Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose–response in mice infected with Escherichia coli

• Published in: Journal of antimicrobial chemotherapy 2007-01, Vol.59 (1), p.66-73
• Main Authors: Tilley, Lucas D., Mellbye, Brett L., Puckett, Susan E., Iversen, Patrick. L., Geller, Bruce L.
• Format: Article
• Language: English
• Subjects: Acyl Carrier Protein - antagonists & inhibitors; Animals; Antibiotics; Antibiotics. Antiinfectious agents. Antiparasitic agents; antisense therapeutics; Apoproteins - antagonists & inhibitors; Biological and medical sciences; Deoxyribonucleic acid; DNA; Dose-Response Relationship, Drug; E coli; Escherichia coli; Escherichia coli Infections - drug therapy; Escherichia coli Proteins - antagonists & inhibitors; Fatty Acid Synthase, Type II; Female; Medical sciences; Membranes; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Morpholines - therapeutic use; Morpholinos; Oligonucleotides, Antisense - therapeutic use; peptide conjugates; Peptides; Pharmacology. Drug treatments; PMOs; Rodents
• ISSN: 0305-7453; 1460-2091
• DOI: 10.1093/jac/dkl444

Objectives: Phosphorodiamidate morpholino oligomers (PMOs) are DNA analogues that inhibit translation by an antisense mechanism. Membrane-penetrating peptides attached to PMOs increase PMO efficacy by enhancing penetration through bacterial membranes. The objectives of these experiments are to demonstrate gene-specific efficacy and establish a dose–response relationship of a peptide-PMO conjugate. Methods: An 11-base PMO (AcpP) targeted at acpP (an essential gene) of Escherichia coli was synthesized and conjugated with the cell-penetrating peptide RFFRFFRFFRXB (X is 6-aminohexanoic acid and B is β-alanine). Mice were infected by intraperitoneal (ip) injection with K-12 E. coli W3110, and treated ip at 15 min and 12 h post-infection with various amounts of AcpP peptide-PMO conjugate, AcpP PMO without attached peptide, scrambled base sequence PMOs or ampicillin. A strain (LT1) of E. coli was constructed by replacing acpP with an allele that has four wobble base substitutions in the region targeted by the PMO. Results: Twelve hours after a single treatment, 30 μg of AcpP peptide-PMO or 3 mg of AcpP PMO reduced bacteraemia by 3 orders of magnitude compared with treatment with water. Neither scrambled base sequence PMO controls nor 30 μg of ampicillin reduced bacteraemia. Two treatments with 30 μg of AcpP peptide-PMO reduced cfu significantly more than four treatments with 15 μg at 15 min, 4, 8 and 12 h. Mice treated with doses of AcpP peptide-PMO >30 μg showed further reductions in plasma cfu. Survival 48 h after treatment with 2 × 30 μg (3 mg/kg) of AcpP peptide-PMO or 2 × 3 mg (300 mg/kg) of AcpP PMO was 100%, compared with 20% for mice treated with water or scrambled base sequence PMO controls. However, survival was reduced to 75% and 0% for mice treated with 2 × 300 μg and 2 × 1 mg of AcpP peptide-PMO, respectively. A conjugate made from the d-isomeric form of each amino acid was less effective than the l-amino acid equivalent, and required 2 × 300 μg treatments for significant reduction in bacteria and survival. Mice infected with LT1 and treated with AcpP peptide-PMO did not survive and had the same amount of bacteria in the blood as mice treated with water, whereas those treated with 2 × 100 μg of AcpPmut4 peptide-PMO (complementary to the mutated allele) survived, and had a 3 orders of magnitude reduction in bacteria in the blood at 24 h post-infection. Conclusions: Both AcpP peptide-PMO and AcpP PMO significantly reduced bacteraemia and promoted survi