Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugate: Dose-Response in Mice Infected with Escherichia coli Lucas Tilley 1, Patrick Iversen 1 and Bruce L. Geller 1,2 1 AVI BioPharma, Inc., and 2 Department of Microbiology, Oregon State University, Corvallis, Oregon F Contact Info: Bruce Geller Tel: ABSTRACT Background: Phosphorodiamidate morpholino oligomers (PMOs) are DNA analogs that inhibit translation by an antisense mechanism. Membrane penetrating peptides attached to PMOs increase PMO efficacy by enhancing penetration through bacterial membranes. Methods: An 11-base PMO (AcpP) targeted to acpP (an essential gene) of E. 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 PMO-peptide conjugate, or PMO without attached peptide. Results: 12 h after a single treatment, 30 µg of AcpP PMO-peptide or 3 mg of AcpP PMO reduced bacteremia by 3 orders of magnitude compared to treatment with water. Neither scrambled base sequence PMO controls nor 30 µg ampicillin reduced bacteremia. Two treatments with 30 µg AcpP PMO-peptide were more effective than four treatments with 15 µg at 15 min, 4 h, 8 h, and 12 h. Survival 48 h after treatment with 2  30 µg AcpP PMO-peptide or 2  3 mg AcpP PMO was 100 %, compared to 25 % for mice treated with water or scrambled base sequence PMO controls. Doses greater than 2  300 µg PMO- peptide apparently caused toxicity and mortality. 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. Conclusion. Both AcpP PMO-peptide and AcpP PMO significantly reduced bacteremia and promoted survival of mice infected with E. coli. The conjugate was about 100  more potent than the PMO without attached peptide. The L-isomeric peptide-PMO was 10  more potent than the D-isomeric equivalent. The conjugate was apparently toxic at doses greater than 2  300 µg/mouse. PMOs produced a sequence-specific antibiotic effect with a reasonable therapeutic index in a mouse model of infection. INTRODUCTION Phosphorodiamidate morpholino oligomers (PMO, Fig. 1) are short (about base), synthetic analogues of DNA that inhibit gene expression by an antisense mechanism of inhibition. PMOs are made with the 4 naturally occurring bases, but have a different linkage between the bases that makes them resistant to nucleases while maintaining the architecture required for complementary base pairing. Entry of PMOs into E. coli is limited by the outer membrane, which excludes solutes greater than about 600 Da. The molecular weight of an 11-base PMO is about ~4 kDa. Improvements in efficacy of antisense oligomers have been made by covalently attaching membrane-penetrating peptides. Our group has shown that attaching peptides such as RFFRFFRFFXB significantly increases the efficacy of PMOs in pure culture. In this report, we demonstrate in infected mice a sigmoidal dose- response of a peptide-PMO, establish a benchmark minimal inhibitory dose, compare different schedules of treatment, compare the D- and L- isomer of the peptide conjugate, and show sequence specificity of the antisense effect. Fig. 1. PMO and Peptide-PMO structures. Base = adenine, thymine, guanine, or cytosine. Peptide-PMO has RFFRFFRFFXB attached by its carboxyl end to the 5’ end of PMO via an amide linkage with piperazine. X is 6-aminohexanoic acid and B is β- alanine. RESULTS Figure 2. Dose-Response AcpP (RFF)3RXB-PMO. Groups of 3-5 BALB/c mice were infected ip with K-12 E. coli W3110 and treated ip at 15 min and 12 h post-infection (arrows) with: H 2 O; AcpP peptide-PMO 10 µg, 30 µg, 100 µg, 300 µg, or 1000 µg; ampicillin 30 µg, or 1000 µg; or scrambled peptide-PMO 10 µg, 30 µg, 100 µg, 300 µg, or 1000 µg. Error bars indicate standard error of the mean. A. Blood was collected and plated for bacteria at the indicated times post-infection. B. Survival was recorded at the indicated times post- infection. Figure 3. Compare 2 x 30 with 4 x 15 µg. Mice (n = 5-9) were infected and treated ip at 15 min and 12 h post-infection (arrow heads) with 30 µg AcpP (RFF) 3 RXB-PMO, or