JAC Advance Access originally published online on October 31, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):66-73; doi:10.1093/jac/dkl444
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Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose–response in mice infected with Escherichia coli
1 AVI BioPharma, Inc. Corvallis, OR, USA 2 The Department of Microbiology, Oregon State University Corvallis, OR, USA
*Correspondence address. Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331-3804, USA. Tel: +1-541-737-1845; Fax: +1-541-737-0496; E-mail: gellerb{at}orst.edu
Received 1 August 2006; returned 10 September 2006; revised 19 September 2006; accepted 8 October 2006
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Abstract |
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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 ß-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 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 µg 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 µg of ampicillin reduced bacteraemia. Two treatments with 30 µg of AcpP peptide-PMO reduced cfu significantly more than four treatments with 15 µg at 15 min, 4, 8 and 12 h. Mice treated with doses of AcpP peptide-PMO >30 µg showed further reductions in plasma cfu. Survival 48 h after treatment with 2 x 30 µg (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 µg 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 µg 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 µg 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 
2 x 300 µg/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.
Keywords: antibiotics , PMOs , E. coli , antisense therapeutics , peptide conjugates
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Introduction |
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Antisense therapeutics are a group of emerging technologies with great potential for drug development. The strategy common to all antisense technologies is to manipulate expression of genes in a sequence-specific manner. Antisense technology provides a flexible platform to target and manipulate any gene with known sequence. Many antisense therapeutics have entered human clinical testing, and already one (Vitravene, Isis Pharmaceuticals, Inc., Carlsbad, CA, USA) has gained United States Food and Drug Administration approval for use in humans.
Antibacterial antisense therapeutics are in the pre-clinical stage of development.1 Recent published reports have focused on two types of antisense compounds: phosphorodiamidate morpholino oligomers (PMO) and peptide nucleic acids (PNA). Both PMOs and PNAs inhibit gene expression in pure cultures.2,3 When antisense oligomers are targeted to genes required for viability, bacterial growth is inhibited.4–6
Recently, an antisense oligomer targeted at an essential bacterial gene was shown for the first time to reduce bacterial infection in mice.7 That report established the efficacy of antisense antibiotics in animal infection and demonstrated sequence specificity. The potency of the antisense PMO was greater than ampicillin on a molar basis. The unexpected result was that the PMO inhibited bacteria better in vivo than in pure culture. Nevertheless, the reduction of bacteria in infected mice was modest (about 1 order of magnitude) and only 1 dose of PMO was tested (300 µg).
Improvements in efficacy of antisense oligomers have been made by covalently attaching membrane-penetrating peptides. Good and colleagues have shown that the peptide KFFKFFKFFK dramatically increases the antisense effects of an attached PNA.5,8 Our group has also demonstrated the beneficial effects of attaching the same peptide, a similar peptide or different peptides to PMOs.2,9 Recently a KFFKFFKFFK-PNA was reported to reduce the level of bacteria in the blood of infected mice.10
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.
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Antisense oligomers and peptides
PMOs, peptides and peptide-PMO conjugates were synthesized, purified and analysed at AVI BioPharma as described previously.9 The sequences of PMOs (5'
3') are: AcpP, CTTCGATAGTG; scrambled base sequence control, TCTCAGATGGT; AcpPmut4, CCTCAATGGTA. The peptide sequence is RFFRFFRFFRXB, where X is 6-aminohexanoic acid and B is ß-alanine.
