Harnessing the PIWI piRNA pathway to silence HIV

项目概要 很明显，cART 治疗的 HIV 感染者中潜伏感染的 CD4 T 细胞是主要的储存库 和根除的障碍。本研究提案特别旨在解决题为“沉默”的 FOA HIV-1 Proviruses”，并由 FOA 提出以下具体目标：“利用 PIWI RNA 抑制艾滋病毒的途径”。与其他小非编码 RNA 沉默途径（siRNA、shRNA、 miRNA），piRNA途径具有通过表观遗传永久沉默基因的理论优势 除了 mRNA 降解活性外，修饰（H3K9 甲基化）也是主要模式 其他小RNA的沉默，从而确保子代细胞中的基因沉默。我们的初步调查结果 表明 PIWI 蛋白在 CD4 T 细胞和 T 细胞系中表达，并且似乎受到 免疫激活。然而，尚不清楚 PIWI-piRNA 通路是否可以被诱导执行 体细胞中的功能与生殖细胞中的功能相似。我们假设 piRNA-PIWI 途径是 在原代 CD4 T 细胞中发挥作用，并且可以被操纵来沉默 HIV。将努力确保 该项目的成功是由基础小非编码 RNA 和 piRNA 生物学领域的专家组成的团队 艾滋病毒专家，他们都在各自的领域拥有长期的记录。在提案的 R66 部分，我们将测试证明 一个概念是，可以在 ACH-2 细胞中诱导针对 HIV 基因的 piRNA 途径，因此，当它们 被细胞因子激活后，它们就停止产生 HIV 病毒粒子。如果成功，R33 部分 该提案将进一步表征 ACH-2 piRNA，这些 ACH-2 piRNA 具有针对广泛的 HIV 毒株的活性， 包括取自 cART 治疗个体的离体原代细胞内的交叉进化枝。作为临床前研究的一部分 将探索开发靶向CD4受体的嵌合适体-piRNA策略。目前的提案 将进一步揭示 piRNA 通路在人类细胞中的作用，以及它们是否可以被用作 HIV 沉默策略作为 HIV 治愈疗法的一部分。

PROJECT SUMMARY It is clear that latently infected CD4+ T cells in cART treated HIV-infected individuals are the major reservoir and obstacle to eradication. This research proposal specifically intends to address the FOA entitled “Silencing of HIV-1 Proviruses” with the following specific objective proposed by the FOA: “ to harness the PIWI RNA pathway to silence HIV”. In contrast to other small non-coding RNA silencing pathways (siRNA, shRNA, miRNA), the piRNA pathway has the theoretical advantage of permanently silencing genes through epigenetic modifications (H3K9 methylation) in addition to mRNA degradation activity, the latter being the primary mode of silencing by other small RNAs, thus, ensuring gene silencing in progeny cells. Our preliminary findings indicate that PIWI proteins are expressed in CD4+ T cells and T cell lines, and appear to be regulated by immune activation. It is unknown, however whether the PIWI-piRNA pathway can be induced to perform similar functions in somatic cells as they do in germ cells. We hypothesize that the piRNA-PIWI pathway is functional in primary CD4+ T-cells and can be manipulated to silence HIV. Attempts will be made to ensure success of the project by teaming experts in fundamental small noncoding RNA and piRNA biology with an HIV expert, all having long track records in their fields. In the R66 portion of the proposal we will test the proof of concept that that one can induce a piRNA pathway against HIV genes in ACH-2 cells, so that, when they are activated by cytokine, they have ceased to produce HIV virions. If successful, the R33 portion of the proposal will further characterize ACH-2 piRNAs that have activity against a broad range of HIV strains, including cross Clades within ex vivo primary cells taken from cART treated individuals. As part of pre-clinical development a CD4 receptor targeting chimeric aptamer-piRNA strategy will be explored. The current proposal will reveal further insights on the role of the piRNA pathway in human cells, whether they can be exploited as an HIV silencing strategy as part of the armamentarium toward HIV cure therapy.