Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency Against Escherichia coli in vitro and in vivo

The potency of antisense peptide-phosphorodiamidate morpholino oligomers (PPMO) was improved by varying peptide composition. An antisense phosphorodiamidate morpholino oligomer (PMO) complementary to mRNA of the essential gene acpP (encodes acyl carrier protein required for lipid biosynthesis) in E. coli was conjugated at the 5'end to various cationic membrane-penetrating peptides. Each peptide had one of three repeating sequence motifs: C-N-N (motif 1), C-N (motif 2), or C-N-C (motif 3), where C is cationic and N is non-polar. Variations in the cationic residue included arginine, lysine, and ornithine (O). Variations in the non-polar residues included phenylalanine, valine, beta-alanine (B), and 6-amino-hexanoic acid (X). The minimal inhibitory concentration (MIC) of PPMOs varied from 0.625 to >80 microM (about 3 to 480 microg/ml). Three of the most potent were (RX)6B-, (RXR)4XB-, and (RFR)4-AcpP PMO, which were tested further in mice infected with E. coli. (RXR)4XB-AcpP PMO was the most potent of the three conjugates tested in mice. Thirty microg (1.5 mg/kg) (RXR)4XB-AcpP PMO given 15 min post-infection reduced CFU/ml in blood by 10(2) to 10(3) within 2 to 12 h compared to water-treated controls. All mice treated with 30 microg/dose of (RXR)4XB-AcpP PMO survived infection, whereas all water-treated mice died 12 h post-infection. The reduction in blood CFU/ml was proportional to the dose of PPMO from 30 to 300 microg/ml. In summary, the C-N-C motif was more effective than the other two motifs, arginine was more effective than lysine or ornithine, phenylalanine was more effective than 6-aminohexanoic acid in vitro but not necessarily in vivo, and (RXR)4XB-AcpP PMO reduced bacterial infection and promoted survival at clinically relevant doses.