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

项目总结

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.