Emery David W, Yannaki Evangelia, Tubb Julie, Nishino Tamon, Li Qiliang, Stamatoyannopoulos George
Department of Medicine, Division of Medical Genetics, Box 357720, HSB K236F, University of Washington, 1705 NE Pacific Street, Seattle, WA 98195-7720, USA.
Blood. 2002 Sep 15;100(6):2012-9. doi: 10.1182/blood-2002-01-0219.
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 the gamma-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.
我们之前曾描述过使用截短的β-珠蛋白启动子、修饰的γ-珠蛋白盒和α-珠蛋白增强子开发用于人γ-珠蛋白的嗜肝性逆转录病毒载体。然而，其中一种载体在基因上不稳定，并且两种载体在培养细胞中均表现出可变的表达模式，这是珠蛋白基因嗜肝性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基因不稳定的载体序列，并用鸡β-珠蛋白HS4染色质绝缘子环绕所得载体，以保护表达免受染色体位置效应的影响。在确定用cHS4元件环绕可使MEL细胞系中γ-珠蛋白表达水平更高、更均匀后，我们使用小鼠骨髓转导和移植模型对这些载体进行了测试。当存在时，未绝缘载体中的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。相比之下，当存在时，绝缘载体中的γ-珠蛋白盒在49%±20%的RBC中长期表达。核糖核酸酶保护分析表明，在转导的RBC中，绝缘的γ-珠蛋白盒每拷贝小鼠α-珠蛋白的表达量为23%±16%。这些结果表明，用cHS4绝缘子环绕珠蛋白载体可使表达可能性增加近10倍，这反过来又使γ-珠蛋白表达接近镰状细胞贫血和β地中海贫血的治疗范围。
