Julie England

Introduction: Pseudomonas aeruginosa is a major cause of healthcare- and ventilator-associated pneumonia (HAP, VAP). This complication produces significant increases in mortality, morbidity, and cost of care.^1,2 20% of Pseudomonas pneumonia cases are caused by multidrug resistant (MDR) strains.³ Importantly, the presence of an MDR strain is the most important predictor of inadequate initial antibiotic therapy (IIAT).⁴ IIAT is an independent risk factor for mortality in the ICU.^4,5 Traditional antibiotics have become increasingly ineffective against MDR P. aeruginosa HAP/VAP, and we are in desperate need of a reliably effective therapy. One potential therapy involves the silencing of bacterial essential genes. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are capable of entering a bacterium, binding the mRNA of the target gene, and blocking its translation.^6,7 The loss of an essential protein product results in death of the bacterium. The purpose of this investigation is to identify PPMOs capable of anti-pseudomonal activity, and to clarify their potential for translation into clinical therapies.  Methods: To identify effective carrier peptides, LacZ expression was induced in P. aeruginosa. Strains were incubated with anti-LacZ and scrambled PMOs, conjugated to peptide 1, KFFKFFKFFK, or peptide 2, (RXR)­­₄. LacZ activity was measured.⁶ To quantify the in vitro efficacy of the acpP-(RXR)­­₄ PPMO, an effective PMO sequence against acpP mRNA was identified, conjugated with the (RXR)­­₄ carrier peptide, and incubated in P. aeruginosa culture at increasing doses.⁷ To investigate the activity of PPMOs against P. aeruginosa pneumonia in vivo, mice were inoculated with P. aeruginosa, incubated 6 hours, treated with anti-acpP PPMOs, saline, or scrambled PPMO, and then sacrificed at 24 hours.  Results: Maekawa, et al. identified (RXR)₄ as an effective carrier peptide against Pseudomonas without off-target toxicities.⁶ Ghosal, et al. determined that an effective anti-acpP PMO was 11 bp long, from -6 bp to +5 bp, across the translation start codon.⁷ This acpP-(RXR)­­₄ PPMO demonstrated activity against P. aeruginosa in vitro.⁷ In the mouse model of acute pneumonia, Howard, et al. demonstrated that the acpP-(RXR)­­₄ PPMO reduced CFU in the lungs by almost 3 logs. Histologically, acpP-(RXR)­­₄ PPMO-treated lungs showed reduced inflammatory infiltrate.⁸  Conclusions: acpP-(RXR)­­₄ PPMOs effectively inhibit growth of P. aeruginosa in vitro and in vivo, and successfully treat P. aeruginosa pneumonia in a murine model. Given these favorable results, and the recent FDA approval of the PMO drug Eteplirsen¹⁰ for muscular dystrophy, PPMOs are a promising option for future clinical studies in Pseudomonas pneumonia.

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