JAC Advance Access originally published online on October 31, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):66-73; doi:10.1093/jac/dkl444
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Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose–response in mice infected with Escherichia coli
1 AVI BioPharma, Inc. Corvallis, OR, USA 2 The Department of Microbiology, Oregon State University Corvallis, OR, USA
Received 1 August 2006; returned 10 September 2006; revised 19 September 2006; accepted 8 October 2006
*Correspondence address. Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331-3804, USA. Tel: +1-541-737-1845; Fax: +1-541-737-0496; E-mail: gellerb{at}orst.edu
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 ß-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 x 30 µg (3 mg/kg) of AcpP peptide-PMO or 2 x 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 x 300 µg and 2 x 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 x 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 x 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 survival of mice infected with E. coli W3110. The conjugate was about 50–100 times more potent than the PMO without attached peptide. The L-isomeric peptide-PMO was 10 times more potent than the D-isomeric equivalent. The conjugate apparently was toxic at doses 
2 x 300 µg/mouse (30 mg/kg). PMOs produced a sequence-specific antibiotic effect and the conjugate had a therapeutic index (toxic dose/effective dose) approximately equal to 10 in a mouse model of infection.
Keywords: antibiotics , PMOs , E. coli , antisense therapeutics , peptide conjugates
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