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

诱导潜伏 HIV-1 感染末端转录沉默的药物的鉴定

• 批准号: 9393866
• 负责人: OLAF KUTSCH
• 金额: $ 65.4万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9393866
• 关键词: ATAC-seq; Address; Algorithms; Antibodies; Antiviral Agents; Antiviral Response; Binding Sites; Biological; Biological Assay; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Catalogs; Cell Line; Cell model; Cells; Chemicals; Clinical; Clone Cells; Complement; DNA; Data; Databases; Defense Mechanisms; Development; Diversity Library; Drug Combinations; Drug Targeting; Elements; Epigenetic Process; Event; Funding; Gene Silencing; Generations; Genetic Transcription; Genome; Genomics; HIV; HIV-1; HIV-2; Human; Immune; Individual; Infection; Intervention; Laboratories; Lead; Libraries; Methods; Molecular; Pathway interactions; Patients; Pattern recognition receptor; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Preclinical Drug Evaluation; Process; Proteins; Proviruses; PubChem; RNA; Reporter; Research; Retrotransposon; Robotics; Severe Acute Respiratory Syndrome; Signal Transduction; Specificity; T-Lymphocyte; TLR3 gene; Testing; Therapeutic; Toxic effect; Viral; Viral Proteins; Viral reservoir; Virus; Virus Diseases; active control; analog; antiretroviral therapy; base; chemical synthesis; clinical translation; clinically relevant; combinatorial; cost; gene therapy; genetic manipulation; genome-wide; high throughput screening; latent infection; lead series; macrophage; overexpression; response; scaffold; screening; small hairpin RNA; small molecule; transcription factor; transcriptome sequencing

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.

因为越来越清楚的是，目前追求HIV-1重新激活策略可能不会导致 在热HIV-1辐射中，需要探索替代的放射分配策略。我们在这里建议 开发针对潜在HIV-1感染事件的终末转录沉默（TTS）策略。 TTS 策略将在复古/慢病毒沉默中具有生物学等效。但是，TT不会针对DE Novo感染事件，但必须在没有病毒的情况下进行转录沉默的潜在病毒 蛋白质，主要是触发抗病毒细胞反应的RNA或非集成DNA。证据 由特异性靶向慢病毒感染的细胞先天免疫机制的存在。 慢病毒沉默是基于慢病毒的基因疗法的主要障碍。先天的存在 可以控制先前存在的病毒式基因组表达的细胞防御机制。 国防机制的存在，反对一种称为“基因组入侵者”的相关的古代阶级 逆转座子。这些防御机制单独演变为响应于每个不同的不同 Retrotransposon类。不幸的是，由于这种特异性，连续主动控制的机制 逆转座子以抑制其基因组扩散，实际上对HIV-1不活跃。 TT的特异性 针对HIV-1感染的策略至关重要，因为除了对任何毒性问题的一般考虑之外 药物干预，TTS策略不会引起（i）对先天控制机制的任何干扰 抑制返回跨座子活性或原因（ii）对控制表观遗传学的机制的任何干扰 在人类细胞中的基因沉默。对于R61阶段，我们提出了一种结合高的迭代方法 内容分析方法（ATAC-SEQ，RNA-SEQ，KINOME阵列分析）与药物扰动 筛选以确定专门控制细胞先天抗病毒对HIV-1感染的药物靶标。 拟议的研究将利用（i）先前存在的大量HIV-1报告基细胞，（ii） 发现某些临床HIV-1，尤其是HIV-2菌株会导致更大的先天TTS 反应比常用的实验室适应HIV-1克隆，以及（iii）巨噬细胞的发现是 与T细胞相比，执行TTS的效率要高得多。 R61阶段的交付将为（1） 鉴定可药理学解决的可药物靶 细胞和（2）与HTS兼容的药物筛查测定法，以鉴定TTS诱导这些药物针对这些药物 目标。在R33阶段，我们将执行实际的药物筛选以识别诱导TTS的化合物。 确定的候选化合物将在潜在感染和细胞材料的主要细胞模型中进行测试 源自HIV-1患者。到R33阶段结束时，我们预计至少已经识别出一个临床 相关的TTS策略完成一种或几种药物。