Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element
Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element
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DOI：
10.1038/mt.2009.1
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发表时间：
2009-04-01
影响因子：
12.4
通讯作者：
Persons, Derek A.
中科院分区：
文献类型：
--
作者：
Hanawa, Hideki;Yamamoto, Motoko;Persons, Derek A.
利用逆转录病毒载体进行造血细胞基因治疗已在临床试验中取得成功。然而，有关载体插入突变的安全问题已经出现。在两个不同的试验中，载体插入导致原癌基因的转录激活。一种可能减少载体插入突变的策略是通过使用自失活慢病毒载体，该载体包含来自鸡β-珠蛋白基因的1.2-kb绝缘元件。然而，使用该元件可以显著降低载体滴度和转基因表达，从而影响其实际应用。在这里，我们研究了在部分缺失的长末端重复序列中，在两个方向上都包含全长1.2kb绝缘子或较小的0.25kb核心元件的慢病毒载体。我们表明，使用0.25-kb的核心绝缘体通过缓解进入后阻止与1.2-kb元件相关的反向转录而挽救了载体滴度。此外，由于转录终止性的改善，0.25kb的核心元件以一种定向依赖的方式显著增加了来自内部启动子的转基因表达。该元件还显示了屏障活性，降低了由于位置效应而导致的表达的可变性。众所周知，0.25kb的核心绝缘子具有增强子封闭活性，这种特殊的绝缘慢病毒载体设计可能有助于临床应用。
Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken beta-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application.