Malik P, Krall W J, Yu X J, Zhou C, Kohn D B
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital, Los Angeles, University of Southern California School of Medicine, USA.
Blood. 1995 Oct 15;86(8):2993-3005.
Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.
将基因导入人类造血干细胞并使其在成熟的骨髓单核细胞后代中表达，这对于影响粒细胞和巨噬细胞的遗传性疾病的基因治疗具有应用价值。我们推测，随着骨髓单核细胞分化而上调的髓系特异性基因的启动子，也会在逆转录病毒载体中上调外源基因的表达。将基于莫洛尼鼠白血病病毒（MoMuLV）的逆转录病毒载体，使用造血基因（CD11b、CD18和CD34）的启动子，与具有病毒启动子（MoMuLV长末端重复序列[LTR]、巨细胞病毒[CMV]和猿猴病毒40[SV40]）的载体进行比较。人类葡萄糖脑苷脂酶（GC）cDNA作为报告基因。用这些载体转导HL60细胞，并比较未分化的HL-60细胞以及使用二甲亚砜分化为粒细胞或使用佛波酯分化为单核细胞/巨噬细胞的相同细胞中载体衍生的GC活性。在未分化的HL-60细胞中，当由MoMuLV LTR控制时，载体衍生的GC活性最高。在分化为粒细胞的HL-60细胞中，从CD11b、MoMuLV LTR和CMV启动子转录的载体衍生的GC活性分别相当于正常内源性GC活性的1.7倍、1.5倍和1.5倍，在分化为巨噬细胞的细胞中分别为正常GC活性的0.8倍、2.0倍和3.6倍。随着粒细胞分化，CD11b启动子在GC活性方面显示出最大诱导（8倍）；随着巨噬细胞分化，CD11b启动子在GC表达方面显示出4倍诱导。CD11b启动子在转导的CD34+细胞的骨髓单核细胞后代中也产生了显著水平的GC活性。与CMV或MoMuLV LTR启动子不同，CD11b启动子的表达相对具有骨髓单核细胞特异性，在Jurkat T细胞或HeLa癌细胞中观察到的表达极少。HL-60细胞中CD11b启动子的表达随分化的诱导与CD11b基因的发育调控相关。使用CD11b启动子的逆转录病毒载体对于影响骨髓单核细胞系的疾病的基因治疗具有潜在用途。
