Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.
Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.
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DOI：
10.1093/nar/gku1312
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发表时间：
2015-01
影响因子：
14.9
通讯作者：
Kohn DB
中科院分区：
文献类型：
--
作者：
Cooper AR;Lill GR;Gschweng EH;Kohn DB
慢病毒载体几乎普遍使用异源内部启动子来表达转基因。最常用的启动子片段之一是来自人泛素C（UBC）基因的1.2-kb序列，包括UBC的启动子、一些增强子、第一外显子、第一内含子和第二外显子的一小部分。由于剪接可以在载体生产过程中载体基因组转录后发生，我们研究了UBC启动子片段内的内含子是否忠实地传递到靶细胞。遗传分析显示，超过80%的前病毒形式缺乏UBC启动子的内含子。人延伸因子1 α（EEF 1A 1）启动子片段内含子在慢病毒包装过程中没有丢失，UBC和EEF 1A 1启动子内含子之间的这种差异是由启动子外显子序列赋予的。UBC启动子内含子缺失导致转基因表达减少4倍。表达盒向相反链的移动防止了内含子丢失并恢复了完全表达。这种表达的增加主要是由于内含子内的非经典增强子活性，并且推定的内含子增强子序列向多个启动子近端位点的移动实际上抑制了表达。UBC启动子的恢复也防止了内含子丢失并恢复了双向慢病毒载体中的完全表达。
Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.
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