Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA
Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA
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DOI：
10.1089/088922203322280928
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发表时间：
2003-08-01
影响因子：
1.5
通讯作者：
Akkina, R
中科院分区：
文献类型：
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作者：
Anderson, J;Banerjea, A;Akkina, R
RNA 干扰 (RNAi) 现象引发了转录后基因沉默方法及其在 HIV-1 基因治疗中的潜在应用领域的新浪潮。一种潜在有前景的策略是利用 siRNA 通过下调必需的细胞表面 HIV-1 辅助受体来防止病毒进入细胞表面。在目前的研究中，我们以 siRNA 破坏 CXCR4 辅助受体为目标，抑制 HIV-1 进入，这是实现将其转化为艾滋病基因治疗这一最终目标的第一步。设计并通过 T7 聚合酶转录在体外合成茎环发夹结构的抗 CXCR4 siRNA。在 U373-Magi-CXCR4 细胞中检测了辅助受体的下调。 FACS 分析显示细胞表面 CXCR4 显着下调，Western blot 分析证实细胞内合成水平降低。当用 X4-tropic HIV-1 NL4-3 攻击时，siRNA 转染的细胞表现出明显的病毒抵抗力。与这些结果一致，siRNA转染的原代淋巴细胞也表现出对HIV-1进入的显着抵抗。这些概念验证研究证明了针对重要细胞辅助受体 CXCR4 的 siRNA 在预防 HIV-1 感染方面的功效。通过慢病毒载体将该 siRNA 递送至造血干细胞可能具有潜在的基因治疗应用。
The phenomenon of RNA interference (RNAi) sparked a new surge in the area of posttranscriptional gene silencing methodologies and their potential application for HIV-1 gene therapy. A potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In the present studies we targeted the CXCR4 coreceptor for disruption with siRNA to inhibit HIV-1 entry as a first step toward the ultimate goal of translating this to gene therapy for AIDS. A stem-loop hairpin structured anti-CXCR4 siRNA was designed and synthesized in vitro by transcription with T7 polymerase. Down-regulation of the coreceptor was assayed in U373-Magi-CXCR4 cells. FACS analysis showed marked down-regulation of CXCR4 on the cell surface and Western blot analysis confirmed the reduced levels of intracellular synthesis. When challenged with X4-tropic HIV-1 NL4-3, the siRNA-transfected cells exhibited marked viral resistance. Consistent with these results, siRNA-transfected primary lymphocytes also exhibited significant resistance to HIV-1 entry. These proof-of-concept studies demonstrated the efficacy of an siRNA targeted to an essential cellular coreceptor CXCR4 in protecting from HIV-1 infection. Delivery of this siRNA into hematopoietic stem cells via lentiviral vectors may have potential gene therapeutic applications.