Aberrant Micro-managing of the Airway Epithelial Transcriptome in HIV-associated COPD

项目总结 这项建议的长期目标是确定HIV相关COPD的病理生理学基础。 艾滋病毒携带者(PLWH)表现出COPD发病率的增加，即使在扣除吸烟状况的情况下也是如此。 MicroRNAs管理细胞转录组，在健康和疾病中发挥重要作用。我们已经展示了 HIVTAT和转化生长因子-β(β)信号对呼吸道微RNA组的调节失调。 MicroRNAome的失调会影响多条信号通路，进而影响细胞 动态平衡。COPD是一种多因素的病理，涉及多种信号通路的失调， 汇聚为呼吸道线粒体功能障碍、昼夜节律失调、重构、炎症等。 β信号是由艾滋病毒TAT和香烟烟雾诱导的，这一点很重要，因为有相当大的比例 的PLWH对尼古丁上瘾并吸烟/吸烟，可能会加剧他们的病情进展到 慢性阻塞性肺疾病（慢阻肺）。因此，TAT和转化生长因子-β诱导的异常microRNA组及其对呼吸道的影响 转录组可以作为一个启动事件，对关键信号产生下游影响 节点和路径。识别这些通路和这些关键信令节点有助于定制干预措施 以改善PLWH患者的长期肺预后。 肺是重要的HIV储存库，我们和其他人已经表明，呼吸道上皮细胞表达 典型的艾滋病毒受体，并可感染艾滋病毒。最近的一项研究表明，大部分艾滋病毒宿主是 即使在抑制车的情况下转录也是活跃的。因此，沉默HIV转录可以减少HIV 像Tat这样的蛋白质在呼吸道中。使用CRISPR/Cas9进行基因编辑在消除HIV方面具有巨大的潜力 水库和报告显示，从受感染的细胞中切除了艾滋病毒。然而，基于CRISPR的艾滋病毒切除是 容易受到病毒逃逸和偏离目标的影响。周期蛋白T1，P-TEFb的一种成分，(异源二聚体 CyClinT1和CDK9)被HIV Tat结合，在HIV转录和其抑制取消中起关键作用 艾滋病毒转录。因此CRISPR介导的细胞周期蛋白T1失活将完全阻断HIV转录、锁定 处于非活性状态的艾滋病毒前病毒DNA。我们将使用我们的专利Pol II HIV LTR-果蝇HSP70融合 仅在HIV中共表达细胞周期蛋白T1 gRNA和Cas9(并影响细胞周期蛋白T1失活)的单启动子 被感染的细胞。表达是自我限制的，因为我们的融合启动子也需要CylinT1，从而将Off- 目标效果。目标1将确定HIVTAT和转化生长因子-β调节呼吸道功能障碍的机制 MicroRNAome及其对转录组的影响以识别与HIV相关的信号通路 慢性阻塞性肺疾病（慢阻肺）。AIM 2将使用可诱导的CRISPR系统来灭活仅在HIV感染细胞中的CylinT1并锁定 处于转录不活跃状态的前病毒DNA。了解促进艾滋病毒的信号通路- 相关的COPD和沉默艾滋病毒转录将防止肺功能下降和 慢性阻塞性肺疾病在PLWH的进展。

PROJECT SUMMARY The long-term goal of this proposal is to identify the pathophysiology underlying HIV associated COPD. People living with HIV (PLWH) show increased incidence of COPD even when compensated for smoking status. MicroRNAs manage the cellular transcriptome and play important roles in health and disease. We have shown that HIV Tat and Transforming Growth Factor-beta (TGF-β) signaling dysregulate the airway microRNAome. Dysregulation of the microRNAome can effect multiple signaling pathways and this can affect cellular homeostasis. COPD is a multifactorial pathology and involves dysregulation of diverse signaling pathways that converge towards airway mitochondrial disfunction, circadian dysregulation, remodeling, inflammation, etc. TGF- β signaling is induced by both HIV Tat and cigarette smoke and this is significant since a significant proportion of PLWH are addicted to nicotine and smoke tobacco/cigarettes, possibly exacerbating their progression to COPD. Hence, Tat and TGF-β-induced aberrant microRNAome and its effects on the airway transcriptome can serve as an initiating events with downstream consequences on critical signaling nodes and pathways. Identifying these pathways and these critical signaling nodes can help tailor interventions to improve long-term pulmonary outcomes in PLWH. Lungs are important HIV reservoirs and we, and others have shown that airway epithelial cells express canonical HIV receptors and can be infected with HIV. A recent study shows that the bulk of HIV reservoirs are transcriptionally active even with suppressive cART. Hence, silencing HIV transcription can decrease HIV proteins like Tat in the airway. Gene editing using CRISPR/Cas9 has tremendous potential in eliminating HIV reservoirs and reports have shown excision of HIV from infected cells. However, CRISPR based HIV excision is vulnerable to viral escape as well as off-target effects. Cyclin T1, a component of P-TEFb, (heterodimer of cyclinT1 and CDK9) is bound by HIV Tat and plays a pivotal role HIV transcription and its inhibition abolishes HIV transcription. Hence CRISPR mediated CyclinT1 inactivation will completely block HIV transcription, locking the HIV proviral DNA in an inactive form. We will use our patented Pol II HIV LTR-drosophila hsp70 fusion monopromoter to co-express CyclinT1 gRNA and Cas9 (and effect CyclinT1 inactivation) only in HIV infected cells. Expression is self-limiting as our fusion promoter also requires CyclinT1 thereby minimizing off- target effects. Aim 1 will determine the mechanism by which HIV Tat and TGF-β dysregulate the airway microRNAome and its effects on the transcriptome to identify signaling pathways involved in HIV associated COPD. Aim 2 will use an inducible CRISPR syste to inactivate CyclinT1 only in HIV infected cells and lock the proviral DNA in a transcriptionally inactive state. Understanding the signaling pathways that promote HIV- associated COPD and silencing HIV transcription in reservoirs will prevent lung function decline and development of COPD in PLWH.