Expression of anti-HIV siRNA in blood cells.

描述(申请人提供)：RNAi是一种强大的细胞机制，它使用短RNA来靶向破坏同源转录。这是一种古老的机制，存在于从真菌、植物到哺乳动物的各种有机体中。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座。在体内，这个过程是由21到23个带有2个碱基3‘突起的核苷酸双链启动的，这些双链是由一种名为Dice的酶从较长的RNA中产生的。这些短双链被称为siRNAs，它们与一种名为RNA诱导沉默复合体(RISC)的蛋白质复合体相关，该复合体将双链的单链定向到互补的靶序列，导致靶序列的降解。几个研究小组最近报告了使用合成的siRNA或细胞内表达的siRNA对艾滋病毒感染的显著抑制作用。RNAi似乎是一种强大的靶向特异性抑制机制，在HIV-1感染的治疗中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siRNAs可以有效地阻断初级淋巴细胞中的HIV感染。该项目的总体目标是验证siRNA可以作为一种抗HIV-1试剂用于造血干细胞基因治疗的假设。本研究的重点是从慢病毒载体骨架中优化siRNA的功能和表达。将测试将siRNAs运送到细胞质的新表达系统，以及siRNAs可以通过DNA甲基化来指导基因沉默的可能性。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。建议的作用机制研究将允许开发在人类基因治疗中部署siRNAs的最有效的策略。最后，将测试siRNAs和其他基于RNA的治疗药物的联合使用，以增强疗效并将病毒耐药性降至最低。潜在毒性的问题将在长期骨髓细胞培养中进行测试，并通过与Alice Tarantal博士合作在胚胎恒河猴身上进行测试来进行测试。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中优化抗HIV siRNAs的表达。2)检测抗HIV siRNAs的作用机制。3)评价siRNAs和siRNAs与抗HIV核酶和诱骗的组合。这些研究的长期目标是开发一种基于siRNA的疗法，用于在基因疗法的背景下治疗艾滋病毒-1感染，利用该计划为基于核酶的基因疗法开发的基础设施。

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.