Transcriptional Gene Silencing of HIV and CCR5

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）已经感染人类约20年了，在不久的将来出现有效疫苗的希望渺茫（Ho 2002）。抑制病毒复制的替代策略，无论是单独还是与目前给予的治疗相结合，都是非常需要的。其中一种策略涉及使用小干扰21-26 bp双链rna (sirna)，这些rna最近被证明是普遍存在的基因表达特异性抑制因子（Finnegan and Matzke 2003）。特别令人感兴趣的是使用sirna专门针对与HIV-1等疾病有关的基因。重要的是，使用这些sirna在启动子控制的表达水平上靶向HIV-1或其趋化因子共受体（即转录基因沉默，TGS）将是一种不可估量的有效治疗剂。直到最近，还没有发表的数据表明TGS在哺乳动物细胞中是可操作的，并且大部分siRNA活性是细胞质的（Zeng 2002）。最近，我们在人类细胞中证明，针对启动子构建的sirna可以有效抑制基因mRNA的表达（Morris, Chan et al. 2004）。此外，本文提供的初步数据进一步支持了siRNA只有在成功将siRNA传递到细胞核时才能在人类细胞中诱导TGS，并且观察到的抑制途径涉及组蛋白去乙酰化（HDAC）。我们假设HIV-1启动子，特别是5' LTR，以及用于HIV-1进入的趋化因子共受体CCR5的启动子可以被sirna特异性靶向（直接传递到细胞核中），并且这些启动子经历特异性的、持久的、显著改变整合病毒或趋化因子共受体转录谱的表观遗传变化。为了验证这一假设，我们提出：1)通过靶向HIV-1 LTR和CCR5启动子中的至少4个位点，确定siRNA介导的抑制的大小、持续时间和特异性；2)表征启动子特异性siRNA介导的HIV-1病毒复制（LTR特异性siRNA）或HIV-1进入（CCR5特异性siRNA）在转染HIV-1的细胞中的抑制；组蛋白乙酰化/磷酸化和甲基化)与HIV-1 LTR和CCR5启动子的siRNA介导的TGS相关，以及4)通过敲除DNMT-1和HDAC来确定siRNA介导的TGS的作用机制，并确定是否与Sin3或Mi2/NuRD蛋白复合物相互作用。本项目概述的工作将进一步加深我们对人类细胞中靶向特异性基因调控的复杂分子途径的理解，特别是HIV-1及其趋化因子共受体CCR5，并提供适用于未来旨在改变HIV-1和共受体表达模式的治疗干预的发现。

DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV-1) has been infecting humans for ~20 years now with little hope for an efficacious vaccine in the near future (Ho 2002). Alternative strategies to inhibit viral replication either alone or in combination with the currently administered therapies are tremendously desirable. One such strategy involves the use of small interfering 21-26 bp double stranded RNAs (siRNAs) which have recently been shown to be ubiquitous and specific repressers of gene expression (reviewed (Finnegan and Matzke 2003)). Of particular interest is the use of siRNAs to specifically target genes involved in diseases such as HIV-1. Importantly, the use of these siRNAs to target HIV-1 or its chemokine co-receptor at the level of promoter-controlled expression (i.e. transcriptional gene silencing, TGS) would be an immeasurable and potent therapeutic agent. Until recently there was no published data suggesting that TGS is operable in mammalian cells and the majority of siRNA activity is cytoplasmic (Zeng 2002). Recently we demonstrated in human cells that siRNAs constructed to target a promoter can effectively suppress a genes mRNA expression (Morris, Chan et al. 2004). Moreover, the preliminary data presented here adds further support to the observation that siRNAs can induce TGS in human cells only upon successful delivery of the siRNA to the nucleus and that the pathway of the observed inhibition involves histone deacetylation (HDAC). We hypothesize that the HIV-1 promoter, specifically the 5' LTR, and the promoter for the chemokine co-receptor CCR5 used for entry by HIV-1 can be specifically targeted by siRNAs (delivered directly into the nucleus) and that these promoters undergo epigenetic changes that are specific, long lasting, and significantly alter the transcriptional profile of the integrated virus or chemokine co-receptor. To test this hypothesis we propose to 1) determine the magnitude, duration, and specificity of siRNA mediated suppression by targeting a minimum of 4 sites in the HIV-1 LTR and CCR5 promoter, 2) Characterize promoter specific siRNA mediated suppression of HIV-1 viral replication (LTR specific siRNAs) or HIV-1 entry (CCR5 specific siRNAs) in transduced cells challenged with HIV-1, 3) characterize the epigenetic changes (DNA methylation, histone acetylation/ phosphorylation and methylation) associated with siRNA mediated TGS of the HIV-1 LTR and CCR5 promoter, and 4) determine the mechanism of action of the siRNA mediated TGS by knocking out DNMT-1 and HDAC's as well as determine if an interaction with Sin3 or Mi2/NuRD protein complexes is involved. The work outlined in this project should further develop our understanding of this complex molecular pathway of target specific gene regulation in human cells, specifically HIV-1 and it's chemokine co-receptor CCR5, and provide findings that will be applicable in future therapeutic interventions aimed at altering patterns of HIV-1 and co-receptor expression.