CRISPRi-based discovery and in vivo validation of drug targets in M. abscessus

项目摘要 摘要非结核分枝杆菌(NTM)是一类新兴的条件致病菌。 引起肺结核样肺部感染，特别是在有潜在合并症的患者中 如艾滋病毒或囊性纤维症。由NTM引起的肺部疾病估计是 在美国比结核病更常见，每年约有15万例。脓肿分枝杆菌 (MAb)导致80%的肺部疾病是由于快速生长的分枝杆菌和 是CF患者的主要NTM病原体。典型的治疗计划包括一种多药 注射和口服抗生素的组合使用，最长可达一年。治疗失败， 发生在50%-70%的病例中，往往使医生别无选择，只能进行肺部手术 切除手术。众所周知，单抗临床分离株对多种抗生素具有抗药性， 包括对其他分枝杆菌有效的药物(例如结核分枝杆菌)。即使是抗生素 体外杀死单抗对体内感染不是很有效。我们假设这是由于 涉及感染期间可能被激活的可诱导耐药机制(即在 巨噬细胞)或暴露在药物中。一个重要的知识鸿沟，阻碍了 发现新的临床有效的治疗单抗感染的药物是缺乏经过验证的药物 目标。这将需要证明基因产品的重要性，至少是有条件的。 体内的重要性，以及对其“可药性”或对抑制的脆弱性的评估。这项建议 寻求在Mab中首次实施强大的遗传工具CRISPRi，以促进诱导 靶向基因的敲除。在目标1中，遵循对CRISPRI的评价和优化 平台，我们将展示它在药物靶点确认和评估方面的效用。 他们易受抑制的弱点。在目标2中，我们将建立CRISPRi中介的概念验证 在一种新的单抗肺部感染小鼠模型中，体内的基因敲除。如果成功，这将是 将为Mab中的快速基因分析提供一个有价值的工具，以及将有助于 克服这种病原体令人印象深刻的抗生素耐药性。

Project Summary Non-tuberculous mycobacteria (NTM) are an emerging group of related opportunistic pathogens that cause TB-like pulmonary infections, particularly in patients with underlying comorbidities such as HIV or cystic fibrosis. Pulmonary disease caused by NTM is estimated to be 10 times more common than tuberculosis in the U.S., with ~150,000 cases per year. Mycobacterium abscessus (Mab) is responsible for >80% of all pulmonary disease due to rapidly growing mycobacteria and is the predominant NTM pathogen in CF patients. Typical treatment plans include a multidrug combination of injectable and oral antibiotics administered for up to one year. Treatment failures, which occur in 50-70% of cases, often leave physicians with no recourse but to perform lung resections. Clinical isolates of Mab are notoriously resistant to many classes of antibiotics, including drugs effective against other mycobacteria (e.g. M. tuberculosis). Even antibiotics that kill Mab in vitro are not very efficacious against in vivo infections. We hypothesize that this is due to inducible drug resistance mechanisms that may be activated during infection (i.e. within macrophages) or upon exposure to the drug. An important knowledge gap that hampers the discovery of new, clinically effective therapeutics for Mab infections is the lack of validated drug targets. This would entail demonstration of the essentiality of a gene product, at least conditional essentiality in vivo, and assessment of its “druggability” or vulnerability to inhibition. This proposal seeks to implement a powerful genetic tool, CRISPRi, for the first time in Mab to facilitate inducible knockdown of targeted genes. In Aim 1, following evaluation and optimization of the CRISPRi platform in Mab, we will demonstrate its utility for validation of drug targets and assessment of their vulnerability of inhibition. In Aim 2, we will establish proof-of-concept for CRISPRi-mediated gene knock-downs in vivo in a novel mouse model of pulmonary Mab infection. If successful, this will yield a valuable tool for rapid genetic analyses in Mab as well as insights that will help to overcome the impressive antibiotic resistance of this pathogen.