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) 是一种强大的新工具，用于序列特异性敲低目标 mRNA。 RNAi 中的活性“触发器”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有义和反义或短发夹 RNA (shRNA)。在我们手中，RNAi 是我们所使用的最强大的 HIV 抑制机制，导致细胞培养中 HIV-1 编码的 p24 抗原的抑制超过 4 对数。尽管 RNAi 具有效力， HIV-1 的突变可以破坏 siRNA 反义链与靶标的碱基配对相互作用，从而避免这种情况。因为最好的抗 HIV-1 疗法结合了组合药物，以最大限度地提高效力并最大限度地减少耐药性。在本提案中，我们将测试以下假设：针对 HIV-1 的 si/shRNA 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。编码针对多个 HIV-1 靶点和细胞的多种不同 siRNA 或 shRNA 的前体转录本 CCR5 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，在 RNAi 中发挥作用。该构建体将通过慢病毒载体转导被递送至造血细胞。我们已经在原代造血细胞中建立了单个 si/shRNA 的稳健表达，从而有效抑制 HIV-1 复制。预计组合转录本将在基因治疗环境中提供针对 HIV-1 感染的持续保护。拟议的研究将测试几种不同的 si/shRNA 组合表达方法。组合构建体将在体外和 SCID-hu 小鼠模型的造血背景下进行测试。这里开发的有效构建体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。该项目的具体目标是： 1) 组合 siRNA 基因的构建和测试 - 针对 HIV-1 多个位点的小干扰 RNA (siRNA) 或短发夹 RNA (shRNA) 的组合 CCR5共受体将从单个转录物共表达；2)测试用于表达组合siRNA构建体的Pol III与Pol II启动子系统-我们将比较多靶向前体si和的表达和抗HIV功效使用 U6 Pol III 启动子、U1 Pol II 启动子和新型 HIV Tat 诱导型启动子系统的 shRNA； 3）SCID-hu小鼠研究。来自特定目标 1 的组合构建体将被转导至 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培养。将表达组合si/shRNA的细胞离体形成红细胞和骨髓细胞集落的能力与模拟转导细胞和载体主链转导细胞进行比较。用组合构建体转导的 CD34+ 细胞也将被输注到 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 挑战以确定以下药物的抗 HIV 功效： 体内环境中的组合构建体。这项研究的长期目标是在 T 细胞和造血干细胞环境中提供针对 HIV-1 的有效细胞内免疫。