Development of Optimized siRNA Inhibition of HIV

HIV 优化 siRNA 抑制的开发

• 批准号: 6850615
• 负责人: John Joseph Rossi
• 金额: $ 24.68万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850615
• 关键词: AIDS therapy; CD34 molecule; CD4 molecule; DNA directed DNA polymerase; Lentivirus; RNA interference; SCID mouse; T lymphocyte; antiAIDS agent; antiviral agents; cell differentiation; flow cytometry; gene therapy; genetic recombination; hematopoietic stem cells; human immunodeficiency virus; human tissue; immunotherapy; macrophage; monocyte; polymerase chain reaction; small interfering RNA; therapy design /development; tissue /cell culture; transfection /expression vector; virus genetics

RNA interference (RNAi) is a powerful new tool for sequence specific knockdown of targeted mRNAs. The active "trigger" in RNAi is a short double stranded molecule of 21 to 23 nucleotides in length termed small interfering RNAs (siRNAs). SiRNAs can be expressed from promoters in cells either as separate sense and antisense or as short hairpin RNAs (shRNAs). In our hands RNAi is the most powerful inhibitory mechanism for HIV we have utilized, resulting in greater than 4 logs of inhibition of HIV-1 encoded p24 antigen in cell culture. Despite its potency, RNAi can be circumvented by mutations in HIV-1 that disrupt base pairing interactions of the siRNA antisense strand with the target. Since the best anti-HIV-1 therapies incorporate combinatorial drugs maximizing potency and minimizing resistance. In this proposal we will test the hypothesis that combinations of si/shRNAs targeting HIV-1 and the cellular co-receptor CCR5 can provide sustained inhibition of HIV-1 replication in cultured and primary cells. Precursor transcripts encoding several different siRNAs or shRNAs against multiple HIV-1 targets and the cellular CCR5 will be transcribed by Pol III and Pol II promoters. These precursor transcripts will be processed into siRNAs where they function in RNAi. The constructs will be delivered to hematopoietic cells via lentiviral vector transduction. We have already established robust expression of single si/shRNAs in primatry hematopoietic cells, resulting in potent inhibition of HIV-1 replication. It is anticipated that the combinatorial transcripts will provide sustained protection from HIV-1 infection in a genet therapy setting. The proposed research will test several different approaches for expression of combinations of si/shRNAs. The combinatorial constructs will be tested in the context of hematopoiesis both in vitro and in the SCID-hu mouse model. Potent constructs developed here will also be provided to other projects in this program for testing both in T-cells and in the primate stem cell transplant models. The specific aims of this project are: 1) Construction and testing of a combinatorial siRNA genes- A combination of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) targeting multiple sites in HIV-1 and the CCR5 co-receptor will be co-expressed from a single transcript;2) Testing of Pol Ill versus Pol II promoter systems for expressing combinatorial siRNA constructs- We will compare expression and anti-HIV efficacies of the multi-targeting precursor si and shRNAs using the U6 Pol III promoter, the U1 Pol II promoter and a novel HIV Tat inducible promoter system; 3) SCID-hu mouse studies. Combinatorial constructs from Specific Aim 1 will be transduced into CD34+ hematopoietic precursor cells for anti-viral evaluation in a stem cell setting. Cells will be cultured under conditions that allow differentiation ex vivo into monocytes and macrophages. The capabilities of cells expressing the combinatorial si/shRNAs to form erythroid and myeloid colonies ex vivo will be compared with mock transduced and vector backbone transduced cells. CD34+ cells transduced with combinatorial constructs will also be infused into thy/liv SCID-hu mice. Development of T-lymphocytes will be monitored and compared with vector -transduced cells. Intra-thymic HIV-1 challenges will be carried out to determine the anit-HIV efficacies of combinatorial constructs in an in vivo setting. The long term goal of this research is to provide potent intracellular immunity against HIV-1 in a T-cell and hematopoietic stem cell setting.

RNA干扰（RNAi）是针对靶向mRNA的序列特异性敲低的强大新工具。 RNAi中的活性“触发”是一个短的双链分子，为21至23个核苷酸的长度为小的小干扰RNA（siRNA）。 siRNA可以从细胞中的启动子中表达为单独的感觉和反义或短发夹RNA（SHRNA）。在我们手中，RNAi是我们使用的最强大的HIV抑制作用机制，导致在细胞培养中抑制HIV-1编码的P24抗原的4个以上。尽管有效力，RNAi HIV-1中的突变可以绕开，从而破坏siRNA反义链与靶标的碱基配对相互作用。由于最佳的抗HIV-1疗法结合了联合药物，从而最大程度地提高了效力并最大程度地减少抗药性。在此提案中，我们将测试以下假设：靶向HIV-1和细胞共受体CCR5的SI/SHRNA的组合可以持续抑制培养的和原代细胞中HIV-1复制。针对多个HIV-1靶标编码几种不同的siRNA或shRNA的前体转录本和细胞 CCR5将由Pol III和Pol II启动子转录。这些前体转录本将被处理到它们在RNAi中起作用的siRNA。这些构建体将通过慢病毒载体转导传递到造血细胞。我们已经建立了在启动造血细胞中单个SI/SHRNA的鲁棒表达，从而导致HIV-1复制有效抑制。可以预计，在遗传治疗环境中，组合转录本将持续保护HIV-1感染。拟议的研究将测试几种不同的SI/SHRNA组合表达的方法。组合构建体将在体外和SCID-HU小鼠模型中的造血作用中进行测试。此处开发的有效构造也将向该计划的其他项目提供，以在T细胞和灵长类动物干细胞移植模型中测试。该项目的具体目的是：1）组合siRNA基因的构建和测试 - 小型干扰RNA（siRNA）或短发夹RNA（SHRNA）的组合，靶向HIV-1中的多个位点 and the CCR5 co-receptor will be co-expressed from a single transcript;2) Testing of Pol Ill versus Pol II promoter systems for expressing combinatorial siRNA constructs- We will compare expression and anti-HIV efficacies of the multi-targeting precursor si and shRNAs using the U6 Pol III promoter, the U1 Pol II promoter and a novel HIV Tat inducible promoter system; 3）SCID-HU小鼠研究。来自特定目标1的组合构建体将被引起到CD34+造血前体细胞中，以在干细胞环境中进行抗病毒评估。细胞将是 在允许离体分化为单核细胞和巨噬细胞的条件下进行培养。表达组合si/shRNA的细胞形成红系和髓样菌落的能力将与模拟转导的和载体骨架转链转导的细胞进行比较。用组合构建体转导的CD34+细胞也将被注入您的/liv scid-hu小鼠中。将监测T淋巴细胞的发展并与载体转导的细胞进行比较。将进行胸腔内HIV-1挑战，以确定 在体内设置中的组合构建体。这项研究的长期目标是在T细胞和造血干细胞的设置中提供有效的对HIV-1的细胞内免疫。