at 15 min, 4 h, 8 h, and 12 h post infection (arrow heads and arrows) with 15 µg AcpP peptide-PMO, or H 2 O. Error bars indicate standard error of the mean. A. Blood was collected and plated for bacteria at the indicated times post-infection. B. Survival was recorded at the indicated times post-infection. Discussion The results establish a minimal effective dose in mice of 1.5 mg/kg (30 µg/20 g mouse) AcpP (RFF)3RXB-PMO. This is equal to 270 nmol/kg (5.4 nmol/mouse). Based on conventional pharmaceutical methods of converting animal dosage, we calculate that 8 mg/day (1.4 µmol/day) may be a feasible minimal therapeutic dose for a 65 kg human. AcpP peptide-PMO promoted survival at some but not all doses tested. At the lower doses tested (10 to 100 µg), length of time to death was either increased, or mice survived to the end of the experiment (48 h). However, at doses of 300 µg or higher, survival decreased inversely with the amount of peptide-PMO given, despite reducing bacteria in the blood. The results suggest that high doses of AcpP (RFF)3RXB-PMO were toxic. In this model, a therapeutic index (toxic dose/effective dose) is about 10. AcpP (RFF)3RXB-PMO was found to be more potent than ampicillin. Clearly 30 µg of AcpP peptide-PMO reduced blood cfu and promoted survival, whereas the same weight of ampicillin did not. Because the molecular weight of AcpP (RFF)3RXB-PMO is 15 times greater than ampicillin, we conclude that the AcpP peptide-PMO is at least 15 times more potent than ampicillin. AcpP PMO without peptide also reduced infection. However, PMO required a minimum of 3 mg (150 mg/kg) doses to reduce blood cfu under the experimental conditions, which is 100 times less potent than the conjugate with the same base sequence. Nevertheless, survival was significantly increased at all doses of non-conjugated PMO compared to mice treated with scrambled base sequence PMO or H2O. Perhaps 1 mg or less of PMO reduced bacterial growth without killing the bacteria, which may have reduced exposure to lipopolysaccharide or other toxic bacterial components. At the highest dose tested (2  3 mg) survival was 100 % and there was no indication of toxicity. The results show that the L -isomer conjugate was significantly more effective than the D -isomeric equivalent. One possible explanation is that proteolytic removal of the L-peptide inside bacterial cell prevents reversal of entry and increases intracellular concentration. Another is that the mechanism of uptake across bacterial or eukaryotic cell membranes favors the L -isomeric form. CONCLUSIONS 1.Both AcpP PMO-peptide and AcpP PMO significantly reduced bacteremia and promoted survival of mice infected with E. coli. The conjugate was about 100  more potent than the PMO without attached peptide. 2.The L-isomeric peptide-PMO was 10  more potent than the D-isomeric equivalent. 3.The conjugate was apparently toxic at doses greater than 2  300 µg/mouse. 4.PMOs produced a sequence-specific antibiotic effect with a therapeutic index approximately equal to 10 in a mouse model of infection. Figure 4. Dose-Response AcpP PMO. Mice (n = 2-4) were infected and treated ip at 15 min and 12 h post- infection (arrows) with 100 µg, 300 µg, 1 mg, or 3 mg AcpP PMO (open symbols), Scr PMO (filled symbols), or H 2 O (open square). A. Blood was collected and plated for bacteria at the various times indicated. B. Survival was recorded at the indicated times post-infection. Error bars indicate standard error of the mean. Figure 5. Dose-Response AcpP D- (RFF)3RXB-PMO. Groups of 3-8 mice were infected ip with K-12 E. coli W3110 and treated ip at 15 min and 12 h post-infection (arrows) with: H 2 O; AcpP D -(RFF)3RXB-PMO at 3 µg, 10 µg, 30 µg, 100 µg, or 300 µg; or scrambled D -(RFF)3RXB-PMO at 3 µg or 300 µg. Error bars indicate standard error of the mean. A. Blood was collected and plated for bacteria at the indicated times post-infection. B. Survival was recorded at the indicated times post-infection.