HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector.
HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector.
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DOI：
10.1186/1742-6405-2-1
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发表时间：
2005-01-13
影响因子：
2.2
通讯作者：
Akkina R
中科院分区：
文献类型：
--
作者：
Anderson J;Akkina R
小干扰RNA（siRNA）介导的RNA干扰（RNAi）是一种高效的基因沉默机制，在HIV/AIDS基因治疗中具有巨大的应用潜力。先前针对细胞辅助受体CXCR 4和CCR 5的siRNA研究表明，下调这些表面分子可以阻止HIV-1进入并赋予病毒抗性。由于靶向单个辅助受体的单特异性siRNA不足以同时保护免受嗜T细胞（X4）和嗜单核细胞（R5）病毒株的侵害，因此需要具有双重特异性的双特异性构建体。为了有效的长程治疗，需要通过整合病毒载体将双特异性构建体稳定地转导到HIV-1靶细胞中。为了实现这一目标，构建了掺入短发夹设计的CXCR 4和CCR 5 siRNA的慢病毒载体。CXCR 4 siRNA由U6启动子驱动，而CCR 5 siRNA由H1启动子驱动。CMV启动子驱动的EGFP报告基因也掺入双特异性构建体中。用慢病毒载体实现了向表达辅助受体的Magi和Ghost细胞系中的高效转导，伴随着相应辅助受体的下调。当表达siRNA的转导细胞用X4和R5嗜性HIV-1攻击时，它们表现出显著的病毒抗性。在双特异性慢病毒载体转导的原代PBMC中也观察到HIV-1抗性。CXCR 4和CCR 5共受体可以同时被靶向以通过掺入各自的抗共受体siRNA的单个组合慢病毒载体下调。两种辅助受体的稳定下调保护细胞免受X4和R5嗜性HIV-1的感染。稳定下调有助于HIV-1感染的细胞分子将是长期HIV基因治疗的有效策略。
RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4) and monocyte tropic (R5) viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.