Bacteria
Escherichia coli W3110 or LT1 were prepared for infection by growing in Luria–Bertani (LB) broth11 at 37°C to an optical density (600 nm) = 0.15, concentrating by centrifugation to 
7 x 109 cfu/mL, and then diluting to a final concentration of 1.5 x 108 cfu/mL (SD = 3.2 x 107, n = 6) in 5% mucin (type III, Sigma Chemical Co., St Louis, MO, USA)/phosphate-buffered saline (PBS) as described previously.7
Allelic replacement
A new strain (LT1) was constructed from strain W3110 by mutating acpP using the lambda Red method as described previously.12 A synthetic DNA was purchased from Blue Heron Biotechnology (Bothell, WA, USA) that encoded 88 bases upstream and 108 bases of the coding region of acpP of K-12 E. coli. Four substitution mutations were encoded at wobble positions as follows: 5'-ATGAGTACCATTGAG-3', where the start codon is shown in italics and the substitutions in bold font. SmaI restriction sites were also included at each end. The synthetic DNA was cut from pUC19, gel purified using QIAquick gel extraction kit (Qiagen, Valencia, CA, USA), and used instead of the PCR product during the lambda Red procedure. After 1 h of recovery, the 100 µL of transformation culture was spread on LB plates (100 mm) to which 50 µL of 3 mM AcpP (RFF)3RXB-PMO had been applied to the surface. AcpP (RFF)3RXB-PMO-resistant mutants were picked from the selection plate and acpP was sequenced as described previously13 using PCR primers 5'-AACGTAAAATCGTGGTAAGACC-3' and 5'-TTACGCCTGGTGGCCGTTGATG-3'.
Mouse infection
An objective criterion of terminal illness (low body temperature) was established as described previously,14 using 6–8 week-old-female BALB/c mice (Simonsen Labs, Inc., Gilroy, CA, USA) and E. coli W3110. A threshold body temperature of 27.9°C was found to predict terminal illness and ensuing death. Body temperature was measured with a Braun Pro 4000 infrared tympanic thermometer (Bethlehem, PA, USA). In all subsequent experiments, terminally ill mice were humanely euthanized if their body temperature decreased below 28.0°C and were scored as a death. This protocol was required and approved by the Oregon State University Institutional Animal Care and Use Committee (approval number 3283) and complies with all state and federal laws for the humane care and treatment of animals.
Groups of 3–6, female, 6–8 week old BALB/c mice were infected by intraperitoneal (ip) injection with 0.1 mL of bacteria (either E. coli W3110 or LT1) containing 1.5 x 107 (SD = 3.2 x 106) cfu in 5% mucin/PBS. Mice were treated at 15 min and 12 h post-infection by ip injection with 0.1 mL of various concentrations of PMO, peptide-PMO, ampicillin or H2O. In one experiment a group of mice was treated at 15 min, 4, 8 and 12 h with AcpP (RFF)3RXB-PMO. Blood (30–50 µL, except at 48 h 350 µL) was collected from the saphenous vein in heparinized tubes as described previously,15 and stored on ice for 1–2 h until plated. Blood was diluted in PBS and spread on LB plates to determine the number of viable bacteria.
MIC
The MIC of each PMO and peptide-PMO was measured according to the broth microdilution method of the CLSI.16
Statistical analysis
Standard deviation, standard error of the mean and non-linear regression analysis were calculated using GraftPad Prism® 6.0 software (GraphPad Software, Inc., San Diego, CA, USA). Non-linear regression analysis of the AcpP (RFF)3RXB-PMO was calculated using sigmoidal dose–response (non-variable slope) without constraints. Non-linear regression analysis of the non-conjugated AcpP PMO was calculated using the sigmoidal dose–response (non-variable slope) with an assumed lower limit of 1.0 log cfu/mL.
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MICs in pure culture
MICs in pure cultures of E. coli W3110 were established for all peptide-PMOs and PMOs used in the mouse experiments described below. In a previous work,9 inhibitory concentrations were reported for peptide-PMO conjugates with a slightly different amino acid sequence (RFFRFFRFFXB) than the one used for this report (RFFRFFRFFRXB). The additional R nearest to the carboxy terminus increased the water-solubility of the conjugate, particularly in media with physiological concentrations of salts. The results show that the MICs of AcpP (RFF)3RXB-PMO and AcpP (RFF)3XB-PMO were the same (2.5 µM), the scrambled base sequence peptide-PMOs did not inhibit growth at any concentrations tested, and the MIC of AcpP PMO without attached peptide was greater than the highest concentration tested (Table 1).
