, Xue J L, Liu Z D
Institute of Genetics, Fudan University, Shanghai, PRC.
Sci China B. 1992 Feb;35(2):183-93.
To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor IX-deficient skin fibroblasts, we constructed four retroviral vectors containing factor IX cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human fibrosarcoma cell line, HT1080 cells, was used to assay the factor IX-virus titers of these four virus-producing PA317 cells, which ranged from 2 x 10(4) to 5 x 10(5) cfu/ml. The factor IX proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor IX at the rate of 584 ng/10(6) cells/24 h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor IX than LTR promoter. The highest expressed retroviral vector XL-IX was used to infect a line of factor IX-deficient human primary skin fibroblasts, FDIX cells. The factor IX secretion rate of the infected FDIX cells was about 549 ng/10(6) cells/24 h and over 75% of secreted factor IX was biologically active. We are convinced that this factor IX-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.
为了研究通过将基因转移至原发性IX因子缺乏的皮肤成纤维细胞来进行血友病B体细胞基因治疗的可能性，我们构建了四种逆转录病毒载体，分别包含由逆转录病毒LTR启动子、SV40早期启动子和小鼠MT-I启动子驱动的IX因子cDNA。通过电穿孔将这些逆转录病毒载体转染至双嗜性包装细胞系PA317细胞中，并用人类纤维肉瘤细胞系HT1080细胞来测定这四种产生病毒的PA317细胞的IX因子病毒滴度，其范围为2×10⁴至5×10⁵ cfu/ml。通过ELISA测定了四种产生病毒的PA317细胞大量培养物所产生的IX因子蛋白。结果显示，LTR启动子指导产生的IX因子最多，速率为584 ng/10⁶细胞/24小时，而SV40早期启动子和MT启动子指导产生的IX因子比LTR启动子少约10倍和20倍。表达量最高的逆转录病毒载体XL-IX用于感染一株IX因子缺乏的人类原发性皮肤成纤维细胞FDIX细胞。被感染的FDIX细胞的IX因子分泌速率约为549 ng/10⁶细胞/24小时，且分泌的IX因子中超过75%具有生物活性。我们确信，通过逆转录病毒介导的基因治疗，这种IX因子缺乏的人类原发性皮肤成纤维细胞在体外已被治愈或基因校正。
