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

HIV 相关 COPD 气道上皮转录组的异常微观管理

• 批准号: 10700300
• 负责人: IRFAN RAHMAN
• 金额: $ 65.39万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700300
• 关键词: Abbreviations; Affect; Aging; Air; Animal Model; Binding; CRISPR/Cas technology; Cells; Chronic Bronchitis; Chronic Obstructive Pulmonary Disease; Cigarette; Circadian Dysregulation; Clustered Regularly Interspaced Short Palindromic Repeats; Compensation; Complement; Continuity of Patient Care; Cyclins; DNA; DNA Polymerase II; Data; Development; Disease; Drosophila genus; Epithelial Cells; Etiology; Event; Excision; Exposure to; Functional disorder; Genes; Genetic Transcription; Genome; Goals; Guide RNA; HIV; HIV Receptors; Health; Homeostasis; Human; Incidence; Inflammation; Intervention; Legal patent; Liquid substance; Lung; Mediating; Mediator; Messenger RNA; MicroRNAs; Mitochondria; Morbidity - disease rate; Mucociliary Clearance; Mutation; Nicotine; Outcome; Pathologic; Pathology; Pathway interactions; Peptide Signal Sequences; Persons; Play; Poly A; Polyadenylation; Positive Transcriptional Elongation Factor B; Proteins; Pulmonary Emphysema; RNA Recognition Motif; Regulation; Reporting; Risk; Risk Factors; Role; Signal Pathway; Signal Transduction; Site; Smoker; Smoking Status; System; Tobacco smoke; Toxic effect; Transforming Growth Factor beta; Translating; United States; Viral; Viral Genome; airway epithelium; bronchial epithelium; cellular targeting; cigarette smoke; circadian regulation; cofactor; comorbidity; cyclin T1; efficacy evaluation; functional decline; gene function; improved; improved outcome; mitochondrial dysfunction; mortality; mouse model; non-smoker; prevent; promoter; pulmonary function; pulmonary function decline; response; senescence; transcriptome; transcriptomics

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.

项目摘要 该提案的长期目标是确定HIV相关COPD的病理生理学基础。 艾滋病毒感染者（PLWH）即使在补偿吸烟状态的情况下，COPD的发病率也会增加。 microRNA管理细胞转录组，并在健康和疾病中发挥重要作用。我们已经表明 HIV达特和转化生长因子-β（TGF-β）信号传导失调气道microRNAome。 微RNA组的调节异常可以影响多种信号通路，从而影响细胞 体内平衡COPD是一种多因素病理学，涉及多种信号通路的失调， 聚集于气道线粒体功能障碍、昼夜节律失调、重塑、炎症等。 HIV达特和香烟烟雾都能诱导β信号传导，这一点很重要，因为 的艾滋病毒携带者对尼古丁和烟草/香烟上瘾，这可能会加剧他们的进展， 慢性阻塞性肺病因此，达特和TGF-β诱导的异常microRNAome及其对气道的影响 转录组可以作为一个启动事件与下游后果的关键信号 节点和路径。识别这些途径和这些关键信号节点可以帮助定制干预措施 改善PLWH患者的长期肺部结局。 肺是重要的HIV储存库，我们和其他人已经证明气道上皮细胞表达 典型的HIV受体，并可感染HIV。最近的一项研究表明，大部分艾滋病病毒储存库是 即使在抑制性cART下也是转录活性的。因此，沉默HIV转录可以减少HIV 像达特这样的蛋白质。使用CRISPR/Cas9进行基因编辑在消除艾滋病毒方面具有巨大潜力 储存库和报告显示从感染的细胞切除HIV。然而，基于CRISPR的HIV切除是 易受病毒逃逸和脱靶效应的影响。细胞周期蛋白T1是P-TEFb的一种成分， 细胞周期蛋白T1和CDK 9）与HIV达特结合，在HIV转录中起关键作用，其抑制作用被消除 艾滋病毒转录。因此，CRISPR介导的CyclinT 1失活将完全阻断HIV转录， HIV前病毒DNA处于非活性形式。我们将使用我们的专利Pol II HIV LTR-果蝇hsp 70融合体 单启动子仅在HIV中共表达CyclinT 1 gRNA和Cas9（并实现CyclinT 1失活） 被感染的细胞表达是自限性的，因为我们的融合启动子也需要细胞周期蛋白T1，从而最大限度地减少了关闭。 目标效应。目的1将确定HIV达特和TGF-β调节气道的机制 microRNAome及其对转录组的影响，以确定HIV相关的信号通路 慢性阻塞性肺病目的2将使用诱导型CRISPR系统仅在HIV感染的细胞中抑制CyclinT 1，并锁定细胞周期蛋白T1。 处于转录非活性状态的前病毒DNA。了解促进艾滋病毒的信号通路- 相关的COPD和水库中的HIV转录沉默将防止肺功能下降， 在PLWH中发生COPD。