HIV-CRISPR: A novel approach to the comprehensive discovery of HIV latency factors

HIV-CRISPR：全面发现HIV潜伏因子的新方法

• 批准号: 10642658
• 负责人: Michael Emerman
• 金额: $ 88万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642658
• 关键词: Adopted; Antiviral Agents; Antiviral Therapy; Basic Science; Cataloging; Cell model; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Combination Drug Therapy; Communities; Dose; Genes; Genetic; Genetic Screening; Genetic screening method; Goals; HIV; HIV Budding; HIV Genome; HIV Infections; HIV/AIDS; Human; Immune system; Immunologics; Individual; Intervention; Maintenance; Methods; Modeling; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Proviruses; Research; Specificity; Techniques; Technology; Time; Virus; Virus Latency; antiretroviral therapy; burden of illness; cell killing; cell type; drug of abuse; high throughput technology; in vivo; innovation; new technology; novel; novel strategies; screening; synergism; transmission process

1 Project Summary/Abstract The availability of simplified and better-tolerated antiretroviral therapy (ART) drug combinations has decreased disease burden in infected individuals and reduced transmission to their contacts. However, treatment for HIV infections requires life-long antiviral therapy since current drug treatments do not lead to “cures”. The single most significant obstacle to total HIV clearance is that of viral latency since the presence of a silent HIV genome in human cells is not recognized as infected by the immune system or by any available antiviral drugs. Thus, the largest challenge to the HIV/AIDS basic sciences research community today is to determine targets and strategies to fully eradicate virus from the body by eliminating this reservoir pool of latently infected cells. One strategy to eliminate latently infected cells involves activating the latent provirus with drugs called latency reversal agents (LRAs) in the presence of suppressive ART, and then using immunological methods to kill the cells newly-producing virus. However, we lack a comprehensive and complete understanding of how HIV latency is established and maintained, and how reactivation occurs across cell types. Importantly, no single genetic or chemical intervention has been able to induce activation of all latent proviruses (or even the majority of them). This proposal describes a novel and powerful approach to understanding HIV latency based on an innovative genetic screening method that we have developed called HIV-CRISPR that uses the packaging of CRISPR guides into budding HIV to serve as a readout for the effects of host genes on HIV. This technique is both high-throughput and comprehensive and our plan is to adapt to models of HIV latency. Because the screening technology is versatile and can be done in many iterations, we will be able to adopt a strategy based on combining genetic screens with low doses candidate latency reversal agents to identify synergism between different pathways of HIV latency maintenance in order to find targets that more broadly activate HIV from latency while at the same time providing a greater degree of specificity for the HIV LTR. Moreover, we will develop methods this technique to primary cell models of HIV latency to better mimic how to reverse latency in vivo. Finally, will also apply this screen to understand how drugs of abuse influence HIV latency pathways.

1 项目总结/摘要 简化和耐受性更好的抗逆转录病毒疗法（ART）药物组合的可用性 减少受感染者的疾病负担，并减少向其接触者的传播。然而，在这方面， HIV感染的治疗需要终身抗病毒治疗，因为目前的药物治疗不会导致 “治愈”完全清除HIV的最大障碍是病毒潜伏期，因为存在 人类细胞中沉默的HIV基因组不会被免疫系统或任何可用的免疫系统识别为感染。 抗病毒药物因此，艾滋病毒/艾滋病基础科学研究界今天面临的最大挑战是 确定目标和策略，通过消除这种储存库， 潜伏感染细胞 消除潜伏感染细胞的一种策略是用药物激活潜伏的前病毒 称为潜伏期逆转剂（LRA），在抑制性ART的存在下，然后使用免疫学 杀死新产生病毒的细胞的方法。然而，我们缺乏一个全面和完整的 了解HIV潜伏期是如何建立和维持的，以及细胞内的再激活是如何发生的 类型重要的是，没有一种单一的遗传或化学干预能够诱导所有潜在的 前病毒（甚至是大多数前病毒）。 该提案描述了一种新颖而强大的方法来了解艾滋病毒的潜伏期， 我们开发了一种创新的基因筛选方法，称为HIV-CRISPR， CRISPR引导进入萌芽的HIV，作为宿主基因对HIV影响的读数。该技术 我们的计划是适应HIV潜伏期的模型。因为 筛选技术是通用的，可以在许多迭代中完成，我们将能够采用一种策略， 基于将遗传筛选与低剂量候选潜伏期逆转剂组合以鉴定协同作用 在HIV潜伏期维持的不同途径之间，以找到更广泛激活HIV的靶点。 从潜伏期，而在同一时间提供了更大程度的特异性，艾滋病毒LTR。 此外，我们将开发这种技术的方法，以原代细胞模型的艾滋病毒潜伏期，以更好地模拟如何 体内逆转潜伏期。最后，也将应用此筛选来了解药物滥用如何影响艾滋病毒 潜伏通路