Influence of insulators on transgene expression from integrating and non-integrating lentiviral vectors.
Influence of insulators on transgene expression from integrating and non-integrating lentiviral vectors.
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DOI：
10.1186/1479-0556-9-1
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发表时间：
2011-01-04
期刊：
影响因子：
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通讯作者：
Sarkis, Chamsy
中科院分区：
文献类型：
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作者：
Grandchamp, Nicolas;Henriot, Dorothee;Sarkis, Chamsy
背景：慢病毒基因转移的有效性和生物安全性受载体设计的影响。为此，慢病毒载体的性质可以通过使用顺式作用元件（如LTR的U3区域的修饰）、中心瓣（cPPT-CTS）元件的结合或转录后调控元件（如土拨鼠转录后调控元件（WPRE））来修饰。最近，一些研究评估了将绝缘子纳入整合慢病毒载体基因组对转基因表达水平和位置效应的影响。方法：本研究采用小鼠免疫球蛋白-kappa （Ig-kappa）或鸡溶菌酶（ChL）基因的基质附着区（MAR）对HeLa、HEK293T、NIH-3T3、Raji、T Jurkat细胞系和原代神经祖细胞中三种hiv -1衍生慢病毒载体：自灭活（SIN）慢病毒载体（LV）、双复制慢病毒载体（DC）和非整合慢病毒载体（nilv）的影响。结果和讨论：我们的研究结果表明，LV背景下的Ig-kappa MAR仅在Hela、NIH-3T3和Jurkat细胞中略微提高转导效率。在双拷贝慢病毒载体的背景下，Ig-kappa MAR对转导效率没有影响，甚至有负面影响。同样，在非整合慢病毒载体的情况下，除了分化的原代神经祖细胞外，Ig-kappa MAR对转导效率没有影响，甚至会产生负面影响。在整合和非整合慢病毒载体的情况下，ChL MAR在所有测试条件下都没有影响或降低转基因表达。结论：本研究表明，MAR序列不一定会增加转基因表达，这些序列的作用可能依赖于环境和/或载体。因此，本研究强调了在给定环境中考虑MAR序列的重要性。此外，最近的其他报道指出了绝缘子随机整合对内源基因表达水平的潜在影响。综上所述，这些结果表明，在载体中使用绝缘体进行基因治疗必须在其将用于的特定治疗环境中进行充分评估，并且必须与其潜在的遗传毒性效应相平衡。
BACKGROUND: The efficacy and biosafety of lentiviral gene transfer is influenced by the design of the vector. To this end, properties of lentiviral vectors can be modified by using cis-acting elements such as the modification of the U3 region of the LTR, the incorporation of the central flap (cPPT-CTS) element, or post-transcriptional regulatory elements such as the woodchuck post-transcriptional regulatory element (WPRE). Recently, several studies evaluated the influence of the incorporation of insulators into the integrating lentiviral vector genome on transgene expression level and position effects.METHODS: In the present study, the influence of the matrix attachment region (MAR) of the mouse immunoglobulin-kappa (Ig-kappa) or the chicken lysozyme (ChL) gene was studied on three types of HIV-1-derived lentiviral vectors: self-inactivating (SIN) lentiviral vectors (LV), double-copy lentiviral vectors (DC) and non-integrating lentiviral vectors (NILVs) in different cell types: HeLa, HEK293T, NIH-3T3, Raji, and T Jurkat cell lines and primary neural progenitors.RESULTS AND DISCUSSION: Our results demonstrate that the Ig-kappa MAR in the context of LV slightly increases transduction efficiency only in Hela, NIH-3T3 and Jurkat cells. In the context of double-copy lentiviral vectors, the Ig-kappa MAR has no effect or even negatively influences transduction efficiency. In the same way, in the context of non-integrating lentiviral vectors, the Ig-kappa MAR has no effect or even negatively influences transduction efficiency, except in differentiated primary neural progenitor cells.The ChL MAR in the context of integrating and non-integrating lentiviral vectors shows no effect or a decrease of transgene expression in all tested conditions.CONCLUSIONS: This study demonstrates that MAR sequences not necessarily increase transgene expression and that the effect of these sequences is probably context dependent and/or vector dependent. Thus, this study highlights the importance to consider a MAR sequence in a given context. Moreover, other recent reports pointed out the potential effects of random integration of insulators on the expression level of endogenous genes. Taken together, these results show that the use of an insulator in a vector for gene therapy must be well assessed in the particular therapeutic context that it will be used for, and must be balanced with its potential genotoxic effects.