ter Brake Olivier, 't Hooft Karen, Liu Ying Poi, Centlivre Mireille, von Eije Karin Jasmijn, Berkhout Ben
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of University of Amsterdam, Amsterdam, The Netherlands.
Mol Ther. 2008 Mar;16(3):557-64. doi: 10.1038/sj.mt.6300382. Epub 2008 Jan 15.
Human immunodeficiency virus type 1 (HIV-1) replication in T cells can be inhibited by RNA interference (RNAi) through short hairpin RNA (shRNA) expression from a lentiviral vector. However, for the development of a durable RNAi-based gene therapy against HIV-1, multiple shRNAs need to be expressed simultaneously in order to avoid viral escape. In this study, we tested a multiple shRNA expression strategy for different shRNAs using repeated promoters in a lentiviral vector. Although highly effective in co-transfection experiments, a markedly reduced activity of each expressed shRNA was observed in transduced cells. We found that this reduced activity was due to recombination of the expression cassette repeat sequences during the transduction of the lentiviral vector, which resulted in deletions of one or multiple cassettes. To avoid recombination, we tested different promoters for multiple shRNA expression. We compared the activity of the human polymerase III promoters U6, H1, and 7SK and the polymerase II U1 promoter. Activities of these promoters were similar, irrespective of which shRNA was expressed. We showed that these four expression cassettes can be combined in a single lentiviral vector without causing recombination. Moreover, whereas HIV-1 could escape from a single shRNA, we now show that HIV-1 escape can be prevented when four shRNAs are simultaneously expressed in a cell.
1型人类免疫缺陷病毒（HIV-1）在T细胞中的复制可通过慢病毒载体表达短发夹RNA（shRNA），利用RNA干扰（RNAi）来抑制。然而，要开发一种持久的基于RNAi的抗HIV-1基因疗法，需要同时表达多个shRNA以避免病毒逃逸。在本研究中，我们在慢病毒载体中使用重复启动子测试了针对不同shRNA的多shRNA表达策略。尽管在共转染实验中非常有效，但在转导细胞中观察到每个表达的shRNA活性明显降低。我们发现这种活性降低是由于慢病毒载体转导过程中表达盒重复序列的重组，导致一个或多个盒的缺失。为避免重组，我们测试了用于多shRNA表达的不同启动子。我们比较了人类聚合酶III启动子U6、H1和7SK以及聚合酶II U1启动子的活性。这些启动子的活性相似，与所表达的shRNA无关。我们表明这四个表达盒可以组合在单个慢病毒载体中而不引起重组。此外，虽然HIV-1可以从单个shRNA逃逸，但我们现在表明当在细胞中同时表达四个shRNA时可以防止HIV-1逃逸。
