Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo

Objectives: Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogues that can inhibit bacterial growth by a gene-specific, antisense mechanism. Attaching cationic peptides to PMOs enables efficient penetration through the Gram-negative outer membrane. We hypothesized that cationic groups attached directly to the PMO would obviate the need to attach peptides. Methods: PMOs with identical 11-base sequence (AcpP) targeted to acpP (an essential gene) of Escherichia coli were synthesized with various numbers of either piperazine (Pip) or N-(6-guanidinohexanoyl)piperazine (Gux) coupled to the phosphorodiamidate linker. Peptide– PMO conjugates were made using the membranepenetrating peptide (RXR)4XB (X is 6-aminohexanoic acid; B is b-alanine). Results: MICs (mM/mg/L) were measured using E. coli :3 þPip –AcpP, 160/653; 6þ Pip–AcpP, 160/673; 2 þGux– AcpP, 20/88; 5þGux –AcpP, 10/49; 8 þGux–AcpP, 10/56; 3 þPip –AcpP–(RXR)4XB, 0.3/2; and 5 þGux–AcpP– (RXR)4XB, 0.6/4. In cell-free protein synthesis reactions, all PMOs inhibited gene expression approximately the same. These results suggested that Pip –PMOs inefficiently penetrated the outer membrane. Indeed, the MICs of 3þPip –AcpP and 6 þPip –AcpP were reduced to 0.6 and 2.5 mM (1.2 and 10.5 mg/L), respectively, using as indicator a strain with a ‘leaky’ outer membrane. In vivo, mice were infected intraperitoneally with E. coli. Intraperitoneal treatment with 50 mg/kg 3 þPip –AcpP, 15 mg/kg 5þ Gux–AcpP or 0.5 mg/kg 3 þPip – AcpP –(RXR)4XB, or subcutaneous treatment with 15 mg/kg 5 þGux– AcpP or (RXR)4XB –AcpP reduced bacteria in blood and increased survival. Conclusions: Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux–PMOs were more effective than Pip–PMOs. However, neither was as effective as the equivalent PMO– peptide conjugates. Subcutaneous treatment showed that 5þGux –AcpP or (RXR)4XB – AcpP entered the circulatory system, reduced infection and increased survival.