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

随着越来越清楚的是，目前推行的HIV-1重新激活策略可能不会产生结果 在治疗性根除艾滋病毒-1方面，需要探索替代根除战略。我们在此提议 制定针对潜伏的HIV-1感染事件的末端转录沉默(TTS)策略。TTS 在逆转/慢病毒沉默方面，这些策略将具有生物学上的等价性。然而，TTS不会以De为目标 Novo感染事件，但在没有病毒的情况下必须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，通常会触发抗病毒细胞反应。证据： 针对慢病毒感染的细胞先天免疫机制的存在是由 慢病毒沉默是慢病毒基因治疗的主要障碍。与生俱来的存在 可以控制预先存在的病毒风格的基因组表达的细胞防御机制由 对一种相关的、进化的古代“基因组入侵者”的防御机制的存在，称为 反转录转座子。这些防御机制分别进化为对每个不同的 反转录转座子类。不幸的是，由于这种特殊性，持续主动控制的机制 用来抑制基因组传播的反转录转座子实际上对HIV-1没有作用。TTS的特异性 预防艾滋病毒-1感染的战略是至关重要的，因为除了对任何 药物干预，TTS策略不能造成(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 人类细胞中的基因沉默。对于R61阶段，我们提出了一种结合HIGH的迭代方法 具有药理扰动的含量分析方法(ATAC-SEQ、RNA-SEQ、基因芯片分析) 筛选特定控制细胞对HIV-1感染的固有抗病毒反应的药物靶点。 拟议的研究将利用(I)预先存在的大量HIV-1报告细胞，(Ii) 发现某些临床上的HIV-1，特别是HIV-2毒株会导致更强的先天TTS 反应比常用的实验室适应的HIV-1克隆更好，以及(Iii)发现巨噬细胞 在执行TTS方面比T细胞效率高得多。R61阶段的交付成果将是(1) 确定药物靶点，如果药理处理，使TTS在潜伏的HIV-1感染T细胞 细胞和(2)HTS相容药物筛选试验用于TTS诱导药物的鉴定 目标。在R33阶段，我们将进行实际的药物筛选，以确定TTS诱导的化合物。 确定的候选化合物将在潜伏感染的原代细胞模型和细胞材料中进行测试 来自HIV-1患者。到R33期结束时，我们预计已经确定了至少一个临床上 由一种或多种药物组成的相关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.