Silencing of HIV-1 with RNA interference: A multiple shRNA approach
Silencing of HIV-1 with RNA interference: A multiple shRNA approach
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DOI：
10.1016/j.ymthe.2006.07.007
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发表时间：
2006-12-01
影响因子：
12.4
通讯作者：
Berkhout, Ben
中科院分区：
文献类型：
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作者：
ter Brake, Olivier;Konstantinova, Pavlina;Berkhout, Ben
双链RNA可以通过一种称为RNA干扰(RNAi)的过程诱导基因沉默。此前，我们已经证明，针对HIV-1Nef基因的单个shRNA的稳定表达强烈地抑制了HIV-1的复制。然而，这还不足以维持抑制。RNAi的特征之一，它的序列特异性，为病毒提供了一条出路，因为靶区的单核苷酸替换取消了抑制。为了开发一种持久的基因疗法来防止病毒逃逸，我们建议结合多个针对HIV-1保守区域的shRNAs。因此，我们筛选了86个针对高度保守区域的不同shRNA。我们发现了多个shRNA，它们可以有效地抑制病毒复制。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个不同shRNA的表达导致了每个shRNA类似的抑制水平。因此，它们的联合表达导致了对病毒生产的更强的抑制。此外，当我们感染转导双shRNA病毒载体的细胞时，病毒逃逸被推迟。这些结果证实了RNAi作为一种抗病毒基因治疗方法的巨大潜力，并支持我们努力开发这一治疗HIV-1感染者的策略。
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.