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

基本信息

批准号：
10205411
负责人：
OLAF KUTSCH
金额：
$ 68.96万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10205411
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 Screening Drug Targeting Elements Epigenetic Process Event Funding Gene Silencing Generations Genome Genomics HIV HIV-1 HIV-2 Human Individual Infection Intervention Laboratories Lead Libraries Methods Molecular Open Reading Frames Pathway interactions Patients Pattern recognition receptor Pharmaceutical Preparations Pharmacology Phase Phenotype Process Proteins Proviruses PubChem RNA Reporter Research Retrotransposon Robotics Severe Acute Respiratory Syndrome Signal Transduction Specificity Structure 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 druggable target gene therapy genetic manipulation genome-wide high throughput screening innate immune mechanisms latent infection lead series macrophage overexpression response scaffold screening small hairpin RNA small molecule transcription factor transcriptome sequencing

项目摘要

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.

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效果在治疗性根除 HIV-1 方面，需要探索替代根除策略。开发针对潜在 HIV-1 感染事件的末端转录沉默 (TTS) 策略。策略在逆转录/慢病毒沉默中具有其生物学等效性，但是，TTS 不会针对 de。新感染事件，但必须在没有病毒的情况下转录沉默预先存在的潜伏原病毒蛋白质，主要是 RNA 或非整合 DNA，通常会引发抗病毒细胞反应。特异性针对慢病毒感染的细胞先天免疫机制的存在是由事实上，慢病毒沉默是基于慢病毒的基因治疗的主要障碍。细胞防御机制可以控制预先存在的病毒型基因组表达针对相关的、古老的进化类别“基因组入侵者”的防御机制的存在，称为这些防御机制分别针对每种不同的情况而进化。不幸的是，由于这种特异性，逆转录转座子类的机制主动控制。逆转录转座子抑制其基因组扩散，实际上对 HIV-1 没有活性。对抗 HIV-1 感染的策略至关重要，因为超出了对任何药物毒性问题的一般考虑药物干预、TTS 策略不会导致 (i) 对先天控制机制的任何干扰抑制逆转录转座子活性或导致 (ii) 对控制表观遗传机制的任何干扰对于 R61 阶段，我们提出了一种结合高水平的迭代方法。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）筛选以确定特异性控制细胞对 HIV-1 感染的先天抗病毒反应的药物靶点。拟议的研究将利用 (i) 现有的大量 HIV-1 报告细胞，(ii) 发现某些临床 HIV-1，特别是 HIV-2 毒株会导致更有效的先天 TTS 反应比常用的实验室适应的 HIV-1 克隆要好，并且 (iii) 巨噬细胞的发现 R61 阶段的 TTS 执行效率更高，其结果是 (1) 识别可药物靶标，如果通过药理学解决，可在潜伏的 HIV-1 感染 T 中启用 TTS 细胞和 (2) HTS 兼容药物筛选测定法，用于识别针对这些细胞的 TTS 诱导药物在 R33 阶段，我们将进行实际的药物筛选，以识别 TTS 诱导化合物。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到 R33 阶段结束时，我们预计将在临床上鉴定出至少一种。相关的 TTS 策略由一种或多种药物组成。