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（RNA interference，RNAi）是一种有效的序列特异性敲除靶基因的新技术。RNAi中的活性“触发物”是长度为21至23个核苷酸的短双链分子，称为小干扰RNA（siRNA）。siRNA可以从细胞中的启动子表达为单独的正义和反义RNA或短发夹RNA（shRNA）。在我们手中，RNAi是我们所利用的最强大的HIV抑制机制，导致细胞培养物中HIV-1编码的p24抗原的抑制大于4个对数。尽管它的效力， 可以通过HIV-1中破坏siRNA反义链与靶标的碱基配对相互作用的突变来规避。由于最好的抗HIV-1疗法包括组合药物，最大限度地提高效力并最大限度地减少耐药性。在该提案中，我们将测试靶向HIV-1的si/shRNAs和细胞共受体CCR 5的组合可以在培养的和原代细胞中提供对HIV-1复制的持续抑制的假设。编码针对多种HIV-1靶点的几种不同siRNA或shRNA的前体转录物和细胞免疫调节剂， CCR 5将由Pol III和Pol II启动子转录。这些前体转录物将被加工成siRNA，它们在RNAi中发挥作用。构建体将通过慢病毒载体转导递送至造血细胞。我们已经在灵长类造血细胞中建立了单一si/shRNAs的稳健表达，从而有效抑制HIV-1复制。预计组合转录物将在基因治疗环境中提供持续的HIV-1感染保护。拟议的研究将测试几种不同的表达si/shRNAs组合的方法。组合构建体将在体外和SCID-hu小鼠模型中的造血背景下进行测试。这里开发的有效结构也将提供给该计划中的其他项目，用于在T细胞和灵长类干细胞移植模型中进行测试。本项目的具体目标是：1）构建和检测一种组合siRNA基因--靶向HIV-1多位点的小干扰RNA（siRNA）或短发夹RNA（shRNA）的组合 并且CCR 5共受体将从单个转录物共表达;2）用于表达组合siRNA构建体的Pol III与Pol II启动子系统的测试-我们将使用U6 Pol III启动子、U1 Pol II启动子和新的HIV达特诱导型启动子系统比较多靶向前体si和shRNA的表达和抗HIV功效; 3）SCID-hu小鼠研究。将来自特异性Aim 1的组合构建体转导到CD 34+造血前体细胞中，用于在干细胞环境中进行抗病毒评价。细胞将被 在允许离体分化成单核细胞和巨噬细胞的条件下培养。将表达组合si/shRNA的细胞离体形成红系和髓系集落的能力与模拟物转导的和载体骨架转导的细胞进行比较。还将用组合构建体转导的⑶ 34+细胞输注到thy/liv SCID-hu小鼠中。将监测T淋巴细胞的发育并与载体转导的细胞进行比较。将进行胸腺内HIV-1激发，以确定 组合构建体在体内环境中的应用。这项研究的长期目标是在T细胞和造血干细胞环境中提供针对HIV-1的有效细胞内免疫。