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)是一种有效的序列特异性敲除靶向mRNAs的新工具。在RNAi中活跃的“触发器”是一个长度为21到23个核苷酸的短双链分子，称为小干扰RNA(SiRNAs)。SiRNAs可通过启动子在细胞内以正、反义或短发夹状RNA的形式表达。在我们手中，RNAi是我们所利用的对HIV最强大的抑制机制，导致在细胞培养中对HIV-1编码的p24抗原的抑制超过4logs。尽管RNAi具有强大的功能，但它 可以通过HIV-1的突变来规避，这些突变破坏了siRNA反义链与靶的碱基配对相互作用。因为最好的抗HIV-1疗法包括组合药物，最大限度地提高效力和最大限度地减少耐药性。在这项建议中，我们将测试针对HIV-1的si/shRNAs和细胞共受体CCR5的组合可以在培养细胞和原代细胞中持续抑制HIV-1复制的假设。针对多个HIV-1靶点编码几种不同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加工成siRNA，在那里它们在RNAi中发挥作用。这些构建物将通过慢病毒载体转导被运送到造血细胞。我们已经建立了单个si/shRNAs在原始造血细胞中的高效表达，从而有效地抑制了HIV-1的复制。预计组合转录本将在基因治疗环境中提供对艾滋病毒-1感染的持续保护。这项拟议的研究将测试si/shRNAs组合的几种不同表达方法。这些组合结构将在体外和SCID-Hu小鼠模型的造血环境中进行测试。这里开发的有效结构也将被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)构建和检测组合siRNA基因--针对HIV-1中多个位点的小干扰RNA(SiRNAs)或短发夹状RNA(ShRNAs)的组合 2)测试表达组合siRNA的Pol Ill和Pol II启动子系统-我们将比较使用U6 Pol III启动子、U1 Pol II启动子和一种新型的HIV Tat诱导启动子系统的多靶向前体si和shRNAs的表达和抗HIV的有效性；3)SCID-Hu小鼠研究。来自特定目标1的组合构建物将被转化为CD34+造血祖细胞，用于在干细胞环境中进行抗病毒评估。单元格将是 在允许体外分化为单核细胞和巨噬细胞的条件下培养。表达组合si/shRNAs的细胞在体外形成红系和髓系集落的能力将与模拟转导和载体骨架转导的细胞进行比较。用组合结构转导的CD34+细胞也将被注入THY/Liv SCID-Hu小鼠。T淋巴细胞的发育将受到监测，并与载体转导细胞进行比较。将进行胸腺内HIV-1挑战，以确定 活体环境中的组合构造。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的强大细胞内免疫。