Expression of anti-HIV siRNA in Blood Cells

抗 HIV siRNA 在血细胞中的表达

• 批准号: 7494914
• 负责人: John Joseph Rossi
• 金额: $ 42.4万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7494914
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Adverse effects; Antiviral Agents; Award; Base Pairing; Blood Cells; CCR5 gene; CD34 gene; Cell Differentiation process; Cells; Combination Drug Therapy; Complex; Cultured Cells; DNA Polymerase II; DNA Polymerase III; Defense Mechanisms; Dendritic Cells; Dose; Drosophila genus; Drug Kinetics; Drug usage; Escape Mutant; Gene Expression; Gene Silencing; Genetic Transcription; Goals; HIV; HIV Infections; Health; Hematopoietic; Heterochromatin; Highly Active Antiretroviral Therapy; Human; Human Engineering; In Vitro; Individual; Mammals; Medical; Medicine; Multi-Drug Resistance; Mus; Nobel Prize; Patients; Physiology; Plants; Point Mutation; Process; Public Health; RNA; RNA Interference; Regulator Genes; Research; Safety; Scientist; Site; Small Interfering RNA; Small RNA; Testing; Therapeutic; Toxic effect; Transfer RNA; Translational Repression; Treatment Protocols; U1 small nuclear RNA; United States; Variant; Viral; design; gene therapy; in vivo; macrophage; mouse model; novel; novel therapeutics; programs; promoter; receptor

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.

描述（由申请人提供）： 艾滋病毒感染和艾滋病仍然是世界性问题。 仅在美国，每年就有超过30，000例新的艾滋病毒感染病例。联合药物治疗或HAART的出现极大地改变了受感染个体向AIDS发展的过程，但对于许多患者来说，这些药物的使用存在相关的问题。 这些包括由于个体之间的药代动力学差异而导致的困难的给药方案，毁容的副作用以及多药耐药变体的出现。 HAART的使用也是终身的，是一项主要的医疗费用。 因此，必须开发和测试治疗艾滋病毒感染的替代方法。 一种这样的方法是使用RNA干扰或RNAi。 RNAi描述了由短RNA双链体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性翻译抑制的基因沉默、mRNA的切割和通过序列特异性异染色质形成触发转录沉默。 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前线抗病毒防御，但所有用于抗病毒防御的RNAi细胞机制都存在，并且在提供小RNA触发剂时可以轻松编程以抑制病毒。 拟议的研究利用了先前发现期间的发现，这些发现建立了在人类造血细胞中触发抗HIV RNAi的原则和方法。 迄今为止，异位触发的RNAi是已知的最强大的抗HIV机制之一。 尽管它的效力，RNAi依赖于沃森-克里克碱基配对用于靶选择的事实意味着HIV可以通过RNAi靶区域中的点突变来逃避这种抑制。规避这一点的最佳方法是使用RNAi触发剂或小干扰RNA（siRNA）的组合，通过造血细胞的基因治疗来治疗HIV感染。 触发转录后基因沉默的siRNA的组合将与使用siRNA指导CCR 5共受体的转录基因沉默的策略多重化。 这些触发物的表达将使用新的但明确定义的tRNA Pol III和U1 snRNA Pol II启动子进行。 siRNA和表达模式的各种组合将在细胞培养中测试安全性和HIV功效，然后在Rag 2-/-？用于人造血细胞分化、成熟和HIV攻击的c-/-小鼠模型。 这些研究的首要假设是，针对与艾滋病毒感染相关的多个位点和功能的异位表达siRNAs的组合可用于规避病毒逃逸突变体。 本研究的具体目的如下：1）siRNA选择和在转录后基因沉默（PTGS）和转录基因沉默（TGS）中的功能的机制研究; 2）探索用于多重RNAi PTGS和TGS触发物的小RNA表达策略;和3）评估组成型表达的shRNA/miRNA在体外的CD 34细胞衍生的巨噬细胞和树突状细胞以及体内人源化小鼠衍生的造血细胞中的稳定性、功效和潜在毒性。 该提案的总体目标是开发用于在造血细胞基因治疗环境中治疗HIV感染的表达siRNA的有效且安全的组合。 与公共卫生的关系：艾滋病毒/艾滋病仍然是全世界人类健康的主要威胁。 持续需要靶向HIV感染的新治疗方法。 RNA干扰（RNA interference，RNAi）是近年来发现的一种强有力的基因表达调控机制。 2006年诺贝尔生理学和医学奖授予发现这一现象的两位科学家，突出了RNAi作为一种可能的治疗方法的重要性。 该提案旨在利用这种自然过程来治疗HIV感染，通过遗传工程改造人类血细胞来产生称为小干扰RNA（siRNA）的RNAi触发物的组合。 这些siRNA将被合理设计以在没有毒性的情况下提供对HIV复制的最大抑制。 这项研究计划的主要目标是验证RNA干扰作为治疗HIV感染的治疗方法。