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Dose–response and toxicity of AcpP peptide-PMO
Mice were infected ip with K-12 E. coli W3110 and treated at 15 min and 12 h post-infection with various doses of AcpP (RFF)3RXB-PMO, scrambled base sequence (RFF)3RXB-PMO or ampicillin. Blood was collected at various intervals and plated to determine viable bacteria in the blood. The results show that bacterial cfu in the blood was inversely proportional to the amount of peptide-PMO given (Figure 1a). The lowest effective dose of AcpP peptide-PMO that was tested was 30 µg, which reduced cfu by 3 orders of magnitude compared with controls at 12 h post-infection. Bacteria in the blood were undetected at 24 h after 2 x 300 µg, or at 12 h after a single dose of 1 mg of AcpP peptide-PMO. In comparison, 2 x 30 µg of ampicillin did not reduce cfu at any time post-infection. Mice treated with 2 x 1 mg of ampicillin showed no detectable cfu in the blood at any time. None of the doses of scrambled peptide-PMO reduced cfu.
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A non-linear regression analysis of the 12 h response as a function of dose is shown in Figure 2. The data form a good fit and are consistent with a sigmoidal curve (R2 = 0.952). The single dose of AcpP peptide-PMO required to reduce cfu in the blood by 1 or 2 orders of magnitude was 6.6 or 19.4 µg/mouse, respectively.
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Survival was significantly affected by treatment (Figure 1b). Survival was 0–20% for mice treated with water or all doses of scrambled peptide-PMO from 2 x 10 µg to 2 x 1 mg. All mice treated with 2 x 30 or 2 x 100 µg of AcpP peptide-PMO survived. Survival of mice treated with 2 x 10 µg, 2 x 300 µg or 2 x 1 mg of AcpP peptide-PMO was 33%, 75% and 0%, respectively. Survival of mice treated with 2 x 30 µg or 2 x 1 mg of ampicillin was 0% and 100%, respectively.
Two versus four treatments
The hypothesis was tested that two treatments of 30 µg each were more effective than four treatments of 15 µg each. Infected mice were treated at either 15 min and 12 h post-infection with 30 µg, or 15 min, 4, 8 and 12 h post-infection with 15 µg of AcpP peptide-PMO. The results show that 2 x 30 µg significantly reduced blood cfu at 12 h post-infection, whereas 4 x 15 µg did not reduce cfu compared with mice treated with H2O (Figure 3a).
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Survival was also increased by treatment with 2 x 30 µg compared with 4 x 15 µg of AcpP peptide-PMO (Figure 3b). By 24 h post-infection, all of the mice treated with 2 x 30 µg survived, whereas 50% of the mice treated with 4 x 15 µg survived. Although one of the three mice treated with 2 x 30 µg died at 48 h, four of the six mice treated with 4 x 15 µg had died at the end of the experiment (48 h).
Dose–response of AcpP PMO
Mice were infected as described above, and then treated with various doses of PMOs without the peptide. The results show that bacterial cfu in the blood was significantly reduced with only the highest dose (2 x 3 mg) of AcpP PMO tested (Figure 4a). AcpP PMO did not significantly reduce cfu in the blood at any dose from 2 x 100 µg to 2 x 1 mg. Scrambled sequence PMOs did not reduce cfu at any dose tested. Non-linear regression analysis (sigmoidal dose–response, R2 = 0.98) predicts a 1 or 2 log reduction of cfu in the blood with 0.38 or 1.32 mg/mouse, respectively (data not shown).
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Survival increased in mice treated with AcpP PMO (Figure 4b). All mice treated with 2 x 3 mg of AcpP PMO survived until the end of the experiment (48 h). All mice treated with any dose of AcpP PMO from 2 x 100 µg to 2 x 1 mg survived for 24 h. All mice treated with any dose of scrambled PMO or H2O did not survive beyond 12 h.
