转染人凝血因子Ⅸ cDNA的血友病B患者皮肤成纤维细胞在小鼠体内的高效表达

本文报道了一个带有增强子的人巨细胞病毒(hCMV)启动子和人凝血因子ⅨcDNA的双拷贝反转录病毒载体(double-copy retroviral vector)-N2CMVIX的构建,转染PA317包装细胞后再离体感染血友病B患者皮肤成纤维细胞,可产生具有凝血活性的Ⅸ因子蛋白高达3420 ng/10~6细胞/24 h。将这些转染人Ⅸ因子cDNA的成纤维细胞包埋于胶原,植入小鼠皮下或腹腔内,人凝血因子Ⅸ蛋白表达最高值达105 ng/ml血浆,可在小鼠体内持续表达12天,其中90％以上的Ⅸ因子具有凝血话性,为血友病B基因治疗临床试验提供了一条可行的技术路线。     

带有人Ⅸ因子cDNA的反转录病毒载体的构建及其在血友病B患者皮肤成纤维细胞中的高效转移和表达

本文报道对血友病B实施基因治疗的可能性,首先将由SV40早期启动子,小鼠MT-1启动子和反转录病毒LTR启动子控制的Ⅸ因子cDNA构建到反转录病毒载体,然后用电穿孔法将构建的4个反转录病毒载体分别转入一株Amphotropic辅助细胞,PA317细胞,再用一株人纤维肉瘤细胞,HT1080细胞,测定这些辅助细胞的产病毒滴度,可得到2×10~4CFU/ml到5×10~5CFU/ml左右的病毒感染颗粒,用ELISA分别测定转有不同病毒载体的PA317细胞的Ⅸ因子蛋白产量,发现LTR启动子的表达效率最高,Ⅸ因子蛋白的分泌速率可达584ng/10~6细胞/24h,而SV40早期启动子和MT-1启动子的表达效率分别只有它的1/10和1/20,将表达效率最高的反转录病毒载体pXL—Ⅸ 1转入一株取自血友病B患者原代培养的皮肤成纤维细胞后同样能产生较高浓度的Ⅸ因子蛋白,其分泌速率可达549ng/10~6细胞/24h,其中75％以上的Ⅸ因子具有凝血活性,从而达到了首先在体外培养细胞纠正Ⅸ因子基因缺陷的目的,实现了血友病B基因治疗的第一步。     

Expression of Human Factor Ⅸ cDNA in Mice by Implants of Genetically Modified Skin Fibroblasts From a Hemophilia B Patient

Double-copy retroviral vector containing human factor Ⅸ cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor Ⅸ at a rate of 3420 ng/10~6 cells/24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. It was demonstrated that human factor Ⅸ was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng/ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B.     

HIGH EFFICIENT TRANSFER AND EXPRESSION OF HUMAN CLOTTING FACTOR Ⅸ cDNA IN CULTURED HUMAN PRIMARY SKIN FIBROBLASTS FROM HEMOPHILIA B PATIENT BY RETROVIRAL VECTORS

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 iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 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 rig/10~6 cells/24h, 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 f