Expression of anti-HIV siRNA in Blood Cells

描述(由申请人提供)： 艾滋病毒感染和艾滋病仍然是世界性的问题。仅在美国，每年就有超过3万例新的艾滋病毒感染病例。联合药物疗法或HAART的出现极大地改变了感染者发展为艾滋病的过程，但许多患者使用这些药物存在相关问题。这些问题包括由于个体之间的药代动力学差异而导致的给药方案的困难、严重的副作用以及出现多药耐药变种。HAART的使用也是终生的，也是一项主要的医疗费用。因此，重要的是开发和测试治疗艾滋病毒感染的替代方法。其中一种方法是使用RNA干扰或RNAi。RNAi描述了由短RNA双链触发的一组复杂的转录后基因调控机制。这些机制包括通过序列特异的翻译抑制、mRNAs的切割和通过序列特异的异染色质的形成触发转录沉默。在植物、蠕虫和果蝇中，RNAi是一种抗病毒防御机制。尽管它从未被正式证明是哺乳动物的一线抗病毒防御，但所有用于抗病毒防御的RNAi细胞机制都是存在的，并且当提供小的RNA触发器时，它很容易被编程为病毒抑制。这项拟议的研究利用了前一个发现期的发现，该发现建立了在人类造血细胞中触发抗艾滋病毒RNAi的原则和方法。到目前为止，异位触发的RNAi是已知的最强大的抗HIV机制之一。尽管RNAi具有效力，但事实上，RNAi依赖Watson-Crick碱基配对进行靶标选择，这意味着HIV可以通过RNAi靶区的点突变来逃避这种抑制。规避这一问题的最好方法是使用RNAi触发器或小干扰RNA(SiRNAs)的组合，通过造血细胞的基因治疗来治疗艾滋病毒感染。触发转录后基因沉默的siRNAs组合将与一种利用siRNAs指导CCR5辅受体转录基因沉默的策略相结合。这些触发器的表达将使用新的但定义良好的tRNA POL III和U1 SnRNA POL II启动子来进行。各种siRNAs和表达模式的组合将在细胞培养中测试安全性和HIV有效性，然后在Rag2-/-？C-/-小鼠模型中用于人类造血细胞的分化、成熟和HIV挑战。这些研究的压倒一切的假设是，针对与艾滋病毒感染相关的多个位置和功能的异位表达的siRNA的组合可以用来避开病毒逃逸突变。本研究的具体目的如下：1)研究siRNA选择及其在转录后基因沉默(PTGS)和转录基因沉默(TGS)中作用的机制；2)探索RNAi转录后基因沉默(PTGS)和转录基因沉默(TGS)触发器的小RNA表达策略；3)评估结构性表达的shRNAs/miRNAs在CD34细胞来源的巨噬细胞、树突状细胞(DC)和体内人源化小鼠来源的造血细胞中的稳定性、有效性和潜在毒性。这项建议的总体目标是开发有效和安全的表达siRNAs组合，用于在造血细胞基因治疗环境中治疗HIV感染。 与公共卫生有关：艾滋病毒/艾滋病仍然是全世界人类健康的主要威胁。不断需要针对艾滋病毒感染的新的治疗方法。RNA干扰(RNAi)是新近发现的一种强大的、自然发生的基因表达调控机制。2006年将诺贝尔生理学和医学奖授予发现这一现象的两位科学家，突显了RNAi作为一种可能的治疗方法的重要性。这项提议旨在利用这一自然过程来治疗艾滋病毒感染，方法是对人类血细胞进行基因工程，以产生称为小干扰RNA(SiRNAs)的RNAi触发器的组合。这些siRNA将被合理设计，以在没有毒性的情况下最大限度地抑制艾滋病毒复制。这项研究计划的主要目标是验证RNA干扰作为治疗艾滋病毒感染的一种方法。

DESCRIPTION (provided by applicant): HIV infection and AIDS continue to be worldwide problems. In the United States alone, there are over 30,000 new cases of HIV infection per year. The advent of combination drug therapy or HAART has greatly changed the course of progression to AIDS for infected individuals, yet there are problems associated with the use of these drugs for many patients. These include difficult dosing regimens due to pharmacokinetic differences among individuals, disfiguring side effects, and the emergence of multi-drug resistant variants. The use of HAART is also lifelong and a major medical expense. It is therefore important that alternate approaches for the treatment of HIV infection be developed and tested. One such approach is the use of RNA interference, or RNAi. RNAi describes a complex set of post-transcriptional gene regulatory mechanisms triggered by short RNA duplexes. These mechanisms include gene silencing via sequence specific translational repression, cleavage of mRNAs and triggering of transcriptional silencing via sequence specific heterochromatin formation. In plants, worms and fruit flies RNAi is an anti-viral defense mechanism. Although it has never formally been proven to be a front line antiviral defense in mammals, all of the RNAi cellular machinery for anti-viral defense is present and readily programmable for viral inhibition when supplied with small RNA triggers. The proposed research capitalizes upon findings from the previous finding period which established principles and approaches for triggering anti-HIV RNAi in human hematopoietic cells. To date, ectopically triggered RNAi is one of the most powerful anti-HIV mechanisms known. Despite its potency, the fact that RNAi relies on Watson-Crick base pairing for target selection means that HIV can escape this inhibition via point mutations in the RNAi target regions. The best way to circumvent this is to use combinations of RNAi triggers, or small interfering RNAs (siRNAs) for the treatment of HIV infection via gene therapy of hematopoietic cells. Combinations of siRNAs triggering post-transcriptional gene silencing will be multiplexed with a strategy for using siRNAs to direct transcriptional gene silencing of the CCR5 co-receptor. The expression of these triggers will be carried out using novel, but well defined tRNA Pol III and U1 snRNA Pol II promoters. Various combinations of siRNAs and expression modes will be tested for safety and HIV efficacy in cell culture and then in a Rag2-/-?c-/- mouse model for human hematopoietic cell differentiation, maturation, and HIV challenge. The overriding hypothesis for these studies is that combinations of ectopically expressed siRNAs, targeting multiple sites and functions relating to HIV infection, can be used to circumvent viral escape mutants. The specific Aims of this study are as follows: 1) Mechanistic studies of siRNA selection and function in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS); 2) Exploring small RNA expression strategies for multiplexing RNAi PTGS and TGS triggers;and:3) Evaluate the stability, efficacy and potential toxicity of constitutively expressed shRNAs/miRNAs in CD34 cell derived macrophages and dendritic cells in vitro, and hematopoietic cells derived from in vivo humanized mice. The overall goal of this proposal is to develop potent and safe combinations of expressed siRNAs for the treatment of HIV infection in a hematopoietic cell gene therapy setting. PUBLIC HEALTH RELEVANCE: HIV/AIDS continues to be a major threat to human health throughout the world. There is a continual need for new therapeutic approaches targeting HIV infection. RNA interference (RNAi) is a recently discovered powerful and naturally occurring mechanism for regulating gene expression. The significance of RNAi as a possible therapeutic is highlighted by the 2006 awarding of the Nobel Prize in Physiology and Medicine to the two scientists who discovered this phenomenon. This proposal is designed to exploit this natural process for the treatment of HIV infection by genetically engineering human blood cells to produce combinations of RNAi triggers called small interfering RNAs (siRNAs). These siRNAs will be rationally designed to provide maximal inhibition of HIV replication in the absence of toxicity. The primary goal of this research program is to validate RNA interference as a therapeutic approach for the treatment of HIV infection.