Sugimoto Yoshikazu, Tsukahara Satomi, Sato Shigeo, Suzuki Mutsumi, Nunoi Hiroyuki, Malech Harry L, Gottesman Michael M, Tsuruo Takashi
Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan.
J Gene Med. 2003 May;5(5):366-76. doi: 10.1002/jgm.362.
Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells.
A bicistronic retrovirus vector, Ha-MDR-IRES-gp91, was constructed for the co-expression of MDR1 and gp91, a gene responsible for X-linked chronic granulomatous disease (X-CGD). Drug-selected co-expression of P-glycoprotein and gp91 was evaluated in transduced cells.
Epstein-Barr virus-transformed B cells from X-CGD patients transduced with Ha-MDR-IRES-gp91 co-expressed human P-glycoprotein and gp91, and acquired superoxide-generating activity. Human CD34-positive cells from an X-CGD patient were transduced with Ha-MDR-IRES-gp91 and subsequently treated with 2 ng/ml vincristine. After 13 days, 20% of Ha-MDR-IRES-gp91-transduced cells were P-glycoprotein- and gp91-positive by FACS analysis. The superoxide-generating activity of the transduced population was 27% of that of normal cells. Mice transplanted with Ha-MDR-IRES-gp91-transduced bone marrow cells showed co-expression of P-glycoprotein and gp91 in peripheral blood mononuclear cells. By administering paclitaxel, the proportions of P-glycoprotein- and gp91-positive cells were increased in all the four mice examined. When mice transplanted with Ha-MDR-IRES-gp91-transduced cells were repeatedly administered paclitaxel, the ratios of P-glycoprotein- and gp91-positive cells were maintained for over 1 year.
These results suggest that MDR1-bicistronic vectors may be useful to select the transduced hematopoietic cells in vivo. This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy.
逆转录病毒转导人造血干细胞是基因治疗中一种有吸引力的策略；然而，在当前方案中转导效率和转基因表达持续时间可能并不令人满意。人多药耐药基因（MDR1）与治疗性基因共表达可赋予基因修饰细胞选择性生长优势。
构建了双顺反子逆转录病毒载体Ha-MDR-IRES-gp91，用于MDR1和gp91（一种与X连锁慢性肉芽肿病（X-CGD）相关的基因）的共表达。在转导细胞中评估药物选择下P-糖蛋白和gp91的共表达情况。
用Ha-MDR-IRES-gp91转导的X-CGD患者的爱泼斯坦-巴尔病毒转化B细胞共表达人P-糖蛋白和gp91，并获得了超氧化物生成活性。一名X-CGD患者的人CD34阳性细胞用Ha-MDR-IRES-gp91转导，随后用2 ng/ml长春新碱处理。13天后，通过流式细胞术分析，20%的Ha-MDR-IRES-gp91转导细胞为P-糖蛋白和gp91阳性。转导群体的超氧化物生成活性为正常细胞的27%。移植了Ha-MDR-IRES-gp91转导骨髓细胞的小鼠在外周血单个核细胞中显示出P-糖蛋白和gp91的共表达。通过给予紫杉醇，在所有检测的四只小鼠中，P-糖蛋白和gp91阳性细胞的比例增加。当移植了Ha-MDR-IRES-gp91转导细胞的小鼠反复给予紫杉醇时，P-糖蛋白和gp91阳性细胞的比例维持超过1年。
这些结果表明，MDR1双顺反子载体可能有助于在体内选择转导的造血细胞。这可能导致接受干细胞基因治疗的患者血细胞中转基因的持续表达。
