ter Brake Olivier, Konstantinova Pavlina, Ceylan Mustafa, Berkhout Ben
Department of Human Retrovirology, University of Amsterdam, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands.
Mol Ther. 2006 Dec;14(6):883-92. doi: 10.1016/j.ymthe.2006.07.007. Epub 2006 Sep 7.
Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against conserved HIV-1 regions. Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication. We show, for the first time, that expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA compared to single shRNA vectors. Thus, their combined expression results in a much stronger inhibition of virus production. Moreover, when we infected cells transduced with a double shRNA viral vector, virus escape was delayed. These results confirm that RNAi has great potential as an antiviral gene therapy approach and support our efforts to develop this strategy for treatment of HIV-1-infected individuals.
双链RNA可通过一种称为RNA干扰（RNAi）的过程诱导基因沉默。此前，我们已经表明，稳定表达靶向HIV-1 Nef基因的单个短发夹RNA（shRNA）可强烈抑制HIV-1复制。然而，这并不足以维持抑制作用。RNAi的一个标志，即其序列特异性，为病毒提供了一条出路，因为靶区域中的单核苷酸替换会消除抑制作用。为了开发一种能够防止病毒逃逸的持久基因疗法，我们提议将多个针对HIV-1保守区域的shRNA组合起来。因此，我们筛选了86种针对高度保守区域的不同shRNA。我们鉴定出了多种可作为病毒复制有效抑制剂的shRNA。我们首次表明，与单个shRNA载体相比，来自单个慢病毒载体的三种不同shRNA的表达导致每个shRNA的抑制水平相似。因此，它们的联合表达对病毒产生的抑制作用要强得多。此外，当我们用双shRNA病毒载体转导的细胞进行感染时，病毒逃逸被延迟。这些结果证实RNAi作为一种抗病毒基因治疗方法具有巨大潜力，并支持我们为治疗HIV-1感染个体而开发这种策略的努力。
