Antisense Phosphorodiamidate Morpholino Oligomers Targeted to an Essential Gene InhibitBurkholderia cepaciaComplex

Abstract

Background: Members of the Burkholderia cepacia complex (Bcc) cause significant morbidity and mortality in patients with chronic granulomatous disease (CGD) and cystic fibrosis (CF). Many Bcc strains are antibiotic resistant requiring the exploration of novel antimicrobial approaches including antisense technologies, such as phosphorodiamidate morpholino oligomers (PMOs). Methods: Peptide-conjugated PMOs (PPMOs) were developed to target the acpP gene, encoding an acyl carrier protein thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models. Results: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (> 4 log reduction), whereas a PPMO with a scrambled base sequence (Scr) had no effect on growth. Human neutrophils (PMN) were infected with B. multivorans, and treated with AcpP PPMO. AcpP PPMO augmented killing compared to PMN alone ± Scr PPMO. CGD mice infected with B. multivorans were treated with AcpP PPMO, Scr PPMO or water at 0, 3 and 6 hours post-infection. Compared to water treated controls, the AcpP PPMO treated mice showed a ~80% reduction in the risk of dying by day 30 and relatively little pathology. Conclusions: AcpP PPMO is active against Bcc infections in vitro and in vivo.

Peptide Conjugated Phosphorodiamidate Morpholino Oligomers Increase Survival of Mice Challenged with AmesBacillus anthracis

“…This suggests that the efficacy of PPMOs may not be entirely gene specific. Off target or nonspecific effects of PPMOs have also been observed in vitro with E. coli (Wesolowski et al, 2011) and Acinetobacter lwoffii (Geller, unpublished data), and in vivo with Burkholderia multivorans (Greenberg et al, 2010). Some of this nonspecific effect may be due to partial complementarity of scrambled sequence controls to other genes (Wesolowski et al, 2011).…”

“…Phosphorodiamidate morpholino oligomers (Geller et al, 2003;Greenberg et al, 2010) have been shown to silence expression of bacterial genes. Gene-silencing oligomers decrease expression of reporter genes such as luciferase, activate endogenous genes such as b-galactosidase, and inhibit growth by targeting essential genes .…”

“…Gene-silencing oligomers decrease expression of reporter genes such as luciferase, activate endogenous genes such as b-galactosidase, and inhibit growth by targeting essential genes . Some of the attractive targets for antisense antibacterial therapy include essential genes such as acyl carrier protein (acpP) encoding the fatty acid biosynthesis protein Greenberg et al, 2010), bacterial RNA polymerases (Bai et al, 2011), fabIencoding enoyl-acyl carrier protein reductase and 16S rRNA (Nielsen, 2010). Prior studies have shown that PMO targeting the acpP gene was able to inhibit E. coli viability in vitro and in vivo in mice .…”

“…To further improve the uptake of the PMOs by the bacteria, PMOs conjugated with various peptides (PPMOs) and targeting the acpP gene were shown to enhance bacterial growth inhibition and viability of E. coli and Salmonella enterica Serovar Typhimurium (Tilley et al, 2006). More recently PPMOs targeting the acpP gene inhibited Burkholderia cepacia complex (Bcc) in vitro and increased survival of mice challenged with a clinical isolate of Bcc (Greenberg et al, 2010).…”

Peptide nucleic acid antisense oligomers open an avenue for developing novel antibacterial molecules

“…The delivery of antibacterial PNA molecules can be achieved by designing of bacterial-specific vehicles. Several peptides, such as (KFF)3K, (R-Ahx-R)4-Ahxβala, (R-Ahx)6-βala, YARVRRRGPRGYARVRRRGPRRC, and RFFRFFRFFRXB, are known for delivering antisense PNA or Morpholino oligomers in bacteria [10,15,22,23]. In addition to their structural and/or sequence dissimilarity, these peptides may also differ in their uptake in bacteria.…”