Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.
Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.
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DOI：
10.1073/pnas.92.15.6728
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发表时间：
1995-07
影响因子：
11.1
通讯作者：
M. Sadelain;C.H.J. Wang;M. Antoniou;F. Grosveld;R. Mulligan
中科院分区：
文献类型：
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作者：
M. Sadelain;C.H.J. Wang;M. Antoniou;F. Grosveld;R. Mulligan
逆转录病毒介导的基因转移到造血细胞中可以提供治疗涉及造血细胞的遗传性和获得性疾病的手段。实施这种方法用于由影响β-珠蛋白基因的突变引起的疾病（例如，β-地中海贫血和镰状细胞性贫血），但是，由于不能产生能够有效和忠实地传递适当基因表达所需序列的重组病毒而受到阻碍。我们已经解决了这个问题，通过仔细检查逆转录病毒和β-珠蛋白基因序列之间的相互作用，影响载体的传输，稳定性和表达。首先，我们研究了大量不同的重组前病毒基因组的传输特性，这些基因组在载体、β-珠蛋白结构基因和基因座控制区（LCR）核心序列的精确性质以及这些序列的位置和方向上都不同。通过该分析，我们鉴定了一种称为M β 6L的特异性载体，其携带人β-珠蛋白基因和来自LCR的核心元件HS 2、HS 3和HS 4，并以大于10（6）/ml的滴度将重组前病毒序列忠实地传递至细胞。由该病毒转导的鼠红白血病（MEL）细胞群体表达的人β-珠蛋白转录物的水平，在每个基因拷贝的基础上，是在MEL衍生的细胞系Hu 11中检测到的水平的78%，Hu 11携带人染色体11，β-珠蛋白基因座的位点。然而，对单个转导的MEL细胞克隆的分析表明，虽然在每个测试的克隆中检测到表达（n = 17），但人β-珠蛋白处理的水平在Hu 11中水平的4%至146%之间变化。这种表达水平的克隆变异表明，小β-珠蛋白LCR序列可能不像以前怀疑的那样提供β-珠蛋白的严格的染色体位置非依赖性表达，至少在逆转录病毒介导的基因转移的背景下是这样。
Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.