Plavec I, Papayannopoulou T, Maury C, Meyer F
Laboratoire d'Oncologie Virale UPR 274, Centre National de la Recherche Scientifique, Villejuif, France.
Blood. 1993 Mar 1;81(5):1384-92.
Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta-globin gene fused to the 4 major regulatory elements of the human beta-globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10-fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.
逆转录病毒介导的人β-珠蛋白基因转移为血红蛋白病的体细胞基因治疗开发提供了一个模型系统。先前的研究表明，接受感染了含有人β-珠蛋白基因的逆转录病毒载体的骨髓细胞移植的小鼠，能够在红系细胞中特异性表达人β-珠蛋白；然而，转导的珠蛋白基因的表达水平较低（每个基因拷贝与内源性小鼠β-珠蛋白基因相比为1%至2%）。我们在此报告构建了一种重组逆转录病毒载体，其编码与人β-珠蛋白基因座控制区（LCR）的4个主要调控元件融合的人β-珠蛋白基因。LCR盒使小鼠红白血病细胞中珠蛋白基因的表达水平提高了10倍。为了研究体内表达水平，用产病毒细胞感染小鼠骨髓细胞，并将转导的细胞注射到经致死剂量照射的受体中。在大多数含有前病毒的小鼠（高达75%）中，移植后至少3至6个月在外周血中检测到了人β-珠蛋白的表达。选择12只代表血液中转导基因表达水平（占内源性小鼠β-珠蛋白RNA的0.04%至3.2%）的动物进行进一步分析。0.4%至12%的循环红细胞对人β-珠蛋白蛋白呈阳性染色。基于这些值，估计每个细胞中转导基因的表达水平为内源性小鼠β-珠蛋白基因的10%至39%。这些数据表明，在逆转录病毒载体中将LCR与β-珠蛋白基因融合可提高小鼠红白血病细胞中β-珠蛋白的表达水平，并表明转导造血干细胞后在体内红系细胞中可获得高水平表达。
