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仍存在一些问题。 其中包括由于个体间药代动力学差异而导致的给药方案困难、毁容副作用以及多重耐药变异体的出现。 HAART的使用也是终生的并且是一项主要的医疗费用。 因此，开发和测试治疗艾滋病毒感染的替代方法非常重要。 其中一种方法是使用 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实现基因沉默、mRNA 裂解以及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中的前线抗病毒防御，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 触发器时易于编程以抑制病毒。 拟议的研究利用了先前发现阶段的发现，该发现建立了在人类造血细胞中触发抗 HIV RNAi 的原理和方法。 迄今为止，异位触发的 RNAi 是已知最强大的抗 HIV 机制之一。 尽管RNAi很有效，但事实上RNAi依赖于Watson-Crick碱基配对来进行目标选择，这意味着HIV可以通过RNAi目标区域中的点突变来逃避这种抑制。规避这一问题的最佳方法是使用 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 组合将与使用 siRNA 指导 CCR5 共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颖但明确的 tRNA Pol III 和 U1 snRNA Pol II 启动子进行。 将在细胞培养物中测试 siRNA 和表达模式的各种组合的安全性和 HIV 功效，然后在 Rag2-/-?c-/- 小鼠模型中测试人类造血细胞分化、成熟和 HIV 攻击。 这些研究的首要假设是，针对与 HIV 感染相关的多个位点和功能的异位表达 siRNA 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）转录后基因沉默（PTGS）和转录基因沉默（TGS）中siRNA选择和功能的机制研究； 2) 探索多重 RNAi PTGS 和 TGS 触发器的小 RNA 表达策略；以及：3) 评估体外 CD34+ 细胞来源的巨噬细胞和树突状细胞以及体内人源化小鼠的造血细胞中组成型表达的 shRNA/miRNA 的稳定性、功效和潜在毒性。 该提案的总体目标是开发有效且安全的表达 siRNA 组合，用于在造血细胞基因治疗环境中治疗 HIV 感染。 公共卫生相关性：艾滋病毒/艾滋病仍然是全世界人类健康的主要威胁。 持续需要针对 HIV 感染的新治疗方法。 RNA 干扰 (RNAi) 是最近发现的一种强大且自然发生的基因表达调节机制。 2006 年，两位发现这一现象的科学家荣获诺贝尔生理学和医学奖，突显了 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治疗 HIV 感染的自然过程，通过对人类血细胞进行基因工程改造，产生称为小干扰 RNA (siRNA) 的 RNAi 触发组合。 这些 siRNA 将经过合理设计，在没有毒性的情况下最大限度地抑制 HIV 复制。 该研究项目的主要目标是验证 RNA 干扰作为治疗 HIV 感染的方法。