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 membrane-penetrating peptide (RXR)(4)XB (X is 6-aminohexanoic acid; B is beta-alanine). RESULTS: MICs (microM/mg/L) were measured using E. coli: 3 + Pip-AcpP, 160/653; 6 + Pip-AcpP, 160/673; 2 + Gux-AcpP, 20/88; 5 + Gux-AcpP, 10/49; 8 + Gux-AcpP, 10/56; 3 + Pip-AcpP-(RXR)(4)XB, 0.3/2; and 5 + Gux-AcpP-(RXR)(4)XB, 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 3 + Pip-AcpP and 6 + Pip-AcpP were reduced to 0.6 and 2.5 microM (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 + Pip-AcpP, 15 mg/kg 5 + Gux-AcpP or 0.5 mg/kg 3 + Pip-AcpP-(RXR)(4)XB, or subcutaneous treatment with 15 mg/kg 5 + Gux-AcpP or (RXR)(4)XB-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 5 + Gux-AcpP or (RXR)(4)XB-AcpP entered the circulatory system, reduced infection and increased survival.

Chemical modifications of antisense morpholino oligomers enhance their efficacy against Ebola virus infection.

Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.

Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity.

Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-alpha amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP-PMOs. Insertion of 6-aminohexanoic acid (X) or beta-alanine (B) residues into oligoarginine R8 decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 microM. Up to 60 microM, only the conjugates with > or = 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP-PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues.

Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate.

OBJECTIVE: Conjugation of arginine-rich cell-penetrating peptide (CPP) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance cytosolic and nuclear delivery of PMO. However, the in vivo disposition of CPP-PMO is largely unknown. In this study, we investigated the pharmacokinetics, tissue distribution, stability, and safety profile of an anti-c-myc PMO conjugated to the CPP, (RXR)4 (X = 6-aminohexanoic acid) in rats. METHODS: The PMO and CPP-PMO were administrated intravenously into rats. The concentrations of the PMO and the CPP-PMO in plasma and tissues were monitored by HPLC. The stability of the CPP portion of the CPP-PMO conjugate in rat plasma and tissue lysates was determined by mass spectrometry. The safety profile of the CPP-PMO was assessed by body weight changes, serum chemistry, and animal behavior. RESULTS: CPP conjugation improved the kinetic behavior of PMO with a 2-fold increase in the estimated elimination half-life, a 4-fold increase in volume of distribution, and increased area under the plasma concentration vs time curve. Consistent with the improved pharmacokinetic profile, conjugation to CPP increased the uptake of PMO in all tissues except brain, varied between organ type with greater uptake enhancement occurring in liver, spleen, and lungs. The CPP-PMO conjugate had greater tissue retention than the corresponding PMO. Mass spectrometry data indicated no observable degradation of the PMO portion, while there was identifiable degradation of the CPP portion. Time-dependent CPP degradation was observed in plasma and tissue lysates, with the degradation in plasma being more rapid. The pattern of degraded products differed between the plasma and lysates. Safety evaluation data showed that the CPP-PMO was well-tolerated at the dose of 15 mg/kg with no apparent signs of toxicity. In contrast, at the dose of 150 mg/kg, adverse events such as lethargy, weight loss, and elevated BUN (p < 0.01) and serum creatinine (p < 0.001) levels were recorded. Supplementation with free L-arginine ad libitum showed improved clearance of serum creatinine (p < 0.05) and BUN (p < 0.01) at the toxicological dose, suggesting that the CPP caused toxicity in kidney. CONCLUSION: This study demonstrates that conjugation of CPP to PMO enhances the PMO pharmacokinetic profile, tissue uptake, and subsequent retention. Therefore, when dosed at < or = 15 mg/kg, CPP is a promising transporter for enhancing PMO delivery in therapeutic settings.

Stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells.

Cell penetrating peptides (CPPs) have been shown to enhance the cellular uptake of antisense oligonucleotides (AOs). However, the effectiveness of the CPPs for cytoplasmic or nuclear delivery of therapeutic AOs must take into account the possible entrapment of the CPP-AO conjugates in endosomes/lysosomes and the overall stability of the CPP-AO conjugates to enzymes. This includes the stabilities of the CPPs and AOs themselves as well as the linkage between them. In this study, we investigated the effects of several structural features of arginine-rich CPPs on the metabolic stability of CPP conjugated to phosphorodiamidate morpholino oligomers (PMOs) in human serum and in cells. Those structural features include amino acid configurations (d or l), incorporation of non-alpha-amino acids, peptide sequences, and types of linkages between CPPs and PMOs. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that the stability of the CPP portion was varied although the PMO portion of the conjugate was completely stable both in cells and in human serum. d-Configuration CPPs were completely stable, while l-CPPs were degraded in both serum and HeLa cells. Insertions of 6-aminohexanoic acid residues (X) into an R8 peptide increased the corresponding CPP's serum stability with the degree of stability being dependent upon the positions of X. However, X-containing CPPs were degraded rapidly intracellularly. Insertions of beta-alanines (B) into the R8 peptide increased its serum stability and intracellular stability. An amide or a maleimide linkage was stable in both serum and cells; however, an unhindered disulfide linkage was not stable in either. By using fluorescent microscopy, flow cytometry, and an antisense splice correction assay, the cellular uptakes of an X-containing conjugate and its fragments were compared to their antisense activities. We found that a large fraction of the conjugate was trapped within vesicles and the degraded fragments cannot escape from the vesicles. This study indicates that the incorporation of non-alpha-amino acids into l-CPPs can increase the metabolic stability of CPP-PMOs without using costly d-CPPs. However, the position and type of non-alpha-amino acids affect the degree of stability extracellularly and intracellularly. In addition, this study reveals that the degradation of an X-containing CPP-PMO conjugate is a more rapid process than degradation of a B-containing conjugate. Last, the endosomal/lysosomal trapping limits the effectiveness of a CPP-PMO conjugate, and the stability of the CPP is one of the factors affecting the ability of the conjugate to escape the endosomes/lysosomes.

Gene-specific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture.

The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is beta-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to luc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 h of growth (50% inhibitory concentration [IC(50)]) were 3.6, 10.8, 9.5, and 7.5 microM, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 microM AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC(50) of either AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 microM. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.