D-(RFF)3RXB
Infected mice were treated with the AcpP D-(RFF)3RXB-PMO conjugate made with D-isomers of R and F. Treatment with the highest dose tested (2 x 300 µg) reduced blood cfu by about 3 orders of magnitude at 2, 6 and 8 h post-infection, and by about 4 orders of magnitude at 24 h post-infection, compared with treatment with H2O (Figure 5a). None of the lower doses of AcpP D-peptide-PMO reduced blood cfu below scrambled D-peptide-PMO controls or H2O.
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Survival results paralleled those of blood cfu. All mice treated with 2 x 300 µg of AcpP D-peptide-PMO survived 48 h (Figure 5b). None of the mice treated with H2O, lower doses of AcpP D-peptide-PMO or scrambled D-peptide-PMO survived beyond 24 h.
LT1 mutant
The gene-specific effect of AcpP peptide-PMO was shown in the following manner: the single chromosomal acpP allele of E. coli W3110, which was used in the previous experiments, was replaced with an allele (acpP10) that has substitutions in four wobble bases of the region complementary to the AcpP peptide-PMO. The AcpP protein in this mutant strain (LT1) has an unaltered amino acid sequence, but its mRNA has a 4-base mismatch with the peptide-PMO. If the effects of AcpP peptide-PMO are sequence specific, then it should have no effect on viability of LT1.
LT1 was grown in pure culture with 20 µM of either AcpP peptide-PMO or AcpPmut4 peptide-PMO, which is complementary to the allele with the 4-base substitutions. The parent strain W3110 (with wild-type acpP) was also grown with one or the other peptide-PMO. Growth was monitored by optical density, and cell viability was measured after 8 h of growth. The results show growth and viability of LT1 was unaffected by AcpP peptide-PMO, but significantly inhibited by AcpPmut4 peptide-PMO (Figure 6a and b). In contrast, growth and viability of W3110 was unaffected by AcpPmut4 peptide-PMO, but significantly inhibited by AcpP peptide-PMO. Neither growth nor viability was inhibited in either strain with 20 µM of a scrambled sequence peptide-PMO (data not shown).
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Mice were infected with LT1 and treated at 15 min and 12 h with 100 µg of either AcpP peptide-PMO or AcpPmut4 peptide-PMO. AcpPmut4 peptide-PMO reduced blood cfu by 2 orders of magnitude at time points from 2 to 12 h post-infection, and about 3 orders of magnitude at 24 h post-infection compared with H2O (Figure 7a). AcpP peptide-PMO did not reduce blood cfu at any time post-infection.
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Survival of mice infected with LT1 and treated with AcpP peptide-PMO was not significantly different from mice treated with H2O, and all mice died by 24 h post-infection (Figure 7b). Seven of eight mice treated with AcpPmut4 peptide-PMO survived to the end of the experiment (48 h).
Groups of mice were infected with W3110 (wild-type acpP allele) instead of LT1 and treated with H2O, AcpP peptide-PMO or AcpPmut4 peptide-PMO. The results show that AcpP peptide-PMO reduced bacterial cfu in the blood by 2–4 orders of magnitude compared with treatment with H2O (Figure 7c). The AcpPmut4 peptide-PMO did not reduce infection.
Survival of mice infected with W3110 showed the opposite pattern as those infected with LT1. Mice treated with AcpP peptide-PMO survived, whereas those treated with AcpPmut4 peptide-PMO or H2O died by 24 h post-infection (Figure 7d).
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The results of dose–response experiments using the mouse model of peritonitis 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,17 we calculate that 8 mg/day (1.4 µmol/day) may be a feasible minimal therapeutic dose for a 65 kg human. Although this pilot study was designed to show feasibility, the results suggest that parenteral administration of PMO or peptide-PMO might be effective for use in bacteraemia caused by E. coli. The efficacy of similar peptide-PMOs in pure cultures of other bacteria such as Salmonella,9 Burkholderia and Pseudomonas (B. Geller, unpublished data) suggests that peptide-PMOs may be developed for a wide range of indications. These data will provide a starting point for pharmacokinetic and toxicological studies.
