PROJECT SUMMARY People living with HIV demonstrate increased incidence of lung inflammation and HIV is an independent risk factor development of COPD. HIV Tat, an immediate early protein of HIV and cigarette smoke suppress the deacetylase SIRT1 that regulates ADAM17, a protease involved in activating Notch signaling as well as proteolytic cleavage and activation of proinflammatory cytokines. This can lead to downstream deleterious effects on ciliogenesis, mucociliary clearance and lung function decline. While chronic inflammation in COPD is a multifactorial etiology, impaired mucociliary clearance leading to recurrent lung infections can play an important role in promoting lung inflammation. Optimal mucociliary clearance requires mucus, cilia, and a thin layer of airway surface liquid to facilitate ciliary beating. Abnormalities in any compartment of the mucociliary system can compromise mucus clearance leading to mucus impaction entrapping bacteria and promoting chronic bacterial infection. HIV Tat promotes an aberrant microRNAome in the primary bronchial epithelium and upregulates miR-142-5p, a microRNA known to suppress SIRT1. This proposal will identify mechanisms involved in HIV Tat promotes mucociliary dysfunction and identifying individual effects on ciliogenesis, mucus hypersecretion and inflammation. Given the causal role for SIRT1 suppression in mediating these effects, we will use a novel CRISPR-mediated gene specific microRNA antagonism approach to disrupt the miR-142-5p target site on the SIRT1 3’UTR. This will preserve SIRT1 expression in the context of HIV Tat and cigarette smoke without affecting other functions of miR-142-5p. Aim 1: HIV Tat upregulates miR-142-5p and suppress SIRT1 exacerbate upregulates ADAM17 levels by HIV and cigarette smoke exposure in NHBE ALI cultures. Given that HIV Tat and cigarette smoke mediated miR-142-5p dysregulation leading to SIRT1 suppression and aberrant Notch signaling. Specifically, we will analyze ADAM17 upregulation, ciliogenesis, goblet cell hyperplasia and ciliary beat frequency in NHBE ALI cultures. Aim 2: Since HIV Tat and cigarette smoke suppresses SIRT1, and SIRT1 suppression impairs mucociliary clearance apparatus, we will determine if gene-specific microRNA antagonism in combination with SIRT1 activator to rescue SIRT1 levels, suppression of inflammation and enhance mucociliary clearance apparatus in primary bronchial epithelial cells treated with HIV Tat and exposed to cigarette smoke.