Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector
Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector
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DOI：
10.1038/sj.mt.6300103
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发表时间：
2007-04-01
影响因子：
12.4
通讯作者：
Nienhuis, Arthur W.
中科院分区：
文献类型：
--
作者：
Evans-Galea, Marguerite V.;Wielgosz, Matthew M.;Nienhuis, Arthur W.
慢病毒载体有效地活化静止干细胞，并正在评估血液疾病的基因治疗。插入诱变导致的遗传毒性风险是一个重要的安全性考虑因素。来自鸡β-珠蛋白基因座的超敏位点4绝缘子（cHS 4）具有染色质屏障和增强子阻断功能。将编码绿色荧光蛋白的对照慢病毒载体与其中cHS 4绝缘子元件侧接绿色荧光蛋白表达盒的载体在培养9天后在转导的人T细胞（Jurkat）的单细胞分离物中进行比较。绝缘子对平均荧光强度的影响最小，并且仅适度降低单个单细胞分离株中绿色荧光蛋白表达的变异性。大多数独特的整合位点在基因内，但与对照载体相比，含有绝缘子的载体有适度的倾向于在转录起始位点附近整合。在含有整合的载体基因组的细胞培养物中形成克隆优势，如含有相同整合位点的多个单细胞分离株的回收所反映的。我们推断，某些整合通过插入诱变影响基因表达，从而导致这种克隆优势，从而赋予增殖或生存优势。通过在载体基因组中包含绝缘子元件，这种效应被减弱。
Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood disorders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hypersensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin barrier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among individual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal dominance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of multiple single cell isolates containing the same integration site. We infer that certain integrations conferred a proliferative or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.