Conditionally replicating vectors and the parasitism of HIV-1

项目摘要 HIV-1通过其整合和持续存在于受感染细胞基因组中的能力，仍然是对全球健康的持续威胁。目前的治疗策略有效地减少了HIV-1的复制并提高了生活质量，然而，病毒变异的存在允许致病性变异的持续存在和最终恢复。 这些变异是HIV-1稳定整合的必然结果，这使得耐药菌株的自然选择和进化成为可能。有趣的是，整合的HIV可以为条件复制载体的繁殖和传播提供理想的环境。我们已经发现，当条件复制载体含有表达靶向于 在LTR的转录活性区域中，对病毒施加了额外的长期表观遗传介导的选择压力。观察到的选择压力与载体的持久性和病毒的显著转录抑制相关，导致病毒适应性降低。此外，我们将利用这些验证的小非编码RNA和最近开发的小RNA靶向基因切除 复合物从人类细胞中切除HIV-1或CCR 5片段，以永久改变病毒和OCRS共受体表达。在这个项目中，我们将开发条件复制载体，既可以靶向HIV-1或CCR 5进行抑制和/或切除，同时劫持病毒机制， 将抗HIV-1载体传播到其他感染HIV-1的细胞中。从本质上讲，我们希望对HIV-1设置一个囚徒困境，并调节病毒的适应性（Morris 2004; Morris and Looney 2005）。我们假设，观察到的HIV-1的长期抑制可以通过这种选择性压力直接调节，并提出通过（1）利用可选择的条件复制HIV-2载体，（2）确定最佳抑制性载体以及靶向HIV或CCR 5和/或靶向基因座的小RNA的数量是否重要，以及（3）表征相应的长期抑制性和/或靶向基因座的表达。或最佳候选载体在人T细胞和巨噬细胞中的切除特性。

PROJECT SUMMARY HIV-1 remains a constant threat to global health through its ability to integrate and persist in the genome of infected cells. Current therapeutic strategies effectively reduce HIV-1 replication and improve quality of life, however, existence of viral variants allows for the persistence and eventual recovery of pathogenic variants. These variants are the inevitable outcome of the stable integration of HIV-1, which allows for the natural selection and evolution of drug resistant strains. Interestingly, integrated HIV can provide an ideal environment for the propagation and dissemination of conditionally replicating vectors. We have found that when conditionally replicating vectors contain antiviral genes expressing small non-coding RNAs targeted to transcriptionally active regions of the LTR, an added long-term epigenetic mediated selective pressure is placed on the virus. The observed selective pressure correlates with both the persistence of the vector and significant transcriptional suppression of the virus resulting in reduced viral fitness. Moreover, we will utilize these validated small non-coding RNAs and a recently developed small RNA targeted gene excision complex to excise fragments of HIV-1 or CCR5 from human cells in an effort to permanently alter viral and OCRS co-receptor expression. In this project we will develop conditionally replicating vectors that can both target HIV-1 or CCR5 for suppression and/or excision while simultaneously hijacking the viral machinery to spread the anti-HIV-1 vector to other cells being infected with HIV-1. In essence we wish to place a prisoners dilemma on HIV-1 and modulate viral fitness (Morris 2004; Morris and Looney 2005). We hypothesize that the observed long-term suppression of HIV-1 can be directly regulated by such selective pressures and propose to test this hypothesis by (1) utilizing selectable conditionally replicating HIV-2 vectors, (2) determining the best suppressive vector and whether the number of small RNAs targeted to HIV or CCR5 and/or the targeted loci is important and (3) characterizing the respective long-term suppressive and/or excision properties of the best candidate vectors in human T cells and macrophages.