Identification of drugs that induce terminal transcriptional silencing of latent HIV-1 infection

越来越清楚的是，目前推行的HIV-1再激活策略可能不会导致 在治疗性HIV-1根除中，需要探索替代根除策略。我们在此提议， 开发针对潜伏HIV-1感染事件的末端转录沉默（TTS）策略。TTS 这些策略在逆转录/慢病毒沉默中具有其生物等效物。然而，TTS不会针对 新的感染事件，但必须在没有病毒感染的情况下转录沉默预先存在的潜伏前病毒。 蛋白质，主要是RNA或非整合的DNA，通常触发抗病毒细胞反应。证据 特异性靶向慢病毒感染的细胞先天免疫机制的存在是由以下提供的： 慢病毒沉默是基于慢病毒的基因治疗的主要障碍。先天的存在 细胞防御机制，可以控制预先存在的病毒风格的基因组表达的建议， 防御机制的存在，以对抗一个相关的，进化的古老类别的“基因组入侵者”，称为 反转录转座子这些防御机制各自进化，以应对每个不同的 逆转录转座子类。不幸的是，由于这种特异性， 逆转录转座子来抑制它们的基因组传播，实际上对HIV-1是无效的。TTS的特异性 针对HIV-1感染的策略是至关重要的，因为除了对任何药物毒性问题的一般考虑之外， 药物干预，TTS策略不会导致（i）对先天控制机制的任何干扰， 抑制反转录转座子活性或引起（ii）对控制表观遗传的机制的任何干扰 人类细胞中基因的沉默。对于R61阶段，我们提出了一种迭代方法， 具有药理学扰动的内容分析方法（ATAC-seq、RNA-seq、激酶组阵列分析） 筛选，以确定药物的目标，特别是控制细胞先天抗病毒反应的HIV-1感染。 拟议的研究将利用（i）预先存在的大量HIV-1报告细胞，（ii） 发现某些临床HIV-1，特别是HIV-2菌株引起更强的先天性TTS， 反应比常用的实验室适应的HIV-1克隆，和（iii）发现巨噬细胞是 比T细胞更有效地执行TTS。R61阶段的可交付成果是：（1） 鉴定可药物化靶点，如果药物化靶点被处理，则可在潜伏HIV-1感染试验中实现TTS 细胞和（2）用于鉴定针对这些细胞的TTS诱导药物的HTS相容性药物筛选测定 目标的在R33阶段，我们将进行实际的药物筛选，以确定TTS诱导化合物。 将在潜伏感染的原代细胞模型和细胞材料中测试鉴定的候选化合物 来自HIV-1患者。到R33阶段结束时，我们预计将在临床上确定至少一种 包括一种或多种药物的相关TTS策略。

As it is becoming increasingly clear that currently pursued HIV-1 reactivating strategies may not result in therapeutic HIV-1 eradication, alternative eradication strategies need to be explored. We here propose to develop terminal transcriptional silencing (TTS) strategies specific for latent HIV-1 infection events. TTS strategies would have their biological equivalent in retro/lentiviral silencing. However, TTS would not target de novo infection events, but have to transcriptionally silence pre-existing, latent provirus in the absence of viral proteins, mostly RNA or non-integrated DNA that usually trigger the antiviral cellular response. Evidence for the existence of cellular innate immune mechanisms that specifically target lentiviral infection is provided by the fact that lentiviral silencing is a major roadblock for lentiviral-based gene therapy. The existence of innate cellular defense mechanisms that can control pre-existing virus-style genome expression is suggested by the existence of defense mechanisms against a related, evolutionary ancient class of “genomic intruders” called retrotransposons. These defense mechanisms individually evolved in response to each different retrotransposon class. Unfortunately, because of this specificity, mechanisms that continuously actively control retrotransposons to suppress their genomic spread, actually are inactive against HIV-1. Specificity of TTS strategies against HIV-1 infection is essential as, beyond the general consideration of toxicity issues of any drug intervention, TTS strategies cannot cause (i) any interference with innate control mechanisms that suppress retrotransposon activity or cause (ii) any interference with mechanisms that control epigenetic silencing of genes in human cells. For the R61 phase, we propose an iterative approach that combines high content analysis methods (ATAC-seq, RNA-seq, kinome array analysis) with pharmacological perturbation screens to identify drug targets that specifically control the cellular innate antiviral response to HIV-1 infection. The proposed research will take advantage of (i) a preexisting, large selection of HIV-1 reporter cells, (ii) the finding that certain clinical HIV-1 and in particular HIV-2 strains cause a much more potent innate TTS response than the commonly used laboratory adapted HIV-1 clones, and (iii) the finding that macrophages are much more efficient in executing TTS than T cells. The deliverable of the R61 phase will be (1) the identification of druggable targets that if pharmacologically addressed, enable TTS in latently HIV-1 infected T cells and (2) HTS-compatible drug screening assays for the identification of TTS-inducing drugs against these targets. In the R33 phase, we will perform the actual drug screens to identify TTS-inducing compounds. Identified candidate compounds would be tested in primary cell models of latent infection and cell material derived from HIV-1 patients. By the end of the R33 phase we expect to have identified at least one clinically relevant TTS strategy comprising of one or several drugs.