Expression of anti-HIV siRNA in blood cells.

血细胞中抗 HIV siRNA 的表达。

• 批准号: 6765938
• 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/6765938
• 关键词: DNA methylation; Lentivirus; RNA interference; Retroviridae; antiAIDS agent; biotechnology; 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; small interfering RNA; 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是一种强大的蜂窝机制，它使用短RNA来靶向同源转录物进行破坏。这是一种在从真菌和植物到哺乳动物的生物中发现的古老机制。它很可能是一种原始的先天免疫反应机制，该机制可以保护生物免受病毒感染和运动的重新转移。该过程由21至23个核苷酸双链体在体内启动，其中2个碱基3'悬垂物是由一种称为dicer的酶从较长的RNA产生的。 这些简短的双链体称为siRNA，它们与称为RNA诱导的沉默复合物或RISC的蛋白质复合物相关联，该复合物将双链体的一条线引导至互补靶序列，从而导致靶标降解。 几个研究组最近报道了使用合成siRNA或细胞内表达的siRNA对HIV感染的令人印象深刻的抑制作用。 RNAi似乎是一种有力的特异性抑制机制，具有潜在的HIV-1感染治疗治疗。本研究利用了非常令人兴奋和有希望的初步发现，这些发现证明了载体驱动的siRNA可以有效阻断原发性淋巴细胞中的HIV感染。该项目的总体目标是检验siRNA可以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对慢病毒载体骨架的siRNA功能和表达的优化。将测试将siRNA传递到细胞细胞质的新表达系统，并可能使用siRNA可用于通过DNA甲基化引导基因沉默。这些抑制分子将针对HIV-1，SHIV中的共同序列和细胞共受体CCR5。 提出了行动研究机制，它将允许发展人类基因治疗中siRNA的最有效策略。最后，将测试siRNA和其他基于RNA的治疗剂的联合使用，以增强病毒抗性的功效和最小化。潜在毒性的问题将在长期的骨髓细胞培养中进行测试，并通过与爱丽丝·塔兰塔尔（Alice Tarantal）博士合作进行胎儿恒河猴进行测试。该项目的具体目的如下， 1）优化慢病毒和逆转录病毒载体骨架的抗HIV siRNA的表达。2）测试抗HIV siRNAS的作用机理。3）评估siRNA和siRNAS和siRNAS组合结合抗HIV核酶和诱饵。这些研究的远距离目标是开发一种基于siRNA的治疗，用于在基因疗法的设置中利用该计划为基于核酶基因疗法开发的基础设施的基因疗法中的治疗。