Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland, USA.
Mol Ther. 2011 Jan;19(1):133-9. doi: 10.1038/mt.2010.218. Epub 2010 Oct 12.
Position effects limit the curative potential of gene transfer strategies for the hemoglobinopathies by inducing clonal variability of transgene expression. We evaluated the mitigating effects of the chicken hypersensitivity site 4 (HS4) insulator among lentiviral vector-transduced human hematopoietic cells. We constructed various lentiviral vectors using a green fluorescent protein (GFP) reporter under the control of a reverse-oriented murine stem cell virus (MSCV)-long-term repeat (LTR) promoter or a reverse-oriented β-globin expression cassette. A full-length HS4, a tandem HS4 core, and a single core insulator were inserted into the 3' LTR in both forward and reverse orientation. All but the reverse single core insulator significantly decreased titers. All reduced %GFP without increasing mean fluorescence intensity (MFI) among erythroid progeny of transduced human CD34(+) cells. A lower coefficient of variation (CV) was observed only among progeny of the full-length vector-transduced cells, yet a fivefold reduction in transduction efficiency was observed. In xenografted mice, the single core insulator decreased both the %GFP and the MFI at 4 and 8 weeks after transplantation with no difference in CVs. These data demonstrate that the inclusion of HS4 insulator elements lowers viral titers, reduces efficiency of transduction, and produces minimal effects on transgene expression among human hematopoietic cells in vitro and in vivo.
位置效应通过诱导转基因表达的克隆变异性，限制了血红蛋白病的基因转移策略的治疗潜力。我们评估了慢病毒载体转导的人类造血细胞中鸡高敏位点 4（HS4）绝缘子的缓解作用。我们构建了各种慢病毒载体，使用绿色荧光蛋白（GFP）报告基因，由反向定向鼠干细胞病毒（MSCV）-长末端重复（LTR）启动子或反向定向β-珠蛋白表达盒控制。全长 HS4、串联 HS4 核心和单个核心绝缘子以正向和反向插入 3'LTR。除了反向单核心绝缘子外，所有其他的都显著降低了滴度。所有的都降低了转导人类 CD34+细胞的红系前体中的 GFP%，而不增加平均荧光强度（MFI）。只有全长载体转导细胞的后代观察到较低的变异系数（CV），但转导效率降低了五倍。在异种移植小鼠中，单核心绝缘子在移植后 4 和 8 周降低了 GFP%和 MFI，而 CV 没有差异。这些数据表明，包含 HS4 绝缘子元件降低了病毒滴度，降低了转导效率，并在体外和体内人类造血细胞中转基因表达产生最小影响。
