Transcriptional Gene Silencing of HIV and CCR5

描述（由申请方提供）：人类免疫缺陷病毒1型（HIV-1）已感染人类约20年，但在不久的将来获得有效疫苗的希望渺茫（Ho 2002）。非常需要单独或与当前施用的疗法组合抑制病毒复制的替代策略。一种这样的策略涉及使用小干扰21-26 bp双链RNA（siRNA），其最近被证明是基因表达的普遍存在的和特异性的阻遏物（综述（Finnegan和Matzke 2003））。特别令人感兴趣的是使用siRNA特异性靶向参与疾病如HIV-1的基因。重要的是，使用这些siRNA在启动子控制的表达水平（即转录基因沉默，TGS）靶向HIV-1或其趋化因子共受体将是不可测量的和有效的治疗剂。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，我们在人细胞中证明了构建的靶向启动子的siRNA可以有效地抑制基因mRNA表达（Morris，Chan等，2004）。此外，本文提供的初步数据进一步支持了以下观察结果：siRNA仅在成功将siRNA递送至细胞核后才能在人细胞中诱导TGS，并且所观察到的抑制途径涉及组蛋白脱乙酰化（HDAC）。我们假设HIV-1启动子，特别是5' LTR，和用于HIV-1进入的趋化因子共受体CCR 5的启动子可以被siRNA特异性靶向（直接递送到细胞核中），并且这些启动子经历特异性、持久的表观遗传变化，并显著改变整合的病毒或趋化因子共受体的转录谱。为了验证这一假设，我们提出1）通过靶向HIV-1 LTR和CCR 5启动子中的最少4个位点来确定siRNA介导的抑制的幅度、持续时间和特异性，2）表征启动子特异性siRNA介导的HIV-1病毒复制的抑制（LTR特异性siRNA）或HIV-1进入（CCR 5特异性siRNA）在用HIV-1攻击的转导细胞中的表达，3）表征表观遗传变化与siRNA介导的HIV-1 LTR和CCR 5启动子的TGS相关的DNA甲基化、组蛋白乙酰化/磷酸化和甲基化，和4）通过敲除DNMT-1和HDAC来确定siRNA介导的TGS的作用机制，以及确定与Sin 3或Mi2/涉及NuRD蛋白复合物。该项目中概述的工作应进一步发展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CCR 5，并提供适用于未来治疗干预的发现，旨在改变HIV-1和共受体表达模式。

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.