Antisense Inhibitors Retain Activity in Pulmonary Models of Burkholderia Infection

Abstract

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens in immunocompromised individuals, such as those with cystic fibrosis (CF) or chronic granulomatous disease (CGD). Burkholderia are intrinsically resistant to many antibiotics and the lack of antibiotic development necessitates novel therapeutics. Peptide-conjugated phosphorodiamidate morpholino oligomers are antisense molecules that inhibit bacterial mRNA translation. Targeting of PPMOs to the gene acpP, which is essential for membrane synthesis, lead to defects in the membrane and ultimately bactericidal activity. Exploration of additional PPMO sequences identified the ATG and Shine-Dalgarno sites as the most efficacious for targeting acpP. The CF lung is a complex microenvironment, but PPMO inhibition was still efficacious in an artificial model of CF sputum. PPMOs had low toxicity in human CF cells at doses that were antibacterial. PPMOs also reduced the bacterial burden in the lungs of immunocompromised CyBB mice, a model of CGD. Finally, the use of multiple PPMOs was efficacious in inhibiting the growth of both Burkholderia and Pseudomonas in an in vitro model of coinfection. Due to the intrinsic resistance of Burkholderia to traditional antibiotics, PPMOs represent a novel and viable approach to the treatment of Burkholderia infections.

Cardiolipin-Based Lipopolyplex Platform for the Delivery of Diverse Nucleic Acids into Gram-Negative Bacteria

“…To evaluate the efficiency of LPN in delivering ASO endowed with effect on bacteria growth inhibition, we delivered ASO targeting the acyl carrier protein (AcpP) mRNA whose sequence is well conserved between E. coli and P. aeruginosa species, with such mRNA targeting yielding effective growth inhibition as previously observed [14,46,47,48,49,50]. Delivery of 11 nt ssASO targeting AcpP mRNA reduced significantly the growth of E. coli (Figure 4A) and P. aeruginosa (Figure 4B).…”

“…Indeed, various delivery systems have been used, until now, to condense, protect, and carry nucleic acids into bacteria. Antisense oligodeoxynucleotides (ASOs) were mainly conjugated to cell-penetrating peptides (CPP) [4,12,13,14]. However, the CPP-dependent approach showed several limitations, including the requirement of designing derivatives when resistance to delivery arises from mutations occurring in bacterial transporter proteins [15].…”

An RNA biology perspective on species‐specific programmable RNA antibiotics

“…tions, suggesting that they may be therapeutically useful for treating sepsis or different diseases of the lung (Barkowsky et al, 2019;Daly et al, 2018;Geller et al, 2018;Sully & Geller, 2016).…”

“…The published work spans Gram‐negative Acinetobacter , Brucella, Burkholderia, Campylobacter, Haemophilus, Klebsiella, Pseudomonas, and Salmonella species, and Gram‐positive Enterococcus , Listeria, Staphylococcus and Streptococcus species (Geller et al, ; Sully & Geller, ). ASOs have shown efficacy in different experimental mouse infections, suggesting that they may be therapeutically useful for treating sepsis or different diseases of the lung (Barkowsky et al, ; Daly et al, ; Geller et al, ; Sully & Geller, ).…”

“…A review of the literature on antibacterial ASOs shows that the site of translation initiation is seen as the optimal mRNA target region for antisense repression. Several studies have tested this more systematically, for example, scanning the 5ʹ regions of different mRNAs in E. coli (Dryselius, Aswasti, Rajarao, Nielsen, & Good, ) or Burkholderia cenocepacia (Daly et al, ) with PPNA or PPMO, respectively. The main conclusion from these studies is that an ASO antisense to the start codon and perhaps part of the Shine‐Dalgarno sequence ( SD ) will be the most potent.…”