Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity
Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity
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DOI：
10.1182/blood-2005-08-024976
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发表时间：
2006-10-15
期刊：
影响因子：
20.3
通讯作者：
Baum, Christopher
中科院分区：
文献类型：
--
作者：
Modlich, Ute;Bohne, Jens;Baum, Christopher
具有长末端重复序列 (LTR) 的逆转录病毒载体在其基因组两端均含有强增强子/启动子序列，广泛用于将基因稳定转移到造血细胞中。然而，最近的临床数据和小鼠模型表明，细胞原癌基因的插入激活是逆转录病毒基因传递的剂量限制副作用，可能诱发白血病。自失活（SIN）逆转录病毒载体不包含增强子/启动子的末端重复，理论上减弱了与邻近细胞基因的相互作用。通过基于原代小鼠造血细胞体外扩增和有限稀释选择的新测定，我们表明使用强内部逆转录病毒增强子/启动子的SIN载体也可以通过插入诱变转化细胞。大多数转化克隆，包括 SIN 载体剂量递增后获得的克隆，显示出 Evi1 第三外显子上游的插入，且插入方向与其转录方向相反。对载体拷贝数进行归一化后，我们发现与相应的 LTR 载体相比，SIN 载体的转化能力显着降低。 SIN 载体的额外修饰可能会进一步提高安全性。改进的细胞培养测定可能在插入诱变的评估中发挥重要作用。
Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.