Insertional Transformation of Hematopoietic Cells by Self-inactivating Lentiviral and Gammaretroviral Vectors
Insertional Transformation of Hematopoietic Cells by Self-inactivating Lentiviral and Gammaretroviral Vectors
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DOI：
10.1038/mt.2009.179
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发表时间：
2009-11-01
影响因子：
12.4
通讯作者：
Baum, Christopher
中科院分区：
文献类型：
--
作者：
Modlich, Ute;Navarro, Susana;Baum, Christopher
基因转移载体可能通过插入上调原癌基因而导致克隆失衡甚至恶性细胞转化。慢病毒载体 (LV) 优先整合在转录基因中，被认为比伽马逆转录病毒载体 (GV) 的基因毒性更小，伽马逆转录病毒载体 (GV) 优先整合在转录起始位点和调控基因区域附近。使用敏感的细胞培养测定和一系列自失活（SIN）载体，我们发现慢病毒插入模式触发原代造血细胞转化的可能性大约是γ逆转录病毒的三倍。然而，慢病毒诱导的突变体也表现出稳健的重铺，这与 Evi1 原癌基因第一个内含子中常见插入位点 (CIS) 的选择一致。因此，这种有效的原癌基因代表了 GV 和 LV 的 CIS，尽管它们的整合机制存在重大差异。改变载体的增强子-启动子元件对安全性的影响比逆转录病毒插入模式更大。临床级LV在其自身启动子的控制下表达Wiskott-Aldrich综合征(WAS)蛋白，没有转化潜力。机制研究支持这样的结论：增强子介导的基因激活是造血细胞插入转化的主要原因，为风险预防开辟了合理的策略。
Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer-promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott-Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.