AcpP peptide-PMO also promoted survival at some but not all doses tested. At the lower doses tested (10–100 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. Similar peptide-PMO conjugates made with arginine and targeted at mouse genes reduce renal function (P. Iversen, unpublished data). However, no toxicity of either the AcpP (RFF)3XB-PMO or the free peptide is evident in tissue culture at 10 µM, which is a higher concentration of peptide-PMO than can reasonably be achieved in serum.9 Preliminary toxicology data show no apparent toxicity and no deaths in uninfected mice treated with 2 x 600 µg of AcpP (RFF)3RXB-PMO (B. Geller, unpublished data).
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. At doses of 2 x 1 mg, AcpP peptide-PMO was nearly as effective as ampicillin in reducing blood cfu. However, because of the apparent toxicity of AcpP (RFF)3RXB-PMO at 2 x 1 mg, a comparison by survival of infection is meaningless.
AcpP (RFF)3RXB-PMO (30 µg equivalent to 5 nmol) was remarkably more potent than a similar peptide-PNA conjugate. Tan et al.10 reported that 100 nmol ip treatment with AcpP (KFF)3K-PNA reduced blood cfu by <1 order of magnitude in mice infected with K-12 E. coli. Moreover, the mice ‘succumbed to infection in a manner similar to (water-treated) controls’. A 500 nmol treatment of AcpP (KFF)3K-PNA resulted in even less reduction of blood cfu than the 100 nmol dose, although survival improved to 60%. The similarity of efficacy in pure cultures between (RFF)3RXB-PMO and (KFF)3K-PNA5,9 and the large apparent difference in efficacy in mice suggests that there might be differences between the two oligomers in serum clearance, tissue distribution, or stability in vivo. Alternatively, differences between the two peptides might account for the difference in efficacies of the peptide-PNA and peptide-PMO in mouse, which for unknown reasons were not reflected in their efficacies in pure cultures.
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. Previously we have shown that 300 µg was effective in reducing bacterial infection in the peritoneum.7 Differences in the bacterial strains used for infection, amounts of inoculum, and methods of measuring cfu most likely account for the difference in effective dose between the two reports.
Despite the lack of reduction in blood cfu at all but the highest dose of PMO tested, survival was significantly increased at all doses compared with mice treated with scrambled base sequence PMO or H2O. One possible explanation is that 1 mg or less of PMO reduced bacterial growth without killing the bacteria, which perhaps reduced exposure to lipopolysaccharide or other toxic bacterial components. At the highest dose tested (2 x 3 mg) survival was 100% and there was no indication of toxicity. Although a therapeutic index cannot be calculated because no apparent toxic dose was achieved, results of toxicity trials on similar PMOs indicate that a therapeutic index is nearly 100.18 These data support the conclusions that the (RFF)3RXB peptide significantly increases the potency of the conjugate and that the toxicity seen with the peptide conjugate is caused by the peptide moiety, not the PMO.
Two 30 µg doses were more effective than 4 x 15 µg. This suggests that higher concentrations of AcpP peptide-PMO are more critical than sustained, lower concentrations.
Further optimization of the dosing regimen will be necessary. It is unclear if the second dose contributed to the lower cfu in the blood or survival. We note that in designing the experiments, timing of the first dose was important. When the first dose was administered 3 h post-infection, all mice died prior to 24 h, including mice treated with 2 x 300 µg of ampicillin (data not shown).
We hypothesized that the peptide made from D-amino acids would increase efficacy of the conjugate. Because D-amino acids are not recognized as substrates for proteases, it was thought that D-isomer conjugates would remain intact longer than their L-isomeric equivalent. However, the results both in pure culture and in infected mice 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 favours the L-isomeric form. Additional tests with D-isomers are warranted, because we have not tested retro-inverso D-peptides, i.e. D-BXR(FFR)3, which would have a different configuration than the isomers tested.
