Self-inactivating retroviral vectors with improved RNA processing
Self-inactivating retroviral vectors with improved RNA processing
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DOI：
10.1038/sj.gt.3302309
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发表时间：
2004-11-01
期刊：
影响因子：
5.1
通讯作者：
Baum, C
中科院分区：
文献类型：
--
作者：
Kraunus, J;Schaumann, DHS;Baum, C
已经鉴定了提高逆转录病毒转基因表达的三个RNA特征：在5 '非翻译区（5' UTR）中的内含子、不存在异常翻译起始密码子和在3 'UTR中土拨鼠肝炎病毒的转录后调节元件（PRE）的存在。为了将这些元件包括到具有潜在改进的安全性的自失活（SIN）载体中，我们从3'长末端重复序列（LTR）的U3区域切除强逆转录病毒启动子，并将其插入逆转录病毒RNA包装信号（Psi）的下游或上游。后一个概念是新的，并且允许在5' UTR中使用内含子，利用Psi周围的逆转录病毒剪接位点。比较了三种LTR和四种SIN载体，以解决RNA元件对滴度、剪接调节和转基因表达的影响。尽管SIN载体的滴度比它们的LTR对应物的滴度低约20倍，但包含PRE允许产生超过106个感染单位/ml而无需进一步的载体优化。与现有技术的LTR载体相比，含内含子的SIN载体显示出极大改善的剪接。关于转基因表达，含内含子的SIN载体在所有研究的细胞类型（胚胎癌细胞、成纤维细胞、原代T细胞和造血祖细胞）中与LTR对应物在很大程度上匹配或甚至超过LTR对应物。
Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5 0 untranslated region (5' UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3 'UTR. To include such elements into self-inactivating ( SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5' UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 106 infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated ( embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).