Expression of anti-HIV siRNA in blood cells.

血细胞中抗 HIV siRNA 的表达。

• 批准号: 6696102
• 负责人: John Joseph Rossi
• 金额: $ 43.75万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6696102
• 关键词: DNA methylation; Lentivirus; RNA; Retroviridae; antiAIDS agent; bone marrow; cytogenetics; gene induction /repression; gene therapy; genetic promoter element; human immunodeficiency virus 1; lymphocyte; nucleic acid chemical synthesis; nucleic acid inhibitor; nucleolus; ribozymes; synthetic nucleic acid; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): RNAi is a powerful cellular mechanism that uses short RNAs to target homologous transcripts for destruction. This is an ancient mechanism found in organisms ranging from fungi and plants to mammals. It most likely is a primitive innate immune response mechanism that can protect organisms from viral infection and retro-transpose on movement. The process is initiated in vivo by 21 to 23 nucleotide duplexes with 2 base 3' overhangs that are generated from longer RNAs by an enzyme called Dicer. These short duplexes are termed siRNAs, and they become associated with a protein complex called the RNA induced silencing complex, or RISC, that directs a single strand of the duplex to a complementary target sequence, resulting in degradation of the target. Several investigative groups have recently reported impressive inhibition of HIV infection using synthetic siRNAs or intracellular expressed siRNAs. RNAi appears to be a powerful, target specific inhibitory mechanism that has potential application for the therapeutic treatment of HIV-1 infection. The present study capitalizes upon very exciting and promising preliminary findings that demonstrate vector driven siRNAs can effectively block HIV infection in primary lymphocytes. The overall goal of this project is to test the hypothesis that siRNA can be used as an anti-HIV-1 agent in hematopoietic stem cell gene therapy. The research focus of this proposal is the optimization of siRNA function and expression from lentiviral vector backbones. New expression systems that deliver siRNAs to the cellular cytoplasm will be tested, along with the possibility that siRNAs can be used to direct gene silencing through DNA methylation. These inhibitory molecules will be targeted to HIV-1, common sequences in SHIV, and the cellular co-receptor CCR5. Mechanisms of action studies are proposed that will allow the development of the most efficacious strategies for deployment of siRNAs in human gene therapy. Finally, the combined use of siRNAs and other RNA based therapeutics will be tested for enhanced efficacy and minimization of viral resistance. The issue of potential toxicity will be tested in long-term bone marrow cell culture and via testing in fetal Rhesus monkeys in collaboration with Dr. Alice Tarantal in an accompanying proposal. The specific aims of this project are as follows, 1) Optimizing expression of anti-HIV siRNAs from lentiviral and retroviral vector backbones.2) Testing the mechanism of action of anti-HIV siRNAs.3) Evaluation of combinations of siRNAs and siRNAs combined with anti-HIV ribozymes and decoys. The long range goal of these studies is to develop an siRNA based therapy for the treatment of HIV-1 infection in a gene therapy setting capitalizing on the infrastructure this program has developed for ribozyme based gene therapy.

描述（由申请人提供）：RNAi 是一种强大的细胞机制，它使用短 RNA 来靶向同源转录物以进行破坏。这是在从真菌、植物到哺乳动物的各种生物体中发现的一种古老机制。它很可能是一种原始的先天免疫反应机制，可以保护生物体免受病毒感染和运动时的逆转录转座。该过程在体内由具有 2 个碱基 3' 突出端的 21 至 23 个核苷酸双链体启动，这些双链体是由称为 Dicer 的酶从较长的 RNA 中生成的。 这些短双链体被称为 siRNA，它们与一种称为 RNA 诱导沉默复合物 (RISC) 的蛋白质复合物相关联，该复合物将双链体的单链引导至互补的靶序列，从而导致靶标降解。 几个研究小组最近报告了使用合成 siRNA 或细胞内表达 siRNA 对 HIV 感染的令人印象深刻的抑制作用。 RNAi 似乎是一种强大的、目标特异性的抑制机制，在治疗 HIV-1 感染方面具有潜在的应用前景。本研究利用了非常令人兴奋和有希望的初步发现，证明载体驱动的 siRNA 可以有效阻止原代淋巴细胞中的 HIV 感染。该项目的总体目标是检验 siRNA 可用作造血干细胞基因治疗中的抗 HIV-1 药物的假设。 该提案的研究重点是慢病毒载体骨架的 siRNA 功能和表达的优化。将测试将 siRNA 传递至细胞质的新表达系统，以及 siRNA 可用于通过 DNA 甲基化指导基因沉默的可能性。这些抑制分子将针对 HIV-1、SHIV 中的常见序列和细胞辅助受体 CCR5。 提出的作用机制研究将有助于开发在人类基因治疗中部署 siRNA 的最有效策略。最后，将测试 siRNA 和其他基于 RNA 的疗法的联合使用，以增强疗效并最大限度地减少病毒耐药性。潜在毒性问题将在长期骨髓细胞培养中进行测试，并在随附的提案中与 Alice Tarantal 博士合作，通过对恒河猴胎儿进行测试。该项目的具体目标如下： 1) 优化慢病毒和逆转录病毒载体主链中抗HIV siRNA的表达。2) 测试抗HIV siRNA的作用机制。3) 评估siRNA和siRNA与抗HIV核酶和诱饵结合的组合。这些研究的长期目标是开发一种基于 siRNA 的疗法，用于在基因治疗环境中治疗 HIV-1 感染，利用该项目为基于核酶的基因疗法开发的基础设施。