Multigene lentiviral vectors based on differential splicing and translational control.
Multigene lentiviral vectors based on differential splicing and translational control.
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基于差异剪接和翻译控制的多基因慢病毒载体。
DOI：
10.1006/mthe.2001.0469
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Planelles,V
中科院分区：
文献类型：
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作者：
Zhu,Y;Feuer,G;Day,SL;Wrzesinski,S;Planelles,V
到目前为止，慢病毒载体已针对单个开放阅读框的表达进行了优化。然而，基因治疗的某些实际应用将需要多个基因的表达。本研究的目的是探索从慢病毒载体引导两个标记基因表达的可行性。我们设计了两种类型的多基因慢病毒载体。首先，我们使用了基于 HIV-1 自然剪接信号的策略，通过该策略从单个转录单元生成多个 mRNA。第二种策略是使用翻译顺反子作用元件、脑心肌炎病毒内部核糖体进入位点（IRES）构建多顺反子 mRNA。我们的研究表明，慢病毒载体中包含多个基因不会导致病毒滴度降低或感染非分裂细胞的能力丧失。我们引入了 intatand/orrev 突变来测试剪接是否调节报告基因的相对表达水平。我们还开发了 atat 的截短版本，它没有凋亡相关结构域。将该tat突变体包含在慢病毒载体中导致产生滴度与表达野生型tat的慢病毒载体相似的病毒。
Lentiviral vectors, so far, have been optimized for the expression of a single open reading frame. Certain practical applications of gene therapy will, however, require expression of multiple genes. The goal of this study was to explore the feasibility of directing expression of two marker genes from a lentiviral vector. We designed two types of multigene lentiviral vectors. First, we used a strategy based on the natural splicing signals of HIV-1, by which multiple mRNAs are generated from a single transcriptional unit. A second strategy was construction of a polycistronic mRNA using a translationalcis-acting element, the encephalomyocarditis virus internal ribosome entry site (IRES). Our studies show that the inclusion of multiple genes in lentiviral vectors does not result in reduction in virus titers or in the loss of ability to infect nondividing cells. We introduced mutations intatand/orrevto test whether splicing modulates the relative levels of expression of reporter genes. We also developed a truncated version oftat, which is devoid of the apoptosis-associated domain. Inclusion of thistatmutant in a lentiviral vector resulted in the generation of virus with titers similar to those of lentivirus vectors expressing wild-typetat.