Sustained HIV Remission via Sequence-Specific Epigenetic Silencing of Latent Proviruses

项目摘要 联合抗逆转录病毒治疗（cART）在临床实践中的引入改变了管理， HIV-1感染，达到完全抑制病毒复制。然而，cART并不针对 潜伏的前病毒或细胞窝藏他们，必须每天采取的生活。艾滋病治疗方法的发展- 1例感染将消除cART毒性，并解决向患者提供cART的经济和后勤负担。 全世界有3000多万患者，其中大多数在发展中国家。尽管在这方面做出了巨大努力， 过去十年来，我们仍然远远没有找到有效的解决办法，这突出表明迫切需要制定新的战略。 我们认为，无cART的HIV-1缓解更有可能通过将潜伏的前病毒限制在更深的 和永久的延迟状态。我们的长期目标是通过表观遗传学开发一种“封锁和锁定”的方法， 沉默HIV-1是序列特异性的，安全的，有效的，并可在世界各地扩展。这种方法 应该旨在以类似于永久沉默的方式诱导HIV-1的不可逆表观遗传失活 我们人类基因组中的组织特异性基因和内源性逆转录因子。 我们的总体目标是实现对潜伏的HIV-1前病毒的强有力的、持久的、序列特异性沉默， 导致其永久转录失活。我们的中心假设是， 天然存在的HIV-1反义转录物（Ast）将导致潜伏HIV-1的序列特异性沉默。我们 已经报道了Ast RNA通过与同源DNA序列结合而天然地损害HIV-1的表达 通过碱基配对和募集多梳阻遏复合物2（PRC 2）， 将抑制性表观遗传标记H3 K27 me 3引入周围的染色质，关闭HIV-1表达， 锡永。此外，我们最近的研究表明，Ast RNA结合了额外的抑制性表观遗传和转录， 这表明这种转录物协调了HIV-1沉默的多种机制。 我们通过三个具体目标（SA）来实现我们的目标。SA 1将研究Ast的修改 RNA，以及Ast招募到HIV-1 5 'LTR的其他表观遗传阻遏物。这些研究将导致发展- 使Ast衍生物具有改进的效力。SA 2将评估Ast RNA及其新衍生物的能力， 在三种体外和离体生物学相关原代细胞中诱导HIV-1的序列特异性沉默 模型这些研究代表了Ast及其衍生物的第一级测试，为以下方面提供了有用的反馈： SA 1研究和选择Ast衍生物用于进一步测试。SA 3将评估Ast RNA的能力及其在细胞中的表达。 在SA 2中选择的衍生物，以在用以下重建的体内人源化小鼠模型中诱导HIV-1沉默： 来自HIV-1患者的CD 4 + T细胞。这些研究是对Ast RNA进行的第二层次且更复杂的测试。 这些研究的完成将提供概念验证，可以推动Ast RNA的开发， 一种新的、有效的和序列特异性的治疗HIV-1的药物。虽然大多数沉默策略具有广泛的 对基因表达的影响可能伴随着毒性，Ast RNA将允许启动靶向， 序列特异性打击潜伏的前病毒，避免显著的毒性脱靶效应。

PROJECT SUMMARY The introduction of combination antiretroviral therapy (cART) in clinical practice has transformed the manage- ment of HIV-1 infection, achieving complete suppression of viral replication. However, cART does not target latent proviruses or the cells harboring them, and must be taken daily for life. The development of a cure for HIV- 1 infection would eliminate cART toxicities, and solve the economic and logistic burden of delivering cART to over 30 million patients worldwide, most of them in the developing world. Despite significant efforts in that direc- tion over the last decade, we are still far from an effective cure, underscoring the urgent need for new strategies. We argue that cART-free HIV-1 remission is more likely achieved by constraining latent proviruses into a deeper and permanent state of latency. Our long-term goal is to develop a “block and lock” approach via epigenetic silencing of HIV-1 that is sequence-specific, safe, effective, and scalable throughout the world. This approach should aim at inducing irreversible epigenetic inactivation of HIV-1, in a manner similar to permanent silencing of tissue-specific genes and endogenous retroelements within our human genomes. Our overall objective is to achieve robust, durable, sequence-specific silencing of latent HIV-1 proviruses that leads to their permanent transcriptional inactivation. Our central hypothesis is that exogenous expression of a naturally occurring HIV-1 antisense transcript (Ast) will lead to sequence-specific silencing of latent HIV-1. We have reported that the Ast RNA naturally impairs HIV-1 expression by binding to homologous DNA sequences in the proviral 5’LTR through base pairing and by recruiting the Polycomb Repressor Complex 2 (PRC2), which introduces the repressive epigenetic mark, H3K27me3 into the surrounding chromatin, turning off HIV-1 expres- sion. In addition, our recent studies show that the Ast RNA binds additional repressive epigenetic and transcrip- tional factors, suggesting that this transcript orchestrates multiple mechanisms of HIV-1 silencing. We propose to achieve our goal through three Specific Aims (SA). SA1 will investigate modifications of the Ast RNA, and additional epigenetic repressors that Ast recruits to the HIV-1 5’LTR. These studies will lead to devel- oping Ast derivatives with improved potency. SA2 will evaluate the ability of the Ast RNA and its new derivatives to induce sequence-specific silencing of HIV-1 in three in vitro and ex vivo biologically relevant primary cell models. These studies represent the first level of testing for Ast and its derivatives, providing helpful feedback to SA1 studies and selecting Ast derivatives for further testing. SA3 will evaluate the ability of Ast RNA and its derivatives selected in SA2 to induce HIV-1 silencing in an in vivo humanized mouse model reconstituted with CD4+ T cells from HIV-1 patients. These studies are a second and more complex level of testing for Ast RNA. Completion of these studies will provide proof-of-concept that could propel the development of the Ast RNA into a novel, potent and sequence-specific therapeutic for HIV-1 cure. While most silencing strategies have a broad impact on gene expression that is likely accompanied by toxicity, the Ast RNA will allow to launch a targeted, sequence-specific strike against latent proviruses, avoiding significant toxic off-target effects.