Expression of anti-HIV siRNA in blood cells.

描述（由申请人提供）：RNAi是一种强大的细胞机制，其使用短RNA靶向同源转录物进行破坏。这是一种古老的机制，存在于从真菌、植物到哺乳动物的生物体中。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，所述双链体由称为Dicer的酶从较长的RNA产生。 这些短的双链体被称为siRNA，它们与称为RNA诱导沉默复合物或RISC的蛋白质复合物结合，该蛋白质复合物将双链体的单链引导至互补靶序列，导致靶的降解。 几个研究小组最近报道了使用合成的siRNA或细胞内表达的siRNA对HIV感染的令人印象深刻的抑制。 RNAi似乎是一种强大的靶特异性抑制机制，在HIV-1感染的治疗性治疗中具有潜在的应用。目前的研究利用了非常令人兴奋和有前途的初步发现，证明载体驱动的siRNA可以有效地阻断HIV感染的初级淋巴细胞。本项目的总体目标是验证siRNA可用作造血干细胞基因治疗中的抗HIV-1药物的假设。 该方案的研究重点是siRNA功能的优化和慢病毒载体骨架的表达。将测试将siRNA递送到细胞质的新表达系统，沿着siRNA可用于通过DNA甲基化指导基因沉默的可能性。这些抑制性分子将靶向HIV-1、SHIV中的常见序列和细胞辅助受体CCR 5。 提出了作用机制研究，这将允许开发用于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的治疗剂的组合使用以增强功效和最小化病毒抗性。潜在毒性问题将在长期骨髓细胞培养中进行测试，并通过与Alice Tarantal博士合作在胎猴中进行测试。该项目的具体目标如下， 1)从慢病毒和逆转录病毒载体骨架优化抗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核酶和诱饵组合的组合。这些研究的长期目标是开发一种基于siRNA的治疗方法，用于在基因治疗环境中治疗HIV-1感染，利用该计划为基于核酶的基因治疗开发的基础设施。

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.