Stable transduction of quiescent CD34+CD38- human hematopoietic cells by HIV-1-based lentiviral vectors
Stable transduction of quiescent CD34+CD38- human hematopoietic cells by HIV-1-based lentiviral vectors
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DOI：
10.1073/pnas.96.6.2988
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发表时间：
1999-03-16
影响因子：
11.1
通讯作者：
Crooks, GM
中科院分区：
文献类型：
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作者：
Case, SS;Price, MA;Crooks, GM
我们比较了由基于HIV-1的慢病毒载体和由Moloney鼠白血病病毒（MLV）逆转录病毒载体的转导效率，使用原始的、静止的人造血祖细胞的严格体外测定。每种构建体含有增强的绿色荧光蛋白（GFP）作为报告基因。转导后48小时，慢病毒载体（而非MLV载体）在未分裂的CD 34（+）细胞（45.5% GFP（+））和CD 34（+）CD 38（-）细胞（G（0））（12.4% GFP（+））中表达GFP。然而，GFP也可以在用含有突变整合酶基因的慢病毒载体转导的CD 34（+）细胞中短期检测到。在延长的长期骨髓培养后，确定来自整合载体的稳定转导水平。MLV载体和慢病毒载体均能有效转导经精氨酸刺激的CD 34（+）细胞。MLV载体不能活化更原始的静止的CD 34（+）CD 38（-）细胞（n = 8）。相反，在超过15周的延长的长期培养中观察到慢病毒载体对CD 34（+）CD 38（-）细胞的稳定转导（9.2 +/-5.2%，n = 7）。来自单个CD 34（+）CD 38（-）细胞的克隆中的GFP表达证实了在29%的早期和晚期增殖细胞中的有效、稳定的慢病毒转导。在转导期间不存在生长因子的情况下，仅慢病毒载体能够使CD 34（+）和CD 34（+）CD 38（-）细胞增殖（分别为13.5 +/-2.5%，n = 11和12.2 +/-9.7，n = 4）。慢病毒载体明显上级MLV载体用于转导静止的原始人造血祖细胞，并且可以提供治疗上有用水平的基因转移到人造血干细胞中。
We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells, Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP(+)) and in CD34(+)CD38(-) cells in G(0) (12.4% GFP(+)), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced vith a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8), In contrast, stable transduction of CD34(+) CD38(-) cells by the lentiviral vector nas seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth th factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.