Efficacy Analysis of Combinatorial siRNAs against HIV Derived from One Double Hairpin RNA Precursor.
Efficacy Analysis of Combinatorial siRNAs against HIV Derived from One Double Hairpin RNA Precursor.
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来自一种双发夹 RNA 前体的组合 siRNA 针对 HIV 的功效分析。
DOI：
10.3389/fmicb.2017.01651
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发表时间：
2017
影响因子：
5.2
通讯作者：
Kong X
中科院分区：
文献类型：
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作者：
Liu C;Liang Z;Kong X
组合小干扰RNA双链体（siRNAs）具有成为抗HIV-1基因治疗的潜力，已有研究报道瞬时组合siRNA表达可抑制HIV复制，但长期siRNA表达对HIV复制的影响尚未见详细研究。在这项研究中，HIV-1复制的影响下，稳定的组合siRNA表达从一个单一的RNA转录进行了分析。首先，构建一系列编码短发夹RNA（shRNA）/长发夹RNA（lhRNA）/双长发夹（dlhRNA）的盒，并进行抑制功效分析。接下来，选择优化的dlhRNA编码盒并插入慢病毒递送载体FG 12中。瞬时dlhRNA表达成功地减少了TZM-bl细胞和CD 4 + T细胞中的HIV-1复制。用表达dlhRNA的慢病毒载体转导HIV-1易感TZM-bl细胞，并通过荧光激活细胞分选进行分选，以获得稳定的表达dlhRNA的细胞。通过茎环RT-PCR测定验证在这些dlhRNA表达细胞中四种抗HIV siRNA的产生。dlhRNA表达不激活非特异性干扰素应答。表达dlhRNA的细胞也用HIV-1 NL 4 -3攻击，这揭示了组合siRNA的稳定表达抑制HIV-1复制8天，之后HIV-1通过表达RNAi靶标的突变形式克服了siRNA表达的抑制作用。本研究评估了组合siRNA对HIV-1的长期抑制作用，为利用组合RNA干扰对抗HIV-1或其他易错病毒的研究人员提供了参考。
Combinatorial small interfering RNA duplexes (siRNAs) have the potential to be a gene therapy against HIV-1, and some studies have reported that transient combinatorial siRNA expression represses HIV replication, but the effects of long-term siRNA expression on HIV replication have not been studied in detail. In this study, HIV-1 replication under the influence of stable combinatorial siRNA expression from a single RNA transcript was analyzed. First, a series of cassettes encoding short hairpin RNA (shRNA)/long hairpin RNA (lhRNA)/double long hairpins (dlhRNA) was constructed and subjected to an analysis of inhibitory efficacy. Next, an optimized dlhRNA encoding cassette was selected and inserted into lentiviral delivery vector FG12. Transient dlhRNA expression reduced replication of HIV-1 in TZM-bl cells and CD4+ T cells successfully. HIV-1 susceptible TZM-bl cells were transducted with the dlhRNA expressing lentiviral vector and sorted by fluorescence-activated cell sorting to obtain stable dlhRNA expressing cells. The generation of four anti-HIV siRNAs in these dlhRNA expressing cells was verified by stem–loop RT-PCR assay. dlhRNA expression did not activate a non-specific interferon response. The dlhRNA expressing cells were also challenged with HIV-1 NL4-3, which revealed that stable expression of combinatorial siRNAs repressed HIV-1 replication for 8 days, after which HIV-1 overcame the inhibitory effect of siRNA expression by expressing mutant versions of RNAi targets. The results of this evaluation of the long-term inhibitory effects of combinatorial siRNAs against HIV-1 provide a reference for researchers who utilize combinatorial RNA interference against HIV-1 or other error-prone viruses.