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

项目总结 联合抗逆转录病毒疗法(CART)在临床实践中的引入，改变了传统的管理方式。 控制HIV-1感染，实现对病毒复制的完全抑制。然而，CART并不针对 潜伏的前病毒或窝藏它们的细胞，必须每天服用。开发一种治疗艾滋病毒的方法-- 1感染将消除购物车的毒性，并解决将购物车运送到 全世界有超过3000万名患者，其中大部分在发展中国家。尽管在这方面做了很大的努力- 在过去十年中，我们仍远未找到有效的治疗方法，这突显出迫切需要制定新的战略。 我们认为，无购物车的HIV-1缓解更有可能是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发展一种“阻断并锁定”的方法 沉默HIV-1是序列特异的、安全、有效和可在世界各地推广的。这种方法 应旨在以类似于永久沉默的方式诱导艾滋病毒-1的不可逆转的表观遗传失活 我们人类基因组中的组织特异性基因和内源性逆转录元件。 我们的总体目标是实现对潜伏的HIV-1前病毒的强大、持久、特定序列的沉默 导致它们的转录永久失活。我们的中心假设是一种外源基因的表达 自然产生的HIV-1反义转录本(Ast)将导致潜伏的HIV-1序列特异性沉默。我们 已有报道称，Ast RNA通过与同源DNA序列结合，天然地损害HIV-1的表达 在前病毒5‘LTR中，通过碱基配对和招募多梳抑制物复合体2(PRC2)，它 将抑制性表观遗传标记H3K27me3引入周围的染色质，关闭HIV-1的表达- 西翁。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- 选择性因素，表明该转录本协调了HIV-1沉默的多种机制。 我们建议通过三个具体目标(SA)来实现我们的目标。SA1将调查对AST的修改 RNA，和其他表观遗传抑制物，AST招募到HIV-15 5‘LTR。这些研究将导致开发- 使Ast衍生物具有更好的效力。SA2将评估Ast RNA及其新衍生物的能力 在三个体外生物学相关的原代细胞中诱导HIV-1的序列特异性沉默 模特们。这些研究代表了对Ast及其衍生品的第一级测试，为 SA1研究并选择Ast衍生物进行进一步测试。SA3将评估Ast RNA和其 在SA2中选择的衍生物在重组的活体人源化小鼠模型中诱导HIV-1沉默 HIV-1患者外周血中的CD4+T细胞。这些研究是对Ast RNA的第二个也是更复杂的测试水平。 这些研究的完成将提供概念证明，可能推动Ast RNA的发展 一种治疗HIV-1病毒的新的、有效的和序列特异性的疗法。虽然大多数沉默策略都有广泛的 对可能伴随毒性的基因表达的影响，Ast RNA将允许发起靶向、 针对潜伏的前病毒的特定序列打击，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.