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

1 项目摘要/摘要 简化和耐受性更好的抗逆转录病毒治疗(ART)药物组合的可用性 减少了感染者的疾病负担，减少了对其接触者的传播。然而， 艾滋病毒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”。彻底清除艾滋病毒的最大障碍是病毒潜伏，因为 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 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 确定目标和战略，通过消除这个蓄水池来完全消除体内的病毒 潜伏感染的细胞。 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 在存在抑制ART的情况下称为潜伏期反转剂(LRAs)，然后使用免疫学 方法杀灭新生病毒细胞。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，以及细胞内重新激活是如何发生的 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伏的激活 普罗鲁斯(甚至是其中的大多数)。 这项建议描述了一种新的、强大的方法来了解HIV潜伏期，该方法基于 我们开发的名为HIV-CRISPR的创新基因筛查方法，它使用包装 CRISPR引导进入萌芽状态的艾滋病毒，作为宿主基因对艾滋病毒影响的读数。这项技术是 我们的计划是适应HIV潜伏期的模型。因为 筛选技术是多才多艺的，可以在很多次迭代中完成，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反转剂相结合来识别协同作用 在HIV潜伏期维持的不同途径之间，以便找到更广泛地激活HIV的靶点 避免潜伏期，同时为HIV LTR提供更大程度的特异性。 此外，我们将开发这项技术的方法来建立HIV潜伏期的原代细胞模型，以更好地模拟如何 体内反转潜伏期。最后，还将应用此屏幕来了解滥用药物如何影响艾滋病毒 潜伏期通路。

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.