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Published on: February 5, 2021
Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose-response in mice infected with Escherichia
Lucas D Tilley1, Brett L Mellbye, Susan E Puckett
1AVI BioPharma, Inc., Corvallis, OR, USA.
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 (i.p.) injection with K-12 E. coli W3110, and treated i.p. 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 microg 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 microg of ampicillin reduced bacteraemia. Two treatments with 30 microg of AcpP peptide-PMO reduced cfu significantly more than four treatments with 15 microg at 15 min, 4, 8 and 12 h. Mice treated with doses of AcpP peptide-PMO > 30 microg showed further reductions in plasma cfu. Survival 48 h after treatment with 2 x 30 microg (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 microg 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 microg 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 microg 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 > or = 2 x 300 microg/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.
Insights
Peptide-conjugated phosphorodiamidate morpholino oligomers (PMOs) show potent antibacterial activity against E. coli, significantly reducing bacterial load and improving survival in mice. The conjugate demonstrated gene-specific efficacy and a therapeutic index of approximately 10.
Area of Science:
- Antimicrobial drug development
- Molecular biology
- Genetics
Background:
- Phosphorodiamidate morpholino oligomers (PMOs) are DNA analogues that inhibit bacterial translation via an antisense mechanism.
- Attaching membrane-penetrating peptides to PMOs enhances their efficacy by improving bacterial membrane penetration.
Purpose of the Study:
- To demonstrate the gene-specific efficacy of a peptide-PMO conjugate.
- To establish a dose-response relationship for the peptide-PMO conjugate.
Main Methods:
- Synthesized an 11-base PMO (AcpP) targeting the essential E. coli gene acpP and conjugated it with the cell-penetrating peptide RFFRFFRFFRXB.
- Infected mice with E. coli and treated them with varying doses of the AcpP peptide-PMO conjugate, AcpP PMO alone, scrambled PMOs, or ampicillin.
- Utilized a modified E. coli strain (LT1) with altered acpP sequence to assess sequence-specific effects.
Main Results:
- A single 30 microg dose of AcpP peptide-PMO reduced bacteraemia by 3 orders of magnitude, outperforming AcpP PMO alone and ampicillin.
- The peptide-PMO conjugate was 50-100 times more potent than the PMO without the peptide.
- Sequence specificity was confirmed, as the conjugate was ineffective against the modified E. coli strain (LT1), while a complementary PMO (AcpPmut4) was effective.
Conclusions:
- AcpP peptide-PMO and AcpP PMO significantly reduced bacteraemia and improved survival in a mouse model of E. coli infection.
- The peptide-PMO conjugate exhibited enhanced potency and sequence-specific antibacterial activity.
- The conjugate demonstrated a therapeutic index of approximately 10, but showed toxicity at higher doses (≥ 30 mg/kg).
