PROJECT SUMMARY/ABSTRACT Gram-negative pathogens are becoming increasingly resistant to many antimicrobials. Furthermore, the pipeline for new antibiotics is small and new therapies are urgently needed. This can be especially problematic in patients who suffer from chronic infections or are immunocompromised. Multidrug-resistant Pseudomonas aeruginosa has been identified by the Centers for Disease Control and Prevention as a serious threat. P. aeruginosa causes healthcare associated infections in a variety of clinical settings and hosts, but is particularly devastating in patients with cystic fibrosis (CF). We have been interested in using antisense molecules called PPMOs as potential therapeutics in these infections. These molecules block messenger RNA and prevent the formation of the target protein. We have demonstrated that PPMOs can be used to target genes that are essential for Pseudomonas to grow, such as acpP, lpxC or rpsJ. We showed that blocking these proteins are essential for Pseudomonas to grow in vitro. We also showed that these PPMOs improve survival in mice that were infected with Pseudomonas. For this project, we propose to further characterize our lead PPMOs in a larger collection of Pseudomonas isolates, both antibiotic-sensitive and multidrug-resistant. In addition, efficacy studies will be performed in both models of pneumonia and bloodstream infection. This process will result in 2-4 PPMOs that will undergo further pre-clinical testing including toxicity, resistance, pharmacodynamic and pharmacokinetic studies. By the end of the proposed project, a lead PPMO will have undergone the needed pre-clinical testing for IND submission to the FDA. This innovative approach to developing novel antibiotics, particularly for P. aeruginosa, could help expand the increasingly shrinking classes of effective antibiotics that are used to treat these severe, life-threatening infections.