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

基于 CRISPRi 的脓肿分枝杆菌药物靶点的发现和体内验证

• 批准号: 10302067
• 负责人: Kyle H Rohde
• 金额: $ 22.8万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-09 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302067
• 关键词: Address; Alleles; Aminoglycosides; Animal Model; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Bacteria; Bacteriophages; Biological Assay; Bronchiectasis; CRISPR interference; Cefoxitin; Chronic; Chronic Obstructive Airway Disease; Clarithromycin; Clinical; Clinical Management; Cloning; Cues; Cystic Fibrosis; Data; Disease; Disease Management; Drug Screening; Drug Targeting; Drug Tolerance; Drug resistance; Enzymes; Essential Genes; Etiology; Evaluation; Excision; Exposure to; Failure; Gene Silencing; Generations; Genes; Genetic; Genomics; Genus Mycobacterium; Goals; HIV; Hypoxia; Imipenem; Immunocompetent; Immunocompromised Host; Immunosuppression; In Vitro; Infection; Injectable; Investigation; Investigational Therapies; Kinetics; Knock-out; Knowledge; Lung; Lung diseases; Lung infections; Mediating; Methods; Modeling; Molecular; Molecular Target; Mutation; Mycobacterium Infections; Mycobacterium abscessus; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Nature; Operative Surgical Procedures; Oral; Pathogenesis; Patients; Pharmaceutical Preparations; Phenotype; Physicians; Predisposition; Quantitative Reverse Transcriptase PCR; Regimen; Reporting; Research; Resistance; Site; Technology; Testing; Therapeutic; Time; Treatment Failure; Tuberculosis; United States National Institutes of Health; Up-Regulation; Validation; Virulence; Virulent; base; comorbidity; cystic fibrosis mouse; cystic fibrosis patients; effective therapy; emerging pathogen; gene function; gene product; genetic analysis; improved; in vivo; insight; interest; knock-down; macrophage; member; microorganism; mouse model; mutant; new therapeutic target; non-tuberculosis mycobacteria; novel; opportunistic pathogen; pathogen; recombinase; resistance gene; resistance mechanism; respiratory; soft tissue; stem; synergism; therapeutically effective; tool; treatment planning; tuberculosis drugs

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.

项目摘要 非结核分枝杆菌（NTM）是一组新兴的相关机会致病菌 导致肺结核样肺部感染，特别是在有潜在合并症的患者中， 艾滋病或囊性纤维化。由NTM引起的肺部疾病估计要多10倍 在美国比肺结核更常见每年约有15万例。脓肿分枝杆菌 (Mab)由于快速生长的分枝杆菌导致>80%的肺部疾病， 是CF患者的主要NTM病原体。典型的治疗计划包括多药 注射和口服抗生素的组合施用长达一年。治疗失败， 发生在50-70%的病例中，通常使医生别无选择，只能进行肺切除术。 切除术众所周知，Mab的临床分离株对许多种类的抗生素具有抗性， 包括对其它分枝杆菌（例如M.肺结核）。即使是抗生素， 体外杀伤Mab对体内感染不是非常有效。我们假设这是由于 可诱导的耐药机制，其可在感染期间被激活（即在 巨噬细胞）或暴露于药物后。一个重要的知识差距，阻碍了 发现新的、临床上有效的单克隆抗体感染治疗方法是缺乏经过验证的药物 目标的这就需要证明基因产品的必要性，至少是有条件的 体内必需性，以及评估其“药物性”或对抑制的脆弱性。这项建议 寻求在Mab中首次实施一种强大的遗传工具CRISPRi，以促进诱导 敲除靶基因。在目标1中，在CRISPRi的评估和优化之后， 平台，我们将展示其在药物靶点验证和评估中的实用性。 他们抑制的脆弱性在目标2中，我们将建立CRISPRi介导的 肺Mab感染的新型小鼠模型中的体内基因敲低。如果成功，这 将为Mab的快速遗传分析提供有价值的工具，并有助于 克服这种病原体令人印象深刻的抗生素耐药性。