Development of virus vectors for gene therapy of β chain hemoglobinopathies:: flanking with a chromatin insulator reduces γ-globin gene silencing in vivo
Development of virus vectors for gene therapy of β chain hemoglobinopathies:: flanking with a chromatin insulator reduces γ-globin gene silencing in vivo
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DOI：
10.1182/blood-2002-01-0219
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发表时间：
2002-09-15
期刊：
影响因子：
20.3
通讯作者：
Stamatoyannopoulos, G
中科院分区：
文献类型：
--
作者：
Emery, DW;Yannaki, E;Stamatoyannopoulos, G
我们先前已经描述了使用截短的β-珠蛋白启动子、修饰的γ-珠蛋白盒和α-珠蛋白增强子的人γ-珠蛋白的肿瘤逆转录病毒载体的开发。然而，这些载体之一是遗传不稳定的，这两个载体在培养细胞中表现出可变的表达模式，珠蛋白基因的肿瘤逆转录病毒载体的共同特征。为了解决这些问题，我们鉴定并去除了导致遗传不稳定性的载体序列，并将所得载体与鸡β-珠蛋白HS 4染色质绝缘子侧接，以保护表达免受染色体位置效应的影响。在确定侧接cHS 4元件允许MEL细胞系中更高、更均匀水平的γ-珠蛋白表达后，我们使用小鼠骨髓转导和移植模型测试了这些载体。当存在时，来自未绝缘载体的γ-珠蛋白盒仅在2%至5%的红细胞（RBC）中长期表达，表明它们对表观遗传沉默高度敏感。相反，当存在来自绝缘载体的γ-珠蛋白盒时，在49% +/-20%的RBC中长期表达。RNA酶保护分析表明，绝缘的γ-珠蛋白盒在转导的RBC中以23% +/-16%/拷贝的小鼠α-珠蛋白表达。这些结果表明，侧接具有cHS 4绝缘子的球蛋白载体使表达的可能性增加近10倍，这进而允许γ-球蛋白表达接近镰状细胞性贫血和β地中海贫血的治疗范围。
We have previously described the development of oncoretrovirus vectors for human gamma-globin using a truncated beta-globin promoter, modified gamma-globin cassette, and alpha-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken beta-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of gamma-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the gamma-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present they-globin cassette from the insulated vector was expressed in 49% +/- 20% of RBCs long term. RNase protection analysis indicated that the insulated gamma-globin cassette was expressed at 23% +/- 16% per copy of mouse alpha-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for gamma-globin expression approaching the therapeutic range for sickle cell anemia and beta thalassemia.