Our results in mice infected with strain LT1 show the gene-specific effect of the antisense technology in vivo. Although the lack of effect with scrambled base sequence peptide-PMOs suggested specificity, off-target inhibition was a formal possibility. However, the complete lack of reduction of blood cfu by the AcpP peptide-PMO in LT1 clearly demonstrates that the reduction of blood cfu by AcpP peptide-PMO in W3110 was sequence-specific.
Non-conjugated PMOs are surprisingly more effective in animals than in pure culture. This has been shown previously,7,9 and is confirmed by the results in Figure 4 and Table 1. The underlying basis for this difference is unknown. However, the environment in vivo is very different to that in pure culture broth. Perhaps the mouse tissues produce a substance that enhances delivery of the PMO into bacteria. Another possibility is that the targeted bacteria express something in animals that they do not in pure culture, and this expression increases the efficacy of the PMO. Regardless of the basis for this, the increased efficacy in the animal was unanticipated from results in pure culture, but a favourable development nonetheless.
In conclusion, a peptide-PMO conjugate has been shown to be a potent antibiotic in a mouse model of infection. A therapeutic index of 10 indicates a 10-fold window of safety at an effective dose. We are optimistic that further refinements in the peptide and target of the PMO can be achieved that will increase the therapeutic index.
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This work was funded by AVI BioPharma, Inc. B. L. G. is an employee of both Oregon State University and AVI BioPharma, Inc. S. E. P. is a student at Oregon State University under the direction of B. L. G.
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Acknowledgements |
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We thank the Chemistry Department at AVI BioPharma for preparing the PMOs and peptide-PMOs. Susan Puckett thanks The Howard Hughes Medical Institute and AVI BioPharma, Inc. for financial support.
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References |
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1 Geller BL. (2005) Antibacterial antisense. Curr Opin Mol Ther 7:109–13.[Web of Science][Medline]
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Geller BL, Deere JD, Stein DA, et al. (2003) Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers. Antimicrob Agents Chemother 47:3233–9.
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Deere J, Iversen P, Geller BL. (2005) Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli. Antimicrob Agents Chemother 49:249–55.
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Tilley LD, Hine OS, Kellogg JA, et al. (2006) 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. Antimicrob Agents Chemother 50:2789–96.
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Tan X-X, Actor JK, Chen Y. (2005) Peptide nucleic acid antisense oligomer as a therapeutic against bacterial infection: proof of principle using mouse intraperitoneal infection. Antimicrob Agents Chemother 49:3203–7.
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18 Iversen. (2001) Phosphorodiamidate morpholino oligomers. In Cooke ST (Ed.). Antisense Drug Technology(Marcel Dekker, Inc., New York) pp. 375–89.
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 J. Meng, H. Wang, Z. Hou, T. Chen, J. Fu, X. Ma, G. He, X. Xue, M. Jia, and X. Luo Novel Anion Liposome-Encapsulated Antisense Oligonucleotide Restores Susceptibility of Methicillin-Resistant Staphylococcus aureus and Rescues Mice from Lethal Sepsis by Targeting mecA Antimicrob. Agents Chemother., July 1, 2009; 53(7): 2871 - 2878. [Abstract] [Full Text] [PDF]
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N. Shen, J.-h. Ko, G. Xiao, D. Wesolowski, G. Shan, B. Geller, M. Izadjoo, and S. Altman Inactivation of expression of several genes in a variety of bacterial species by EGS technology PNAS, May 19, 2009; 106(20): 8163 - 8168. [Abstract] [Full Text] [PDF]
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N. Woodford, D. W. Wareham, and on behalf of the UK Antibacterial Antisense Study Tackling antibiotic resistance: a dose of common antisense? J. Antimicrob. Chemother., February 1, 2009; 63(2): 225 - 229. [Abstract] [Full Text] [PDF]
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B. L. Mellbye, S. E. Puckett, L. D. Tilley, P. L. Iversen, and B. L. Geller Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency against Escherichia coli In Vitro and In Vivo Antimicrob. Agents Chemother., February 1, 2009; 53(2): 525 - 530. [Abstract] [Full Text] [PDF]
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