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

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 beta-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 mu g 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 mu g of ampicillin reduced bacteraemia. Two treatments with 30 mu g of AcpP peptide-PMO reduced cfu significantly more than four treatments with 15 mu g at 15 min, 4, 8 and 12 h. Mice treated with doses of AcpP peptide-PMO > 30 mu g showed further reductions in plasma cfu. Survival 48 h after treatment with 2 x 30 mu g (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 mu g 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 mu g 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 mu g 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 mu g/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.