Combinatorial incorporation of enhancer-blocking components of the chicken beta-globin 5'HS4 and human T-cell receptor alpha/delta BEAD-1 insulators in self-inactivating retroviral vectors reduces their genotoxic potential.
Combinatorial incorporation of enhancer-blocking components of the chicken beta-globin 5'HS4 and human T-cell receptor alpha/delta BEAD-1 insulators in self-inactivating retroviral vectors reduces their genotoxic potential.
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在自失活逆转录病毒载体中组合掺入鸡 β 球蛋白 5'HS4 的增强子阻断成分和人 T 细胞受体 α/δ BEAD-1 绝缘子可降低其潜在的基因毒性。
DOI：
10.1634/stemcells.2008-0258
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发表时间：
2008-12
期刊：
影响因子：
5.2
通讯作者：
Hawley, Robert G.
中科院分区：
文献类型：
--
作者：
Ramezani, Ali;Hawley, Teresa S.;Hawley, Robert G.
关键词：
逆转录病毒载体的插入突变已成为广泛应用造血干细胞基因转移治疗血液病的严重障碍。在这里，我们报道了一个77 bp的元件FII/BEAD-A （FB）的开发，它包含鸡β-珠蛋白5'HS4绝缘子的最小增强子阻断成分和来自人t细胞受体α/δ BEAD-1绝缘子的同源区域。通过一种新的基于流式细胞术的检测，我们发现FB元件在增强子阻断活性方面与典型的1.2 kb 5'HS4绝缘子片段一样有效。当整合到自灭活(SIN) γ -逆转录病毒载体的3 ‘长末端重复序列（LTR）的残余U3区域时，FB元件在逆转录过程中稳定地转移到整合的转基因表达盒两侧的5 ’ LTR上。值得注意的是，使用最近建立的涉及原代小鼠造血细胞的体外插入诱变试验，我们发现，与未屏蔽的对应物相比，含有FB元件的SIN γ逆转录病毒载体和SIN慢病毒载体在实验条件下的转化潜力大大降低至背景水平。这些结果表明，FB元件介导的增强子阻断修饰是一种有希望的方法，可以显著提高逆转录病毒载体用于治疗性基因转移的安全性。
Insertional mutagenesis by retroviral vectors has emerged as a serious impediment to the widespread application of hematopoietic stem cell gene transfer for the treatment of hematologic diseases. Here we report the development of a 77-bp element, FII/BEAD-A (FB), which contains the minimal enhancer blocking components of the chicken β-globin 5′HS4 insulator and a homologous region from the human T-cell receptor α/δ BEAD-1 insulator. With a new flow cytometry-based assay, we show that the FB element is as effective in enhancer blocking activity as the prototypical 1.2-kb 5′HS4 insulator fragment. When incorporated into the residual U3 region of the 3′ long terminal repeat (LTR) of a self-inactivating (SIN) gammaretroviral vector, the FB element was stably transferred to the 5′ LTR during reverse transcription, flanking the integrated transgene expression cassette. Notably, using a recently established in vitro insertional mutagenesis assay involving primary murine hematopoietic cells, we found that SIN gammaretroviral vectors as well as SIN lentiviral vectors containing the FB element exhibited greatly reduced transforming potential—to background levels under the experimental conditions used—compared to their unshielded counterparts. These results suggest that the FB element-mediated enhancer blocking modification is a promising approach to dramatically improve the safety of retroviral vectors for therapeutic gene transfer.
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DOI：
10.1073/pnas.94.2.575
发表时间：
1997-01-21
影响因子：
11.1
作者：
Chung, JH;Bell, AC;Felsenfeld, G
通讯作者：
Felsenfeld, G
影响因子：
20.3
作者：
Du, Y;Jenkins, NA;Copeland, NG
通讯作者：
Copeland, NG
影响因子：
3.6
作者：
Hawley, Robert G.
通讯作者：
Hawley, Robert G.
影响因子：
5.1
作者：
Kraunus, J;Schaumann, DHS;Baum, C
通讯作者：
Baum, C
影响因子：
56.9
作者：
Kustikova, O;Fehse, B;Baum, C
通讯作者：
Baum, C